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Dissertations Theses and Dissertations
2010
Polyomavirus Enhancer Activator 3 (PEA3), a Member of the Ets Polyomavirus Enhancer Activator 3 (PEA3), a Member of the Ets
Family of Transcription Factors, Is a Transcriptional Activator of Family of Transcription Factors, Is a Transcriptional Activator of
Notch-1 and Notch-4 in Breast Cancer: An Opportunity for Novel Notch-1 and Notch-4 in Breast Cancer: An Opportunity for Novel
Combinational Therapy Combinational Therapy
Anthony George Clementz Loyola University Chicago
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Recommended Citation Recommended Citation Clementz, Anthony George, "Polyomavirus Enhancer Activator 3 (PEA3), a Member of the Ets Family of Transcription Factors, Is a Transcriptional Activator of Notch-1 and Notch-4 in Breast Cancer: An Opportunity for Novel Combinational Therapy" (2010). Dissertations. 81. https://ecommons.luc.edu/luc_diss/81
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LOYOLA UNIVERSITY CHICAGO
POLYOMAVIRUS ENHANCER ACTIVATOR 3 (PEA3),
A MEMBER OF THE ETS FAMILY OF TRANSCRIPTION FACTORS,
IS A TRANSCRIPTIONAL ACTIVATOR OF NOTCH-1 AND NOTCH-4
IN BREAST CANCER: AN OPPORTUNITY
FOR NOVEL COMBINATIONAL THERAPY
A DISSERTATION SUBMITTED TO
THE FACULTY OF THE GRADUATE SCHOOL
IN CANDIDACY FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
PROGRAM IN MOLECULAR AND CELLULAR BIOCHEMISTRY
BY
ANTHONY G. CLEMENTZ
CHICAGO, ILLINOIS
DECEMBER 2010
Copyright by Anthony G. Clementz, 2010 All rights reserved.
ACKNOWLEDGEMENTS
I am grateful to Dr. Clodia Osipo for her continual support, guidance, and
excellent mentorship. She has taught me how to be a strong scientist and person. When I
entered the laboratory, one of the first things she said to me was, “I am going to teach you
to be a good scientist, and above all else, as a young researcher, I will teach you
patience.” Not only have I learned patience, but the importance of integrity,
communication, and dedication. I must say that Dr. Osipo has gone above and beyond in
her mentorship and is herself an amazing scientist. I am very honored to be her first Ph.D.
graduate student.
I would like to thank my committee members, Dr. Mary Druse-Manteuffel, Dr.
Richard Schultz, Dr. Maurizio Bocchetta, and Dr. Paola Rizzo, for their support and
direction during my doctoral research studies. I would like to recognize Dr. Paola Rizzo,
a dear friend that has guided me not only in science, but also in many areas of my life; I
am ever grateful for our long talks and many laughs. I also would like to thank Dr.
Osipo’s lab personnel, former master’s student Parul Patel, Kinnari Pandya, Allison
Rogowski, and Kathleen Meeke, who are all excellent scientists, great friends, and made
my experience within the lab fun and unforgettable.
I would like to thank the Department of Cell Biology, Neurobiology, and
Anatomy: Division of Molecular and Cellular Biochemistry for educating and assisting
iii
me throughout their program. I would also like to thank Loyola University, the many
faculty, and friends both at Loyola and outside Loyola. I have met many friends and
colleagues that have made my experience here memorable.
Most importantly, I must recognize and thank my dearest parents, Peter and Toni
Clementz, and family, Gina, Peter, Mia, and Andria, who stuck with me through it all,
listened to my stories, and continues to give endless support and love. I have been
blessed to have learned from the greatest people and to them I am truly grateful. I
attribute my gifts to four amazing people in my life and I have carried with me a part of
each of them: among so many other things, my father has taught me determination,
caring, and problem solving; my mother, respect, openness, and consideration. To my
incomparable grandfather, who taught me strength, passion, art, and always saw my
potential before I could even see it in myself; and my grandmother, for her wisdom,
prayer, and hope.
As I evolve into a young scientist, I find that the more I study concerning the vast
molecular infrastructure of the human body and life in general, I find myself humbled by
the intelligent design innate in us. It is within all of us –the beauty and blessings of life.
iv
To my Father and Mother, And for Dorothy and Sebastian
Considering the wonderful frame of the human body, this infinitely complicated engine, in which, to the due performance of the several functions and offices of life, so many strings and springs, so many receptacles and channels are necessary, and all to be in their right frame and order; and in which, besides the infinite, imperceptible and secret ways of mortality, there are so many sluices and flood-gates to let death in, and life out, it is next to a miracle we survived the day we were born.
J. Puckle, 1798
I awoke to the reality that I did not choose science; it chose me.
A.G.Clementz, 2010
vii
PREFACE
Breast cancer continues to be the second leading cause of cancer-related deaths
among women. It is a disease that transcends gender boundaries, arising in both women
and men. Currently, according to the statistics acquired by the National Institute of
Health, there are approximately 200,000 female and 2,000 male new breast cancer cases
in the United States of America. Roughly 41,000 females and 450 males will parish this
year from the disease. However, with the advancement of current research, these
numbers are substantially lower than the past, and the percentage of patients reaching
their five year disease free survival is increasing. The advent of new surgical and
oncological techniques enables patients to receive a lumpectomy, radiation treatment,
targeted chemotherapeutics, and reconstruction or breast-conserving surgery, which not
only leads to increased survival rate, but also aims to regain physically and
psychologically the patient’s former quality of life. Nevertheless, breast cancer is an
aggressive and devastating illness. With continued support from global governments,
commercial industries, and personal conquests, the prospect for a cure is on the horizon.
Eradication of breast cancer is no longer a hopeful wish, but an emerging and tangible
promise.
viii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS………………………………………………………………iii
PREFACE………………………………………………………………………………..vii
LIST OF TABLES…………………………………………………………..……..……xi
LIST OF FIGURES……………………………………………………………………...xii
LIST OF ABBREVIATIONS………………………………………………………….xv
ABSTRACT…………………………………………………………………………….xix
CHAPTER I: AN INTRODUCTION TO BREAST CANCER.…………………….1 Introduction……………………………………………………......………………1 Categorization and Features of Breast Cancers..………………….………….….3
1. Luminal A Breast Cancers…………………………………………….4 2. Luminal B Breast Cancers…………………………………………….5 3. HER2+ Breast Cancers………………………………...……………...6 4. Basal-like, Triple Negative Breast Cancers……………………….…7 5. Normal-like Breast Cancers……………………………………..…..9 6. Claudin-low Breast Cancers………………………………………....10 7. Molecular Apocrine Breast Cancers…………………………………10
Risk Factors for Breast Cancer………………………………………………….11 Treatment Strategies for Breast Cancer…………………………..……………..11
1. Surgery…………………………………………………………….…12 2. Radiation Therapy……………………………………………………13 3. Systemic Therapy……………………………………………….……13
A. Hormone Therapy……………………………………………14 B. Targeted Biological Therapy…………………………...…....16 C. Cytotoxic Chemotherapy……………………………………..17
Conclusions: Breast Cancer Now and the Future……………………………….18
CHAPTER II: NOTCH, PEA3, AND AP-1: A LITERATURE REVIEW……………...21 Notch Signaling……………………………………………………………..21
1. An Overview of Notch Signaling………………………………...….22 2. Notch Receptors…………………………………………………...…25
A. Structural Analysis of the Notch Receptors……….…….25 B. Notch Receptors and Breast Cancer……………………..….27
3. Notch Ligands……………………………………………….…….29 4. Inhibitors of Notch Signaling……………………………..……...29 5. Regulation of Notch Signaling…………………………….…….32
ix
A. Post-transcriptional Regulation of Notch...........................32 B. Transcriptional Regulation of Notch…………….……...33
Polyomavirus Enhancer Activator 3 (PEA3)…………………………………….34 1. Ets Transcription Factors…………………………………………….34 2. Polyomavirus Enhancer Activator 3 (PEA3)………………………...35
A. The Structure of PEA3……………………………………….35 B. Activity and Regulation of PEA3…………………………….36
3. PEA3 and Breast Cancer……………………………………………..36 Activating Protein 1 (AP-1)……………………………………….……...40
1. The c-JUN Family………………………………………………42 A. c-JUN……………………………………………..……...42 B. JunB……………………………………………………….45 C. JunD……………………………………………………….45
2. The c-FOS Family………………………………………………46 A. c-FOS……………………………………………………...46 B. Fra-1………………………………………………………49 C. Fra-2………………………………………………………50 D. FosB……………………………………………………….50
3. AP-1 and PEA3…………………………………………………51 A Systems Approach: Linking Notch, PEA3, & AP-1……………………….…52
CHAPTER III: MATERIALS AND METHODS……..………………………...53
Cell Culture……………………………………………………………….…53 Drugs and Chemicals……………………………………………………….53 Expression Vectors………………………………………………………….54 RNA Interference and Reagents…………………………………………….54 Antibodies………………………………………………………………..…..54 Real-Time RT-PCR……………………………………………………….…56 Western Blot Analysis…………………………………………………….…57 Luciferase Constructs and Assay…………………………………………...59 Chromatin Immunoprecipitation (ChIP)…………………………………...60 Co-Immunoprecipitation (Co-IP)…………………………………………..62 Cell Cycle Analysis………………………………………………………..64 Annexin-V Apoptosis Assay……………………………………………....65 Cell Viability Assay…………………………………………………………66 Colony Formation Assay…………………………………………………..67 Statistical Analysis………………………………………………………….68
CHAPTER IV: RESULTS……………..………………………….……………..69
Hypothesis…………………………………………………….…………….69 Specific AIMS………………………………………………………………70 Specific AIM-1: To Determine Whether PEA3 is an Upstream
Mediator of the Notch Signaling Pathway………………..…..…72 1. PEA3 Regulates Notch-1 and Notch-4 Transcripts…………...75 2. PEA3 Regulates Notch-1Protein Expression…………..........75
x
3. Classic Gene Targets of PEA3 and Notch are Decreased by PEA3 Knockdown………..…………………………………...79
Specific AIM-2: To Elucidate Whether PEA3 Acts as a Direct Positive Regulator of Notch-1 and Notch-4 Transcription………81 1. PEA3 is Enriched on the Notch-1 and Notch-4 Promoters…………..83 2. PEA3 and AP-1 Regulate the Notch-4 Promoter…………………....89 3. PEA3 and AP-1 Dynamically Regulate Notch-4 Transcription……101
Specific AIM-3: To Establish a Biological Significance by Determining Whether Dual Inhibition of Notch and PEA3 Leads to Reduced Cell Proliferation, Tumorigenicity, Migration, and Invasion in vitro………………………………………………107 1. Dual inhibition of Notch and PEA3 Inhibits Cell Proliferation……109 2. Dual Inhibition of Notch and PEA3 Induces Apoptosis……………109 3. Dual Inhibition of Notch and PEA3 Reduces Cell Viability……….111 4. Notch-1 and Notch-4 Overexpression Rescues the Effect of
Dual Notch and PEA3 Inhibition…………….…………….…..111 5. PEA3 Regulates Notch-1 and Notch-4 Expression in other
Subtypes of Breast Cancer………………………………….….117 CHAPTER V: DISCUSSION……………………………………………….….124
The Analysis………………………………………………………………125 The Model………………………………………………………………….135
1. Production…………………………………………………....135 2. Activation…………………………………………………....137
Future Investigations………………………………………………….....138
CHAPTER VI: CLOSING REMARKS…………………..………………….…140
BIBLIOGRAPHY……………………………………………………………...…142
VITA………………………………………………………………………………163
xi
LIST OF TABLES
TABLE PAGE
1. Properties of the Classic Subtypes of Breast Cancer…………………………….20
2. Relative Levels of AP-1 Family Members in Breast Cancer…………………….41
3. Sequences of siRNA(s)…………………………………………………………..55
4. Sequences of PCR Primers………………………………………………………55
5. Chromatin Immunoprecipitation Primers………………………………………..63
LIST OF FIGURES
FIGURE PAGE
1. Anatomy of the Breast…………………………………………………………….2
2. Chemical Structures of Common Estrogenic and Aromatase Inhibitors………...15
3. The Notch Signaling Pathway…………………………………………………...24
4. The Four Mammalian Notch Receptors, Their Ligands, and Their Domains…...26
5. Chemical Structures of Common GSI(s)…………………………………….…..31
6. Protein Structure of Polyomavirus Enhancer Activator 3 (PEA3)………………37
7. AP-1 Members and Dynamic Partnerships………………………………………41
8. Protein Structure of c-JUN and its Dominant Negative Form, TAM-67………...43
9. Protein Structure of c-FOS and its Dominant Negative Form, A-FOS………….47
10. Schematic Representation of Notch-1 and Notch-4 Promoters Labeled to Designate Areas of Primer Design……………………………………….63
11. Hypothesis of the Investigation……………………………………………….…71
12. PEA3 Transcripts Upon RNA Interference……………………………………...73
13. Endogenous PEA3 Protein Upon RNA Interference…………………………….74
14. Effects of PEA3 siRNA on Notch Transcripts…………………………………..76
15. Protein Levels of Notch-1 Upon PEA3 RNA Interference………………………77
16. Protein Levels of Notch-4 Upon PEA3 RNA Interference………………………78
xii
17. Transcript Levels of Classic PEA3 and Notch Targets Upon PEA3 siRNA…….80
18. Schematic Representation of Notch-1 and Notch-4 Promoters………………….82
19. Exogenous Expression of PEA3 and Its Effects on Target Genes……………….84
20. Immunoprecipitation of Endogenous and Exogenous PEA3…………………….85
21. Chromatin Immunoprecipitation of Endogenous and Exogenous PEA3 on the Notch-1 and Notch-4 Promoter…………………………………...86
22. Chromatin Immunoprecipitation of PEA3 on the Notch-1 Promoter……………87
23. Chromatin Immunoprecipitation of PEA3 on the Notch-4 Promoter……………88
24. Western Blot Analysis of PEA3 Expression……………………………………..90
25. PEA3 Enrichment Specificity on the Notch-1 and Notch-4 Promoters as Measured by Chromatin Immunoprecipitation…………………….…….91
26. Wildtype and Mutant Notch-4 Promoter Luciferase Constructs……………...92
27. Luciferase Assay of Wildtype and AP-1 Mutant Notch-4 Promoter………….94
28. PEA3 Does Not Regulate AP-1 Activity………………………………………...95
29. Dominant Negative c-JUN Ablates PEA3 Recruitment on Notch-4 and Has No Effect on PEA3 Recruitment on Notch-1……………………….96
30. Dominant Negative c-JUN (TAM-67) Reduces AP-1 Activity………………….98
31. c-JUN is Required for PEA3 Enrichment on the Notch-4 Promoter…………...99
32. Co-Immunprecipitation of PEA3 in Complex with c-JUN……...……………...100
33. Dominant Negative c-FOS (A-FOS) Has No Effect on PEA3 Recruitment on the Notch-1 and Notch-4 Promoters………………………………...102
34. Dominant Negative c-FOS (A-FOS) Reduces AP-1 Activity………………….103
35. PEA3 and AP-1 Dynamically Regulate Notch-4 Transcription……………….105
36. Notch-1 Transcripts Are Not Effected by AP-1 siRNA Interference………….106
xiii
37. RNA Interference Knockdown of PEA3 and AP-1 Members…………………108
38. Effects of Dual Notch and PEA3 on Cell Proliferation………………………..110
39. Dual Inhibition of Notch and PEA3 Increases Apoptosis……………………...112
40. Dual Inhibition of Notch and PEA3 Decreases Cell Viability…………………113
41. Notch-1 and Notch-4 Overexpression Reduces the Effect of Dual Notch and PEA3 Inhibition …………………………………………….115
42. Quantification of Notch-1 and Notch-4 Rescue on Colony Formation....……...116
43. Relative PEA3 Levels and Knockdown Efficiency in Breast Cancer Subtypes………………………………………………...120
44. PEA3 Regulates Notch in Other Breast Cancer Subtypes ……………………..121
45. Relative AP-1 Activity in Different Breast Cancer Subtypes…………………..122
46. PEA3 Regulates Notch-4 in BT474 and SKBr3 Breast Cancer Subtypes……...123
47. Model of the Transcriptional Regulation of Notch-1 and Notch-4 by PEA3…..136
xiv
xv
LIST OF ABBREVIATIONS
ADAM A disintegrin and metalloprotease
A-FOS Dominant negative c-FOS
AI Aromatase inhibitor
ANK Ankyrin repeats
AP-1 Activator protein 1
APBI Accelerated partial breast irradiation
bZIP Basic leucine zipper domain
ChIP Chromatin immunoprecipitation
CoA Co-activator
Co-IP Co-immunoprecipitation
CoR Co-repressor
COX2 Cyclooxygenase 2
CR Cysteine rich region
Ct Cycle number at threshold
DBD DNA binding domain
DCIS Breast ductal carcinoma in situ
DMEM Dulbecco’s Modified Eagle Medium
DMEM/F12 Dulbecco’s Modified Eagle Medium: Nutrient Mixture F12
xvi
DMSO Dimethyl sulfoxide
EGF-like Epidermal growth factor like repeats
EGFR Epidermal growth factor receptor
ER Estrogen receptor alpha
FBS Fetal bovine serum
FL Notch full length
GSI Gamma-secretase inhibitor
HC Heavy chain IgG isotype control
HER2 Epidermal growth factor receptor 2
HPRT Hypoxanthine-guanine phosphoribosyl transferase
IC Notch intracellular domain
IL-8 Interleukin 8
IMEM Iscove’s Minimal Essential Media
IP Immunoprecipitation
LC Long chain IgG isotype control
LNR Lin12 repeats
MAML-1 Mastermind-like 1
MAPK Mitogen activating pathway kinase
MMP Matrix metalloprotease
MMTV Mouse mammary tumor virus
Mut Mutation
NCR Cytokine response element
xvii
NEAA Non-essential amino acids
NEC Notch extracellular domain
NIC Notch intracellular domain
NLS Nuclear localization signal
NTM Notch transmembrane domain
O-fut O-fucosyl transferase
PARP Poly-ADP-ribose polymerase
PBS Phosphate buffer saline
PCR Polymerase chain reaction
PEA3 Polyomavirus Enhancer Activator 3
PEA3i PEA3 siRNA
PEST Proline glutamic acid serine threonine
PI Propidium iodide
PR Progestrone receptor
PVDF Polyvinylidene fluoride
RFU Relative fluorescence units
RIPA Radioimmunoprecipitation assay buffer
RT-PCR Realtime polymerase chain reaction
SCRBi Scrambled control random siRNA
SDS Sodium dodecyl sulfate
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SERM Selective estrogen receptor modulator
xviii
siRNA Small interfering RNA
SUMO Sumoylation
TACE TNF-alpha converting enzyme
TAD Transactivation domain
TAM-67 Dominant negative c-JUN
TBST Tris buffer saline tween
TM Notch transmembrane portion
uPA Urokinase-type plasminogen activator
VEGF Vascular endothelial growth factor
VEGFR Vascular endothelial growth factor receptor
Wt Wildtype
xix
ABSTRACT
Women diagnosed with triple-negative breast cancer have the worst overall
prognosis and frequently present with metastatic tumors. To date, there are no targeted
therapies available to combat this aggressive form of breast cancer due to the lack of
expression of well-known targets such as ERα, PR, or HER2/neu. Therefore, there is an
immediate need to identify novel targets that are responsible for the proliferation,
survival, and invasive phenotype. Notch-1 and Notch-4, both potent breast oncogenes,
are overexpressed in triple-negative breast cancers-associated with the poorest overall
survival. PEA3 (polyomavirus enhancer activator 3), a member of the Ets family of
transcription factors, is overexpressed in triple-negative breast cancer and also correlates
with aggressive behavior and poor overall survival. Here, we provide new evidence for
transcriptional regulation of Notch in triple negative breast cancer and other subtypes of
breast cancer. Our results showed that PEA3 is a transcriptional activator of both Notch-
1 and Notch-4 genes using a PEA3 siRNA and measuring both transcripts and proteins in
MDA-MB-231 cells where PEA3 levels are endogenously high. In SKBr3 and BT474
breast cancer cells where PEA3 levels are low, exogenous overexpression of PEA3
significantly increased Notch-4 transcripts. Chromatin immunoprecipitation confirmed
enrichment of PEA3 within the promoter regions of both Notch-1 and Notch-4. Notch-1
xx
recruitment appears to be AP-1 independent, whereas PEA3 recruitment on the Notch-4
promoter is dependent upon c-JUN. Furthermore, results showed that either c-Jun or Fra-
1 are required for Notch-4 transcription while c-FOS was a repressor. Importantly, the
combined inhibition of Notch signaling via a gamma-secretase inhibitor (GSI) and
knockdown of PEA3 (via siRNA) significantly growth arrested breast cancer cells in G1
and decreased both anchorage dependent and independent growth in vitro. In correlation,
a significant decrease in cell viability and tumorigenicity, as well as an increase in
apoptotic cells were observed when a GSI was combined with PEA3 siRNA.
Interestingly, a combined inhibition of Notch signaling and PEA3 showed no significant
changes in invasion and migration. Taken together, results from this study suggest for
the first time that Notch-1 and Notch-4 are novel transcriptional targets of PEA3 in breast
cancer cells. PEA3-mediated Notch-1 transcription is AP-1 independent while Notch-4
transcription requires both PEA3 and AP-1 most probably composed of the c-Jun:Fra-1
complex. Moreover, both PEA3 and Notch signaling are essential for proliferation and
survival of MDA-MB-231 breast cancer cells. Thus, dual targeting of both PEA3 and
Notch pathways might provide a new therapeutic strategy for triple-negative breast
cancer as well as additional therapeutic targeting in other breast cancer subtypes.
CHAPTER I
AN INTRODUCTION TO BREAST CANCER
Introduction
Breast cancers are thought to originate from a small number of cancer cells that
are capable of self renewal and pluripotency. These breast cancer “stem cells” are capable
of initiating and sustaining breast tumor growth. The cancer-initiating cells or cancer
stem cells were originally identified in hematological malignancies but is now being
recognized in breast and several other solid tumors (Subramaniam et al., 2010). The
normal breast anatomy contains lobular glands that produce milk, the luminal ducts that
carry the milk to the nipple, and the surrounding environment consisting of adipose,
connective, and lymphatic tissue (Figure 1). Transformed stem cells lose their internal
proliferation checkpoints and develop into a benign palpable mass or neoplastic tumor.
When tumor cells further transform into migratory cells or under epithelial-to-
mesenchymal transition (EMT), they become invasive or infiltrating breast cancers
distinguishing them from benign or breast cancer in situ. The breast undergoes multiple
remodeling events similar to EMT throughout a woman’s lifetime and most notably
during pregnancy. Multiple steps during development and pregnancy could be targets of
mutation and subsequent transformation where cancer may develop. Consequently,
1
2
DuctsAreolaNipple
Connective tissueAdipose tissue
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Connective tissueAdipose tissue
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Figure 1: Anatomy of the Breast.
3
breast cancer assumes diverse intrinsic gene patterns leading to the formation,
identification, and classification of particular molecular subtypes (Micalizzi et al., 2010)
Categorization and Features of Breast Cancers
Breast cancer is a heterogeneous disease. Molecular and pathological portraits of breast
cancer provide insight into the vast genetic and phenotypic varieties that may present in
the clinic. A hierarchical division among breast cancer using a both pathological and
genetic analysis of expression have been formulated (Perou et al., 2000). They
subdivided breast cancer into four minimal subtypes: luminal, HER2, normal breast-like,
and basal-like. Soon after, further investigations divided the luminal subtype into luminal
A and luminal B based on gene profiling and clinical outcomes (Sorlie et al., 2001). The
classification of tumors stemming from the lumen or the myoepithelial (basal) layer was
determined by immunohistochemistry on sections of patient samples using antibodies to
keratin 5,8,17, and 18 (Perou et al., 2000). Those tumors that showed positive keratin 8
and 18 staining were considered of luminal origin and those that were positive for keratin
5 and 17 were derived from the basal-epithelial layer. Additionally, whether or not there
was presence of gene expression patterns of the estrogen receptor and its corresponding
hormone responsive genes, aided in the categorization of luminal versus basal-like
cancers, respectively. Further classifications are continuing to emerge such as the
molecular apocrine and claudin-low breast cancer subtypes. Although segregated into
different categories, sometimes the classification of breast cancer subtypes share common
gene expression and tend to overlap. However, four classic subtypes have
4
withstood the test of time, and are commonly referenced: luminal A, luminal B, HER2,
and basal-like (including triple negative) (Table 1) (Kao et al., 2009).
1. Luminal A Breast Cancers
The luminal A subtype makes up approximately 60% of breast cancers and
originates from the cells that nest within the lumen of the breast that have undergone
oncogenic transformation. They are classically hormone receptor positive, that is
estrogen receptor alpha positive (ERα+) and progesterone receptor positive (PR+) (Sorlie
et al., 2001). They are negative for the overexpression or gene amplification of HER2, a
member of the EGFR family, and tend to express low levels of genes responsible for
proliferation. They lack expression of basal markers such as cytokeratin 5/6, but usually
express GATA3 (Chou et al., 2010; Usary et al., 2004), a 13% rate of TP53 mutations
(Langerod et al., 2007; Sorlie et al., 2001), and have a distinct characteristic gene
expression profile (Perou et al., 2000). Luminal A tumors usually present with a low
histological grade and have overall good prognostics when detected early. Patients with
luminal A breast cancer are six-times less likely to die from the disease when compared
to the aggressive basal-like subtype (Langerod et al., 2007). This overall good survival
may be due in part to the availability of targeted therapy directed at ERα. Common
hormonal treatments given to these patients to treat early and advanced breast cancer
include tamoxifen, a selective estrogen receptor modulator (SERM), often given to pre-
menopausal women, and/or letrozole, an aromatase inhibitor (AI), often a better choice
for post-menopausal women (Swaby and Jordan, 2008). Luminal A breast cancer tends
5
to have a 29% rate of relapse 15 years after initial diagnosis and have the longest survival
rates when compared to the other subtypes (Kennecke et al., 2010; Peppercorn et al.,
2008). However, intrinsic and acquired resistance to adjuvant anti-hormonal therapy is
still a major problem showing a 63% rate of recurrence (Peppercorn et al., 2008).
2. Luminal B Breast Cancers
Like luminal A, the luminal B subtype makes up 55% of breast cancers and
originates from luminal cells nested in the breast. However, the luminal B gene profile
differs from the luminal A, thus creating its own subcategory in breast cancer. They are
classically hormone receptor positive (ERα+ / PR+), however, a key aspect of luminal B
tumors is the presence of ErbB-2/HER2/neu (HER2+) (Sorlie et al., 2001). These tumors
are more aggressive in nature than luminal A tumors and often co-express EGFR and/or
cyclin E1 (Sorlie et al., 2003). They tend to have high expression of proliferation-
associated genes and high histological grade. They too lack expression of basal markers
such as cytokeratin 5/6, but express GATA3 (Usary et al., 2004), and have a 40%-70%
rate of TP53 mutation status (Langerod et al., 2007; Peppercorn et al., 2008). Much of
current research is devoted to attacking this aggressive form of luminal B breast cancer.
If presented in the clinic as a non-metastatic lesion, these patients have overall good
survival due to combinatory treatment strategies involving pharmacological drugs
targeted at the estrogen receptor (tamoxifen or AI), and the humanized monoclonal
antibody directed against HER2 (trastuzumab) or a small molecule tyrosine kinase
inhibitor against the EGFR/HER2 receptor (lapatinib) (Amar et al., 2008; Bedard et al.,
6
2009; Dean-Colomb and Esteva, 2008). Compared to luminal A, HER2+, or basal-like
subtypes, 50% of patients with luminal B breast cancer have a five year disease-free
survival (Sorlie et al., 2001). However, intrinsic or acquired resistance to anti-hormonal
or anti-HER2 therapy remains problematic as evidenced by a 81% rate of recurrence
(Peppercorn et al., 2008).
3. HER2+ Breast Cancers
Approximately 15-25% of breast cancers have acquired an overexpression or gene
amplification for HER2 on chromosome 17q12 (Rouzier et al., 2005), whose transcripts
can be elevated by two-fold to greater than twenty-fold (Slamon et al., 1989).
Interestingly, the HER2+ subtype is hormone receptor negative showing no positive
expression of the estrogen or progesterone receptors, and low levels of their hormone
responsive genes (Sorlie et al., 2001). HER2+ breast cancers tend to have aggressive
clinical behavior acquiring 4.2 fold increase in expression of proliferation-associated
genes when compared to luminal A tumors (Peppercorn et al., 2008). They also have
high histological grade and harbor a 71% rate of TP53 mutations (Peppercorn et al.,
2008; Weigelt et al., 2009). Although they exhibit luminal features, they are associated
with only a 30% rate of disease-free survival at 5 years (Cheang et al., 2008; Sorlie et al.,
2001). HER2+ tumors are associated with early relapse, high risk of recurrence, and
more likely to involve metastasis to the axillary lymph nodes before diagnosis (Sorlie et
al., 2001). However, current anti-HER2 targeted therapy, such as the monoclonal
antibody, trastuzumab, or the EGFR/HER2 tyrosine kinase inhibitor, lapatinib, has
7
reduced the risk of relapse and dramatically improved survival outcomes (Romond et al.,
2005). In the adjuvant setting, the overall response rate of 26% to lapatinib treatment
was increased to 90% when lapatinib was combined with chemotherapeutic agents in
HER2+ breast cancers (Amar et al., 2008; Bedard et al., 2009; Dean-Colomb and Esteva,
2008). Although responsive to chemotherapy and regimented targeted therapy, 10-15%
of women acquire resistance, particularly those that present with metastatic tumors (Amar
et al., 2008; Romond et al., 2005).
4. Basal-like, Triple Negative Breast Cancers
The basal-like subtype makes up approximately 15-20% of breast cancer cases.
They are defined as those tumors which lack ER, PR, and HER2 expression (Sorlie et al.,
2001). They are prevalent among pre-menopausal, African American women: 20.8% in
African American women versus 10.4% other races including those harboring a BRCA1
mutation (Morris et al., 2007; Peppercorn et al., 2008). Basal-like tumors are very
aggressive in nature, have high rates of metastasis (Nielsen et al., 2004), and lead to poor
overall survival (van de Rijn et al., 2002). They are considered basal-like because the
tumor expresses genes usually associated with the normal breast basal/myoepithelial cells
(Jones et al., 2004), such as cytokeratin 5 and 17 (Perou et al., 2000). These tumors are
highly proliferative, exhibit elevated expression of proliferation-associated genes, have a
high histological grade, up to 85% tend to harbor TP53 mutations (Sorlie et al., 2001;
Weigelt et al., 2009), and approximately 60% overexpress EGFR (Nielsen et al., 2004;
Reis-Filho et al., 2006). Pathology of basal-like tumors demonstrates high central
8
necrotic zones, typical and atypical medullary features, and metaplastic areas (Fulford et
al., 2006; Livasy et al., 2006). It is important to note, that not all basal-like tumors are
triple negative. In fact, a small percentage (<5%) of basal-like tumors contain ER, PR, or
HER2 gene expression, implying that the idea of “triple-negative breast cancer” may not
be synonymous with basal-like (Reis-Filho and Tutt, 2008). Reis-Filho made it quite
clear the importance of this separation in the clinic:
Patients with triple negative cancers expressing a basal phenotype had a
significantly shorter disease-free survival than those with triple-negative
cancers lacking the expression of basal markers. Therefore, caution
should be exercised not to equate a triple negative phenotype with a basal-
like profile. Triple-negative cancer is not a synonym for basal-like cancer
(Reis-Filho and Tutt, 2008).
This is a clear testament of the diverse signaling pathways present within the cell, the
intrinsic differences between breast cancers, and the need to better comprehend and target
aberrant factors controlling this transformation.
The interest in basal-like, triple-negative breast cancer is in part due its highly
aggressive nature and the fact that currently there is no known targeted therapy.
Cytotoxic chemotherapeutics such as taxenes (62% initial response rate), epidermal
growth factor receptor (EGFR) inhibitors (49% initial response rate), vascular endothelial
growth factor (VEGF) inhibitors (63% initial response rate), and poly-ADP-ribose
polymerase (PARP) inhibitors (48% initial response rate) are currently under
investigation for the treatment of triple-negative breast cancers (Bartsch et al., 2010).
9
Although the percent response rates appear high, unfortunately the risk of resistance and
recurrence is also high usually developing between the first and third years following
therapy (Dent et al., 2007). The majority or patients never make their five year survival
mark. The standard of care is a combination of cytotoxic drugs: cyclophosphamide,
doxorubicin, and fluorouracil (Hortobagyi, 1998) or recently shown to be more effective
in triple negative breast cancer was the combination of cyclophosphamide, methotrexate,
and fluorouracil (Colleoni et al., 2010). Their rate of survival is shorter than those who
present with luminal type tumors (Sorlie et al., 2001). Thus, there is an immediate need
for the elucidation of novel targets to treat these patients and increase their survival.
5. Normal-like Breast Cancers
Normal-like breast cancer is still in its infancy regarding lucid categorization. It
is known to be ER positive or negative, PR status undetermined (most likely negative),
and HER2 negative. It does show signs of basal markers, leading some to believe it
stems from the branch of the basal-like subtype. The grade, presence of TP53 mutations,
and relative levels of genes responsible for proliferation are low. Interestingly, Normal-
like breast tumors have been shown to consistently cluster with fibroadenoma and normal
breast samples. These Normal-like breast tumors contain genes normally associated with
adipose tissue (Peppercorn et al., 2008).
