GSK-3Β Governs Inflammation-Induced Nfatc2 Signaling Hubs to Promote Pancreatic Cancer Progression

Published OnlineFirst January 28, 2016; DOI: 10.1158/1535-7163.MCT-15-0309

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics GSK-3b Governs Inﬂammation-Induced NFATc2 Signaling Hubs to Promote Pancreatic Cancer Progression Sandra Baumgart1, Nai-Ming Chen2, Jin-San Zhang3, Daniel D. Billadeau3, Irina N. Gaisina4, Alan P. Kozikowski4, Shiv K. Singh5, Daniel Fink1, Philipp Strobel€ 6, Caroline Klindt1, Lizhi Zhang7, William R. Bamlet8, Alexander Koenig2, Elisabeth Hessmann2, Thomas M. Gress1, Volker Ellenrieder2, and Albrecht Neesse2

Abstract

We aimed to investigate the mechanistic, functional, and stabilization of NFATc2–STAT3 complexes independent of SP2 therapeutic role of glycogen synthase kinase 3b (GSK-3b)inthe phosphorylation. For NFATc2–STAT3 complex formation, regulation and activation of the proinflammatory oncogenic GSK-3b–mediated phosphorylation of STAT3 at Y705 is transcription factor nuclear factor of activated T cells (NFATc2) required to stimulate euchromatin formation of NFAT target in pancreatic cancer. IHC, qPCR, immunoblotting, immuno- promoters, such as cyclin-dependent kinase-6, which promotes fluorescence microscopy, and proliferation assays were used to tumor growth. Finally, preclinical experiments suggest that analyze mouse and human tissues and cell lines. Protein– targeting the NFATc2–STAT3–GSK-3b module inhibits prolif- protein interactions and promoter regulation were analyzed eration and tumor growth and interferes with inflammation- by coimmunoprecipitation, DNA pulldown, reporter, and ChIP induced pancreatic cancer progression in KrasG12D mice. In assays. Preclinical assays were performed using a variety of conclusion, we describe a novel mechanism by which GSK- pancreatic cancer cells lines, xenografts, and a genetically engi- 3b fine-tunes NFATc2 and STAT3 transcriptional networks to neered mouse model (GEMM). GSK-3b–dependent SP2 phos- integrate upstream signaling events that govern pancreatic phorylation mediates NFATc2 protein stability in the nucleus of cancer progression and growth. Furthermore, the therapeutic pancreatic cancer cells stimulating pancreatic cancer growth. In potential of GSK-3b is demonstrated for the first time in a addition to protein stabilization, GSK-3b also maintains relevant Kras and inflammation-induced GEMM for pancreatic NFATc2 activation through a distinct mechanism involving cancer. Mol Cancer Ther; 15(3); 1–12. 2016 AACR.

Introduction health problem at the beginning of the twenty-first century (1). Mouse models and genetic studies have highlighted that pan- Pancreatic ductal adenocarcinoma (PDAC) is among the most creatic carcinogenesis occurs through chronologic accumula- lethal of solid malignancies with more than 40,000 new cases tion of genetic alterations, of which activating mutations of the diagnosed in the United States each year, thus posing a significant Kras oncogene represent a signature event that governs a multitude of mitogenic signaling events driving tumor initiation and progression (2). Importantly, preneoplastic cells and 1Department of Gastroenterology, Endocrinology, Infectiology and pancreatic cancer cells show a marked upregulation of inflam- Metabolism, University of Marburg, Marburg, Germany. 2Department of Gastroenterology and Gastrointestinal Oncology, University Med- matory cytokines and transcription factors and are surrounded ical Center Gottingen,€ Gottingen,€ Germany. 3Schulze Center for Novel by a pronounced inflammatory microenvironment that enables Therapeutics, Division of Oncology Research, Mayo Clinic, Rochester, Kras-mutant cells to progress toward frank malignancy (3–8). 4 Minnesota. Drug Discovery Program, Department of Medicinal Moreover, chronic pancreatitis represents an important risk Chemistry and Pharmacognosy, University of Illinois at Chicago, Chi- cago, Illinois. 