Upload
others
View
4
Download
0
Embed Size (px)
Citation preview
I
M 2015
ROLE OF mTOR PATHWAY IN DIFFERENTIATED
THYROID CANCER
MARIA JOÃO DE GUSMAN GERALDES CORREIA COELHO DISSERTAÇÃO DE MESTRADO APRESENTADA AO INSTITUTO DE CIÊNCIAS BIOMÉDICAS ABEL SALAZAR DA UNIVERSIDADE DO PORTO EM ONCOLOGIA
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
II
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
III
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
IV
MARIA JOÃO DE GUSMAN GERALDES CORREIA COELHO ROLE OF mTOR PATHWAY IN DIFFERENTIATED THYROID CANCER
Dissertação de Candidatura ao grau de Mestre
em Oncologia – Especialização em Oncologia
Molecular submetida ao Instituto de Ciências
Biomédicas Abel Salazar da Universidade do
Porto
Orientadora – Professora Doutora Ana Paula
Soares Dias Ferreira
Categoria – Professora Auxiliar na Faculdade de
Medicina da Universidade do Porto,
Coordenadora do Grupo Cancer Biology no
Instituto de Investigação e Inovação em
Saúde/Instituto de Patologia e Imunologia
Molecular da Universidade do Porto
Afiliações – Faculdade de Medicina da
Universidade do Porto, Instituto de Investigação e
Inovação em Saúde/Instituto de Patologia e
Imunologia Molecular da Universidade do Porto
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
V
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
VI
AGRADECIMENTOS Em primeiro lugar, gostaria de agradecer ao Professor Sobrinho-Simões,
na qualidade de director do Instituto de Patologia e Imunologia Molecular da
Universidade do Porto, por ter aberto as suas portas para me receber num local
de referência para a investigação em oncologia. À Professora Doutora Paula Soares
pela sua orientação. Não podia também deixar de agradecer à Catarina Tavares por todo o apoio e
ensinamentos que me transmitiu durante este último ano. E finalmente, ao grupo Cancer
Biology, pela amabilidade de todos e prontidão em ajudar.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
VII
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
VIII
INDEX
RESUMO XI
ABSTRACT XIV
List of Figures XVII
List of Tables XX
Abbreviations XXIV
INTRODUCTION 3
THE THYROID GLAND 3
1.1 Anatomical Features 3
1.2 Thyroid Physiology 4
1.3 The Sodium/iodide Symporter (NIS) 7
THE BASICS OF THYROID CANCER 9
2.1 Epidemiology 9
2.2 Disease Presentation and Management 11
2.3 Genetic Alterations 12
SIGNALING PATHWAYS IN THYROID CANCER 16
3.1 MAPK Signaling Pathway 16
3.2 PI3K/AkT/mTOR Signaling Pathway 17
3.3 Interaction between the PI3K/Akt/mTOR and MAPK Pathways 21
3.4 Impaired Iodide Metabolism in Cancer 22
AIMS OF THE STUDY 26
METHODOLOGY 30
MATERIAL 30
1.1 Samples 30
1.2 Antibodies 31
METHODS 31
1.1 Sample Preparation 31
1.2 Hematoxylin-Eosin Staining 31
1.3 Immunohistochemistry 32
1.4 Classification of the Immunohistochemical staining 33
1.5 Statistical Analysis 33
RESULTS 37
SERIES 1 37
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
IX
1.1 Distribution of Pathological and Molecular Characteristics 37
1.2 Association of p-S6 and p-mTOR with Pathological and Molecular Characteristics
in PTC 41
1.3 Cellular localization of staining 45
SERIES 2 45
2.1 Distribution of Clinico-pathological and Molecular Characteristics 45
2.2 Association of p-S6 and p-mTOR with Clinico-pathological and Molecular
Characteristics 52
2.3 Cellular localization of staining 57
DISCUSSION 61
mTOR PATHWAY ACTIVATION 61
mTOR PATHWAY AND GENETIC ALTERATIONS 63
mTOR PATHWAY AND TREATMENT FOR THYROID CANCER 63
CONCLUSIONS 68
FUTURE PERSPECTIVES 68
BIBLIOGRAPHIC REFERENCES 70
SUPPLEMENTARY TABLES 80
SUPPLEMENTARY FIGURES 86
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
X
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XI
RESUMO
As mutações activadoras nos genes BRAF e rat sarcoma viral oncogene (RAS) e
o rearranjo rearranged during transfection (RET/PTC) são responsáveis pela activação da
via mitogen-activated protein kinase (MAPK) nas células tumorais da tireoide. Esta
activação constitutiva da via de sinalização conduz a processos de proliferação e
resistência à apoptose celulares, assim como perda de expressão de genes da tireoide,
incluindo o NIS, cuja função da proteína é indispensável para o funcionamento da
tireoide. Por outro lado, a activação da via phosphoinositide 3-kinase/protein kinase
B/mammalian target of rapamycin (PI3K/Akt/mTOR) também tem um papel importante na
transformação das células. Mutações nos seus efectores, juntamente com mutações na
via MAPK, são responsáveis pela activação constitutiva desta via.
A molécula mTOR, uma cinase serina/treonina, é activada pela via
PI3K/Akt/mTOR. É constituída por dois complexos, o complexo 1 (mTORC1) e o 2
(mTORC2). O mTORC1 participa na biogénese dos ribossomas por activação da
ribosomal protein S6 (S6) e na tradução do ácido ribonucleico mensageiro (mRNA). Por
sua vez, o mTORC2 participa na organização do citoesqueleto por activação da protein
kinase Cα (PKCα), assim como na migração e sobrevivência celulares por activação da
Akt na serina 473.
Aproximadamente 10-15% dos carcinomas papilares da tireoide (PTC) perdem
expressão do NIS, tornando-se indetectáveis no diagnóstico e resistentes ao tratamento
com 131I, agravando significativamente o seu prognóstico. Estes casos merecem uma
atenção especial, uma vez que requerem terapias alternativas. Um dos alvos nestas
terapias é a via mTOR.
O objectivo geral deste trabalho foi investigar a activação da via mTOR nos PTC.
Para tal, estudámos a activação da via mTOR em duas séries, uma delas com 73 e a
outra com 114 PTC, e avaliámos a expressão de dois marcadores moleculares da via –
mTOR fosforilado (p-mTOR) na serina 2448 e S6 fosforilado (p-S6) na serina 235/236, o
seu efector imediato. Para além disso, investigámos também possíveis associações entre
esses marcadores e características moleculares e clinico-patológicas.
Os nossos resultados mostraram que a expressão de p-mTOR tinha uma
associação significativa com a extensão extra-tireoideia e metástases à distância, assim
como com a persistência de doença no final do follow-up nas variantes clássicas de PTC
(cPTC). Este marcador estava também preferencialmente localizado na periferia e na
frente invasiva dos tumores. Por sua vez, a expressão de p-S6 revelou ser inconsistente
entre as duas séries, uma vez que mostrou estar associada a factores de bom
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XII
prognóstico numa das séries e à invasão vascular na outra. Adicionalmente, não
encontrámos nenhuma correlação entre a expressão dos dois marcadores.
Estes resultados sugerem que o p-mTOR é um marcador mais promissor nos
tumores da tireoide. Confirmámos ainda a activação da via mTOR em PTC, e propomos
que essa activação ocorra por via do mTORC2, dado as associações com características
de agressividade e a inexistência de correlação entre a expressão de p-mTOR e p-S6.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XIII
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XIV
ABSTRACT
Gain-of-function mutations in BRAF and rat sarcoma viral oncogene (RAS) and
rearranged during transfection fusion gene (RET/PTC) rearrangement are responsible for
activation of the mitogen-activated protein kinase (MAPK) signaling pathway in thyroid
cancer cells. Constitutive activation of this pathway leads to uncontrolled cell proliferation
and resistance to apoptosis, as well as loss of thyroid-differentiated gene expression,
including the gene coding for NIS, which is indispensable for thyroid function. On the other
hand, activation of the phosphoinositide 3-kinase/protein kinase B/mammalian target of
rapamycin (PI3K/Akt/mTOR) pathway also plays a major role in transformation of cells.
Mutations in its effectors, along with mutations in the MAPK cascade, are responsible for
constitutive activation of the pathway.
mTOR, a serine/threonine kinase, is activated by the PI3K/Akt/mTOR pathway. It
comprises mTOR complex 1 (C1), which participates in ribosomal biogenesis via
activation of ribosomal protein S6 (S6), and messenger ribonucleic acid (mRNA)
translation; and mTOR complex 2 (C2), which participates in cytoskeleton organization via
activation of protein kinase Cα (PKCα), and cell migration and survival via activation of
Akt on serine 473.
Approximately 10-15% of papillary thyroid carcinoma (PTC) lose expression of NIS
and become undetectable to diagnosis and resistant to treatment with 131I, which worsens
their prognosis significantly. These cases require special attention because alternative
treatment options are required. One of the targets in such cases is the mTOR pathway.
The general aim of this work was to investigate the mTOR pathway activation in
PTC. In order to do that, we studied the mTOR pathway activation in two series, one with
73 and the other with 114 PTC, and evaluated the immune-expression of two molecular
markers of the mTOR pathway – phosphorylated mTOR (p-mTOR) on serine 2448 and
phosphorylated S6 (p-S6) on serine 235/236, its immediate effector. Furthermore, we
investigated possible associations between these markers and clinico-pathological and
molecular features.
Our results showed p-mTOR expression to be significantly associated with
extrathyroid extension and distant metastases, as well as persistence of disease after
follow-up in the classical variant of PTC (cPTC). p-mTOR was also preferentially localized
in the periphery of tumors and in their invasive front. In turn, p-S6 expression was not
consistent between series, being associated with factors of good prognosis in one and
with vascular invasion in the other. Furthermore, we did not find any correlation between
p-mTOR and p-S6 expression.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XV
These results suggest that p-mTOR is a more promising marker in thyroid tumors.
Our results confirmed the activation of mTOR pathway in PTC, and suggest its activation
to occur via mTORC2, given the associations with clearly aggressive features and the lack
of correlation between expression of p-mTOR and p-S6.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XVI
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XVII
List of Figures
Figure 1 – Macro- and microscopic structure of the thyroid gland. Adapted from Boron et
al. (2012) Section VIII: The endocrine system, in Medical Physiology: A cellular and
molecular approach.
Figure 2 – Hypothalamic-pituitary-thyroid axis. Adapted from Hill et al. (1998) Risk
assessment of thyroid follicular cell tumors, Environ Health Persp.
Figure 3 – Synthesis of thyroid hormones. Adapted from Cohen-Lehman et al. (2010)
Effects of amiodarone therapy on thyroid function, Nat Rev Endocrinol.
Figure 4 – Signaling pathways involved in NIS regulation. Adapted from Riesco-
Eizaguirre et al. (2006) A perspective view of sodium iodide symporter research and its
clinical implications, Eur J Endocrinol.
Figure 5 – Genetic events of thyroid cancer and the effect on the MAPK signaling
pathway. Adapted from Nikiforov et al. (2011) Molecular genetics and diagnosis of thyroid
cancer, Nat Rev Endocrinol.
Figure 6 – MAPK signaling pathway activation. Adapted from Oikonomou et al. (2014)
BRAF vs RAS oncogenes: are mutations of the same pathway equal? Differential
signaling and therapeutic implications, Oncotarget.
Figure 7 – PI3K/Akt/mTOR signaling pathway activation. Adapted from Oikonomou et al.
(2014) BRAF vs RAS oncogenes: are mutations of the same pathway equal? Differential
signaling and therapeutic implications, Oncotarget.
Figure 8 - The mTOR protein. Adapted from Cargnello et al. (2015) The expanding role of
mTOR in cancer cell growth and proliferation, Mutagenesis.
Figure 9 – PI3K/Akt/mTOR signaling pathway. Adapted from Populo et al. (2012) The
mTOR pathway signaling in cancer, Int J Mol Sci.
Figure 10 - Diagram of the interactions between the MAPK and PI3K/Akt/mTOR
pathways. Adapted from De Luca et al. (2012) The RAS/RAF/MEK/ERK and the
PI3K/Akt signaling pathways: role in cancer pathogenesis and implications for therapeutic
approaches, Expert Opin Ther Targets.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XVIII
Supplementary Data
Figure 1 - Positive controls. The tissue used as a positive control is a lymph node
invaded by cancer cells. We can see in a brown coloration the cells stained for the
marker and in blue the lymphocytes (200x magnification). A – p-S6 ; B – p-mTOR.
Figure 2 - Representative case of the excluded cases from Series 2. This case is a cPTC
with cancer cells surrounding the follicles and lymphocytes infiltrating the tissue (200x
magnification).
Figure 3 – Staining of p-S6. p-S6 shows a preferential cytoplasmic localization (brown
coloration) in tumor cells. A – cPTC from Series 2; B – fvPTC from Series 1 (200x
magnification).
Figure 4 – Representative image of p-mTOR staining. p-mTOR was localized both in
cytoplasm and membrane of tumor cells (brown coloration). A – Warthin like PTC; B –
cPTC (200x magnification).
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XIX
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XX
List of Tables Table 1 – Physiological effects of thyroid hormones in a context of hypo- and
hyperthyroidism. Adapted from Boron et al. (2012) Section VIII: The endocrine system, in
Medical Physiology: A cellular and molecular approach.
Table 2 – Diagnosis of the tumors present in Series 1 (n=90)
Table 3 – Pathological and molecular characteristics of the whole series (n=90), of PTC
(n=73) and FTC (n=13) in Series 1
Table 4 – Diagnosis of cases of papillary thyroid carcinoma (n=73) in Series 1
Table 5 – Pathological and molecular characteristics of cPTC (n=39) and fvPTC (n=23) in
Series 1
Table 6 – Distribution of p-S6 score in PTC (n=73) in Series 1
Table 7 – Associations between p-S6 score in PTC and pathological and molecular
characteristics (n=73) in Series 1
Table 8 – Distribution of p-mTOR score in PTC (n=73) in Series 1
Table 9 – Associations between p-mTOR score and pathological characteristics in PTC
(n=73) in Series 1
Table 10 – Distribution of p-S6 score in cPTC (n=39) and fvPTC (n=23) in Series 1
Table 11 – Distribution of p-mTOR score in cPTC (n=39) and fvPTC (n=23) in Series 1
Table 12 – Associations between p-mTOR score and pathological characteristics in cPTC
(n=39) in Series 1
Table 13 – Associations between p-S6 score and pathological characteristics in fvPTC
(n=23) in Series 1
Table 14 – Distribution of the tumors present in Series 2 (n=127)
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XXI
Table 15 – Clinico-pathological and molecular characteristics of the whole series (n=127),
PTC (n=114) and FTC (n=6) in Series 2
Table 16 – Distribution of PTC histotypes (n=114) in Series 2
Table 17 – Clinico-pathological and molecular characteristics in cPTC (n=77) and fvPTC
(n=28) in Series 2
Table 18 – Distribution of p-S6 score in PTC (n=76) in Series 2
Table 19 – Associations between p-S6 score and pathological characteristics in PTC
(n=76) in Series 2
Table 20 – Distribution of p-mTOR score in PTC (n=107) in Series 2
Table 21 – Associations between p-mTOR score and clinico-pathological characteristics in
PTC (n=107) in Series 2
Table 22 – Distribution of p-S6 scores in cPTC (n=51) and fvPTC (n=18) in Series 2
Table 23 – Distribution of p-mTOR score in cPTC (n=73) and fvPTC (n=25) in Series 2
Table 24 – Associations between p-S6 and pathological characteristics in cPTC (n=51) in
Series 2
Table 25 – Associations between p-S6 and pathological characteristics in fvPTC (n=18) in
Series 2
Table 26 – Associations between p-mTOR and clinico-pathological and molecular
characteristics in cPTC (n=73) in Series 2
Table 27 – Associations between p-mTOR score and pathological data in fvPTC (n=25) in
Series 2
Supplementary Tables
Table 1 – Cases with missing information within the whole series (n=90), in PTC (n=73)
and in FTC (n=13) in Series 1
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XXII
Table 2 – Cases with missing information within cPTC (n=39) and fvPTC (n=23) in Series
1
Table 3 – Distribution of p-S6 scores in each variant of PTC (n=73) in Series 1
Table 4 – Distribution of p-mTOR scores in each variant of PTC (n=73) in Series 1
Table 5 – Cases with missing information within the whole series (n=127), in PTC (n=114)
and in FTC (n=6) in Series 2
Table 6 – Cases with missing information within cPTC (n=77) and in fvPTC (n=28) in
Series 2
Table 7 – Distribution of p-S6 scores in each variant of PTC (n=) in Series 2
Table 8 – Distribution of p-mTOR scores in each variant of PTC (n=) in Series 2
Table 9 – Correlation between p-S6 and p-mTOR in Series 1
Table 10 – Correlation between p-S6 and p-mTOR in Series 2
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XXIII
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XXIV
Abbreviations
131I Radioiodine
A Adenine
Akt Protein kinase B
ATC Anaplastic thyroid carcinoma
ATP Adenosine triphosphate
ATPase Adenosine triphosphatase
C Cytosine
Ca2+ Calcium ion
cAMP Cyclic adenine monophosphatase
CNS Central nervous system
cPTC Classical variant of papillary thyroid carcinoma
CT Computed tomography
Deptor DEP domain-containing mTOR-interacting protein
DIT Diiodotyrosine
DNA Deoxyribonucleic acid
DTC Differentiated thyroid carcinoma
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
eIF4E/4EBP1 Eukaryotic translation initiation factor 4E-binding protein 1
eIF4E/4A/4G Eukaryotic translation initiation factor 4E/ 4A/4G
ERK Extracellular signal-regulated kinases
ETS E26 transformation-specific
FFPE Formalin-fixed paraffin-embedded
FKBP12 FK506 binding protein
FTA Follicular thyroid adenoma
FTC Follicular thyroid carcinoma
fvPTC Follicular variant of papillary thyroid carcinoma
G Guanine
GDP Guanosine diphosphate
GRB2 Growth factor receptor-bound protein 2
GTP Guanosine triphosphate
GTPase Guanosine triphosphatase
HIF-1α Hypoxia-inducible factor 1α
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XXV
HPT Hypothalamic-pituitary-thyroid axis
HRAS Harvey rat sarcoma viral oncogene homolog
Hsp70 Heat shock protein 70
I- Iodide
I0 Iodine
IFN-γ Interferon γ
IGF-1 Insulin-like growth factor 1
IL-1α Interleukin 1α
K+ Potassium ion
KRAS Kirsten rat sarcoma viral oncogene homolog
MAPK Mitogen-activated protein kinase
MEK Mitogen/Extracellular signal-regulated kinase
MIT Monoiodotyrosine
mLST8 Mammalian lethal with sec13 protein 8
MMP Matrix metalloproteinase
MRI Magnetic resonance imaging
mRNA Messenger ribonucleic acid
mSin1 Mammalian stress-activated protein kinase interacting protein 1
MTC Medullary thyroid carcinoma
mTOR Mammalian target of rapamycin
mTORC1 Mammalian target of rapamycin complex 1
mTORC2 Mammalian target of rapamycin complex 2
Na+ Sodium ion
NF-κB Nuclear factor κB
NIS Sodium/iodide symporter
NRAS Neuroblastoma rat sarcoma viral oncogene homolog
O2 Oxygen
oPTC Oncocytic variant of papillary thyroid carcinoma
p-Akt Phosphorylated protein kinase B
p-mTOR Phosphorylated mammalian target of rapamycin
p-S6 Phosphorylated S6
PAX8/PPARγ Paired box gene 8/peroxisome proliferator-activated receptorγ
fusion gene/protein
PDGF Platelet-derived growth factor
PDK1 Phosphoinositide-dependent kinase 1
PDTC Poorly differentiated thyroid carcinoma
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XXVI
PdtIns Phosphatidylinositol
PI3K Phosphoinositide 3-kinase
PIKK Phosphatidylinositol 3-kinase-related kinase
PIP2 Phosphatidylinositol 4,5-biphosphate
PIP3 Phosphatidylinositol 3,4,5-triphosphate
PKA Protein kinase A
PKCα Protein kinase C-α
PRAS40 Proline-rich Akt substrate of 40kDa
Protor Protein observed with rictor
PTC Papillary thyroid carcinoma
PTEN Phosphatase and tensin homologue deleted on chromosome ten
RAF Rapidly accelerated fibrosarcoma gene/protein
Raptor Regulatory-associated protein of mTOR
RAS Rat sarcoma viral oncogene
REDD1 Protein regulated in development and DNA damage response 1
RET/PTC Rearranged during transfection fusion gene/protein
rhTSH Recombinant human thyroid-stimulating hormone
Rictor Rapamycin-insensitive companion of mTOR
RORENO Registo Oncológico Regional do Norte
RSK p90 ribosomal S6 kinase
rT3 Reverse triiodothyronine
RTK Receptor tyrosine kinase
S6 Ribosomal protein S6
S6K p70 ribosomal S6 kinase
Ser Serine
SOS Son of sevenless
sPTC Sclerosing variant of papillary thyroid carcinoma
stPTC Solid trabecular variant of papillary thyroid carcinoma
T Thymine
T3 Triiodothyronine
T4 Thyroxine
TBG Thyroxine-binding globulin
TC Thyroid cancer
tcPTC Tall cell variant of papillary thyroid carcinoma
TERT Telomerase reverse transcriptase
Tg Thyroglobulin
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XXVII
TGF-β1 Transforming growth factor β1
Thr Threonine
TMA Tissue microarray
TNF-α Tumor necrosis factor α
TPO Thyroid peroxidase
TRH Thyrotropin-releasing hormone
TSC1/TSC2 Tuberous sclerosis protein 1/2
TSH Thyroid-stimulating hormone
TSHR Thyroid-stimulating hormone receptor
TTR Transthyretin
VEGF Vascular endothelial growth factor
wPTC Warthin-like papillary thyroid carcinoma
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
XXVIII
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
1
INTRODUCTION
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
2
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
3
INTRODUCTION
THE THYROID GLAND
1.1 Anatomical Features
The thyroid gland (Figure 1-A), situated in the lower anterior neck in front of the
trachea, is composed of right and left lobes, connected in the middle by an isthmus and
encapsulated in a thin fibrous connective tissue [1]. It has an extensive lymphatic system
network, which comprises regional lymph nodes that belong to levels I to VII (anterior
triangle, upper jugular, middle jugular, lower jugular, posterior triangle, central
compartment and upper mediastinal, respectively) [2].