10
6. Claudin-low Breast Cancers
Claudin-low breast cancer acquires its name because of the reduced expression of
genes involved in tight junctions and cell-cell adhesion. It could be considered a branch
of the triple negative subtype in that it lacks expression of ER, PR, or HER2+. The
histological grade and relative levels of genes responsible for proliferation are high. The
TP53 status is unknown. However, what differentiates this type from the triple negative
is that the transcriptomic features of the tumors are suggestive of ‘cancer stem cell-like’
(Hennessy et al., 2009). Investigations into this subtype may prove to be advantageous
and interesting to the field of cancer stem cell research.
7. Molecular Apocrine Breast Cancers
Molecular apocrine breast cancer has recently surfaced as a category, which is
believed to be close to the HER2 positive, ER negative, and PR negative subtype. What
distinguishes it from the HER2+ subtype and all other ER-negative tumors is the
presence of an active androgen receptor (AR) and its subsequent target genes (Farmer et
al., 2005). However, the relative levels of genes responsible for proliferation are high.
With these gene signatures, apocrine tumors represent 8–14% of breast tumors. The
knowledge of elevated AR may provide a novel strategy for targeted therapy in this type
of breast cancer.
11
Risk Factors for Breast Cancer
Age, race, family history, breast density, early menarche, age of first pregnancy,
and late menopause can all contribute to the risk of breast cancer (Peppercorn et al.,
2008). These are factors over which there is limited personal control. However, certain
hormonal therapies such as hormone replacement therapy, obesity, alcohol consumption,
inactivity, diet, smoking, and radiation exposure are other risk factors that can be
monitored and/or avoided. Similarly, increase instances in ER positive over ER negative
breast cancers have been associated with an increase in alcohol consumption (Suzuki et
al., 2005). Long-term aspirin use has been associated with decreased development of ER
positive tumors, but increased ER negative tumors (Marshall et al., 2005). Poor diets,
even ones primarily rich in vegetables, and inactivity has been associated with the
development of ER negative breast cancers (Fung et al., 2006). Along with a healthy
balanced diet, folate (Vitamin B9) has been linked to a decreased risk of ER negative
breast cancer (Zhang et al., 2005). Even though correlations have been observed, it is
important to understand that there are multiple factors that are required to develop breast
cancer and that none of these alone can be solely attributed to breast cancer development.
Nevertheless, there is no doubt that overall psychological and physiological well being
helps to provide a better defense against breast cancer development.
Treatment Strategies for Breast Cancer
Breast cancer is a diverse and aggressive disease. When presented at time of
diagnosis, each patient is unique and their breast cancer varies according to their grade,
12
type, and response to treatment. With such individualization, the ultimate decision of
proper treatment lies with the patient, their doctors, and their families. Many times,
multiple treatment strategies are administered to help eradicate the disease. Currently,
the most common strategies to treat breast cancer are surgery, radiation therapy, systemic
therapy, and a low-fat/low-alcohol diet.
1. Surgery
When a patient presents in the clinic, a biopsy is taken, and surgery usually
follows after diagnosis. Surgery can be minimal such as a lumpectomy, or as extensive
as radical mastectomy (Maughan et al., 2010). A lumpectomy is the first choice of
surgery for early stage breast cancers, where the surgeon will remove the tumor and a
layer of lining stroma. After surgery, there is usually a five to seven week follow-up with
radiation therapy. A second surgical choice is simple or total mastectomy, where there is
removal of the entire breast. Later stage breast cancers may have infiltrated to the
sentinel and/or axillary lymph nodes. A biopsy of the sentinel and then possibly the
axillary lymph nodes is taken to determine the extent of metastasis. A modified radical
mastectomy may be administered in which the total breast is removed as well as the
lymph nodes. An extremely aggressive breast cancer that has infiltrated to the lymph
nodes and chest wall requires a total radical mastectomy, where the breast, lymph nodes,
and chest wall muscle are all removed (Fisher et al., 2002a; Fisher et al., 2002b; Veronesi
et al., 2002). Normally, surgery is not a sole means of treatment. As the grade and stage
13
of the disease increases, the more combinatory strategies are taken (e.g. surgery with
chemotherapy and radiation).
2. Radiation Therapy
Radiation therapy is either given before surgery to help reduce the tumor burden,
or after surgery to insure the annihilation of remaining cancer cells. There are two
methods of administering radiation therapy: external or internal. In external radiation, a
biophysical machine directs a beam of a radioactive isotope towards the affected breast
and/or tumor. The external radiation is administered across an average five to seven
weeks and is effective at eliminating and/or reducing tumorigenic cells. In internal
radiation, the radioactive isotope is dispensed directly into the tumor through thin
capillary wires or needle injection. This method is also termed brachytherapy and is
increasingly becoming more effective. Some patients undergo both forms of radiation
treatment, but the type and stage of the cancer may favor one method over the other.
Currently, accelerated partial breast irradiation (APBI) is a method being developed to
reduce doses, area, and duration of treatment (Dirbas, 2009). Although reduced tumor
burden has been observed, radiation is rarely used as a sole form of treatment. Its
effectiveness relies on both surgery and systemic therapy.
3. Systemic Therapy
Systemic therapy is a means to treat breast cancer through oral administration or
injection, which consequently circulates through the entire body. Treatment may be
14
given before surgery (neoadjuvant), after surgery (adjuvant), or when surgery is not an
option. Currently, hormone therapy, targeted biological therapy, and cytotoxic
chemotherapy are the most common forms of systemic therapy.
A. Hormone Therapy
The estrogen receptor has emerged as a critical hormonal target to modulate in ER
positive breast cancers and has been shown to promote tumor growth. Tamoxifen, a
selective estrogen receptor modulator (SERM) and partial agonist, is a leading triphenyl-
ethylene compound and medicinally modified moiety that binds and competitively
inhibits the estrogen receptor, dampens the estrogenic signaling, and consequently
reduces the estrogenic responsive effects (Figure 2) (Jordan et al., 1981). Tamoxifen is
given orally to both pre- and post-menopausal women for a period of five years (Swaby
and Jordan, 2008). It has been demonstrated that following treatment, there is an
approximately 33% reduction in death rate and 41% reduction in recurrence (Coates et al.,
2007). Tamoxifen has undesirable thromboembolism events and increased endometrial
cancer risk as major side effects (Hind et al., 2007). Another anti-estrogen is Fulvestrant
(Faslodex), a complete antagonist and competitive inhibitor of the estrogen receptor
which is given as an intramuscular injection (Diagram 2) (Buzdar, 2008). It binds to the
estrogen receptor and destabilizes the three dimensional structure thus facilitating E3
ubiquitin ligase/proteasome-mediated receptor degradation (Berry et al., 2008; Howell,
2006). Consequently, estrogenic signaling and tumor growth is significantly reduced.
Patients taking Faslodex have reported gastrointestinal side effects.
15
CH3
ON
CH3
CH3
S
OH
OH
CH3
O
F
FF
F
F
NN
N
NN
N
NN
CH3
CH3CH3
CH3
CH3CH3
CH3
CH3
CH2
O
O
Tamoxifen Fulvestrant
Letrozole Anastrozole Exemestane
Aromatase Inhibitors (AI)s
Estrogenic Inhibitors
Figure 2: Chemical Structures of Common Estrogenic and Aromatase Inhibitors
16
Aromatase Inhibitors (AIs) are a class of drugs that inhibit the aromatase enzyme
that converts either androstenedione into 17β-estradiol, or directly testosterone into 17β-
estradiol. Current drugs include letrozole (Femara), anastrozole (Arimidex), and
exemestane (Aromasin) (Figure 2). They are effective in post-menopausal women where
small amounts of estrogen are produced in the adipose tissue surrounding the tumor.
Current research has shown that AIs alone or interspersed with tamoxifen treatments is
more advantageous than tamoxifen alone (Buzdar et al., 2006). AIs are relatively more
expensive and possess undesirable side-effects such as osteopenia and bone pain (Hind et
al., 2007).
B. Targeted Biological Therapy
Since approximately 30% of breast cancers contain a gene amplification or
overexpression of the HER2 receptor linked to tumor survival, HER2 has become a
critical target to inhibit. In 2006, trastuzumab (Herceptin) was approved by the FDA to
treat patients with HER2 positive breast cancer (O'Mahony and Bishop, 2006).
Trastuzumab is a humanized, monoclonal antibody that binds to the extracellular portion
of the HER2 receptor (Carter et al., 1992). HER2 dimerization is disrupted and its ability
to promote survival is dampened. Studies have shown that treatment with trastuzumab in
combination with cytotoxic chemotherapy such as a taxane have reduced patient death
rate by 33% and risk of recurrence by 52% (Romond et al., 2005). Lapatinib, a small
molecule tyrosine kinase inhibitor targeted against EGFR and HER2, is also used to
target the signaling cascade from the EGFR family of receptors (McArthur, 2009).
17
Another current form of targeted biological therapy is bevaxizumab (Avastin), which is a
humanized monoclonal antibody against the vascular endothelial growth factor (VEGF)
(Sachdev and Jahanzeb, 2008). VEGF is responsible for angiogenesis, and since a tumor
needs an enriched blood supply, a VEGF inhibitor prevents tumor vascularization and
angiogenesis. Avastin was approved by the FDA in 2008 as an added chemotherapeutic
strategy for advanced breast cancer (Spalding, 2008). Other small molecules or
antibodies are being developed, tested, and currently are in clinical trials. Targeted
biological therapy shows much promise particularly when combined with surgery and
cytotoxic chemotherapy.
C. Cytotoxic Chemotherapy
Some breast cancers require a more general systemic treatment. Cytotoxic
chemotherapy is designed to target rapidly dividing cells, which is a hallmark of a highly
proliferative tumor. Since cytotoxic chemotherapy target all rapidly dividing cells,
several non-cancerous cells may unfortunately be targets as well. Rapidly dividing
benign cells are found in the hair shaft, intestines, and in the bone marrow, which are
why many patients experience side effects such as hair loss, gastrointestinal disturbances,
and anemia (Chan and Giaccia, 2008). Some examples of cytotoxic drugs include
taxanes (paclitaxel, Taxol), cyclophosphamide, fluorouracil, and cisplatin (Mouridsen,
1990; Rowinsky et al., 1992; Sledge, 1992). Taken together, systemic therapy when
combined with other targeted drugs, surgery, and radiation have been shown to be more
advantageous than with one form of treatment (Hortobagyi, 1998; Mauri et al., 2008). As
18
medicines are being developed and studied, future research needs to understand and
elucidate novel aspects of breast cancer biology.
Conclusions: Breast Cancer Now and the Future
Breast cancer continues to be one of the major causes of cancer-related death in
women. Presently, there are four classic subtypes of breast cancer: luminal A, luminal B,
HER2/neu, and basal-like, but more novel categorizations are being developed as the
systems biology and gene arrays uncover new patterns of expression. Currently,
tamoxifen still remains a leading chemotherapeutic choice for hormone positive breast
cancers. In addition, the advent of novel aromatase inhibitors and more potent
medicinally modified estrogenic inhibitors are becoming an additional choice for
treatment. However, approximately half of the women treated will develop resistance to
the anti-estrogens, driving current research to develop targeted combinational strategies.
Today, trastuzumab (Herceptin) and lapatinib, are chemotherapeutic strategies to treat
patients with HER2+ breast cancer. Unfortunately, the triple negative basal-like subtype
still manifests high mortality, high recurrence, and no known targeted treatment to date.
Therefore, there is an immediate need to identify novel targets that are responsible for the
proliferation and invasive phenotype for these cancers so as to increase overall patient
survival. Taken together, the future of breast cancer lies within the very biochemical
infrastructure of the transformed cells, their stem-likeness, and their microenvironment,
which still remains relatively elusive. As each compromised cell responds to its extrinsic
signaling cascades and its downstream aberrant intrinsic transcriptional effects,
19
progressive research will uncover and exploit the molecular mechanisms and novel
targets that drive its progression, aggression, and survival.
SUBTYPE EXAMPLE CELL LINE ER PR HER2 TP53
(MUT) GRADE PROLIFERATIONGENE PROFILE
CLINICAL FEATURES
TREATMENT STRATEGIES
LUMINAL A
MCF-7
TYPE: Metastatic
Adenocarcinoma
+ + – LOW LOW LOW
Most common form of breast cancer
Lower risk of reoccurrence
Endocrine therapy responsive
Less responsive to chemotherapy
Tamoxifen SERM(s)
Letrozole
Exemestane AI(s)
LUMINAL B
BT474
TYPE: Invasive Ductal
Carcinoma
+ + + MODERATE MODERATE HIGH
Endocrine therapy responsive
Moderate response to chemotherapy
Tamoxifen SERM(s)
Letrozole
Exemestane AI(s)
Trastuzumab
Lapatinib
ERBB2/HER2+
SKBR3
TYPE: Metastatic
Adenocarcinoma
– – + HIGH HIGH HIGH
Likely involvement of axillary lymph nodes upon diagnosis
High risk of reoccurrence
Responsive to chemotherapy
Trastuzumab
Lapatinib
BASAL-LIKE
MDAMB231
TYPE: Adenocarcinoma
– – – HIGH HIGH HIGH
Association with BRCA-1 mutations
Common in pre-menopausal African American women
High risk of reoccurrence
Responsive to chemotherapy
No known targeted treatment
No targeted treatment
PARP inhibitors
VEGF inhibitors
Taxanes
Cytotoxic
chemotherapy Abbreviations: ER, estrogen receptor; PR, progesterone receptor; MUT, mutations. References: (Peppercorn et al. 2008, Kao 2009)
Table 1: Properties of the Classic Subtypes of Breast Cancer.
20
CHAPTER II
NOTCH, PEA3, AND AP-1: A LITERATURE REVIEW
The molecular profile of breast cancer involves a series of interconnected
pathways that when perturbed, disrupts normal cellular homeostasis and drives the cell
towards transformation. The cell discovers new ways of utilizing signaling cascades and
transcriptional events to become hyper-sensitive through amplification or enhanced
activity. In addition, elimination of genes, often termed “tumor suppressors,” is exploited
so the cell can acquire resistance to apoptosis, immortality, self-sufficiency, and
migratory capabilities. Oncogenic pathways that increase proliferation and prevent
apoptosis have emerged as potential targets for the treatment of breast cancer. For
example, Notch signaling, PEA3 transcriptional effects, and AP-1 dynamic partnership
all have been associated with aggressive breast cancers, particularly the triple negative
subtype, and are pathways the cancer cell has manipulated to drive its survival and
progression.
Notch Signaling
In the early twentieth century, the discovery of “Notch” and its name came after
an investigation of genetic gene mutants in Drosophila. Upon exploration of the
mutations, 21
22
one particular mutant resulted in a “notch” of missing tissue at the tip of the fly wing
blades (Morgan, 1917). This identification eventually led to the discovery of the Notch
pathway. As years passed, Notch signaling became very interesting to scientists both in
development –implicated in insulin-secreting pancreatic beta-cells (Apelqvist et al.,
1999), the inner ear hair cell development (Lanford et al., 1999), and intestinal crypt and
goblet cells (van Es et al., 2005) –and eventually cancer. Notch has emerged as a potent
oncogene and is aberrantly expressed in breast cancer (Stylianou et al., 2006). Elevated
levels of Notch-1 and one of its ligands, Jagged-1, confer poor prognosis and overall
diminished survival (Dickson et al., 2007; Reedijk et al., 2005). Currently, Notch is
becoming an advantageous pathway to explore and exploit in neoplastic cells as well as
potential cancer stem cells having been implicated in proliferation of tumor initiating or
mammary cancer stem cells (Harrison et al., 2010). Currently, Notch is becoming an
advantageous pathway to explore and exploit in neoplastic cells as well as potential
cancer stem cells having been implicated in proliferation of tumor initiating or mammary
cancer stem cells (Harrison et al., 2010).
1. An Overview of Notch Signaling
Notch signaling has emerged as a target for the treatment of breast cancer. In the
mammalian system, there are four Notch receptors (Notch-1,2,3,4) which are all type-I
transmembrane proteins (Blaumueller and Artavanis-Tsakonas, 1997) and five known
ligands (Delta-like 1,2,4, and Jagged 1,2) (Dunwoodie et al., 1997; Lindsell et al., 1995;
Shawber et al., 1996). Cell-to-cell contact activates Notch signaling, which subsequently
23
enables the pathway to modulate genes involved in cell fate, proliferation and
differentiation (Callahan and Raafat, 2001). Notch is processed in the trans-Golgi
apparatus, where it undergoes the first of three proteolytic cleavages (Figure 3)
responsible for transcriptional activation. The first cleavage (S1) takes place in the trans-
Golgi by furin-like convertase converting the single polypeptide into the mature
heterodimeric Notch receptor held together by di-cations (Ca2+). The receptor is
trafficked to the cellular membrane where it awaits physical contact with its ligand
(Kopan and Ilagan, 2009). When ligand binds through EGF-like repeats and electrostatic
interactions, the extracellular portion of Notch is co-endocytosed with ligand into the
neighboring, ligand-expressing cell thus exposing a second cleavage site (S2) on the
transmembrane portion of the Notch (NTM) which is then cleaved by TNF-alpha
converting enzyme (TACE), a disintegrin and metalloproteinase (ADAM10/17) (Brou et
al., 2000). Consequently, a third cleavage site (S3) within the transmembrane domain of
Notch is now susceptible to proteolysis by the membrane associated aspartyl proteinase,
the γ-secretase complex (presenilin, nicastrin, APH1, PEN2) (Kopan and Ilagan, 2004),
liberating the intracellular portion of Notch (NIC) (Saxena et al., 2001). Notch (NIC)
translocates to the nucleus and binds to CBF-1/Serrate/Lag-1, a constitutive repressor on
target genes that binds the core sequence CGTGGGAA and other related sequences. NIC
then displaces co-repressors (SMRT, N-coR, SKIP, and histone deacetylases) (Hsieh et
al., 1999), and recruits co-activators such as p300, histone acetyl transferases, and
mastermind-like 1 (MAML1-3) (Wu et al., 2000). Notch is known to activate many
genes including, the pro-differentiation transcription factors, HES and HEY family of
CBF-1
CoR
CoACoA
Golgi
Furin-likeConvertase
HEYHES
Cyclin D1
++
ADAM
Gamma-secretase
Jagged-1
NIC
1
2 3
Notch-1
Endocytosis
PEN2
NicastrinPresenilin
APH-1
MAML-1
NCoRSMRT
CBF-1
CoR
CoACoA
Golgi
Furin-likeConvertase
HEYHES
Cyclin D1
++
ADAM
Gamma-secretase
Jagged-1
NIC
1
2 3
Notch-1
Endocytosis
PEN2
NicastrinPresenilin
APH-1
MAML-1
NCoRSMRT
24
RECEPTORS: LIGANDS: Notch-1 Jagged-1 Notch-2 Jagged-2 Notch-3 Delta-like 1 Notch-4 Delta-like 2
Delta-like 4
Figure 3: The Notch Signaling Pathway. Abbreviations- a disintegrin and metalloproteases, ADAM; Notch intracellular protein, NIC; co-activators, CoA; co-repressors, CoR; mastermind-like 1, MAML-1.
25
basic helix-loop-helix transcription factors, genes implicated in angiogenesis (Fischer et
al., 2004) and in epithelial-to-mesenchymal transition (Iso et al., 2003; Maier and Gessler,
2000; Zavadil et al., 2004), cyclin D1 (Ronchini and Capobianco, 2001), c-Myc (Weng et
al., 2006), NF-κB (Cheng et al., 2001), and the IGF-1R (Eliasz et al., 2010).
2. Notch Receptors
A. Structural Analysis of Notch Receptors
In the mammalian system, there are four Notch receptors (Notch-1,2,3,4)
(Blaumueller and Artavanis-Tsakonas, 1997). The structure of the receptor is conserved,
but emerging evidence demonstrates distinct properties among the different receptors
(Figure 4). The region of Notch that extends into the intercellular space (NEC) consists of
a series of epidermal growth factor-like (EGF-like) repeats. These EGF-like repeats
make up the ligand binding surface. The number of EGF-like repeats varies among the
receptors: Notch-1 having the most and Notch-4 the least. The EGF-like repeats contain
sites for O-linked glycosylation by the O-fucosyl transferase (O-fut) enzyme (Haines and
Irvine, 2003). Glycosylation plays a vital role in the function, folding, and ligand binding
preference of Notch (Sasamura et al., 2003; Shi and Stanley, 2003). Juxtaposed to the
cellular membrane, there are three Lin12 Notch repeats (LNR) that help stabilize and
direct appropriate ligand binding.
The next portion of Notch is the transmembrane region (NTM), which transverses
the lipid bilayer and contains the cleavage site for the γ-secretase complex. The
intracellular portion (NIC) contains a RAM domain (a domain involved in protein
26 RECEPTORS
RAMANKNCRTAD
PEST
NLS
NLS
LNR
EGFR
NOTCH-1 NOTCH-2 NOTCH-3 NOTCH-4
EXTRACELLULARNOTCH (NEC)
INTRACELLULARNOTCH (NIC)
RAMANKNCRTAD
PEST
NLS
NLS
LNR
EGFR
NOTCH-1 NOTCH-2 NOTCH-3 NOTCH-4
EXTRACELLULARNOTCH (NEC)
INTRACELLULARNOTCH (NIC)
LIGANDS
EGF
JAGGED FAMILY DELTA-LIKE FAMILY
Jagged-1Jagged-2
Delta-like 1Delta-like 2Delta-like 4
CR
DSL
EGF
JAGGED FAMILY DELTA-LIKE FAMILY
Jagged-1Jagged-2
Delta-like 1Delta-like 2Delta-like 4
CR
DSL
JAGGED FAMILY DELTA-LIKE FAMILY
Jagged-1Jagged-2
Delta-like 1Delta-like 2Delta-like 4
JAGGED FAMILY DELTA-LIKE FAMILY
Jagged-1Jagged-2
Delta-like 1Delta-like 2Delta-like 4
CR
DSL
Figure 4: The Four Mammalian Notch Receptors, Their Ligands, and Their Domains.
Abbreviations- epidermal growth factor repeats, EGF; Lin12, LNR; ankyrin repeats, ANK; nuclear localization signal, NLS; cytokine response element, NCR; transactivation domain, TAD; proline glutamic acid serine threonine, PEST; Notch extracellular region, NEC; Notch intracellular region, NIC; cysteine rich region, CR.
27
interactions), ankyrin repeats (site of protein-protein interactions), two nuclear
localization sequence (NLS; Notch-1 contains three), and a C-terminal PEST domain (a
signal region for protein degradation). Notch-1 and Notch-2 contain transactivation
domains (TAD) and cytokine response elements (NCR), where as Notch-3 only contains
a NCR region (Kopan and Ilagan, 2009). All these elements together are vital to the
successful activity of Notch signaling.
B. Notch Receptors and Breast Cancer
The Notch receptors have been linked to many types of cancers and are found to
play a vital role in the development and progression of mammary tumorigenesis (Miele et
al., 2006). For example, elevated expression of Notch-1 and its ligand Jagged-1 in
ERalpha negative, basal-like, triple negative, or HER2 positive cells are associated with
poorest overall patient survival (Dickson et al., 2007; Reedijk et al., 2005).
Using a xenograft MDA-MB-231 model expressing active Notch-2 (N2IC), there
was overall decreased tumor take and increased apoptosis (O'Neill et al., 2007). In
agreement in endothelial cells, expression Notch-2 (N2IC) stimulated pro-apoptotic genes
such as caspases 3 and 7, as well as PARP cleavage (Quillard et al., 2009). These studies
imply that unlike Notch-1, Notch-3, or Notch-4, Notch-2 has a pro-apoptotic and possibly
tumor suppressor function. This brings to question whether pan-pharmacological Notch
inhibitors should be used, or should more specific targeting be explored.
Notch-3 has also been implicated in mammary tumor formation as well as
mammary gland development. Similar to studies performed using Notch-1, MMTV-
28
driven Notch-3 (N3IC) transgenic mice showed similar exacerbation of mammary
tumorigenesis (Hu et al., 2006). In HER2 negative breast cancers, downregulation of
Notch-3 resulted in decreased proliferation and increased apoptosis even more than
Notch-1 (Yamaguchi et al., 2008). Similar to Notch-4, Notch-3 promotes self-renewal
and expansion of mammary gland stem and progenitor cells (Sansone et al., 2007). Thus,
Notch-3 like Notch-1 and Notch-4 plays a critical role in the transformation and
progression of breast cancer.
The Notch-4 gene contains a site of integration (int-3) for retroviral insertion such
as mouse mammary tumor virus (MMTV) (Uyttendaele et al., 1996). This site occurs
downstream of the Lin12 portion, yet upstream of the transmembrane portion of Notch
(see Figure 4). Loss of the extracellular portion from Lin12 to the N-terminus results in a
truncated C-terminus form of Notch-4 that is constitutively active. Notch-1 has similar
integration sites and is found to also be constitutively active (Dievart et al., 1999). In a
separate study, active intracellular Notch-1 or Notch-4 (N1IC and N4IC) was demonstrated
to form spontaneous mammary tumors in mice (Callahan and Raafat, 2001). Recently,
Notch-4 has been implicated in survival and proliferation of tumor initiating or mammary
cancer stem cells (Harrison et al., 2010). Aberrant Notch signaling has become evident
not only in tumor initiation, but also progression playing a role in undifferentiated stem-
like and more terminally differentiated neoplastic cells.
29
3. Notch Ligands
In the mammalian system, there are five known ligands (Delta-like 1,2,4, and
Jagged 1,2) (Dunwoodie et al., 1997; Lindsell et al., 1995; Shawber et al., 1996). The
ligands are divided into two groups depending upon whether or not they contain a
cysteine rich region (Delta-class or Jagged, respectively) (see Figure 4). In common,
both classes have a DSL domain and a series of EGF-like repeats, which are essential for
Notch receptor binding. Notch receptor binding to its ligand on an adjacent cell is critical
for proteolysis and signaling events. However in Drosophila, cleaved and/or soluble
ligands have been found to activate or downregulate the signaling cascade independent of
cell to cell contact depending upon context (Klueg et al., 1998; Sun and Artavanis-
Tsakonas, 1997). Interestingly during hematopoesis, these “decoy” ligands provide an
exciting new strategy to modulate the Notch pathway, which could be exploited in other
biological systems (Masuya et al., 2002). High Jagged-1 expression has been correlated
with the poorest overall patient survival in ERalpha negative breast cancer subtypes
including the basal-like, triple negative, and HER2 positive tumors (Dickson et al., 2007;
Reedijk et al., 2005).
4. Inhibitors of Notch Signaling
With the understanding that ligand engagement is critical for Notch activation,
receptor decoys could be a means to interrupt the ligand-receptor mediated activation
(Nickoloff et al., 2002). Disruption of the O-linked glycosylation required for mature
receptor formation could also be used to inhibit the activity of Notch. Specific Notch
30
inhibition could be achieved by using siRNA, antisense, or monoclonal antibodies
directed at specific receptors (Miele et al., 2006). Other methods could be mastermind
decoys, RAS inhibitors, or even transcription factor-stapled peptides that disrupt the co-
activation complex in the nucleus (Moellering et al., 2009).
Enzymatic inhibition has shown promise in dampening Notch signaling. Anyone
of the three enzymatic cleavage steps that process Notch (furin-like convertase [S1],
ADAM10/17 [S2], or γ-secretase [S1]) (see Figure 3 for more detail) may be used as a
means to attenuate signaling. Of great interest is the final cleavage step required for the
liberation of the active, intracellular form of Notch (NIC) to activate Notch signaling
(Kopan and Ilagan, 2004; Kopan and Ilagan, 2009). Its inhibition can be exploited
through emerging pharmacological drugs identified as γ-secretase inhibitors (GSIs),
which inhibit signaling from all four receptors. These can be either peptide-mimics or
small molecule inhibitors. One such GSI is the compound developed by Merck
Pharmaceuticals known as MRK-003 (Figure 5) and used in this investigation (Lewis et
al., 2007). This compound is a small medicinally modified chemical that binds to the
ATP-dependent binding pocket of presenilin, the catalytic subunit of the γ-secretase
complex. Since this is an experimental drug designed by Merck Pharmaceuticals, the
rates, pharmacokinetics, and pharmacodynamics are confidential information secured by
the company. Other GSI compounds include LY 411,575, a reversible competitive
inhibitor, Calbiochem, an irreversible covalent suicide inhibitor (see Figure 5 for
chemical structures), and Z-Leu-Leu-Nle-CHO, a tripeptide inhibitor (Curry et al., 2005).
Recent studies demonstrate that GSI treatment in combination with tamoxifen inhibits
31
F
F
OHOON
NH
NH
CH3 O
CH3
LY 411,575
OO NH
NHNH
O
O
O
CH3
CH3
(CH2)3CH3CH3
CH3
Calbiochem (C26H41N3O5)
S
N
NH
N
O
O
F
FF
F
FF
MRK-003
Figure 5: Chemical Structures of Common GSI(s).
32
ER+ breast tumor growth in athymic, nude mice (Rizzo et al., 2008) providing evidence
of novel therapeutic approaches to enhancing current treatments. Certain GSI(s) are
currently in clinical trials for the treatment of breast cancer.
5. Regulation of Notch Signaling
A. Post-transcriptional Regulation of Notch
Notch signaling is regulated post-transcriptionally by several different methods.
Fringe glycosylation of the extracellular portion of Notch determines the relative affinity
for contact with its ligands (Okajima and Irvine, 2002). Phosphorylation events on Notch
also aid its signaling sustainability such as phosphorylation by GSK-3beta (Foltz et al.,
2002). Ubiquitination by E3 ligases such as Neuralized and Mindbomb are important for
endocytosis of Notch ligands and subsequent activation events (Le Borgne et al., 2005;
Wang and Struhl, 2005). Alpha-adaptin mediated endocytosis by negative regulator
Numb also plays a critical role in the regulation of Notch signaling (Berdnik et al., 2002).
Numb is shown to interact with the intracellular Notch domain and recruit the Nedd4
family of E3-ubiquitin ligases. This leads to the ubiquitination and degradation of Notch
IC (McGill et al., 2009; McGill and McGlade, 2003). Other post-transcriptional events
are currently being investigated and could be manipulated as a means to dampen Notch
signaling in cancer. However, Notch is already processed and reaching maturity by this
time. A better option could be to understand the transcriptional regulation of Notch and
terminate its transcription prior to protein synthesis.
33
B. Transcriptional Regulation of Notch
Notch begins its life in the nucleus as a gene awaiting transcription. The factors
that are transcriptional regulators of the Notch receptors are still widely unknown. It was
shown in human keratinocytes that p53 regulates the Notch-1 receptor (Alimirah et al.,
2007). In vascular endothelial cells, AP-1 was shown to be important in the regulation of
Notch-4 transcription (Wu et al., 2005). Demonstrated by the same group, AP-1 was
shown to stabilize the glucocorticoid receptor on the Notch-4 promoter in vascular
endothelial cells and aid in its expression (Wu and Bresnick, 2007). During T-cell
development, NKAP was shown to be a Notch transcriptional repressor (Pajerowski et al.,
2009). During hair shaft differentiation, both Msx2 and Foxn1 were demonstrated to be
responsible for Notch-1 expression (Cai et al., 2009). However, more detailed
transcriptional regulators are left widely understudied, particularly in breast cancer. Thus,
novel regulators of the Notch pathway will prove to be significant in the fight against
breast cancer.
Promoter analysis of Notch genes reveals evidence of p53, AP-1, SP-1, Ets, as
well as several other consensus sites that may all play a role in the transcriptional
regulation of Notch which is most likely cell context-dependent. In breast cancer,
particularly the basal-like/triple negative breast cancer, used in this investigation, p53
mutations are known to exist, which raises the question of what else is regulating these
transcripts. Evidence of Ets consensus sequences is exciting, due to the fact that PEA3, a
well known member of the Ets family of transcription factors, is overexpressed in
34
aggressive breast cancers. Therefore, could the transcription factor PEA3 be responsible
for the regulation of Notch in breast cancer?
Polyomavirus Enhancer Activator 3 (PEA3)
1. Ets Transcription Factors
Ets proteins are a family of transcription factors of which there are over 30 known
members. The members are categorized into 13 groups: Ets, TEL, YAN, SPI, ERG,
PEA3, ELF, DETS4, ELK, GABP, ER71, ERF, and ESE. These transcription factors
share a common DNA Ets domain which binds through its winged helix-turn-helix motif
(Graves and Petersen, 1998; Laudet et al., 1999) to the canonical 11 base pair DNA
sequences with the core being GGAA/T on target genes (Sharrocks, 2001). The affinity
of binding relies on the proximal sequences surrounding the Ets binding site. The
domain is important for protein-protein co-regulatory interactions. Although there is
marked similarities among the Ets domain, differences in a single amino acid within the
C-terminal portion of the Ets domain can lead to altered DNA specificity and interactions
with other transcription factors (Fitzsimmons et al., 1996; Shore et al., 1996). Activation
and/or repression domains are often present in these factors, which aid their
transcriptional control based on context (Oikawa and Yamada, 2003). This further
individualizes and separates the Ets transcription factors into unique groups.
35
2. Polyomavirus Enhancer Activator 3 (PEA3)
Polyomavirus enhancer activator 3, commonly known as PEA3, E1AF, or ETV4,
is a member of the Ets family of transcription factors and located on chromosome 17q21
(Oikawa and Yamada, 2003). Other family members with PEA3 are ER81 and Erm.
