Inﬂ ammation-Induced NFATc1–STAT3 ... - Cancer Discovery€¦ · mechanistically links inﬂ...

RESEARCH ARTICLE

Infl ammation-Induced NFATc1–STAT3 Transcription Complex Promotes Pancreatic Cancer Initiation by Kras G12D Sandra Baumgart 1 , Nai-Ming Chen 1 , 2 , Jens T. Siveke 3 , Alexander König 1 , 2 , 7 , Jin-San Zhang 7 , Shiv K. Singh 11 , Elmar Wolf 5 , Marek Bartkuhn 6 , Irene Esposito 4 , Elisabeth Heßmann 1 , 2 , Johanna Reinecke 1 , 2 , Julius Nikorowitsch 1 , Marius Brunner 1 , Garima Singh 11 , Martin E. Fernandez-Zapico 7 , Thomas Smyrk 8 , William R. Bamlet 9 , Martin Eilers 5 , Albrecht Neesse 1 , Thomas M. Gress 1 , Daniel D. Billadeau 7 , David Tuveson 12 , Raul Urrutia 10 , and Volker Ellenrieder 2

on March 15, 2021. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst April 2, 2014; DOI: 10.1158/2159-8290.CD-13-0593

http://cancerdiscovery.aacrjournals.org/

JUNE 2014�CANCER DISCOVERY | 689

ABSTRACT Cancer-associated infl ammation is a molecular key feature in pancreatic ductal

adenocarcinoma. Oncogenic KRAS in conjunction with persistent infl ammation is

known to accelerate carcinogenesis, although the underlying mechanisms remain poorly understood.

Here, we outline a novel pathway whereby the transcription factors NFATc1 and STAT3 cooperate in

pancreatic epithelial cells to promote Kras G12D - driven carcinogenesis. NFATc1 activation is induced by

infl ammation and itself accelerates infl ammation-induced carcinogenesis in Kras G12D mice, whereas

genetic or pharmacologic ablation of NFATc1 attenuates this effect. Mechanistically, NFATc1 com-

plexes with STAT3 for enhancer–promoter communications at jointly regulated genes involved in

oncogenesis, for example, Cyclin, EGFR and WNT family members. The NFATc1–STAT3 cooperativity is

operative in pancreatitis-mediated carcinogenesis as well as in established human pancreatic cancer.

Together, these studies unravel new mechanisms of infl ammatory-driven pancreatic carcinogenesis

and suggest benefi cial effects of chemopreventive strategies using drugs that are currently available

for targeting these factors in clinical trials.

SIGNIFICANCE: Our study points to the existence of an oncogenic NFATc1–STAT3 cooperativity that

mechanistically links infl ammation with pancreatic cancer initiation and progression. Because NFATc1–

STAT3 nucleoprotein complexes control the expression of gene networks at the intersection of infl am-

mation and cancer, our study has signifi cant relevance for potentially managing pancreatic cancer and

other infl ammatory-driven malignancies. Cancer Discov; 4(6); 688–701. ©2014 AACR.

Authors’ Affi liations: 1 Signaling and Transcription Laboratory, Depart-ment of Gastroenterology, Philipps University, Marburg; 2 Department of Gastroenterology and Gastrointestinal Oncology, University Medical Center Göttingen, Göttingen; 3 II. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität; 4 Institute of Pathology, Helmholtz Zentrum, Munich; 5 Theodor Boveri Institute, University of Würzburg, Würzburg; 6 Institute for Genetics, Justus-Liebig-University, Giessen, Germany; 7 Schulze Center for Novel Therapeutics, Division of Oncology Research; Divisions of 8 Anatomic Pathology and 9 Biostatistics, College of Medi-cine; 10 Laboratory of Epigenetics and Chromatin Dynamics, Department of Medicine, Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota; 11 Barrow Brain Tumor Research Center, St. Joseph’s Hospital and Medical Center, Phoenix, Arizona; and 12 Cold Spring Harbor Labora-tory, Cold Spring Harbor, New York

Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).

Corresponding Authors: Raul Urrutia, Laboratory of Epigenetics and Chromatin Dynamics, GIH Division, Department of Medicine, Biochemistry and Molecular Biology, Guggenheim 10, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Phone: 507-5385636; Fax: 507-2556138; E-mail: [email protected] ; and Volker Ellenrieder, Department of Gastroenter-ology and Gastrointestinal Oncology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany. Phone: 49-6421-58-62766; Fax: 49-6421-58-68922; E-mail: [email protected]

doi: 10.1158/2159-8290.CD-13-0593

©2014 American Association for Cancer Research.

INTRODUCTION Commonly diagnosed at advanced and incurable stages,

pancreatic ductal adenocarcinoma (PDA) represents the

fourth leading cause of cancer-related death in Western

countries, rendering it one of the most lethal human can-

cers ( 1, 2 ). PDA evolves through a series of histopathologic

changes referred to as acinar-to-ductal metaplasia and pro-

gressive pancreatic intraepithelial neoplasia (PanIN), which

are accompanied by a recurrent pattern of genetic alterations;

the earliest and most prevalent of which is oncogenic activa-

tion of KRAS ( 3 ). The relevance of the Kras G12D mutation for

pancreatic carcinogenesis has been elegantly demonstrated

in genetically engineered mouse models (GEM) with condi-

tional activation of this oncogene in the embryonic pancreas.

Of note, as originally described by Hingorani and colleagues

( 4 ), Kras G12D activation in pancreatic epithelial cells induces

the development of PanIN precursor lesions, which eventu-

ally progress to invasive PDA after a long latency. Collectively,

these studies in mice and humans suggest that PDA originates

from Kras G12D -initiated cells, which need long-time exposure

to either cell-autonomous or environmental clues that act

as tumor promoters. Importantly, pancreatic cancer cells

are surrounded by a pronounced proinfl ammatory microen-

vironment that is driven by the secretion of tumor-derived

proinfl ammatory cytokines ( 5, 6 ). Furthermore, recent

fi ndings unraveled that infl ammatory cytokines, such as

tumor-derived granulocyte macrophage colony-stimulating

factor (GM-CSF), can exert cancer-promoting effects in vivo

by directly modifying gene expression networks in pancreatic

epithelial cells, rather than exclusively turning on and off

these pathways in infl ammatory cell populations from the

tumor microenvironment ( 5–7 ).

Moreover, chronic pancreatitis is regarded as a major risk

factor for the development of pancreatic cancer, further

highlighting the key role of infl ammation in the patho-

physiology of pancreatic cancer development ( 8, 9 ). To this

end, Guerra and colleagues ( 10–13 ) recently established a

new experimental GEM, whereby induction of a mild form

of pancreas infl ammation synergizes with Kras G12D to initi-

ate early PanIN lesions and promote their rapid progression

toward invasive PDA. This model highlighted the crucial role

of infl ammation in the process of malignant transformation

in the pancreas.




690 | CANCER DISCOVERY�JUNE 2014 www.aacrjournals.org

Baumgart et al.RESEARCH ARTICLE

However, the mechanisms linking infl ammation and malig-

nant transformation and progression in pancreatic epithelial

cells are still poorly understood. As oncogenic activation

of the Kras G12D signaling pathways is still deemed undrug-

gable, interaction partners that promote and cooperate with

Kras G12D -driven carcinogenesis may open new avenues for

novel drugs in prevention and therapy ( 4 , 14 , 15 ). Here,

we demonstrate that NFATc1, a transcription factor origi-

nally discovered in T lymphocytes ( 16 ), is strongly induced

upon infl ammatory stimuli and dramatically accelerates

malignant transformation in the pancreas when concomitant

Kras G12D mutation is present. We also fi nd that NFATc1 forms

chromatin-bound complexes with STAT3 in epithelial cells,

another well-characterized and infl ammation-induced tran-

scription factor. The generation of genome-wide chromatin

immunoprecipitation sequencing (ChIP-seq ) and expression

profi ling datasets reveal that the NFATc1–STAT3 coopera-

tivity regulates genome areas involved in the transcriptional

activation of cancer-associated gene networks. Combined,

these data provide robust evidence for the existence of a novel

interaction between two important transcription factors (the

NFATc1–STAT3 complex) in pancreatic epithelial cells. More

importantly, these transcriptional pathways, which exert dis-

tinct functions in infl ammatory cells, act in concert in pan-

creatic epithelial cells to mediate growth-promoting effects

upon infl ammation in the setting of Kras mutations. The rel-

evance of these fi ndings is underscored by the fact that small

molecules that target these pathways are being tested in early

clinical trials. Consequently, our fi ndings not only advance

our understanding of how infl ammation drives the progres-

sion of pancreatic cancer but may also open new avenues

for the rational design of future combinatorial therapies for

patients with chronic infl ammatory conditions that are at risk

to develop malignancies.