The basic constitutive unit of this endocrine gland is the follicle. A single layer of
polarized follicular epithelial cells outlines each of these follicles and colloid material is
present in the lumen of the follicles. Normal follicles range from 50 to 500µm in size
(200µm average). The follicular cells can vary in shape, from flat to columnar, depending
on their degree of hormone synthesis activity, with flat follicular cells being less active than
the columnar cells. Their nuclei are round and basally located, with diffuse chromatin. In
the basal membrane of the follicular cells are the molecules which transport iodide (I-) into
the follicular cells (Figure 1-B). These molecules are called the sodium/iodide symporter
(NIS), and they uptake iodide in exchange for sodium (Na+), against a concentration and
an electrical gradient, therefore using energy in the form of adenosine triphosphate (ATP)
[1, 2].
Figure 1 – Macro- and microscopic structure of the thyroid gland. A – The gland is located in the lower anterior neck across the front of the trachea. A single layer of follicle cells outlines each of these basic units, and colloid is present in their lumen. In the interfollicle space there can be seen blood vessels and a couple of C cells grouped together [4]. B – the sodium/iodide symporter is located in the basal membrane of the follicular cells, and is responsible for the uptake of iodide into the follicular cells. Adapted from Boron et al. (2012) Section VIII: The endocrine system, in Medical Physiology: A cellular and molecular approach.
A B
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
4
Figure 2 – Hypothalamic-pituitary-thyroid axis. The levels of circulating thyroid hormones T4 and T3.are maintained by a negative feedback loop. The hypothalamus responds to low circulating levels of T4 and T3 by releasing TRH, which in turn stimulates the production of TSH by the anterior pituitary gland. TSH then stimulates the thyroid gland to synthesize T4 and T3. Adapted from Hill et al. (1998) Risk assessment of thyroid follicular cell tumors, Environ Health Persp.
Also present in the thyroid parenchyma are C cells, or parafollicular cells, but they
are not part of the follicle. They are present in small numbers, between or peripheral to
follicular cells. C cells produce calcitonin, a hormone involved in calcium (Ca2+) and
phosphate homeostasis [2].
1.2 Thyroid Physiology
The thyroid is an endocrine gland, and its role in the organism is to produce
hormones. The thyroid hormones include thyroxine (T4) and triiodothyronine (T3), whose
precursors are constitutive part of the hormone thyroglobulin (Tg), a macromolecule
present in the colloid structure in the follicle [3]. Hormone synthesis is very complex,
involving not only the thyroid gland for the production of hormones, but also the central
nervous system (CNS), which regulates their synthesis, and T4-T3 peripheral conversion.
As a matter of fact, the thyroid is part of the hypothalamic-pituitary-thyroid (HPT) axis
(Figure 2), which is responsible for the regulation of circulating levels of T4 and T3 by
classical negative feedback – if T4 and T3 are circulating in low levels, the hypothalamus
reacts by releasing thyrotropin-releasing hormone (TRH), which in turn stimulates the
anterior pituitary gland to secrete thyroid-stimulating hormone (TSH); by interaction with
thyroid-stimulating hormone receptor (TSHR), TSH will then stimulate the thyroid gland to
synthesize T4 and T3, thus replenishing their levels in the circulation [4]. The TSH activity
stimulates the cyclic adenine monophosphatase (cAMP) cascade, which is responsible for
transcriptional and post-transcriptional regulation of Tg and thyroid peroxidase (TPO) [5].
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
5
Thyroid hormones are synthesized only if iodide uptake occurs (Figure 3). Until
then, their precursors remain inactive while bound to Tg in the colloid. The synthesis of
thyroid hormones can be divided into 6 stages: (1) iodide trapping, (2) iodination, (3)
conjugation, (4) endocytosis and proteolysis, and (5) secretion [6]. TSH stimulates each of
these steps, except for the exocytosis of iodide to the colloid. If TSH is present in high
concentrations, the rate at which hormone synthesis and release occurs is much faster
than when TSH is in low concentrations.
(1) Iodide trapping – in this first stage, the anterior pituitary gland secretes TSH, which
will stimulate the activity of NIS (basolateral membrane of the follicular cells), thus
increasing the concentration of iodide in the follicular cells. Iodide uptake occurs
against a concentration and an electrical gradient, and its transport is coupled with
sodium transport, therefore requiring energy and oxidative metabolism. At this point,
the follicular cells secrete Tg into the lumen;
(2) Iodination – iodide diffuses to the apical surface of the follicular cells, and leaves
them presumably via pendrin, in an exocytosis vesicle formed in the follicular cells,
entering the colloid of the follicle. Inside this vesicle, iodide is oxidized to iodine (I0)
Figure 3 – Synthesis of thyroid hormones. TSH stimulates the synthesis of thyroid hormones, and its concentration affects the rates at which synthesis and release of the hormones occurs. T4 and T3 synthesis only begins when I- is co-transported into the follicular cells by NIS, via active transport. Once inside the follicular lumen, I- is oxidized to I0 by TPO in a process called iodination. I0 then binds to tyrosine residues in Tg, producing DIT and MIT. The conjunction of two DIT and a MIT with a DIT yields T4 and T3, respectively. At this stage, they are still bound to Tg, but ready to be transported from the colloid to the follicular cells. Once in the follicular cells, Tg is hydrolyzed, resulting in the release of both hormones into the blood circulation. The hormones become ready to be transported to target cells. Adapted from Cohen-Lehman et al. (2010) Effects of amiodarone therapy on thyroid function, Nat Rev Endocrinol.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
6
by TPO. I0 then binds to tyrosine residues of Tg, resulting in the formation of
diiodotyrosine (DIT) and monoiodotyrosine (MIT);
(3) Conjugation – the conjugation of iodinated tyrosine residues (DIT and MIT) results in
the formation of T4 and T3 linked to Tg. T4 is the result of conjugation between two
DIT molecules, and T3 is formed by a DIT molecule conjugated with a MIT
molecule;
(4) Endocytosis – for T4 and T3 to be able to enter circulation, iodinated Tg is
incorporated by endocytosis in the follicular cells in the form of colloid droplets.
These will fuse with lysosomes, so that Tg is hydrolyzed to active T4 and T3;
(5) Secretion – finally, the free hormones are secreted into the blood circulation, where
they bind to carrier proteins for transport to target cells.
Approximately 90% of thyroid hormones are released as T4 and the remaining 10%
as T3. Of the released T4, roughly 80% is converted by deiodinases to T3 in the liver and
kidney (peripheral conversion), and the rest to reverse triiodothyronine (rT3). T3 is the
more active form, being 10 times more active than T4, whereas rT3 has no significant
biological function [1].
Once they reach the circulation, T4 and T3 tightly bind to plasma proteins, such as
thyroxine-binding globulin (TBG), albumin and transthyretin (TTR), and circulate mostly
bound to proteins. As soon as they are freed from these proteins, they carry out their
actions on the target tissues [3]. There, they are transported inside the cells either by
membrane proteins or by diffusion and head towards the nucleus, where they bind to
nuclear receptors, which in turn bind to chromatin regulating the expression of specific
genes, either by inhibition or stimulation of transcription [7].
Thyroid hormones play a role in the homeostasis and metabolism of the organism
as a whole, including the cardiovascular system, respiratory system, central nervous
system, gut, muscle, skin/hair, skeleton, kidneys, bone marrow, gonads. In infants, thyroid
hormones are especially important in brain and somatic development, whereas in adults
they are essential for the metabolic activity. Either excess (hyperthyroidism) or deficiency
(hypothyroidism) of thyroid hormones has a critical impact. Iodine deficiency, which results
in thyroid hormone deficiency, is compensated by secretion of TSH, increasing the iodide
trapping, and therefore the concentration of thyroid hormones. Contrarily, excess iodide
generally results in inhibitory effects in the secretion of TSH (Table 1) [3, 5].
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
7
Table 1 – Physiological effects of thyroid hormones in a context of hypo- and hyperthyroidism.
Adapted from Boron et al. (2012) Section VIII: The endocrine system, in Medical Physiology: A cellular and
molecular approach.
1.3 The Sodium/iodide Symporter (NIS)
NIS is an integral plasma membrane glycoprotein that co-transports two Na+ along
with one iodide, a mechanism that relies on the electrochemical gradient provided by the
Na+/potassium (K+)-adenosine triphosphatase (ATPase) pump [8]. Besides thyroid
follicular cells, low levels of NIS expression can be detected in salivary glands, stomach,
small intestine and lactating mammary glands, and its regulation is tissue-specific [8].
As mentioned previously, thyroid hormone production depends on the active
transport of iodide into the follicular cells. This is a process carried out by NIS, thus
making it a fundamental structure in the thyroid gland. Although adequate NIS function is
important, the supply of iodide is just as essential for the synthesis of T4 and T3 [9]. When
thyroid function is normal, the iodide trapping mechanism is responsible for the
accumulation of iodide, which is 30 to 60-fold more concentrated in the follicular cells than
in the plasma [10].
NIS expression and function regulation (Figure 4) are carried out by several
molecules and pathways, at the transcriptional and post-transcriptional levels [11]. The
Parameter Low level of thyroid hormones (Hypothyroid)
High level of thyroid hormones (Hyperthyroid)
Basal metabolic rate ↓ ↑
Carbohydrate metabolism
↓ Gluconeogenesis
↓ Glucogenolysis
Normal serum [glucose]
↑ Gluconeogenesis
↑ Glucogenolysis
Normal serum [glucose]
Protein metabolism ↓ Synthesis
↓ Proteolysis
↑ Synthesis
↑ Proteolysis
Muscle wasting
Lipid metabolism
↓ Lipogenesis
↓ Lipolysis
↑ Serum [cholesterol]
↑ Lipogenesis
↑ Lipolysis
↓ Serum [cholesterol]
Thermogenesis ↓ ↑
Autonomic nervous system
Normal levels of serum catecholamines
↑ Expression of β adrenoreceptors (increased
sensitivity to catecholamines, which remain at
normal levels)
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
8
primary hormonal regulator is TSH, which exerts its function via stimulation of TSHR,
activating the TSH-TSHR-cAMP-protein kinase A (PKA) signaling pathway [12, 13]. The
TSH-TSHR-cAMP-PKA signaling pathway has long been studied as a regulatory pathway
for thyroid differentiation and proliferation both PKA-dependent and –independent [14].
Overall, TSH is a positive regulator, as it increases iodide uptake and accumulation into
the follicular cells, which is mediated by NIS, in a protein synthesis-dependent manner
[15]. This mechanism, which was shown in both in vitro and in vivo models, demonstrated
that increased iodide transport into thyroid cells was a result of positive regulation of NIS
[16, 17]. Post-transcriptionally, NIS activity was shown to be modulated by modifications
at the protein level, namely the phosphorylation patterns, induced by TSH, which are
distinct from those in the absence of TSH [18], suggesting an involvement in NIS
subcellular distribution. Besides this hormonal control, iodide itself is considered to be one
of the main regulators of NIS function, dubbing as a homeostatic regulator of thyroid
hormone synthesis. In 1948, Wolff and Chaikoff [19] demonstrated in rats that high doses
of iodide caused a decrease in thyroid function. They stated that if a critically high level of
organic iodide in the plasma was reached, an inhibitory effect in the organic binding of
iodide would be observed; this mechanism is known as the acute Wolff-Chaikoff effect.
Furthermore, they showed that when plasma organic iodide went down to a normal basal
level, the effect would be reversed, and iodide organification restored. This phenomenon
can be explained in light of an intrinsic autoregulatory mechanism, in which the thyroid
protects itself from high doses of iodide while at the same time providing enough iodide for
hormone synthesis.
On the contrary, cytokines and growth factors have been shown to exert a
negative regulation on NIS, via activation of phosphoinositide 3-kinase/protein kinase
B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway [14, 20]. This
pathway seems to counterbalance the activity of the TSH-TSHR-cAMP-PKA pathway, by
downregulating NIS expression. Tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and
interleukin-1α (IL-1α) have been implicated in both the inhibition of NIS messenger
ribonucleic acid (mRNA) expression and TSH-induced NIS expression in FRTL-5 cells
[20-22], whereas insulin growth factor-1 (IGF-1) was associated only with the former [20].
Similarly, transforming growth factor-β1 (TGF-β1) showed a decrease in TSH-induced NIS
expression and TSH-induced iodide uptake [22], in addition to reducing Na+/K+-ATPase
pump activity [23]. Moreover, Tg was shown to suppress NIS mRNA levels and of some
thyroid-specific genes, suggesting that Tg acts as a negative feedback regulator,
counteracting TSH [24]. This hypothesis was corroborated by a study from the same
authors that showed that Tg significantly suppressed TSH-induced NIS promoter activity,
NIS protein and NIS-dependent iodide uptake into the follicular cells [25].
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
9
THE BASICS OF THYROID CANCER
2.1 Epidemiology
Thyroid cancer (TC) is the most common of endocrine malignancies and accounts
for 1% of all cancers, having an incidence higher in women than in men, with a 3:1 ratio
[26]. Overall, the incidence has been gradually increasing at a rate of about 6% [27].
Mortality rates have remained more or less stable over the years, with a reported 0.5
cases per 100 000 individuals, although a slight increase has been noted, predominantly
in men (+1,6% annual percent change in men versus +0,9% annual percent change in
women) [28, 29]. If we do an analysis by histological type, papillary thyroid cancer (PTC)
is the most common (70-80%), followed by follicular thyroid cancer (FTC) (10-15%) and
then undifferentiated thyroid cancers (<5%) [30].The 10-year survival rate has increased
and is now at about 93% in PTC and 85% in FTC. In undifferentiated anaplastic thyroid
carcinoma (ATC) the 10-year survival rate is approximately 14% [31].
In Portugal, the same trends can be observed. In the North region, where this work
took place, the Registo Oncológico Regional do Norte (RORENO) has revealed the
Figure 4 – Signaling pathways involved in NIS regulation. Upregulation of NIS is carried out by TSH via TSH-TSHR-
cAMP-PKA pathway, which can be dependent or independent of PKA. On the other hand, downregulation of NIS expression
occurs via PI3K/AKT/mTOR signaling pathway and by molecules such as TNF-α, IFN-γ, IL-1α, IGF-1 and TGF-β1. Adapted
from Riesco-Eizaguirre et al. (2006) A perspective view of sodium iodide symporter research and its clinical implications, Eur
J Endocrinol.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
10
incidence of thyroid cancer to be 21.2 cases per 100 000 individuals, and the second
most common cancer in women (34.5 cases per 100 000 individuals, which represents
11.8% of all cancers) [32]. Overall mortality rate is at 0.9 per 100 000 individuals, with a
slight increase in women [33]. The 5-year survival rate is at 97.9%, and lower in men [34].
All of this data is in accordance to the worldwide incidence and mortality rates.
Over the years, the normal evolution of cancers dictates an increase in incidence,
mainly because of the growing population and extended life expectancy, as well as tumor
etiology itself, and thyroid cancer is no exception. However, it should be noted that this
increase is happening in PTC, given that it is the main histologic subtype, while the other
types of thyroid cancer have not been substantially affected [27]. Nowadays,
overdiagnosis also accounts for this trend, which does not reflect a real increase in the
disease, since mortality rates are not necessarily accompanying this tendency [28].
O’Grady et al. [35] performed a retrospective study in the United States comprised of
individuals who were diagnosed between 1981 and 2011, and estimated that
overdiagnosis resulted in 82 000 cases of PTC, that most likely would not have resulted in
clinical symptoms, in the year of 2011. The authors estimated that the percentage of
diagnosed cases attributable to overdiagnosis in men aged 20-49 and +50 years, were
4% and 42%, respectively, and in women aged 29-40 and 50+ years, 47% and 61%,
respectively. Similarly, Korean researchers have documented a 15-fold increase in thyroid
cancer in the past two decades, which they concluded was due to overdiagnosis [36]. A
similar tendency has been observed throughout Europe [27], including Portugal, in which
an estimate of an incidence rate of 28.6 per 100 000 individuals should be met in 2020
[37].
The primary cause for this problem is the improvement in medical imaging
detection by technologies such as magnetic resonance imaging (MRI), computed
tomography (CT), ultrasonography and nuclear medicine. This means that smaller tumors
(<1cm) with irrelevant clinical significance are being diagnosed, resulting in overtreatment
and putting patients at risk for complications from the invasive procedures, otherwise
avoidable [38, 39].