5Barrow Brain Tumor Research Center, St. Joseph's factor for the development of pancreatic cancer, underscoring Hospital and Medical Center, Phoenix, Arizona. 6Institute of Pathology, the crucial role of inflammation in the process of malignant University Medical Center Gottingen,€ Gottingen,€ Germany. 7Division of – 8 transformation in the pancreas (9 11). Blockade of these Anatomic Pathology, Mayo Clinic, Rochester, Minnesota. Division of fl Biostatistics, College of Medicine, Mayo Clinic, Rochester, Minnesota. in ammatory factors, either directly or indirectly via interfer- ence with relevant signaling nodes, is likely to be important for Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). effective Kras-directed therapy. Interestingly, a recent study discovered Kras-dependent epige- S. Baumgart and N.-M. Chen are co-first authors of this article. netic induction of the glycogen synthase kinase 3b (GSK-3b)ina Corresponding Author: Albrecht Neesse, University Medical Center Gottingen,€ genetically engineered mouse model (GEMM) of PDAC. The € Robert Koch Str. 40, Gottingen 37075, Germany. Phone: 0049-5513-966326; authors showed that Kras signaling recruits the ETS/p300 tran- Fax: 0049-55139-19125; E-mail: [email protected] scriptional complex to the GSK-3b promoter, whose increased doi: 10.1158/1535-7163.MCT-15-0309 expression stimulated cancer cell growth (12). GSK-3b is a con- 2016 American Association for Cancer Research. stitutive active and multifunctional Ser/Thr kinase, which, due to

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its broad substrate specificity, is involved in numerous cellular ionomycin, and IL6 were from Sigma-Aldrich. GSK-3b inhibitor processes (13, 14). GSK-3b was initially recognized as an impor- 9-ING-41 has been previously described (33). tant tumor suppressor in various cancers predominantly acting through destabilization of the oncogene b-catenin (15). However, GSK-3b also plays important protumorigenic roles in several Plasmids human malignancies, most notably in pancreatic cancer, regulat- The full-length human NFATc2 expression vector as well as ing proliferation and survival (16–20). Increased expression of the bacterial expression vector pENTR11 NFATc2 were kindly active GSK-3b and nuclear accumulation of active GSK-3b signif- provided by A. Rao (Department of Signaling and Gene Expres- icantly correlated with a dedifferentiated state in pancreatic cancer sion, La Jolla Institute for Allergy and Immunology, La Jolla, b cells (18). Furthermore, GSK-3b is commonly involved in inflam- CA). GSK-3 S9A was from J. Woodgett (Lunenfeld-Tanenbaum matory responses such as enhanced cytokine production driven Research Institute, Mount Sinai Hospital, Toronto, Ontario, by NF-kB and STAT3 pathways (21–24), and inhibition was Canada). The wt STAT3 and STAT3 Y705F constructs were from shown to inhibit tumor cell growth via downregulation of NF- J. Darnell (Laboratory of Molecular Cell Biology, The Rock- b kB–mediated expression of antiapoptotic and proinflammatory efeller University, New York, NY). pBabe V5-GSK-3 S9A was genes (18, 25). Thus, GSK-3b has recently received increasing from B. Manning (Department of Genetics and Complex Dis- attention as a therapeutic target in preclinical and clinical ther- eases, Harvard T.H. Chan School of Public Health, Boston, MA). apeutics. However, the downstream effects of GSK-3b are complex The NFAT-luc reporter construct harboring three consensus sites and by far not restricted to the NF-kB pathway. Therefore, further for NFAT fused to a luciferase gene was purchased from Stra- studies are needed to fully comprehend the multiple molecular tagene. The HA-tagged NFATc2 construct was generated by þ and cellular mechanisms by which GSK-3b contributes to the cloning of HA-wt NFATc2 into the pcDNA3.1 ( )vectorsup- pathogenesis of cancer. plied by Invitrogen into HindIII and XbaI restriction sites. The NFAT (nuclear factor of activated T cells) proteins are oncogenic pSP2 mutation was generated as previously described (31). transcription