RESULTS The Transcription Factor NFATc1 Cooperates with Kras G12D to Give Rise to Highly Aggressive Pancreatic Cancer

This work was prompted by recent observations suggest-

ing that activation of transcription factor pathways in pan-

creatic epithelial cells through environmental infl ammatory

conditions can promote carcinogenesis, specifi cally in

the presence of oncogenic Kras mutations ( 17 ). We initially

focused our attention on the transcription factor NFATc1,

which, though absent in healthy human and murine pan-

creas, becomes highly induced in Kras G12D -expressing neoplas-

tic pancreatic cells when these mice were treated with daily

doses of cerulein to induce infl ammation (Supplementary Fig.

S1A–S1C), strengthening our own previous observations of

nuclear NFATc1 activation in pancreatitis-associated human

PDA ( 18 ). Thus, we tested whether recapitulating the induc-

tion of NFATc1 in pancreatic epithelial cells exerts oncogenic

functions in cooperation with Kras G12D . For this purpose, we

fi rst generated an inducible transgenic Nfatc1 mouse model

( Fig. 1A ) by introducing a loxP–STOP–loxP hemagglutinin

(HA)-tagged c.n.NFATc1 cDNA into the mouse ROSA26 locus

by homologous recombination and crossed it with p48-Cre and

Pdx1-Cre mice to obtain animals that express a form of nuclear-

localized NFATc1 that is transcriptionally active (Supplementary

Figure 1. NFATc1 accelerates Kras G12D -driven pancreatic carcinogenesis. A, generation of Pdx1/ p48-Cre-Nfatc1 and Pdx1/p48-Cre;Kras G12D ;Nfatc1 mice after Pdx1/p48-Cre –mediated excision-recombination. B, Kaplan–Meier curves displaying survival of Pdx1/p48-Cre ; Kras G12D ;Nfatc1 mice compared with Pdx1/p48-Cre ; Kras G12D and Pdx1/p48-Cre ; Nfatc1 mice. (***, P < 0.0001 for Kras G12D ;Nfatc1 vs. Pdx1/p48-Cre;Kras G12D cohorts, log-rank test, for pairwise combination). C, gross anatomy of Pdx1/p48-Cre;Kras G12D ;Nfatc1 mice before (top) and after (bottom) pancreatic tumor extraction. D, hematoxylin and eosin (H&E )–stained section from Pdx1/p48-Cre;Kras G12D ;Nfatc1 mice demonstrating the presence of acinar-to-ductular metaplasia (1I), PanIN lesion (1II-III and 2II–III), atypical fl at lesions (2I), invasive cancer (3I–III), and liver metastases (3III). Scale bars, 200 μm (1I and 3I) and 100 μm (1II–III, 2I–III, and 3II–III).

1

A D

B C

1

Surv

ival (%

)

p48(Pdx1)-Cre;Nfatc1 (n = 28)

p48(Pdx1)-Cre;KrasG12D (n = 16)

Pdx1-Cre;KrasG12D;Nfatc1 (n = 7)

p48-Cre;KrasG12D;Nfatc1 (n = 34)

150

100

50

Time (d)

100 200 300

***

400

Median survival

140/161 d

2 3

2 3

0I II III

0

1*

1*

2 3 4

1

2*

3

2 3 4

STOP

Kras

KrasG12D;Nfatc1

KrasG12D;Nfatc1

Rosa 26

Rosa 26

+

Pdx1/p48-Cre

SA-Nfatc1-pA LSL-KrasG12D





NFATc1–STAT3 Complexes in Pancreatic Cancer RESEARCH ARTICLE

Fig. S2A and S2B). Nfatc1 mice were born at the expected

Mendelian ratio and did not display gross abnormalities in

the pancreas. Increased cell proliferation was observed in pan-

creata of young mice, but despite the early proliferative effect

on pancreatic cells, Nfatc1 mice failed to develop advanced

PanIN lesions within a 1-year observation span (data not

shown). Therefore , though NFATc1 activation promotes cell

growth, it does not cause cancer by itself, and instead may

synergize with oncogenic Kras G12D to promote neoplastic cell

growth in response to infl ammation. This intriguing hypoth-

esis is particularly attractive as the majority of human PDAs

are characterized by the combined expression of both pro-

teins ( 18 ). Consequently, we mimicked this situation by gen-

erating Kras G12D ; Nfatc1 mice carrying transgenic expression

of both proteins ( Fig. 1A and Supplementary Fig. S2A and

S2B), a genetic manipulation that dramatically shortened ani-

mal survival (140 of 161 days; P < 0.0001) when compared

with littermates expressing Kras G12D or Nfatc1 alone ( Fig. 1B ).

Kras G12D ; Nfatc1 mice developed severe cachexia and abdominal

distension caused by the accumulation of sanguineous ascites

and bile duct obstruction highly resembling clinical features

of human PDA ( Fig. 1C ). At necropsy, the pancreata from

Kras G12D ; Nfatc1 mice were enlarged by tumor masses, which

invariably contained both solid and cystic regions ( Fig. 1C ).

At the histologic level, the pancreas of Kras G12D ; Nfatc1 animals

at 4 weeks of age displayed substantial replacement of acinar

cell areas by numerous acinar-to-ductular metaplasia (ADM;

Fig. 1D , 1I), PanIN precursors ( Fig. 1D , 1II–III), and atypical

fl at lesions (AFL; Fig. 1D , 2I). At 8 weeks, the full spectrum of

preinvasive lesions ranging from ADM to early- and late-stage

PanIN1-3 lesions ( Fig. 1D , 2II–III and Supplementary Fig. S2C)

was observed, and by 36 weeks of age, all animals showed

invasive and metastatic cancers ( Figs. 1D , 3I–III and 2A ) rang-

ing from well-differentiated (G1) and moderately differenti-

ated (G2) PDA to poorly differentiated G3 tumors with

anaplastic and/or adenosquamous components and low lev-

els of cytokeratin-19 expression ( Fig. 2B and Supplementary

Fig. S2D). Notably, equivalent to human PDA, pancreata from

Kras G12D ; Nfatc1 mice showed robust nuclear NFATc1 expression

throughout carcinogenesis ( Fig. 2C ). Tumor progression in

Kras G12D ; Nfatc1 mice was further characterized by an increased

proliferative index in epithelial cells as assessed by Ki67 quan-

tifi cation (5% vs. 17%; P < 0.05; Fig. 2D and Supplementary

Fig. S2E), which positively correlated with the upregulation

KrasG12D;Nfatc1

KrasG12D;Nfatc1

HA-NFATc1

4 wks 8 wks

p16INK4a

CDK4

β-Actin

KrasG12D;Nfatc1KrasG12D KrasG12D;

Nfatc1

Kra

sG

12D ;N

fatc

1

Kra

sG

12D ;N

fatc

1

Kra

sG

12D

Kra

sG

12D

KrasG12D

150

A C

D E

B

100

50

10

G2

H&

EC

K-1

9

G3 Anaplastic

20Time (wks)

Tum

or

incid

ence (

%)

30

n = 16

n = 26

40

30

20

*

10P

rolif

era

tion index (

%)

P < 0.001

NFATc1

Human pancreatic cancer***

Figure 2. Characteristic features of Kras G12D ;Nfatc1 mice tumors. A, tumor onset in cohorts of p48-Cre;Kras G12D ;Nfatc1 and p48-Cre;Kras G12D mice. Note that 100% of p48-Cre;Kras G12D ;Nfatc1 mice develop PDA at 36 weeks. ***, P < 0.001. B, hematoxylin and eosin (H&E; top) and corresponding cyto-keratin (CK)-19 stainings (bottom) of representative Kras G12D ;Nfatc1 mice tumors illustrating G2, G3, and anaplastic PDAs. C, NFATc1 staining in PanIN precursor and invasive pancreatic cancer lesions from p48-Cre;Kras G12D ;Nfatc1 mice and human PDA samples. D, proliferation index was measured in Ki67-stained pancreatic sections ( n ≥ 3; means ± SE). *, P < 0.05. E, pancreas lysates from 4- and 8-week-old p48-Cre;Kras G12D ;Nfatc1 mice were tested for p16 INK4A and CDK4 expression. Scale bars, 100 μm.






of cell cycle–promoting genes (e.g., Cdk4 ), and silencing of the

p16 INK4a tumor suppressor ( Fig. 2E and Supplementary Fig. S2F).