There are several risk factors associated with the development of thyroid cancer.
The most well-studied cause is exposure to ionizing radiation, particularly during
childhood. Studies have concluded that there is a significant dose-response risk
association for exposure and that radiation-induced thyroid cancer has an inverse relation
with age at exposure and a direct relation with background iodine deficiency [40]. Genetic
susceptibility is also associated with the risk of developing thyroid cancer. A few studies
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
11
have revealed an increased relative risk when thyroid carcinoma was diagnosed in direct
relatives of TC patients, especially in first-degree relatives [41, 42]. Benign thyroid
conditions, such as goiter, benign nodules/ adenomas and hyperthyroidism, have been
shown to be the most important factor as a risk to develop TC [43]. This relationship is
stronger in men, although these conditions are more common in women [44]. Hormonal
and reproductive factors are weakly associated with the risk of developing thyroid cancer,
but deserve attention since women are 3 times more likely than men to develop thyroid
cancer [45]. Among dietary risk factors, iodine is by far the most important, considering
thyroid function depends on it. Iodine deficiency is associated with FTC, whereas iodine
sufficiency is associated with PTC [46]. Overall, as a result of iodine deficiency, TSH
secretion is increased, suggesting this overstimulation by TSH as the mechanism behind
the association [47]. More controversially, one study reported bigger height and higher
body mass index to have an influence by increasing the risk of differentiated thyroid
carcinoma (DTC) [48], whereas a different study found no associations [49]. These factors
may suggest a potential influence of growth factors and hormones during development of
the body.
2.2 Disease Presentation and Management
Thyroid carcinomas can arise from thyroid follicular cells, which represent the
majority of cancers, or from parafollicular cells. The former originate PTC, FTC, poorly
differentiated thyroid carcinoma (PDTC) and ATC, and the latter originates medullary
thyroid carcinoma (MTC) [50]. PDTC and ATC can develop de novo or arise from a
process of dedifferentiation of pre-existing PTC and FTC [51].
Thyroid tumors usually present with a single nodule, and diagnosis is made mainly
by cytological examination of fine needle aspirate [52]. These tumors are generally slow
growing, although they can metastasize in the regional lymph nodes, but also in the lungs,
bone, and occasionally the brain, via hematogenous dissemination. In general, primary
treatment consists of total thyroidectomy for most cases of DTC, although tumors less
than 1 centimeter (cm) in diameter can be treated by unilateral lobectomy [53]. The
decision to perform a total thyroidectomy is made based on 3 rationales: 60-85% of PTC
cases present foci in both lobes [54], 5-10% of PTC cases recur after unilateral surgery in
the contralateral lobe [55], and effectiveness of radioiodine ablative treatment is higher
when resection is total, therefore increasing the sensitivity and specificity of serum Tg as a
tumor marker [53]. Given the particularity of the thyroid gland, it is possible to take
advantage of the iodide uptake to aid in a therapy setting. Therefore, postoperative care
consists of adjuvant ablative therapy with radioiodine (131I), which is chemically identical to
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
12
the non-radioactive form, to eliminate any thyroid remnants, and radioiodine treatment for
residual disease and metastases. When the follicular cells uptake 131I and it concentrates
in the cells, the radionuclide undergoes β decay, resulting in a high-energy release, which
is responsible for the cytotoxic effects. γ rays are also released and used for surveillance
scanning procedures, given that they are detectable. As usual, there are exceptions:
tumors less than 1 cm are not given radioiodine ablation, unless evidence of extrathyroid
extension or metastases exists [53]. Before radioiodine ablation, it is important that the
serum levels of TSH are sufficiently elevated to ensure that NIS expression is increased to
optimize 131I uptake. For that purpose, withdrawal of thyroid hormone or use of
recombinant human TSH (rhTSH) is advised to optimize 131I uptake before patients begin
therapy [56].
Follow-up monitoring consists of whole body scanning with radioiodine every 6-12
months after initial ablation, and more frequently if there is an indication of recurrent
disease. Locoregional metastases can be identified by ultrasonography, as well as
recurring tumors measuring several millimeters in diameter [57]. Metastatic disease
should be confirmed with a fine needle aspiration-assisted ultrasonography [58].
Ultrasonography should also be used in the routine monitoring of patients with
extrathyroid extension and locoregional nodal metastases. Serum Tg levels measurement
is also recommended to aid in the detection of recurrent and metastatic disease [59].
2.3 Genetic Alterations
Treatment of DTC is standardized, but tumor response is not. This variability
comes from tumor heterogeneity, such as histological type, stage, presence of
metastases, molecular and genetic alterations, and ability to trap iodine, as well as patient
age and gender. All of these factors contribute to the aggressiveness of the tumor, and
consequently response to therapy. Knowing the genetic and molecular mechanisms
through which these tumors develop has brought insight into how treatment can be
modeled to each case. In this subchapter, genetic alterations in thyroid cancer will be
discussed to outline the importance they have on the clinical course of the disease.
Similar to other cancers, thyroid cancer emerges from an accumulation of genetic
mutations, which also dictate how the tumor will progress. In the case of thyroid cancer,
there are critical genes that frequently undergo mutation, and thus enable characterization
of the tumor (Figure 5).
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
13
BRAF Mutations
The BRAF gene encodes for BRAF protein, a member of the rapidly accelerated
fibrosarcoma (RAF) protein family, which are intracellular elements of the mitogen-
activated protein kinase (MAPK) pathway. In this cascade, rat sarcoma viral oncogene
(RAS) binds and activates BRAF, which results in phosphorylation and subsequent
activation of mitogen/extracellular signal-regulated kinase (MEK), followed by activation of
extracellular signal-regulated kinases (ERK) [60]. The BRAF protein is important in cell
proliferation, differentiation and apoptosis.
The most frequent mutation that occurs in this gene is the substitution of valine to
glutamate at codon 600 (BRAF V600E), which is an activating point mutation. This
mutation leads to constitutive activation of BRAF, resulting in induction of the MAPK
signaling pathway [61]. BRAF V600E occurs in approximately 45% of PTC, being more
common in the classic (45%) and tall cell variants (80-100%), and less in the follicular
variant (15%) [51].
BRAF V600E is associated with an overall poor clinical prognosis, including
presence of extrathyroid extension and lymph node metastases, as well as a high rate of
tumor recurrence [62] due to treatment failure because of a deficient NIS targeting to the
Figure 5 – Genetic events of thyroid cancer and the effect on the MAPK signaling pathway. Mutations of RAS, BRAF,
TERT promoter and the rearrangements RET/PTC and PAX8/PPARγ are all genetic events that occur in the pathogenesis of
thyroid cancer. RAS and BRAF mutations and RET/PTC rearrangement will result in activation of the MAPK signaling
pathway, ultimately leading to transcription of genes involved in tumor cell differentiation, proliferation and survival.
Furthermore, RAS mutation and RET/PTC rearrangement also lead to activation of the PI3K/Akt/mTOR signaling pathway
(not pictured). TERT promoter mutation and the PAX8/PPARγ rearrangement do not participate in the activation of this
cascade, but exert their functions through other mechanisms. The PAX8/PPARγ rearrangement directly inhibits PPARγ
protein, located in the nucleus. Adapted from Nikiforov et al. (2011) Molecular genetics and diagnosis of thyroid cancer, Nat
Rev Endocrinol.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
14
membrane [63]. Evidence suggests this mutation to be an early event in tumorigenesis
[60].
RAS Mutations
The RAS genes, which comprise neuroblastoma rat sarcoma viral oncogene
homolog (NRAS), Harvey rat sarcoma viral oncogene homolog (HRAS) and Kirsten rat
sarcoma viral oncogene homolog (KRAS), encode G-proteins located at the inner surface
of the cell membrane, and are responsible for intracellular signal transduction. In a normal
setting, RAS is bound to guanosine diphosphate (GDP) in its inactive state. Once it
becomes active, GDP is released and RAS binds to guanosine triphosphate (GTP),
activating the MAPK and PI3K/Akt/mTOR signaling pathways. This active state in which
the complex RAS-GTP is formed is transient, though, and RAS quickly becomes inactive
again due to its guanosine triphosphatase (GTPase) activity and cytoplasmic GTPase-
activating proteins [64].
When a point mutation occurs in a discrete domain, it can have one of two
outcomes – increased affinity for GTP, due to mutations in codons 12 and 13 (KRAS), or
inactivation of the intrinsic GTPase, due to mutations in codon 61 (NRAS). All of these
mutations switch on the active state of the gene permanently, and consequently activate
constitutively the downstream signaling pathways.
RAS mutations are more frequent in FTC (30-45%), whereas in PTC they are
rather infrequent (10%) and are almost always present in the follicular variant of PTC
(fvPTC) (30-45%) [60]. They are associated with more aggressive behavior in FTC (tumor
dedifferentiation and less favorable prognosis) [65], but correlated with more frequent
encapsulation, less prominent nuclear features and low rate of lymph node metastases in
PTC [66]. However, RAS mutations are not specific for malignant lesions, as they also
occur in follicular thyroid adenomas (FTA) [67].
TERT Promoter Mutations
Telomerase is responsible for maintaining the genomic integrity of cells. The
catalytic subunit responsible for this is called the telomerase reverse transcriptase
(TERT). The TERT gene encodes this protein that, together with an integral RNA
component, is responsible for telomere elongation [68]. In normal cells, TERT is
repressed in somatic cells, and in a context of cellular transformation this protein has an
increased expression, due to mutations in the promoter region of the gene.
TERT promoter mutations occur in two hotspot positions – the -124 and -146
upstream from the ATG start site. In the former, there is a guanine (G) to adenine (A)
substitution, and in the latter there can be a substitution of a G to A or cytosine (C) to
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
15
thymine (T) [69]. These mutations create a sequence that harbors a consensus binding
site (GGAA) for E26 transformation-specific (ETS) transcription factors, which lead to an
increase in expression of TERT [70].
TERT promoter mutations are present only in malignant thyroid lesions. In
differentiated tumors, the majority is in FTC (17.1%) followed by PTC (7.5%). The lesions
with the highest percentage of these mutations are PDTC (29%) and ATC (33.3%) [71].
The -124 G>A mutation is the most common of the two. In DTC, the mutations are
associated with distant metastases, worse response to treatment and poor outcome,
including decreased survival [71].
RET/PTC Rearrangement
Rearranged during transfection (RET) is a proto-oncogene that encodes a tyrosine
kinase cell membrane receptor that transduces signals for cell growth and differentiation.
In follicular thyroid cells, this gene can be activated by a chromosomal rearrangement with
a partner gene, which results in a RET/PTC fusion gene [60].
The most frequent types of rearrangement are the RET/PTC1 (60-70%) and
RET/PTC3 (20-30%), but all of the fusion genes have the intact tyrosine kinase domain of
RET receptor. The rearrangement enables the chimeric protein to activate the MAPK
cascade, promoting and contributing to tumorigenesis [64]. More recent evidence with
FRTL-5 cell line points to the activation of the PI3K/Akt/mTOR signaling pathway as well
[72].
The rearrangement is associated with younger age and lymph node metastases in
PTC [66]. Our group has also reported a significant association between RET/PTC
rearrangements and FTC [73].
PAX8/PPARγ Rearrangement
The paired box 8 (PAX8) is a gene that belongs to the PAX family genes and
codes for the thyroid-specific paired domain transcription factor, which means that it is
involved in expression of thyroid-specific genes [64]. The gene is a target for
rearrangement with the peroxisome proliferator-activated receptor γ (PPARγ), which
encodes nuclear receptors, resulting in the PAX8/PPARγ fusion gene. The rearrangement
is a result of the chromosomal translocation t(2;3)(q13;p25) [74].
The mechanism by which the fusion gene leads to cell transformation is not
established, but it is known that PAX8/PPARγ leads to a strong overexpression of the
chimeric protein, inhibiting the tumor suppressor activity of PPARγ [51].
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
16
Figure 6 – MAPK signaling pathway activation. Activation of this pathway occurs through binding of molecules, such as growth factors, to their receptors in the cytoplasmic membrane. This activates RAS, initiating the signaling cascade that culminates with activation by phosphorylation of ERK1/2. This protein complex is responsible for activating genes in the nucleus that are involved in differentiation, proliferation and survival. Adapted from Oikonomou et al. (2014) BRAF vs RAS oncogenes: are mutations of the same pathway equal? Differential signaling and therapeutic implications, Oncotarget.
The frequency of the rearrangement is approximately 30-35% for FTC and lower in
Hürthle cell carcinomas, and fvPTC has a frequency of 1-5% [75]. Evidence indicates that
this rearrangement is associated with younger age, vascular invasion and smaller tumors
[76].
SIGNALING PATHWAYS IN THYROID CANCER
3.1 MAPK Signaling Pathway
The activation of MAPK cascade (Figure 6) begins when growth factors, hormones
or chemokines bind to their respective tyrosine kinase receptors (RTK), like the epidermal
growth factor receptor (EGFR), on the cell membrane. Upon binding, the extracellular
domain of the RTK dimerizes and induces kinase activity in the cytoplasmic domain via
phosphorylation of tyrosine residues. The receptor is now able to provide docking sites for
proteins, such as the growth factor receptor-bound protein 2 (GRB2). The binding of
GRB2 induces recruitment of an effector protein, which can be the guanine-nucleotide
exchange factor son of sevenless (SOS), responsible for the nucleotide exchange of GDP
to GTP. This exchange occurs while GDP/GTP is bound to RAS, in the cytoplasm. From
here on, RAS is activated, giving rise to a series of activation of other effector proteins
(RAF, MEK, ERK), culminating in activation genes encoding for transcription factors
associated with cell differentiation, proliferation and survival [77].
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
17
The gain-of-function mutations in BRAF and RAS and RET/PTC rearrangement
are responsible for activation of the MAPK signaling pathway in cancer cells. Constitutive
activation of this pathway leads to uncontrolled cell proliferation and resistance to
apoptosis (or cell survival), contributors to transformation and progression of cancer, as
well as loss of thyroid-differentiated gene expression, including NIS, TPO and Tg, via
methylation of their promoter [78, 79]. The tumorigenic expression of this cascade is
amplified by secondary molecular alterations such as genome-wide hypermethylation and
hypomethylation [80]. The activation of MAPK pathway ultimately leads to upregulation of
several oncogenic genes encoding for proteins such as chemokines [81], vascular
endothelial growth factor (VEGF) [82], nuclear factor-κB (NF-κB) [83], matrix
metalloproteinases (MMPs) [84], vimentin [85], hypoxia-inducible factor-1α (HIF-1α) [86]
and TGF-β1 [87], which are all known to play a role in tumorigenesis by inducing tumor
cell proliferation, growth, migration and survival and tumor angiogenesis, invasion and
metastasis.
3.2 PI3K/AkT/mTOR Signaling Pathway
PI3Ks are a family of lipid kinases that phosphorylate the 3’-hydroxyl group of
phosphatidylinositol (PtdIns) on the plasma membrane. There are different isoforms that
act in several physiological processes such as inflammation, metabolism, migration and
survival, as well as cancer progression, and can be differentiated by their structure, lipid
substrate specificity and action mechanism [88].
Class I PI3Ks are the best characterized and most important in cancer. It has two
subunits that are activated by RTKs upon binding – p85 and p110. Activation of this
pathway occurs in the same way as activation of MAPK pathway – binding of growth
factors or chemokines to their RTKs. This binding is responsible for the activation of RAS,
which regulates the activation of PI3K, much like it does in the MAPK pathway to BRAF.
The p110 subunit of PI3K is also activated, and phosphorylates phosphatidylinositol-4,5-
biphosphate (PIP2) to produce phosphatidylinositol-3,4,5-triphosphate (PIP3). At this point,
PIP3 can translocate Akt to the cell membrane, where it becomes active by
phosphorylation. Akt activates downstream effectors, among which is mTOR, in order to
target molecules that regulate cell functions [89]. A schematic representation of pathway
is pictured in Figure 7.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
18
Figure 7 – PI3K/Akt/mTOR signaling pathway activation. Activation of PI3K/Akt/mTOR pathway occurs by phosphorylation of RAS in the MAPK pathway. This causes activation of effectors PIP2 and 3, which in turn activates Akt. Akt activates its downstream effectors, including mTOR, in order to target genes in the nucleus responsible for ribosomal biogenesis and mRNA translation, as well as cytoskeleton organization. PTEN is the negative regulator of this cascade, and it exerts its function at the PIP2 level. Adapted from Oikonomou et al. (2014) BRAF vs RAS oncogenes: are mutations of the same pathway equal? Differential signaling and therapeutic implications, Oncotarget.
The protein phosphatase and tensin homolog deleted on chromosome ten (PTEN)
is also involved in this cascade by exerting a negative regulation through
dephosphorylation of PIP3, therefore terminating the signal of the cascade [90].
Genetic Events Contributing to PI3K/Akt/mTOR Pathway Activation
Genetic alterations in genes coding for the proteins involved in the PI3K/Akt/mTOR
pathway have serious implications in the development of cancer, and in conjunction with
BRAF mutations, the alterations in the pathway effectors have been shown to increase the
aggressiveness of tumors [91]. Wu et al. suggested that amplification of a gene coding for
a catalytic subunit of PI3K, largely present in differentiated thyroid cancer tissue and in
thyroid cancer cell lines, could be an activating mechanism of the PI3K/Akt/mTOR
signaling pathway in thyroid cancer [92]. Furthermore, in a study using thyroid cancer cells
lines and tissues, it was shown that levels of phosphorylated total Akt were increased in
FTC [93]. Alterations in the genes Akt1, 2 and 3 have been reported in different cancers
[77, 94]. Mutation of Akt1 has been identified in breast, colorectal and ovarian carcinomas,
and amplification of the gene in prostatic carcinomas. Akt2 mutations were reported in
breast carcinomas and its amplification in pancreatic, ovarian and breast carcinomas.
Akt3 mutation has been reported in melanomas and the amplification in breast
carcinomas. The mutations in these genes cause constitutive activation of its protein
downstream effectors, which results in evasion of apoptosis and transformation of cells
contributing to tumor invasion and metastasis [95].