PEA3 is important in embryogenesis and is rarely expressed at elevated levels during
adulthood. Interestingly, PEA3 levels re-emerge during remodeling events and cancer
progression. Approximately 76% of human breast tumors contain elevated levels of
PEA3; the highest found in the triple negative breast cancers conferring overall poor
prognosis (Trimble et al., 1993). Interestingly, mRNA levels of PEA3 demonstrated
marked over-expression, yet endogenous PEA3 protein is rarely detectable (Baert et al.,
1997). This may be impart do to the rapid turnover rate and protein stability (Baert et al.,
1997). Several studies associate PEA3 in the regulation of enzymes involved in tissue
remodeling and invasion such as matrix metalloproteases (MMP-1, MMP-3, MMP-7,
MMP-9) (Benbow and Brinckerhoff, 1997), urokinase-type plasminogen activator (uPA)
(Evans et al., 2001), cyclooxygenase (COX-2) (Subbaramaiah et al., 2002), HER2/ErbB-
2 (Matsui et al., 2006), and IL-8 (Davidson et al., 2003) implicating it as a potential target
for therapeutics in a diseased state. However, little concurring evidence of a mechanistic
role of PEA3 in breast cancer has left the field widely controversial.
A. The Structure of PEA3
The gene is made up of 13 exons spanning approximately 15 kilobases on
chromosome 17q21 (Coutte et al., 1999). The 62 kD protein contains an Ets DNA
36
binding domain with a tryptophan repeat and a conserved trans-activation domain flanked
by negative regulatory domains (Figure 6) (Bojovic and Hassell, 2001). It also contains
an acidic region and glutamine-rich region, which have activation capabilities (Higashino
et al., 1993). The C-terminus domain of PEA3 has been found to be important to regulate
its stability (Takahashi et al., 2005).
B. Activity and Regulation of PEA3
Activation of PEA3 is through phosphorylation of serine and threonine residues
by the mitogen activation protein kinase pathway (MAPK: Ras, Raf-1, MEK, ERK-1, and
ERK-2) (Wasylyk et al., 1998). The protein is relatively unstable exhibiting a turnover
half-life of of about 30 minutes via the ubiquitin-proteasome pathway (Takahashi et al.,
2005). Recently, PEA3 was suggested to be negatively regulated in part by sumoylation
(Bojovic and Hassell, 2008; Takahashi et al., 2005). SUMO-E3 ligase PIASy has been
implicated directly in sumoylating PEA3 (Nishida et al., 2007). Sumoylation sites have
been identified as lysines 96, 222, and 256 (Bojovic and Hassell, 2008), and have been
demonstrated to either transiently enhance the activity of PEA3 or mark it for degradation
by the ubiquitin-proteasome pathway (Guo and Sharrocks, 2009). Therefore, there is a
direct connection and interplay between both sumoylation and ubiquitinylation pathways.
3. PEA3 and Breast Cancer
PEA3 is overexpressed in 76% of breast cancers. The highest expression is
observed in invasive breast cancers particularly the triple negative subtype (Trimble et al.,
37
Figure 6: Protein Structure of Polyomavirus Enhancer Activator 3 (PEA3). Abbreviations- phosphorylation motif, P; transactivation domain, TAD; threonine, T; serine, S; lysine, K; sumoylation moiety, SUMO.
38
1993), which is associated with overall poor prognosis, and is also highly expressed in
HER2 positive tumors (Benz et al., 1997). PEA3 is present during embryogenesis and
expressed within the epithelial buds of the mammary gland (Chotteau-Lelievre et al.,
1997; Chotteau-Lelievre et al., 2003). Later studies confirmed the presence of PEA3 in
undifferentiated epithelial cap cells of the terminal end buds (Shepherd and Hassell,
2001). In a mouse model of MMTV-neu-induced mammary primary adenocarcinomas,
overexpression of PEA3 resulted in enhanced metastatic lesions (Trimble et al., 1993).
Conversely, expression of a dominant negative form of PEA3 resulted in a delay of tumor
onset and progression in a MMTV-neu mouse model (Shepherd et al., 2001). PEA3 also
enhances transcriptional activity of important genes involved in breast cancer metastasis
including, but not limited to cyclooxygenase-2 (Subbaramaiah et al., 2002), MUC4
(Perez et al., 2003), and osteopontin (El-Tanani et al., 2004). Taken together, PEA3 has
been correlated with tumor initiation, progression, and invasiveness and appears to play a
vital role in breast cancer.
Interestingly, PEA3 has been implicated recently in multipotent progenitor
regulation of lineage-specific differentiation (Kurpios et al., 2009) implying a potential
function in mammary stem cells. This is similar to the recent study mentioned previously
regarding Notch-4 in survival of tumor initiating cells/cancer stem cells (Harrison et al.,
2010). This may provide an exciting correlation between PEA3 and Notch-4 expression.
Conflicting studies also have been shown regarding the role of PEA3 in regulation
of HER2 and its clinical correlations. Originally, it was identified that the HER2
promoter contained a conserved Ets consensus site that when mutated dampened HER2
39
transcription (Scott et al., 1994). Shortly after, PEA3 was shown to be upregulated in
93% of HER2/Neu-overexpressing human breast tumors and correlated with breast
cancer initiation and progression (Benz et al., 1997). Again in 2001, PEA3 was linked to
the positive expression of HER2 helping confirm the potential of PEA3 as a therapeutic
target (Shepherd et al., 2001).
However, other studies demonstrated that PEA3 repressed HER2 expression and
that PEA3 does not positively correlate with HER2 expression in breast cancer (Xia et al.,
2006; Xing et al., 2000). Although the field presents a “gray area” regarding the role of
PEA3 in HER2 regulation, this controversy actually provokes the notion of dynamic
molecular variability. The role of PEA3 on other genes in different systems implies the
importance of cellular context. In fact, further studies demonstrated that PEA3 and c-
JUN independently had slight roles in the regulation of HER2. However, their coordinate
expression along with histone acetyltransferase p300 stimulated transcription of the
HER2 gene by 20-fold in the MCF-7 breast cancer cell line (Matsui et al., 2006). This
stresses the importance of transcription factors such as c-JUN working in tandem with
PEA3 to appropriately regulate PEA3 target genes. Moreover, PEA3 directly interacts
with p300 (Liu et al., 2004) and USF-1 (Firlej et al., 2005), and many other co-regulatory
factors to enhance gene transcription. Therefore could PEA3 be responsible with other
factors such as AP-1 for the regulation of Notch transcription in breast cancer?
40
Activating Protein 1 (AP-1)
A common player in transcriptional control and implicated in various cellular
reactions is the homo- or heterodimeric transcription factor complex AP-1. The dimeric
complex classically consists of the FOS (c-FOS, FosB, Fra-1, Fra-2) and JUN (c-JUN,
JunB, and JunD) families (Curran and Franza, 1988). Other dimeric complexes can also
include ATF and MAF protein families. c-JUN:c-JUN homodimers can form, but are
less stable than c-JUN:c-FOS heterodimers (Smeal et al., 1989). Homodimers of c-FOS
have yet to be identified (Figure 7). AP-1 contains a leucine zipper motif and binds
through its basic DNA binding domain (DBD) to a canonical DNA binding element:
TGAg/cTCA. This is known as the TPA-responsive element (TRE) named after its
enhanced DNA binding to this consensus site upon treatment with 12-O-
tetradecanoylphorbol-13-acetate (TPA), a tumor promoter. AP-1 is activated through
phosphorylation by the mitogen activating pathway (MAPK) or the stress pathway (JNK)
(Chen et al., 1996). It is negatively regulated by the ubiquitin-proteasome degradation
pathway.
AP-1 cooperates with other proteins including, but not limited to, NFκB,
CBP/p300, Rb, and PEA3 (Hesselbrock et al., 2005; Matthews et al., 2007). AP-1
members generally favor a dynamic “switch of partners” to mediate targeted effects as
opposed to a “static predetermined partner” model. Expression profiles and partnerships
of the different members vary among cell types, even in different breast cancers (Table 2).
The functional role of AP-1 is to promote proliferation, differentiation, inflammation,
41
Jun-Jun Jun-Fos Jun-ATF
c-Fos Fra-1JunB JunD FosBc-Jun Fra-2
MDA-MB-231
JUN FAMILY FOS FAMILY
Jun-Jun Jun-Fos Jun-ATFJun-Jun Jun-Fos Jun-ATF
c-Fos Fra-1JunB JunD FosBc-Jun Fra-2
MDA-MB-231
JUN FAMILYJUN FAMILY FOS FAMILYFOS FAMILY
Figure 7: AP-1 Members and Dynamic Partnerships.
AP-1 Reference Transformed Invasive Non-invasive Members Normal HBL-100 MDAMB231 T47D
c-FOS ---- High High Low FosB Low ---- ---- High Fra-1 ---- Medium High ---- Fra-2 ---- Low Low ---- c-Jun ---- Low High Low JunB Low ---- Low Low
junD Low High High High
Table 2: Relative Levels of AP-1 Family Members in Breast Cancers. AP-1 expression pattern was determined in separate cell types: HBL-100 cells, transformed mammary epithelial cells derived from a normal lactating breast, MDA-MB-231 cells which are highly invasive, and T47D cells which are weakly or non-invasive as compared to normal breast tissue as a base reference (Bamberger et al. 1999).
42
and/or apoptosis. The perturbed expression and activity of AP-1 varies among cancers,
and is positively linked to tumor promotion and progression.
1. The c-JUN Family
A. c-JUN
c-JUN was originally identified and characterized as the AP-1 founding member.
The protein is approximately 330 amino acids in length and harbors many domains
important for its function (Hattori et al., 1988). c-JUN contains a N-terminal
transactivation domain (TAD), a domain consisting of a stretch of basic residues
considered the DNA binding domain (DBD), and the C-terminal leucine zipper domain
(ZIP) (Figure 8). The basic DNA binding region allows for appropriate DNA binding by
interaction with the negatively charged DNA backbone. The C-terminal leucine zipper
motif aids in protein-protein interactions and dimerization between AP-1 partners.
Activation of c-JUN occurs through phosphorylation events by MAPK or JNK within the
transactivation domain at serines 63 and 73, and threonines 91 and 93 (Smeal et al., 1991).
Phosphorylation events are important for the stability and interaction with other proteins.
This transactivation domain has been completely removed to create a dominant negative
form of c-JUN and consequently the AP-1 complex itself, known as TAM-67 (Figure 8).
This was originally created by Brown et al. in 1993 (Brown et al., 1993). TAM-67 is a
transdominant inhibitor of AP-1, which can bind DNA but quenches AP-1 complexes by
competing for wildtype c-JUN and c-FOS members (Brown et al., 1994; Brown et al.,
1996) thus dampening AP-1 activity. Like PEA3, ubiquitinylation directs c-JUN
43
H N3 COOH
TAD DBD ZIP
H N3 COOH
DBD ZIP
c-JUN
TAM-67
TTSS
63 73 91 93H N3 COOH
TAD DBD ZIP
H N3 COOHH N3H N3 COOH
TAD DBD ZIP
H N3 COOH
DBD ZIP
H N3 COOHH N3H N3 COOH
DBD ZIP
c-JUN
TAM-67
TTSS
63 73 91 93
Figure 8: Protein Structure of c-JUN and its Dominant Negative Form, TAM-67. Abbreviations- serine, S; threonine, T; transactivation domain, TAD; DNA binding domain, DBD; basic leucine zipper domain, bZIP.
44
proteasome-mediated degradation. Interestingly, c-JUN knockout mice are embryonic
lethal showing effects on liver, heart, and hepatoblasts (Eferl and Wagner, 2003).
The critical member of the AP-1 family, c-JUN is associated with cancer acting as
a major transcription factor. It is overexpressed in triple negative breast cancer cells,
MDA-MB-231 cells (Bamberger et al., 1999). c-JUN is associated with proliferation
showing marked proliferation alterations in c-JUN deficient fibroblasts (Wisdom et al.,
1999). Also, c-JUN cooperates with p300 and PEA3 to promote HER2 mediated
transcription in MCF-7 cells, luminal A HER2 expressing breast cancer cells (Matsui et
al., 2006). Correlation studies determined that c-JUN is involved in both proliferation,
hyperphosphorylated Rb, angiogenesis, and vascular endothelial growth factor (VEGF),
showing expression on 38% of invasive breast cancers in 103 breast cancer cases
(Vleugel et al., 2006). Interestingly, high expression of c-JUN has been associated with
insensitivity to chemotherapy and drug resistance in MCF-7 breast cancer cells (Daschner
et al., 1999). Other associations regarding cellular proliferation have shown that c-JUN
expression and activity upregulates cyclin D1, a critical mediator of cell cycle
progression from G1-S phase (Albanese et al., 1995; Bakiri et al., 2000; Herber et al.,
1994) and downregulates p16, an important cell cycle G1/S checkpoint inhibitory protein
(Passegue and Wagner, 2000). In contrast, another member of the JUN family, JunB,
downregulates cyclin D1 (Bakiri et al., 2000) and upregulates p16 (Passegue and Wagner,
2000), which limits cell proliferation and provides an interesting and distinctive role
among each AP-1 member.
45
B. JunB
JunB has a similar structure to c-JUN, but lacks the important serines 63 and 73
phosphorylation sites on (Kallunki et al., 1996), which may be responsible for the
interesting and opposite role JunB has on cellular transformation. Many studies have
reported an antagonism between c-JUN and JunB (Bakiri et al., 2000; Passegue and
Wagner, 2000); their expression tend to be inversely proportional (Shaulian and Karin,
2002). Its expression in MDA-MB-231 cells is very low (Bamberger et al., 1999). JunB
is less active than c-JUN. c-JUN and c-FOS heterodimers are much more potent
activators of gene transcription than c-JUN and JunB dimers (Deng and Karin, 1993).
This opposite effect is illustrated in overexpression studies in which c-JUN expression
alone can transform immortalized rat fibroblasts, but JunB expression has no
transformation capability (Schutte et al., 1989). This interesting opposition only stresses
the notion that cellular context and dynamic partnership between AP-1 members and their
interacting protein partners determines the appropriate cellular response.
C. JunD
JunD has a similar structure as the other JUN family members, but unlike JunB,
JunD contains the serines 63 and 73 phosphorylation sites as c-JUN. The functional role
of JunD is still being discerned showing both positive and negative effects on cell
proliferation and apoptosis (Pfarr et al., 1994; Weitzman et al., 2000). JunD null mice do
not form spontaneous tumors, yet inhibition of JunD suppresses neoplastic growth
indicative of a tumor promoting effect (Agarwal et al., 1999; Thepot et al., 2000). In a
46
study investigating 53 mammary carcinomas and three cell lines, relative expression of
JunD varied greatly with no distinct pattern (Bamberger et al., 1999). It is expressed in
MDA-MB-231 cells (Bamberger et al., 1999), but its functional role is relatively
unknown. To further demonstrate the spurious nature of JunD, studies have shown that
when the interaction with JunD and menin, a known tumor suppressor, is abolished, JunD
changes its activity from a tumor suppressive function to one that is tumor promoting
(Agarwal et al., 2003). However, in Ras transformed fibroblasts, JunD protects cells
from oxidative stress and reduces angiogenesis (Gerald et al., 2004). This further
demonstrates the necessity of comprehending appropriate cellular context and not
limiting AP-1 members to a specific function, namely tumor suppressive or tumor
promoting.
2. The c-FOS Family
A. c-FOS
c-FOS was originally identified and characterized when the viral protein bound to
the same consensus as c-JUN (Rauscher et al., 1988). It contains 381 amino acids
consisting of a basic DNA binding domain (DBD), a transactivation domain (TAD), and
a leucine zipper domain (ZIP) (Figure 9). Similarly to c-JUN, c-FOS has been exploited
to create a dominant negative AP-1 known as A-FOS. A-FOS is mutant form of c-FOS
in which the DNA binding domain has been mutated (Figure 9) (Olive et al., 1997). The
dominant negative mutant incorporates an acidic extension made up of many negatively
charged glutamate amino acids (Olive et al., 1997). This charge reversal
47
H N3 COOH
DBD ZIPTAD
H N3 COOHH N3H N3 COOH
DBD ZIPTAD
H N3 COOH
DBD ZIPTAD
H N3 COOHH N3H N3 COOH
DBD ZIPTAD
A-FOS
c-FOS
TTS
232 325 331 374
T
LEQRAEE LARENEE LEKEAEE LEQELAE LE
ACIDIC EXTENSION
Figure 9: Protein Structure of c-FOS and its Dominant Negative Form, A-FOS. Abbreviations- serine, S; threonine, T; leucine, L; transactivation domain, TAD; DNA binding domain, DBD; basic leucine zipper domain, bZIP.
48
destroys the intermolecular charge attraction between the negative DNA backbone and
the DNA binding domain of A-FOS, thus dampening AP-1 activity (Ransone et al., 1990).
c-FOS heterodimerizes with c-JUN to form a stable dimeric transcription factor capable
of regulating genes important in cell proliferation, differentiation, and apoptosis
(Shaulian and Karin, 2001). Homodimers of c-FOS do not exist, but additional binding
partners of c-FOS are MAF proteins (Kataoka et al., 1996), CBP (Bannister and
Kouzarides, 1995), and GATA-4 (McBride et al., 2003). Similar to c-JUN, c-FOS is
phosphorylated on threonine 232 by MAP kinases (Deng and Karin, 1994) and by ERKs
on threonines 325, 331, and 374 (Monje et al., 2003) to enhance its activity. c-FOS relies
on dimerization for its activity, since it is highly unstable and rapidly degraded by the
ubiquitin-meditated proteasome degradation (Bossis et al., 2003).
In cancer, c-FOS upregulates genes responsible for angiogenesis such as VEGF
(Marconcini et al., 1999) and MMP-1 and MMP-3 (Hu et al., 1994). It has also been
shown that c-FOS upregulates DNA 5-methylcytosine transferase responsible for
methylating and silencing tumor suppressor genes (Bakin and Curran, 1999).
Alternatively, c-FOS upregulates FAS ligand (FASL) to induce apoptosis through the
extrinsic death-receptor pathway in T-lymphocytes (Kasibhatla et al., 1998). It is highly
expressed in triple negative breast cancer, MDA-MB-231 cells (Bamberger et al., 1999).
Even though the majority of the literature associates active c-FOS with cancer
progression, the specific role of c-FOS depends on cell type and cellular context. With
this in mind, c-FOS becomes a more dynamic partner relying greatly on other cellular
signals.
49
B. Fra-1
Elevated levels of AP-1, especially Fra-1, are commonly found in the most
aggressive estrogen receptor negative mammary tumors when compared to less
metastatic estrogen receptor positive mammary tumors (Zajchowski et al., 2001).
Exogeneous overexpression of Fra-1 in the non-invasive MCF-7 breast cancer cells
disturbs cell morphology and increases the invasive potential of the cells. Conversely, if
endogenous Fra-1 is inhibited using RNA interference in MDA-MB-231 cells, overall
motility and aggressiveness are diminished (Belguise et al., 2005). In a cDNA array of
breast cancer subtypes, increased expression of both Fra-1 and c-JUN partners was
correlated to overall breast tumor aggressiveness (Zajchowski et al., 2001). Similarly,
Fra-1 expression in epithelioid adenocarcinoma cells demonstrated a positive effect on
motility and invasiveness (Kustikova et al., 1998). In MDA-MB-231 cells, motility was
not affected by Fra-1, but its invasiveness potential was highly enhanced (Milde-
Langosch et al., 2004). Recently, Baan et al. demonstrated that invasive breast cancer
cells including MDA-MD-231 favored the c-JUN:Fra-1 heterodimer, compared to less
invasive breast cancer cells, which favored the c-JUN:c-FOS heterodimer (Baan et al.,
2010). Fra-1 is known to regulate genes involved in proliferation (p19-ARF) (Ameyar-
Zazoua et al., 2005), invasion (MMP-1,9,) (Milde-Langosch et al., 2004), and
angiogenesis (IL-8) (Freund et al., 2004). Taken together, AP-1 complexes involving
Fra-1 most probably are critical for mammary tumorigenesis and invasion.
50
C. Fra-2
Most focus on AP-1 has encompassed c-JUN, c-FOS, and Fra-1 leaving the other
members like Fra-2 in the shadows. Nevertheless, Fra-2 does play an interesting role in
development and cancer. Fra-2 is involved in cellular differentiation during development.
The expression pattern of Fra-2 is observed in the epithelia and central nervous system
during mouse development (Carrasco and Bravo, 1995). Its expression in triple negative
breast cancer cells MDA-MB-231 is relatively low (Bamberger et al., 1999). Fra-2
overepression in mouse mammary adenocarinoma cells (CSML0) had no affect on
motility, but in a fibroblastoid cell line, its overexpression effected motility (Tkach et al.,
2003). In MDA-MB-231 cells, Fra-2 expression had a mild positive effect on cellular
invasion (Milde-Langosch et al., 2004). Fra-2 tends to be a modular protein partner not
only relying on cellular context, but also the AP-1 core complex.
D. FosB
FosB is the last member of the FOS family and possesses an opposite function
than the other members. An inverse correlation between FosB and Fra-1 has been
observed. In tumor tissues with high Fra-1 expression, FosB is low, but in tissues where
FosB is elevated, low expression of Fra-1 is observed. Also, association of FosB with
well-differentiated and estrogen positive breast cancer has been observed (Bamberger et
al., 1999). The expression of FosB is relatively undetectable in poorly-differentiated
steroid receptor negative clinical breast cancer tissues, but highly expressed in normal
ductal mammary epithelial cells (Milde-Langosch et al., 2004). Opposite of the effects
51
demonstrated by expression of c-FOS, Fra-1, and Fra-2, FosB has an inhibitory effect on
cell proliferation in correlation studies using breast cancer samples (Milde-Langosch et
al., 2000). Although FosB has an inverse role than its siblings, the AP-1 partner choice
and directed cellular signals could tip the balance and potentially change its function role.
3. AP-1 and PEA3
AP-1 interacts with partners on the promoter of many genes which is dependent
on cellular context to determine function, Similarly, PEA3 exists in a dynamic
equilibrium relying on partners to determine its function (Kurpios et al., 2003).
Interestingly, PEA3 has been shown to partner with AP-1 on several genes such as MMP-
1, MMP-3, MMP-7, MMP-9 (Benbow and Brinckerhoff, 1997), urokinase-type
plasminogen activator (uPA) (Evans et al., 2001), cyclooxygenase (COX-2)
(Subbaramaiah et al., 2002), HER2/ErbB-2 (Matsui et al., 2006), IL-8 (Davidson et al.,
2003), and mammaglobin (Hesselbrock et al., 2005). AP-1 and PEA3 DNA binding
elements are generally within relative close proximity to each other and generally to the
transcriptional start site (Benbow and Brinckerhoff, 1997; Davidson et al., 2003; Evans et
al., 2001; Hesselbrock et al., 2005; Matsui et al., 2006; Subbaramaiah et al., 2002).
Taken together, AP-1 and PEA3 are most probably dynamic transcriptional partners that
tend to share a common gene regulatory unit.
52
A Systems Approach: Linking Notch, PEA3, & AP-1
Biological systems are made up of interconnecting networks of cellular
communication. These pathways involve a plethora of proteins that when stretched
beyond homeostasis produces a diseased state. Notch signaling, PEA3, and AP-1 are
shown to be atypically regulated in breast carcinomas. Studies have shown that elevated
levels and/or misdirected signaling of these proteins lead to abnormal cell proliferation,
increased aggressive phenotypes, and overall reduced patient survival (Angel and Karin,
1991; Coutte et al., 1999; Kopan and Ilagan, 2009). In our investigations, initial
promoter scanning indicated proximal AP-1 and PEA3 sites on both the Notch-1 and
Notch-4 genes. With vast research independently correlating Notch, PEA3, and AP-1
with breast cancer, we provide, to our knowledge, for the first time a link between these
signaling pathways, with aim at exploiting and manipulating their function to create
innovative strategies for better targeted treatment of triple-negative breast cancer.
Women diagnosed with triple-negative breast cancer have the worst overall prognosis,
frequently present with metastatic tumors, and have few targeted therapy options to date.
And therefore, there is an immediate need to identify and understand the novel targets
that are responsible for the proliferation, survival, and invasive phenotype of breast
cancer.
CHAPTER III
MATERIALS AND METHODS
Cell Culture
MDA-MB-231, MCF-7, SKBr3 and BT474 breast cancer cells were purchased
from American Type Culture Collection. All cell lines were supplemented with 100 μM
nonessential amino acid and 1% L-glutamine (2 mM). SKBr3 cells [supplemented with
10% fetal bovine serum (FBS)] and MDA-MB-231 cells (5% FBS) were maintained in
Iscove’s Minimal Essential Media (IMEM). MCF-7 cells (10% FBS) were maintained in
Dulbecco's Modified Eagle Medium: Nutrient Mixture F12 (DMEM/F12). BT474 cells
(10% FBS) were maintained in Dulbecco's Modified Eagle Medium (DMEM). All cells
were maintained at 37°C with 95% O2 and 5% CO2.
Drugs and Chemicals
Gamma-secretase inhibitor (MRK-003) was kindly provided by Merck Oncology
International, Inc. (see Diagram 5 for chemical structure). MRK-003 was dissolved in
the solvent dimethylsulfoxide (DMSO) and stored at -80°C. Lactacystin (L6785) was
purchased from Sigma Aldrich (St. Louis, MO, USA), dissolved in water, and stored at -
20°C until use.
53
54
Expression Vectors
pcDNA3.1 and pcDNA3.1-PEA3 expression vectors were kindly provided by Dr.
Mein-Chie Hung (The University of Texas, MD Anderson Cancer Center, Houston, TX,
USA). Contol CMV-500, CMV-500-AFOS, Control pHMB, and pHMB-TAM67 vectors
were kindly provided by Dr. Richard Schultz (Loyola University Medical Center,
Maywood, IL, USA).
RNA Interference and Reagents
All small interfering RNA (siRNA) reagents were purchased from Santa Cruz
Biotechnologies (Santa Cruz, CA, USA). Transfection reagents were Lipofectamine
2000 and Lipofectamine RNAiMAX purchased from Invitrogen (Carlsbad, CA, USA),
and Fugene 6 was purchased from Roche Diagnostics Corporation (Indianapolis, IN,
USA). Protocols were performed according to the manufacturer’s instructions. Co-
transfections of DNA plasmid and siRNA were performed with Lipofectamine 2000.
The siRNA sequences are shown in Table 3.
Antibodies
Notch-1(C-20)R: (sc-6014-R), Notch-4(H-225): (sc-5594), PEA3 (16): sc-113, c-
FOS(H-125): (sc-7202), c-JUN(G-4): sc-74543, FRA-1(R-20): (sc-605) were purchased
from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Beta-actin (AC-15): (A5441)
was purchased from Sigma Aldrich (St. Louis, MO, USA) and used as the internal
55
siRNA Catalog # Sequence Control sc-37007 5’-UUCUCCGAACGUGUCACGU-3’
Notch-1 sc-36095 5’-CACCAGUUUGAAUGGUCAA-3’ 5’-CCCAUGGUACCAAUCAUGA-3’ 5’-CCAUGGUACCAAUCAUGAA-3’
PEA3 sc-36205 5’-CCAGACAAAUCGCCAUCAA-3’ 5’-CGCUCCGAUACUAUUAUGA-3’ 5’-CCAACCACAGAGAACAAGA-3’
c-JUN sc-29223
5’-GUGACGGACUGUUCUAUGA-3’ 5’-CCAGAAAGGAUAUUUAAGA-3’ 5’-GAUGGCCUUUGCUUAUGAA-3’ 5’-GCAUCAUCUGUAGAUACUA-3’
c-FOS sc-29221
5’-CAAGGUGGAACAGUUAUCU-3’ 5’-GGCUUCCCUUGAUCUGACU-3’ 5’-CCGAGCCCUUUGAUGACUU-3’ 5’-GGCAAUAGUGUGUUCUGAU-3’
Fra-1 sc-35405 5’-CCAGCAACUUCUUCUCCAU-3’ 5’-CUGACCAUAUUGUGCUUCA-3’ 5’-CGGAUCUCAGCUUUGAGAA-3’
Table 3: Sequences of siRNA(s).
TARGETS FORWARD PRIMERS REVERSE PRIMERS Notch-1 5'-GTCAACGCCGTAGATGACC-3' 5'-TTGTTAGCCCCGTTCTTCAG-3'
Notch-2 5'-TCCACTTCATACTCACAGTTGA-3' 5'-TGGTTCAGAGAAAACATACA-3'
Notch-3 5'-GGGAAAAAGGCAATAGGC-3' 5'-GGAGGGAGAAGCCAAGTC-3'
Notch-4 5'-AACTCCTCCCCAGGAATCTG-3' 5'-CCTCCATCCAGCAGAGGTT-3'
Hes-5 5'-ATGGCCCCCAGCACTGTGGCC-3' 5'-CTGACAGCCATCTCCAGGATG-3'
Hey-1 5'-TGGATCACCTGAAAATGCTG-3' 5'-TTGTTGAGATGCGAAACCAG-3'
cIAP2 5'-GGACTCAGGTGTTGGGAATCTG-3' 5'-CCTTTAATTCTTATCAAGTACTCACACCTT-3'
HPRT 5'-ATGAACCAGGTTATGACCTTGAT-3' 5'-CCTGTTGACTGGTCATTACAATA-3'
PEA3 5'-AGGAGACGTGGCTCGCTGA-3' 5'-GGGGCTGTGGAAAGCTAGGTT-3'
COX-2 5'-CAGGATACAGCTCCACAGCA-3' 5'-ATCACAGGCTTCCATTGACC-3'
MMP-9 5'-TCGTGGTTCCAACTCGGTTT-3' 5'-GCGGCCCTCGAAGATGA-3'
IL-8 5’-CACCGGAAGGAACCATCTCACT-3’ 5’-TCAGCCCTCTTCAAAAACTTCTCC-3’
Table 4: Sequences of PCR Primers.
56
control. Secondary antibodies include donkey anti-mouse IgG-HRP (sc-2314) and
donkey anti-rabbit IgG-HRP (sc-2313) (Santa Cruz, CA, USA).
Real-Time RT-PCR
MDA-MB-231 (4x105), MCF-7 (4x105), SKBr3 (5x105), and BT474 (5x105) cells
were plated in a 6-well tissue culture treated dish. Twenty-four hours later, the cells were
transfected with appropriate reagents (7 μL of lipofectamine 2000 for plasmids or 5 μL of
lipofecatime RNAiMAX for siRNA and 5 μL of siRNA reagents (16 nM) and/or 3 μg of
pcDNA3.1-PEA3) and maintained at 37°C 95% O2 and 5% CO2. Forty-eight hours later,
cells were harvested. Total RNA was extracted from the cells using the RNA extraction
kit: RNeasy® Mini Kit (Qiagen, Valencia, CA, USA). Total RNA recovered was then
quantified using the NanoDrop Spectrophotometer (Thermo Scientific). The cDNA was
reverse transcribed (RT) from 1 μg total RNA extracted in a total 100 μL volume
containing 1X RT buffer, 5.5 mM MgCl2, 500 μM dNTPs, 2.5 μM random hexamers, 0.4
U/μL RNase inhibitors, and 1.25 U/μL RT enzyme (MultiScribe™ Reverse Transcriptase
Kit, Applied Biosystems, Foster City, CA, USA). The parameters were as follows: 10
minutes at 25°C, 30 minutes at 48°C, 5 minutes at 95°C, and held at 25°C until use.
Analysis of mRNA relative fold copy number adjusted to hypoxanthine-guanine
phosphoribosyl transferase (HPRT), an endogenous control, was carried out by real-time,
quantitative PCR using iTaq™ SYBR® Green Supermix with ROX (BioRad, Hercules,
CA, USA). In a 96-optical PCR plate, 2.5 μL of cDNA (10 ng/μL) was added to 22.5 μL
of mastermix (2X Syber green Universal Master Mix, and 1 μM forward/reverse primers).
57
The quantitative PCR parameters were as follows: the initial denature temperature was
95°C for 10minutes; PCR cycling for 40 cycles was carried out at 95°C for 10 seconds,
and 60°C for 45 seconds. A melt curve was added after completion of the 40 cycles set
by the StepOnePlus thermocycler manufacturer (Applied Biosystems). Cycle number at
threshold (Ct) values were subsequently determined relative to the value for the
quantified cDNA that was amplified. Analysis of mRNA copy number adjusted to
hypoxanthine-guanine phosphoribosyl transferase (HPRT), an endogenous control, was
used to calculate relative fold induction or decrease compared to a control sample. The
PCR primers that were used for detection of specific transcripts are shown in Table 4.
Western Blot Analysis
MDA-MB-231 (4x105) cells were plated in a 6-well tissue culture treated dish.
Twenty-four hours later, the cells were transfected with appropriate reagents (7 μL of
lipofectamine 2000 with plasmid addition or 5 μL of lipofecatime RNAiMAX with
siRNA addition and 5 μL of siRNA (16 nM) reagents and/or 3 μg of pcDNA3.1-PEA3)
and maintained at 37°C with 95% O2 and 5% CO2. Forty-eight hours later, cells were
harvested. The cells were lysed in radioimmunoprecipitation assay buffer (RIPA, pH 8.0
containing 50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate, 25 mM β-glycerophosphate, 1 mM sodium
orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 1 mg/mL
aprotinin and 1 mg/mL leupeptin). Lysates were sonicated six times for three seconds
each and incubated on ice for 30 minutes. Lysates were then centrifuged at 10,000 g for
58
5 minutes at 4°C. The supernatant was isolated for further testing. Protein within the
supernatant was measured using the BCA protein concentration assay (Pierce Chemicals,
Rockford, IL), incubated for 30 minutes at 37°C with 95% O2 and 5% CO2, and
measured using a 96 plate UV/Visible spectrophotometer (PolarStar Omega, BMG
Labtech, Durham, NC). Samples were treated with an equal volume of sample buffer
containing NuPAGE 1X LDS Sample buffer, 1X NuPAGE reducing agent or 1 mM DTT.
