Nuove molecole e peptidi quali farmaci regolatori del ciclo cellulare e della risposta a chemioterapici nei tumori epiteliali e cutaneiCoordinatore del Progetto: Prof. Gerry Melino
PROGRAMMA 3Trasferimento delle conoscenze allo sviluppo di interventi volti a prevenire, diagnosticare e trattare il cancro.
• UO 1: Prof. Gerry Melino (IDI-IRCCS)E.Candi (Universita’ Tor Vergata)
• UO 2: Dott. Giovanni Blandino (Ist. Naz. Tumori Regina Elena)Prof.ssa MG Santoro (Universita’ di Tor Vergata)Dott.ssa L Lanfrancone (Istituto Europeo di Oncologia)
• UO 3: Dott.ssa Stefania D’Atri (IDI-IRCCS)Dott.ssa E Alvino (CNR, Ist. Neurobiologia e Med. Molec.)Dott. U Pfeffer (Ist. Naz. Per la Ricerca sul Cancro)Prof. Golino (Universita’ della Sapienza)
OBIETTIVO PRINCIPALE del PROGETTO:Identificare nuove molecole e peptidi quali farmaci regolatori del ciclo cellulare e della risposta a chemioterapici nei tumori epiteliali e cutanei
• Sviluppare nuove molecole inibitori della E3 ubiquitin ligasi ITCH e di validare tali molecole nella via di p63/p73 e di Hedgehog/Gli;
• Studio del potenziale terapeutico di pepidi capaci di disassemblare complessi proteici (mp53/p73) ad attivita’oncogenica;
• Studio del potenziale terapeutico di inibitori selettivi di NF-kB/IKK e delle cicline-chinasi dipendenti (CDK), come singoli agenti ed in associazione agli inibitori di ITCH e ai chemoterapici;
• Studio dell’espressione della proteina chinasica RaLP nei melanomi e caratterizzazione del pathway di trasduzione del segnale.
OBIETTIVI SPECIFICI:
ab
c
The p53 family
Epidermal
Neural
lethal
severe
minimalCancer
91% 83%
80%
McKeon F & Melino G Fog of war: the emerging p53 family. Cell Cycle. (2007) 6, 3: 229-232Melino G, De Laurenzi V, Vousden KH p73: friend or foe in tumorigenesis. Nature Review Cancer. (2002) 2: 605-615Melino G, Lu X, Crook T, Knight RA Functional studies of p73 and p63: Development and Cancer TIBS. (2003) 28: 663
DNA damage Replicative Stress
Cell Cyle ArrestApoptosis
Sensors
Effectors
Signals
EFFECTS
ALTERATIONS
ATM ATR
p53chk1
chk2
p21gadd45 ….
mdm2
c-abl
bax CD9514-3-3Noxa ….
MRE11rad50
BRCA1NSB1
DNA-PK PARP
cdc25
G1 S G2
Ku70/Ku86
14-3-3cdc25
Cdc2 ….
DNA Repair
HRNHEJ
NERMMRBERp73p63
Gressner et al Embo J 2005. 24: 2458Mueller et al CDD 2005. 12: 1564Flinterman et al. JBC 2005. 280: 5945
Gong et al Nature 1999. 399: 806Melino et al. JBC 2004. 279: 8076Bernassola et al JEM 2004. 199: 1545
Vigano et al Embo J 2006. 25: 5105Rossi et al PNAS 2006. 103: 12753Sayan et al. JBC 2006. 281: 13566
p53
Senescence Apoptosis Cell Cycle Arrest
ONCOGENES
DNA damage
Ub E3 ligase inhib.nutlin
activatorsPRIMA-1
DNA methyltransferase inhibitors ARF
mdm2
ITCH E3 ligase regulates p63 stability
Itch
Ubiquitin E3 ligase (NEDD4 family)
18H Mice natural Knockout (immune defects)
Interacts with Atrophin
Degrades (transcription factor):Jun-B, c-Jun, Notch, FLIP
HECTcWW WWC2Ca2+ dependent lipids interaction
protein-protein interacting domain
catalytic domain transfering ubiquitin to substrate
CN
Phage Display
TA DNA Binding Domain OD SAM PR PRCN TAp63
UbiquitylationBinding
ITCH wtITCH mut
p63 p53
IP: p63/p53 WB: HA(Ub)
Ubiquitylatedp63
pcDNA Cyclo-hexamide (h) 0 1 2 4 6 8
< p63
< tubulinpcDNA
ITCH
< p63
< tubulin
Degraded p63
Degradation
ITCH wtITCH mut
p63 p53pcDNA
IP: Itch WB: p63/p53
p63 bound
Can we control p63/p73 protein levels?