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
19
Gain-of-function mutations in RAS and loss-of-function mutations in PTEN are also
contributors to the activation of PI3K/Akt/mTOR pathway in thyroid cancer. PTEN is
mutated in hereditary (Cowden Syndrome) and sporadic thyroid cancers [96] and its
mutations are most likely associated with constitutive activation of Akt [95].
mTOR: The Protein and the Pathway
mTOR is a serine/threonine kinase that belongs to the PI3K-related protein kinase
(PIKK) family [97], and the gene is located on chromosome 1, 1p36.2 [98]. In eukaryotic
cells, mTOR comprises two complexes – mTOR complex 1 (mTORC1) and mTOR
complex 2 (mTORC2). mTORC1 is rapamycin-sensitive, and consists of mTOR,
regulatory-associated protein of mTOR (raptor), mammalian lethal with sec13 protein 8
(mLST8) and the negative regulators proline-rich Akt substrate of 40kDa (PRAS40) and
DEP domain-containing mTOR-interacting protein (deptor); its activity is regulated by
raptor. On the other hand, mTORC2 is rapamycin-insensitive, and consists of mTOR,
rapamycin-insensitive companion of mTOR (rictor), mLST8, mammalian stress-activated
protein kinase interacting protein 1 (mSin1), protein observed with rictor (protor), heat
shock protein 70 (Hsp70) and the negative regulator deptor; this complex is regulated by
rictor [99] (Figure 8).
mTORC1 is activated by the PI3K/Akt/mTOR pathway and is inhibited by the
tuberous sclerosis protein 1/2 complex (TSC1/TSC2). It is responsible for the regulation of
ribosomal biogenesis by phosphorylation and activation of p70 ribosomal S6 kinase
(S6K), which then activates ribosomal protein S6 (S6). Its other role is to regulate protein
synthesis by phosphorylation and inhibition of eukaryotic translation initiation factor 4E-
binding protein 1 (eIF4E/4EBP1), which causes its release to bind to other proteins in
order to initiate mRNA translation [100]. mTORC2 is regulated by rictor. Its activation
depends on the presence of growth factors, and once active, it phosphorylates Akt on
serine (Ser) 473, via Sin1, while phosphoinositide-dependent kinase 1 (PDK1)
Figure 8 – The mTOR protein. mTOR is part of two complexes – complex 1 (mTORC1) consists of its main regulator raptor and three other molecules, mLST8 and the negative regulators deptor and PRAS40. This complex is responsible for regulating ribosomal biogenesis and mRNA translation; the complex 2 (mTORC2) is comprised of its main regulator rictor and the molecules mLST8, mSin1, Hsp70 protor, as well as the negative regulator deptor. mTORC2 is responsible for regulating cytoskeleton organization as well as cellular metabolism. Adapted from Cargnello et al. (2015) The expanding role of mTOR in cancer cell growth and proliferation, Mutagenesis.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
20
Figure 9 – PI3K/Akt/mTOR signaling pathway. Growth factors, amino acids and ATP and O2 levels, which are responsible for starting this cascade, will activate PI3K (also activated by RAS in the MAPK pathway). In turn, Akt is phosphorylated by mTORC2, disrupting the TSC1/TSC2 complex, and activates mTORC1. This complex phosphorylates and activates S6K1 and phosphorylates and inhibits the complex eIF4E/4EBP1. S6K1 phosphorylates S6, which is involved in ribosome biogenesis. On the other hand, eIF4E is released from its complex, and binds to other molecules such as eIF4A and eIF4A, in order to initiate mRNA translation. A number of other cellular processes are regulated by this pathway – protein synthesis, cellular growth, proliferation and survival. Adapted from Populo et al. (2012) The mTOR pathway signaling in cancer, Int J Mol Sci.
phosphorylates it on Threonine (Thr) 308. Akt is involved in cell survival and migration.
Protein kinase Cα (PKCα) is also phosphorylated by mTORC2 to regulate activity of small
GTPases in order to act on cytoskeleton organization [101]. Sin1 is also responsible for
targeting mTORC2 to the membrane.
This pathway (Figure 9) is activated and regulated by several growth factors, such
as epidermal growth factor (EGF), IGF-1, platelet-derived growth factor (PDGF) and
VEGF, amino acids and ATP and oxygen (O2) levels, as well as the MAPK pathway [102].
Signaling through mTOR modulates several downstream pathways that regulate cell-cycle
progression, translation initiation, transcriptional stress responses, protein stability and
survival of cells [103]. While we know that mTORC1 has several regulators, regulation of
its counterpart mTORC2 is largely unknown.
One of the hormones that regulates mTORC1 is insulin. It is responsible for the
activation of class I PI3Ks and the downstream effector Akt, which when active reverses
the inhibitory effect that TSC1/TSC2 has on the cascade [104]. Amino acids present in
nutrients also exert a positive regulation on mTORC1 by inducing S6K and 4E-BP1
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
21
phosphorylation [105]. ATP is equally important – under ATP deprivation, mTORC1 in
inhibited by TSC2 in order to prevent cell apoptosis; conversely, under high levels of
energy, Akt is phosphorylated and activates mTORC1 [106]. Oxygen deprivation is
another negative regulator of mTORC1 – in response to energy stress, the hypoxia-
inducible gene regulated in development and deoxyribonucleic acid (DNA) damage
responses 1 (REDD1) is induced, therefore inhibiting mTORC1 and cell growth [107].
Additionally, MAPK pathway can also activate mTORC1, and it does so by activating
ERK1/2, which results in dissociation of the TSC1/TSC2 complex, and thus activation of
mTORC1 [108].
3.3 Interaction between the PI3K/Akt/mTOR and MAPK Pathways
Between PI3K/Akt/mTOR and MAPK pathways there is an interplay in which a
downstream member of one pathway is directly involved in the activation/inhibition of an
upstream member of the other (Figure 10). This means that both pathways can activate or
inhibit each other.
Figure 10 – Diagram of the interactions between the MAPK and PI3K/Akt/mTOR pathways. The first interaction occurring between these pathways is via RAS and PI3K, which results in activation of the PI3K pathway. At the TSC1/TSC2 level in the PI3K/Akt/mTOR pathway, ERK1/2 can inactivate that complex, enabling the progression of the PI3K/Akt/mTOR cascade. Raptor can also be activated by both ERK1/2 and RSK, which also dissociate the TSC1/TSC2 complex. Adapted from De Luca et al. (2012) The RAS/RAF/MEK/ERK and the PI3K/Akt signaling pathways: role in cancer pathogenesis and implications for therapeutic approaches, Expert Opin Ther Targets.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
22
This crosstalk seems to be important to promote and maintain growth and survival
of transformed cells, although it is dependent of context. At the very beginning of the
cascade, there is an important interaction, which happens between RAS and PI3K.
Activation of RAS can have a positive effect in the other pathway, seeing that RAS is also
responsible for activating PI3K, and thus initiating this signaling cascade. Besides, the
MAPK pathway can also control the PI3K/Akt/mTOR pathway at the TSC1/TSC2 level, by
inactivating the complex via ERK1/2 activity. This results in dissociation of the complex,
which we already know enables the progression of PI3K/Akt/mTOR pathway [108]. Lastly,
raptor can also be activated by both ERK1/2 and p90 ribosomal kinase (RSK) (which also
dissociates the TSC1/TSC2 complex) [109, 110].
Although there are plenty of interactions between pathways, they mostly occur in
the early stages of the PI3K/Akt/mTOR pathway and go down until S6K. This means that
S6 can only be activated by mTOR (via S6K).
3.4 Impaired Iodide Metabolism in Cancer
The ability of thyroid cells to uptake iodide from the bloodstream through NIS
makes possible an efficient targeted therapy in thyroid cancer. In this context, 131I is
administrated systemically and captured by thyroid cells, causing their death.
Unfortunately, 10-15% of PTC lose NIS expression/functionality, becoming undetectable
to diagnosis and resistant to 131I treatment, which worsens significantly their prognosis [9].
The molecular mechanisms underlying NIS underexpression are not fully understood.
However, it is well established that tumors harboring BRAFV600E mutation have loss of NIS
expression [8]. It is also described that mTOR pathway activation impairs NIS expression
[111] and that treatment of thyroid cancer cell lines with rapamycin is able to restore NIS
expression and 131I uptake [112].
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
23
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
24
AIMS OF THE STUDY
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
25
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
26
AIMS OF THE STUDY
The general aim of this work was to investigate the mTOR pathway activation in
PTC.
In order to do that, we evaluated the immune-expression of two molecular markers
of mTOR pathway – phosphorylated mTOR and phosphorylated S6, its immediate
effector. Furthermore, we investigated possible associations between those markers and
clinico-pathological and molecular features.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
27
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
28
METHODOLOGY
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
29
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
30
METHODOLOGY
MATERIAL
1.1 Samples
Tumor samples were obtained from Centro Hospitalar de São João (CHSJ) and
Centro Hospitalar e Universitário de Coimbra (CHUC), comprising two different series,
with approval from the ethic committee of both institutions. CHSJ series consists of
consecutive cases and CHUC series of cases selected according to outcomes. From now
on, CHSJ series will be designated as Series 1 and CHUC as Series 2.
The samples were stored at Instituto de Patologia e Imunologia da Universidade
do Porto (Ipatimup) as formalin-fixed paraffin-embedded (FFPE) tissue samples.
Diagnosis and clinicopathological data were obtained from CHSJ and CHUC medical
records of each patient. Clinicopathological data was reviewed before any statistical
analysis was performed and an experienced certified pathologist reviewed the diagnosis
of each case before the beginning of this work. Series 1 is comprised of 90 patients and
Series 2 of 127 patients, each case with one sample.
The clinico-pathological variables studied, both series combined, were gender,
age, lymph node metastases (yes/no), distant metastases (yes/no), tumor size (<2cm,
≥2cm), tumor capsule (yes/no), tumor capsule invasion (yes/no), vascular invasion
(yes/no), tumor margins (well defined/infiltrative), thyroid capsule invasion (yes/no),
extrathyroid extension (yes/no), multifocality (single/multiple), lymphocytic infiltrate
(yes/no), staging (I, II, III, IVa and IVc), BRAF mutation (V600E), NRAS mutation (Q61R,
Q16R and Q61K), TERT promoter mutation (-124G>A and -146G>A), RET/PTC
rearrangement (RET/PTC1 and RET/PTC3), PAX/8PPARγ rearrangement, one year
disease-free survival (yes/no), number of 131I therapies (<2, ≥2), cumulative dose of 131I
therapies (<150mCi, ≥150mCi), number of additional therapies (<2, ≥2), type of additional
therapies (<2, ≥2; these include additional 131I therapy, surgery, radiotherapy and tyrosine
kinase inhibitors), disease-free survival after follow-up (yes/no), evidence of disease
during follow-up (yes/no), recurrence of disease (yes/no) and mortality (yes/no).
Frequency and percentage of yes/no variables and of genetic alterations are present in
the tables only for yes and mutated or rearranged, respectively.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
31
1.2 Antibodies
Antibodies for phosphorylated mTOR (p-mTOR) and phosphorylated S6 (p-S6)
were purchased from Cell Signaling Technology® – phospho-mTOR Ser2448 49F9
rabbit mAb #2976 and phospho-S6 ribosomal protein Ser235/236 D57.2.2E XP® rabbit
mAb #4858.
METHODS
1.1 Sample Preparation
FFPE samples were cut by a professional pathology technician using a microtome
(Shandon™ Finesse™ 325, Thermo Scientific, MA, USA) into 3µm thick sections. The
sections were mounted onto positively charged slides and labeled with the corresponding
case number. Slides were placed in an incubator at 37ºC for 24 hours to dry, and at 65ºC
for 10-15 minutes, to make sure tissue sections adhered correctly to the slides, right
before immunohistochemistry.
1.2 Hematoxylin-Eosin Staining
The paraffin from FFPE sections was completely removed by immersion in xylene
(Atom Scientific Ltd, United Kingdom) for 2 x 10 minutes and rehydrated in a series of
decreasing alcohol (Aga – Álcool e Géneros Alimentares, S.A., Portugal) concentrations –
2 x 100%, 95% and 70% – for 5 minutes in each concentration, followed by 5 minutes in
running water, all at room temperature.
Samples were then stained with Mayer’s hematoxylin (DiaPath, Italy) for 4 minutes
and rinsed in running water for 5 minutes, followed by 5 minutes in 95% alcohol (Aga –
Álcool e Géneros Alimentares, S.A., Portugal), both at room temperature. Eosin (Eosin-Y
Alcoholic, Richard-Allan Scientific™, Thermo Scientific, MA, USA) staining followed for 4
minutes at room temperature.
The samples were dehydrated in a series of increasing alcohol (Aga – Álcool e
Géneros Alimentares, S.A., Portugal) concentrations – 95% and 2 x 100% - for 5 minutes
in each condition at room temperature. The subsequent step consisted of diaphonization
with xylene (Atom Scientific Ltd, United Kingdom) for 2 x 10 minutes at room temperature.
Finally, the slides were mounted using a toluene-based mounting medium
(Richard-Allan Scientific™Mounting Medium, Thermo Scientific, MA, USA).
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
32
1.3 Immunohistochemistry
Immunohistochemistry was performed using specific markers of the mTOR
pathway.
The paraffin from FFPE sections was completely removed by immersion in xylene
(Atom Scientific Ltd, United Kingdom) for 2 x 10 minutes and rehydrated in a series of
decreasing alcohol (Aga – Álcool e Géneros Alimentares, S.A., Portugal) concentrations –
2 x 100%, 95% and 70% – for 5 minutes in each concentration, followed by 5 minutes in
running water.
Antigen retrieval was performed through heat-induced epitope retrieval, slides
placed in a slide rack inside a small container, using citrate (#s/3320/60, Thermo Fisher
Scientific Inc., UK) buffer 10mM pH6 for p-mTOR and EDTA (#4050, OmniPur® EDTA,
Calbiochem, Merck Millipore, MA, USA) buffer 10mM pH9 for p-S6 in a microwave in
decreasing power increments – 600W, 450W, 300W and 150W – for a total of 10 minutes.
Slides were left to cool for 30 minutes at room temperature and then rinsed 2 x 5 minutes
in PBS-Tween 0,02% (PBS-T) pH7.
Endogenous activity was blocked in three phases. First, endogenous peroxidase
was blocked using a solution of 30% peroxidase with methanol (EMPLURA®, Merck
Millipore, MA, USA) – 10% peroxidase and 90% methanol – incubated for 10 minutes at
room temperature followed by a 2 x 5 minute rinse with PBS-T. Secondly, avidin and
biotin were blocked using the Avidin/Biotin Blocking System (#TA-015-BB, Thermo
Scientific, MA, USA), for a 10 minute incubation period at room temperature and 2 x 5
minutes PBS-T rinse afterwards, for each of the proteins. Thirdly, for reduction of non-
specific background, the UltraVision Protein Block (#TA-125-PBQ, Thermo Scientific, MA,
USA) was used for a 10 minute incubation period at room temperature.
The tissue sections were then incubated with the primary antibodies p-S6 and p-
mTOR. Antibodies were diluted with the UltraAb Diluent (#TA-125-UD, Thermo Scientific,
MA, USA), with dilution ratios of 1:100 and 1:400 for p-mTOR and p-S6, respectively.
Incubation was done overnight at 4ºC.
On the following day, the slides were rinsed for 2 x 5 minutes with PBS-T.
Incubation with secondary antibody was performed using the Biotinylated Goat Anti-
Polyvalent solution (#TP-125-BN, Thermo Scientific, MA, USA) for 10 minutes followed by
a 2 x 5 minute rinse with PBS-T.
Streptavidin/peroxidase was labeled using the Streptavidin/Peroxidase solution
(#TS-125-HR, Thermo Scientific, MA, USA), and incubated for 10 minutes at room
temperature and rinsed for 2 x 5 minutes PBS-T.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
33
Chromogenic detection was based on peroxidase activity using 3,3’ –
Diaminobenzidine (DAB) (#K3468, Dako, Denmark), and incubating the samples for 2
minutes at room temperature.
Samples are subsequently counterstained with Mayer’s hematoxilyn (DiaPath,
Italy) for 4 minutes, followed by a 5 minute rinse in running water and dehydration in a
series of increasing alcohol (Aga – Álcool e Géneros Alimentares, S.A., Portugal)
concentrations – 95% and 2 x 100% - for 5 minutes in each condition at room
temperature. The subsequent step consisted of diaphonization with xylene (Atom
Scientific Ltd, United Kingdom) for 2 x 10 minutes at room temperature.
The last step in this technique consisted of mounting the slides using a toluene-
based mounting medium (Richard-Allan Scientific™Mounting Medium, Thermo Scientific,
MA, USA).
1.4 Classification of the Immunohistochemical staining
Slides were observed under an optical microscope and each case classified
semiquantitatively by an experienced pathologist in terms of percentage of stained cells
(0, <5%; 1, 5-25%; 2, 25-50%; 3, 50-75%; 4, >75%), intensity of staining (0, negative; 1,
weak; 2, intermediate; 3, strong) and cellular localization of the staining (membrane and/or
cytoplasmic) An immunohistochemical score was obtained by multiplying the percentage
of stained cells by the intensity of the staining, with 12 being the maximum score. This
classification system was previously used in studies of our group at Ipatimup [113]. We
considered negative as 0, low score from 1 to 3, intermediate 4 and high from 6 to 12.
1.5 Statistical Analysis
Statistical analysis was carried out using IBM SPSS Statistics Version 23 (IBM,
NY, USA). The relationship between clinico-pathological characteristics and expression of
p-S6 and p-mTOR was examined by an unpaired Student’s t test, and the results
expressed in mean ± standard deviation. A non-parametric Mann-Whitney U-test was
applied for sample sizes smaller than 30, and results expressed in mean rank and U-test.
Furthermore, we obtained frequency tables for each of the variables, expressed in
frequency and percentage. We considered statistically significant P-value < 0.05.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
34
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
35
RESULTS
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
36
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
37
RESULTS
SERIES 1
1.1 Distribution of Pathological and Molecular Characteristics
This series included a total of 90 cases of thyroid carcinoma, comprised of 73
PTC, 13 FTC, 2 PDTC and 2 cases of PTC metastasis. Subdividing the PTC into its
variants, there are 39 classical variants of PTC (cPTC), 23 follicular variants of PTC
(fvPTC), 5 mixed PTCs, 3 sclerosing variants of PTC (sPTC), 1 tall cell variant of PTC
(tcPTC), 1 oncocytic variant of PTC (oPTC) and 1 solid trabecular PTC (stPTC) – Table 2.
The pathological data of the series lacked information in some of the cases, which were
presented as missing information in the database.
Table 2 – Diagnosis of the tumors present in Series 1 (n=90)
Diagnosis Frequency Percentage (%)
PTC
cPTC 39 43.3
fvPTC 23 25.6
mixed PTC 5 5.6
sPTC 3 3.3
tcPTC 1 1.1
oPTC 1 1.1
stPTC 1 1.1
FTC 13 14.4
PDTC 2 2.2
Metastasis PTC 2 2.2
Total 90 100
Of the 90 cases, the mean age was 44, ranging from 11 to 82 years. The
majority were younger than 45 years (52.2%). 75 cases (83.3%) were female, with a
mean age of 44 (13-82) years and 14 cases (15.6%) were male, with a mean age of 41
(11-80) years.
Tumors had mainly more than 2 centimeters (cm) in their biggest diameter
(66.7%), had a single nodule at presentation (51.1%) and were encapsulated (58.9%). Of
those that had a tumor capsule (n=53), 62.3% had invasion of the capsule. Furthermore,
51.1% of the tumors had vascular invasion and 67.8% extended beyond the thyroid. The
majority of tumors had no sign of lymph node metastasis (56.7%) or lymphocytic infiltrate
(52.2%). The majority of cases were stage I (61.1%) – Table 3.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
38
As far as genetic alterations go, 26.7% of the tumors were mutated for BRAF,
12.2% for NRAS, 4.4% for TERT promoter, and 12.2% and 2.2% had rearrangements of
RET/PTC and PAX8/PPARγ, respectively.