Samples were heated at 70°C for 10 minutes. Total denatured proteins in lysates were
added to wells of a 4-12% NuPAGE® Bis-Tris Gels in 3-(N-morpholino)
propanesulfonic acid running buffer (MOPS). The gel was run at 175V for 1.5 hours and
separated proteins were transferred to PVDF membranes at 38V for 2 hours in Transfer
Buffer (25 mM Tris, 192 mM glycine, 10% methanol). The membranes were then
blocked in 5% non-fat milk in TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl,
0.05% Tween-20, and 0.2%NP-40 at pH 8.0) for one hour at room temperature while
constantly shaking. Primary antibodies were added (1:200) overnight in 5% non-fat milk
in TBST at 4°C. Blots were washed three times in TBST at intervals of 10 minutes at
room temperature while constantly shaking. Secondary antibodies conjugated to
horseradish peroxidase were added (1:2000) to 5% non-fat milk in TBST for one hour at
room temperature while constantly shaking. Blots were then subjected to a second wash
cycle: washed three times in TBST at intervals of 10 minutes at room temperature while
constantly shaking. Substrate for protein detection was SuperSignal® West Dura
(Thermo Scientific, Rockford, IL, USA) added in equal parts, incubated for 5 minutes at
room temperature while rocking, and visualized by the FUGIFILM™ Las-300 imager.
59
To re-probe western blots, the membranes washed three times in TBST at intervals of 10
minutes at room temperature while vigorously shaking. Restore Plus Western Blot
Stripping buffer (Thermo Scientific) was added to cover the membrane and incubated for
1 hour at 47°C. The blots were then rocked at room temperature for 30 minutes followed
by three quick rinses with deionized water. The membranes were then rocked at room
temperature in deionized water for 5 minutes, and then in TBST for 5 minutes.
Membranes were then stripped and Western blot analysis was repeated as described
above.
Luciferase Constructs and Assay
Notch-4 luciferase constructs were generously provided by Dr. Emery Bresnick
(University of Wisconsin at Madison, Madison, WI, USA) and previously described (Wu
et al., 2005). Notch-1 luciferase constructs were generously provided by Dr. Paolo Dotto
(Massachusetts General Hospital: Cutaneous Biology Research Center, Charlestown, MA,
USA). The AP-1 luciferase construct was generously provided by Dr. Richard Schultz
(Loyola University Medical Center, Maywood, IL, USA). MDA-MB-231 (1x105) cells
were plated in a 6-well tissue culture treated dish. Twenty-four hours later, the cells were
co-transfected in each well with appropriate reagents: 7 μL of lipofectamine 2000 with
plasmid addition 4 μg of Firefly luciferase (AP-1 or Notch-4 wildtype or mutant
reporters) and 0.4 μg pTL-TK (Renilla luciferase and internal control), and/or 5 μL (16
nM) of PEA3 siRNA reagent. The cells were incubated at 37°C with 95% O2 and 5%
CO2. Forty-eight hours later media was removed from the cells, they were washed with
60
ice cold PBS, and harvested in 1X passive lysis (300 μL) for 30 minutes while rocking at
room temperature. The plates were then frozen at -80°C for 1 hour to over overnight.
The plates were then removed from the freezer and rocked at room temperature for 15
minutes. Ten microliters of each sample was added to a white coated luminescent 96-
well plate and subjected to the manufactured dual-luciferase assay (Promega, Madison,
WI, USA). Luminescence was measured using the Veritas Microplate Luminometer
(Turner Biosystems, Sunnyvale, CA, USA). The range of luminescence was between
350-650 nm; Firefly luciferase read at 560 nm and pTL-TK at 480 nm. The graphs
represent the ratio of Firefly luciferase to Renilla luciferase (560 nm / 480 nm).
Chromatin Immunoprecipitation (ChIP)
MDA-MB-231 cells (3x106) were plated in 150 cm2 petri dishes for 24 hours.
The cells were transfected with 3 μg of pcDNA3.1, 3 μg of pcDNA3.1-PEA3, and 3 μg
of pcDNA3.1-PEA3 and 5 μL of 10 μM solution of PEA3 siRNA for 48 hours at 37°C
with 95% O2 and 5% CO2. The total volume in the wells was 3 mL. Media was
aspirated and the cells washed once with ice-cold PBS. The cells were cross-linked with
1% formaldehyde in fresh media and incubated at 37°C with 95% O2 and 5% CO2 for 10
minutes. A final concentration of 0.125 M glycine in the 150 cm2 petri dishes was added
while rocking at room temperature for 5 minutes to stop the reaction. The media was
aspirated while on ice, and washed twice with ice-cold PBS. Cells were then scraped,
centrifuged at 2000 rpm at 4°C, and lysed in 500 μL SDS lysis buffer (1% SDS, 10 mM
EDTA, 50 mM Tris-HCl, pH 8.1) containing a protease inhibitor cocktail tablet (Roche
61
Diagnostics, Indianapolis, IN). The samples were allowed to incubate on ice for 10
minutes. Following incubation, the lysates were sonicated using the Branson Sonifier
250 at output 4.5, duty cycle 50, and pulsed 10 times. The lysates were then diluted in IP
dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, 167
mM NaCl, pH 8.1) to a achieve a 1 to 10 dilution ratio and separated into two tubes: 1)
IgG mouse isotype control, and 2) specific PEA3 antibody. Lysates were pre-cleared
with 30 μL of protein G-plus beads (sc-2002, Santa Cruz) for 1 hour at 4°C with agitation.
Supernatants were isolated and 4 μg of PEA3 (sc-113X) antibody or mouse IgG isotype
control were added and incubated at 4°C overnight with agitation. Fifty microliters of
protein G-plus beads (sc-2002, Santa Cruz) were added to the immune complexes for 2
hours while gently rocking. Immune complexes/beads were washed in low salt buffer
(0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 150 mM NaCl, pH 8.1),
high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 500 mM
NaCl, pH 8.1), LiCl buffer (1% sodium deoxycholate, 1% NP-40, 0.25 M LiCl, 1 mM
EDTA, 10 mM Tris-HCl, pH 8.1), and TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH
8.0). The protein/antibody complexes from beads were eluted in freshly prepared elution
buffer (1% SDS, 0.1 M sodium bicarbonate, pH 8.0). Protein/DNA was reversed
crosslinked by heating at 65°C overnight while gentle rocking. The protein was degraded
using a proteinase solution (0.5 M EDTA, 1 M Tris-HCl, 10 mg/mL proteinase K, pH
8.0) and incubated at 52°C for 1 hour. DNA was isolated using the Qiagen PCR
purification kit (Qiagen, Valencia, CA, USA ). 20 μL of total lysate was set as an input
control. Enrichment at promoter sites was detected quantitative PCR using iTaq™
62
SYBR® Green Supermix with ROX (BioRad, Hercules, CA, USA). Ct values were
normalized to the IgG control. Quantitative real-time PCR was carried out as described
under the Real-Time PCR method section. The following primers flanking specific
promoter regions used in detection are shown in Table 5. A schematic representation of
the Notch-1 and Notch-4 promoters is shown in Diagram 10.
Co-Immunoprecipitation (Co-IP)
MDA-MB-231 cells (3x106) were plated in 150 cm2 petri dishes for 24 hours.
The cells were transfected with 3 μg of pcDNA3.1, 3 μg of pcDNA3.1-PEA3, and 3 μg
of pcDNA3.1-PEA3 and 5 μL of 10 uM solution of PEA3 siRNA for 48 hours at 37°C
with 95% O2 and 5% CO2. The total volume in the wells was 3 mL. Media was
aspirated and the cells were washed once with ice-cold PBS. The cells were cross-linked
with 1% formaldehyde in fresh media and incubated at 37°C with 95% O2 and 5% CO2
for 10 minutes. A final concentration of 0.125 M glycine in the 150 cm2 petri dishes was
added while rocking at room temperature for 5 minutes to stop reaction. The media was
aspirated while on ice, and washed twice with ice-cold PBS. Cells were then scraped,
centrifuged at 2000 rpm at 4°C, and lysed in 500 μL SDS lysis buffer (1% SDS, 10 mM
EDTA, 50 mM Tris-HCl, pH 8.1) containing a protease inhibitor cocktail tablet. The
samples were allowed to incubate on ice for 10 minutes. Following incubation, the
lysates were sonicated using the Branson Sonifier 250 at output 4.5, duty cycle 50, and
pulsed 10 times. The lysate concentration was ascertained using the protein BCA protein
assay (Pierce Chemicals, Rockford, IL), incubated for 30 minutes at 37°C with 95% O2
63
ChIP Primers # FORWARD REVERSE
A) Notch-1 Promoter
Negative Control D 5'-GTGCACACGGCTGTCCG-3' 5'-GCGACAACTGGCGACTGAA-3' 2 x PEA3(+Ctrl) A 5'-GCTGCAAGAGCCAAGATGAA-3' 5'-GGTGCCTGTGTTGAAAGCTCT-3' c-ETS E 5'-CTCCTGGCGCTTAACCAGG-3' 5'-CCAGAAAGCACAAACGGGTC-3' AP-1 (A) B 5'-GCCTCCTTAGCTCACCCTGA-3' 5'-TCTTCAGAGGCCCCCTGC-3' AP-1 (B) C 5'-TCCGCAAACCAGGCTCTG-3' 5'-ATTGGGGTGCAGTGCCG-3'
B) Notch-4 Promoter Negative Control D 5'-AGTGGGAGCAGAGGAGGTG-3' 5'-TGGGTCTGACCACTGAGACA-3' CBF-1 (+Ctrl) A 5'-TGGTACCACCCTGGTCAGTAT-3' 5'-GCTCAGGCATTATGAGCTATG-3' AP-1 (A) B 5'-AGCTGCCACTGACACCTTCT-3' 5'-CAGGGAATGCCAGTCAGAAT-3' AP-1 (B) C 5'-ACTTCCCCAGGGGTTGTC-3' 5'-CTTCCTCCTCGGCCTGCT-3'
Table 5: Chromatin Immunoprecipitation Primers.
NOTCH-4 PROMOTER
NOTCH-1 PROMOTER
ETS ETS ETS ETS ETSAP-1 AP-1NOTCH-1
-3.3 kb -2.9 kb -2.7 kb -1.1 kb
A B C D E
ETS ETS ETS ETS ETSAP-1 AP-1NOTCH-1
-3.3 kb -2.9 kb -2.7 kb -1.1 kb
A B C D E
CBF-1 ETS ETSAP-1 AP-1
A B C D
-3.5 kb -700 bp -70 bp
NOTCH-4
CBF-1 ETS ETSAP-1 AP-1
A B C D
-3.5 kb -700 bp -70 bp
NOTCH-4
Figure 10: Schematic Representation of Notch-1 and Notch-4 Promoters Labeled to Designate Areas of Primer Design.
64
and 5% CO2, and measured using a 96 plate UV/Visible spectrophotometer. The lysates
were equally diluted in IP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM
EDTA, 16.7 mM Tris-HCl, 167 mM NaCl, pH 8.1) to a achieve a 1 to 10 dilution ratio
and separated into two tubes: 1) IgG mouse isotype control, and 2) specific PEA3
antibody. Lysates were pre-cleared with 30 μL of protein G-plus beads (sc-2002, Santa
Cruz) for 1 hour at 4°C with agitation. Precleared lysates were incubated with 3 μg of
PEA3 (sc-113X) or mouse IgG overnight. Thirty microliters of protein G-plus beads (sc-
2002, Santa Cruz) were added to the immune complexes for 2 hours while gently rocking.
Immune complexes/beads were washed three times in phosphate buffered saline (PBS).
The pellet was resuspeneded in Laemmli sample buffer supplemented with beta-
mercaptoethanol (BioRad, Hercules, CA, USA) and heated for 5 minutes at 95°C while
vigorously shaking. Western blot technique was used for detection of co-
immunoprecipitation proteins as described in the Western Blot section of these methods.
Antibodies used for detection were: PEA3 (16), c-FOS (H-125), c-JUN (G-4), and FRA-1
(R-20) from Santa Cruz Biotechnologies, CA.
Cell Cycle Analysis
MDA-MB-231 cells (1x105) were seeded in a 6-well plate. After 24 hours, the
cells were treated/transfected with either DMSO + scrambled siRNA, DMSO + PEA3
siRNA, MRK-003 (10 μM) + scrambled siRNA, or MRK-003 (10 μM) + PEA3 siRNA.
After 24 and 48 hours post-treatment/transfection at 37°C with 95% O2 and 5% CO2, the
cells were trypinized, and the media was isolated. Cell and media were centrifuged at
65
4°C, 500 rpm for 5 minutes. The cell pellet was resuspended in 2 mL ice-cold PBS.
From the 2 mL single cell suspension, 500 μL of solution was placed into FACS tubes.
Cells were pelleted at 1200 rpm (4°C) for 5 minutes. The supernatant was removed by
inverting and decanting and washed by adding 2 mL of ice cold 5% bovine calf serum
(BCS) in PBS. Cells were again pelleted at 1200 rpm (4°C) for 5 minutes and fixed: cells
were resuspend cells in 100 μL ice cold 5% BCS and 600 μL of ice cold 100% EtOH was
slowly added while gently mixing. The tube was incubated on ice for 30 minutes and
then washed by adding 2 mL of ice cold 5% BCS. Cells were then centrifuged at 1200
rpm (4°C) for 5 minutes and resuspended in 500 μL of 10 μg/mL RNase A. They were
incubated at 37°C for 15 minutes and then allowed to incubate at room temperature for 5
minutes. To the 500 μL volume, 500 μL of 100 μg/mL Propidium Iodide (P4170, Sigma
Aldrich) was added, gently mixed, and incubated at room temperature for at least one
hour in a dark area void of light. The samples were measured by flow cytometry (FACS
Canto, BD Biosciences).
Annexin-V Apoptosis Assay
MDA-MB-231 cells (1.5x105) were seeded in a 6-well plate for 24 hours. Cells
were treated and transfected with either DMSO + scrambled siRNA, DMSO + PEA3
siRNA, MRK-003 (10 μM) + scrambled siRNA, and MRK-003 (10 μM) + PEA3 siRNA.
After 24 and 48 hours post-treatment/transfection at 37°C with 95% O2 and 5% CO2,
cells were trypsinized, washed twice with ice-cold PBS, and resuspended in a 1X
Annexin-V binding buffer (0.1 M HEPES/NaOH, 140 mM NaCl, 25 mM CaCl2, pH 7.4).
66
From that suspension, 100 μL of the solution was placed into a FACS compatible tube.
To the tube, 5 μL of 1 μg/mL FITC-Annexin-V stain (556420, BD Biosciences) and 5 μL
of 50 μg/mL propidium iodide (P4170, Sigma Aldrich) were added, incubated for 15
minutes at room temperature in the dark. Then, 400 μL of 1X Annexin-V binding buffer
was added back and immediately within the hour the samples were measured by flow
cytometry (FACS Canto, BD Biosciences) using an Annexin-V specific antibody.
Control tubes were used for compensation and background normalization: 1) a tube
containing cell suspension in Annexin-V binding buffer alone, 2) a tube containing cell
suspension in Annexin-V binding buffer with FITC-conjugated Annexin-V antibody, 3) a
tube containing cell suspension in Annexin-V binding buffer with Propidium Iodide, and
4) a tube containing cell suspension in Annexin-V binding buffer, FITC-conjugated
Annexin-V antibody, and Propidium Iodide.
Cell Viability Assay
MDA-MB-231 cells (1x105) were seeded in a 6-well plate. After 24 hours, the
cell were treated and transfected with either DMSO + scrambled siRNA, DMSO + PEA3
siRNA, MRK-003 (10 μM) + scrambled siRNA, and MRK-003 (10 μM) + PEA3 siRNA.
After 24 and 48 hours post-treatment and transfection at 37°C with 95% O2 and 5% CO2,
the cells were trypsinized, and the media was collected. Cell and media were centrifuged
at 4°C, 500 rpm for 5 minutes. The cell pellet was resuspended in 2 mL ice-cold PBS.
From the 2 mL single cell suspension, 500 μL of solution was placed into 1.5 mL
Eppendorf tube. 500 μL of Trypan Blue was added, mixed gently, and incubated at room
67
temperature for 20 minutes. Then, 20 μL of the solution was place on a heamocytometer.
Stained cells were counted at 40X magnification under a standard light microscope
(Leica DMIL, Leica Microsystems), normalized, and graphed as percent viability as
compared to control.
Colony Formation Assay
A 1.75% solution of Nobel agar was autoclaved, allowed slightly to cool, and
added in equal parts [1:1] to IMEM supplemented with 100 μM nonessential amino acid,
1% L-glutamine (2 mM), and 5% FBS (making a final agar solution of 0.8%). To a 6-
well plate, 1 mL of the agar/media solution was added and allowed to completely solidify.
Once cooled, 2 mL of a 1% methylcellulose (M0512, Sigma Aldrich)/IMEM solution
(supplemented with 5% FBS, 1% nonessential amino acids, and 1% L-glutamine) was
added on top of the agar. The methylcellulose/IMEM solution was made as follows: A
2% methylcellulose solution in serum-free media was made by stirring vigorously for 2-3
hours at room temperature and transferred/stirred at 4°C overnight. Then, 5%FBS, 1%
NEAA, and 1% L-glutamine was added back to the solution, stirred at room temperature
for 30 minutes, and transferred/stirred at 4°C for 1-2 hours depending upon incorporation
rate. Once incorporated completely, the solution was centrifuged at 5500 rpm for 30
minutes at 4°C. The solution was then warmed, to accommodate the transfected/treated
cells to 37°C. MDA-MB-231 (1x103 cells/ml) were treated/transfected with either
DMSO + scrambled siRNA, DMSO + PEA3 siRNA, Mrk-003 (5 μM) + scrambled
siRNA, and Mrk-003 (5 μM) + PEA3 siRNA for 24 hours at 37°C and 5% CO2. Forty-
68
eight hours later, the transfected/treated cells were trypsinized, counted, and added
directly to the methylcellulose solution and 2 mL of 10,000 cells/well were added atop of
the formerly solidified agar coated plates. The assay was left untouched for 14 days at
37°C and 5% CO2. After 14 days, 20 μL of crystal violet stock (0.2% crystal violet and
2% ethanol diluted in deionized water) was added to each well and gently swirled to
make a final stain concentration around 0.002%. The plate was then incubated for 1 hour
in a 37°C, 5% CO2 incubator. Colonies were photographed (4X magnification) and
counted under a standard light microscope. Nine fields were counted per well and
averaged.
Statistical Analysis
The statistics of these experiments are represented by means plus or minus
standard deviations on graphs and numbers in parenthesis that denote standard deviation.
The statistical significance of three or more independent studies in these experiments was
determined by a two-tailed, unpaired Student’s t-test for two comparisons. Statistical
significance of pairwise comparisons of more than two experiments was discerned using
a one-way ANOVA.
CHAPTER IV
RESULTS
Even with the advent of modern technologies, breast cancer still remains an area
of extensive research with hopes of uncovering novel strategies to help treat patients.
Previous investigations have determined that overexpression of Notch-1 and Notch-4
play a critical role in breast tumorigenesis and PEA3 overexpression is associated with
aggressive breast cancers particularly the triple-negative subtype. Preliminary research
via promoter scanning using NCBI Entrez Gene bank identified on the Notch-1 and
Notch-4 promoters several putative Ets binding sites, some independent with no known
consensus sequences nearby, or interestingly some Ets sites adjacent to AP-1 consensus
sites. Notch-2 and Notch-3 also have putative Ets binding sites, but preliminary real-time
PCR and Western blot data directed the focus on Notch-1 and Notch-4 for these studies.
Knowing the role of both these aberrant pathways in breast cancer, this investigation aims
at determining a link between the Notch pathway and PEA3 signaling. The following
hypothesis was proposed:
PEA3 is a transcriptional activator of Notch-1 and Notch-4 in MDA-MB-231
triple-negative breast cancer cells. Dual inhibition of PEA3 and Notch
signaling will result in a reduction in cell proliferation and survival implicating
better therapeutic targeting. 69
70
The following aims were formulated to address the hypothesis in MDA-MB-231 cells:
Specific Aim 1. To determine whether PEA3 is an upstream mediator of the
Notch signaling pathway.
Specific Aim 2. To elucidate whether PEA3 acts a direct positive regulator of
Notch-1 and Notch-4 transcription.
Specific Aim 3. To establish a biological significance by determining whether
dual inhibition of Notch and PEA3 leads to reduced cell proliferation and
tumorigenicity in vitro.
This research strategy provides novel evidence of a link between two pathways that are
overexpressed in breast cancer. PEA3 is a transcriptional activator of both Notch-1 and
Notch-4 mRNA in MDA-MB-231 cells. PEA3-mediated Notch-1 transcription is AP-1
independent while Notch-4 transcription requires both PEA3 and AP-1 (Figure 11).
PEA3 and Notch signaling are essential for proliferation and survival in MDA-MB-231
cells. Furthermore, PEA3 is a transcriptional activator of both Notch-1 and Notch-4
mRNA in other breast cancer cells. Thus, the hypothesis remains that dual targeting of
both PEA3 and Notch pathways might provide a new therapeutic strategy for triple-
negative breast cancer as well as possibly therapeutic targeting in other breast cancer
subtypes where PEA3 regulates Notch-1 and Notch-4.
Our novel findingsPreviously knownTherapeutic strategy
HYPOTHESIS
PEA3
Notch-1
Notch-4
Migration
HESHEY-1
Cyclin-D1
MMP-9COX-2
IL-8
Proliferation
GSI
GSIsiRNA
AP-1
c-JUNFRA-1
AP-1
c-JUNFRA-1
Malignancy
71
Figure 11: Hypothesis of the Investigation. PEA3 is a novel transcriptional activator of Notch-1 and Notch-4 in basal-like breast cancer. Dual inhibition of PEA3 and Notch signaling will result in a reduction in cell proliferation, tumorigenicity, and malignancy implicating better therapeutic targeting and overall survival.
72
SPECIFIC AIM-1:
To Determine Whether PEA3 is an Upstream Mediator of the
Notch Signaling Pathway.
MDA-MB-231 cells are a representative cell line of triple-negative breast cancer.
MDA-MB-231 cells lack expression of estrogen receptor, progesterone receptor, or
HER2 overexpression. These cells are the model system used for the subsequent studies.
The investigation began by determining the efficiency of PEA3 knockdown by
RNA interference to determine its resulting effects in vitro. Acquiring SMART-POOL of
three specific PEA3 siRNAs from Santa Cruz Biotechnologies (Santa Cruz, CA), siRNA
transfection in a dose dependent manner was performed to evaluate the optimal
concentration of PEA3 siRNA. Using lipofectamine as the transfecting vehicle, 5 μL of
siRNA (16 nM) was transfected into MDA-MB-231 cells. After 48 hours, cells were
harvested for RNA and protein. Real-time PCR of PEA3 transcripts indicated that 5 μL
of siRNA (16 nM) was sufficient to reduce PEA3 transcripts by ~90% (Figure 12).
Endogenous PEA3 is extremely difficult to detect; the protein is relatively unstable
exhibiting a turnover rate of about 30 minutes via the ubiquitin-proteasome pathway on
the C-terminus domain of PEA3 (Takahashi et al., 2005). It is rare to capture the
endogenous PEA3 protein levels (Baert et al., 1997). A Western blot analysis of
endogenous PEA3 protein levels demonstrated the corresponding effect of PEA3 siRNA
73
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
Rel
ativ
e m
RN
A L
evel
s
PEA3 mRNA
* p<0.0001
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
Rel
ativ
e m
RN
A L
evel
s
PEA3 mRNA
* p<0.0001
Figure 12: PEA3 Transcripts Upon RNA Interference. MDA-MB-231 cells were transfected with either scrambled siRNA (SCRBi) or 5 μL of PEA3 siRNA (PEA3i) corresponding to overall siRNA concentrations of 16nM. Relative mRNA levels of PEA3 were measured 48 hours later as a knockdown control using quantitative PCR. Transcripts were normalized to HPRT and means were plotted. Error bars represent standard deviation. Statistical significance (p<0.0001) of three experiments was determined by two-tailed, unpaired Student’s t- test.
74
PEA3
Actin
SCRBi
PEA3i+ –– +
PEA3
Actin
SCRBi
PEA3i+ –– +
1.00 0.55
Figure 13: Endogenous PEA3 protein upon RNA Interference. MDA-MB-231 cells were transfected with either scrambled siRNA (SCRBi) or 5 μL of PEA3 siRNA (PEA3i) corresponding to overall siRNA concentrations of 16nM. Protein levels of PEA3 were detected by Western blot analysis after 48 hours as described in the materials and methods. 50 μg of total protein lysate in each lane was subjected to SDS-PAGE 4-12% gel. Beta-actin was detected as an endogenous control.
75
on its protein levels (reduced by 50% upon siRNA transfection as measured by
densitometry) (Figure 13).
1. PEA3 Regulates Notch-1 and Notch-4 Transcripts.
To investigate the role of PEA3 on Notch expression, the effect of PEA3 RNA
interference on the expression of all four Notch receptor (1,2,3,4) mRNAs was examined.
Cells were transfected with either scrambled control siRNA or PEA3 siRNA for 48 hours.
As measured by real-time PCR, Notch-1 and Notch-4 transcripts were decreased by 50%
and 70%, respectively upon PEA3 knockdown. Notch-3 transcript levels were
unchanged. Interestingly, Notch-2 levels showed a moderate but significant increase
upon PEA3 knockdown, which upon further investigation may prove to be advantageous
since it has been correlated with pro-apoptotic and breast tumor suppressive function
[(O'Neill et al., 2007) and discussed in Chapter III] (Figure 14).
2. PEA3 Regulates Notch-1 Protein Expression.
To determine if the effect on Notch-1 and Notch-4 transcripts by PEA3 correlated
with protein expression, we measured steady protein levels using Western blotting in
cells transfected with either control (SCRBi) or PEA3 siRNA for 48 hours. The results
showed a reduction of Notch-1 full length (FL) and transmembrane (TM) receptor protein
levels by 40% and 50%, respectively (Figure 15) agreeing with its corresponding
transcripts. In contrast to Notch-1, PEA3 siRNA had no noticeable effects on steady-
state Notch-4 IC protein levels (Figure 5, lanes 1 and 2). However, it is known that the
NOTCH-1
0.00.20.40.60.81.01.21.41.61.82.0
SCRBi PEA3i
Rel
ativ
e m
RN
A L
evel
s
*p<0.001
NOTCH-1
0.00.20.40.60.81.01.21.41.61.82.0
SCRBi PEA3i
Rel
ativ
e m
RN
A L
evel
s
*p<0.001
SCRBi PEA3i0.00.20.40.60.81.01.21.41.61.82.0
NOTCH-4R
elat
ive
mR
NA
Lev
els
*p<0.001
SCRBi PEA3i0.00.20.40.60.81.01.21.41.61.82.0
NOTCH-4R
elat
ive
mR
NA
Lev
els
*p<0.001
NOTCH-2
0.00.20.4
0.60.81.01.21.41.61.82.0
SCRBi PEA3i
Rel
ativ
e m
RN
A L
evel
s
* p<0.05
NOTCH-2
0.00.20.4
0.60.81.01.21.41.61.82.0
SCRBi PEA3i
Rel
ativ
e m
RN
A L
evel
s
NOTCH-2
0.00.20.4
0.60.81.01.21.41.61.82.0
SCRBi PEA3i
Rel
ativ
e m
RN
A L
evel
s
* p<0.05
SCRBi PEA3i0.00.2
0.40.60.81.01.21.41.61.8
2.0NOTCH-3
Rel
ativ
e m
RN
A L
evel
s
SCRBi PEA3i0.00.2
0.40.60.81.01.21.41.61.8
2.0NOTCH-3
Rel
ativ
e m
RN
A L
evel
s
SCRBi PEA3i0.00.2
0.40.60.81.01.21.41.61.8
2.0NOTCH-3
Rel
ativ
e m
RN
A L
evel
s
76
Figure 14: Effects of PEA3 siRNA on Notch Transcripts. MDA-MB-231 cells were transfected with either scrambled siRNA (SCRBi) or PEA3 siRNA (PEA3i) for 48 hours. Relative mRNA levels of Notch receptors (NOTCH-1,2,3,4) were measured by quantitative PCR. Transcripts were normalized to HPRT and means were plotted. Error bars represent standard deviation. Statistical significance of three or more experiments was determined by two-tailed, unpaired Student’s t- test.
77
NOTCH-1
β-ACTIN
FL
TM
SCRBi + –– +PEA3i
NOTCH-1
β-ACTIN
FL
TM
SCRBi + –– +PEA3i
0.0
0.2
0.4
0.6
0.8
1.0
1.2
p<0.01
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
*
p<0.001
FULL LENGTHNOTCH-1 (FL)
TRANSMEMBRANEPORTION NOTCH-1 (TM)
SCRBi PEA3i SCRBi PEA3i0.0
0.2
0.4
0.6
0.8
1.0
1.2
p<0.01
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
*
p<0.001
FULL LENGTHNOTCH-1 (FL)
TRANSMEMBRANEPORTION NOTCH-1 (TM)
SCRBi PEA3i SCRBi PEA3i
Den
sito
met
ry (N
orm
aliz
ed to
Act
in)
Den
sito
met
ry (N
orm
aliz
ed to
Act
in)
Figure 15: Protein Levels of Notch-1 Upon PEA3 RNA Interference. MDA-MB-231 cells were transfected with either scrambled siRNA (SCRBi) or 5 μL (16nM) of PEA3 siRNA (PEA3i). Protein levels of Notch-1 were detected by Western blot analysis after 48 hours as described in the materials and methods. 10 μg of total protein lysate in each lane was subjected to SDS-PAGE 10% gel. Both transmembrane portion (TM) and full length portion (FL) of Notch-1 were detected by the antibody. Beta-actin was detected as an endogenous control. Graphs represent quantification of changes in full length and transmembrane length Notch upon transfection with either scrambled or PEA3 siRNA as measured by densitometry. Statistical significance of three or more experiments was determined by two-tailed, unpaired Student’s t-test.
78
SCRBi + – + –– + – +– – + +
β-ACTIN
NOTCH-4
PEA3i
LACTACYSTIN
SCRBi + – + –– + – +– – + +
β-ACTIN
NOTCH-4
PEA3i
LACTACYSTIN
Figure 16: Protein Levels of Notch-4 Upon PEA3 RNA Interference. MDA-MB-231 cells were transfected with either scrambled siRNA (SCRBi) or 5 μL (16nM) of PEA3 siRNA (PEA3i). Protein levels of Notch-4 were detected by Western blot analysis after 48 hours as described in the materials and methods. Cells were treated with or without 7 μM lactacystin after 24 hours of transfection for an additional 18 hours. 50 μg of total protein lysate in each lane was subjected to SDS-PAGE 4-12% gel. Beta-actin was detected as an endogenous control. Western Blot is representative of more than three experiments.
79
Notch-4 receptor is sensitive to stress and has a rapid turnover rate with a half life of 1.25
hours which is mediated by the proteosome (Wu et al., 2001). Thus, to determine if
protein stability is critical for regulation of Notch-4 protein by PEA3, a proteasome
inhibitor (lacacystin) was used to treat the cells prior to transfection with control or PEA3
siRNA. The protein levels of Notch-4 IC were increased almost two fold (Figure 16, lane
3) with lacacystin treatment and decreased by almost 90% upon PEA3 knockdown
(Figure 16, lane 4). This result suggests that Notch-4IC protein expression is critically
regulated by protein stability and not necessarily at the transcriptional level. Since PEA3
siRNA demonstrated little effects on Notch-2 and Notch-3 transcripts, their
corresponding protein levels by Western blot analysis were not pursued.
3. Classic Gene Targets of PEA3 and Notch are Decreased by PEA3
Knockdown.
To confirm the effect of PEA3 siRNA on the activity of PEA3, its classical
downstream targets were measured by real-time PCR after transfection with control or
PEA3 siRNA for 48 hours. PEA3 target genes, namely IL-8 and MMP-9, were
significantly reduced by 80% and 60%, respectively (Figure 17). To address whether the
Notch signaling pathway was affected by PEA3 RNA interference, a classic downstream
target of the Notch, HEY-1, was also significantly reduced upon PEA3 knockdown by
40% (Figure 6). Taken together, these results suggest that PEA3 positively regulates
Notch-1 and Notch-4 receptor transcripts, Notch-1 protein and, Notch signaling in MDA-
MB-231 cells.
80
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
HEY-1
Rel
ativ
e m
RN
A L
evel
s
* p<0.05
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
HEY-1
Rel
ativ
e m
RN
A L
evel
s
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
HEY-1
Rel
ativ
e m
RN
A L
evel
s
* p<0.05
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
MMP-9*
Rel
ativ
e m
RN
A L
evel
s
p<0.01
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
MMP-9*
Rel
ativ
e m
RN
A L
evel
s
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
MMP-9*
Rel
ativ
e m
RN
A L
evel
s
p<0.01
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
IL-8*
Rel
ativ
e m
RN
A L
evel
sp<0.001
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
IL-8*
Rel
ativ
e m
RN
A L
evel
s
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
IL-8*
Rel
ativ
e m
RN
A L
evel
sp<0.001
Figure 17: Transcript Levels of Classic PEA3 and Notch Targets Upon PEA3 siRNA. MDA-MB-231 cells were transfected with either scrambled siRNA (SCRBi) or PEA3 siRNA (PEA3i) for 48 hours. Downstream targets of PEA3 (IL-8, MMP-9) and Notch (HEY-1) were measured by quantitative PCR. Transcripts were normalized to HPRT and means were plotted. Error bars represent standard deviation. Statistical significance of three or more experiments was determined by two-tailed, unpaired Student’s t-test.
81
SPECIFIC AIM-2:
To Elucidate Whether PEA3 Acts as a Direct Positive Regulator of
Notch-1 and Notch-4 Transcription.