Together, the Kras G12D ; Nfatc1 model not only recapitulates key

features of human PDA but also demonstrates profound pro-

oncogenic properties of NFATc1. Moreover, this observation

suggests that stimuli mediating the transition of pancreatitis to

pancreatic cancer in the background of Kras G12D may proceed,

at least in part, via this transcription factor pathway.

NFATc1–STAT3 Cooperativity Contributes to Kras G12D -Induced Pancreatic Carcinogenesis

Here, we sought to elucidate the mechanism of NFATc1 to

accelerate pancreatic carcinogenesis. First, we generated pri-

mary cell lines derived from Kras G12D ; Nfatc1 tumors (hereafter

referred to as KNC 1–6 cell lines), and used microarray-based

expression profi ling analyses as genome-wide reporter assays

for determining the effects of inactivating NFATc1 in KNC

cells by RNAi. The data of these experiments were subjected

to gene set enrichment analysis (GSEA) for the identifi cation

of NFATc1-dependent gene signatures ( 19 ). GSEA pathway

analysis revealed enrichment of NFATc1 signatures including

target genes implicated in transformation, growth, and infl am-

mation, such as Cyclin D1 and D3 and CDK1 and CDK4

( Fig. 3A and B ). Most notably, we identifi ed an enrichment

of STAT3 and related infl ammatory pathways in NFATc1-

expressing cells, and, consequently, NFATc1 depletion was

accompanied by a massive loss of STAT3 expression ( Fig.

3A–C and Supplementary Fig. S3A–S3C). This observation is

important, as earlier studies had suggested that high STAT3

expression and activity levels associate with the development of

PDA in an infl ammatory setting in both humans and Kras G12D

mice ( 17 , 20 ). Therefore, we hypothesized the existence of a

pro-oncogenic NFATc1 and STAT3 cooperativity during PDA

development. In line with this, we found high levels of STAT3

expression and activation (indicated by Y705 phosphorylation)

in Kras G12D ; Nfatc1 tumors compared with Kras G12D litterma-

tes, and in human and murine PDA cells with concomitant

high NFATc1 expression levels and Kras G12D mutation ( Fig.

3D–F and Supplementary Fig. S3D). Furthermore, immuno-

histochemistry staining of PDA samples from 217 patients

identifi ed nuclear NFATc1 expression in 70.2% (151 of 217)

and, most importantly, the vast majority (86.7%) of NFATc1-

positive PDA showed coexpression of nuclear phosphorylated

(p) STAT3 (Y705; Fig. 3G and H ; further details are provided

in Supplementary Data). Consistent with the observed posi-

tive correlation of nuclear NFATc1 and STAT3 activation lev-

els, immunofl uorescence microscopy in PDA cells revealed

accumulation of NFATc1 and pSTAT3 (Y705) in euchro-

matic regions of tumor cell nuclei, suggestive of a functional

cooperation of both transcription factors in gene activation

( Fig. 3I ). Correspondingly, coimmunoprecipitation identifi ed

endogenous NFATc1–STAT3 complexes in human and murine

PDA cells ( Fig. 3J ) and demonstrated that successful complex

formation requires STAT3 activation at Y705 [pSTAT3 (Y705);

Fig. 3K and L ]. In fact, mutational disruption of the Y705

activation site or treatment with the STAT3 inhibitor WP1066

disrupted complex formation with NFATc1 in cancer cells

( Fig. 3K and L ). Thus , our combined cell biologic, biochemical,

and molecular datasets derived from studying both mice and

humans support the notion of an NFATc1–STAT3 interplay

in the nucleus of pancreatic cancer cells that functionally pro-

motes Kras G12D -induced carcinogenesis.

STAT3-Dependent NFATc1 Binding at Enhancer-Specifi c Target Sites

To investigate the function of the NFATc1–STAT3 inter-

action in gene regulation and carcinogenesis, we generated

KNC cells with stable STAT3 knockdown (KNC–shSTAT3;

Supplementary Fig. S4A) and performed ChIP to enrich

DNA fragments bound by NFATc1, followed by direct high-

throughput sequencing (ChIP-seq). Data analysis using the

PeakRanger algorithm [with negative binomial P < 10 −4 at

a false discovery rate (FDR ) < 5 × 10 −2 ] identifi ed 1,798

NFATc1-binding genomic regions. Multiple EM for Motif

Elicitation (MEME)-ChIP de novo identifi cation of motifs

overrepresented within peak regions ( 21 ) disclosed highest

enrichment of the previously established NFAT consensus

motif GGAAA ( Fig. 4A ). Furthermore, the MEME algorithm

identifi ed a signifi cant accumulation of the restricted STAT3

consensus site (GGAA for monomeric STAT3 vs. TTCN 3 GAA

for all STAT dimers), which was centered in the NFATc1 peak

summit ( P = 2 × 10 −12 ; Supplementary Fig. S4B), suggesting

a heterodimeric binding of both factors on sites of NFATc1

enrichment. Interestingly, we found a striking accumulation

of NFATc1 binding sites distant to transcription start sites

(TSS; within 50–500 kb upstream and downstream; Fig. 4A ;

Supplementary Fig. S4C), arguing that NFATc1 may prefer-

entially operate through long-range chromatin interactions

to control target gene expression in pancreatic cancer cells. In

fact, specifi c histone modifi cations, for example, H3K4me1

and H3K27ac ( 22 ), have been shown to mark features of

active enhancer regions. To determine whether predicted

NFATc1 enrichment sites overlap with active enhancer regu-

latory regions, we used ENCODE consortium datasets ( 23 ).

Congruently, we found that 1,155 of 1,789 NFATc1 bind-

ing peaks fall within genomic regions typically marked by

H3K4me1 (5.18-fold enriched over control regions, empirical

P after 1,000 simulations < 5 × 10 −324 ) and H3K27ac ( Fig.

4B ). In contrast, H3K4me3, a landmark for TSS binding, was

not enriched in NFATc1 binding sites (76 of 1,789; Fig. 4B ).

In line with these fi ndings, ChIP experiments at randomly

selected regions indeed confi rmed highly enriched NFATc1

binding at sites of high H3K4me1 and H3K27ac levels, indic-

ative of active enhancers (Supplementary Fig. S4E and S4F).

Importantly, more than two thirds of the identifi ed NFATc1

peaks were explicitly regulated by STAT3, as inferred from a

pairwise comparison between average binding levels across all

1,798 peaks in KNC–shControl versus KNC–shSTAT3 cells

( Fig. 4C–E ). Single ChIP experiments confi rmed that STAT3

plays a critical role in NFATc1 recruitment and, therefore,

RNAi-mediated depletion of this transcription factor dimin-

ished NFATc1 enrichment at active enhancer regions ( Fig.