Table 3 – Pathological and molecular characteristics of the whole series (n=90), PTC (n=73) and FTC (13) in Series 1
Variables All cases
(n=90) PTC
(n=73) FTC
(n=13)
Age (%)
<45 years 47 (52.2)
41 (56.2)
5 (38.5)
≥45 years 42 (46.7)
31 (42.5)
8 (61.5)
Gender (%)
Female 75 (83.3)
61 (83.6)
11 (84.6)
Male 14 (15.6)
11 (15.1)
2 (15.4)
Tumor size (%)
<2cm 29 (32.2)
28 (38.4)
1 (7.7)
≥2cm 60 (66.7)
44 (60.3)
12 (92.3)
Tumor capsule (%)
53 (58.9)
38 (52.1)
13 (100)
Tumor capsule invasion (%)
33 (62.3)
17 (44.7)
11 (84.6)
Multifocality (%)
Single 46 (51.1)
39 (53.4)
5 (38.5)
Multiple 42 (46.7)
32 (43.8)
8 (61.5)
Vascular invasion (%)
46 (51.1)
33 (45.2)
10 (76.9)
Lymph node metastasis (%)
24 (26.7)
22 (30.1)
1 (7.7)
Extrathyroid extension (%)
25 (27.8)
23 (31.5)
1 (7.7)
Lymphocytic infiltrate (%)
40 (44.4)
35 (47.9)
4 (30.8)
BRAF mutation (%)
24 (26.7)
24 (32.9)
0
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
39
Note: the number of cases with missing information is presented in Table 1 in the Supplementary Tables
Our interest for this work was mainly in PTC, so we conducted a separate analysis
for those cases.
There were 73 PTC in total – Table 4. Of the whole PTC cases, 83.6% were
female and 15.1% male, and the majority of the cases were younger than 45 years
(56.2%), with a mean of 43 (11-82) years.
Table 4 – Diagnosis of the cases of papillary thyroid carcinoma (n=73) in Series 1
Diagnosis Frequency Percentage (%)
cPTC 39 53.4
fvPTC 23 31.5
mixed PTC 5 6.8
sPTC 3 4.1
tcPTC 1 1.4
oPTC 1 1.4
stPTC 1 1.4
Total 73 100
The same variables mentioned above were also analyzed for this group (n=73).
For the majority of the variables, the same tendency observed when analyzing all the
cases was found – i.e. tumors ≥2cm (60.3%), tumor encapsulation (52.1%), invasion of
NRAS mutation (%)
11 (12.2)
7 (9.6)
3 (23.1)
TERT promoter mutation (%)
4 (4.4)
2 (2.7)
1 (7.7)
RET/PTC rearrangement (%)
11 (12.2)
10 (13.7)
1 (7.7)
PAX8/PPARγ rearrangement (%)
2 (2.2)
1 (1.4)
1 (7.7)
Staging (%)
I 55 (61.1)
48 (65.8)
6 (46.2)
II 9 (10.0)
4 (5.5)
4 (30.8)
III 14 (15.6)
11 (15.1)
2 (15.4)
IVa 6 (6.7)
5 (6.8)
1 (7.7)
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
40
tumor capsule (44.7% of the tumors with capsule), single nodule at presentation (53.4%),
no lymph node metastasis (54.8%), no lymphocytic infiltrate (50.7%) and stage I disease
(65.8%). On the other hand, the majority of PTC do not present vascular invasion (54.8%)
nor extrathyroid extension (63.0%) – Table 3.
We also did a comparative analysis between cPTC and fvPTC to see the
distribution of the clinico-pathological characteristics within those variants – Table 5. Table 5 – Pathological and molecular characteristics of cPTC (n=39) and fvPTC (n=23) in Series 1
Variables cPTC (n=39) fvPTC (n=23)
Age (%)
<45 years 22
(56.4)
11 (47.8)
≥45 years 17
(43.6)
11 (47.8)
Gender (%)
Female 35
(89.7)
18 (78.3)
Male 4
(10.3)
4 (17.4)
Tumor size (%)
<2cm 15
(38.5)
8 (34.8)
≥2cm 23
(59.0)
15 (65.2)
Tumor capsule (%)
17 (43.6)
13 (56.5)
Tumor capsule invasion (%)
11 (64.7)
3 (23.1)
Multifocality (%)
Single 21
(53.8)
11 (47.8)
Multiple 16
(41)
12 (52.2)
Vascular invasion (%)
23 (59)
4 (17.4)
Lymph node metastasis (%)
14 (35.9)
4 (17.4)
Extrathyroid extension (%)
12 (30.8)
4 (17.4)
Lymphocytic infiltrate (%)
25 (64.1)
6 (26.1)
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
41
Note: the number of cases with missing information is presented in Table 2 in the Supplementary Tables
cPTC turned out to be the variant with the most features associated with
aggressive behavior – 53.8% of tumors without capsule, and of those with tumor capsule,
64.7% had tumor capsule invasion, 59.0% of tumors with vascular invasion and 64.1%
with lymphocytic infiltrate. This variant also presented with more tumors with a single
nodule (53.8%) and in younger patients (56.4%).
fvPTC presented tumors with a more indolent behavior – 56.5% with tumor
capsule, and of those, only 23.1% with invasion of the capsule, 82.6% of tumors without
vascular invasion and 69.6% without lymphocytic infiltrate. Tumors were equally
distributed between ages (47.8% for patients <45 years and ≥45 years), and were
multifocal at presentation (52.2%).
1.2 Association of p-S6 and p-mTOR with Pathological and Molecular
Characteristics in PTC
p-S6 expression in PTC
The distribution of p-S6 scores is presented in Table 6.
BRAF mutation (%)
19 (48.7)
3 (13)
NRAS mutation (%)
1 (2.6)
3 (13)
TERT promoter mutation (%)
1 (2.6)
1 (4.3)
RET/PTC rearrangement (%)
7 (17.9)
2 (8.7)
PAX8/PPARγ rearrangement
(%) 0
1 (4.3)
Staging (%)
I 27
(69.2)
14 (60.9)
II 2
(5.1)
1 (4.3)
III 6
(15.4)
3 (13)
IVa 3
(7.7)
2 (8.7)
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
42
Table 6 – Distribution of p-S6 score in PTC (n=73) in Series 1
Score Frequency Percentage (%)
0 17 23.3
1 9 12.3
2 7 9.6
3 5 6.8
4 10 13.7
6 9 12.3
8 4 5.5
9 6 8.2
12 6 8.2
Total 73 100
Note: the distribution of the p-S6 scores in each variant of PTC is presented in Table 3 in the Supplementary Tables
There is a considerable number of negative cases (23.3%) or with a low (from 0-3)
score (42.1%). Only 13.7% of cases had an intermediate score of 4, and about one third
(34.2%) of the cases scored 6 or above.
In Table 7 are presented the statistically significant results obtained in the
comparison of pathological and molecular characteristics. We included BRAF and NRAS
mutations, which had a borderline statistically significant P-value.
Table 7 – Associations between p-S6 score in PTC and pathological and molecular characteristics (n=73) in Series 1
Variables Frequency P-value Mean Standard deviation
Tumor capsule invasion Yes 20
0.011 2.65 3.233
No 17 5.71 3.378
Extrathyroid extension Yes 23
0.018 2.57 2.873
No 46 4.61 4.025
Lymphocytic infiltrate Yes 35
0.001 2.43 2.779
No 37 5.30 4.027
BRAF mutation WT 49
0.071 4.49 3.964
Mutated 24 2.92 3.134
NRAS mutation WT 66
0.087 3.73 3.813
Mutated 7 6.29 2.360
There were statistically significant associations between p-S6 score and tumor
capsule invasion, extrathyroid extension and lymphocytic infiltrate. Cases without tumor
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
43
capsule invasion (P-value=0.001), without extrathyroid extension (P-value=0.018) and
without lymphocytic infiltrate (P-value=0.001) had a greater p-S6 score.
A tendency was observed between p-S6 score and BRAF and NRAS mutations.
Wild type (WT) BRAF (P-value=0.071) and NRAS mutated (P-value=0.087) cases had
higher p-S6 score.
p-mTOR expression in PTC
For p-mTOR, we found that the majority of cases scored above 6 (67.1%),
whereas only 21.9% scored negative or low – Table 8.
Table 8 – Distribution of p-mTOR score in PTC (n=73)
Score Frequency Percentage (%)
0 5 6.8
1 3 4.1
2 4 5.5
3 4 5.5
4 8 11.0
6 13 17.8
9 11 15.1
12 25 34.2
Total 73 100
Note: the distribution of the p-mTOR scores in each variant of PTC is presented in Table 4 in the Supplementary Tables
We only observed one statistical significant association. Cases with extrathyroid
extension had higher p-mTOR score (P-value=0.027) – Table 9.
Table 9 – Associations between p-mTOR score and pathological characteristics in PTC (n=73) in Series 1
cPTC and fvPTC
We also did the same analysis considering cPTC and fvPTC separately. For both
markers, we had 39 cPTC cases and 23 fvPTC cases analyzed. Distribution of p-S6
scores for both variants is presented in Table 10.
Variables Frequency P-value Mean Standard deviation
Extrathyroid extension Yes 23
0.027 8.87 3.548
No 46 6.52 4.299
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
44
Table 10 – Distribution of p-S6 score in cPTC (n=39) and fvPTC (n=23) in Series 1
cPTC (n=39) fvPTC (n=23)
Score Frequency Percentage (%) Frequency Percentage (%)
0 11 28.2 3 13
1 4 10.3 4 17.4
2 5 12.8 0 0
3 4 10.3 1 4.3
4 5 12.8 3 13
6 3 7.7 5 21.7
8 2 5.1 2 8.7
9 2 5.1 2 8.7
12 3 7.7 3 13
Total 39 100 23 100
In cPTC (n=39), the majority of cases stained with p-S6 scored negative or low
(61.6%) and 25.6% had a high score. On the other hand, fvPTC (n=23) had high scores in
52.1% of cases and negative or low scores in 34.7%.
Table 11 – Distribution of p-mTOR score in cPTC (n=39) and fvPTC (n=23) in Series 1
cPTC (n=39) fvPTC (n=23)
Score Frequency Percentage (%) Frequency Percentage (%)
0 2 5.1 3 13
1 1 2.6 2 8.7
2 1 2.6 2 8.7
3 2 5.1 2 8.7
4 5 12.8 2 8.7
6 10 25.6 1 4.3
9 6 15.4 3 13
12 12 30.8 8 34.8
Total 39 100 23 100
For p-mTOR, 15.4% cPTC cases had a negative or low score and 71.8% had a
high score. When looking at fvPTC, a little more than one third (39.1%) of cases had a
negative or low score and about half (52.1%) of the cases had a high score – Table 11.
No significant differences in p-S6 expression were found for cPTC. p-mTOR score
showed a significant association with two variables – multifocality and extrathyroid
extension – Table 12.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
45
Table 12 – Associations between p-mTOR score and pathological characteristics in cPTC (n=39) in Series 1
Variables Frequency P-value Mean Standard deviation
Multifocality Single 21
0.018 8.87 3.651
Multiple 16 5.63 3.739
Extrathyroid extension Yes 12
0.028 9.42 3.175
No 26 6.46 3.901
Cases with a single nodule at presentation (P-value=0.018) and those with
extrathyroid extension (P-value=0.028) had a higher p-mTOR score.
For fvPTC (n=23), there were no significant differences in p-mTOR expression, but
p-S6 score associated with lymphocytic infiltrate – Table 13.
Table 13 – Associations between p-S6 score and pathological characteristics in fvPTC (n=23)
Variables Frequency P-value Mean Rank U-test
Lymphocytic infiltrate Yes 6
0.027 6.50
18.000 No 16 13.38
Cases with no lymphocytic infiltrate had higher p-S6 score (P-value=0.027).
1.3 Cellular localization of staining
Within the tumors, we observed that both p-S6 and p-mTOR had a more intense
staining in the periphery, a result that was more consistent with p-mTOR. We also
observed that that staining was located mainly on the membrane and cytoplasm for p-
mTOR and cytoplasmic only for p-S6. The staining was highly heterogeneous within the
tumors (Supplementary Figures 1, 3-B and 4-B).
SERIES 2
2.1 Distribution of Clinico-pathological and Molecular Characteristics
This series included a total of 127 cases of thyroid carcinoma, comprised of 114
PTC, 6 FTC and 7 PDTC. Subdividing the PTC into its variants, there are 77 cPTC, 28
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
46
fvPTC, 4 sPTC, 1 oPTC, 1 tcPTC and 3 Warthin-like variant of PTC (wPTC) – Table 14.
The clinicopathological data of Series 2 lacked information in some of the cases, which
were presented as missing information in the database.
Table 14 – Distribution of the cases present in Series 2 (n=127)
Diagnosis Frequency Percentage (%)
PTC
cPTC 77 60.6
fvPTC 28 22
sPTC 4 3.1
oPTC 1 0.8
tcPTC 1 0.8
wPTC 3 2.4
FTC 6 4.7
PDTC 7 5.5
Total 127 100
Of the 127 cases, the mean age was 47, ranging from 13 to 79 years, and the
majority of patients being 45 years or older (51.2%). The majority of cases (77.2%) were
female, with a mean age of 45 (13-74) years and 29 cases (22.8%) were male, with a
mean age of 50 (17-79) years.
Tumors had mainly more than 2 cm in their biggest diameter (58.3%), had a single
nodule at presentation (55.9%), had infiltrative margins (61.4%) and were encapsulated
(59.1%). Of those that had a tumor capsule (n=75), 92% had invasion of the capsule.
Furthermore, 44.1% had lymph node metastases and 19.7% had distant metastases. In
terms of peritumoral invasiveness, 28.3%, 57.7% and 40.2% had vascular invasion,
thyroid capsule invasion and extrathyroid extension, respectively. All of this characteristics
contributed to place the majority of tumors at stage I (54.3%).
Table 15 – Clinico-pathological and molecular characteristics of the whole series (n=127), PTC (n=114) and FTC (n=6) in Series 2
Variables All cases (n=127)
PTC (n=114)
FTC (n=6)
Age (%)
<45 years 61
(48)
58 (50.9)
2 (33.3)
≥45 years 65
(51.2)
55 (48.2)
4 (66.7)
Gender (%)
Female 98
(77.2)
90 (78.9)
4 (66.7)
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
47
Male 29
(22.8)
24 (21.1)
2 (33.3)
Lymph node metastases (%)
56 (44.1)
54 (47.4)
0
Distant metastases (%)
25 (19.7)
15 (13.2)
4 (66.7)
Tumor size (%)
<2cm 43
(33.9)
43 (37.7)
-
≥2cm 74
(58.3)
65 (57)
4 (66.7)
Tumor capsule (%)
75 (59.1)
65 (57)
5 (83.3)
Tumor capsule invasion (%)
70 (93.3)
60 (92.3)
5 (100)
Vascular invasion (%)
36 (28.3)
28 (24.6)
3 (50)
Tumor margins (%)
Well defined
42 (33.1)
34 (29.8)
4 (66.7)
Infiltrative
78 (61.4)
76 (66.7)
1 (16.7)
Thyroid capsule invasion (%)
73 (57.5)
70 (61.4)
3 (50)
Extrathyroid extension (%)
51 (40.2)
50 (43.9)
-
Multifocality (%)
Single 71
(55.9)
64 (56.1)
3 (50)
Multiple 47
(37)
44 (38.6)
2 (33.3)
Lymphocytic infiltrate (%)
77 (60.6)
71 (62.3)
3 (50)
Staging (%)
I 69
(54.3)
66 (57.9)
1 (16.7)
II 9
(7.1)
6 (5.3)
2 (33.3)
III 28
(22)
26 (22.8)
1 (16.7)
IVa 6
(4.7)
6 (5.3)
-
IVc 6 4 1
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
48
Note: the number of cases with missing information is presented in Table 5 in the Supplementary Tables.
With reference to genetic alterations, 46.5% were mutated for BRAF, 2.4% for
NRAS and 11% for TERT promoter.
Regarding the therapeutic aspect, the majority of cases had less than two 131I
therapies (52%) and a cumulative dose of 131I lower than 150 mCi (50.4%). One third of
(4.7) (3.5) (16.7)
BRAF mutation (%)
59 (46.5)
56 (49.1)
-
NRAS mutation (%)
3 (2.4)
3 (2.6)
-
TERT promoter mutation (%)
14 (11)
8 (7)
4 (66.7)
One year disease-free survival (%)
70 (55.1)
64 (56.1)
1 (16.7)
Number of 131I therapies (%)
<2 66
(52)
61 (53.5)
1 (16.7)
≥2 57
(44.9)
49 (43)
5 (83.3)
Cumulative dose (%)
<150mCi 64
(50.4)
60 (52.6)
0
≥150mCi 58
(45.7)
49 (43)
6 (100)
Number of additional therapies (%)
<2 16
(12.6)
15 (13.2)
1 (16.7)
≥2 47
(37)
39 (34.2)
5 (83.3)
Type of additional therapies (%)
1 type 34
(53.9)
32 (59.3)
2 (33.3)
More than 1 type 29
(46.1)
22 (40.7)
4 (66.7)
Disease-free survival after follow-up (%)
74 (58.3)
70 (61.4)
1 (16.7)
Evidence of disease during follow-up (%)
41 (77.4)
35 (76.1)
4 (66.7)
Recurrence (%)
66 (52)
56 (49.1)
6 (100)
Mortality (%)
9 (7.1)
5 (4.4)
1 (16.7)
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
49
the cases had two or more additional therapies (37%), and of those that needed additional
therapies, 53.9% had undergone one type of therapy. Concerning clinical outcome, 55.1%
of cases were free of disease after 1 year and 58.3% free of disease after follow-up, which
had a mean of 101.27 (3-467) months, but the majority had recurrence of disease (52%).
Of those that had persistence of disease after one year, we combined them with the
results from the variable Disease free after follow-up, and observed that 77.4% of those
cases had evidence of disease during follow-up. The mortality rate observed was 7.1% –
Table 15.
In the separate analysis for PTC, there were 114 cases in total – Table 16.
Table 16 – Distribution of PTC histotypes (n=114)
Diagnosis Frequency Percentage (%)
PTC
cPTC 77 67.5
fvPTC 28 24.6
sPTC 4 3.5
oPTC 1 0.9
tcPTC 1 0.9
wPTC 3 2.6
Total 114 100
The mean age in PTC was 46, ranging from 13 to 79 years, and the majority being
younger than 45 years (50.9%). Ninety cases (78.9%) were female, with a mean age of 45
(13-74) years and 24 cases (21.1%) were male, with a mean age of 50 (17-79) years.
We observed in PTC (n=114) the same tendencies as we did for the whole series.
Tumors were ≥2cm (57%), presented as a single nodule (56.1%), had infiltrative margins
(66.7%), lymphocytic infiltrate (62.3%), presence of tumor capsule (57%), with 90.8% of
those invading the capsule, and invasion of thyroid capsule (61.4%).
In comparison with the whole series, there were less tumors with vascular invasion
(24.6%), extrathyroid extension (43.9%), lymph node metastases (47.4%) and distant
metastases (13.2%). For the most part, tumors were also staged at stage I (57.9%).
Mutation of BRAF was the most common genetic alteration (49.1%), whereas
NRAS mutation and TERT promoter mutation were present in only a small part of tumors
(2.6% and 7%, respectively).
In most cases, it was shown that they were free of disease after 1 year (56.1%)
and at the end of follow-up as well (61.4%), but 76.1% had evidence of disease during
follow-up and about half had disease recurrence (49.1%). Follow-up mean was 93.7 (3-
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
50
325) months. Mortality rate was at 4.4%. With regards to therapeutic data, the great part
of these cases underwent less than 2 131I therapies (53.5%) with a cumulative dose of less
than 150 mCi (52.6%). About a third (34.2%) had to receive two or more additional
therapies, with 59.3% of those requiring only 1 type of therapy – Table 15.
