NID1 and NUPR1 are ETV5 Regulated

Genes Involved in Endometrial Tumor

Dissemination

Núria Pedrola Montero Research Unit in Biomedicine and Translational and Pediatric Oncology

URB-Institut de Recerca HVdH

0

5

10

15

20

25

30

All Rac

es

White

Black

Asian

/Pac

ific Is

land

er

Amer

ican

Indian

/Ala

ska

Nat

ive

Hispa

nic

per

100.0

00 w

om

en

Incidence Rates by Race

Death Rates by Race

Localized Regional Distant All Stages0

20

40

60

80

100All Races

White

American African

5-y

ear

Su

rviv

al

(%)

Colas et. al., 2012 Monge et. al., 2007

ER

M/E

TV

5

Planagumà et. al., 2005

Monge et. al., 2007

Symbol Entrez Gene Name p-value Fold Chg

CYP2B6 cytochrome P450, family 2, subfamily B, polypeptide 6 5,10E-16 5,06830513

BMPR1B bone morphogenetic protein receptor, type IB 9,16E-17 3,61813172

DKFZp686O24166 1,50E-13 3,4421192

PLOD2 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 8,64E-17 3,16526154

ANGPT2 angiopoietin 2 8,77E-15 3,18148719

NUPR1 nuclear protein, transcriptional regulator, 1 3,31E-14 2,80059159

SLC16A1solute carrier family 16, member 1 (monocarboxylic

acid transporter 1)7,54E-16 2,59079947

C18orf19 chromosome 18 open reading frame 19 7,34E-12 2,55831426

NFIA nuclear factor I/A 1,12E-13 2,41906714

CNTN4 contactin 4 1,43E-11 2,31793924

TEX15 testis expressed 15 3,73E-08 2,29350537

EFNA5 ephrin-A5 9,33E-10 2,33677419

HSPB8 heat shock 22kDa protein 8 8,72E-12 2,29284644

MPDZ multiple PDZ domain protein 3,91E-10 2,21259852

LOC728212 hypothetical LOC728212 1,06E-08 2,13268826

LYST lysosomal trafficking regulator 6,43E-11 2,20050468

TTYH3 tweety homolog 3 (Drosophila) 5,78E-14 2,12336247

LOC728212 hypothetical LOC728212 4,41E-08 1,94093484

LOC728212 hypothetical LOC728212 7,26E-09 2,00399353

TSPAN7 tetraspanin 7 1,79E-11 2,02812977

SERPINE2serpin peptidase inhibitor, clade E (nexin,

plasminogen activator inhibitor type 1), member 23,82E-12 2,02378228

MTUS1 microtubule associated tumor suppressor 1 3,97E-09 2,01750099

IFI16 interferon, gamma-inducible protein 16 4,00E-10 1,95679486

FLJ26056 hypothetical protein LOC375127 6,00E-09 1,90133332

SATB1 SATB homeobox 1 2,27E-11 1,97480899

FOLH1B folate hydrolase 1B 1,88E-10 1,85821335

CDGAP Rho GTPase activating protein 31 1,24E-10 1,8730086

ETV5 ets variant 5 1,98E-12 1,93163908

CDH2 cadherin 2, type 1, N-cadherin (neuronal) 2,80E-12 1,85674924

FOLH1folate hydrolase (prostate-specific membrane antigen)

11,22E-10 1,81033416

NPR3natriuretic peptide receptor C/guanylate cyclase C

(atrionatriuretic peptide receptor C)1,09E-12 1,78402339

MCTP1 multiple C2 domains, transmembrane 1 1,04E-11 1,82716336

NAP1L3 nucleosome assembly protein 1-like 3 6,87E-10 1,81692158

NID1 nidogen 1 1,74E-11 1,80873287

LOC25845 hypothetical LOC25845 2,11E-11 1,72485245

FLJ26056 hypothetical protein LOC375127 1,98E-07 1,64985815

FLJ26056 hypothetical protein LOC375127 1,98E-07 1,64985815

EMID2 EMI domain containing 2 3,43E-09 1,63933874

CWH43cell wall biogenesis 43 C-terminal homolog (S.

cerevisiae)2,88E-12 1,64138057

LOC728212 hypothetical LOC728212 5,55E-09 1,68221155

JPH1 junctophilin 1 5,90E-08 1,7094977

APOC1 apolipoprotein C-I 1,88E-10 1,70687871

HIST1H2BK histone cluster 1, H2bk 1,76E-12 1,70470976

ICAM1 intercellular adhesion molecule 1 6,13E-06 1,68695716

PCDH24 cadherin-related family member 2 4,03E-10 1,63832951

.....