HighTroughPut screening
Open question
Itch inhibitors search by HTP
48 Hits cut off < 50%287 Hits cut off < 70%
Ar ArX
S
O O
N Ar
R
Rigel X= O, S
Stability/Itch binding studies
PNAS, 2006. 103, 34: 12753-12758
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
PNAS, 2007. 104, 27: 11280-11285
YAP, WwoxJNK
p63ITCH Ub, degradation
N4BP1
p73
- p63 is involved in DNA repair/Cancer - p63 regulates chemosensitivity in cancer - p63 is transcriptionally regulated by PML- p63 is Ub & degraded by Itch- Itch is regulated by N4BP1- low MW Itch inhibitors are under development
ITCH is a candidatetherapeutical target
(to regulate p63/p73)
perspective
THERAPY Conclusion 1
E1= 10
E2= 100
E3= 1000
Proteasome= 1
human
Non-Apoptotic Function
p63 function ?
wt p63-/- p63-/-;ΔN
+/+ -/-
TIFF (Uncompressed) dare needed to see thecompressor
is picture
Failure ofstem cells
D Roop F McKeon
Failure ofdifferentiation
CornifiedEnvelope
Granular
Basal
Spinous
Membr TAp63ΔNp63 stem
cell
CE
ΔNp63
TAcell
keratinocytes
TAp63
K14K14integrinsintegrins
IKKaIKKa
EtsEts--11PERP, Gata3PERP, Gata3
p63 function ?
p63 mutations in human syndromes
TA DBD OD SAM
D312H I541TQ540L
T537PG534VC526W
C526G
L518FL518V
Q634X
C308S
R304WR304QR279C
R279HR279Q
S272N P309S
C306R
R280CR280HR280S
R204WR204Q C269YR227Q
K193EK194E
R298Q
N CPR TA2 TI
EEC, (SHFM) SHFMAEC
EECADULTAEC
Ectrodactyly, Ectodermal dysplasia and Cleft lip/palate syndrome
Acro-Dermato-Ungual-Larimal-Tooth syndrome
Split Hand-split Foot Malformations
Ankyloblepharon-Ectodermal dysplasia-Clefting (or Hay-Wells Syndrome)
SHFMLimb-Mammary Syndrome LMS
p63 regulation: miR
miR are essential for epithelial development
K14:Dicer1 Knockout
- Few Hair follicle (invaginated)- Abnormal Epithelial Layers (thin, granules)
miR expressed in skin
miRs expressedin epidermis and hair follicle
miRs detected with high signal intensityin microarray of control mouse skin(Andl et al, CB 2006 and in Yi et al,Nature Genetics 2006)
mouse
Table 1. miRs expressed in epidermis and hair follicle miRs expressed in hair follicle
miRs expressed in epidermis
Expression and/or biological function
Target [ref]
miR-15a nd Leukemia miR-15b miR-15b Leukemia, antigen incuced T cell
differentiation Bcl-2 [26]
miR-16 miR-16 Leukemia, antigen incuced T cell differentiation
miR-17-5p miR-17-5p T cells, monocytes differentiation AML-1 [27] miR-18 miR-18 ? nd miR-19b T cells, leukemia Mylip, Rbp1like
[28] miR-20 miR-20 Megakaryiocytopoiesis miR-21 miR-21 Invasion, metastasis, Squamous Cell
Carcinoma, antigen incuced T cell differentiation
TPM1, PDCD4, maspin [29]
miR-24 miR-24 Myogenic differentiation nd miR-27a Breast cancer Myt-1, ZBTB10
[30] miR-27b miR-27b Angiogenesis CYP1B1 [31] nd miR-30b ? nd miR-34a Cancer apoptosis Bcl-2 [32] miR-92 miR-92 Lymphoma, B cell development PTEN, Bim [33] nd miR-93 Gastric cancer p21,E2F1, Bim
[34] miR-99b miR-99b Megakaryiocytopoiesis miR-106b miR-106b Gastric cancer p21,E2F1, Bim
[34] nd miR-125a Breast cancer ERBB2, ERBB3
[35] miR-125b miR-125b Breast cancer, ovarian cancer ERBB2, ERBB3