We performed a comparative analysis between cPTC and fvPTC in order to see
the distribution of the clinico-pathological and molecular characteristics within those
variants – Table 17.
Table 17 – Clinico-pathological and molecular characteristics in cPTC (n=77) and fvPTC (n=28) in Series 2
Variables cPTC (n=77)
fvPTC (n=28)
Age (%)
<45 years 41
(53.2)
14 (50)
≥45 years 36
(46.8)
14 (50)
Gender (%)
Female 60
(77.9)
24 (85.7)
Male 17
(22.1)
4 (14.3)
Lymph node metastases (%)
41 (53.2)
8 (28.6)
Distant metastases (%)
9 (11.7)
4 (14.3)
Tumor size (%)
<2cm 27
(35.1)
12 (42.9)
≥2cm 44
(57.1)
16 (57.1)
Tumor capsule (%)
37 (48.1)
23 (82.1)
Tumor capsule invasion (%)
20 (86.9)
35 (94.6)
Vascular invasion (%)
22 (28.6)
5 (17.9)
Tumor margins (%)
Well defined
16 (20.8)
15 (53.6)
Infiltrative
57 (74)
13 (46.4)
Thyroid capsule invasion 53 11
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
51
(%) (68.8) (39.3)
Extrathyroid extension (%)
40 (51.9)
7 (25)
Multifocality (%)
Single 36
(46.8)
22 (78.6)
Multiple 36
(46.8)
6 (21.4)
Lymphocytic infiltrate (%)
49 (63.6)
19 (67.9)
Staging (%)
I 47
(61)
15 (53.6)
II 3
(3.9)
3 (10.7)
III 20
(26)
4 (14.3)
IVa 3
(3.9)
2 (7.1)
IVc - 3
(10.7)
BRAF mutation (%)
43 (55.8)
8 (28.6)
NRAS mutation (%)
8 (10.4)
2 (7.1)
TERT promoter mutation (%)
5 (6.5)
3 (10.7)
1 year disease-free survival (%)
42 (54.5)
19 (67.9)
Number of 131I therapies (%)
<2 39
(50.6)
18 (64.3)
≥2 36
(46.8)
10 (35.7)
Cumulative dose (%)
<150mCi 38
(49.4)
18 (64.3)
≥150mCi 36
(46.8)
10 (35.7)
Number of additional therapies (%)
<2 11
(14.3)
3 (10.7)
≥2 30
(39)
7 (25)
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
52
Note: the number of cases with missing information is presented in Table 6 in the Supplementary Tables.
cPTC turned out to be associated with more aggressive features – presence of
lymph node metastases (53.2%), infiltrative tumor margins (74%), invasion of thyroid
capsule (68.8%) and extrathyroid extension (51.9%). Single and multifocal nodules had
the same distribution (46.8%) and the majority of patients were younger than 45 years
(53.2%). Follow-up mean was 105.23 (9-467) months.
On the other hand, fvPTC (n=28) were associated with less aggressive features –
71.4% of tumors had no signs of lymph node metastases, 53.6% of tumors had well
defined margins, 57.1% had no thyroid capsule invasion, 75% did not extend beyond the
thyroid and 78.6% of tumors presented with a single nodule. Age was equally distributed
between groups (50%). Follow-up median was 71 (3.325) months.
2.2 Association of p-S6 and p-mTOR with Clinico-pathological and
Molecular Characteristics
We had difficulties classifying some cases, mainly for p-S6, because there was no
positive staining of internal tissue control, which deemed these samples unviable for the
study, and therefore excluded from the analysis. The best hypothesis to support this was
poor fixation of tissue. For this reason, we had to exclude from the statistical analysis 38
cases of p-S6 and 7 cases of p-mTOR (Supplementary Data Figure 2).
p-S6 in PTC
The distribution of p-S6 score in PTC (n=76) is presented in Table 18.
Type of additional therapies (%)
1 type 23
(56.1)
7 (70)
More than 1 type 18
(43.9)
3 (30)
Disease-free survival after follow-up (%)
44 (57.1)
20 (71.4)
Evidence of disease during follow-up (%)
26 (81.5)
7 (77.8)
Recurrence (%)
41 (53.2)
10 (35.7)
Mortality (%)
2 (2.6)
2 (7.1)
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
53
Table 18 – Distribution of p-S6 score in PTC (n=76) in Series 2
Score Frequency Percentage (%)
0 34 44.7
1 12 15.8
2 11 14.5
3 7 9.2
4 3 3.9
6 5 6.6
9 2 2.6
12 2 2.6
Total 76 100
Note: the distribution of p-S6 scores in each variant of PTC is presented in Table 7 of Supplementary Tables
We had 38 cases excluded for this analysis, thus totaling 76 cases, and not the
total 114. The majority of cases (84.2%) scored negative or low and 11.8% scored high.
Table 19 presents the statistically significant results. We also included tumor
margins and thyroid capsule invasion, which had a borderline statistically significant P-
value.
Table 19 – Associations between p-S6 score and pathological characteristics in PTC (n=76) in Series 2
Variables Frequency P-value Mean Standard deviation
Tumor capsule No 35
0.001 0.74 1.379
Yes 38 2.87 3.223
Vascular invasion No 53
0.045 1.38 2.132
Yes 19 3.26 3.664
Tumor margins Infiltrative 53
0.053 1.47 2.636
Well defined 20 2.85 2.739
Thyroid capsule invasion
No 26 0.069
2.65 3.224
Yes 45 1.42 2.360
Cases with invasion of tumor capsule (P-value=0.001) and vascular invasion (P-
value=0.045) were found to have higher scores of p-S6. We also saw a tendency between
higher scores of p-S6 and absence thyroid capsule invasion (P-value=0.069) and well-
defined tumor margins (P-value=0.053) – Table 19.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
54
p-mTOR in PTC
We analyzed 107 cases of PTC stained for p-mTOR, and not the 114, due to
exclusion of 7 cases.
Table 20 – Distribution of p-mTOR score in PTC (n=107) in Series 2
Score Frequency Percentage (%)
0 33 30.8
1 7 6.5
2 15 14
3 8 7.5
4 9 8.4
6 9 8.4
8 3 2.8
9 11 10.3
12 12 11.2
Total 107 100 Note: the distribution of the p-mTOR scores in each variant is presented in Table 8 of Supplementary Tables
We had 58.8% of cases with a negative or low score and 32.7% with a high score
– Table 20.
Table 21 presents the statistically significant results. We also included
multifocality, one year disease-free survival and cumulative dose, which had a borderline
statistically significant P-value.
Table 21 – Associations between p-mTOR score and clinico-pathological characteristics in PTC (n=107) in Series 2
Variables Frequency P-value Mean Standard deviation
Distant metastases No 92
0.049 3.61 4.087
Yes 14 5.93 3.912
Tumor capsule No 43
0.024 5.00 4.342
Yes 61 3.16 3.782
Multifocality Single 61
0.066 3.30 3.909
Multiple 41 4.83 4.347
One year disease-free survival
No 42 0.053
4.95 4.742
Yes 61 3.26 3.521
Cumulative dose <150 58
0.090 3.45 3.752
>=150 44 4.89 4.489
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
55
Higher p-mTOR scores were found in cases presenting distant metastases (P-
value=0.049) and in those without a tumor capsule (P-value=0.024). The presence of
multiple nodules showed a tendency to be associated with higher p-mTOR score (P-
value=0.066), as well as persistence of disease after one year (P-value=0.053) and higher
doses of 131I (P-value=0.090).
cPTC and fvPTC
For p-S6, we analyzed only 51 cPTC cases, instead of 77, and 18 fvPTC cases,
instead of 28, because of the cases that were excluded.
Table 22 – Distribution of p-S6 scores in cPTC (n=51) and fvPTC (n=18) in Series 2
cPTC (n=51) fvPTC (n=18)
Score Frequency Percentage (%) Frequency Percentage (%)
0 25 49 7 38.9
1 8 15.7 2 11.1
2 7 13.7 4 22.2
3 3 5.9 3 16.6
4 2 3.9 0 0
6 3 5.9 1 5.6
9 1 2 1 5.6
12 2 3.9 0 0
Total 51 100 18 100
The majority (84.3%) of cPTC (n=51) had a negative or low p-S6 score and 11.8%
had a high score. In fvPTC (n=18), the vast majority (88.8%) had a negative or low p-S6
score and only 11.2% presented a high score – Table 22.
For p-mTOR, we only analyzed 73 cPTC, instead of 77, and 25 fvPTC, instead of
28, because of the cases excluded.
Table 23 – Distribution of p-mTOR score in cPTC (n=73) and fvPTC (n=25) in Series 2
cPTC (n=73) fvPTC (n=25)
Score Frequency Percentage (%) Frequency Percentage (%)
0 23 31.5 8 32
1 7 9.6 0 0
2 11 15.1 3 12
3 3 4.1 3 12
4 6 8.2 3 12
6 7 9.6 2 8
8 1 1.4 1 4
9 7 9.6 3 12
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
56
12 8 10.9 2 8
Total 73 100 25 100
Approximately two thirds of cPTC (60.3%) and fvPTC (68%) had a negative or low
p-mTOR score and 31.5% of cPTC and 32% of fvPTC had a high p-mTOR score – Table
23.
We only found a significant association between high p-S6 score and presence of
tumor capsule (P-value=0.017) in cPTC.
Table 24 – Associations between p-S6 and pathological characteristics in cPTC (n=51) in Series 2
Variables Frequency P-value Mean Standard deviation
Tumor capsule No 27
0.017 0.81 1.520
Yes 21 3.05 3.761
On the other hand, in fvPTC, cases with higher p-S6 score were significantly
associated with presence of distant metastases (P-value=0.013) and vascular invasion (P-
value=0.005). Furthermore, cases with higher p-S6 scores show more frequently well-
defined tumor margins (P-value=0.083) and single nodules at presentation (P-
value=0.061) – Table 25.
Table 25 – Associations between p-S6 and pathological characteristics in fvPTC (n=18) in Series 2
Variables Frequency P-value Mean Rank U
Distant metastases No 16
0.013 8.50
0.000 Yes 2 17.50
Vascular invasion No 15
0.005 8.07
1.000 Yes 3 16.67
Tumor margins Well-defined 10
0.083 12.00
20.000 Infiltrative 8 7.59
Multifocality Single 22
0.061 10.75
10.500 Multiple 6 5.13
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
57
Regarding p-mTOR in cPTC, there were statistically significant associations with
presence of distant metastases (P-value=0.025) and persistence of disease after follow-
up (P-value=0.048). We also obtained a borderline significance with multifocal tumors (P-
value=0.068), absence of TERT promoter mutation (P-value=0.095) and persistence of
disease after one year (P-value=0.099) – Table 26.
Table 26 – Associations between p-mTOR and clinico-pathological and molecular characteristics in cPTC (n=73) in Series 2
Variables Frequency P-value Mean Standard deviation
Distant metastases No 64
0.025 3.31 4.011
Yes 9 6.56 3.644
Multifocality Single 36
0.068 2.83 3.768
Multiple 33 4.64 4.314
TERT promoter mutation
WT 53 0.095
4.04 4.206
Mutated 5 2.20 1.789
One year disease free survival
No 28 0.099
4.86 4.882
Yes 42 3.02 3.339
Disease free survival after follow-up
No 28 0.048
5.11 4.841
Yes 43 2.98 3.327
In fvPTC, we only found a significant association between p-mTOR score and
absence of tumor capsule (P-value=0.035) – Table 27.
Table 27 - Associations between p-mTOR score and pathological data in fvPTC (n=25) in Series 2
Variables Frequency P-value Mean Rank U
Tumor capsule No 5
0.035 19.10
19.500 Yes 20 11.48
2.3 Cellular localization of staining
In this series, we observed p-S6 and p-mTOR with intense peripheral staining,
which was more consistent with p-mTOR. It was preferentially located on the membrane
and cytoplasm for p-mTOR and cytoplasmic only for p-S6. Staining was highly
heterogeneous within the tumors (Supplementary Figures 1, 3-A and 4-A).
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
58
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
59
DISCUSSION
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
60
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
61
DISCUSSION
mTOR PATHWAY ACTIVATION
mTOR is a central regulator of several cellular processes, such as cell growth,
proliferation and survival, and it is also involved in regulating cell migration.
Understandably, this pathway is overactivated in several cancers, including thyroid
cancer, and has been linked to its pathogenesis. PTC are usually indolent, have a good
prognosis and ablative treatment is efficient. Unfortunately, a subset of cases will develop
recurrence and metastases [114].
The main goal of this work was to evaluate the activation of the mTOR pathway in
PTC, and we accomplished this by confirming its overactivation in such cases.
Furthermore, we have found p-mTOR to be associated with aggressive behavior and
poorer outcomes. p-mTOR expression was significantly higher in PTC with extrathyroid
extension and distant metastases; there was also a tendency to higher expression in
those cases that required higher doses of 131I. Considering cPTC only, we observed a
significant association with persistence of disease after follow-up.
Additionally, p-mTOR was preferentially localized in the periphery of tumors, as
well as in their invasive front, and the majority of cases had a membrane staining. Similar
findings were also reported in renal cell carcinoma [115].
The consistent results with p-mTOR, even taking into consideration the differences
between series, strongly suggests that p-mTOR is highly expressed in aggressive PTC,
and thus would be useful to evaluate behavior in these tumors. Additionally, unpublished
data from our group reveals a significant negative correlation between p-mTOR and NIS,
which is in accordance with what is reported on papillary thyroid cancer lineages [111].
Since patients with low NIS expression do not respond to ablative 131I therapy [9], once
more, p-mTOR seems to be associated with poor prognosis.
Regarding p-S6, the results were not consistent between series. In Series 1, we
found expression of p-S6 to be associated with factors of good prognosis (absence of
invasion of tumor capsule, absence of extrathyroid extension and absence of lymphocytic
infiltrate) and in Series 2 to be associated with vascular invasion. Interestingly, we found a
borderline significance between high p-S6 expression and wild-type BRAF and NRAS
mutation in PTC in Series 1. We feel that p-S6 is not a robust marker to evaluate the
outcome in PTC.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
62
Activation of the mTOR pathway has been reported in a variety of cancers. These
studies evaluated expression of p-S6, p-mTOR and other effectors of the pathway and
found significant associations with aggressive behavior and poorer outcomes. Activation
of mTOR pathway via overexpression of p-mTOR was found to be associated in
glioblastoma [116] and melanoma [117] with poor prognosis, in pancreatic
neuroendrocrine tumors with aggressive characteristics [118] , in extrahepatic
cholangiocarcinoma [119] and in head and neck small cell carcinoma [120]. On the
contrary, overexpression of p-mTOR was found in luminal breast cancer [121] and small
cell lung carcinoma [122] with good prognosis. Some authors have also found the mTOR
pathway to be activated via overexpression of p-S6 in lung adenocarcinoma [123] and
renal cell carcinoma [124].
In thyroid cancer, a few studies on mTOR pathway also demonstrated its
activation. Faustino et al. reported p-mTOR expression in PTC, and its expression was
associated with BRAF mutation [113]. Ahmed et al. [125] also found activation of mTOR
pathway in PTC, although they did not find any associations with aggressive features. In
FTC, Kirschner et al. showed activation of the mTOR pathway as well [126]. Moreover,
overexpression of p-S6 leading to activation of the mTOR pathway was reported in
medullary thyroid carcinoma associated with lymph node metastases [127] and RAS
mutation [128]. With different markers, Kouvaraki et al. [129] observed activation of the
pathway via overactivation of phosphorylated 4E-PB1 in 8 cases with aggressive histology
as compared to cPTC.
In order to better understand our results, we analyzed the correlation between p-
mTOR and p-S6 expression, and did not find any correlation (Supplementary Tables 6
and 7). However, a previous study in our group by Populo et al. [117] showed a positive
correlation between p-S6 and p-mTOR in melanoma cases. This was also reported in
glioblastoma [130]. Taking this into account and the associations we found with p-mTOR
and its localization in the tumors, we hypothesized that activation of the mTOR pathway
was occurring through activation of mTORC2.
Through the canonical way, mTOR activation goes through mTORC1, and in this
case it is expected a positive correlation between expression of p-mTOR and p-S6, given
that there are no other activators of p-S6 besides mTORC1. If the expression of p-mTOR
does not correlate in any way with p-S6, which is what we observed, one can hypothesize
that activation of mTOR in PTC is occurring mainly via mTORC2 (to be noted, our
antibody recognized p-mTOR on Ser2448, which is present on both mTORC1 and
mTORC2, whereas phosphorylation on Ser2481 is exclusive for mTORC2 [131]).
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
63
One of the roles of mTORC2 is the phosphorylation of Akt on Ser473, which takes
place in the cytoplasmic membrane. Curiously, we observed p-mTOR membrane staining
in the majority of our cases. Besides that, mTORC2 also controls cytoskeletal remodeling
and cell migration through protein kinase Cα (PKCα) activation [101], which can be
related with the overexpression of mTOR in cases with distant metastases that we found
in our results.
A previous study in thyroid tumors reported an overexpression of Sin1, a
component of mTORC2, and phosphorylated Akt (p-Akt) on Ser473 in aggressive variants
of PTC compared to cPTC [132]. In other models, mTORC2 seems to be associated with
characteristics of aggressiveness, invasion and poor prognosis. This complex has been
implicated in bladder cancer as being critical for its invasive behavior, as seen in cell lines
and tissue specimens [133]; in colorectal cancer, Gulhati et al. [134] observed an
overexpression of mTORC2 in tissue specimens and an association of that expression
with invasion and migration in cell lines; in gliomas [135] it has also been reported an
increase in mTORC2 activity in cell lines and tissue specimens, correlating with increased
levels of rictor, which translates into proliferation of tumor cells and invasive potential; in
erbB2-positive breast cancer, the authors detected high expression of p-Akt (Ser473)
associated with metastases in tumor cells [136]. Furthermore, mTORC2 seems to be a
central player in cancer cell metabolism, with probable effect on epigenetics [137, 138].
mTOR PATHWAY AND GENETIC ALTERATIONS
Regarding genetic alterations, we did not find p-S6 or p-mTOR expression to be
associated with the mutational status of the genes that were screened. However, in
previous studies by Faustino et al. [113] and Ahmed et al. [125], expression of p-S6 and
p-mTOR was associated with BRAF mutation in PTC.
Mutations in RAS genes, namely HRAS and KRAS, were also found to be
associated with higher expression of p-S6 in medullary thyroid carcinoma [128].
mTOR PATHWAY AND TREATMENT FOR THYROID CANCER
mTOR inhibitors are classified as rapamycin and rapamycin analogs, such as
temsirolimus and everolimus. These drugs act by forming a complex with FK506 binding
protein (FKBP12), and this complex acts as an allosteric inhibitor of mTORC1. The acute
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
64
inhibition occurs because raptor dissociates from p-mTOR [139]. Recent discoveries
showed that mTORC2 can also be inhibited after prolonged treatment with rapamycin in a
subset of cell lines, by dissociation of rictor from the complex, and inhibition of Akt
phosphorylation on Ser473 [140]. This mTORC2 inhibition was also observed in different
tissues of mice, with decreased expression of PKCα Ser657 and Akt Ser473 [141].