factors, which are induced upon inflammatory conditions and act as crucial promoters of Kras-induced pancre- Generation of viral vectors and stable cell lines atic cancer progression and cellular plasticity (26). Moreover, Retroviral vectors and stable cell lines were generated as nuclear NFAT is highly overexpressed in the majority of invasively described previously (28). Briefly, HA-tagged NFATc2 constructs growing pancreatic cancers (27–30). Interestingly, recent data were cloned into a pQCXIP vector (BD Biosciences) and trans- suggest that NFATc2 is regulated by GSK-3b (31, 32); however, fected into the packaging cell line LinxA. The supernatant contain- the exact mechanism and subsequent functional implications on ing retroviral particles was filtered through a 0.45-mm filter and tumor growth and inflammation-induced tumor progression are supplemented with 2 mg/mL polybrene (Millipore) to infect the unknown. target cells PaTu8988t, Suit-028, and Suit-007. Spin infection was In this study, we sought to investigate the mechanistic role of carried out at 2,500 g and at 37 C. Transduced cells were GSK-3b in the regulation and transcriptional activation of onco- selected with 1 mg/mL puromycin (Sigma-Aldrich) for at least genic NFATc2. Furthermore, we use pharmacologic as well as two weeks. The successful overexpression of the NFATc2 con- genetic approaches in vitro and in several mouse models of structs was tested by Western blotting. pancreatic cancer to address the therapeutic potential of this oncogenic-inflammatory signaling axis. qPCR analysis Materials and Methods RNA from cell lines was isolated using RNeasy Mini Kit (Qia- gen), and first-strand cDNA was synthesized from 2 mg total Cell culture RNA using random primers and Omniscript Reverse Transkriptase The human pancreatic adenocarcinoma cell line IMIM-PC1 was Kit (Qiagen). xs-13 was used as housekeeping gene. The following provided by FX Real (Institute Municipale de Investigacion Med- primer pairs were used: xs-13; 50-gtcggaggagtcggacgag-30,50- ica, Barcelona, Spain), Suit-028 and Suit-007 were kindly gifted gcctttatttccttgttttgcaaa-30, CDK-6; 50-tcccgttccactgcgtcc-30,50- from T. Iwamura (Miyazaki Medical College, Miyazaki, Japan). caagccgcttaatccttcctggtt-30. All primers were supplied by Biomers. PaTu8988t were obtained from H.P. Elsaesser (Department of Real-time PCR experiments were performed in triplicates and are Cell Biology, Marburg, Germany). LinxA cells were purchased displayed SD. from Open Biosystems. All cell lines were obtained between 2004 to 2010. Mouse KrasG12D;p53 / cells were established from mouse Pdx1-KrasG12D;p53 / pancreatic tumors and generously Transient transfection and siRNA gifted by J. Siveke (Technische Universit€at Munchen,€ Munich, The transient transfection of expression constructs was carried Germany) in 2012. Testing and authentication of human cell lines out 24 hours after seeding using TransFast (Promega) and Lipo- were not performed by the authors. All human pancreatic cancer fectamine 2000 (Invitrogen) as transfection reagents following cells were cultured in DMEM (Gibco, Invitrogen Corp.), supple- manufacturer's instructions. siRNA was transfected using the mented with 10% FCS (Gibco) and 50 mg/mL gentamicin siLentFect Lipid Reagent (Bio-Rad) according to the manufac- (Gibco). LinxA cells were maintained in DMEM (PAA Laborato- turer's protocol. The RNAi oligonucleotide sequences were as ries GmbH), supplemented with 10% FCS and 100 mg/mL follows: NFATc2, 50-ccauuaaacaggagcagaatt-30; STAT3, 50-gca- hygromycin (Carl Roth GmbH). Mouse KrasG12D;p53 / cells caaucuacgaagaauctt-30 (Applied Biosystems); GSK-3b, siGEN- were grown in DMEM, 10% FCS, 1% nonessential amino acids OME ON-TARGETplus SMARTpool duplex (Thermo Scientific). (Gibco), and 1% penicillin/streptomycin (Gibco). GSK-3b inhib- The Silencer Negative Control from Ambion (Applied Biosys- itor AR-A014418 was purchased from Calbiochem, caerulein, tems) was used as a negative control.