[35] miR-126 nd Endotelial cells/leukocytes adherence,
breast cancer VCAM1, SOX4, V-CRK [36;37]
miR-127 miR-127 Tumor suppression BCL6 [38] miR-130a miR-130a Angiogenesis, Megakaryiocytopoiesis GAX, HOXA5
[39] nd miR-133b Colorectal cancer nd miR-141 Placenta, EMT ZEB1, ZEB2
[40] miR-143 nd Adipocytes differentiation, B-cell
cancer ERK5 [41;42]
miR-152 nd Breast cancer nd miR-191 Leukemia miR-196 nd Limb development, myeloid
differentiation HOXB8 [43]
miR-199a nd Leukemia, ovarian cancer miR-199b nd ? nd miR-200a Ovarian cancer, EMT ZEB1, ZEB2
[40] nd miR-200b Ovarian cancer, brain, EMT Zfhx1b [44]
nd miR-200c Ovarian cancer, leukemia ZEB1, ZEB2 [40;45]
nd miR-203 Skin, hair, ovary, Squamous Cell Carcinoma
p63 [22;23] Zfp281 [22] SOCS-3 [46]
nd miR-205 Ovarian cancer, Squamous Cell Carcinoma
miR-206 nd Skeletal muscle Pol-a , Cx43, ERa, Ftl1 [47]
miR-214 nd Ovarian cancer PTEN [48] miR-351 nd ? nd miR-429 EMT ZEB1, ZEB2
[40;45] Let-7a Let-7a Lymphome Myc [49] Let-7b Let-7b Acute lymphoblastic leukemia Let-7c Let-7c ? Let-7d nd ? Let-7f Let-7f Angiogenesis, antigen induced Tcell
differentiation
Let-7g Let-7g ? Let-7i Let-7i Immune responce TLRs [50]
miR expressed in skin
miRs showing more than 1.7 fold changebetween normal skin and psoriasis andnormal skin and atopic eczema(Sonkoy et al PLoS ONE 2006)
miRs expression in psoriasis and atopic eczema
human
Psoriasis Atopic eczema miRs up regulated
miRs down regulated
miRs up regulated
miRs down regulated
miR-17-5p miR-10a miR-17-5p miR-122a miR-20a miR-22 miR-20a miR-133a miR-21 miR-30c miR-21 miR-133b miR-30e-5p miR-99b miR-24 miR-215 miR-31 miR-100 miR-27a miR-326 miR-106a miR-122a miR-29a miR-335 miR-142-3p miR-125b miR-106b miR-483 miR-146b miR-133a miR-146a miR-515-5p miR-146a miR-133b miR-193a miR-519 miR-200a miR-197 miR-199a miR-141 miR-215 miR-146 miR-203 miR-326 miR-365 miR-381 miR-518b miR-524 let-7e
miR-203 inversely correlates to p63
miR-203a 0 1 3 5nt days diff.
0 1 3 5p63
involucrin
actin
Mw(KDa) days diff.
56 -
80 -
48 -
U2snRNA
22 -
185 -
Arb
itrar
y un
its
0 1 3 50 1 3 50
8
16
24miR-203
0
40
80
120 p63
days diff.
1 2 3 4 lanes
1 2 3 4 lanes
b 0 3 5 7 days diff.nt
miR-203
U2snRNA
22 -
185 -
p63
K10
actin
days diff.0 3 5 7
56 -
80 -
48 -
Mw(KDa)
0 1 3 50
4
8
12
Arb
itrar
y un
its miR-203
0
40
80
120
0 1 3 5
p63
days diff.
1 2 3 4 lanes
1 2 3 4 lanes
humanmouse
b5’- AUGUUAAAGAGAAUGAGUCCUUG--AUUUCAAAGUUUUGUUGUAC -3’ p63-3’UTR
5’- AUGUUAAAGAGAAUGAGUCCUUG---------AGUUUUGUUGUAC -3’ p63-3’UTRdel 203
3’- GAUCACCAGGAUUUGUAAAGUG -5’ hsa-miR-203
.1970......1980......1990......2000.......2010....bp Hs AUGUUAAAGAGAAUGAGUCCUUGAUUUCAAAGU-UUUGUUGUACUUAAAUMm AUGCAAAAGCAAAUGAGUCCUUAAUUUCAACAU-UUUGUUGUGUUUAAAURn AUACUAAAGAGAAUGAGUCCUUAAUUUCAACAU-UCUGUUGCAUUUAAAUCf AUGUUAAAGAGAGUAAGUCCUUGAUUUCAAAGU-UUUAUUGUGCUUAAAUGg ACUUCAAAGAGCA-GCGUCCUUGAUUUCAAAGUAUUUAACGUGCUCA---
Human TP73L NM_003722 3’UTR length:2772 bp
1K
miR-203
2KamiR-203 targets p63
hsa-2
03
mmu-203
p63-3’UTRdel 203
Ctr
Luc
activ
ity (
%)
0
20
40
60
80
100
120
140
hsa-2
03
mmu-203Ctr