Several studies have been performed to evaluate mTOR inhibitors in aggressive
forms of cancer, namely in treatment-refractory metastatic pancreatic cancer [142],
treatment-refractory metastatic renal cell carcinoma [143], recurrent Hodgkin lymphoma
[144], recurrent aggressive lymphoma [145], recurrent/metastatic breast cancer [146],
recurrent/metastatic endometrial cancer [147] and recurrent/refractory mantle cell
lymphoma [148]. While they seemed promising at first, increasing the time to tumor
progression, their effect was short-lasting, but effective in the breast cancer study, due to
development of therapy resistance after failure to inhibit mTORC2.
The second-generation of mTOR inhibitors are aimed to inhibit both complexes, by
binding to the ATP-binding site of mTOR and interfering with some of its effectors. They
became important because rapamycin inhibits preferentially mTORC1, while mTORC2
continues to phosphorylate Akt and maintaining the pathway active – a mechanism of
resistance to rapamycin. They have been shown to surpass rapamycin and its analogs’
inhibition potential. Torin1 and Torin2, two of those inhibitors tested in vitro and in vivo,
showed a decrease in tumor size and cell viability, as well as inactivation of the effectors
S6, 4E-BP1 and Akt [125, 149]. So far, these drugs have not been approved for
therapeutic purposes.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
65
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
66
CONCLUSIONS AND FUTURE PERSPECTIVES
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
67
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
68
CONCLUSIONS
We confirm activation of the mTOR pathway in PTC. We report an association
between expression of p-mTOR and aggressive features, such as distant metastases and
extrathyroid extension. Being highly expressed in aggressive PTC, p-mTOR would be
useful to evaluate behavior in these tumors. Furthermore, p-mTOR was located in the
periphery and invasive front of tumors, as well as in the membrane in the majority of
cases.
Expression of p-S6 was not consistent between series, showing to be associated
with both good prognosis and vascular invasion. We feel that p-S6 is not a robust marker
to evaluate the outcome in PTC.
Given our results, we hypothesize that activation of the mTOR pathway in PTC
occurs via phosphorylation of mTORC2.
FUTURE PERSPECTIVES
In light of our results, we should further investigate the lack of correspondence
between p-mTOR and p-S6 in thyroid tumors, studying, for example, mTORC2
downstream effectors, such as p-Akt Ser473 in tumor samples as an mTORC2 activation
readout. Additionally, in vitro studies would be performed with thyroid cancer cell lines. We
intend to evaluate their response to treatment with rapamycin, taking into account different
genetic backgrounds, as well as markers for the activation of the PI3K/Akt/mTOR pathway
– such as mTOR, S6, Akt. NIS will also be evaluated in cell lines.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
69
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
70
BIBLIOGRAPHIC REFERENCES
1. Nussey, S. and S. Whitehead, Chapter 3:The thyroid gland, in Endocrinology: an integrated approach. 2001, BIOS Scientific Publishers Limited: Oxford.
2. Nikiforov, Y.E., P.W. Biddingger, and L.D.R. Thompson, Diagnostic pathology and molecular genetics of the thyroid: a comprehensive guide for practicing thyroid pathology. 1st ed. 2009: Lippincott Williams & Wilkins.
3. Barrett, E.J., Section VIII: The endocrine system, in Medical Physiology: A cellular and molecular approach, W.F. Boron and E.L. Boulpaep, Editors. 2012, Saunders: Philadelphia, PA, USA.
4. Dietrich, J.W., G. Landgrafe, and E.H. Fotiadou, TSH and thyrotropic agonists: key actors in thyroid homeostasis. J Thyroid Res, 2012. 2012(351864).
5. Cavalieri, R.R., Iodine metabolism and thyroid physiology: current concepts. Thyroid, 1997. 7(2): p. 177-181.
6. Van Herle, A.J., G. Vassart, and J.E. Dumont, Control of thyroglobulin synthesis and secretion (first of two parts). N Engl J Med, 1979. 301(5): p. 239-249.
7. Ichikawa, K. and K. Hashizume, Cellular binding proteins of thyroid hormones. Life Sci, 1991. 49(21): p. 1513-1522.
8. Portulano, C., M. Paroder-Belenitsky, and N. Carrasco, The Na+/I- symporter (NIS): mechanism and medical impact. Endocr Rev, 2014. 35(1): p. 106-49.
9. Dohan, O., et al., The sodium/iodide Symporter (NIS): characterization, regulation, and medical significance. Endocr Rev, 2003. 24(1): p. 48-77.
10. Kogai, T. and G.A. Brent, The sodium iodide symporter (NIS): regulation and approaches to targeting for cancer therapeutics. Pharmacol Ther, 2012. 135(3): p. 355-70.
11. Riesco-Eizaguirre, G. and P. Santisteban, A perspective view of sodium iodide symporter research and its clinical implications. Eur J Endocrinol, 2006. 155(4): p. 495-512.
12. Armstrong, R., et al., A refractory phase in cyclic AMP-responsive transcription requires down regulation of protein kinase A. Mol Cell Biol, 1995. 15(3): p. 1826-1832.
13. Taki, K., et al., A thyroid-specific far-upstream enhancer in the human sodium/iodide symporter gene requires Pax-8 binding and cyclic adenosine 3',5'-monophosphate response element-like sequence binding proteins for full activity and is differentially regulated in normal and thyroid cancer cells. Mol Endocrinol, 2002. 16(10): p. 2266-82.
14. Cass, L.A. and J.L. Meinkoth, Ras signaling through PI3K confers hormone-independent proliferation that is compatible with differentiation. Oncogene, 2000. 19(7): p. 924-932.
15. Weiss, S.J., et al., Thyrotropin-stimulated iodide transport mediated by adenosine 3',5'-monophosphate and dependet on protein synthesis. Endo, 1984. 114(4): p. 1099-1107.
16. Ohno, M., et al., The paired-domain transcription factor Pax8 binds to the upstream enhancer of the rat sodium/iodide symporter gene and participates in both thyroid-specific and cyclic-AMP-dependent transcription. Mol Cell Biol, 1999. 19(3): p. 2051-2060.
17. Kogai, T., et al., Regulation by thyroid-stimulating hormone of sodium/iodide symporter gene expression and protein levels in FRTL-5 cells. Endo, 1997. 138(6): p. 2227-2232.
18. Riedel, C., O. Levy, and N. Carrasco, Post-transcriptional regulation of the sodium/iodide symporter by thyrotropin. J Biol Chem, 2001. 276(24): p. 21458-63.
19. Wolff, J. and I.L. Chaikoff, Plasma inorganic iodide as a homeostatic regulator of thyroid function. J Biol Chem, 1948. 174: p. 555-564.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
71
20. García, B. and P. Santisteban, PI3K is involved in the IGF-1 inhibition of TSH-induced sodium/ioded symporter gene expression. Mol Endocrinol, 2002. 16(2): p. 342-352.
21. Ajjan, R.A., et al., The sodium iodide symporter gene and its regulation by cytokines found in autoimmunity. J Endocrinol, 1998. 158(3): p. 351-358.
22. Pekary, A.E. and J.M. Hershman, Tumor necrosis factor, ceramide, transforming growth factor-beta1, and aging reduce Na+/I- symporter messenger ribonucleic acid levels in FRTL-5 cells. Endo, 1997. 139(2): p. 703-712.
23. Pekary, A.E., et al., Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta 1 (TGF-beta 1) inhibit the expression and activity of Na+/K(+)-ATPase in FRTL-5 rat thyroid cells. J Interferon Cytokine Res, 1997. 17(4): p. 185-195.
24. Suzuki, K., et al., Autoregulation of thyroid-specific gene transcription by thyroglobulin. Proc Natl Acad Sci, 1998. 95(14): p. 8251-8256.
25. Suzuki, K., et al., Follicular thyroglobulin suppresses iodide uptake by suppressing expression of the sodium/iodide symporter gene. Endo, 1999. 140(11): p. 5422-5430.
26. Curado, M.P., et al., Cancer incidence in five continents, vol. IX. IARC Scientific publications, No.160, IARC, Lyon, France, 2007.
27. Pellegriti, G., et al., Worldwide increasing incidence of thyroid cancer: update on epidemiology and risk factors. J Cancer Epidemiol, 2013. 2013: p. 965212.
28. Davies, L. and H.G. Welch, Increasing incidence of thyroid cancer in the United States, 1973-2002. JAMA, 2006. 295(10): p. 2164-2167.
29. Siegel, R., et al., Cancer statistics, 2014. CA Cancer J Clin, 2014. 64(1): p. 9-29. 30. Lal, G., et al., Cancer of the endocrine system, in Abeloff's Clinical Oncology, 4th
edition, M.D. Abeloff, et al., Editors. 2008, Churchill Livingstone Elsevier: Philadelphia, PA.
31. Hundahl, S.A., et al., A National Cancer Data Base report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985-1995. Cancer, 1998. 83(12): p. 2638-2648.
32. RORENO, Registo Oncológico Regional do Norte 2009. Instituto Português de Oncologia do Porto, ed. Porto, 2015.
33. RORCentro, Registo Oncológico Nacional 2008. Instituto Português de Oncologia de Coimbra Franscisco Gentil-EPE, ed. ROR-Centro, 2014.
34. RORENO, Sobrevivência Global. Instituto Português de Oncologia do Porto, ed. Porto, 2007-2008.
35. O'Grady, T.J., M.A. Gates, and F.P. Boscoe, Thyroid cancer incidence attributable to overdiagnosis in the United States 1981-2011. Int J Cancer, 2015. [E-pub ahead of print].
36. Ahn, H.S., H.J. Kim, and H.G. Welch, Korea's thyroid-cancer "epidemic" - screening and overdiagnosis. N Engl J Med, 2014. 371: p. 1765-1767.
37. RORENO, Projecções da incidência de cancro na Região Norte - 2012, 2015 e 2020. Instituto Português de Oncologia do Porto, ed. Porto, 2013.
38. Kent, W.D., et al., Increased incidence of differentiated thyroid carcinoma and detection of subclinical disease. CMAJ, 2007. 177(11): p. 1357-1361.
39. Esserman, L.J., I.M. Thompson, and B. Reid, Overdiagnosis and overtreatment in cancer: an opportunity for improvement. JAMA, 2013. 310(8): p. 797-798.
40. Ron, E., et al., Thyroid cancer after exposure to external radiation: a pooled analysis of seven studies. Radiat Res, 1995. 141(3): p. 259-277.
41. Hemminki, K., C. Eng, and B. Chen, Familial risks for nonmedullary thyroid cancer. J Clin Endocrin Metab, 2005. 90(10): p. 5747-5753.
42. Prazeres, H., et al., The familial counterparts of follicular cell-derived thyroid tumors. Int J Surg Pathol, 2010. 18(4): p. 233-242.
43. Franceschi, S., et al., A pooled analysis of case-control studies of thyroid cancer. IV. Benign thyroid diseases. Cancer Causes Control, 1999. 10(6): p. 583-595.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
72
44. Knudsen, N., et al., Risk factors for goiter and thyroid nodules. Thyroid, 2002. 12(10): p. 879-888.
45. Negri, E., et al., A pooled analysis of case-control studies of thyroid cancer. II. Menstrual and reproductive factors. Cancer Causes Control, 1999. 10(2): p. 143-155.
46. Lind, P., et al., Epidemiology of thyroid diseases in iodine sufficiency. Thyroid, 1998. 8(12): p. 1179-1183.
47. Franceschi, S., Iodine intake and thyroid carcinoma - a potential risk factor. Exp Clin Endocrinol Diabetes, 1998. 106 (Suppl. 3): p. S38-S44.
48. Engeland, A., et al., Body size and thyroid cancer in two million Norwegian men and women. Br J Cancer, 2006. 95(3): p. 366-370.
49. Iribarren, C., et al., Cohort study of thyroid cancer in a San Francisco Bay area population. Int J Cancer, 2001. 93(5): p. 745-750.
50. DeLellis, R.A., et al., World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Endocrine Glands. IARC Press, Lyon, 2004.
51. Nikiforov, Y.E. and M.N. Nikiforova, Molecular genetics and diagnosis of thyroid cancer. Nat Rev Endocrinol, 2011. 7(10): p. 569-80.
52. Ravetto, C., L. Colombo, and M.E. Dottorini, Usefulness of fine-needle aspiration in the diagnosis of thyroid carcinoma: a retrospective study in 37,895 patients. Cancer, 2000. 90: p. 357-363.
53. Luster, M., et al., Guidelines for radioiodine therapy of differentiated thyroid cancer. Eur J Nucl Med Mol Imaging, 2008. 35(10): p. 1941-1959.
54. Katoh, R., et al., Multiple thyroid involvement (intraglandular metastasis) in papillary thyroid carcinoma. A clinicopathologic study of 105 consecutive patients. Cancer, 1992. 70(6): p. 1585-1590.
55. Tollefsen, H.R., J.P. Shah, and A.G. Huvos, Papillary carcinoma of the thyroid. Recurrence in the thyroid gland after initial surgical treatment. Am J Surg, 1972. 124(4): p. 468-472.
56. Cooper, D.S., et al., Management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid, 2006. 16(2): p. 109-142.
57. Force, T.C.T., AACE/AAES medical/surgical guidelines for clinical practice: management of thyroid carcinoma. Endocr Pract, 2001. 7(3): p. 202-220.
58. Zhao, L., et al., Ultrasound-guided fine-needle aspiration biopsy of thyroid bed lesions from patients with thyroidectomy for thyroid carcinomas. Cancer Cytopathol, 2013. 121(2): p. 101-107.
59. Spencer, C.A., et al., Detection of residual and recurrent differentiated thyroid carcinoma by serum thyroglobulin measurement. Thyroid, 1999. 9(5): p. 435-441.
60. Nikiforov, Y.E., Thyroid carcinoma: molecular pathways and therapeutic targets. Mod Pathol, 2008. 21 Suppl 2: p. S37-43.
61. Xing, M., Molecular pathogenesis and mechanisms of thyroid cancer. Nat Rev Cancer, 2013. 13(3): p. 184-99.
62. Xing, M., et al., BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrin Metab, 2005. 90(12): p. 6373-6379.
63. Riesco-Eizaguirre, G., et al., The oncogene BRAF V600E is associated with a high risk of recurrence and less differentiated papillary thyroid carcinoma due to the impairment of Na+/I- targeting to the membrane. Endocr Relat Cancer, 2006. 13(1): p. 257-69.
64. Nikiforova, M.N. and Y.E. Nikiforov, Molecular diagnostics and predictors in thyroid cancer. Thyroid, 2009. 19(12): p. 1351-1361.
65. Garcia-Rostan, G., et al., ras mutations are associated with aggressive tumor phenotypes and poor prognosis in thyroid cancer. J Clin Oncol, 2003. 21(17): p. 3226-3235.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
73
66. Adeniran, A.J., et al., Correlation between genetic alterations and microscopic features, clinical manifestations, and prognostic characteristics of thyroid papillary carcinomas. Am J Surg Pathol, 2006. 30(2): p. 216-222.
67. Karga, H., et al., Ras oncogene mutations in benign and malignant thyroid neoplasms. J Clin Endocrin Metab, 1991. 73(4): p. 832-836.
68. Heidenreich, B., et al., TERT promoter mutations in cancer development. Curr Opin Genet Dev, 2014. 24: p. 30-37.
69. Vinagre, J., et al., Frequency of TERT promoter mutations in human cancers. Nat Commun, 2013. 4(2185): p. 1-6.
70. Hoang-Vu, C., et al., Expression of telomerase genes in thyroid carcinoma. Int J Oncol, 2002. 21(2): p. 265-272.
71. Melo, M., et al., TERT promoter mutation are a major indication of poor outcome in differentiated thyroid carcinomas. J Clin Endocrin Metab, 2014. 99(5): p. E754-E765.
72. Miyagi, E., et al., Chronic expression of RET/PTC 3 enhances basal and insulin-stimulated PI3 kinase/Akt signaling and increases IRS-2 expression in FRTL-5 thyroid cells. Mol Carcinog, 2004. 41(2): p. 98-107.
73. de Vries, M.M., et al., RET/PTC rearrangement is prevalent in follicular Hurthle cell carcinomas. Histopathology, 2012. 61(5): p. 833-843.
74. Kroll, T.G., et al., PAX8-PPARgamma1 fusion oncogene in human thyroid carcinoma. Science, 2000. 289(5483): p. 1357-1360.
75. Omur, O. and Y. Baran, An update on molecular biology of thyroid cancers. Crit Rev Oncol Hematol, 2014. 90(3): p. 233-52.
76. Nikiforova, M.N., et al., RAS point mutations and PAX8-PPARg rearrangement in thyroid tumors: evidence for distinct molecular pathways in thyroid follicular carcinomas. J Clin Endocrin Metab, 2003. 88(5): p. 2318-2326.
77. De Luca, A., et al., The RAS/RAF/MEK/ERK and the PI3K/AKT signaling pathways: role in cancer pathogenesis and implications for therapeutic approaches. Expert Opin Ther Targets, 2012. 16(S2): p. S17-S27.
78. Durante, C., et al., BRAF mutations in papillary thyroid carcinomas inhibit genes involved in iodine metabolism. J Clin Endocrinol Metab, 2007. 92(7): p. 2840-3.
79. Chien, W. and H.P. Koeffler, Molecular Biology of Thyroid Cancer. Thyroid Cancer, Endocrine Updates 30, 2012. 32: p. 35-43.
80. Hou, P., D. Liu, and M. Xing, Genome-wide alterations in gene methylation by the BRAF V600E mutation in papillary thyroid cancer cells. Endocr Relat Cancer, 2011. 18(6): p. 687-697.
81. Oler, G., et al., Gene expression profiling of papillary thyroid carcinomas identifies transcripts correlated with BRAF mutational status and lymph node metastasis. Clin Cancer Res, 2008. 14(15): p. 4735-4742.
82. Jo, Y.S., et al., Influence of the BRAF V600E mutation on expression of vascular endothelial growth factor in papillary thyroid cancer. J Clin Endocrin Metab, 2006. 91(9): p. 3667-3670.
83. Palona, I., et al., BRAFV600E promotes invasiveness of thyroid cancer cells through nuclear factor kB activation. Endocrinology, 2006. 147(12): p. 5699-5707.
84. Mesa, C., et al., Conditional activation of RET/PTC3 and BRAFV600E in thyroid cells is associated with gene expression profiles that predict a preferential role of BRAF in extracellular matrix remodeling. Cancer Res, 2006. 66(13): p. 6521-6529.
85. Watanabe, R., et al., Possible involvement of BRAFV600E in altered gene expression in papillary thyroid cancer. Endocr J, 2009. 56(3): p. 407-414.
86. Zerilli, M., et al., BRAF(V600E) mutation influences hypoxia-inducible factor-1a expression levels in papillary thyroid cancer. Mod Pathol, 2010. 23(8): p. 1052-1060.
87. Riesco-Eizaguirre, G., et al., The BRAFV600E oncogene induces transforming growth factor beta secretion leading to sodium iodide symporter repression and increased malignancy in thyroid cancer. Cancer Res, 2009. 69(21): p. 8317-25.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
74
88. Vanhaesebroeck, B., et al., The emerging mechanisms of isoform-specific PI3K signaling. Nat Rev Mol Cell Biol, 2010. 11(5): p. 329-341.
89. Xing, M., Genetic alterations in the phosphatidylinositol-3 kinase/Akt pathway in thyroid cancer. Thyroid, 2010. 20(7): p. 697-706.