4F ). Taken together, these data provide evidence for the exist-

ence of nuclear NFATc1–STAT3 transcription complexes that

exert a mutually dependent binding to regulatory regions

within the genome in pancreatic cells. To better understand

the potential impact of this new transcriptional complex on

PDA progression, these fi ndings led us to subsequently defi ne

the gene networks that are regulated by these factors using

genome-wide expression profi les.






Figure 3. Existence of a nuclear NFATc1–STAT3 complex in pancreatic cancer. A, genome-wide expression and GSEA analysis in p48-Cre;Kras G12D ; Nfatc1 tumor cells. Negative normalized enrichment score (NES) indicates loss of gene enrichment upon NFATc1 knockdown (additional information in Supplementary Table S1). B, heatmap showing selection of differentially regulated genes in p48-Cre;Kras G12D; Nfatc1 tumor cells depending on NFATc1 expression. Fold change relative to control cells is displayed in a blue–white–red pseudo color scheme for selected genes with FClog 2 < 1.5 or FClog 2 > −1.5. STAT3 expression changes are highlighted in red (details in Supplementary Table S2). C, qRT-PCR displaying Stat3 expression upon NFATc1 depletion in p48-Cre;Kras G12D ;Nfatc1 –derived tumor cell clones. D and E, pancreatic lysates from p48-Cre;Kras G12D; Nfatc1 and p48-Cre;Kras G12D mice were assessed for Stat3 mRNA expression (D) or total STAT3 protein expression and phosphorylation of STAT3 at Y705 [pSTAT3 (Y705); E]. F and G, immunohistochemical analysis for STAT3 and pSTAT3 (Y705) in p48-Cre;Kras G12D; Nfatc1 mice tumors (F) and NFATc1 and pSTAT3 (Y705) in human PDA (G). Scale bars, 100 μm. H, statistical illustration of tissue microarray (TMA) analysis ( n = 215 patients) demonstrating high correlative expression levels of nuclear NFATc1 and pSTAT3 in human PDA tissues. I, immunofl uorescence staining displays intracellular localization of STAT3 (green) and NFATc1 (red) in p48-Cre;Kras G12D; Nfatc1 tumor cells. Nuclei are visualized by Hoechst staining (blue). J, coimmunoprecipitation of endogenous NFATc1 and STAT3 was performed in murine Kras G12D; Trp53 −/− PDA cells and human Panc1 cells upon TGFβ and IL6 treatment. K and L, coimmunoprecipitation for NFATc1 and STAT3 in p48-Cre;Kras G12D; Nfatc1 –derived cells transfected with FLAG-tagged wild-type (wt)-STAT3 and (K) FLAG-STAT3 (Y705F) or (L) treated with 1 μmol/L WP1066 for 3 hours [blocking STAT3 (Y705) phosphorylation].

Inflammatory signaling

A

F

I J K L

E

H

B C D

Human pancreatic cancer

NFATc1

Nuclear NFATc1

(n = 215)

Negative

64/215

29.8%

Positive

151/215

70.2%

Negative

20/151

13.3%

P < 0.0007

Positive

131/151

86.7 %

Nuclear pSTAT3

(NFATc1 pos. n = 151)

STAT3

pSTAT3 (Y705)

STAT3

DAPI Merge

NFATc1

pSTAT3 (Y705)

IgG

IgG

IgG

DM

SO

WP

1066

wt S

TAT3

IgG

STA

T3 (Y

705F)

NFA

Tc1

NFA

Tc1

pSTAT3 (Y705)

NFATc1

Cell-cycle progression

Gene set

Gene set

FUNG_IL2_SIGNALING_1

CROONQUIST_NRAS_SIGNALING_DN

–1.91

–2.20 0.004

–1,5 1,50

0.059

0.121

Color key

siRNA

control

siRNA

NFATc1

siRNA

NFATc1

Ccnd14

3

2

1

STAT3

pSTAT3

(Y705)

β-Actin

β-Actin β-Actin

KrasG12D;

Nfatc1 #2

KrasG12D;Nfatc1

KrasG12D

KrasG12D

KrasG12D;

Nfatc1

KrasG12D;

Nfatc1

KrasG12D;Nfatc1 #2 KrasG12D;Nfatc1 #2KrasG12D;

Trp53–/–

KrasG12D;

Nfatc1 #51

0.75

0.5

0.25

– –+ +

Fold

STAT

3 m

RN

Aexpre

ssio

n

Fold

STAT

3 m

RN

Aexpre

ssio

n

Ccnd3

Ccn25a

Cdk1

Cdk4

Ezh2

H2afx

Pcna

Pold3

Rcan1

Rfc3

Rpa1

Sox9

Tert

Tk1

Wnt1

Wnt10a

Stat3

0.174

0.204

0.211

0.223

–1.77

–1.70

–1.68

–1.67

–1.66

ST_STAR3_PATHWAY

CROONQUIST_IL6_DEPRIVATION_DN

ST_INTERLEUKIN_4_PATHWAY

MORI_LARGE_PRE_BII_LYMPHOCYTE_UP

NES FDR

NES FDR

NFATc1FLAG-STAT3

HA-NFATc1

FLAG-STAT3

HA-NFATc1

FLAG-STAT3pSTAT3

(Y705)

IP: NFATc1

Input Input Input Input

IP: FLAG IP: FLAG

Panc-1

IP: NFATc1

STAT3

STAT3

NFATc1

FUNG_IL2_TARGETS_WITH_STAT5_

BINDING_SITES

–2.17 0.002REACTOME_TELOMERE_MAINTENANCE

–2.07 0.008REACTOME_EXTENSION_OF _TELOMERES

–2.05 0.011KEGG_DNA_REPLICATION

–1.98 0.030KOBAYASHI_EGFR_SIGNALING_24HR_DN

–1.97 0.033REACTOME_DNA_STRAND_ELONGATION

–1.90 0.057YU_MYC_TARGETS_UP

–1.78 0.114SA_G1_AND_S_PHASES

G

–1.77 0.124CHANG_CYCLING_GENES






Figure 4. STAT3-dependent NFATc1 binding at enhancer-specifi c target sites. A, ChIP-seq analysis and region–gene association studies revealed preferential NFATc1 long-distance binding from annotated transcriptional start sites with particular enrichment between 50 kb and 500 kb upstream and downstream of TSS. De novo identifi cation of overrepresented motifs using the MEME algorithm revealed the published NFAT consensus site GGAAA (displayed in inset) as best hit ( http://meme.sdsc.edu/meme/cgi-bin/meme-chip.cgi ). B, superposition for enhancer-specifi c (H3K27ac and H3K4me1) and promoter-specifi c (H3K4me3) histone modifi cations shows enrichment of enhancer marks peaking with a typical bimodal distribution centered on NFATc1 peak positions. C, DESeq statistics reveals STAT3 dependence of genome-wide NFATc1 binding (bar chart). The average binding across the 1,798 NFATc1 peak intervals was determined in Kras G12D ;Nfatc1 and Kras G12D ;Nfatc1-shSTAT3 cells. Signifi cance for lost NFATc1 binding in STAT3-depleted cells is demonstrated by Wilcoxon signed-rank test: P = 0. D, a region map of a 10-kb window is shown displaying genomic NFATc1 binding derived from ChIP-seq in stable Kras G12D ;Nfatc1 scramble and shSTAT3 tumor cells. K-means clustering identifi ed a large group of STAT3-dependent NFATc1-binding sites (gray bar). E, the average binding across the 1,798 NFATc1 peak intervals was determined in Kras G12D ;Nfatc1 scramble and shSTAT3 cells. Overall, NFATc1 binding is signifi cantly reduced in cells with decreased STAT3 levels (*** Wilcoxon signed-rank test: P = 2.225074 × 10 −308 ). F, ChIP analysis dis-plays NFATc1 binding at randomly selected enhancer regions in STAT3-depleted cells. Mean ± SD are shown from one out of three independent experiments.