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Subcellular fractionation and coimmunoprecipitation and the acid-insoluble fraction was dissolved by incubation with Subcellular fractionation was performed as described previ- 1 mmol/L NaOH for 30 minutes at 37C. Radioactivity was ously (2). Briefly, cells were washed with ice-cold PBS and evaluated with a scintillation counter (Pharmacia). For BrdUrd centrifuged at 2,500 g at 4C. Pellets were subsequently resus- assays, (transfected) cells were seeded in 96-well plates and pended in buffer A (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, treated as indicated. Incorporation of BrdUrd was assayed with 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, and Cell Detection ELISA, BrdU Kit (Roche) according to the manu- protease inhibitors) for 30 minutes and consequently centrifuged facturer's instructions. The intensity of incorporation was mea- for 20 minutes at 5,000 g. Supernatants were transferred to new sured with a DLReady Luminometer (Berthold Technologies). All tubes and centrifuged for 20 minutes at 12,000 g. Pellets were proliferation assays were performed in triplicate in at least three resuspended in buffer C (20 mmol/L HEPES, pH 7.9, 0.4 mmol/L independent experiments. NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, and protease inhibitors) and incubated on ice for additional Mouse strains and in vivo experiments 30 minutes. The final centrifugation step was carried out at Chronic pancreatitis was induced in 8-week-old Pdx1-Cre; 12,000 g for 10 minutes. KrasG12D mice by single daily intraperitoneal injections of caer- For coimmunoprecipitation, cells were lysed with lysis buffer ulein (0.2 mg/kg BW; Sigma-Aldrich) and the GSK-3b inhibitor 9- (50 mmol/L HEPES; pH 7.5–7.9, 150 mmol/L NaCl, 1 mmol/L ING-41 (20 mg/kg BW) 3 days per week for a period of 4 weeks EGTA, 10% glycerin, 1% Triton X-100, 100 mmol/L NaF, and 10 (33). Mice were sacrificed at 12 weeks of age. mmol/L Na4P2O7 10 H20) containing protease inhibitors. Five For the nude mice studies, 6- to 8-week-old pathogen-free hundred micrograms of the total lysates or 100 mg of nuclear athymic female nu/nu mice were obtained from Harlan Winkel- lysates were immunoprecipitated with the indicated antibodies mann. A total of 1 106 IMIM-PC1 was mixed with Matrigel (BD and protein G- or A-agarose (Roche Diagnostics). Biosciences) and injected subcutaneously in both flanks of For isolation of proteins from murine tissues, the tissues were 12 nude mice. After the establishment of visible tumors, mice homogenized using a mortar and subsequently mixed with lysis were randomized into two groups and were treated 3 times weekly buffer. After sonication, cells were centrifuged at 12,000 g for 10 with either 10% DMSO or 10 mg/kg of AR-A014418 (Calbio- minutes and subjected to immunoblotting. chem). Mice and tumor sizes were monitored twice weekly. Furthermore, we injected 1 106 parental PaTu8988t control Chromatin immunoprecipitation cells or PaTu8988t cells stably overexpressing wt NFATc2 and Chromatin immunoprecipitation assays using IgG (Upstate NFATc2 pSP2 subcutaneously into the flank of 6 nude mice each Biotechnology) or antibodies specific to NFATc2, H3K4me3 (all and weekly measured the tumor sizes. The tumor volumes (V) Abcam), RNA Polymerase II (Millipore), or STAT3 (Cell Signaling were estimated using the formula V ¼ 4/3 p (L/2) (i/2) Technology) were performed as described previously (27). Bound (H/2) where L ¼ length, W ¼ width, and H ¼ height. After 4 weeks, regions were detected by qPCR using the following primer: CDK-6 mice were sacrificed and tumors were explanted, analyzed by size, promoter; 50-tcccgttccactgcgtcc-30,50-caagccgcttaatccttcctggtt-30 and subjected to IHC and qPCR or Western Blot analyses. Animal (Biomers). experiments were carried out using protocols approved by the Institutional Animal Care and Use Committee at the University of Reporter gene assays Marburg (Marburg, Germany). Cells were seeded in 12-well plates and were transfected after 24 hours with the indicated constructs. Luciferase activity was mea- Ki67 proliferation analysis sured using the Lumat LB 9501 Luminometer (Berthold Technol- High-power images (20 objective) were taken from Ki67- ogies) and the Dual-Luciferase Reporter Assay System (Promega) stained slides from at least 3 animals for each group. Nuclei according