90. Cantley, L.C. and B.G. Neel, New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci, 1999. 96(8): p. 4240-4245.
91. Costa, A.M., et al., BRAF mutation associated with other genetic events identifies a subset of agressive papillary carcinoma. Clin Endocrinol (Oxf), 2008. 68(4): p. 618-634.
92. Wu, G., et al., Uncommon mutation, but common amplifications, of the PIK3CA gene in thyroid tumors. J Clin Endocrin Metab, 2005. 90(8): p. 4688-4693.
93. Ringel, M.D., et al., Overexpression and overactivation of Akt in thyroid carcinoma. Cancer Res, 2001. 61(16): p. 6105-6111.
94. Bartholomeusz, C. and A.M. Gonzalez-Angulo, Targeting the PI3K signaling pathway in cancer therapy. Expert Opin Ther Targets, 2012. 16(1): p. 121-130.
95. Keniry, M. and R. Parsons, The role of PTEN signaling perturbations in cancer and in targeted therapy. Oncogene, 2008. 27(41): p. 5477-5485.
96. Di Cristofano, A., et al., Pten is essential for embryonic development and tumour suppression. Nat Genet, 1998. 19(4): p. 348-355.
97. Keith, C.T. and S.L. Schreiber, PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints. Science, 1995. 270(5233): p. 50-51.
98. Huang, S. and P.J. Houghton, Targeting mTOR signaling for cancer therapy. Curr Opin Pharmacol, 2003. 3(4): p. 371-377.
99. Loewith, R., et al., Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth. Mol Cell, 2002. 10(3): p. 457-468.
100. Hay, N. and N. Sonenberg, Upstream and downstream of mTOR. Genes Dev, 2004. 2004(18): p. 16.
101. Sarbassov, D.D., et al., Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol, 2004. 14(14): p. 1296-1302.
102. Populo, H., J.M. Lopes, and P. Soares, The mTOR signalling pathway in human cancer. Int J Mol Sci, 2012. 13(2): p. 1886-918.
103. Meric-Bernstam, F. and A.M. Gonzalez-Angulo, Targeting the mTOR signaling network for cancer therapy. J Clin Oncol, 2009. 27(13): p. 2278-87.
104. Vander Haar, E., et al., Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol, 2007. 9(3): p. 316-323.
105. Dann, S.G. and G. Thomas, The amino acid sensitive TOR pathway from yeast to mammals. FEBS Lett, 2006. 580(12): p. 2821-2829.
106. Inoki, K., T. Zhu, and K.L. Guan, TSC2 mediates cellular energy response to control cell growth and survival. Cell, 2003. 115(5): p. 577-590.
107. Sofer, A., et al., Regulation of mTOR and cell growth in response to energy stress by REDD1. Mol Cell Biol, 2005. 25(14): p. 5834-5845.
108. Ma, L., et al., Phosphorylation and functional activation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell, 2005. 121(2): p. 179-193.
109. Carriere, A., et al., Oncogenic MAPK signalling stimulates mTORC1 activity by promoting RSK-mediated raptor phosphorylation. Curr Biol, 2008. 18(17): p. 1269-1277.
110. Carriere, A., et al., ERK1/2 phosphorylate Raptor to promote Ras-dependent activation of mTOR complex 1 (mTORC1). J Biol Chem, 2011. 286(1): p. 567-577.
111. de Souza, E.C., et al., MTOR downregulates iodide uptake in thyrocytes. J Endocrinol, 2010. 206(1): p. 113-20.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
75
112. Plantinga, T.S., et al., mTOR Inhibition promotes TTF1-dependent redifferentiation and restores iodine uptake in thyroid carcinoma cell lines. J Clin Endocrinol Metab, 2014. 99(7): p. E1368-75.
113. Faustino, A., et al., mTOR pathway overactivation in BRAF mutated papillary thyroid carcinoma. J Clin Endocrinol Metab, 2012. 97(7): p. E1139-49.
114. Shaha, A.R., Recurrent differentiated thyroid cancer. Endocr Pract, 2012. 18(4): p. 600-603.
115. Lin, F., et al., Morphoproteomic and molecular concomitants of an overexpressed and activated mTOR pathway in renal cell carcinomas. Ann Clin Lab Sci, 2006. 36(3): p. 283-293.
116. Pelloski, C.E., et al., Prognostic associations of activated mitogen-activated protein kinase and Akt pathways in glioblastoma. Clin Cancer Res, 2006. 12(13): p. 3935-3941.
117. Populo, H., et al., mTOR pathway activation in cutaneous melanoma is associated with poorer prognosis characteristics. Pigment Cell Melanoma Res, 2011. 24(1): p. 254-257.
118. Komori, Y., et al., Mammalian target of rapamycin signaling activation patterns in pancreatic neuroendocrine tumors. J Hepatobiliary Pancreat Sci, 2014. 21(4): p. 288-295.
119. Chung, J.Y., et al., The expression of phospho-AKT, phospho-mTOR, and PTEN in extrahepatic cholangiocarcinoma. Clin Cancer Res, 2009. 15(2): p. 660-7.
120. Clark, C., et al., Teasing out the best molecular marker in the AKT/MTOR pathway in HNSCC patients. Laryngoscope, 2010. 120(6): p. 1159-1165.
121. Beca, F., et al., p-mTOR expression is associated with better prognosis in luminal breast cancer. J Clin Pathol, 2014. 67(11): p. 961-967.
122. Ikeda, S., et al., Expression of phosphorylated Akt in patients with small cell carcinoma of the lung indicates good prognosis. Pathol Int, 2010. 60(11): p. 714-719.
123. McDonald, J.M., et al., Elevated phospho-S6 expression is associated with metastasis in adenocarcinoma of the lung. Clin Cancer Res, 2008. 14(23): p. 7832-7837.
124. Pantuck, A.J., et al., Prognostic relevance of the mTOR pathway in renal cell carcinoma: implications for molecular patient selection for targeted therapy. Cancer, 2007. 109(11): p. 2257-2267.
125. Ahmed, M., et al., High prevalence of mTOR complex activity can be targeted using Torin2 in papillary thyroid carcinoma. Carcinogenesis, 2014. 35(7): p. 1564-72.
126. Pringle, D.R., et al., Follicular thyroid cancers demonstrate dual activation of PKA and mTOR as modeled by thyroid-specific deletion of Prkar1a and Pten in mice. J Clin Endocrinol Metab, 2014. 99(5): p. E804-12.
127. Tamburrino, A., et al., Activation of the mTOR pathway in primary medullary thyroid carcinoma and lymph node metastases. Clin Cancer Res, 2012. 18(13): p. 3532-40.
128. Lyra, J., et al., mTOR activation in medullary thyroid carcinoma with RAS mutation. Eur J Endocrinol, 2014. 171(5): p. 633-640.
129. Kouvaraki, M.A., et al., Activation of mTOR signaling in medullary and aggressive papillary thyroid carcinomas. Surgery, 2011. 150(6): p. 1258-65.
130. Choe, G., et al., Analysis of the phosphatidylinositol 3'-kinase signaling pathway in glioblastoma patients in vivo. Cancer Res, 2003. 63(11): p. 2742-2746.
131. Copp, J., G. Manning, and T. Hunter, TORC-specific phosphorylation of mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR signaling complex 2. Cancer Res, 2009. 69(5): p. 1821-7.
132. Moraitis, D., et al., SIN1, a critical component of the mTOR-Rictor complex, is overexpressed and associated with AKT activation in medullary and aggressive papillary thyroid carcinomas. Surgery, 2014. 156(6): p. 1542-1548.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
76
133. Gupta, S., et al., Mammalian target of rapamycin complex 2 (mTORC2) is a critical determinant of bladder cancer invasion. PLoS One, 2013. 8(11): p. e81081.
134. Gulhati, P., et al., mTORC1 and mTORC2 regulate EMT, motility, and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways. Cancer Res, 2011. 71(9): p. 3246-3256.
135. Masri, J., et al., mTORC2 activity is elevated in gliomas and promotes growth and cell motility via overexpression of rictor. Cancer Res, 2007. 67(24): p. 11712-11720.
136. Qiao, M., J.D. Iglehart, and A.B. Pardee, Metastatic potential of 21T human breast cancer cells depends on Akt/Protein kinase B activation. Cancer Res, 2007. 67(11): p. 5293-5299.
137. Khan, M.W., et al., mTORC2 controls cancer cell survival by modulating gluconeogenesis. Cell Death Discovery, 2015. 1: p. 15016.
138. Masui, K., W.K. Cavenee, and P.S. Mischel, mTORC2 is the center of cancer metabolic reprogramming. Trends Endocrinol Metab, 20141. 25(7): p. 364-373.
139. Zoncu, R., D.M. Sabatini, and A. Efeyan, mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol, 2011. 12(1): p. 21-35.
140. Sarbassov, D.D., et al., Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell, 2006. 21(22): p. 159-168.
141. Lamming, D.W., et al., Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. Science, 2012. 335(6076): p. 1638-1643.
142. Wolpin, B.M., et al., Oral mTOR inhibitor everolimus in patients with gemcitabine-refractory metastatic pancreatic cancer. J Clin Oncol, 2009. 27(2): p. 193-198.
143. Atkins, M.B., et al., Levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma. J Clin Oncol, 2004. 22(5): p. 909-918.
144. Johnston, P.B., et al., A phase II trial of the oral mtor inhibitor everolimus in relapsed Hodgkin lymphoma. Am J Hematol, 2010. 85(5): p. 320-324.
145. Witzig, T.E., et al., A phase II trial of the oral mTOR inhibitor everolimus in relapsed aggressive lymphoma. Leukemia, 2011. 25(2): p. 341-347.
146. Ellard, S.L., et al., Randomized phase II study comparing two schedules of everolimus in patients with recurrent/metastatic breast cancer: NCIC clinical trials group IND.163. J Clin Oncol, 2009. 27(27): p. 4536-4541.
147. Oza, A.M., et al., Phase II study of temsirolimus in women with recurrent or metastatic endometrial cancer: a trial of the NCIC clinical trials group. J Clin Oncol, 2011. 29(24): p. 3278-3285.
148. Hess, G., et al., Phase III study to evaluate temsirolimus compared with investigator's choice therapy for the treatment of relapsed or refractory mantle cell lymphoma. J Clin Oncol, 2009. 27(23): p. 3822-3829.
149. Thoreen, C.C., et al., An ATP-competitve mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem, 2009. 284(12): p. 8023-8032.
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
77
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
78
SUPPLEMENTARY DATA
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
79
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
80
SUPPLEMENTARY TABLES Table 1 – Cases with missing information within the whole series (n=90), in PTC (n=73) and in FTC (n=13) in Series 1
Table 2 – Cases with missing information within cPTC (n=39) and fvPTC (n=23) in Series 1
Variables All cases (n=90)
PTC (n=73)
FTC (n=13)
Age (%)
1 (1.1)
1 (1.4)
-
Gender (%)
1 (1.1)
1 (1.4)
-
Tumor size (%)
1 (1.1)
1 (1.4)
-
Tumor capsule (%)
2 (2.2)
2 (2.7)
-
Tumor capsule invasion (%)
3 (5.7)
1 (2.6)
2 (15.4)
Multifocality (%)
2 (2.2)
2 (2.7)
-
Vascular invasion (%)
- - -
Lymph node metastasis (%)
15 (16.7)
11 (15.1)
3 (23.1)
Extrathyroid extension (%)
4 (4.4)
4 (5.5)
-
Lymphocytic infiltrate (%)
3 (3.3)
1 (1.4)
2 (15.4)
TERT promoter mutation (%)
- - 1 (7.7)
NRAS mutation (%)
- - 1 (7.7)
BRAF mutation (%)
- - 1 (7.7)
PAX8/PPARγ rearrangement
(%)
1 (1.1)
1 (1.4)
1 (7.7)
RET/PTC rearrangement (%)
- - 1 (7.7)
Staging (%)
6 (6.7)
5 (6.8)
-
Variables cPTC (n=39)
fvPTC (n=23)
Age (%)
- 1
(4.3)
Gender (%)
- 1
(4.3)
Tumor size (%)
1 (2.6)
-
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
81
Table 3 – Distribution of p-S6 scores in each variant of PTC (n=73) in Series 1
Score cPTC fvPTC sPTC tcPTC oPTC stPTC mixed PTC
0 11 3 2 0 0 0 1
1 4 4 0 0 1 0 0
2 5 0 0 0 0 0 2
3 4 1 0 0 0 0 0
4 5 3 0 1 0 1 0
6 3 5 0 0 0 0 1
8 2 2 0 0 0 0 0
9 2 2 1 0 0 0 1
12 3 3 0 0 0 0 0
Total 39 23 3 1 1 1 5
Table 4 – Distribution of p-mTOR scores in each variant of PTC (n=73) in Series 1
Score cPTC fvPTC sPTC tcPTC oPTC stPTC mixed PTC
0 2 3 0 0 0 0 0
1 1 2 0 0 0 0 0
2 1 2 1 0 0 0 0
3 2 2 0 0 0 0 0
Tumor capsule (%)
1 (2.6)
1 (4.3)
Tumor capsule invasion (%)
- 1
(7.7)
Multifocality (%)
2 (5.1)
-
Vascular invasion (%)
- -
Lymph node metastasis (%)
6 (15.4)
4 (17.4)
Extrathyroid extension (%)
1 (2.6)
2 (8.7)
Lymphocytic infiltrate (%)
- 1
(4.3)
TERT promoter (%)
- -
NRAS (%)
- -
BRAF (%)
- -
PAX8/PPARγ
(%)
1 (2.6)
-
RET/PTC (%)
- -
Staging (%)
1 (2.6)
3 (13)
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
82
4 5 2 0 0 0 1 0
6 10 1 1 0 0 0 1
9 6 3 0 1 0 0 1
12 12 8 1 0 1 0 3
Total 39 23 3 1 1 1 5
Table 5 – Cases with missing information within the whole series (n=127), in PTC (n=114) and in FTC (n=6) in Series 2
Variables All cases (n=127) PTC (n=114) FTC (n=6)
Age (%)
1 (0.8)
- -
Gender (%)
- 1
(0.9) -
Lymph node metastases (%)
1 (0.8)
1 (0.9)
-
Distant metastases (%)
1 (0.8)
1 (0.9)
-
Tumor size (%)
10 (7.9)
6 (5.3)
2 (33.3)
Tumor capsule (%)
7 (5.5)
4 (3.5)
1 (16.7)
Vascular invasion (%)
10 (7.9)
9 (6.1)
1 (16.7)
Tumor margins (%)
7 (5.5)
4 (3.5)
1 (16.7)
Thyroid capsule invasion (%)
11 (8.7)
7 (6.1)
1 (16.7)
Extrathyroid extension (%)
8 (6.3)
4 (3.5)
1 (16.7)
Multifocality (%)
9 (7.1)
6 (5.3)
1 (16.7)
Lymphocytic infiltrate (%)
10 (7.9)
7 (6.1)
2 (33.3)
Staging (%)
9 (7.1)
6 (5.3)
1 (16.7)
TERT promoter mutation (%)
26 (20.5)
24 (21.1)
2 (33.3)
NRAS mutation (%)
16 (12.6)
14 (12.3)
2 (33.3)
BRAF mutation (%)
4 (3.1)
4 (3.5)
-
One year disease free survival (%)
4 (3.1)
4 (3.5)
1 (16.7)
Evidence of disease during follow-up (%)
- - 1
(16.7)
Number of 131I therapies (%)
4 (3.1)
4 (3.5)
-
Cumulative dose (%)
5 (3.9)
5 (4.4)
-
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
83
Table 6 – Cases with missing information within cPTC (n=77) and in fvPTC (n=28) in Series 2
Disease free survival after follow-up (%)
3 (2.4)
3 (2.6)
-
Variables cPTC (n=77) fvPTC (n=28)
Tumor size (%)
6 (7.8)
-
Tumor capsule (%)
4 (5.2)
-
Vascular invasion (%)
6 (7.8)
1 (3.6)
Tumor margins (%)
4 (5.2)
-
Thyroid capsule invasion (%)
6 (7.8)
1 (3.6)
Extrathyroid extension (%)
4 (5.2)
-
Multifocality (%)
5 (6.5)
-
Lymphocytic infiltrate (%)
6 (7.8)
1 (3.6)
Staging (%)
4 (5.2)
1 (3.6)
TERT promoter mutation (%)
15 (19.5)
5 (17.9)
NRAS mutation (%)
- 5
(17.9)
BRAF mutation (%)
2 (2.6)
2 (7.1)
One year disease free survival (%)
3 (3.9)
-
Number of 131I therapies (%)
2 (2.6)
-
Cumulative dose (%)
3 (3.9)
-
Disease free survival after follow-up (%)
2 (2.6)
-
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
84
Table 7 – Distribution of p-S6 scores in each variant of PTC (n=76) in Series 2
Score cPTC fvPTC sPTC oPTC wPTC
0 25 7 0 1 1
1 8 2 1 0 1
2 7 4 0 0 0
3 3 3 1 0 0
4 2 0 1 0 0
6 3 1 1 0 0
9 1 1 0 0 0
12 2 0 0 0 0
Total 51 18 4 1 2
Table 8 – Distribution of p-mTOR scores in each variant of PTC (n=107) in Series 2
Score cPTC fvPTC sPTC oPTC tcPTC wPTC
0 23 8 0 0 1 1
1 7 0 0 0 0 0
2 11 3 1 0 0 0
3 3 3 2 0 0 0
4 6 3 0 0 0 0
6 7 2 0 0 0 0
8 1 1 1 0 0 0
9 7 3 0 0 0 1
12 8 2 0 0 0 1
Total 73 25 4 1 1 3
Table 9 – Correlation between p-S6 and p-mTOR in Series 1
All cases (n=90) PTC (n=73) cPTC (n=39) fvPTC (n=23)
P-value 0.680 0.962 0.528 0.967
Pearson Correlation 0.044 -0.006 0.104 -0.009
Table 10 – Correlation between p-S6 and p-mTOR in Series 2
All cases (n=127) PTC (n=114) cPTC (n=77) fvPTC (n=28)
P-value 0.988 0.753 0.700 0.360
Pearson Correlation -0.002 -0.038 -0.057 0.237
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
85
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
86
SUPPLEMENTARY FIGURES
Figure 2 – Representative case of the excluded cases from Series 2. This case is a cPTC with cancer cells surrounding the follicles and lymphocytes infiltrating the tissue (200x magnification).
Figure 1 – Positive controls. The tissue used as a positive control is a lymph node invaded by cancer cells. We can see in a brown coloration the cells stained for the marker and in blue the lymphocytes (200x magnification). A – p-S6 ; B – p-mTOR.
A B
A B
Figure 3 – Staining of p-S6. p-S6 shows a preferential cytoplasmic localization (brown coloration) in tumor cells. A – cPTC from Series 2; B – fvPTC from Series 1 (200x magnification).
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
87
A B
Figure 4 – Representative image of p-mTOR staining. p-mTOR was localized both in cytoplasm and membrane of tumor cells (brown coloration). A – Warthin like PTC from Series 2; B – cPTC from Series 1 (200x magnification).
Role of mTOR Pathway in Differentiated Thyroid Cancer MSc in Oncology Thesis Dissertation
88
UNIVERSIDADE DO PORTO