1,500

A

D E F

B C

8

H3K27ac

Regulated by STAT3

Not regulated by STAT3

% 0

20

15

10

5

0

shcontrol

shSTAT3

***

1,000

900

800

700

600

400

200

40

0

20

chr1

4:47

5073

53-4

7507

518

chr6

:134

8668

79-1

3486

7051

chr4

:134

9959

87-1

3499

6175

chr1

8:80

8284

27-8

0828

599

chr1

4:79

8262

40-7

9826

409

chr3

:954

5459

0-95

4547

90

chr1

0:92

7720

99-9

2772

257

chr1

1:11

7801

206-

1178

0137

8

–3,000 –1,000 0 1,000 3,000

10 20

1798 NFATc1-bound peaks

NFATc1 occupancy

30 40 50

1219 579

60 70 80 90 100

shcontrol

shSTAT3

H3K4me1

H3K4me3

6

4

2

0

10

8

6

4

2

0

shcontrol shSTAT3

–3,000 –1,000Distance from NFATc1 peak center (bp)

Distance from NFATc1 peak center (bp)

0 1,000 3,000

1,000

< –

500

–500 to –

50

–50 to –

5 –

5 to 0

0 to 5

5 to 5

050 to 5

00

> 5

00

500

Distance to TSS (kb)

shcontrol

–3,000 0 3,000

Peak center

–3,000 0 3,000–3,000 0 3,000

Peak r

egio

ns

Peak b

indin

g leve

l

shSTAT3 Input

Regio

n–gene a

ssocia

tion

Bin

din

g

Ave

rage N

FAT

c1 b

indin

g

Fold

enri

chm

ent

NFATc1–STAT3 Complexes Regulate a Defi ned Gene Expression Network Involved in Cancer Progression

To gain insight into the gene-regulatory functions of

the NFATc1–STAT3 interplay, we fi rst used the Genomic

Regions Enrichment of Annotations Tool (GREAT; ref. 24 )

and analyzed the genome-wide ChIP-seq data as these relate

to pathway affi liations and disease relevance. Numerous

NFATc1 peaks coincided with gene signatures with func-

tional implications for cell motility, cell migration, and

extracellular matrix regulation (Supplementary Fig. S5A).

We then matched ChIP-seq data with results from expression

profi ling to identify direct target genes of NFATc1–STAT3

complexes. Comparison of datasets identifi ed distinct target

gene subsets, of which selected candidates were chosen for

further validation. Among these targets was EGFR, a recep-

tor tyrosine kinase that has been reported to be a key player

in infl ammation-associated carcinogenesis with implications

for transformation, tumor cell growth, and metastasis ( 25,

26 ). Furthermore , our target gene analyses revealed a promi-

nent member of the cyclin protein family (Cyclin D3) that

exerts essential growth-stimulating functions in pancreatic

cancer ( 27, 28 ) as a target. Of note, gene signatures that

associate to activate EGFR signaling as well as Cyclin D3

were also among the most signifi cantly regulated NFATc1






downstream targets in KNC tumor cells identifi ed by GSEA

( Fig. 3A ). Other subsets of direct target genes encompass the

NFAT pathway regulator Rcan1 , matrix metalloproteinase

13 ( Mmp13 ), and the Wnt family members Wnt1 and Wnt10a

( Fig. 5A ). Consistent with ChIP-seq data, NFATc1 specifi cally

regulates these direct target genes through interaction with

nearby enhancer regions, as indicated by NFATc1 binding

to sites of enriched H3K4me1 and H3K27ac modifi cations

( Fig. 5A and B ), low promoter-occupancy levels, increased

DNAse hypersensitivity, and site-specifi c recruitment of

Figure 5. NFATc1–STAT3 complexes regulate gene networks involved in cancer progression. A and B, ChIP analysis determines NFATc1 binding (A) or H3K4me1 and H3K27ac (B) at identifi ed enhancer regions of selected target genes. Mean ± SD are shown from one out of three independent experi-ments. C, histograms of ChIP fragment coverage for STAT3-dependent NFATc1 binding at the Egfr genomic region (chromosome 7:92436000-92444000). D, Kras G12D ;Nfatc1 cells stably depleted for STAT3 expression were transfected with wild-type (wt) -STAT3 or STAT3 (Y705F) and ChIP was performed to assess NFATc1 binding at selected targets. E, Kras G12D ;Nfatc1 cells were transfected with increasing amounts of STAT3 (200–500 ng) along with a Rcan1 promoter + enhancer reporter construct which harbors a wt or mutant NFATc1 binding site within the enhancer (as illustrated in the upper cartoon). Note that disruption of the NFAT enhancer binding sequence abolishes STAT3-mediated transactivation. Results in D and E are shown as mean ± SD from triplicates. F, murine p48-Cre;Kras G12D ;Nfatc1 and human PDA tissues were analyzed for EGFR expression. Scale bars, 100 μm. G, Western blot analysis demonstrating time-dependent decrease of EGFR expression in Kras G12D ;Nfatc1 PDA cells upon cyclosporin A (CsA) treatment. Displayed are measured expression intensities (%) related to the untreated control. H, relative expression of respective mRNAs in Kras G12D ;Nfatc1 tumor cells with and without transient NFATc1 knockdown. Data are shown as fold change compared with controls. Representative results from at least three independent experi-ments are shown. Mean ± SD. I, reduced EGFR protein expression levels in murine Kras G12D ; Trp 53 −/− PDA cells upon genetic Nfatc1 depletion. Mean ± SD. J, effect of NFAT inhibition by CsA (24 hours) on mRNA expression of target genes in human Panc1 cells. Data are shown as fold change compared with controls. Representative results from at least three independent experiments are shown. Mean ± SD.

1 200 60

40

20

60

40

20

60

40

20

KrasG12D;Nfatc1

KrasG12D;Nfatc1

KrasG12D;Trp53 mutKrasG12D;Nfatc1

Invasive cancerPanIN

EG

FR

EG

FR

92436000 92438000 92440000 92442000 92444000

150

100

50

20

15

10

5

1.5

1

0.5

IP: IgG IP: IgG

IP: H3K4me1

IP: H3K27ac

IP: NFATc10.8

NFATc1 occupancy

A B C

D

G H I J

E F

Enhancer signatures Egfr genomic regionP

erc

enta

ge o

f in

put

Perc

enta

ge o

f in

put

shS

TAT

3S

cra

mble

Input

Fold

enri

chm

ent

Contr

ol

Fold

mR

NA

expre

ssio

n

Fold

mR

NA

expre

ssio

n

0.6

0.4

0.2

60

0.5

Rela

tive

lucife

rase a

ctivity

Enhancer

NFATc1consensus site

Promoter

Luc

0.4

0.3

0.2

0.1

shSTAT3

NFATc1 occupancy

RCAN1 reporter construct

Human pancreatic cancer

Panc-1

wt NFATc1

binding siteMutant NFATc1

binding site

Ccnd3

CsA

0.5

100 90 82 77 45

3.5 18

1 1

0.8

0.6

0.4

0.2

Mmp13 Wnt1 Egfr

DMSO

CsA

0.5

(h)

EGFR

Ccnd3 Rcan1 Wnt1

siRNA

control

siRNA

NFATc1NFATc1

EGFR

β-Actin β-Actin

1

Rcan1STAT3

siR

NA

cont

rol

siR

NA

NFA

Tc1

STAT3

shSTAT3 +wt STAT3shSTAT3 +STAT3 (Y705F)40

20

Ccn

d3

Mm

p13

Rca

n1

Wnt

1

Wnt

10a

Egf

r

Ccn

d3

Mm

p13

Rca

n1

Wnt

1

Wnt

10a

Egf

r






histone acetyltransferase p300 (Supplementary Fig. S5B–

S5D). In addition, we confi rmed that NFATc1 recruitment

to the enhancers of these genes required STAT3 interac-

tion, as its genetic depletion or mutational inactivation

[STAT3 (Y705)] diminished NFATc1 recruitment and target

gene transcription ( Fig. 5C and D and Supplementary Fig.