to the manufacturer's instructions. Firefly luciferase positive for Ki67 were counted as actively proliferating cells in values were normalized to Renilla luciferase activity and are shown 20 metaplastic lesions and were related to total numbers of nuclei as mean values SD. per lesion. Data were expressed as percentage of positive nuclei per total nuclei. DNA pulldown assays For DNA pulldown assays, 100 mgto150mgofnuclear Fluorescence microscopy, IHC, and Western blotting protein per sample was incubated for 3 hours with 1 mgof Immunofluorescence and IHC were performed on formalin- biotinylated double-stranded oligonucleotides containing the fixed, paraffin-embedded tumor sections as described previously consensus NFAT-binding site (50-tctaaggaggaaaaactgtttcatg-30 (28). The following antibodies were used for immunofluores- and 50-catgaaacagtttttcctccttaga-30). DNA–protein complexes cence: HA (1:1,000) and STAT3 (1:200; all Cell Signaling Tech- were further incubated with streptavidin–agarose beads (Sig- nology) and for IHC: NFATc2 (1:200; Abcam), p-NFATc2 (S213/ ma-Aldrich) for 1 hour, washed twice with lysis buffer, and 217/221; 1:50, Santa Cruz Biotechnology), STAT3 (1:100, Cell subjected to immunoblotting. Signaling Technology), p-STAT3 (Y705; 1:50, Cell Signaling Tech- nology), p-GS (S641/645; 1:25, Cell Signaling Technology), Ki67 Proliferation assays (1:600, NeoMarker), GSK-3b (1:200, Epitomics), and CD45 (BD Cell proliferation was measured by 3Hthymidine and bromo- Pharmingen, 1:100). Immunoblotting was performed using stan- deoxyuridine (BrdUrd) incorporation. Cells were seeded in dard methods on uncultured, macrodissected tumor protein 24-well plates and cultured in medium containing 10% FCS. lysates or lysates from cultured cell lines. Membranes were probed Nineteen hours after the indicated treatment or transfection, with NFATc2 (Abcam), p-NFATc2 (S213/217/221; Abcam), Flag 3Hthymidine (0.5 mCi/well) was added during the last 5 hours (Sigma-Aldrich), HA, STAT3, GSK-3b, CDK-6, p-STAT3 (Y705), of incubation. The cells were washed with 5% trichloroacetic acid, p-GS (S641/645), GS (all Cell Signaling Technology), V5

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(Invitrogen), and b-actin (Sigma-Aldrich) antibodies. Densito- region, we aimed to assess the functional relevance of this post- metric analysis was carried out with ImageQuant 5.1. translational modification. Importantly, a phospho-specific SP2 antibody detected wild-type NFATc2, but not a mutant NFATc2 TMA staining and analysis lacking these phosphorylation sites (Supplementary Fig. S1C). All staining carried out on human specimens was approved by Notably, whereas GSK-3b depletion resulted in diminished SP2 the Mayo Clinic Institutional Review Board (Rochester, MN). phosphorylation, expression of a constitutively active mutant Written confirmation was obtained from each patient. Tissue (GSK-3b S9A) increased SP2 phosphorylation (Supplementary microarrays (TMA) containing samples from PDAC patients were Fig. S1D and S1E). Immunoblot and immunohistologic analyses stained as indicated and analyzed for NFATc2 and p-STAT3 from GSK-3i–treated xenograft tumors revealed reduced levels of expression in the Pathology Research Core. TMA slides were p-NFATc2, p-glycogen synthase (GS), and Ki67 (P < 0.01; Fig. 1G placed in the BOND III stainer (Leica Biosystems) for online and Supplementary Fig. S1F and 1G). Moreover, nuclear NFATc2 processing. They were treated with Epitope Retrieval 2 Solution protein levels were substantially reduced in pancreatic cancer cells for 20 minutes and stained with NFATc2 (Abcam) or p-STAT3 treated with the GSK-3i, whereas overexpression of an NFATc2 (Cell Signaling Technology) for 15 minutes, and detection was mutant mimicking constitutive phosphorylation (NFATc2 pSP2) achieved using the Polymer Refine Detection Kit per manufac- rescued the effect of GSK-3i treatment on NFATc2 expression turer's instructions (Leica Biosystems). Counterstaining was per- (Fig. 1H and Supplementary Fig. S1H). formed for 5 minutes with hematoxylin. Slides were dehydrated It is noteworthy that in immune cells, GSK-3b–mediated through increasing concentrations of alcohol, cleared in xylene, phosphorylation of the SP2 region terminated DNA binding of and cover-slipped in xylene-based mounting media. NFAT proteins and induced shuttling of NFAT proteins to the