To determine the mechanism by which PEA3 regulates Notch-1 and Notch-4
transcription, the promoter regions of Notch-1 and Notch-4 were scanned using NCBI
Entrez Gene bank and identified several putative Ets binding sites (Figure 18).
Interestingly both promoters contained two putative AP-1 sites adjacent to Ets sites. A
classic CBF-1 site was also previously identified in the Notch-4 promoter and used as a
control. Primers were subsequently designed to include these identified regions. Primers
flanking promoter regions containing no known consensus sites were used as negative
controls (Figure 18). Notch-2 and Notch-3 also contain minimal putative Ets sites. The
quantitative PCR results on these promoters indicated mild changes in transcript levels
(Figure 14) and therefore were not pursued. However, future studies on these sites may
prove to be significant and/or may be used as negative controls.
Emphasizing the notion that PEA3 is highly unstable, endogenous protein levels
are difficult to detect (Baert et al., 1997). Consequently, exogenous expression of PEA3
was required to perform subsequent chromatin immunoprecipitations (ChIPs). Empty
vector (pcDNA3.1) and PEA3 expression vector (pcDNA3.1-PEA3) were tested in
MDA-MB-231 cells in a dose dependent manner to determine an optimal concentration.
Upon transfection of pcDNA3.1-PEA3 at concentrations of 0.5 μg, 1.0 μg, 2.0 μg, and
82 NOTCH-4 PROMOTER
NOTCH-1 PROMOTER
ETS ETS ETS ETS ETSAP-1 AP-1NOTCH-1
-3.3 kb -2.9 kb -2.7 kb -1.1 kb
A B C D E
ETS ETS ETS ETS ETSAP-1 AP-1NOTCH-1
-3.3 kb -2.9 kb -2.7 kb -1.1 kb
A B C D E
CBF-1 ETS ETSAP-1 AP-1
A B C D
-3.5 kb -700 bp -70 bp
NOTCH-4
CBF-1 ETS ETSAP-1 AP-1
A B C D
-3.5 kb -700 bp -70 bp
NOTCH-4
Figure 18: Schematic Representation of Notch-1 and Notch-4 Promoters.
83
4.0 μg, transcript levels of PEA3 increased in a dose dependent manner (Figure 19).
Moreover, a classic target gene of PEA3, COX-2, was measured and demonstrated a
similar dose dependent increase (Figure 19). Discerning that the maximal effect was at a
concentration of 4 μg, 4 μg concentration of pcDNA3.1-PEA3 and pcDNA3.1 was used
in the subsequent ChIP analyses.
To determine if exogenous PEA3 expression was compatible with
immunoprecipitation, 4 μg of pcDNA3.1 and pcDNA3.1-PEA3 vectors were transfected
in MDA-MB-231 cells for 48 hours prior to being subjected to immunoprecipitation.
Endogenous levels of PEA3 were not detected by immunoprecipitation, however
exogenous expression successfully immunoprecipitated PEA3 protein (Figure 20).
1. PEA3 is Enriched on the Notch-1 and Notch-4 Promoters.
To address whether PEA3 is recruited to the Notch-1 and Notch-4 promoters,
chromatin immunoprecipitation experiments were performed on lysates from MDA-MB-
231 cells that were transfected with PEA3 expression plasmid (pcDNA3.1-PEA3).
Comparable to the immunoprecipitation studies, ChIP analysis on endogenous levels of
PEA3 was not measurable (Figure 21) most likely a result of PEA3 protein instability,
which it is rapidly turned over and undetectable (Baert et al., 1997).
Five Ets sites on the Notch-1 promoter and four on the Notch-4 promoter were
identified and pursued via chromatin immunoprecipitation of PEA3. As a result of ChIP
analysis, PEA3 enrichment was found on both Notch-1 (Figure 22) and Notch-4 (Figure
23) promoter regions within 1.1 kilobase or -70 nuclotides, respectively, upsteam of the
84
0
10
20
30
40
50
60
70
80
pcDNA3.1 0.5ug 1.0ug 2.0ug 4.0ug
pcDNA3.1-PEA3
Rel
ativ
e m
RN
A L
evel
s
PEA3
0
10
20
30
40
50
60
70
80
pcDNA3.1 0.5ug 1.0ug 2.0ug 4.0ug
pcDNA3.1-PEA3
Rel
ativ
e m
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A L
evel
s
PEA3
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
pcDNA3.1 0.5ug 1.0ug 2.0ug 4.0ug
pcDNA3.1-PEA3
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
pcDNA3.1 0.5ug 1.0ug 2.0ug 4.0ug
pcDNA3.1-PEA3
COX-2
Rel
ativ
e m
RN
A L
evel
s
Figure 19: Exogenous Expression of PEA3 and Its Effects on Target Genes. MDA-MB-231 cells were transfected with either vector control plasmid (pcDNA3.1) or PEA3 expression vector (pcDNA3.1-PEA3) in a dose dependent manner for 48 hours. Relative mRNA levels of PEA3 and a classic target gene (COX-2) were measured by quantitative PCR. Transcripts were normalized to HPRT and means were plotted.
85
Heavy IgG
Light IgG
pcDNA3.1 pcDNA3.1-PEA3
IgG PEA3 IgG PEA3
PEA3
Antibodies:
Vectors: I.P.
Conjugated
Heavy IgG
Light IgG
pcDNA3.1 pcDNA3.1-PEA3
IgG PEA3 IgG PEA3
PEA3
Antibodies:
Vectors: I.P.
Heavy IgG
Light IgG
pcDNA3.1 pcDNA3.1-PEA3
IgG PEA3 IgG PEA3
PEA3
Antibodies:
Vectors: I.P.
Conjugated
Figure 20: Immunoprecipiation of Endogenous and Exogenous PEA3. MDA-MB-231 cells were transfected with either pcDNA3.1 or pcDNA3.1-PEA3 vectors. Immunoprecipitation of PEA3 and its isotype IgG control were performed after 48 hours as described in the materials and methods. Total immunoprecipitated protein lysates were subjected to SDS-PAGE 4-12% gel. Heavy and light chain IgG were detected as an endogenous loading control.
86
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Rel
ativ
e Fo
ld In
duct
ion
* p<0.0001
IgG PEA3
Vector Alone
PEA3 over-expression
NOTCH-4
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Rel
ativ
e Fo
ld In
duct
ion
p<0.0001*
Vector Alone
PEA3 over-expression
IgG PEA3
NOTCH-1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Rel
ativ
e Fo
ld In
duct
ion
* p<0.0001
IgG PEA3
Vector Alone
PEA3 over-expression
NOTCH-4
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Rel
ativ
e Fo
ld In
duct
ion
p<0.0001*
Vector Alone
PEA3 over-expression
IgG PEA3
NOTCH-1
Figure 21: Chromatin Immunoprecipitation of Endogenous and Exogenous PEA3 on the Notch-1 and Notch-4 Promoter.
MDA-MB-231 cells were transfected with either pcDNA3.1 (Vector alone) or PEA3-pcDNA3.1 overexpression plasmid. Lysates were subjected to chromatin immunoprecipitation using either PEA3 antibody or isotype control IgG on the Notch-1 and Notch-4 promoter. PEA3 enrichment was measured by quantitative PCR and normalized to IgG control. Below graphs is a schematic representation of the Notch-1 and Notch-4 promoters containing the identified enriched Ets sites. Means and standard deviation of more than three experiments were plotted. Statistical significance was determined by two-tailed unpaired Student’s t test.
IgG PEA3 IgG PEA3
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Rel
ativ
e Fo
ld In
duct
ion
*
A B C D E
NOTCH-1 PROMOTER
p<0.0001
87
Figure 22: Chromatin Immunoprecipitation of PEA3 on the Notch-1 Promoter. MDA-MB-231 cells were transfected with PEA3 expression plasmid (pcDNA3.1). Lysates were subjected to chromatin immunoprecipitation using either PEA3 antibody or isotype control IgG on the Notch-1. PEA3 enrichment was measured by quantitative PCR and normalized to IgG control. Below graphs is a schematic representation of the Notch-1 promoter containing multiple AP-1 consensus sites and Ets sites. Means and standard deviation of more than three experiments were plotted. Statistical significance was determined by two-tailed unpaired Student’s t test.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Rel
ativ
e Fo
ld In
duct
ion
*IgG PEA3
A B C D
NOTCH-4 PROMOTER
p<0.0001
88
Figure 23: Chromatin Immunoprecipitation of PEA3 on the Notch-4 Promoter. MDA-MB-231 cells were transfected with PEA3 expression plasmid (pcDNA3.1). Lysates were subjected to chromatin immunoprecipitation using either PEA3 antibody or isotype control IgG on the Notch-4 promoter. PEA3 enrichment was measured by quantitative PCR and normalized to IgG control. Below graphs is a schematic representation of the Notch-4 promoters containing multiple AP-1 consensus sites and Ets sites. Means and standard deviation of more than three experiments were plotted. Statistical significance was determined by two-tailed unpaired Student’s t test.
89
transcriptional start site. The other identified sites via promoter scanning showed no
measurable PEA3 enrichment (Figure 22 and Figure 23).
To address the specificity of the PEA3 antibody to detect recruitment of PEA3 to
these promoter regions, a SMART-POOL of three specific PEA3 siRNAs was used.
Western blot analysis was performed to determine the efficiency of PEA3 siRNA on
exogenous PEA3 expression (Figure 24). As a result from ChIP analysis, PEA3
enrichment on the -1.1 kb site on the Notch-1 promoter and the -70 bp site on the Notch-4
promoter was abrogated upon specific PEA3 knockdown (Figure 25).
2. PEA3 and AP-1 Regulate the Notch-4 Promoter.
Previous studies in vascular endothelial cells performed by Wu et al. at the
University of Wisconsin at Madison indicated the importance of AP-1 on the regulation
of Notch-4 (Wu et al., 2005). To assess if the -70 nucleotide region upstream of the start
site was indeed necessary in MDA-MB-231 cells for the regulation of the Notch-4
promoter by PEA3 as identified by ChIP and potentially AP-1, a wild type AP-1 or
mutant AP-1-containing Notch-4 promoter luciferase reporter constructs were obtained
from the Dr. Emery Bresnick’s laboratory and previously described (Wu et al., 2005).
The wild type construct contained an intact AP-1 and Ets consensus sites at -70
nucleotides. The mutant AP-1 construct contained an ablated AP-1 consensus site using
site-directed mutagenesis; the adjacent Ets binding site was left preserved (Wu et al.,
2005) (Figure 26).
90
pcDNA3PEA3 vector
Scrambled siRNAPEA3 siRNA
PEA3
+ – –– + + – + –– – +
Actin
pcDNA3PEA3 vector
Scrambled siRNAPEA3 siRNA
PEA3
+ – –– + + – + –– – +
Actin
W.Blot
Figure 24: Western Blot Analysis of PEA3 Expression.
MDA-MB-231 cells were transfected with either pcDNA3.1 and scrambled siRNA (SCRBi), pcDNA3.1-PEA3, PEA3 siRNA, or a combination of pcDNA3.1-PEA3 and PEA3 siRNA (PEA3i). Protein levels of PEA3 were detected by Western blot analysis after 48 hours as described in the materials and methods. Total protein lysate in each lane was subjected to SDS-PAGE 4-12% gel. Beta-actin was detected as an endogenous control.
91
0.0
0.5
1.0
1.5
2.0
2.5
3.0
NOTCH-4
AP-1
*
PEA3
PEA3 siRNAPEA3
PEA3 siRNA
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
NOTCH-1
c-ETS
*
PEA3-pcDNA3.1PEA3-pcDNA3.1
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
NOTCH-4
AP-1
*
PEA3
PEA3 siRNAPEA3
PEA3 siRNA
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
NOTCH-1
c-ETS
*
PEA3-pcDNA3.1PEA3-pcDNA3.1
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
p<0.001 p<0.001
Figure 25: PEA3 Enrichment Specificity on the Notch-1 and Notch-4 Promoters as Measured by Chromatin Immunoprecipitation.
MDA-MB-231 cells were co-transfected with PEA3 overexpression plasmid (pcDNA3.1) and either scrambled siRNA (SCRBi) or PEA3 siRNA (PEA3i) for 48 hours. Lysates then were subjected to chromatin immunoprecipitation using either PEA3 antibody or isotype control IgG on the Notch-1 and Notch-4 promoter. PEA3 enrichment was measured by quantitative PCR and normalized to IgG control. Below graphs is a schematic representation of the Notch-1 and Notch-4 promoters containing the identified enriched Ets sites. Means and standard deviation of more than three experiments were plotted. Statistical significance was determined by two-tailed unpaired Student’s t test.
92
TTGTGTGACTCAGGAAACAGCTCAGACGTG
TTGTGGCTAGACGGAAACAGCTCAGACGTGAP-1 ETS
AP-1 (wt)
AP-1 (mut)
LUCIFERASEETSAP-1~650 bp -70 bp
TTGTGTGACTCAGGAAACAGCTCAGACGTG
TTGTGGCTAGACGGAAACAGCTCAGACGTGAP-1 ETS
AP-1 (wt)
AP-1 (mut)
LUCIFERASEETSAP-1~650 bp -70 bp
Figure 26: Wildtype and Mutant Notch-4 Promoter Luciferase Constructs
Abbreviations: AP-1 consensus site construct, wt; Site-directed AP-1 consensus site mutant construct, mut;
93
The wildtype or mutant Notch-4 promoter Luciferase constructs were co-
transfected with either scrambled or PEA3 siRNA in our in vitro triple-negative MDA-
MB-231 model system. PEA3 siRNA significantly decreased wild type AP-1-containing
or mutant AP-1-containing reporter activity compared to the scrambled control (Figure
27). These results suggest that either PEA3 or AP-1 is critical for the regulation of the -
70 region within the Notch-4 promoter in MDA-MB-231 cells. The mutant ablated AP-1
Luciferase results suggest that PEA3 may play an independent role from AP-1. However,
the construct does contain an additional, but minimal Ets site upstream of the AP-1:Ets
site, which may account for the further decrease in Luciferase on the mutant AP-1
construct. As a control study, we observed no significant difference in AP-1 luciferase
reporter activity upon PEA3 knockdown indicating that PEA3 is not mediating effects on
the Notch-4 promoter through AP-1 regulation (Figure 28).
To address if PEA3 acts directly with AP-1 on Notch-1 and Notch-4 promoter
regions, since it is known to work in accordance with c-JUN on several other genes
(referenced in Chapter III), transfected lysates with either pHMB empty vector or pHMB-
TAM-67, a dominant negative form of c-JUN missing the transactivation domain (Figure
8) (Liu et al., 2002), were subjected to chromatin immunoprecipitation. PEA3
enrichment on the Notch-1 promoter was not affected by TAM-67 (Figure 29). However,
PEA3 recruitment to an Ets site adjacent to an AP-1 site approximately -70 nucleotides
upstream of the start site of the Notch-4 promoter was abrogated by expression of TAM-
67 (Figure 29) indicating that the transactivation domain of c-JUN is required for PEA3
recruitment. As a control, dual Firefly-Renilla luciferase assays using the AP-1 reporter
TTGTG
Figure 27: Luciferase Assay of Wildtype and AP-1 Mutant Notch-4 Promoter.
Above is a schematic representation of the Notch-4 luciferase construct indicating mutated AP-1 consensus site and Ets site. MDA-MB-231 cells were co-transfected with either wildtype AP-1 or mutated AP-1 Notch-4 luciferase and either scrambled or PEA3 siRNA. Firefly luciferase (pGL2) was measured normalized to Renilla as an internal transfection control. Means and standard deviation of three or more experiments were plotted. Statistical significance was determined by two-tailed unpaired Student’s t test.
0.0
0.2
0.4
0.6
0.8
1.0
SCRBi PEA3i SCRBi PEA3i
FIR
EFLY
/ R
ENIL
LA 0.0
0.2
0.4
0.6
0.8
1.0
*
SCRBi PEA3i
FIR
EFLY
/ R
ENIL
LA
Notch-4 (Wt) Luciferase Notch-4 (Mut) Luciferase
*p<0.001
p<0.01
0.0
0.2
0.4
0.6
0.8
1.0
SCRBi PEA3i SCRBi PEA3i
FIR
EFLY
/ R
ENIL
LA 0.0
0.2
0.4
0.6
0.8
1.0
*
SCRBi PEA3i
FIR
EFLY
/ R
ENIL
LA
Notch-4 (Wt) Luciferase Notch-4 (Mut) Luciferase
*p<0.001
p<0.01
TGACTCA
GCTAGAC
GGAAACAGCTCAGACGTG
TTGTG GGAAACAGCTCAGACGTGAP-1 ETS
AP-1 (wt)
AP-1 (mut)
LUCIFERASEETSAP-1~650 bp -70 bp
ETSAP-1~650 bp -70 bp
LUCIFERASE
TTGTG GGAAACAGCTCAGACGTG
TTGTG GGAAACAGCTCAGACGTG
TGACTCA
GCTAGAC
94
AP-1 ETSAP-1 (wt)
AP-1 (mut)
95
LUCIFERASEAP-1 AP-1 AP-1 AP-1
TGAGTCA TGAGTCA TGAGTCA TGAGTCA
LUCIFERASEAP-1 AP-1 AP-1 AP-1
TGAGTCA TGAGTCA TGAGTCA TGAGTCA
2468
101214
SCRBi PEA3i0
FIR
EFLY
/ R
ENIL
LA
2468
101214
SCRBi PEA3i0
FIR
EFLY
/ R
ENIL
LA
Figure 28: PEA3 Does Not Regulate AP-1 Activity.
Above is a schematic representation of the AP-1 luciferase construct. MDA-MB-231 cells were co-transfected with an AP-1 luciferase and either scrambled or PEA3 siRNA. Firefly luciferase (pGL2) was measured normalized to Renilla as an internal transfection control. Means and standard deviation of three or more experiments were plotted.
96
c-ETS
NOTCH-1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
PEA3
PEA3 + TAM-67
0.0
0.5
1.0
1.5
2.0
2.5
AP-1
NOTCH-4
* p<0.01
PEA3
PEA3 +TAM-67
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
c-ETS
NOTCH-1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
PEA3
PEA3 + TAM-67
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
1.0
1.5
2.0
2.5
AP-1
NOTCH-4
* p<0.01
PEA3
PEA3 +TAM-67
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
Figure 29: Dominant Negative c-JUN Ablates PEA3 Recruitment on Notch-4, And has No Effect on PEA3 Recruitment on Notch-1.
MDA-MB-231 cells were co-transfected with PEA3 expression plasmid (pcDNA3.1) and with either pHMB-vector or TAM-67. Lysates were subjected to chromatin immunoprecipitation using either PEA3 antibody or isotype control IgG on the Notch-1 and 4 promoters, respectively. PEA3 enrichment was measured by quantitative PCR and normalized to IgG control. Means and standard deviation of more than three experiments were plotted. Statistical significance was determined by two-tailed unpaired Student’s t test.
97
construct containing four tandem AP-1 consensus sites (Figure 28) were performed to
determine the efficiency of TAM-67 on AP-1 activity. Upon co-transfection with TAM-
67 and AP-1 luciferase construct, AP-1 activity was diminished by 50% (Figure 30).
To determine if the c-JUN protein specifically is required for PEA3 enrichment
on the identified Notch-4 promoter region, transfected lysates with either scrambled or a
smart-pool of specific c-JUN siRNAs were subjected to chromatin immunoprecipitation.
In agreement, PEA3 enrichment on the Notch-4 promoter region was abrogated upon c-
JUN knockdown (Figure 31, top) suggesting that c-JUN is required for PEA3 enrichment
on the identified Notch-4 promoter region. As a control, c-JUN siRNA was transfected
in MDA-MB-231 cells and subjected to quantitative real-time PCR. As a result, c-JUN
siRNA reduced endogenous c-JUN transcripts by 45% (Figure 31, bottom). Chromatin
immunoprecipitation of c-JUN on Notch-1 and Notch-4 promoters was performed, but
resulted in inconclusive findings. Other investigators have completed successful ChIP(s)
of c-JUN (Zhao et al., 2010), which in our system will require optimization and increased
sensitivity.
To determine whether PEA3 is in a complex with c-JUN since c-JUN is required
for PEA3 enrichment as observed by the previous chromatin immunoprecipitation
experiments, PEA3 was immunoprecipitated and Western blotting was performed to
detect PEA3 and c-JUN proteins. Co-immunoprecipitation demonstrated that PEA3 was
in complex with c-JUN (Figure 32). Taken together, these results strongly indicate that
PEA3 and c-JUN are required to regulate the Notch-4 promoter.
98
H N3 COOH
TAD DBD ZIP
H N3 COOH
DBD ZIP
c-JUN
TAM-67
TTSS
63 73 91 93H N3 COOH
TAD DBD ZIP
H N3 COOHH N3H N3 COOH
TAD DBD ZIP
H N3 COOH
DBD ZIP
H N3 COOHH N3H N3 COOH
DBD ZIP
c-JUN
TAM-67
TTSS
63 73 91 93
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
phMB TAM-67
Fire
fly L
ucife
rase
/ R
enilla
AP-1 LuciferaseTAM-67
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
phMB TAM-67
Fire
fly L
ucife
rase
/ R
enilla
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
phMB TAM-67
Fire
fly L
ucife
rase
/ R
enilla
AP-1 LuciferaseTAM-67
p<0.05*
Figure 30: Dominant Negative c-JUN (TAM-67) Reduces AP-1 Activity.
Above is a schematic representation of the wildtype and dominant negative c-JUN construct. MDA-MB-231 cells were co-transfected with AP-1 luciferase and either phMB-vector or TAM-67. Firefly luciferase (pGL2) was measured normalized to Renilla as an internal transfection control. Means and standard deviation of three or more experiments were plotted.
99
PEA3
PEA3 +c-JUNi
AP-1
* p<0.01
NOTCH-4
0.00.5
1.0
1.5
2.0
2.5
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
PEA3
PEA3 +c-JUNi
AP-1
* p<0.01
NOTCH-4
0.00.5
1.0
1.5
2.0
2.5
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
PEA3
PEA3 +c-JUNi
AP-1
* p<0.01
NOTCH-4
0.00.5
1.0
1.5
2.0
2.5
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi c-JUNi
Rel
ativ
e m
RN
A L
evel
s
c-JUN
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi c-JUNi
Rel
ativ
e m
RN
A L
evel
s
c-JUN
p<0.01*
Figure 31: c-JUN is Required for PEA3 Enrichment on the Notch-4 Promoter. MDA-MB-231 cells were co-transfected with PEA3 expression plasmid (pcDNA3.1) and with either scrambled or c-JUN siRNA. Lysates were subjected to chromatin immunoprecipitation using either PEA3 antibody or isotype control IgG on the Notch-4 promoter. PEA3 enrichment was measured by quantitative PCR and normalized to IgG control. Means and standard deviation of more than three experiments were plotted. Below: Quantitative PCR was used to measure the efficiency of c-JUN siRNA knockdown. Transcripts were normalized to HPRT and means were plotted. Error bars represent standard deviation. Statistical significance was determined by two-tailed unpaired Student’s t test.
100
Co-IP: PEA3
c-JUN
IgG (LC)
PEA3
IgG α-PEA3
Figure 32: Co-Immunprecipitation of PEA3 in Complex with c-JUN.
MDA-MB-231 cells were transfected with PEA3 expression plasmid (pcDNA3.1). Lysates were subjected to chromatin immunoprecipitation and subsequent Western blot analysis using either PEA3 antibody or isotype control IgG. Following Western blot analysis, PEA3 and c-JUN proteins were detected by immunoblotting. Western blot are representative of three independent experiments.
101
Since c-JUN is known to heterodimerize with the FOS family and to understand if
c-FOS is required for PEA3 enrichment on Notch-1 and Notch-4 promoter regions,
transfected lysates with either CMV-500 empty vector or CMV-500-AFOS, a dominant
negative form of c-FOS (Figure 9), were subjected to chromatin immunoprecipitation.
PEA3 enrichment on either the Notch-1 or Notch-4 promoter was not affected by A-FOS
(Figure 33). Although c-JUN and c-FOS are preferred partners, c-JUN homodimers
indeed exist as well as other FOS family members, which may contribute to PEA3
promoter enrichment. The dominant-negative effect of A-FOS on AP-1 activity was
confirmed by co-transfecting A-FOS and AP-1 luciferase construct and performing a dual
Firefly-Renilla luciferase assays (Figure 34). The results demonstrated a 40% reduction
in AP-1 activity upon A-FOS transfection (Figure 34). Taken together, the results
indicate that AP-1, with emphasis on c-JUN, and PEA3 regulate the Notch-4 promoter in
MDA-MB-231 cells.
3. PEA3 and AP-1 Dynamically Regulate Notch-4 Transcription.
AP-1 members c-JUN, Fra-1, and c-FOS are overexpressed in MDA-MB-231
cells (Bamberger et al., 1999). To address which member(s) of the AP-1 complex is
responsible for the regulation of Notch-4 gene transcription with PEA3, MDA-MB-231
cells were transfected with PEA3, Fra-1, c-JUN, or c-FOS siRNA individually for 48
hours.
As measured by quantitative real-time PCR, Notch-4 transcripts were reduced
upon individual knockdown of PEA3, c-JUN, or Fra-1 and that the siRNA combinations
102
NOTCH-1
0.0
0.51.0
1.52.0
2.53.0
3.54.0
4.5
c-ETS
PEA3
PEA3 +A-FOS
NOTCH-4
AP-1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
PEA3
PEA3 +A-FOS
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
NOTCH-1
0.0
0.51.0
1.52.0
2.53.0
3.54.0
4.5
c-ETS
PEA3
PEA3 +A-FOS
NOTCH-4
AP-1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
PEA3
PEA3 +A-FOS
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
Rel
ativ
e Fo
ld In
duct
ion
(spe
cific
Ab
/ IgG
)
Figure 33: Dominant Negative c-FOS ( A-FOS) Has No Effect on PEA3 Recruitment on Notch-1 and Notch-4 Promoters.
MDA-MB-231 cells were co-transfected with PEA3 expression plasmid (pcDNA3.1) and with either CMV-500-vector or A-FOS. Lysates were subjected to chromatin immunoprecipitation using either PEA3 antibody or isotype control IgG on the Notch-1 and 4 promoters, respectively. PEA3 enrichment was measured by quantitative PCR and normalized to IgG control. Means and standard deviation of more than three experiments were plotted. Statistical significance was determined by two-tailed unpaired Student’s t test.
103
H N3 COOH
DBD ZIPTAD
H N3 COOHH N3H N3 COOH
DBD ZIPTAD
H N3 COOH
DBD ZIPTAD
H N3 COOHH N3H N3 COOH
DBD ZIPTAD
A-FOS
c-FOS
TTS
232 325 331 374
T
LEQRAEE LARENEE LEKEAEE LEQELAE LE
ACIDIC EXTENSION
0.00.20.4
0.60.81.01.2
cmv-500 A-FOS
Fire
fly L
ucife
rase
/ R
enill
a
0.00.20.4
0.60.81.01.2
cmv-500 A-FOS
Fire
fly L
ucife
rase
/ R
enill
a
p<0.05*
Figure 34: Dominant Negative c-FOS (A-FOS) Reduces AP-1 Activity.
Above is a schematic representation of the wildtype and dominant negative c-FOS construct. MDA-MB-231 cells were co-transfected with AP-1 luciferase and either CMV-500-vector or A-FOS. Firefly luciferase (pGL2) was measured normalized to Renilla as an internal transfection control. Means and standard deviation of three or more experiments were plotted.
104
of either c-JUN:PEA3, Fra-1:PEA3, Fra-1:c-JUN, or PEA3:Fra-1:c-JUN had no
additional effect (Figure 35). Interestingly, in the presence of c-FOS siRNA, Notch-4
transcripts were increased suggesting that c-FOS may act as repressor in our MDA-MB-
231 breast cancer in vitro model. The combination of c-JUN and c-FOS siRNAs had no
measurable effect on Notch-4 transcripts compared to control siRNA suggesting the role
of c-FOS as a potential repressor of Notch-4 (Figure 35). The combination of c-FOS and
c-JUN siRNAs had the tendency to restore the transcript levels of Notch-4 to basal levels.
This result would suggest that c-FOS could be a transcriptional repressor of Notch-4
mRNA. A one-way ANOVA resulted in no statistical significance because of the
pairwise multiple comparisons and minimal repeated measurements (n=3). However,
there is a clear trend and a Power Analysis would help to determine how many
independent studies need to be performed to achieve statistical significance.
As a control experiment, Notch-1 transcripts were measured by quantitative real-
time PCR under the same conditions as mentioned above. As a result, Notch-1
transcripts were not affected by AP-1 knockdown using c-JUN, Fra-1, or c-FOS
siRNA(s). However, PEA3 siRNA reduced Notch-1 transcript levels as seen previously
by 50%, indicating that PEA3 acts most probably independently of AP-1 to regulate the
Notch-1 promoter (Figure 36). Interestingly, c-FOS in combination with PEA3 siRNA
did not reduce Notch-1 transcripts. This suggests that there is a trend that c-FOS may
serve as a repressor upstream or downstream on the Notch-1 promoter. Again, statistical
analysis using one-way ANOVA resulted in no significance because of the pairwise
105
NOTCH-4
0.0
0.5
1.0
1.5
2.0
2.5
SCRBi PEA3i FRA1i cJUNi cJUNiPEA3i
cJUNiFRA1iPEA3i
FRA1iPEA3i
cJUNiFRA1i
Rel
ativ
e m
RN
A L
evel
s
0.0
0.5
1.0
1.5
2.0
2.5
SCRBi PEA3i cFOSi cJUNi cJUNiPEA3i
cJUNicFOSiPEA3i
cFOSiPEA3i
cJUNicFOSi
Rel
ativ
e m
RN
A L
evel
sNOTCH-4
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
1.0
1.5
2.0
2.5
SCRBi PEA3i FRA1i cJUNi cJUNiPEA3i
cJUNiFRA1iPEA3i
FRA1iPEA3i
cJUNiFRA1iSCRBi PEA3i FRA1i cJUNi cJUNi
PEA3i
cJUNiFRA1iPEA3i
FRA1iPEA3i
cJUNiFRA1i
Rel
ativ
e m
RN
A L
evel
s
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
1.0
1.5
2.0
2.5
SCRBi PEA3i cFOSi cJUNi cJUNiPEA3i
cJUNicFOSiPEA3i
cFOSiPEA3i
cJUNicFOSiSCRBi PEA3i cFOSi cJUNi cJUNi
PEA3i
cJUNicFOSiPEA3i
cFOSiPEA3i
cJUNicFOSi
Rel
ativ
e m
RN
A L
evel
s
Figure 35: PEA3 and AP-1 Dynamically Regulate Notch-4 Transcription. MDA-MB-231 cells were transfected individually or with various combinations of PEA3, Fra-1, c-JUN, or c-FOS siRNA. Notch-4 transcripts were normalized to HPRT and measured by quantitative PCR. Means and standard deviation of three or more experiments were plotted. Statistical significance was determined by one-way ANOVA.
NOTCH-1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
SCRBi PEA3i FRA1i cJUNi cJUNiPEA3i
cJUNiFRA1iPEA3i
FRA1iPEA3i
cJUNiFRA1i
Rel
ativ
e m
RN
A L
evel
s
SCRBi PEA3i cFOSi cJUNi cJUNiPEA3i
cJUNicFOSiPEA3i
cFOSiPEA3i
cJUNicFOSi
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Rel
ativ
e m
RN
A L
evel
sNOTCH-1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
SCRBi PEA3i FRA1i cJUNi cJUNiPEA3i
cJUNiFRA1iPEA3i
FRA1iPEA3i
cJUNiFRA1i
Rel
ativ
e m
RN
A L
evel
s
SCRBi PEA3i cFOSi cJUNi cJUNiPEA3i
cJUNicFOSiPEA3i
cFOSiPEA3i
cJUNicFOSi
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Rel
ativ
e m
RN
A L
evel
s
106
Figure 36: Notch-1 Transcripts Are Not Affected by AP-1 siRNA Interference.
MDA-MB-231 cells were transfected individually or with various combinations of PEA3, Fra-1, c-JUN, or c-FOS siRNA. Notch-1 transcripts were normalized to HPRT and measured by quantitative PCR. Means and standard deviation of three or more experiments were plotted. Statistical significance was determined by one-way ANOVA.
107
multiple comparisons and minimal repeated measurements (n=3). More independent
experiments might be needed to achieve statistical significance.
It is also important to mention that none of the siRNA treatments had any
significant effect on each other, confirming that siRNA effects are not interacting with
gene regulation (data not shown). As a final control, quantitative real-time PCR and
Western blot analysis were performed on PEA3, c-JUN, Fra-1, and c-FOS to determined
their individual siRNA knockdown efficiency. After 48 hours, PEA3, c-JUN, Fra-1, and
c-FOS transcripts and protein levels were reduced by approximately 90%, 40%, 80%,
60% after their specific siRNA transfections, respectively (Figure 37).
SPECIFIC AIM-3:
To Establish a Biological Significance by Determining Whether
Dual Inhibition of Notch and PEA3 Leads to Reduced
Cell Proliferation and Tumorigenicity in vitro.
