The document summarizes key findings from a study on genes regulated by the transcription factor ETV5 that are involved in endometrial tumor dissemination. The study identified two genes, NID1 and NUPR1, as being upregulated in an ETV5-dependent manner. Chromatin immunoprecipitation experiments confirmed that ETV5 directly binds to the regulatory regions of NID1 and NUPR1. Knockdown experiments showed that NID1 and NUPR1 promote endometrial cancer cell dissemination and invasion.

1. 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

2. per 100.000 women
0
5
10
15
20
25
30
Al
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ac
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W
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Bl
As ac
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Pa
Am ci
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ic la
an nd
In e r
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an
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la
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a
N
at
iv
e
H
is
pa
ni
c
Death Rates by Race
Incidence Rates by Race

3. 100
All Races
80 White
Survival (%)
American African
60
5-year
40
20
0
Localized Regional Distant All Stages

5. ERM/ETV5
Monge et. al., 2007
Planagumà et. al., 2005
Colas et. al., 2012
Monge et. al., 2007

6. 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
solute carrier family 16, member 1 (monocarboxylic
SLC16A1 7,54E-16 2,59079947
acid transporter 1)
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
serpin peptidase inhibitor, clade E (nexin,
SERPINE2 3,82E-12 2,02378228
plasminogen activator inhibitor type 1), member 2
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
folate hydrolase (prostate-specific membrane antigen)
FOLH1 1,22E-10 1,81033416
1
natriuretic peptide receptor C/guanylate cyclase C
NPR3 1,09E-12 1,78402339
(atrionatriuretic peptide receptor C)
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
cell wall biogenesis 43 C-terminal homolog (S.
CWH43 2,88E-12 1,64138057
cerevisiae)
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
LAMP3
fibronectin 1
lysosomal-associated membrane protein 3
2,20E-06
2,89E-09
1,31768141
1,3278793
Colas et. al., 2012

7. ETV5
AcH3
noAb
Input
ETV5
AcH3
noAb
IgG
Input
C+
C-
IgG
C+
C-
NUPR1
NID1
444bp
378bp
5 ’ …ACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCTGGCC
5 ’ ...ACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCT
TAACTGGCCGGTACCTGAGCTCGCTAGCGAACCTCCACAATAGGGAGCACAGGACAGGCACTCAAAAA
GGCCTAACTGGCCGGTACCTGAGCTCGCTAGCGAACCTGGGAGGAGGAGGTTGCAGGTTGCA
A
NUPR1 PromoterForward
NID1 Promoter Forward
GTGAGCCAAGATCACACCACTGCACTCCAGCCGGGCGACAGAGACTCCATTTCAAAAAAAAAAA
CGCGCAATGAATGGGTGGATGAGCCCATGAACGTGTTGGTGGCTCTGTCCCTTGGCTGATTCCCAGCT
AGAAGACAAGCAAGATGTTGAGTGTGACTGGGTGGAAGCCACTGACTTGTCCCTGGCACAAGTG
CCCACCCCGTCTTCCTACCAGGGTCAAGCGAATTGGACCCCGGACGCGGCCGAGCGGCAGTGGGGTG
TGCTGATATCCCTTCCCCCTCCTTCCTCACGTCCTGCATCTCAGCGAGAATTGGGGTGCCGGGG
GGCGCCGTTCCTGAATCCGGAGCGTTTCCACGTCGCCGGCTCTCCACGACCCAACCCTAATCAGAGGA
TTAGCTGTGTGGCCGTCATCCCAGGCCCAGGTGAAGCCCTCGATCAGGTGAGGCCATGATCCC
CCACGGTGCGGGTCCCGCGCTCTGCTCCCTCTCCCGGAGGCGCCGTTCGCTGGGAGTCTGGCTGGTT
AACGAGTGTTTTAGCCCAGATCCTGCCTCTTCCCTGCTTTCTGCCGAGAAGCGGGACCTCCTCTA
TCGAGAATCGCAGGCACCGGCGCCCAAGGCGGGAGGTTCGGCTTCGCCCCTCGCCCTCCCCCTCGCG
TGGAAAATATCCAGCTGGGTGAGCCTGGGAGGGAGGAGGTGAGTTGGGCTGGACTCAGGGAC
GCCATTGGGCTGCCCCGCGGCGCGCCCGCTGGACGGGGCGGGGCCCTCCGCTCTCCCCTCCGCTCC
CGACTCTTCCCGTCTCATGACTGTGTTTACTGGGCTGGATTTTGGGAAGGGGCCAGATTGCATC
CCTCCCCTATTTCCCGGGGTGGGAACGCCGGGAGGCGGGAGGAGAGGGGGCTGCCAGGGGCGTCCG
AGACAGGGCCTGATGGGCTGGAGCCAGACTGTGGTCTGAGGAGGAGACACAGCCTTATAAGCT
GTTACATCCCCGCCTTCCTCTGTCCTGGCCGCGGGACCGGGTTTGCGGGACCGCAGTTCGGGAACAT
GAGGGAGTGGAGAGGCCCGGGGCCAGGAAAGCAGAGACAGACAAAGCGTTAGGAGAAGAAGA
GAAGCTTGGCAATC NID1 Promoter Reverse
GAGGCAGGGAAGACAAGCCAGGCACGATGAAGCTTGGCAATCCGGTACTGTTGGTAAAGCCA
NUPR1Promoter Reverse
CGGTACTGTTGGTAAAGCCACCATGG...3´
CCATGG...3´