cytoplasm, followed by inactivation of NFAT activity (34–36). Data analysis To address this issue in pancreatic cancer, we performed immu- The TMAs were evaluated for NFATc2 and p-STAT3 expression noblot analysis and DNA pulldown assays that detected high by a trained pancreatic pathologist and were scored as positive NFATc2 pSP2 levels in the nucleus as well as efficient binding to (score 1, 2, or 3) or negative (score 0). Stain positivity across the the NFAT consensus sequence GGAAA (Fig. 1I). Moreover, multiple evaluable cores per patient was reduced to 1 observation reporter gene assays demonstrated profound activation of an per unique subject by using the core, which stained with the NFAT-responsive "cisNFAT" promoter by NFATc2 pSP2 com- maximum intensity for each stain. pared with wild-type NFATc2 (Fig. 1J). Functionally, stable NFATc2 pSP2 expression significantly stimulated tumor growth Statistical analyses of PaTu8988t xenograft tumors after 4 weeks (Fig. 1K). Immu- Data were presented as averages SD or SE as noted and were nohistochemical analysis of human PDA tissues showed nucle- analyzed by built-in t test using Microsoft Excel. P < 0.05 was ar NFATc2 expression (Fig. 1L). To extend and quantify this fi considered significant. nding, we analyzed a human TMA consisting of 217 human pancreatic cancer tissues for NFATc2 expression and found a positive maximum nuclear tumor staining intensity in 96 of Results 217 (44.2%) of subjects, indicating the presence of active p- GSK-3b confers pancreatic tumor growth stimulation by NFATc2, especially in neoplastic regions (Supplementary Table maintenance of NFAT expression levels through specific S1A). Collectively, our data suggest that contrary to the func- phosphorylation tion in immune cells, SP2 phosphorylation by GSK-3b in To confirm and extend existing data on GSK-3b inhibition in pancreatic cancer cells maintains active NFATc2 protein in the pancreatic cancer, we applied a GSK-3b inhibitor (hereafter des- nucleus, resulting in accelerated pancreatic cancer growth in ignated GSK-3i) to block kinase activity in different established vivo. pancreatic cancer cell lines. As expected, diminished GSK-3b activity was accompanied by a time- and dose-dependent decrease GSK-3b regulates NFATc2 activity through stabilization of of cancer cell proliferation (Fig. 1A and B). We next treated nude complex formation with STAT3 mice bearing IMIM-PC1 xenograft tumors 3 weekly with GSK-3i Next, we assessed whether NFATc2 activity is also regulated by and observed a significant tumor growth inhibition (Fig. 1C and GSK-3b or whether the observed elevated NFATc2 pSP2 activation Supplementary Fig. S1A), supporting the notion of a growth- levels were due to increased protein levels following nuclear promoting role of GSK-3b in pancreatic cancer. stabilization. Activation of the NFAT-responsive "cisNFAT" pro- Interestingly, previous work by our group had pointed toward moter correlated well with GSK-3b activity (Fig. 2A and B; Sup- an oncogenic GSK-3b/NFATc2 stabilization pathway in pancre- plementary Fig. S2A and S2B). However, expression of the atic and breast cancer cells (31). To further investigate the GSK-3b/ NFATc2 pSP2 construct, which is resistant to degradation upon NFATc2 axis, we assessed NFATc2 expression levels in pancreatic GSK-3b inhibition, could not activate the cisNFAT promoter cancer cell lines after pharmacologic inhibition or genetic deple- following GSK-3 depletion (Fig. 2A and Supplementary tion of GSK-3b. Treatment with the GSK-3i reduced nuclear Fig. S2B). This was surprising, as we expected that recombinant NFATc2 protein expression in a dose-dependent fashion, as did NFATc2 pSP2 expression levels would overrule GSK-3b depletion. transfection with a GSK-3b–specific siRNA (Fig. 1D and E and Moreover, expression of constitutively active GSK-3b (S9A) Supplementary Fig. S1B). Similarly, tissues derived from nude resulted in enhanced activity of the cisNFAT promoter when in mice following GSK-3i treatment showed substantially less the presence of wt or pSP2 NFATc1. Therefore, our data suggest NFATc2 (Fig. 1F). that GSK-3b not only regulates NFATc2 protein stability through As GSK-3b was reported to specifically phosphorylate three SP2 phosphorylation but also stimulates transcriptional activa- conserved serines in the NFATc2 SP2 (S213, S217, and S221) tion by a distinct mechanism.