Notch is vital for proliferation and cell fate determination (Miele et al., 2006)
whereas PEA3 is critical for migration and invasion (Hida et al., 1997; Kaya et al., 1996;
Miele et al., 2006). Both aberrant and unregulated activities can lead to malignancy and
overall poor survival (Reedijk et al., 2005; Shepherd et al., 2001). To understand the
biological significance and their effect in our breast cancer model, we explored dual
108
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi c-JUNi
Rel
ativ
e m
RN
A L
evel
s
c-JUN
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi c-JUNi
Rel
ativ
e m
RN
A L
evel
s
c-JUN
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
Rel
ativ
e m
RN
A L
evel
s
PEA3
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi PEA3i
Rel
ativ
e m
RN
A L
evel
s
PEA3
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi FRA-1i
Rel
ativ
e m
RN
A L
evel
s
FRA-1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi FRA-1i
Rel
ativ
e m
RN
A L
evel
s
FRA-1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi c-FOSi
Rel
ativ
e m
RN
A L
evel
sc-FOS
0.0
0.2
0.4
0.6
0.8
1.0
1.2
SCRBi c-FOSi
Rel
ativ
e m
RN
A L
evel
sc-FOS
PEA3
ACTIN
c-JUN
ACTIN
FRA-1
ACTIN
c-FOS
ACTIN
p<0.01*
p<0.0001*
p<0.05*
p<0.001*
Figure 37: RNA interference Knockdown of PEA3 and AP-1 Members.
MDA-MB-231 cells were transfected individually PEA3, Fra-1, c-JUN, or c-FOS siRNA. Their transcripts were normalized to HPRT and measured by quantitative PCR to determine siRNA knockdown efficiency. Means and standard deviation of three or more experiments were plotted. Statistical significance was determined by two-tailed unpaired Student’s t test.
109
inhibition of PEA3 and Notch using a specific PEA3 siRNA and the pre-clinical γ-
secretase inhibitor (GSI: MRK-003), respectively.
1. Dual inhibition of Notch and PEA3 Inhibits Cell Proliferation.
To determine the effects of dual Notch and PEA3 inhibition on cell proliferation,
MDA-MB-231 cells were transfected with scrambled or PEA3 siRNA alone, or treated
with vehicle or GSI (10 μM), or a combination for 48 hours. Independently, PEA3
knockdown and/or Notch inhibition all had effects on cell cycle (Figure 38). PEA3
knockdown or GSI treatment of MDA-MB-231 cells alone showed an increase in G1
arrest. A significant increase (p<0.05) in G1 arrest was determined in the combination
group of Notch inhibition and PEA3 knockdown as compared to control (Figure 38).
PEA3 knockdown and GSI treatment alone showed a reduction in S-phase, while the
combination of Notch inhibition and PEA3 knockdown significantly reduced S-phase
(p<0.05) as compared to control (Figure 38). PEA3 siRNA and GSI treatment alone
demonstrated a reduction in G2/M cell cycle, but the combination showed a significant
reduction (p<0.05) as compared to control in G2/M (Figure 38). These results indicate
that both PEA3 and Notch, whether working together or independently at the various
stages of the cell cycle, are critical for proliferation of MDA-MB-231 cells.
2. Dual Inhibition of Notch and PEA3 Induces Apoptosis.
To address whether dual inhibition using both PEA3 knockdown and Notch
inhibition affected cell viability potentially through increased apoptosis, MDA-MD-231
110
0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00
100.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
% of Cells in
Figure 38: Effects of Dual Notch and PEA3 on Cell Proliferation.
MDA-MB-231 cells were transfected with scrambled or PEA3 siRNA alone, or treated with vehicle or a gamma-secretase inhibitor (GSI: MRK-003, 10 μM), or a combination thereof for 48 hours. Cell cycle analysis was performed by flow cytometry. Mean percentage and standard deviation of cells in each experiment were plotted. Statistical significance was determined by one-way ANOVA.
G1-phase
Perc
ent o
f Cel
ls in
G1
* p<0.05
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
Perc
ent o
f Cel
ls in
S
% of Cells in S-phase
* p<0.05
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
Perc
ent o
f Cel
ls in
G2/M
% of Cells in G2/M-phase
* p<0.05
111
cells were transfected with scrambled or PEA3 siRNA alone, or treated with vehicle or
GSI (10 μM), or a combination, and subjected to Annexin-V staining, and subsequently
measured by flow cytometry 24 hours later as an early marker of apoptosis.
Independently, PEA3 knockdown showed no change in apoptosis (Figure 39). GSI
treatment alone increased apoptosis by 30%. Importantly, the combination of PEA3
knockdown and GSI treatment significantly increased apoptosis by almost 40% as
measured by Annexin-V positive stained cells (Figure 39).
3. Dual Inhibition of Notch and PEA3 Reduces Cell Viability.
To confirm the effects of treatment/transfection on cell viability, a Trypan blue
exclusion assay was performed after MDA-MD-231 cells were transfected with
scrambled or PEA3 siRNA alone, or treated with vehicle or GSI (10 μM), or a
combination for 48 hours. PEA3 siRNA alone showed no change in cell viability, but
GSI alone resulted in decreased viability (Figure 40). In agreement with previous
experiments, the combination of PEA3 knockdown and GSI treatment significantly
diminished percent cell viability by more than 50% (Figure 40). These results taken
together with the cell cycle data indicate that both PEA3 and Notch activities together are
essential for cell proliferation and survival in MDA-MB-231 breast cancer cells.
4. Notch-1 and Notch-4 Overexpression Rescues the Effect of Dual Notch and
PEA3 Inhibition.
112
% o
f Ann
exin
-V +
Cel
ls
05
10152025303540
24 Hours
p<0.05
VEHICLEPEA3 siRNAGSI (Mrk-003)
+ – – –– + – + – – + +
*%
of A
nnex
in-V
+ C
ells
05
10152025303540
24 Hours
p<0.05
VEHICLEPEA3 siRNAGSI (Mrk-003)
+ – – –– + – + – – + +
*
Figure 39: Dual Inhibition of Notch and PEA3 Increases Apoptosis
MDA-MB-231 cells were transfected with scrambled or PEA3 siRNA alone, or treated with vehicle or a gamma-secretase inhibitor (GSI: MRK-003, 10 μM), or a combination thereof for 24 hours. Annexin-V staining was performed and measured by flow cytometry. Mean percentage and standard deviation of cells in each experiment were plotted. Statistical significance was determined by one-way ANOVA.
113
0102030405060708090
100
48 Hours
p<0.05%
Cel
l Via
bilit
y
VEHICLEPEA3 siRNAGSI (Mrk-003)
+ – – –– + – + – – + +
*
0102030405060708090
100
48 Hours
p<0.05%
Cel
l Via
bilit
y
VEHICLEPEA3 siRNAGSI (Mrk-003)
+ – – –– + – + – – + +
*
Figure 40: Dual Inhibition of Notch and PEA3 Decrease Cell Viability. MDA-MB-231 cells were transfected with scrambled or PEA3 siRNA alone, or treated with vehicle or a gamma-secretase inhibitor (GSI: MRK-003, 10 μM), or a combination thereof for 48 hours. Percent viable cells were measured by Trypan blue exclusion study using a standard light microscope (10X magnitude). Mean percentage and standard deviation of cells in each experiment were plotted. Statistical significance was determined by two-tailed unpaired Student’s t test.
114
To address whether dual inhibition of PEA3 and Notch activities have effects on
anchorage independent growth as a measure of cell tumorigenicity, a colony formation
assay was performed with MDA-MB-231 cells that were transfected with either
scrambled or PEA3 siRNA and treated with vehicle or a gamma-secretase inhibitor (GSI:
MRK-003, 5 μM) independently or in combination. Forty-eight hours after transfection,
cells were suspended in methylcellulose and allowed to grow into colonies over 14 days.
Colonies were photographed (Figure 41) and counted under a 40X magnification using a
standard light microscope, and quantified (Figure 42). As a result, there was a reduction
in the number of colonies to almost 60% after PEA3 siRNA knockdown or by 40% when
Notch was inhibited using a GSI in the presence of vector alone. Furthermore, the percent
number of colonies was reduced to 40% upon the combination of PEA3 knockdown and
GSI treatment (Figure 41, 42).
GSI inhibits cleavage of all four Notch receptors (Kopan and Ilagan, 2009). To
confirm the hypothesis that the effects of PEA3 inhibition on cell growth and survival are
mediated primarily through Notch-1 and/or Notch-4, in MDA-MB-231 cells expression
plasmids for the Notch-1 or Notch-4 intracellular domain (N1IC or N4IC) (10 ng) were
simultaneously co-transfected independently or in combination in all four treatment
groups (i.e. vehicle control, PEA3 siRNA, GSI, or GSI plus PEA3 siRNA) (Figure 41,
column 2, 3, 4, quantified Figure 42). When Notch-1IC, Notch-4IC, or both were
expressed in the absence of PEA3 siRNA, GSI treatment, or their combination, there was
no measurable difference in percent number of colonies compared to control (Figure 41,
row 1: E, I, M compared to control A, quantified Figure 42). The addition of Notch-1IC
115
NOTCH-1 (IC) NOTCH-4 (IC) NOTCH-1 (IC)NOTCH-4 (IC)
VEHICLE
PEA3i
GSI (5uM)
PEA3iGSI (5uM)
VECTOR
ROW-1
ROW-2
ROW-3
ROW-4
COLUMN-1 COLUMN-2 COLUMN-3 COLUMN-4
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Figure 41: Notch-1 and Notch-4 Overexpression Rescues the Effect of Dual Notch and PEA3 Inhibition.
MDA-MB-231 cells were transfected with either scrambled or PEA3 siRNA and treated with vehicle or gamma-secretase inhibitor (GSI: MRK-003, 5 μM) independently or in combination for 48 hours. Simultaneously, cells were co-transfected with expression plasmids for Notch-1 and Notch-4 intracellular domain (IC) independently or in combination. Colony formation assay was preformed using methylcellulose and incubated for 14 days. Colonies were photographed using a standard light microscope (40X magnitude) representative of three or more experiments.
116
0.00
20.00
40.00
60.00
80.00
100.00
120.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
VECTOR NOTCH-1 (IC) + NOTCH-4 (IC)
Perc
ent #
of C
olon
ies
0.00
20.00
40.00
60.00
80.00
100.00
120.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
VECTOR NOTCH-1 (IC) + NOTCH-4 (IC)
0.00
20.00
40.00
60.00
80.00
100.00
120.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
0.00
20.00
40.00
60.00
80.00
100.00
120.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
VECTOR NOTCH-1 (IC) + NOTCH-4 (IC)
Perc
ent #
of C
olon
ies
0.00
20.00
40.00
60.00
80.00
100.00
120.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
VECTOR NOTCH-1 (IC)
Perc
ent #
of C
olon
ies
0.00
20.00
40.00
60.00
80.00
100.00
120.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
VECTOR NOTCH-1 (IC)
0.00
20.00
40.00
60.00
80.00
100.00
120.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
0.00
20.00
40.00
60.00
80.00
100.00
120.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
VECTOR NOTCH-1 (IC)
Perc
ent #
of C
olon
ies
0.00
20.00
40.00
60.00
80.00
100.00
120.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
VECTOR NOTCH-4 (IC)
Perc
ent #
of C
olon
ies
0.00
20.00
40.00
60.00
80.00
100.00
120.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
VECTOR NOTCH-4 (IC)
0.00
20.00
40.00
60.00
80.00
100.00
120.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
0.00
20.00
40.00
60.00
80.00
100.00
120.00
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
GSI +DMSOSCRBi
GSISCRBi PEA3i
DMSOPEA3i
VECTOR NOTCH-4 (IC)
Perc
ent #
of C
olon
ies
**
p<0.05p<0.05
**
p<0.05p<0.05
**
p<0.05p<0.05
Figure 42: Quantification of Notch-1 and Notch-4 Rescue on Colony Formation. MDA-MB-231 cells were transfected with either scrambled or PEA3 siRNA and treated with vehicle or gamma-secretase inhibitor (GSI: MRK-003, 5μM) independently or in combination for 48 hours. Simultaneously, cells were co-transfected with expression plasmids for Notch-1 and Notch-4 intracellular domain (IC) independently or in combination. Colony formation assay was performed using methyl-cellulose and incubated for 14 days. Nine fields were counted and quantified per well and means plotted. Statistical significance was determined by one-way ANOVA.
117
alone and partially rescued the effects mediated by PEA3 siRNA, GSI, and their
combination on percent colony formation (Figure 42, top). Notch-4IC alone partially
rescued the effects in anchorage independent growth in the presence of GSI treatment
alone and in combination with PEA3 siRNA. Notch-4IC rescue effects on PEA3 siRNA
transfection alone were minimal (Figure 42, middle). In the presence of dual inhibition of
PEA3 and Notch, Notch-1IC expression was able to restore anchorage independent
growth by 80% than Notch-4IC in which only 70% of colony number was restored (Figure
41, row 4: H and L compared to D, respectively, Figure 42). When both Notch-1IC and
Notch-4IC were co-expressed in this combination treatment group, complete restoration of
percent number of colonies was observed by 93% (Figure 41, row 4: P compared to D,
Figure 42, bottom). This result suggests that PEA3 mediates its effect primarily through
expression of Notch-1 and Notch-4. While Notch-1IC had a greater colony restoration
effect than Notch-4IC, both receptors are required to fully restore both the phenotypic
appearance and percent number of colonies. Taken together, the results suggest that
transcription of Notch-1 and Notch-4 genes by PEA3 and the activation of Notch-1 and
Notch-4 receptors (after gamma-secretase cleavage leading to the formation of active
Notch-1IC and Notch-4IC) are both critical biologic events required for proliferation and
survival of triple negative, MDA-MB-231 breast cancer cells in vitro.
5. PEA3 Regulates Notch-1 and Notch-4 mRNA Expression in other
Subtypes of Breast Cancer.
118
Our investigations are the first to show that PEA3 is an activator of Notch-1 mRNA and
protein expression and Notch-4 mRNA in the triple negative subtype, MDA-MB-231
breast cancer cells. To determine whether this is a common mechanism among the other
subtypes of breast cancer cells, MDA-MB-231 (triple-negative), MCF-7 (Luminal A),
BT474 (Luminal B), and SKBr3 (HER2+) cells were transfected with either scrambled or
PEA3 siRNA. Normalized to MDA-MB-231 cells, MCF-7 cells had slightly less PEA3
transcript levels followed by BT474 cells and then SKBr3 cells (Figure 43). Notch-1 and
Notch-4 transcript levels were compared with either scrambled or PEA3 siRNA among
the cell lines. PEA3 regulation of Notch-1 mRNA was seen in MDA-MB-231, SKBr3,
and MCF-7 cells but not BT474 cells (Figure 44, top). However, PEA3-mediated effects
on Notch-4 transcripts were only observed in MDA-MB-231 (Figure 44, bottom). This
could be in part due to differences in AP-1 activity (Figure 45) or that PEA3 basal
transcript levels were very low in other cell types (Figure 43). Provoked by this notion,
we asked if we could drive the expression of Notch-4 by increasing levels of PEA3 in
breast cancer cells that express low endogenous PEA3 levels. To address this, BT474
and SKBr3 cells were transfected with either pcDNA3.1 empty vector or pcDNA3.1-
PEA3 expression plasmid. Notch-1 transcripts remained unchanged when PEA3 was
overexpressed (Figure 46). Notch-4 transcript levels were significantly increased by nine
fold in BT474 and sixteen fold in SKBr3 breast cancer cells (Figure 46). As a control,
quantitative PCR was performed on PEA3 transcript levels after transfection of
pcDNA3.1 and pcDNA3.1-PEA3 to determine PEA3 overexpression efficiency in both
BT474 and SKBr3 cells (Figure 46, bottom). Taken together, PEA3 is required for
119
Notch-1 transcription in at least three subtypes of breast cancer cells: MDA-MB-231,
SKBr3, and MCF-7. However, PEA3 is an activator of Notch-4 transcription in MDA-
MB-231 cells where endogenous PEA3 levels are high. Exogenous expression of PEA3
in BT474 and SKBr3 cells provides evidence that it is also a potential activator of Notch-
4 expression in other breast cancer subtypes.
Taken together, results from this study demonstrate for the first time that PEA3 is
a novel activator of Notch-1 and Notch-4 transcription in different subtypes of breast
cancer cells and could prove to be an important therapeutic target (targeted by
nanotechnology, directed siRNAs, or staple interface masking peptides) when combined
with a Notch inhibitor such as a GSI, particularly in triple negative breast cancer.
120
MDAMB231 SKBr3MCF-7 BT474
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3
i0.00.10.20.30.40.50.60.70.80.91.01.1
PEA3R
ELA
TIVE
mR
NA
LEVE
LSp<0.0001*
p<0.0001*
p<0.001*
p<0.001*
MDAMB231 SKBr3MCF-7 BT474
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3
i0.00.10.20.30.40.50.60.70.80.91.01.1
PEA3R
ELA
TIVE
mR
NA
LEVE
LSp<0.0001*
p<0.0001*
p<0.001*
p<0.001*
Figure 43: Relative PEA3 levels and Knockdown Efficiency
in Breast Cancer Subtypes. MDA-MB-231, MCF-7, BT474, and SKBr3 cells were transfected with either scrambled or PEA3 siRNA. Normalized to MDA-MB-231 cells, the transcript levels of PEA3 were compared and measured by quantitative PCR. Means and standard deviation of three experiments were plotted. Statistical significance was determined by two-tailed unpaired Student’s t test.
121
0123456789
10111213
REL
ATI
VE m
RN
A LE
VELS
NOTCH-1
MDAMB231 SKBr3 MCF-7 BT474
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
0123456789
10111213
REL
ATI
VE m
RN
A LE
VELS
NOTCH-1
MDAMB231 SKBr3 MCF-7 BT474
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
MDAMB231 SKBr3 MCF-7 BT474
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
0.00.20.40.60.81.01.21.41.61.82.02.22.42.6
REL
ATI
VE m
RN
A L
EVEL
S
NOTCH-4
MDAMB231 SKBr3 MCF-7 BT474
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
0.00.20.40.60.81.01.21.41.61.82.02.22.42.6
REL
ATI
VE m
RN
A L
EVEL
S
NOTCH-4
MDAMB231 SKBr3 MCF-7 BT474
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
MDAMB231 SKBr3 MCF-7 BT474
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
SCRBi
PEA3i
p<0.001*
p<0.001
**
*
p<0.01
p<0.01
Figure 44: PEA3 Regulates Notch in Other Breast Cancer Subtypes. MDA-MB-231, SKBr3, MCF-7, and BT474 cells were transfected with either scrambled or PEA3 siRNA. Normalized to MDA-MB-231 cells, the transcript levels of Notch-1 and Notch-4 were compared and measured by quantitative PCR. Means and standard deviation of three experiments were plotted. Statistical significance was determined by two-tailed unpaired Student’s t test.
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Relative AP-1 Activity in Cell Lines
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Figure 45: Relative AP-1 Activity in Different Breast Cancer Subtypes. BT474, MDA-MB-231, MCF-7, and SKBr3 cells were transfected with an AP-1 luciferase reporter construct (pGL2). After 48 hours, 10uL of each sample was added to a white coated luminescent 96-well plate and subjected to the manufactured dual-luciferase assay. Luminescence was measured using the Veritas Microplate Luminometer. The range of luminescence was between 350-650nm; Firefly luciferase read at 560nm and pTL-TK at 480nm. Firefly luciferase was measured and normalized to Renilla as an internal transfection control. Means and standard deviation of three or more experiments were plotted.
123
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PEA3 PEA3 Figure 46: PEA3 Regulates Notch-4 in BT474 and SKBr3 Breast Cancer Subtypes.
BT474 and SKBr3 cells were transfected with either empty vector or PEA3 expression plasmid (pcDNA3.1). Notch-1, Notch-4, and PEA3 mRNA levels were normalized to empty vector and measured by quantitative PCR. Means and standard deviation of three experiments were plotted. Statistical significance was determined by two-tailed unpaired Student’s t test.
CHAPTER V
DISCUSSION
PEA3 was originally identified as a member of the Ets family of transcription
factors (Xin et al., 1992). Since then, it has been observed that PEA3 is expressed during
breast development, is quickly lost upon maturation, and yet re-emerges in metastatic
breast cancers (Benz et al., 1997; Chotteau-Lelievre et al., 2003). Similarly, Notch-1 has
been implicated in breast cancer demonstrating elevated expression and activity in breast
tumors (Reedijk et al., 2005). Little is known about the factors that influence Notch gene
expression, and why its levels are elevated in breast cancer.
MDA-MB-231 cells are an in vitro model of triple negative breast cancer and
therefore lack expression of estrogen receptor α, progesterone receptor, or HER2
overexpression (Sorlie et al., 2001). They also contain a p53 mutation (Weigelt et al.,
2009). Triple negative breast cancers have no known FDA approved, targeted treatment
strategies, and as of yet have limited direct targeted chemotherapeutics. Their standard of
care is a combination of cytotoxic drugs: cyclophosphamide, doxorubicin, and
fluorouracil (Hortobagyi, 1998) or recently shown to be more effective in triple-negative
breast cancer was the combination of cyclophosphamide, methotrexate, and fluorouracil
(Colleoni et al., 2010). Therefore, it is imperative to understand the molecular
infrastructure of triple-negative breast cancers to uncover and provide novel targeted
124
125
methods to help these patients reach their five year and beyond disease-free survival rate.
The Analysis
The initial results indicating that PEA3 is a transcriptional activator of Notch-1
and Notch-4 in triple-negative MDA-MB-231 cells provides new insight into targeted
therapeutic options for patients afflicted with this illness. Targeting PEA3 in
combination with inhibition of Notch shows promise in vitro to decrease MDA-MB-231
cellular viability (Figure 40), reduce cell proliferation (Figure 38), and increase apoptosis
(Figure 39). What is even more intriguing is the result that PEA3 inhibition slightly, but
significantly increased the transcript levels of Notch-2 (Figure 14). Notch-2 recently has
been implicated as a tumor suppressor (O'Neill et al., 2007; Quillard et al., 2009).
Minimal putative PEA3 Ets sites, although less abundant, were identified on the Notch-2
promoter (data not shown). This could indicate that targeting PEA3 in MDA-MB-231
could be beneficial in two ways: abrogating Notch-1 and Notch-4 transcripts and
stimulating expression of Notch-2, thus increasing the chances of a complete reduction in
proliferation and survival of the cancerous cells. This notion stems from other
investigations of PEA3, which demonstrate it to be both an activator and repressor of
gene transcription (Benz et al., 1997; Scott et al., 1994; Xia et al., 2006; Xing et al.,
2000).
It was demonstrated that PEA3 siRNA alone showed no effect on cell
proliferation (Figure 38), apoptosis (Figure 39), and viability (Figure 40) in vitro
indicating that it is not efficient enough to inhibit PEA3 alone. Firstly, endogenous levels
126
of Notch-4 protein were not changed after PEA3 siRNA but were altered in the presence
of lactacystin showing that inhibition of proteasome-mediated protein degradation
inhibited pathways that resulted in transcription becoming rate controlling. Secondly,
Notch-2 transcripts, although modest, were significantly increased in the presence of
PEA3 siRNA and Notch-3 transcripts unaffected. A gamma-secretase inhibitor inhibits
the third proteolytic cleavage responsible for the formation of active intracellular Notch-
1,2,3, and 4 (Weijzen et al., 2002). The results shown here suggest that PEA3 only
activates Notch-1 and Notch-4 transcripts. Therefore, one could speculate that the Notch-
3 receptor could compensate for the dampening of Notch-1 and Notch-4 mRNA, and
consequently maintain cell proliferation and viability, which were observed in these
studies.
Notch-4 transcripts were positively regulated by PEA3 (Figure 14), however
steady-state Notch-4 protein levels were not. In the presence of lactacystin, a specific
proteasome inhibitor, the transcriptional effects of PEA3 on Notch-4 were observed
(Figure 14). A speculative explanation could be that the rate of protein degradation could
be decreased upon knockdown of PEA3, which may be a result of PEA3 regulating
components of the proteasome. Another hypothesis could be that post-transcriptional
steps regulating Notch-4 protein levels must be important since the endogenous Notch-4
protein levels do not correlate with Notch-4 mRNA levels. Examples of post-
transcriptional steps include translation regulation, vesicular transport of Notch-4 protein
through the endoplasmic reticulum and Golgi apparatus, ligand activation and gamma-
secretase cleavage regulating plasma membrane Notch-4 protein levels, and proteasome
127
degradation of ubiquitinated Notch protein or other proteins involved in Notch-4 turnover.
However, several experiments are necessary to answer these speculations. Nevertheless,
the results suggest that Notch-4 is primarily regulated by the proteasome, but when
compromised, the transcriptional effects of PEA3 on Notch-4 protein levels are
observable.
To date, PEA3 has been implicated in regulating various genes in a complex with
additional transcription factors (Firlej et al., 2005; Hesselbrock et al., 2005; Liu et al.,
2004; Matsui et al., 2006). It was observed that AP-1 and PEA3 regulate Notch-4
transcription (Figures 27, 29, and 32). The results of this investigation suggest the
importance of c-JUN, Fra-1, c-FOS, and PEA3 on the regulation of Notch-4 transcription.
Interestingly, Fra-1 and c-FOS also regulate the Notch-4 promoter indicating that they
either activate or dampen the transcription of Notch-4, respectively (Figure 35). This
provides a unique future direction of understanding the partner preference on the Notch-4
promoter and the homeostatic balance needed for appropriate expression of Notch-4 in
response to cellular stimuli. Recently, Baan et al. demonstrated that invasive breast
cancer cells including MDA-MB-231 favored the c-JUN:Fra-1 heterodimer, compared to
less invasive breast cancer cells, which favored the c-JUN:c-FOS heterodimer (Baan et
al., 2010). Preference towards the c-JUN:Fra-1 AP-1 dimer may be responsible for
Notch-4 mRNA modulation and subsequent tumor aggressiveness of MDA-MB-231
triple negative breast cancer cells. The data suggests that PEA3 expression could tip the
balance and aid in this partner choice.
128
The partner choice of the AP-1 complex can be a heterodimer of JUN and FOS
families or a homodimer of the JUN family (Eferl and Wagner, 2003). It was interesting
to find that c-JUN was required for PEA3 recruitment to the Notch-4 promoter and that c-
FOS was not as shown by the dominant negative ChIP studies (Figure 29 and Figure 33).
Dominant negative c-JUN and c-FOS experiments may answer distinct questions. c-JUN
is highly expressed in triple-negative MDA-MB-231 cells (Bamberger et al., 1999).
TAM-67 only reduced AP-1 function by 50% (Figure 29). However, JUN:FOS is the
preferred partnership, with c-JUN acting as an important member capable of homo- and
hetero-dimerization among family members leading to subsequent AP-1 signaling effects
(Eferl and Wagner, 2003). Thus upon transfection, TAM-67 has the capability to partner
with JUN and FOS families limiting the cellular pool of potent gene regulators (Brown et
al., 1994; Brown et al., 1996). A-FOS on the other hand can only partner the JUN family
and not the FOS family (Olive et al., 1997). Therefore, important FOS family members
are potentially left available to still regulate the Notch-4 promoter, namely Fra-1.
Since dynamic transcription factor partnering is plausible (Beckett, 2004), other
putative regulatory factors must be critical for the regulation of Notch genes, because
PEA3 is not solely responsible for the regulation of Notch-1 and Notch-4 transcription.
This is evident by the observation that PEA3 RNA interference decreased Notch-1 and
Notch-4 transcripts by 50% and 70% (Figure 14). If PEA3 was the sole transcription
factor necessary, the transcripts would have been completely eliminated upon PEA3
knockdown. The results of this investigation demonstrate that PEA3 is one of many
factors involved in the regulation of Notch transcription. A very dynamic interplay by
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many additional factors may be responsible for the appropriate regulation of Notch-1 and
Notch-4 transcripts. Moreover, key areas flanking the start site of the Notch-1 and
Notch-4 gene including downstream intronic and extronic regions, unidentified cryptic
sites, or regulatory transcriptional hubs within the gene (areas of three dimensional gene
locus looping as seen in the CFTR gene (Ott et al., 2009)) could be putative sites of
regulation. Endogenous genes are highly coiled, compact, and require chromatin
remodeling events (Zhao et al., 2009). Therefore, PEA3 in addition with other putative
factors potentially carrying histone acetyltransferases (Liu et al., 2004; Matsui et al.,
2006) activity may be required to regulate Notch gene transcription.
These putative factors are yet to be identified especially on the Notch-1 promoter
where the investigations indicate that PEA3 works independently of AP-1 (Figure 36).
However, PEA3 is known in the literature to work in tandem with other transcription
factors at variable genomic positions to regulate its target genes (Firlej et al., 2005; Liu et
al., 2004). Therefore, it is highly unlikely that PEA3 independently regulates Notch-1
without the aid of other factors. Other consensus sites on the Notch-1 promoter near
PEA3 sites are putative NFκB and SP-1 sites (data not shown) as determined by promoter
scanning using NCBI Entrez Gene bank. These factors have not been significantly tested
in vitro to discern if they interact with PEA3, but are emerging as transcription factors of
interest in the regulation of Notch-1. Moreover, if in addition to directly regulating
Notch-1 transcripts, PEA3 carries factors with chromatin remolding capabilities, then the
dynamics of PEA3 regulation becomes more fascinating allowing for remodeling events
(Clapier and Cairns, 2009; Zhao et al., 2009) to occur, exposing silent regulatory sites,
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and recruiting other transcription factors possibly the factors mentioned above to regulate
the Notch-1 receptor or even other Notch receptors as well. This requires further studies
to address, but becomes an additional hypothesis. Taken together, PEA3 harbors very
interesting properties in response to cellular stimuli, not only limited to its effects on gene
regulation, but also its effects mediated by pharmacological intervention.
It was demonstrated that dual inhibition of Notch via a gamma-secretase inhibitor
(MRK-003) and PEA3 via siRNA, reduced proliferation (Figure 38), anchorage-
independent growth (Figure 41 and Figure 42), and increased apoptosis in MDA-MB-231
cells (Figure 39). The enhanced sensitivity of MDA-MB-231 cells towards the GSI in
the presence of PEA3 siRNA could be a result of targeting both the transcription and
activity of the Notch pathway (Figure 47). GSI treatment inhibits the third proteolytic
cleavage and attenuates the signaling cascade (Weijzen et al., 2002). However, more
receptors are continually being transcribed at the gene level without control. Thus, it
could be hypothesized that inhibiting at the level of gene transcription and at the level of
receptor cleavage could greatly enhance the inhibition of Notch signaling overall. It was
observed that PEA3 inhibition alone was not sufficient to reduce anchorage dependent
and affect anchorage independent proliferation (Figure 38 and Figure 42) most likely due
the fact that other regulatory factors (Alimirah et al., 2007; Cai et al., 2009; Pajerowski et
al., 2009; Wu and Bresnick, 2007; Wu et al., 2005) including AP-1 (Wu and Bresnick,
2007; Wu et al., 2005) may be important in regulation of Notch transcripts.
PEA3 regulation of Notch-1 and Notch-4 transcripts was observed among other
subtypes of breast cancer (Figure 44) in vitro. Notch-4 transcripts were affected in the
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presence of exogenous PEA3 expression (Figure 44). This could be in part due to the
differences in AP-1 activity in the cell types (Figure 45), or that the endogenous levels of
PEA3 differ (Figure 43). BT474 cells have endogenously high activity of AP-1, and
SKBr3 have lower activity (Figure 45). However, in SKBr3 and BT474 cells, in which
PEA3 is endogenously low, exogenous expression of PEA3 increased Notch-4 transcripts
(Figure 46). This suggests together with the Luciferase results seen prior (Figure 27) that
PEA3 is a potent activator of Notch-4 transcription and the levels of AP-1 activity,
although important, may have a secondary role in Notch-4 transcription.
It was observed that PEA3 or GSI alone was able to reduce colony formation by
approximately 40-50% (Figure 42), but the combination was able to reduce colony
formation by more than 60% (Figure 42). When Notch-1IC was transfected into MDA-
MB-231 cells, restoration of colony formation number was quantified (Figure 42),
indicating that Notch-1 may be sufficient to restore the effects of PEA3, GSI, and dual
inhibition. Notch-4IC transfection also restored colony formation but not as sufficiently
as Notch-1IC (Figure 42). Initially, the effects of treatment/transfection appear to be
mediated through Notch-1. However upon closer observation, although Notch-1
exogenous expression produced similar colony numbers as compared to control, the
colonies were smaller in size when visualized under a 40X magnification of a light
microscope (Figure 41). This observation was not quantified, but merely observed.
Notch-4 exogenous expression did not rescue colony number as sufficiently as Notch-1,
but the phenotype of the colonies (not quantified data, mere observation) were similar to
control; the colonies were repeatedly observed to appear more robust and similar in size
132
to control (Figure 41). Interestingly, dual exogenous expression not only restored the
colony formation number, but also the phenotype of the colonies when compared to
control (not quantified data, mere observation) (Figure 41). This could be explained by
the notion that Notch-1 along with its ligand, Jagged-1, are highly regulated receptors and
have been implicated in cell proliferation and growth (Dickson et al., 2007; Reedijk et al.,
2005). Notch-4 has been implicated in mammary cancer stem cells (Harrison et al.,
2010). Taken together, Notch-1 exogenous expression could be responsible for
proliferation of the cells thus acquiring similar colony numbers (in a agreement to what
was observed using a peptidyl γ-secretase inhibitor, z-Leu-Leu-Nle-CHO reviewed in
Chapter II and (Clementz and Osipo, 2009), in MDA-MB-231 cells (Lee et al., 2008)),
but Notch-4 could be responsible for stimulating the mammary cancer stem cells
(Harrison et al., 2010) creating a more robust colony from mere observation. Together,
they restore the effects of PEA3 and Notch inhibition by possibly stimulating both
proliferating cells and stem cells.
In addition, Harrison et al. has recently implicated Notch-4 in mammary tumor
stem cell survival and self-renewal in which they demonstrated that targeting Notch-4 via
siRNA interference specifically was more effective than a GSI in inhibiting the Notch
pathway (Harrison et al., 2010). In these studies, Notch-4 gene transcription was more
sensitive to PEA3 inhibition. Given this fact, the sensitivity obtained in our MDA-MB-
231 system by the dual inhibition of PEA3 and Notch towards reduced viability (Figure
40) may be explained by the notion that we may be not only targeting proliferation and
133
survival of bulk cancer cell populations but also the cancer stem cell population (Harrison
et al., 2010; Korkaya and Wicha, 2007; Korkaya and Wicha, 2009).