p63-3’UTRc
Ad-Scr Ad-203 CaCl2
Rel
ativ
e Ex
pres
sion
Lev
el (%
)
0
20
40
60
80
100
120
48h
0h24h
d Ad-Scr0 24 0 24 hrs
Ad-miR-203
p63
actin
MwKDa80 -
48 -
0
0.5
1.0
0 24 0 24 hrsAd-Scr Ad-miR-203
Arb
itrar
y un
its
1 2 3 4 lanes
e
miR-203 seems insufficient to promote differentiation
0
10
20
30
40
50
60
70
80
mock Ad-Scr Ad-203 CaCl2
TG5
0
12
3
45
6
7
8
9TG1
mock Ad-Scr Ad-203 CaCl20
0.5
1.0
1.5
2.0
2.5
3.0
3.5
mock Ad-Scr Ad-203 CaCl2
Involucrin
Rel
ativ
e Ex
pres
sion
Lev
el (%
)
48h
0h24h
48h
0h24h
48h
0h24h
b
a
Arb
itrar
yun
its
020406080
100120 p63
0 1 2 0 1 2 0 1 2 daysscramble miR-203 scramble
+ CaCl2
0
1
2
3
4 involucrin
Arb
itrar
yun
its
0 1 2 0 1 2 0 1 2 daysscramble miR-203 scramble
+ CaCl2
involucrin
0 1 2 daysmiR-203scramble scramble + CaCl2
0 1 2
p63
actin
Mw(KDa)
80 -
48 -
80 -
0 1 2
1 2 3 4 5 6 7 8 9 lanes
miR-203: clonogenicity
(1.7% +/-0.10) (0.7% +/- 0.18) (2.9% +/- 0.16)p < 0.019 p < 0.018
miR-203 antagomiR-203cont
aES neural
epidermal epithelialK8/K18 K5/K14
GFPABMP4
ES neuralSerum
i.
ii.
miR-203: ES differentiation
b
ES
neuralBMP4
epidermal
epithelial
serum
c
dFo
ld c
hang
e (A
U)
K14
K18
1 2 3 7 8 9 10 11 12 13 14 150.1
1
10
100
1000
days of differentiation64 5
Serum
Fold
cha
nge
(AU
)
days of differentiation
0.1
1
10
100
1000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
ΔNp63
miR-203
BMP-4
CYCLE ARREST
ADHESION
LIPIDS METABOLISM
TG & SUBSTRATES
PROTEASES
p63SOCS-3Zfp281
“S T E M N E S S”
QUIES-CENT
PROLIFE-RATIVE
D I F F E R E N T I A T I O N
miR-203
Effects of UVC irradiation on miR-203 and ΔNp63α
(0.005 J/cm2) for 30 secJHU-012 cells (SCC)
b
Arb
it rar
y u n
its
0
1
2
3
4
0 12 24 36 hrs
miR-203
Cel
l num
ber (
%)
0
20
40
60
G1 G2 S
0 hrs12 hrs24 hrs36 hrs
c
0 12 24 36 hrs
% S
ubG
1 ev
ents
d
0
5
10
15
20
c-jun
actin
PARP
0 12 24 36 hrs
48 -
48 -
75 -116 -
e Mw(KDa)
1 2 3 4 lanes
ΔNp63
actin
80 -
48 -
0 12 24 36 hrs
Arb
it rar
y u n
its
0
40
80
120
1 2 3 4
a Mw(KDa)
1 2 3 4 lanes
- The miR-203 / ΔNp63α pathway is retained in Squamus Cell Carcinoma- miR-203 is induced by UV stress
c
Cel
l num
ber (
%)
0
20
40
60
G1 G2 S
mockAd.scrAd-h203
b
% S
ubG
1 ev
ents
0
5
10
15
20
mockAd-sc
r
Ad-h203
p63
actin
80 -
48 -
aMw
(KDa) mockAd-sc
r
Ad-h203
1 2 3
Arb
it rar
y u n
its
0
0.5
1.0
1.5
1 2 3
miR-203 alone is sufficient to down regulate ΔNp63α and trigger death
JHU-012 cells (SCC)
Project 3: MELINO
Definition of the role of the p53 (p63/p73) family
Studies of p63 & Stem Cells
Analysis of miR in epidermal morphogenesis
Development of Ub E3 (Itch) inhibitors
Validation of Ub E3 (HECT) inhibitors for Cancer
CONCLUSION
Incidence of p73 alterations in cancer
0.41% 21.76%
p73 in hepatoma cells
ΔNp73 is a negative
Prognostic Factor
p73 as prognostic factor?
Hepathoma patients
N TA PR DBD OD CPR SAMTI
N PR DBD OD CPR SAMTI
TAp73 ISOFORM
ΔNp73 ISOFORM
ΔNp73 inhibits TAp73 and p53 apoptosis
ΔNp73 is induces by TAp73 and p53
ΔNp73 acts as an oncogenehypothesis
TAp73 induces apoptosis TAp73 acts as a tumour suppressorhypothesis
The ratio TAp73/ΔNp73 dictates the response to chemotherapy