S5E and S5F). Conversely, increased NFATc1 recruitment

at specifi c enhancer sites was observed when STAT3 was

activated upon IL6 treatment of pancreatic cancer cells, and

this was accompanied by recruitment of RNA polymerase

II at the corresponding promoter (Supplementary Fig. S5G

and S5H). Interestingly, STAT3 is preferentially recruited

to corresponding promoter sites rather than to enhancers

upon activation (Supplementary Fig. S5I), in a manner that

requires the presence of NFATc1 binding to nearby enhanc-

ers. Hence, increasing target promoter transactivation upon

STAT3 titration occurred only in the presence of an intact

NFAT consensus site within the corresponding enhancer

( Fig. 5E ). Thus, these data suggest that both proteins sup-

port the type of chromatin looping that is characteristic of

enhancer–promoter communications at regulatory regions

that mediate robust gene activation ( 29, 30 ).

Finally, the relevance of this novel pathway was confi rmed

by expression studies, showing strong induction of EGFR dur-

ing pancreatic carcinogenesis in both human and mouse PDA

cells, in which treatment with NFAT inhibitors (cyclosporin

A) or genetic Nfatc1 suppression diminished expression of the

identifi ed target genes ( Fig. 5F–J ). Together, these data dem-

onstrate for the fi rst time that nuclear interactions between

the transcription factors NFATc1 and STAT3 regulate genes

known to promote cancer initiation and progression in vivo

and identify enhancer-to-promoter communication as one

of the putative mechanisms by which these proteins achieve

their functions.

Targeting NFATc1–STAT3 Complexes Interferes with the Induction of Infl ammation-Induced Carcinogenesis

The studies described above have thus far revealed that

NFATc1, after being induced by infl ammatory conditions,

accelerates Kras G12D -mediated initiation of pancreatic car-

cinogenesis by forming a complex with STAT3, and identifi ed

their direct target genes using two complementary genome-

wide methods (ChIP-seq and expression profi ling). Thus,

to determine the signifi cance of this novel pathway in the

original context of infl ammation-induced cancer promotion

as well as to evaluate the chemopreventive potential of its

targeting, we treated Kras G12D mice with cerulein daily for

4 weeks to induce a mild and persistent infl ammation, as

described recently ( 10 ). Consistent with previous reports,

infl ammation accelerated Kras G12D -driven carcinogenesis and

caused rapid formation of ADM with subsequent progres-

sion to high-grade PanIN2 and PanIN3 lesions ( Fig. 6A ). The

promotion of these Kras G12D -initiated neoplastic lesions was

characterized by a strong induction of NFATc1 and pSTAT3

(Y705) in neoplastic epithelial cells and subsequent induc-

tion of oncogenes that function as a downstream target of

this cooperativity, as demonstrated for EGFR tyrosine kinase

and Wnt10a ( Fig. 6A and B ). The results are congruent with

our recently proposed model for the role of self-reinforcing

loops in the pathobiology of pancreatic cancer that occurs in

the presence of persistent infl ammation ( 31 ), as they indicate

that the NFATc1–STAT3 complex transduces signals acti-

vated by cerulein from the cell membrane to the nucleus to

turn on genes encoding proteins that reinforce cell growth

stimulation.

Finally, to determine the extent to which the induction of

the NFATc1–STAT3 cooperativity contributes to infl amma-

tory-driven cancer promotion by KRAS G12D , we used both

pharmacologic and genetic strategies to inactivate this com-

plex. Consequently, we specifi cally inactivated NFATc1 in

pancreatic epithelial cells by interbreeding Kras G12D mice with

NFATc1 fl /fl ; Pdx1-Cre animals ( Kras G12D ;Nfatc1 Δ/Δ mice; Supple-

mentary Fig. S6A–S6C). Here, cerulein treatment failed to

induce STAT3 activation and subsequent target gene expres-

sion ( Fig. 6A and B ). Although genetic depletion of Nfatc1 did

not affect ADM and PanIN formation in untreated 3-month-

old Kras G12D mice (data not shown), it signifi cantly antago-

nized the cerulein-induced proliferation rates in Kras G12D

epithelial cells ( Fig. 6C and Supplementary Fig. S6D) and

signifi cantly blocked ADM, as evidenced by restored normal

duct levels in Nfatc1 -null tissues following cerulein challenge

( Fig. 6D ). This supports the hypothesis that disruption of

NFATc1–STAT3 cooperativity rather than Nfatc1 ablation in

progenitor cells itself protects from pancreatic cancer initia-

tion and progression.

On the basis of these results, we hypothesized that drugs

that are currently used in the clinical setting to inhibit

NFAT might have a similar effect on infl ammation-associ-

ated PanIN formation. To test this hypothesis, we suppressed

NFATc1 activity in Kras G12D mice in vivo by daily treatment

with cyclosporin A along with cerulein for 3 months and

examined the biochemical and pathobiologic effect of this

intervention on preneoplastic epithelial cells. Congruent with

the genetic data described above, cyclosporin A treatment

blocked STAT3 activation, signifi cantly reduced cell pro-

liferation, and prevented ADM in 12-week-old mice ( Fig.

6A–D ). Noteworthy, the disruption of the NFATc1–STAT3

interaction (by either genetic or pharmacologic approaches)

was paralleled by the lack of EGFR induction in both geneti-

cally modifi ed mice ( Fig. 6A ) and acinar cell explants ( Fig.

6E ). Similar fi ndings were obtained for other cancer-related

NFATc1–STAT3-regulated oncogenic targets identifi ed by

our genome-wide approaches ( Fig. 6A and B and Supplemen-

tary Fig. S6E).

DISCUSSION Persistent infl ammation is a hallmark feature of PDA that

promotes the transition of this disease from its preneoplas-

tic state to frank PDA in the context of KRAS mutations.

The goal of the current study has been to provide insight

into mechanisms of cancer progression driven by infl am-

mation. Our guiding hypothesis has been that transcription

factors, which were originally discovered in infl ammatory

cells and thought to act in the tumor microenvironment,

are activated in preneoplastic and neoplastic cells through

infl ammatory stimuli, thus executing their function within

the epithelial compartment to promote infl ammation-

associated carcinogenesis in the presence of KRAS mutations.






Our study led to the following fi ndings: (i) the pro-oncogenic

transcription factors NFATc1 and STAT3 are activated by

infl ammatory stimuli and subsequently cooperate to govern

a defi ned oncogene expression profi le in neoplastic epithe-

lial cells; (ii) activation of the NFATc1–STAT3 cooperativity

in GEMs promotes Kras G12D -driven carcinogenesis, whereas

their inactivation has the opposite effect; (iii) mechanis-

tically, NFATc1–STAT3 complexes control gene expression

through enhancer-to-promoter communication, a powerful

epigenetic regulatory mechanism in the fi eld of gene expres-

sion ( 29, 30 ); (iv) identifi ed NFATc1–STAT3-regulated genes,

for example, those encoding EGFR and Wnt family members,

which are targets of novel drugs being tested in the setting of

experimental therapeutics; (v) pharmacologic disruption of

the NFATc1–STAT3 complex hampers its tumor-promoting

effects; and (vi) ectopic coexpression of NFATc1 and STAT3

is observed in human pancreatic cancer tissues, suggesting a

possibility of immediate translation of our fi ndings.

Figure 6. NFATc1 activation is required for pancreatitis-promoted carcinogenesis. A, immunohistochemical hematoxylin and eosin (H&E), NFATc1, pSTAT3, EGFR, and Wnt10a staining in Pdx1;Kras G12D and Pdx1-Kras G12D ;Nfatc1 Δ/Δ mice after indicated treatment showing an NFAT-dependent target gene induction during Kras G12D -driven carcinogenesis. Scale bars, 100 μm. B, Western blot analysis of Pdx1;Kras G12D and Pdx1-Kras G12D ;Nfatc1 Δ/Δ mice tissues for NFATc1, pSTAT3, EGFR, and Wnt10a expression upon treatment with cerulein and cyclosporin A (CsA) as indicated. ERK1/2 serves as a loading control. C, proliferation index was measured in Ki67-stained pancreatic sections ( n ≥ 3). Mean ± SE. P values are related to Pdx1 - Kras G12D control cohorts or treated Kras G12D cohorts as indicated. *, P < 0.05. D, quantifi cation of normal and preneoplastic ducts in Pdx1 - Kras G12D and Pdx1 - Kras G12D ;Nfatc1 Δ/Δ mice upon treatment as indicated. Mean ± SD ( n ≥ 4), P values are calculated in relation to untreated Kras G12D control cohorts or treated Kras G12D mice as indicated. ***, P < 0.0001; n.d., not detectable. E, qRT-PCR illustrating reduced Egfr mRNA expression in cultured acinar cell explants with NFATc1 inactivation ( Kras G12D ;Nfatc1 Δ/Δ vs. Kras G12D ).

