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