Notch is a cellular fate determinant, can induce proliferation, and/or
differentiation depending on the cellular environment (Kopan and Ilagan, 2009). PEA3
has been linked to invasion, migration, and aggressiveness of tumor cells (Coutte et al.,
1999). The dual inhibition of Notch by the GSI, and the inhibition of PEA3 by siRNA
could act by preventing two vital arms of cancer progression, namely proliferation and
possibly metastatic potential in vivo. Emerging nanotechnology as a means to direct
siRNA therapies (Del Pino et al., 2010), recent advances in targeted siRNA treatments in
vivo (Ueyama et al., 2010), and the advent of stapled interface peptides (Moellering et al.,
2009) that disrupt transcription factor complexes transforms the notion for specific
targeting of PEA3 into a potential reality.
Here, these studies have provided evidence of a novel combination treatment to
be exploited for the treatment of triple-negative breast cancer, and potentially other breast
cancer subtypes where PEA3 regulates Notch-1 and Notch-4. The findings offer a
potential mechanism that has identified better strategies for future pre-clinical, in vivo
studies which are currently underway. Enhanced sensitivity towards current GSIs in
future clinical trials by inhibition of PEA3 by nanoparticles (Del Pino et al., 2010), small
molecule inhibitors, or future siRNA approaches (Ueyama et al., 2010) may increase
patient response to treatment and could reduce or eliminate recurrence if stem cell
populations are eliminated (Korkaya and Wicha, 2009). Inhibiting PEA3 in combination
with the GSI may also allow for a larger therapeutic window for the use of a GSI,
134
enabling the reduction of pharmacological doses given to the patient and thus limiting the
resulting undesirable side effects such as gastrointestinal toxicity (Staal and Langerak,
2008; van Es et al., 2005).
Furthermore, the implications of these results lead to many areas of active
research within the clinic. PEA3 expression in breast cancer could become a prognostic
indicator of how a patient may respond to Notch inhibition by a GSI since endogenous
levels of PEA3 (high or low) could determine whether or not a combination treatment is
desirable. From the results observed when PEA3 was endogenously elevated, inhibition
of PEA3 sensitized the cells towards the GSI (Figures 38-42). Therefore, if a patient
presents in the clinic with elevated endogenous levels of PEA3, it may be beneficial to
inhibit both PEA3 and Notch to achieve tumor regression. Inversely, if a patient presents
with endogenously low levels of PEA3, Notch inhibition via a GSI may be only required
as signal targeted therapeutic strategy. Taken together, a patient may acquire a more
unique chemotherapeutic strategy and reduce unnecessary combinatory strategies as
demonstrated with these results. Oncogenic gene profiling acquired by a primary biopsy
is currently measured to decide what appropriate treatment is beneficial to the patient
(Zanetti-Dallenbach et al., 2006). Diagnosis of HER2, estrogen receptor α, and
progesterone receptor expression are already used in the clinic (Carter et al., 1989; Galea
et al., 1992; Mirza et al., 2002). Thus, PEA3 could be another protein to screen to
determine the necessary therapeutic strategy if methods of PEA3 are available. Moreover,
AP-1 activity independently may also prove to be significant in deciding treatment, since
the results indicate that PEA3 and AP-1 are interactive partners in certain breast cancers.
135
The Model
This investigation has lead to the development of a model of targeting Notch both
at production (transcription of Notch) and activation (formation of the active intracellular
Notch) (Figure 47):
1. Production
It was determined that PEA3 transcriptionally activates Notch-1 independently
where as PEA3 partners with c-JUN on Notch-4 (Figure 22 and Figure 31, 32). The
results also suggest that Fra-1 is an activator and c-FOS a repressor of Notch-4
transcription (Figure 35). This stage involves direct regulation at the level of the Notch
gene. Many events including transcriptional complex formation, active transcriptional
hubs (areas of three dimensional gene locus looping as seen in the CFTR gene (Ott et al.,
2009)), and chromatin remodeling (Zhao et al., 2009) are brought to fruition to
successfully regulate the production of the mature Notch receptor. The dynamic
interplay of these factors identified here, such as AP-1 and PEA3 as well as other
potential players unidentified including histone acetyltransferases (Liu et al., 2004;
Matsui et al., 2006), SP-1 (determined by promoter scanning using NCBI Entrez Gene
bank), and/or basic transcriptional machinery (Firlej et al., 2005; Hesselbrock et al.,
2005) are working to tightly regulate Notch gene transcription. This transcriptional stage
provides the first area for potential targeted therapy, namely inhibition of PEA3, to
abrogate Notch production. Once cells acquire the appropriate cellular stimuli, Notch is
136
CoR
CoA
NIC
MAML-1
NCoRSMRT
ENDOCYTOSIS
Gamma-SECRETASE
PEN2
NicastrinPresenilin
APH-1
NOTCH-4
TTGTG
Figure 47: Model of the Transcriptional Regulation of Notch-1 and Notch-4 by PEA3. I. Production: PEA3 transcriptionally activates Notch-1 independently where as Notch-4 in a dynamic partnership with AP-1 notably c-JUN, Fra-1 (Activators) and c-FOS (Repressor). Notch is then processed and shuttled to the cellular membrane. II. Activation: Upon engagement of ligand, Notch undergoes two subsequent cleavages. The last cleavage by the gamma-secretase complex is inhibited by GSI, MRK-003, to attenuate Notch signaling and prevent the formation of intracellular Notch (NIC), responsible for displacing co-repressors and recruiting co-activators on CBF-1 driving target genes involved in cell fate, proliferation, and differentiation. Dual targeting of Notch via GSI and PEA3 via siRNA, reduces tumorigenicity by attenuation of Notch signaling at both its production and activation stages resulting in overall growth arrest and increased apoptosis. This combinatory inhibition may provide a more effective targeted therapy for those with triple negative breast cancers, and could provide addition means of targeting in other breast cancer subtypes.
TGACTCAGGAAACAGCTCAGACGTGETSAP-1
~70 bps
Fra-1 c-JUN
c-FOS
NOTCH-1
AATAAATCAGCC AGCACGTGETS
~1.1 kbGGAAGG GGAAGG
NOTCH-1or
NOTCH-4
GROWTH
GSI
siRNA
siRNA
LL ONE
LL TWO
II. ACTIVATION
CE
LIGAND INTERCELLULAR SPACE
TACECE
I. PRODUCTION
PEA3
PEA3
CBF-1
CoA+ + +
NUCLEUS
CYTOSOL
137
then processed and shuttled to the cellular membrane where it awaits engagement with its
ligand (Kopan and Ilagan, 2009).
2. Activation
Upon engagement of ligand, Notch undergoes two subsequent cleavages. The last
cleavage by the gamma-secretase complex is inhibited by GSI (MRK-003) to attenuate
Notch signaling and prevent the formation of intracellular Notch (NIC), responsible for
displacing co-repressors and recruiting co-activators such as Mastermind on CBF-1
driving target genes involved in cell fate, proliferation, and differentiation (Callahan and
Raafat, 2001). This step is critical for activation of Notch at the post-translational level
and provides a second area for targeted treatment, namely GSI treatment (Weijzen et al.,
2002).
The hypothesis remains that two critical functional areas are open for direct
targeting: transcription and activation. GSI targets the mature receptor (Kopan and
Ilagan, 2004; Weijzen et al., 2002), but production of the receptor is continuing to form.
PEA3 inhibition was only successful at reducing transcripts by 50-80% (Figure 14)
leaving some receptor formation to continue to be produced. If both function areas are
successfully targeted, Notch activation can be attenuated by a GSI and more importantly
the continual production of Notch is dampened by PEA3 siRNA. Therefore, the “global”
abrogation of Notch signaling will reduce the oncogenic effects more successfully than
targeting one or the other independently. Dual targeting of Notch (GSI) and PEA3
(siRNA) reduces tumorigenicity by attenuation of Notch signaling at both production and
138
activation resulting in overall growth arrest and increased apoptosis. This combinatory
inhibition may provide a more effective targeted therapy for those with triple negative
breast cancers, and could provide additional means of targeting in other breast cancer
subtypes where PEA3 regulates Notch-1 and Notch-4.
Future Investigations
In summary, several areas can be investigated to understand the regulation of
Notch by PEA3. It would be interesting to explore further the notion of PEA3 acting as a
repressor on the Notch-2 promoter. Properly designing primers flanking the Ets sites and
performing subsequent chromatin immunoprecipitations will determine if PEA3 directly
regulates Notch-2. Further confirmation regarding the necessity of certain Ets sites could
be explored by designing promoter luciferase constructs. Some may contain the intact
Ets sites and the other ablated Ets sites via site-directed mutagenesis. Dual luciferase
could determine the importance of each particular site in regulating Notch-2.
PEA3 enrichment on the Notch-1 promoter was independent from AP-1. Other
sites have been identified on the Notch-1 promoter including SP-1 and NFκB. It would
be interesting to understand what additional factors may be regulating Notch-1 in
conjunction with PEA3. Co-immunoprecipitation studies could determine if PEA3 is in
complex with SP-1, NFkB, or p300, for example. Moreover, an in depth analysis of the
Notch-1 promoter may reveal other sites of importance. Continuing this idea, it would be
interesting to determine what other partners work with PEA3 and AP-1, including classic
transcriptional machinery, co-activators, co-repressors, etc.
139
With the effects observed on cellular proliferation and apoptosis upon dual
inhibition of PEA3 and Notch in vitro, an in vivo study could be formulated. Xenografts
of MDA-MB-231 transfected with PEA3 siRNA could be injected into the mammary fat
pads of nude mice and immediately administered MRK-003 GSI via oral gavage. Tumor
burden and size could be recorded to determine if dual inhibition has the same effects on
tumor proliferation in vivo.
These are just a few areas that could be explored in the future to continue
understanding the role of PEA3 on Notch regulation. What is clear from these studies is
that PEA3 is a transcriptional activator of Notch-1 and Notch-4 in MDA-MB-231 cells,
an example of triple-negative breast cancer. PEA3 inhibition could help MDA-MB-231
cells respond better to a gamma-secretase inhibitor in combination as indicated by these
studies (Figures 38–42). Dual inhibition reduces both anchorage dependent and
independent proliferation (Figure 38, 41, 42) and increases apoptosis (Figure 39). PEA3
may be a great candidate in addition with Notch inhibition for the targeted treatment of
triple-negative breast cancers.
CHAPTER VI
CLOSING REMARKS
As of yet, there is no known FDA approved, targeted therapy for women
diagnosed with triple-negative breast cancer. They frequently present with metastatic
tumors and have the worst overall prognosis. These studies provide the first evidence of
transcriptional regulation of Notch-1 and Notch-4 by PEA3 in triple-negative and other
breast cancer subtypes. It was demonstrated that Notch-4 gene regulation is in part due to
the presence of AP-1, specifically c-JUN and Fra-1, and PEA3 depending on cellular
context. PEA3 regulates the Notch-1 promoter, but the additional factors that aid in the
regulation are still being investigated. Further evidence reveals that PEA3 inhibition
helps sensitize MDA-MB-231 cells to a gamma-secretase inhibitor, showing promise in
significantly reducing both anchorage dependent and independent growth and increasing
apoptosis. Thus, PEA3 emerges as potential target of inhibition in combination with
inhibiting Notch activity to provide a novel and combined targeted therapy for triple-
negative cancer.
The building blocks of human life, their innate blueprint, rests within the genome.
The genome is a dynamic world of regulation involving opening and closing of DNA
segments, chromatin remolding, histone modifications, and epigenetics. This research
provides insight into gene regulation that can be applied to other genes, systems, and
140
141
disease states. Complex interplay of transcription factors and cellular stimuli when
altered significantly can lead to the disruption of homeostasis and the development of
human disease. PEA3, AP-1, and NOTCH are just a few proteins among myriads of
human disease. PEA3, AP-1, and NOTCH are just a few proteins among myriads of
others aberrantly expressed during disease. Many thousands of proteins are known to be
active within the cell and their genomic regulation is just as vast. This research aims to
provide another stepping stone to understand gene regulation and breast cancer disease
state. Eradication of the breast cancer disease begins here –at the transcriptome.
BIBLIOGRAPHY Agarwal SK, Guru SC, Heppner C, Erdos MR, Collins RM, Park SY, Saggar S,
Chandrasekharappa SC, Collins FS, Spiegel AM, Marx SJ, Burns AL. 1999. Menin interacts with the AP1 transcription factor JunD and represses JunD-activated transcription. Cell 96(1):143-152.
Agarwal SK, Novotny EA, Crabtree JS, Weitzman JB, Yaniv M, Burns AL,
Chandrasekharappa SC, Collins FS, Spiegel AM, Marx SJ. 2003. Transcription factor JunD, deprived of menin, switches from growth suppressor to growth promoter. Proc Natl Acad Sci U S A 100(19):10770-10775.
Albanese C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A, Pestell RG. 1995.
Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem 270(40):23589-23597.
Alimirah F, Panchanathan R, Davis FJ, Chen J, Choubey D. 2007. Restoration of p53
expression in human cancer cell lines upregulates the expression of Notch1: implications for cancer cell fate determination after genotoxic stress. Neoplasia 9(5):427-434.
Amar S, Moreno-Aspitia A, Perez EA. 2008. Issues and controversies in the treatment of
HER2 positive metastatic breast cancer. Breast Cancer Res Treat 109(1):1-7. Ameyar-Zazoua M, Wisniewska MB, Bakiri L, Wagner EF, Yaniv M, Weitzman JB.
2005. AP-1 dimers regulate transcription of the p14/p19ARF tumor suppressor gene. Oncogene 24(14):2298-2306.
Angel P, Karin M. 1991. The role of Jun, Fos and the AP-1 complex in cell-proliferation
and transformation. Biochim Biophys Acta 1072(2-3):129-157. Apelqvist A, Li H, Sommer L, Beatus P, Anderson DJ, Honjo T, Hrabe de Angelis M,
Lendahl U, Edlund H. 1999. Notch signalling controls pancreatic cell differentiation. Nature 400(6747):877-881.
Baan B, Pardali E, Ten Dijke P, van Dam H. 2010. In situ proximity ligation detection of
cJun/AP-1 dimers reveals increased levels of cJun/Fra1 complexes in aggressive breast cancer cell lines in vitro and in vivo. Mol Cell Proteomics.
142
143 Baert JL, Monte D, Musgrove EA, Albagli O, Sutherland RL, de Launoit Y. 1997.
Expression of the PEA3 group of ETS-related transcription factors in human breast-cancer cells. Int J Cancer 70(5):590-597.
Bakin AV, Curran T. 1999. Role of DNA 5-methylcytosine transferase in cell
transformation by fos. Science 283(5400):387-390. Bakiri L, Lallemand D, Bossy-Wetzel E, Yaniv M. 2000. Cell cycle-dependent variations
in c-Jun and JunB phosphorylation: a role in the control of cyclin D1 expression. Embo J 19(9):2056-2068.
Bamberger AM, Methner C, Lisboa BW, Stadtler C, Schulte HM, Loning T, Milde-
Langosch K. 1999. Expression pattern of the AP-1 family in breast cancer: association of fosB expression with a well-differentiated, receptor-positive tumor phenotype. Int J Cancer 84(5):533-538.
Bannister AJ, Kouzarides T. 1995. CBP-induced stimulation of c-Fos activity is
abrogated by E1A. Embo J 14(19):4758-4762. Bartsch R, Ziebermayr R, Zielinski CC, Steger GG. 2010. Triple-negative breast cancer.
Wien Med Wochenschr 160(7-8):174-181. Beckett D. 2004. Functional switches in transcription regulation; molecular mimicry and
plasticity in protein-protein interactions. Biochemistry 43(25):7983-7991. Bedard PL, Cardoso F, Piccart-Gebhart MJ. 2009. Stemming resistance to HER-2
targeted therapy. J Mammary Gland Biol Neoplasia 14(1):55-66. Belguise K, Kersual N, Galtier F, Chalbos D. 2005. FRA-1 expression level regulates
proliferation and invasiveness of breast cancer cells. Oncogene 24(8):1434-1444. Benbow U, Brinckerhoff CE. 1997. The AP-1 site and MMP gene regulation: what is all
the fuss about? Matrix Biol 15(8-9):519-526. Benz CC, O'Hagan RC, Richter B, Scott GK, Chang CH, Xiong X, Chew K, Ljung BM,
Edgerton S, Thor A, Hassell JA. 1997. HER2/Neu and the Ets transcription activator PEA3 are coordinately upregulated in human breast cancer. Oncogene 15(13):1513-1525.
Berdnik D, Torok T, Gonzalez-Gaitan M, Knoblich JA. 2002. The endocytic protein
alpha-Adaptin is required for numb-mediated asymmetric cell division in Drosophila. Dev Cell 3(2):221-231.
144 Berry NB, Fan M, Nephew KP. 2008. Estrogen receptor-alpha hinge-region lysines 302
and 303 regulate receptor degradation by the proteasome. Mol Endocrinol 22(7):1535-1551.
Blaumueller CM, Artavanis-Tsakonas S. 1997. Comparative aspects of Notch signaling
in lower and higher eukaryotes. Perspect Dev Neurobiol 4(4):325-343. Bojovic BB, Hassell JA. 2001. The PEA3 Ets transcription factor comprises multiple
domains that regulate transactivation and DNA binding. J Biol Chem 276(6):4509-4521.
Bojovic BB, Hassell JA. 2008. The transactivation function of the Pea3 subfamily Ets
transcription factors is regulated by sumoylation. DNA Cell Biol 27(6):289-305. Bossis G, Ferrara P, Acquaviva C, Jariel-Encontre I, Piechaczyk M. 2003. c-Fos proto-
oncoprotein is degraded by the proteasome independently of its own ubiquitinylation in vivo. Mol Cell Biol 23(20):7425-7436.
Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens JR, Cumano A, Roux P, Black
RA, Israel A. 2000. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell 5(2):207-216.
Brown PH, Alani R, Preis LH, Szabo E, Birrer MJ. 1993. Suppression of oncogene-
induced transformation by a deletion mutant of c-jun. Oncogene 8(4):877-886. Brown PH, Chen TK, Birrer MJ. 1994. Mechanism of action of a dominant-negative
mutant of c-Jun. Oncogene 9(3):791-799. Brown PH, Kim SH, Wise SC, Sabichi AL, Birrer MJ. 1996. Dominant-negative mutants
of cJun inhibit AP-1 activity through multiple mechanisms and with different potencies. Cell Growth Differ 7(8):1013-1021.
Buzdar AU. 2008. Fulvestrant--a novel estrogen receptor antagonist for the treatment of
advanced breast cancer. Drugs Today (Barc) 44(9):679-692. Buzdar AU, Baum M, Cuzick J. 2006. Letrozole or tamoxifen in early breast cancer. N
Engl J Med 354(14):1528-1530; author reply 1528-1530. Cai J, Lee J, Kopan R, Ma L. 2009. Genetic interplays between Msx2 and Foxn1 are
required for Notch1 expression and hair shaft differentiation. Dev Biol 326(2):420-430.
Callahan R, Raafat A. 2001. Notch signaling in mammary gland tumorigenesis. J
Mammary Gland Biol Neoplasia 6(1):23-36.
145 Carrasco D, Bravo R. 1995. Tissue-specific expression of the fos-related transcription
factor fra-2 during mouse development. Oncogene 10(6):1069-1079. Carter CL, Allen C, Henson DE. 1989. Relation of tumor size, lymph node status, and
survival in 24,740 breast cancer cases. Cancer 63(1):181-187. Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, Rowland AM, Kotts
C, Carver ME, Shepard HM. 1992. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A 89(10):4285-4289.
Chan DA, Giaccia AJ. 2008. Targeting cancer cells by synthetic lethality: autophagy and
VHL in cancer therapeutics. Cell Cycle 7(19):2987-2990. Cheang MC, van de Rijn M, Nielsen TO. 2008. Gene expression profiling of breast
cancer. Annu Rev Pathol 3:67-97. Chen RH, Juo PC, Curran T, Blenis J. 1996. Phosphorylation of c-Fos at the C-terminus
enhances its transforming activity. Oncogene 12(7):1493-1502. Cheng P, Zlobin A, Volgina V, Gottipati S, Osborne B, Simel EJ, Miele L, Gabrilovich
DI. 2001. Notch-1 regulates NF-kappaB activity in hemopoietic progenitor cells. J Immunol 167(8):4458-4467.
Chotteau-Lelievre A, Desbiens X, Pelczar H, Defossez PA, de Launoit Y. 1997.
Differential expression patterns of the PEA3 group transcription factors through murine embryonic development. Oncogene 15(8):937-952.
Chotteau-Lelievre A, Montesano R, Soriano J, Soulie P, Desbiens X, de Launoit Y. 2003.
PEA3 transcription factors are expressed in tissues undergoing branching morphogenesis and promote formation of duct-like structures by mammary epithelial cells in vitro. Dev Biol 259(2):241-257.
Chou J, Provot S, Werb Z. 2010. GATA3 in development and cancer differentiation: cells
GATA have it! J Cell Physiol 222(1):42-49. Clapier CR, Cairns BR. 2009. The biology of chromatin remodeling complexes. Annu
Rev Biochem 78:273-304. Clementz AG, Osipo C. 2009. Notch versus the proteasome: what is the target of gamma-
secretase inhibitor-I? Breast Cancer Res 11(5):110. Coates AS, Keshaviah A, Thurlimann B, Mouridsen H, Mauriac L, Forbes JF, Paridaens
R, Castiglione-Gertsch M, Gelber RD, Colleoni M, Lang I, Del Mastro L, Smith I,
146 Chirgwin J, Nogaret JM, Pienkowski T, Wardley A, Jakobsen EH, Price KN, Goldhirsch A. 2007. Five years of letrozole compared with tamoxifen as initial adjuvant therapy for postmenopausal women with endocrine-responsive early breast cancer: update of study BIG 1-98. J Clin Oncol 25(5):486-492.
Colleoni M, Cole BF, Viale G, Regan MM, Price KN, Maiorano E, Mastropasqua MG,
Crivellari D, Gelber RD, Goldhirsch A, Coates AS, Gusterson BA. 2010. Classical cyclophosphamide, methotrexate, and fluorouracil chemotherapy is more effective in triple-negative, node-negative breast cancer: results from two randomized trials of adjuvant chemoendocrine therapy for node-negative breast cancer. J Clin Oncol 28(18):2966-2973.
Coutte L, Monte D, Baert J, de Launoit Y. 1999. Genomic organization of the human
e1af gene,a member of Ets transcription factors. Gene 240(1):201-207. Curran T, Franza BR, Jr. 1988. Fos and Jun: the AP-1 connection. Cell 55(3):395-397. Curry CL, Reed LL, Golde TE, Miele L, Nickoloff BJ, Foreman KE. 2005. Gamma
secretase inhibitor blocks Notch activation and induces apoptosis in Kaposi's sarcoma tumor cells. Oncogene 24(42):6333-6344.
Daschner PJ, Ciolino HP, Plouzek CA, Yeh GC. 1999. Increased AP-1 activity in drug
resistant human breast cancer MCF-7 cells. Breast Cancer Res Treat 53(3):229-240.
Davidson B, Goldberg I, Gotlieb WH, Kopolovic J, Risberg B, Ben-Baruch G, Reich R.
2003. Coordinated expression of integrin subunits, matrix metalloproteinases (MMP), angiogenic genes and Ets transcription factors in advanced-stage ovarian carcinoma: a possible activation pathway? Cancer Metastasis Rev 22(1):103-115.
Dean-Colomb W, Esteva FJ. 2008. Her2-positive breast cancer: herceptin and beyond.
Eur J Cancer 44(18):2806-2812. Del Pino P, Munoz-Javier A, Vlaskou D, Rivera Gil P, Plank C, Parak WJ. 2010. Gene
Silencing Mediated by Magnetic Lipospheres Tagged with Small Interfering RNA. Nano Lett.
Deng T, Karin M. 1993. JunB differs from c-Jun in its DNA-binding and dimerization
domains, and represses c-Jun by formation of inactive heterodimers. Genes Dev 7(3):479-490.
Deng T, Karin M. 1994. c-Fos transcriptional activity stimulated by H-Ras-activated
protein kinase distinct from JNK and ERK. Nature 371(6493):171-175.
147 Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA,
Rawlinson E, Sun P, Narod SA. 2007. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 13(15 Pt 1):4429-4434.
Dickson BC, Mulligan AM, Zhang H, Lockwood G, O'Malley FP, Egan SE, Reedijk M.
2007. High-level JAG1 mRNA and protein predict poor outcome in breast cancer. Mod Pathol 20(6):685-693.
Dievart A, Beaulieu N, Jolicoeur P. 1999. Involvement of Notch1 in the development of
mouse mammary tumors. Oncogene 18(44):5973-5981. Dirbas FM. 2009. Accelerated partial breast irradiation: where do we stand? J Natl
Compr Canc Netw 7(2):215-225. Dunwoodie SL, Henrique D, Harrison SM, Beddington RS. 1997. Mouse Dll3: a novel
divergent Delta gene which may complement the function of other Delta homologues during early pattern formation in the mouse embryo. Development 124(16):3065-3076.
Eferl R, Wagner EF. 2003. AP-1: a double-edged sword in tumorigenesis. Nat Rev
Cancer 3(11):859-868. El-Tanani M, Platt-Higgins A, Rudland PS, Campbell FC. 2004. Ets gene PEA3
cooperates with beta-catenin-Lef-1 and c-Jun in regulation of osteopontin transcription. J Biol Chem 279(20):20794-20806.
Eliasz S, Liang S, Chen Y, De Marco MA, Machek O, Skucha S, Miele L, Bocchetta M.
2010. Notch-1 stimulates survival of lung adenocarcinoma cells during hypoxia by activating the IGF-1R pathway. Oncogene 29(17):2488-2498.
Evans CP, Stapp EC, Dall'Era MA, Juarez J, Yang JC. 2001. Regulation of u-PA gene
expression in human prostate cancer. Int J Cancer 94(3):390-395. Farmer P, Bonnefoi H, Becette V, Tubiana-Hulin M, Fumoleau P, Larsimont D,
Macgrogan G, Bergh J, Cameron D, Goldstein D, Duss S, Nicoulaz AL, Brisken C, Fiche M, Delorenzi M, Iggo R. 2005. Identification of molecular apocrine breast tumours by microarray analysis. Oncogene 24(29):4660-4671.
Firlej V, Bocquet B, Desbiens X, de Launoit Y, Chotteau-Lelievre A. 2005. Pea3
transcription factor cooperates with USF-1 in regulation of the murine bax transcription without binding to an Ets-binding site. J Biol Chem 280(2):887-898.
148 Fischer A, Schumacher N, Maier M, Sendtner M, Gessler M. 2004. The Notch target
genes Hey1 and Hey2 are required for embryonic vascular development. Genes Dev 18(8):901-911.
Fisher B, Anderson S, Bryant J, Margolese RG, Deutsch M, Fisher ER, Jeong JH,
Wolmark N. 2002a. Twenty-year follow-up of a randomized trial comparing total mastectomy, lumpectomy, and lumpectomy plus irradiation for the treatment of invasive breast cancer. N Engl J Med 347(16):1233-1241.
Fisher B, Jeong JH, Anderson S, Bryant J, Fisher ER, Wolmark N. 2002b. Twenty-five-
year follow-up of a randomized trial comparing radical mastectomy, total mastectomy, and total mastectomy followed by irradiation. N Engl J Med 347(8):567-575.
Fitzsimmons D, Hodsdon W, Wheat W, Maira SM, Wasylyk B, Hagman J. 1996. Pax-5
(BSAP) recruits Ets proto-oncogene family proteins to form functional ternary complexes on a B-cell-specific promoter. Genes Dev 10(17):2198-2211.
Foltz DR, Santiago MC, Berechid BE, Nye JS. 2002. Glycogen synthase kinase-3beta
modulates notch signaling and stability. Curr Biol 12(12):1006-1011. Freund A, Jolivel V, Durand S, Kersual N, Chalbos D, Chavey C, Vignon F, Lazennec G.
2004. Mechanisms underlying differential expression of interleukin-8 in breast cancer cells. Oncogene 23(36):6105-6114.
Fulford LG, Easton DF, Reis-Filho JS, Sofronis A, Gillett CE, Lakhani SR, Hanby A.
2006. Specific morphological features predictive for the basal phenotype in grade 3 invasive ductal carcinoma of breast. Histopathology 49(1):22-34.
Fung TT, Hu FB, McCullough ML, Newby PK, Willett WC, Holmes MD. 2006. Diet
quality is associated with the risk of estrogen receptor-negative breast cancer in postmenopausal women. J Nutr 136(2):466-472.
Galea MH, Blamey RW, Elston CE, Ellis IO. 1992. The Nottingham Prognostic Index in
primary breast cancer. Breast Cancer Res Treat 22(3):207-219. Gerald D, Berra E, Frapart YM, Chan DA, Giaccia AJ, Mansuy D, Pouyssegur J, Yaniv
M, Mechta-Grigoriou F. 2004. JunD reduces tumor angiogenesis by protecting cells from oxidative stress. Cell 118(6):781-794.
Graves BJ, Petersen JM. 1998. Specificity within the ets family of transcription factors.
Adv Cancer Res 75:1-55.
149 Guo B, Sharrocks AD. 2009. Extracellular signal-regulated kinase mitogen-activated
protein kinase signaling initiates a dynamic interplay between sumoylation and ubiquitination to regulate the activity of the transcriptional activator PEA3. Mol Cell Biol 29(11):3204-3218.
Haines N, Irvine KD. 2003. Glycosylation regulates Notch signalling. Nat Rev Mol Cell
Biol 4(10):786-797. Harrison H, Farnie G, Howell SJ, Rock RE, Stylianou S, Brennan KR, Bundred NJ,
Clarke RB. 2010. Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor. Cancer Res 70(2):709-718.
Hattori K, Angel P, Le Beau MM, Karin M. 1988. Structure and chromosomal
localization of the functional intronless human JUN protooncogene. Proc Natl Acad Sci U S A 85(23):9148-9152.
Hennessy BT, Gonzalez-Angulo AM, Stemke-Hale K, Gilcrease MZ, Krishnamurthy S,
Lee JS, Fridlyand J, Sahin A, Agarwal R, Joy C, Liu W, Stivers D, Baggerly K, Carey M, Lluch A, Monteagudo C, He X, Weigman V, Fan C, Palazzo J, Hortobagyi GN, Nolden LK, Wang NJ, Valero V, Gray JW, Perou CM, Mills GB. 2009. Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics. Cancer Res 69(10):4116-4124.
Herber B, Truss M, Beato M, Muller R. 1994. Inducible regulatory elements in the
human cyclin D1 promoter. Oncogene 9(7):2105-2107. Hesselbrock DR, Kurpios N, Hassell JA, Watson MA, Fleming TP. 2005. PEA3, AP-1,
and a unique repetitive sequence all are involved in transcriptional regulation of the breast cancer-associated gene, mammaglobin. Breast Cancer Res Treat 89(3):289-296.
Hida K, Shindoh M, Yoshida K, Kudoh A, Furaoka K, Kohgo T, Fujinaga K, Totsuka Y.
1997. Expression of E1AF, an ets-family transcription factor, is correlated with the invasive phenotype of oral squamous cell carcinoma. Oral Oncol 33(6):426-430.
Higashino F, Yoshida K, Fujinaga Y, Kamio K, Fujinaga K. 1993. Isolation of a cDNA
encoding the adenovirus E1A enhancer binding protein: a new human member of the ets oncogene family. Nucleic Acids Res 21(3):547-553.
Hind D, Ward S, De Nigris E, Simpson E, Carroll C, Wyld L. 2007. Hormonal therapies
for early breast cancer: systematic review and economic evaluation. Health Technol Assess 11(26):iii-iv, ix-xi, 1-134.
150 Hortobagyi GN. 1998. Treatment of breast cancer. N Engl J Med 339(14):974-984. Howell A. 2006. Pure oestrogen antagonists for the treatment of advanced breast cancer.
Endocr Relat Cancer 13(3):689-706. Hsieh JJ, Zhou S, Chen L, Young DB, Hayward SD. 1999. CIR, a corepressor linking the
DNA binding factor CBF1 to the histone deacetylase complex. Proc Natl Acad Sci U S A 96(1):23-28.
Hu C, Dievart A, Lupien M, Calvo E, Tremblay G, Jolicoeur P. 2006. Overexpression of activated murine Notch1 and Notch3 in transgenic mice blocks mammary gland development and induces mammary tumors. Am J Pathol 168(3):973-990.
Hu E, Mueller E, Oliviero S, Papaioannou VE, Johnson R, Spiegelman BM. 1994.
Targeted disruption of the c-fos gene demonstrates c-fos-dependent and -independent pathways for gene expression stimulated by growth factors or oncogenes. Embo J 13(13):3094-3103.
Iso T, Kedes L, Hamamori Y. 2003. HES and HERP families: multiple effectors of the
Notch signaling pathway. J Cell Physiol 194(3):237-255. Jones C, Mackay A, Grigoriadis A, Cossu A, Reis-Filho JS, Fulford L, Dexter T, Davies
S, Bulmer K, Ford E, Parry S, Budroni M, Palmieri G, Neville AM, O'Hare MJ, Lakhani SR. 2004. Expression profiling of purified normal human luminal and myoepithelial breast cells: identification of novel prognostic markers for breast cancer. Cancer Res 64(9):3037-3045.