IGF2BP1 insulin-like growth factor 2 mRNA binding protein 1 2,10E-10 1,60842788

CYP2J2 cytochrome P450, family 2, subfamily J, polypeptide 2 8,95E-10 1,33794636

RNMT RNA (guanine-7-) methyltransferase 2,22E-07 1,29670691

CDK6 cyclin-dependent kinase 6 5,02E-09 1,2950852

CT45A1 cancer/testis antigen family 45, member A1 2,33E-07 1,36560822

DDR2 iscoidin domain receptor tyrosine kinase 2 0,00018295 1,31222367

BIRC7 baculoviral IAP repeat containing 7 1,00E-07 1,31377666

FN1 fibronectin 1 2,20E-06 1,31768141

LAMP3 lysosomal-associated membrane protein 3 2,89E-09 1,3278793Colas et. al., 2012

NUPR1

444bp

AcH

3

ET

V5

IgG

noA

b

C-

Input

C+

NID1

378bp

AcH

3

ET

V5

IgG

noA

b

C-

Input

C+

5 ’…ACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCTGGCC

TAACTGGCCGGTACCTGAGCTCGCTAGCGAACCTCCACAATAGGGAGCACAGGACAGGCACTCAAAAA

A

CGCGCAATGAATGGGTGGATGAGCCCATGAACGTGTTGGTGGCTCTGTCCCTTGGCTGATTCCCAGCT

CCCACCCCGTCTTCCTACCAGGGTCAAGCGAATTGGACCCCGGACGCGGCCGAGCGGCAGTGGGGTG

GGCGCCGTTCCTGAATCCGGAGCGTTTCCACGTCGCCGGCTCTCCACGACCCAACCCTAATCAGAGGA

CCACGGTGCGGGTCCCGCGCTCTGCTCCCTCTCCCGGAGGCGCCGTTCGCTGGGAGTCTGGCTGGTT

TCGAGAATCGCAGGCACCGGCGCCCAAGGCGGGAGGTTCGGCTTCGCCCCTCGCCCTCCCCCTCGCG

GCCATTGGGCTGCCCCGCGGCGCGCCCGCTGGACGGGGCGGGGCCCTCCGCTCTCCCCTCCGCTCC

CCTCCCCTATTTCCCGGGGTGGGAACGCCGGGAGGCGGGAGGAGAGGGGGCTGCCAGGGGCGTCCG

GTTACATCCCCGCCTTCCTCTGTCCTGGCCGCGGGACCGGGTTTGCGGGACCGCAGTTCGGGAACAT

GAAGCTTGGCAATC

CGGTACTGTTGGTAAAGCCACCATGG...3´

NID1 Promoter Forward

NID1 Promoter Reverse

5 ’ ...ACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCT

GGCCTAACTGGCCGGTACCTGAGCTCGCTAGCGAACCTGGGAGGAGGAGGTTGCAGGTTGCA

GTGAGCCAAGATCACACCACTGCACTCCAGCCGGGCGACAGAGACTCCATTTCAAAAAAAAAAA

AGAAGACAAGCAAGATGTTGAGTGTGACTGGGTGGAAGCCACTGACTTGTCCCTGGCACAAGTG

TGCTGATATCCCTTCCCCCTCCTTCCTCACGTCCTGCATCTCAGCGAGAATTGGGGTGCCGGGG

TTAGCTGTGTGGCCGTCATCCCAGGCCCAGGTGAAGCCCTCGATCAGGTGAGGCCATGATCCC

AACGAGTGTTTTAGCCCAGATCCTGCCTCTTCCCTGCTTTCTGCCGAGAAGCGGGACCTCCTCTA

TGGAAAATATCCAGCTGGGTGAGCCTGGGAGGGAGGAGGTGAGTTGGGCTGGACTCAGGGAC

CGACTCTTCCCGTCTCATGACTGTGTTTACTGGGCTGGATTTTGGGAAGGGGCCAGATTGCATC