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Figure 1. GSK-3b stabilizes NFATc2 in vitro and in vivo. A and B, PaTu8988t (A) and IMIM-PC1 (B) cells were treated with indicated concentrations of GSK-3b inhibitor AR-A014418 (hereafter referred to as GSK-3i) and analyzed for BrdUrd incorporation after 24 hours and 48 hours. C, nude mice bearing IMIM-PC1 xenograft tumors were treated 3 weekly with 10 mg/kg BW GSK-3i (n ¼ 5) or 10% DMSO (n ¼ 5) intraperitoneally. Tumors were extracted after 4 weeks. The tumor growth was normalized to the respective starting volumes. Means SE. , P < 0.05; , P < 0.01. D, PaTu8988t cells were starved and nuclear translocation of NFATc2 was induced by ionomycin treatment (0.5 mmol/L). Cells were then treated for 1 hour with 2.5, 25, and 50 mmol/L of GSK-3i and analyzed for NFATc2 and p-GS expression. Reduction of the well-described GSK-3b target p-GS serves as treatment control. E, PaTu8988t cells were transfected with GSK-3b siRNA, and lysates were tested for NFATc2 and GSK-3b expression after 48 hours. F and G, tumor lysates from three representative mice treated with 10% DMSO or GSK-3i were assessed for NFATc2 and p-GS (F) or NFATc2 and p-NFATc2 (G) expression as indicated. H, nuclear extracts from Suit-028 cells with stable expression of wt NFATc2 and NFATc2 pSP2 were treated for 3 hours with 25 mmol/L GSK-3i and then subjected to immunoblot analysis with the indicated antibodies. Lamin a/c serves as loading control. I, nuclear lysates from Suit-007 cells with stable expression of wt NFATc2 and NFATc2 pSP2 were subjected to DNA pulldown assays with oligonucleotides bearing the NFAT consensus sequence. DNA-bound NFATc2 was then analyzed by immunoblotting. J, PaTu8988t cells with stable wt NFATc2 and NFATc2 pSP2 expression or untransfected control cells were transfected with calcineurin and a reporter construct harboring three NFAT consensus binding sites (cisNFAT). Luciferase activity was measured after 24 hours. Displayed are means SD related to the control (ﬁrst bar). K, nude mice were injected with PaTu8988t cells with stable wt NFATc2 or NFATc2 pSP2 expression or control cells, and tumor volumes were estimated after 4 weeks (n 5). Shown are means SE. , P < 0.05; , P < 0.01. L, representative IHC pictures of human PDA showing nuclear p-NFATc2 expression.

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Figure 2. SP2-independent regulation of NFATc2 activity by GSK-3b. Luciferase reporter gene assay demonstrating regulation of the cisNFAT promoter in PaTu8988t cells with stable wt NFATc2 and NFATc2 pSP2 expression or control cells following transfection with GSK-3b siRNA (A) or GSK-3b S9A (B). Means SD. C, PaTu8988t cells with stable expression of indicated constructs were transfected with wt STAT3 and cisNFAT and subjected to reporter gene assay. Shown are means SD normalized to controls. D, PaTu8988t cells with stable expression of indicated constructs were treated with 25 mmol/L GSK-3i, and after 3 hours nuclear lysates were subjected to coimmunoprecipitation and subsequent immunoblotting as indicated. E, reporter gene assay of Suit-007 cells with stable expression of indicated constructs after transfection with wt STAT3 and cisNFAT and subsequent administration of 25 mmol/L GSK-3i for 6 hours. Means SD. Successful knockdown of STAT3 is shown on right.