Jordan VC, Haldemann B, Allen KE. 1981. Geometric isomers of substituted
triphenylethylenes and antiestrogen action. Endocrinology 108(4):1353-1361. Kallunki T, Deng T, Hibi M, Karin M. 1996. c-Jun can recruit JNK to phosphorylate
dimerization partners via specific docking interactions. Cell 87(5):929-939. Kao J, Salari K, Bocanegra M, Choi YL, Girard L, Gandhi J, Kwei KA, Hernandez-
Boussard T, Wang P, Gazdar AF, Minna JD, Pollack JR. 2009. Molecular profiling of breast cancer cell lines defines relevant tumor models and provides a resource for cancer gene discovery. PLoS One 4(7):e6146.
Kasibhatla S, Brunner T, Genestier L, Echeverri F, Mahboubi A, Green DR. 1998. DNA
damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-kappa B and AP-1. Mol Cell 1(4):543-551.
Kataoka K, Noda M, Nishizawa M. 1996. Transactivation activity of Maf nuclear
oncoprotein is modulated by Jun, Fos and small Maf proteins. Oncogene 12(1):53-62.
151 Kaya M, Yoshida K, Higashino F, Mitaka T, Ishii S, Fujinaga K. 1996. A single ets-
related transcription factor, E1AF, confers invasive phenotype on human cancer cells. Oncogene 12(2):221-227.
Kennecke H, Yerushalmi R, Woods R, Cheang MC, Voduc D, Speers CH, Nielsen TO,
Gelmon K. 2010. Metastatic behavior of breast cancer subtypes. J Clin Oncol 28(20):3271-3277.
Klueg KM, Parody TR, Muskavitch MA. 1998. Complex proteolytic processing acts on Delta, a transmembrane ligand for Notch, during Drosophila development. Mol Biol Cell 9(7):1709-1723.
Kopan R, Ilagan MX. 2004. Gamma-secretase: proteasome of the membrane? Nat Rev
Mol Cell Biol 5(6):499-504. Kopan R, Ilagan MX. 2009. The canonical Notch signaling pathway: unfolding the
activation mechanism. Cell 137(2):216-233. Korkaya H, Wicha MS. 2007. Selective targeting of cancer stem cells: a new concept in
cancer therapeutics. BioDrugs 21(5):299-310. Korkaya H, Wicha MS. 2009. HER-2, notch, and breast cancer stem cells: targeting an
axis of evil. Clin Cancer Res 15(6):1845-1847. Kurpios NA, MacNeil L, Shepherd TG, Gludish DW, Giacomelli AO, Hassell JA. 2009.
The Pea3 Ets transcription factor regulates differentiation of multipotent progenitor cells during mammary gland development. Dev Biol 325(1):106-121.
Kurpios NA, Sabolic NA, Shepherd TG, Fidalgo GM, Hassell JA. 2003. Function of
PEA3 Ets transcription factors in mammary gland development and oncogenesis. J Mammary Gland Biol Neoplasia 8(2):177-190.
Kustikova O, Kramerov D, Grigorian M, Berezin V, Bock E, Lukanidin E, Tulchinsky E.
1998. Fra-1 induces morphological transformation and increases in vitro invasiveness and motility of epithelioid adenocarcinoma cells. Mol Cell Biol 18(12):7095-7105.
Lanford PJ, Lan Y, Jiang R, Lindsell C, Weinmaster G, Gridley T, Kelley MW. 1999.
Notch signalling pathway mediates hair cell development in mammalian cochlea. Nat Genet 21(3):289-292.
Langerod A, Zhao H, Borgan O, Nesland JM, Bukholm IR, Ikdahl T, Karesen R,
Borresen-Dale AL, Jeffrey SS. 2007. TP53 mutation status and gene expression profiles are powerful prognostic markers of breast cancer. Breast Cancer Res 9(3):R30.
152 Laudet V, Hanni C, Stehelin D, Duterque-Coquillaud M. 1999. Molecular phylogeny of
the ETS gene family. Oncogene 18(6):1351-1359. Le Borgne R, Bardin A, Schweisguth F. 2005. The roles of receptor and ligand
endocytosis in regulating Notch signaling. Development 132(8):1751-1762. Lee CW, Raskett CM, Prudovsky I, Altieri DC. 2008. Molecular dependence of estrogen
receptor-negative breast cancer on a notch-survivin signaling axis. Cancer Res 68(13):5273-5281.
Lewis HD, Leveridge M, Strack PR, Haldon CD, O'Neil J, Kim H, Madin A, Hannam JC,
Look AT, Kohl N, Draetta G, Harrison T, Kerby JA, Shearman MS, Beher D. 2007. Apoptosis in T cell acute lymphoblastic leukemia cells after cell cycle arrest induced by pharmacological inhibition of notch signaling. Chem Biol 14(2):209-219.
Lindsell CE, Shawber CJ, Boulter J, Weinmaster G. 1995. Jagged: a mammalian ligand
that activates Notch1. Cell 80(6):909-917. Liu Y, Borchert GL, Phang JM. 2004. Polyoma enhancer activator 3, an ets transcription
factor, mediates the induction of cyclooxygenase-2 by nitric oxide in colorectal cancer cells. J Biol Chem 279(18):18694-18700.
Liu Y, Ludes-Meyers J, Zhang Y, Munoz-Medellin D, Kim HT, Lu C, Ge G, Schiff R,
Hilsenbeck SG, Osborne CK, Brown PH. 2002. Inhibition of AP-1 transcription factor causes blockade of multiple signal transduction pathways and inhibits breast cancer growth. Oncogene 21(50):7680-7689.
Livasy CA, Karaca G, Nanda R, Tretiakova MS, Olopade OI, Moore DT, Perou CM.
2006. Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod Pathol 19(2):264-271.
Maier MM, Gessler M. 2000. Comparative analysis of the human and mouse Hey1
promoter: Hey genes are new Notch target genes. Biochem Biophys Res Commun 275(2):652-660.
Marconcini L, Marchio S, Morbidelli L, Cartocci E, Albini A, Ziche M, Bussolino F,
Oliviero S. 1999. c-fos-induced growth factor/vascular endothelial growth factor D induces angiogenesis in vivo and in vitro. Proc Natl Acad Sci U S A 96(17):9671-9676.
Marshall SF, Bernstein L, Anton-Culver H, Deapen D, Horn-Ross PL, Mohrenweiser H,
Peel D, Pinder R, Purdie DM, Reynolds P, Stram D, West D, Wright WE, Ziogas
153 A, Ross RK. 2005. Nonsteroidal anti-inflammatory drug use and breast cancer risk by stage and hormone receptor status. J Natl Cancer Inst 97(11):805-812.
Masuya M, Katayama N, Hoshino N, Nishikawa H, Sakano S, Araki H, Mitani H, Suzuki
H, Miyashita H, Kobayashi K, Nishii K, Minami N, Shiku H. 2002. The soluble Notch ligand, Jagged-1, inhibits proliferation of CD34+ macrophage progenitors. Int J Hematol 75(3):269-276.
Matsui K, Sugimori K, Motomura H, Ejiri N, Tsukada K, Kitajima I. 2006. PEA3 cooperates with c-Jun in regulation of HER2/neu transcription. Oncol Rep 16(1):153-158.
Matthews CP, Colburn NH, Young MR. 2007. AP-1 a target for cancer prevention. Curr
Cancer Drug Targets 7(4):317-324. Maughan KL, Lutterbie MA, Ham PS. 2010. Treatment of breast cancer. Am Fam
Physician 81(11):1339-1346. Mauri D, Polyzos NP, Salanti G, Pavlidis N, Ioannidis JP. 2008. Multiple-treatments
meta-analysis of chemotherapy and targeted therapies in advanced breast cancer. J Natl Cancer Inst 100(24):1780-1791.
McArthur H. 2009. An overview of HER-targeted therapy with lapatinib in breast cancer.
Adv Ther 26(3):263-271. McBride K, Charron F, Lefebvre C, Nemer M. 2003. Interaction with GATA
transcription factors provides a mechanism for cell-specific effects of c-Fos. Oncogene 22(52):8403-8412.
McGill MA, Dho SE, Weinmaster G, McGlade CJ. 2009. Numb regulates post-endocytic
trafficking and degradation of Notch1. J Biol Chem 284(39):26427-26438. McGill MA, McGlade CJ. 2003. Mammalian numb proteins promote Notch1 receptor
ubiquitination and degradation of the Notch1 intracellular domain. J Biol Chem 278(25):23196-23203.
Micalizzi DS, Farabaugh SM, Ford HL. 2010. Epithelial-mesenchymal transition in
cancer: parallels between normal development and tumor progression. J Mammary Gland Biol Neoplasia 15(2):117-134.
Miele L, Golde T, Osborne B. 2006. Notch signaling in cancer. Curr Mol Med 6(8):905-
918. Milde-Langosch K, Bamberger AM, Methner C, Rieck G, Loning T. 2000. Expression of
cell cycle-regulatory proteins rb, p16/MTS1, p27/KIP1, p21/WAF1, cyclin D1
154
and cyclin E in breast cancer: correlations with expression of activating protein-1 family members. Int J Cancer 87(4):468-472.
Milde-Langosch K, Roder H, Andritzky B, Aslan B, Hemminger G, Brinkmann A,
Bamberger CM, Loning T, Bamberger AM. 2004. The role of the AP-1 transcription factors c-Fos, FosB, Fra-1 and Fra-2 in the invasion process of mammary carcinomas. Breast Cancer Res Treat 86(2):139-152.
Mirza AN, Mirza NQ, Vlastos G, Singletary SE. 2002. Prognostic factors in node-
negative breast cancer: a review of studies with sample size more than 200 and follow-up more than 5 years. Ann Surg 235(1):10-26.
Moellering RE, Cornejo M, Davis TN, Del Bianco C, Aster JC, Blacklow SC, Kung AL,
Gilliland DG, Verdine GL, Bradner JE. 2009. Direct inhibition of the NOTCH transcription factor complex. Nature 462(7270):182-188.
Monje P, Marinissen MJ, Gutkind JS. 2003. Phosphorylation of the carboxyl-terminal
transactivation domain of c-Fos by extracellular signal-regulated kinase mediates the transcriptional activation of AP-1 and cellular transformation induced by platelet-derived growth factor. Mol Cell Biol 23(19):7030-7043.
Morgan TH. 1917. The theory of the gene. American Naturalist 51(609):513-544. Morris GJ, Naidu S, Topham AK, Guiles F, Xu Y, McCue P, Schwartz GF, Park PK,
Rosenberg AL, Brill K, Mitchell EP. 2007. Differences in breast carcinoma characteristics in newly diagnosed African-American and Caucasian patients: a single-institution compilation compared with the National Cancer Institute's Surveillance, Epidemiology, and End Results database. Cancer 110(4):876-884.
Mouridsen HT. 1990. New cytotoxic drugs in treatment of breast cancer. Acta Oncol
29(3):343-347. Nickoloff BJ, Qin JZ, Chaturvedi V, Denning MF, Bonish B, Miele L. 2002. Jagged-1
mediated activation of notch signaling induces complete maturation of human keratinocytes through NF-kappaB and PPARgamma. Cell Death Differ 9(8):842-855.
Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z, Hernandez-Boussard T,
Livasy C, Cowan D, Dressler L, Akslen LA, Ragaz J, Gown AM, Gilks CB, van de Rijn M, Perou CM. 2004. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 10(16):5367-5374.
155 Nishida T, Terashima M, Fukami K, Yamada Y. 2007. PIASy controls ubiquitination-
dependent proteasomal degradation of Ets-1. Biochem J 405(3):481-488. O'Mahony D, Bishop MR. 2006. Monoclonal antibody therapy. Front Biosci 11:1620-
1635. O'Neill CF, Urs S, Cinelli C, Lincoln A, Nadeau RJ, Leon R, Toher J, Mouta-Bellum C,
Friesel RE, Liaw L. 2007. Notch2 signaling induces apoptosis and inhibits human MDA-MB-231 xenograft growth. Am J Pathol 171(3):1023-1036.
Oikawa T, Yamada T. 2003. Molecular biology of the Ets family of transcription factors. Gene 303:11-34.
Okajima T, Irvine KD. 2002. Regulation of notch signaling by o-linked fucose. Cell
111(6):893-904. Olive M, Krylov D, Echlin DR, Gardner K, Taparowsky E, Vinson C. 1997. A dominant
negative to activation protein-1 (AP1) that abolishes DNA binding and inhibits oncogenesis. J Biol Chem 272(30):18586-18594.
Ott CJ, Blackledge NP, Kerschner JL, Leir SH, Crawford GE, Cotton CU, Harris A. 2009.
Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus. Proc Natl Acad Sci U S A 106(47):19934-19939.
Pajerowski AG, Nguyen C, Aghajanian H, Shapiro MJ, Shapiro VS. 2009. NKAP is a
transcriptional repressor of notch signaling and is required for T cell development. Immunity 30(5):696-707.
Passegue E, Wagner EF. 2000. JunB suppresses cell proliferation by transcriptional
activation of p16(INK4a) expression. Embo J 19(12):2969-2979. Peppercorn J, Perou CM, Carey LA. 2008. Molecular subtypes in breast cancer
evaluation and management: divide and conquer. Cancer Invest 26(1):1-10. Perez A, Barco R, Fernandez I, Price-Schiavi SA, Carraway KL. 2003. PEA3
transactivates the Muc4/sialomucin complex promoter in mammary epithelial and tumor cells. J Biol Chem 278(38):36942-36952.
Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross
DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO, Botstein D. 2000. Molecular portraits of human breast tumours. Nature 406(6797):747-752.
156 Pfarr CM, Mechta F, Spyrou G, Lallemand D, Carillo S, Yaniv M. 1994. Mouse JunD
negatively regulates fibroblast growth and antagonizes transformation by ras. Cell 76(4):747-760.
Quillard T, Devalliere J, Chatelais M, Coulon F, Seveno C, Romagnoli M, Barille Nion S,
Charreau B. 2009. Notch2 signaling sensitizes endothelial cells to apoptosis by negatively regulating the key protective molecule survivin. PLoS One 4(12):e8244.
Ransone LJ, Visvader J, Wamsley P, Verma IM. 1990. Trans-dominant negative mutants
of Fos and Jun. Proc Natl Acad Sci U S A 87(10):3806-3810. Rauscher FJ, 3rd, Sambucetti LC, Curran T, Distel RJ, Spiegelman BM. 1988. Common
DNA binding site for Fos protein complexes and transcription factor AP-1. Cell 52(3):471-480.
Reedijk M, Odorcic S, Chang L, Zhang H, Miller N, McCready DR, Lockwood G, Egan
SE. 2005. High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival. Cancer Res 65(18):8530-8537.
Reis-Filho JS, Pinheiro C, Lambros MB, Milanezi F, Carvalho S, Savage K, Simpson PT,
Jones C, Swift S, Mackay A, Reis RM, Hornick JL, Pereira EM, Baltazar F, Fletcher CD, Ashworth A, Lakhani SR, Schmitt FC. 2006. EGFR amplification and lack of activating mutations in metaplastic breast carcinomas. J Pathol 209(4):445-453.
Reis-Filho JS, Tutt AN. 2008. Triple negative tumours: a critical review. Histopathology
52(1):108-118. Rizzo P, Miao H, D'Souza G, Osipo C, Song LL, Yun J, Zhao H, Mascarenhas J, Wyatt
D, Antico G, Hao L, Yao K, Rajan P, Hicks C, Siziopikou K, Selvaggi S, Bashir A, Bhandari D, Marchese A, Lendahl U, Qin JZ, Tonetti DA, Albain K, Nickoloff BJ, Miele L. 2008. Cross-talk between notch and the estrogen receptor in breast cancer suggests novel therapeutic approaches. Cancer Res 68(13):5226-5235.
Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE, Jr., Davidson NE, Tan-Chiu E,
Martino S, Paik S, Kaufman PA, Swain SM, Pisansky TM, Fehrenbacher L, Kutteh LA, Vogel VG, Visscher DW, Yothers G, Jenkins RB, Brown AM, Dakhil SR, Mamounas EP, Lingle WL, Klein PM, Ingle JN, Wolmark N. 2005. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 353(16):1673-1684.
157 Ronchini C, Capobianco AJ. 2001. Induction of cyclin D1 transcription and CDK2
activity by Notch(ic): implication for cell cycle disruption in transformation by Notch(ic). Mol Cell Biol 21(17):5925-5934.
Rouzier R, Wagner P, Morandi P, Pusztai L. 2005. Gene expression profiling of primary
breast cancer. Curr Oncol Rep 7(1):38-44. Rowinsky EK, Onetto N, Canetta RM, Arbuck SG. 1992. Taxol: the first of the taxanes,
an important new class of antitumor agents. Semin Oncol 19(6):646-662. Sachdev JC, Jahanzeb M. 2008. Evolution of bevacizumab-based therapy in the
management of breast cancer. Clin Breast Cancer 8(5):402-410. Sansone P, Storci G, Giovannini C, Pandolfi S, Pianetti S, Taffurelli M, Santini D,
Ceccarelli C, Chieco P, Bonafe M. 2007. p66Shc/Notch-3 interplay controls self-renewal and hypoxia survival in human stem/progenitor cells of the mammary gland expanded in vitro as mammospheres. Stem Cells 25(3):807-815.
Sasamura T, Sasaki N, Miyashita F, Nakao S, Ishikawa HO, Ito M, Kitagawa M,
Harigaya K, Spana E, Bilder D, Perrimon N, Matsuno K. 2003. neurotic, a novel maternal neurogenic gene, encodes an O-fucosyltransferase that is essential for Notch-Delta interactions. Development 130(20):4785-4795.
Saxena MT, Schroeter EH, Mumm JS, Kopan R. 2001. Murine notch homologs (N1-4)
undergo presenilin-dependent proteolysis. J Biol Chem 276(43):40268-40273. Schutte J, Minna JD, Birrer MJ. 1989. Deregulated expression of human c-jun transforms
primary rat embryo cells in cooperation with an activated c-Ha-ras gene and transforms rat-1a cells as a single gene. Proc Natl Acad Sci U S A 86(7):2257-2261.
Scott GK, Daniel JC, Xiong X, Maki RA, Kabat D, Benz CC. 1994. Binding of an ETS-
related protein within the DNase I hypersensitive site of the HER2/neu promoter in human breast cancer cells. J Biol Chem 269(31):19848-19858.
Sharrocks AD. 2001. The ETS-domain transcription factor family. Nat Rev Mol Cell Biol
2(11):827-837. Shaulian E, Karin M. 2001. AP-1 in cell proliferation and survival. Oncogene
20(19):2390-2400. Shaulian E, Karin M. 2002. AP-1 as a regulator of cell life and death. Nat Cell Biol
4(5):E131-136.
158 Shawber C, Boulter J, Lindsell CE, Weinmaster G. 1996. Jagged2: a serrate-like gene
expressed during rat embryogenesis. Dev Biol 180(1):370-376. Shepherd T, Hassell JA. 2001. Role of Ets transcription factors in mammary gland
development and oncogenesis. J Mammary Gland Biol Neoplasia 6(1):129-140. Shepherd TG, Kockeritz L, Szrajber MR, Muller WJ, Hassell JA. 2001. The pea3
subfamily ets genes are required for HER2/Neu-mediated mammary oncogenesis. Curr Biol 11(22):1739-1748.
Shi S, Stanley P. 2003. Protein O-fucosyltransferase 1 is an essential component of Notch
signaling pathways. Proc Natl Acad Sci U S A 100(9):5234-5239. Shore P, Whitmarsh AJ, Bhaskaran R, Davis RJ, Waltho JP, Sharrocks AD. 1996.
Determinants of DNA-binding specificity of ETS-domain transcription factors. Mol Cell Biol 16(7):3338-3349.
Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG,
Udove J, Ullrich A, et al. 1989. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244(4905):707-712.
Sledge GW, Jr. 1992. Cisplatin and platinum analogues in breast cancer. Semin Oncol
19(1 Suppl 2):78-82. Smeal T, Angel P, Meek J, Karin M. 1989. Different requirements for formation of Jun:
Jun and Jun: Fos complexes. Genes Dev 3(12B):2091-2100. Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M. 1991. Oncogenic and
transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature 354(6353):494-496.
Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van
de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein Lonning P, Borresen-Dale AL. 2001. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 98(19):10869-10874.
Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, Deng S, Johnsen H,
Pesich R, Geisler S, Demeter J, Perou CM, Lonning PE, Brown PO, Borresen-Dale AL, Botstein D. 2003. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A 100(14):8418-8423.
Spalding BJ. 2008. Thumbs up for Avastin. Nat Biotechnol 26(4):365.
159 Staal FJ, Langerak AW. 2008. Signaling pathways involved in the development of T-cell
acute lymphoblastic leukemia. Haematologica 93(4):493-497. Stylianou S, Clarke RB, Brennan K. 2006. Aberrant activation of notch signaling in
human breast cancer. Cancer Res 66(3):1517-1525. Subbaramaiah K, Norton L, Gerald W, Dannenberg AJ. 2002. Cyclooxygenase-2 is
overexpressed in HER-2/neu-positive breast cancer: evidence for involvement of AP-1 and PEA3. J Biol Chem 277(21):18649-18657.
Subramaniam D, Ramalingam S, Houchen CW, Anant S. 2010. Cancer stem cells: a
novel paradigm for cancer prevention and treatment. Mini Rev Med Chem 10(5):359-371.
Sun X, Artavanis-Tsakonas S. 1997. Secreted forms of DELTA and SERRATE define
antagonists of Notch signaling in Drosophila. Development 124(17):3439-3448. Suzuki R, Ye W, Rylander-Rudqvist T, Saji S, Colditz GA, Wolk A. 2005. Alcohol and
postmenopausal breast cancer risk defined by estrogen and progesterone receptor status: a prospective cohort study. J Natl Cancer Inst 97(21):1601-1608.
Swaby RF, Jordan VC. 2008. Low-dose estrogen therapy to reverse acquired
antihormonal resistance in the treatment of breast cancer. Clin Breast Cancer 8(2):124-133.
Takahashi A, Higashino F, Aoyagi M, Yoshida K, Itoh M, Kobayashi M, Totsuka Y,
Kohgo T, Shindoh M. 2005. E1AF degradation by a ubiquitin-proteasome pathway. Biochem Biophys Res Commun 327(2):575-580.
Thepot D, Weitzman JB, Barra J, Segretain D, Stinnakre MG, Babinet C, Yaniv M. 2000.
Targeted disruption of the murine junD gene results in multiple defects in male reproductive function. Development 127(1):143-153.
Tkach V, Tulchinsky E, Lukanidin E, Vinson C, Bock E, Berezin V. 2003. Role of the
Fos family members, c-Fos, Fra-1 and Fra-2, in the regulation of cell motility. Oncogene 22(32):5045-5054.
Trimble MS, Xin JH, Guy CT, Muller WJ, Hassell JA. 1993. PEA3 is overexpressed in
mouse metastatic mammary adenocarcinomas. Oncogene 8(11):3037-3042. Ueyama K, Ikeda K, Sato W, Nakasato N, Horie-Inoue K, Takeda S, Inoue S. 2010.
Knockdown of Efp by DNA-modified small interfering RNA inhibits breast cancer cell proliferation and in vivo tumor growth. Cancer Gene Ther 17(9):624-632.
160 Usary J, Llaca V, Karaca G, Presswala S, Karaca M, He X, Langerod A, Karesen R, Oh
DS, Dressler LG, Lonning PE, Strausberg RL, Chanock S, Borresen-Dale AL, Perou CM. 2004. Mutation of GATA3 in human breast tumors. Oncogene 23(46):7669-7678.
Uyttendaele H, Marazzi G, Wu G, Yan Q, Sassoon D, Kitajewski J. 1996. Notch4/int-3, a
mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development 122(7):2251-2259.
van de Rijn M, Perou CM, Tibshirani R, Haas P, Kallioniemi O, Kononen J, Torhorst J,
Sauter G, Zuber M, Kochli OR, Mross F, Dieterich H, Seitz R, Ross D, Botstein D, Brown P. 2002. Expression of cytokeratins 17 and 5 identifies a group of breast carcinomas with poor clinical outcome. Am J Pathol 161(6):1991-1996.
van Es JH, van Gijn ME, Riccio O, van den Born M, Vooijs M, Begthel H, Cozijnsen M,
Robine S, Winton DJ, Radtke F, Clevers H. 2005. Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 435(7044):959-963.
Veronesi U, Cascinelli N, Mariani L, Greco M, Saccozzi R, Luini A, Aguilar M,
Marubini E. 2002. Twenty-year follow-up of a randomized study comparing breast-conserving surgery with radical mastectomy for early breast cancer. N Engl J Med 347(16):1227-1232.
Vleugel MM, Greijer AE, Bos R, van der Wall E, van Diest PJ. 2006. c-Jun activation is
associated with proliferation and angiogenesis in invasive breast cancer. Hum Pathol 37(6):668-674.
Wang W, Struhl G. 2005. Distinct roles for Mind bomb, Neuralized and Epsin in
mediating DSL endocytosis and signaling in Drosophila. Development 132(12):2883-2894.
Wasylyk B, Hagman J, Gutierrez-Hartmann A. 1998. Ets transcription factors: nuclear
effectors of the Ras-MAP-kinase signaling pathway. Trends Biochem Sci 23(6):213-216.
Weigelt B, Kreike B, Reis-Filho JS. 2009. Metaplastic breast carcinomas are basal-like
breast cancers: a genomic profiling analysis. Breast Cancer Res Treat 117(2):273-280.
Weijzen S, Rizzo P, Braid M, Vaishnav R, Jonkheer SM, Zlobin A, Osborne BA,
Gottipati S, Aster JC, Hahn WC, Rudolf M, Siziopikou K, Kast WM, Miele L. 2002. Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells. Nat Med 8(9):979-986.
161 Weitzman JB, Fiette L, Matsuo K, Yaniv M. 2000. JunD protects cells from p53-
dependent senescence and apoptosis. Mol Cell 6(5):1109-1119. Weng AP, Millholland JM, Yashiro-Ohtani Y, Arcangeli ML, Lau A, Wai C, Del Bianco
C, Rodriguez CG, Sai H, Tobias J, Li Y, Wolfe MS, Shachaf C, Felsher D, Blacklow SC, Pear WS, Aster JC. 2006. c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma. Genes Dev 20(15):2096-2109.
Wisdom R, Johnson RS, Moore C. 1999. c-Jun regulates cell cycle progression and
apoptosis by distinct mechanisms. Embo J 18(1):188-197. Wu G, Lyapina S, Das I, Li J, Gurney M, Pauley A, Chui I, Deshaies RJ, Kitajewski J.
2001. SEL-10 is an inhibitor of notch signaling that targets notch for ubiquitin-mediated protein degradation. Mol Cell Biol 21(21):7403-7415.
Wu J, Bresnick EH. 2007. Glucocorticoid and growth factor synergism requirement for
Notch4 chromatin domain activation. Mol Cell Biol 27(6):2411-2422. Wu J, Iwata F, Grass JA, Osborne CS, Elnitski L, Fraser P, Ohneda O, Yamamoto M,
Bresnick EH. 2005. Molecular determinants of NOTCH4 transcription in vascular endothelium. Mol Cell Biol 25(4):1458-1474.
Wu L, Aster JC, Blacklow SC, Lake R, Artavanis-Tsakonas S, Griffin JD. 2000.
MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors. Nat Genet 26(4):484-489.
Xia WY, Lien HC, Wang SC, Pan Y, Sahin A, Kuo YH, Chang KJ, Zhou X, Wang H, Yu
Z, Hortobagyi G, Shi DR, Hung MC. 2006. Expression of PEA3 and lack of correlation between PEA3 and HER-2/neu expression in breast cancer. Breast Cancer Res Treat 98(3):295-301.
Xin JH, Cowie A, Lachance P, Hassell JA. 1992. Molecular cloning and characterization
of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells. Genes Dev 6(3):481-496.
Xing X, Wang SC, Xia W, Zou Y, Shao R, Kwong KY, Yu Z, Zhang S, Miller S, Huang
L, Hung MC. 2000. The ets protein PEA3 suppresses HER-2/neu overexpression and inhibits tumorigenesis. Nat Med 6(2):189-195.
Yamaguchi N, Oyama T, Ito E, Satoh H, Azuma S, Hayashi M, Shimizu K, Honma R,
Yanagisawa Y, Nishikawa A, Kawamura M, Imai J, Ohwada S, Tatsuta K, Inoue J, Semba K, Watanabe S. 2008. NOTCH3 signaling pathway plays crucial roles in
162 the proliferation of ErbB2-negative human breast cancer cells. Cancer Res 68(6):1881-1888.
Zajchowski DA, Bartholdi MF, Gong Y, Webster L, Liu HL, Munishkin A, Beauheim C,
Harvey S, Ethier SP, Johnson PH. 2001. Identification of gene expression profiles that predict the aggressive behavior of breast cancer cells. Cancer Res 61(13):5168-5178.
Zanetti-Dallenbach R, Vuaroqueaux V, Wight E, Labuhn M, Singer G, Urban P,
Eppenberger U, Holzgreve W, Eppenberger-Castori S. 2006. Comparison of gene expression profiles in core biopsies and corresponding surgical breast cancer samples. Breast Cancer Res 8(4):R51.
Zavadil J, Cermak L, Soto-Nieves N, Bottinger EP. 2004. Integration of TGF-beta/Smad
and Jagged1/Notch signalling in epithelial-to-mesenchymal transition. Embo J 23(5):1155-1165.
Zhang SM, Hankinson SE, Hunter DJ, Giovannucci EL, Colditz GA, Willett WC. 2005.
Folate intake and risk of breast cancer characterized by hormone receptor status. Cancer Epidemiol Biomarkers Prev 14(8):2004-2008.
Zhao C, Gao H, Liu Y, Papoutsi Z, Jaffrey S, Gustafsson JA, Dahlman-Wright K. 2010.
Genome-wide mapping of estrogen receptor-beta-binding regions reveals extensive cross-talk with transcription factor activator protein-1. Cancer Res 70(12):5174-5183.
Zhao R, Bodnar MS, Spector DL. 2009. Nuclear neighborhoods and gene expression.
Curr Opin Genet Dev 19(2):172-179.
VITA
The author, Anthony G. Clementz, was born in Aurora, IL on July 14th, 1983. He
is the last child of five children of Peter and Toni Clementz. He has four amazing
siblings all sharing interest in the sciences: Gina, a doctor of dermatology; Peter, an
electrician; Mia, a science teacher; and Andria, a registered nurse.
Anthony graduated as a Cadet First Lieutenant at Marmion Military Academy in
2001. He graduated from Northern Illinois University with high recognition with his
bachelors in Chemistry and Biochemistry in 2005. Anthony received numerous awards
while in school including the American Chemical Society Award in Analytical Chemistry,
Scholastic Excellence Award, Golden Key International Honor Society, National Society
of Collegiate Scholars, CRC Press Frosh Chemistry Achievement Award, and the
National Deans List.
After graduation, he worked for Flavors of North America (FONA) as a flavor scientist
and technician. Anthony entered the doctoral program in the Department of Cell Biology,
Neurobiology, and Anatomy in the Division of Molecular and Cellular Biochemistry at
Loyola University Medical Center in August 2006. He joined Dr. Clodia Osipo’s
laboratory in June 2007 where he began his work on novel crosstalk between Notch and
ErBB2 signaling and transcriptional regulation of Notch receptors. While at Loyola
163
164
University he has been a graduate school council representative for the Division of
Molecular and Cellular Biochemistry, and has participated in assisting students with
disabilities. Anthony has taught lectures on Hormone Signaling in both graduate student
courses: Molecular Oncogenesis and Signal Transduction. He also was an active
facilitator for the first year medical students in their course: Cell Molecular Biology and
Genetics. Anthony is an active member as an Associate Member of American
Association for Cancer Research and an American Chemical Society affiliate. He has
also been an active judge for the Chicago Public Schools Science Fairs. Anthony’s
research presented here has been submitted and is under review for a novel patent with
Merck Pharmaceuticals.
Anthony G. Clementz also has a musical artistic side for he is an accomplished
pianist and singer having played and sung various musicals and events throughout the
Chicagoland area. He is also a graduate of iO!, formly Improv Olympic!, and Chicago’s
Second City and actively performs improv and sketch comedy.
Anthony G. Clementz has recently accepted a position as adjunct professor in the
Department of Chemistry at DePaul University. Also, following completion of his
dissertation, he will continue his career in transcription regulation as a post-doctoral
researcher at Northwestern University Children’s Memorial Center in the laboratory of
Dr. Ann Harris, professor and director of the Human Molecular Genetics Program. He
plans to investigate the “transcription hubs” of the CFTR gene known to be responsible
for the development of Cystic Fibrosis. He also plans to use his knowledge of
165
oncogenesis to elucidate the role of Collagen XV as a tumor suppressor in pancreatic
cancer both in vitro and in vivo.
DISSERTATION APPROVAL SHEET
The dissertation submitted by Anthony G. Clementz has been read and approved by the
following committee:
Clodia Osipo, Ph.D., Assistant Professor of Pathology Loyola University Chicago Mary Druse-Manteuffel, Ph.D., Professor of Cell Biology, Neurobiology, and Anatomy Division of Molecular and Cellular Biochemistry Loyola University Chicago Richard Schultz, Ph.D., Professor of Cell Biology, Neurobiology, and Anatomy Division of Molecular and Cellular Biochemistry Loyola University Chicago Maurizio Bocchetta, Ph.D., Associate Professor of Pathology Loyola University Chicago Paola Rizzo, Ph.D., Assistant Professor of Pathology Loyola University Chicago
The final copies have been examined by the director of the dissertation and the signature
which appears below verifies the fact that any necessary changes have been incorporated
and that the dissertation is now given final approval by the committee with reference to
content and form.
The dissertation is therefore accepted in partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
_______________________ ____________________________________
Date Director’s Signature