AGACAGGGCCTGATGGGCTGGAGCCAGACTGTGGTCTGAGGAGGAGACACAGCCTTATAAGCT

GAGGGAGTGGAGAGGCCCGGGGCCAGGAAAGCAGAGACAGACAAAGCGTTAGGAGAAGAAGA

GAGGCAGGGAAGACAAGCCAGGCACGATGAAGCTTGGCAATCCGGTACTGTTGGTAAAGCCA

CCATGG...3´

NUPR1 PromoterForward

NUPR1Promoter Reverse

HHG

HGE

0

5

10

15

20

25 ***

Fo

ld C

han

ge (

2-

Ct )

HHG

HGE

0

100

200

300

400

500***

Fo

ld C

han

ge (

2-

Ct )

Ponceau

HG

HGE

0.00

0.02

0.04

0.06

0.08

NID1 activation

*

Lu

cif

era

se A

cti

vit

y

NUPR1 activation

HG

HGE

0.00

0.02

0.04

0.06

0.08

**

Lu

cif

era

se A

cti

vit

y

ETV5

NID1 promoter Luciferase

ETV5

NUPR1promoter

HGE

HGEsh

C

HGEsh

NID

1a

HGEsh

NID

1b

HGEsh

NID

1c

0.0

0.5

1.0

1.5

Fo

ld C

han

ge (

2-

Ct )

HGE

HGEsh

C

HGEsh

NUPR1a

HGEsh

NUPR1b

HGEsh

NUPR1c

0.0

0.5

1.0

1.5

Fo

ld C

han

ge (

2-

Ct )

HGE

HGEsh

C

HGEsh

nid1a

HGEsh

nid1b

HGEsh

nid1c

HGEsh

nupr1a

HGEsh

nupr1b

HGEsh

nupr1c

0.0

0.5

1.0

1.5

2.0

2.5

Fo

ld C

han

ge (

2-

Ct )

Videomicroscopy migration assay

HHG

HGE

HGEsh

C

HGEsh

NID

1

HGEsh

NUPR

1

0

50

100

150

200

**

**

MR

DO

(A

rbit

rari

ty U

nit

s)

Migration Transwell Assay

HHG

HGE

HGEsh

C

HGEsh

NUPR1

0.0

0.5

1.0

1.5

2.0

**

*

Ab

so

rban

ce

Hec1A

HG

HGE

HGEshC

HGEsh

NID1

HGEsh

NUPR1

Field 1 Field 2 Field 3 Field 4 Field 5 Field 6

HHG

HGE

HGEsh

C

HGEsh

NID

1

HGEsh

NUPR

1

0.0

0.2

0.4

0.6

0.8

1.0

10

20

30

40

**

*

Fo

ld C

han

ge H

no

rmali

zed

(1/1

0)

shC

shNID

1

shNUPR1

0.0

5.0×1011

1.0×1012

1.5×1012

Tumor distribution

*

GF

P F

luo

res

ce

nt

(Ra

dia

nt

Eff

icie

nc

y)

HGEsh

C

HGEsh

NID

1

HGEsh

NUPR1

0

2

4

6

8

**

*

Average number of metastases

(me

tas

tas

es

/ m

ice

wit

h m

eta

sts

es

)

Superficial Part Invasion Front (of the

myometrium)

myometrium

Colas et al., 2012

NUPR1

Superficial Invasion Front0

2

4

6

8

10

*

Fo

ld C

han

ge

NID1

Superficial Invasion Front0.0

0.5

1.0

1.5

2.0

Fo

ld C

han

ge

Thematic Network

of Cooperative

Research on

Cancer

Spanish Association Against Cancer