9. Fold Change (2-  Ct)
0
5
10
15
20
25
Ponceau
H
H
G
H
G
E
***
Fold Change (2-  Ct)
0
100
200
300
400
500
H
H
G
H
G
E
***

10. NID1 activation
NUPR1 activation
0.08 * 0.08
**
Luciferase Activity
Luciferase Activity
0.06 0.06
0.04 0.04
0.02 0.02
0.00 0.00
G E
E
G
G
G
H
H
H
H
ETV5
NID1 promoter Luciferase
ETV5
NUPR1promoter

11. Fold Change (2-  Ct)
0.0
0.5
1.0
1.5
H
G
E
H
G
Es
hC
H
G
Es
hN
ID
1a
H
G
Es
hN
ID
1b
H
G
Es
hN
ID
1c
Fold Change (2-  Ct)
0.0
0.5
1.0
1.5
H
G
E
H
G
Es
hC
H
G
Es
hN
U
PR
1a
H
G
Es
hN
U
PR
1b
H
G
Es
hN
U
PR
1c
Fold Change (2-  Ct)
0.0
0.5
1.0
1.5
2.0
2.5
H
G
E
H
G
Es
H hC
G
Es
hn
id
H 1a
G
Es
hn
id
H 1b
G
Es
hn
H id
G 1c
Es
hn
up
H
G r1
a
Es
hn
up
H r1
G b
Es
hn
up
r1
c

12. Migration Transw ell Assay
Videomicroscopy migration assay
2.0
200
**
MRDO (Arbitrarity Units)
1.5
**
Absorbance
150
1.0 *
100
**
0.5
50
0.0
0
G
H
E
hC
1
PR
G
H
Es
H
1
E
1
hC
G
H
U
ID
PR
G
G
hN
H
H
Es
H
hN
Es
U
G
G
hN
H
Es
H
Es
G
H
G
H

13. Field 1 Field 2 Field 3 Field 4 Field 5 Field 6
Hec1A
HG
Fold Change H normalized
40
30 ** HGE
20
10
*
(1/10)
HGEshC
1.0
0.8
0.6 HGEsh
0.4 NID1
0.2
0.0 HGEsh
NUPR1
E
G
H
hC
1
1
ID
PR
G
H
Es
H
hN
U
G
hN
Es
H
Es
G
H
G
H

15. Tumor distribution
1.5×10 12
(Radiant Efficiency)
GFP Fluorescent
1.0×10 12
5.0×10 11 *
0.0
C 1 1
sh N
ID PR
U
sh N
sh
Average number of metastases
(metastases/ mice with metastses)
8
6
*
4
**
2
0
1
hC
1
ID
PR
Es
hN
U
G
hN
Es
H
Es
G
H
G
H

17. NUPR1
10
8
*
Fold Change
6
4
myometrium
2
0
Invasion Front Superficial Invasion Front
Superficial Part
(of the
myometrium)
NID1
2.0
1.5
Fold Change
1.0
0.5
0.0
Superficial Invasion Front
Colas et al., 2012

19. Spanish Association Against Cancer
Thematic Network
of Cooperative
Research on
Cancer