KrasG12D

Cerulein

CsA

A B

C

E

D

H&

EDMSO

Cerulein

CsA

Nfatc1

mRNA

Egfr

mRNA

Cerulein Cerulein

NFATc1

pSTAT3

EGFR

Wnt10a

Norm. duct

DMSO Cerulein

n.d.n.d. n.d. n.d

Cerulein

+ CsA

Cerulein

PanIN lb

PanIN ll

PanIN lll

PanIN la

ERK1/2

30

1.2

100

80

60

40

20

1

0.8

0.6

0.4

0.2

*

******

*

20

10

Cerulein

+ CsAN

FAT

c1

Pro

lifera

tion index (

%)

Fold

mR

NA

expre

ssio

n

pS

TAT

3E

GF

RW

nt1

0a

Ducta

l le

sio

ns/H

PF

(%

)– + +

+

+

– – –

–– – –

+++

+

KrasG12D

KrasG12D

KrasG12D

Kra

sG

12D

KrasG12D KrasG12D

KrasG12D

KrasG12D

KrasG12D;Nfatc1Δ/Δ

KrasG12D;Nfatc1Δ/Δ

KrasG12D;

Nfatc1Δ/Δ

Kra

sG

12D ;

Nfa

tc1

Δ/Δ

Kra

sG

12D

Kra

sG

12D ;

Nfa

tc1

Δ/Δ

KrasG12D;

Nfatc1Δ/Δ






Our fi ndings elucidate fundamental biochemical proper-

ties displayed by transcription factors under infl ammatory

conditions to achieve their tumor-promoting effect. For

instance, we fi nd that at the transcriptional level, NFATc1

binds to GGAAA consensus sequences on DNA, albeit with

weak affi nity ( 32 ). Effi cient NFATc1 DNA binding, however,

can be mediated and maintained through interactions with

other partner proteins ( 32–34 ). Therefore, we identifi ed that

other infl ammatory transcription factors, such as STAT3,

partner with NFATc1 in the nucleus of pancreatic epithelial

cells. Like NFATc1, STAT3 activation translates infl amma-

tory signals from the tumor microenvironment into the

expression of specifi c gene networks involved in carcino-

genesis ( 35 ). Aberrant expression and activation of STAT3

is frequently observed in human pancreatic carcinoma and

can favor the progression of PanIN lesions in the transgenic

Kras G12D model ( 36 ). Activating STAT3 mutations are not

observed in PDA ( 17 , 20 , 36, 37 ); however, the mechanisms

of enhanced STAT3 expression are poorly understood. One

proposed mechanism is a feed-forward loop that main-

tains STAT3 expression through (IL6-mediated) elevated

activation levels of pSTAT3 (Y705; 37 ). It is worth underscor-

ing that our studies demonstrate that NFATc1 stimulates

STAT3 expression in primary tumor cells, whereas genetic

loss or pharmacologic inhibition of NFATc1 by cyclosporin A

diminishes expression of STAT3. Likewise , extensive analyses

of human PDA samples found a signifi cant positive correla-

tion between nuclear STAT3 and NFATc1 expression levels,

although we cannot fully exclude correlations between nega-

tive staining for NFATc1 and STAT3 that can be driven arti-

fi cially in a few samples in which inadequate antigen retrieval

or autolysis has occurred. However, it remains to be eluci-

dated whether NFATc1 directly infl uences STAT3 expression

or whether it stimulates STAT3 expression by maintain-

ing STAT3 activation, as we and others have observed a

regulatory impact on IL6 expression and STAT3 pathway

activation by NFATc1 ( 38–40 ; data not shown). Even more

striking, we fi nd that this new NFATc1 pathway is not lim-

ited to mediating regulation of STAT3 expression, but also

leads to the formation of NFATc1–STAT3 nucleoprotein

complexes, which are essential for the transcription of gene

networks that account, at least in part, for tumor progres-

sion in PDA. Interestingly, ChIP-seq analyses for genome-

wide identifi cation of genes regulated by the NFATc1–STAT3

complex revealed more than 1,100 putative NFATc1 target

sites, whose binding intensity was signifi cantly regulated

by nuclear STAT3. Although NFATc1–STAT3 interactions

on target gene promoters that infl uence cell migration and

proliferation have been identifi ed ( 40 ), our further analyses,

in contrast, demonstrated that our identifi ed binding sites

are mostly located at putative enhancer regions located

upstream, downstream, within, or even several thousand

bases away from their corresponding target genes ( 41 ). These

enhancer regions have been described as specialized areas in

the nucleus where protein–DNA complexes are responsive

to signal-regulated transcription factors and translate envi-

ronmental stimuli into the regulation of gene expression

networks, thus constituting at least one type of regulatory

modules within genomes that support environmental–gene

interactions. Enhancer activation defi nes time point, dura-

tion, and intensity of gene expression via complex mecha-

nisms of chromatin regulation ( 42 ). Enhancers may spread

stimulating signals as a result of acetylation and rearrange-

ment processes of nucleosomes along the chromatin or

induce the transcription of downstream genes through loop

formation and promoter communications ( 41 , 43 ). Our

data support a model in which NFATc1–STAT3 complexes

regulate target gene transcription through highly specifi c

enhancer–promoter interactions, presumably via formations

of chromatin loops. Our comparative analyses of genome-

wide ChIP-seq studies and expression analysis confi rmed

this model and, moreover, provided evidence for the exist-

ence of distinct gene expression networks that are regulated

via NFATc1–STAT3 complexes. We fi nd that most of the

NFATc1–STAT3 targets identifi ed hereby exert functions in

oncogenic processes such as cell-cycle propagation, migra-

tion, and invasion, as well as remodeling of the extracellular

matrix of the pancreas. Some relevant examples include the

EGFR, an oncogenic tyrosine kinase with critical implica-

tions for pancreatitis-promoted pancreatic carcinogenesis in

mice and humans, as well as proproliferative cyclin D3 and

MMP-13 ( 44 ), a central component of the MMP activation

cascade and mediator of tumor cell invasion. The identifi ca-

tion of Wnt1 and Wnt10a, ligands of the classical Wnt-β–cat-

enin pathway and important regulators of growth, stemness,

and differentiation, extends the scope of these investigations

and suggests important cross-talk interactions between the

NFATc1–STAT3 network and the Wnt pathway in pancreatic

cancer progression. Interestingly, these data are in agreement

with the recently proposed model whereby pancreatic cancer

proceeds by the establishment of positive feedback loops

that are self-reinforcing ( 31 ).

In conclusion, our results support the notion that tran-

scription factors, previously known to regulate the function

of immune cells, are activated by infl ammatory stimuli and

operate as nucleoprotein complexes within epithelial cells to

promote Kras -driven carcinogenesis. Moreover, we unravel

detailed mechanisms as to how these novel transcriptional

complexes form and execute genome-wide instruction by

binding and activating cancer-associated gene expression

networks. These investigations led to the discovery of sev-

eral proteins (e.g., EGFR) that, together with their regula-

tors, such as Wnt family members or STAT3, are currently

being evaluated as drug targets in early clinical trials. For

instance, the oral small-molecule Wnt signaling inhibitor

LGK974 is currently being tested in a phase I, open-label,

dose-escalation study in several solid malignancies, includ-

ing pancreatic cancer (NCT01351103). Furthermore, several

STAT3 inhibitors (OPB-31121 and OPB-51602) are clinically

tested in solid malignancies (NCT00955812, NCT01423903,

and NCT01867073). Thus, the new knowledge provided

by the current study helps to build the rationale for the

future design of combinatorial therapies that should be

more effi cient for controlling infl ammation-associated can-

cer progression. Finally, the fi nding that modulation of tran-

scriptional networks that work via epigenetic mechanisms

(e.g., NFATc1–STAT3 enhancer–promoter communications)

can modulate oncogenic KRAS function in infl ammation-

promoted carcinogenesis expands our mechanistic under-

standing of this disease beyond the genetic-centric model






that dominated the last two decades of research in this fi eld.

In light of the failure to translate these fi ndings into success-

ful therapies, for example through gene therapy, our data

also highlight the possibility that therapeutic strategies that

target transcriptional and epigenetic changes may be more

benefi cial for the management of infl ammatory-driven pre-

neoplastic diseases, such as in patients with chronic pancrea-

titis who are known to have an increased risk of developing

pancreatic cancer.

METHODS Cell Culture

Panc-1 and PaTu8988t cells were obtained from the European Col-

lection of Animal Cell Cultures (ECACC ) and HP Elsaesser (Philipps

University, Marburg, Germany); L3.6 cells were a gift from Dan

Billadeau (Mayo Clinic, Rochester, MN). Murine TD-2 cells were

described previously ( 18 ). Testing and authentication of human

cell lines were not performed by the authors. Primary pancreatic

cancer cell lines were derived from murine Kras G12D ;Trp53 −/− and p48-

Cre;Kras G12D ; Nfatc1 pancreatic tumors. A detailed description can be

found in the Supplementary Methods.

Mouse Strains and In Vivo Experiments P48-Cre , Pdx1-Cre , and LSL-Kras G12D mice have been described

previously ( 45–47 ). Nfatc1 fl / fl mice were kindly provided by Lau-

rie Glimcher ( 48 ). The c.n.Nfatc1 knockin strain (C57BL/6 back-

ground) was generated by cloning an N-terminal HA-tagged

constitutively active version of NFATc1 containing serine to

alanine substitutions in the conserved serine-rich domain and

all three serine–proline repeats into the ROSA26 promoter

locus (Artemis Pharmaceuticals). The strains were interbred

to generate Pdx1/p48-Cre;c.n.Nfatc1 , Pdx1/p48-Cre;Kras G12D , Pdx1/

p48-Cre;c.n.Nfatc1;Kras G12D , Pdx1-Cre; Nfatc1 Δ/Δ , and Pdx1-Cre;

Nfatc1 Δ/Δ ;Kras G12D cohorts. Mutant mouse strains were genotyped

by PCR as previously described by the laboratories that gener-

ated them. The following primers were used to genotype Nfatc1 :

5′-catgtctgggagatggaagc-3′. Chronic pancreatitis was induced by

single daily intraperitoneal injections of cerulein (0.2 mg/kg body

weight; Sigma-Aldrich) 3 days/week for a period of 4 weeks ( 10 ).

Mice were sacrifi ced after 12 weeks of treatment. All procedures

were conducted in accordance with the regulatory standards of and

were approved by the Regierungspräsidium Gießen.

ChIP-seq ChIP-seq analysis was done as previously described ( 49 ). ChIP

DNA was end-repaired and A-tailed. Illumina adaptors were ligated

to the ChIP DNA fragments. Fractions (175-bp to 225-bp size) were

cut out from a gel, eluted by Qiagen gel extraction kit, and enriched

by 20 cycles of PCR amplifi cation. The library size was controlled

with the Experion-system (Bio-Rad) and subsequently quantifi ed

by PicoGreen assay and subjected to Illumina GAIIx sequencing

according to the manufacturer’s instructions. Only high-quality

reads passing the internal Illumina-Raw data-fi lter (PF-cluster) were

considered.

TMA Staining and Analysis All studies carried out on human specimens were approved by the

Mayo Clinic Institutional Review Board. Ten adenocarcinoma tissue

microarrays (TMA ) containing samples from 217 patients were ana-

lyzed and stained for NFATc1 and pSTAT3 expression in the Pathol-

ogy Research Core. More details about staining procedures and data

analysis can be found in the Supplementary Methods.

Statistical Analyses Data are presented as averages ± standard deviations (SD) or

standard errors (SE) as noted and were analyzed by built-in t test

using Microsoft Excel. P < 0.05 was considered signifi cant. Tumor

incidences and survivals were calculated with GraphPad Prism4. For

the overall survival analysis, Kaplan–Meier curves were analyzed by

log-rank test. In all cases, we chose a group size that produced statis-

tically unambiguous results.

Disclosure of Potential Confl icts of Interest No potential confl icts of interest were disclosed.

Authors’ Contributions Conception and design: S. Baumgart , J.T. Siveke, M.E. Fernandez-

Zapico, M. Eilers, T.M. Gress, R. Urrutia, V. Ellenrieder

Development of methodology: S. Baumgart, J.-S. Zhang, S.K. Singh,

I. Esposito, G. Singh, V. Ellenrieder

Acquisition of data (provided animals, acquired and managed

patients, provided facilities, etc.): S. Baumgart, N.-M. Chen,

J.T. Siveke, A. König, J.-S. Zhang, S.K. Singh, E. Wolf, I. Esposito,

J. Reinecke, J. Nikorowitsch, M. Brunner, G. Singh, T. Smyrk

Analysis and interpretation of data (e.g., statistical analysis,

biostatistics, computational analysis): S. Baumgart, N.-M. Chen,

J.T. Siveke, A. König, S.K. Singh, E. Wolf, M. Bartkuhn, I. Esposito,

E. Heßmann, J. Reinecke, J. Nikorowitsch, G. Singh, W.R. Bamlet,

A. Neesse, R. Urrutia, V. Ellenrieder

Writing, review, and/or revision of the manuscript: S. Baum-

gart, N.-M. Chen, J.T. Siveke, E. Heßmann, M.E. Fernandez-Zapico,

T. Smyrk, W.R. Bamlet, A. Neesse, T.M. Gress, D.D. Billadeau,

D. Tuveson, R. Urrutia, V. Ellenrieder

Administrative, technical, or material support (i.e., reporting or

organizing data, constructing databases): S. Baumgart, E. Heßmann,

G. Singh, T.M. Gress, R. Urrutia

Study supervision: S. Baumgart, S.K. Singh, R. Urrutia, V. Ellenrieder

Acknowledgments The authors thank Dr. Laurie Glimcher (Weill Cornell Medical

College, Provost of Medical Affairs, Cornell University, Ithaca, NY)

for NFATc1 Δ/Δ mice. The authors are grateful to Kristina Reutlinger,

Bettina Geisel (Philipps University), and Susanne Haneder (Tech-

nische Universität, Munich, Germany) for technical support, and

Dr. Lukas Rycak (GenXPro GmbH) for the statistical analyses.

Grant Support This work was generously supported by the Deutsche For-

schungsgemeinschaft (KFO210 and SFB-TR17, to V. Ellenrieder); the

German Cancer Research Foundation (no. 109423 “Infl ammation

and Cancer” and AK “Mildred Scheel” Fellowship, to V. Ellenrieder;

“Max Eder” Fellowship, to A. Neesse); the University Medical Centre

Giessen and Marburg (to A. Neesse); Mayo Foundation for Medical

Research, NIH grants DK52913 and P30DK084567 (to R. Urrutia);

and NCI Pancreas SPORE Grant P50 CA102701 (to M.E. Fernandez-

Zapico and D.D. Billadeau).

Received September 2, 2013; revised March 26, 2014; accepted

March 28, 2014; published OnlineFirst April 2, 2014.

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2014;4:688-701. Published OnlineFirst April 2, 2014.Cancer Discovery Sandra Baumgart, Nai-Ming Chen, Jens T. Siveke, et al.

G12DKrasPromotes Pancreatic Cancer Initiation by STAT3 Transcription Complex−Inflammation-Induced NFATc1

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