We recently established an oncogenic NFATc1-STAT3 coop- NFATc2 physically and functionally cooperates with canonical erativity governing pancreatic cancer progression (27). Thus, we p-STAT3 (Y705) signaling to maintain cell growth were interested whether other NFAT family members such as We next sought to characterize the NFATc2–STAT3 complex NFATc2 also interact and cooperate with STAT3 to promote and its functional implication in pancreatic cancer. Immunohis- cancer growth and whether this cooperation is regulated by tochemical costaining of 217 human pancreatic cancer tissues GSK-3b. We detected comparable and robust physical interac- revealed that 81 (37.3%) of cases were positive for both nuclear tion of endogenous STAT3 with all NFATc2 SP2 mutants NFATc2 and nuclear p-STAT3 (Fig. 3A; Supplementary Table S1B). (Supplementary Fig. S2C). Correspondingly, STAT3 overexpres- Remarkably, those tumors that showed NFATc2 expression sion remarkably elevated NFATc2 activation of the cisNFAT stained positive for p-STAT3 in 84% (Fig. 3A). These observations promoter, irrespective of NFATc2 phosphorylation on SP2 (Fig. were confirmed by immunofluorescence studies detecting simul- 2C). We next assessed whether this NFATc2/STAT3 complex taneous nuclear NFATc2 and STAT3 expression levels in pancre- formation is regulated by GSK-3b in pancreatic cancer. Signif- atic cancer cells (Fig. 3B). Interestingly, the inflammatory cytokine icantly, GSK-3b inhibition disrupted the physical interaction of IL6 could induce a robust physical STAT3–NFATc2 interaction STAT3 with both wt and pSP2 NFATc2 (Fig. 2D). In addition, that was further enhanced by the addition of serum (Fig. 3C) and depletion of STAT3 abrogated the ability of GSK-3b S9A to that required STAT3 Y705 phosphorylation (Fig. 3D). In addition, enhance the transactivation of the cisNFAT promoter in the DNA pulldown assays demonstrated an interaction of the tran- presence of wt NFATc2 (Fig. 2E). Moreover, STAT3 could not scription factors on the NFAT consensus sequence that was also transactivate NFATc2 without GSK-3b activity (Supplementary dependent on STAT3 phosphorylation (Fig. 3E). The expression of Fig. S2D). Taken together, these data suggest that a functional mutant STAT3 Y705F most notably antagonized the activating NFATc2–STAT3 transcriptional complex requires the presence effect of wt STAT3 on the NFAT-responsive "cisNFAT" promoter, of GSK-3b activity not only to prevent NFATc2 nuclear degra- indicating that STAT3 phosphorylation is not only required for dation but also to engender NFATc2–STAT3 complex formation complex formation but also for efficient NFATc2 transactivation and transcriptional activity. (Fig. 3F). It is noteworthy that GSK-3i treatment reduced STAT3

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Figure 3. STAT3 Y705 phosphorylation is required for efficient physical and functional cooperation with NFATc2. A, TMA analysis of 217 cases of PDACs shows nuclear NFATc2 expression in 44% of cases, of which 84% also stained positive for p-STAT3. B, immunofluorescence staining displays nuclear colocalization of STAT3 (green) and HA-tagged NFATc2 (red) in PaTu8988t cells with stable expression of HA-NFATc2. Nuclei are visualized by DAPI staining (blue). C, Suit-007 cells with stable wt NFATc2 expression were transfected with Flag-tagged wt STAT3 and incubated with serum-free or serum medium for 24 hours with subsequent stimulation with IL6 (50 ng/mL). Lysates were subjected to coimmunoprecipitation and Western blotting as indicated. D, coimmunoprecipitation demonstrating reduced interaction of nuclear HA-tagged wt NFATc2 with Flag-tagged STAT3 Y705F compared with wt STAT3 in Suit-028 cells with stable HA-NFATc2 expression. E, PaTu8988t cells were transfected with STAT3 constructs as indicated and subjected to DNA pulldown assays with oligonucleotides bearing the NFAT consensus sequence. F, reporter gene assay demonstrating the inducibility of cisNFAT upon transfection of wt STAT3 and STAT3 Y705F in Suit-028 control cells and cells with stable expression of wt NFATc2 under serum-free conditions. Means SD. G, proliferation of Suit-007 cells with and without stable expression of wt NFATc2 following 72 hours of STAT3 siRNA transfection. Means SD. Efficient knockdown was tested by immunoblotting and is shown in Fig. 2E.

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Figure 4. Stimulation of euchromatin formation on CDK-6 target promoter through NFATc2–STAT3 complexes. A, transcription start site (TSS) binding motifs for NFAT and STAT3. B, human CDK-6 promoter with putative NFATc2 and STAT3 binding sites. C, chromatin immunoprecipitation analysis determines NFATc2, STAT3, RNA-polymerase II (RNA-Pol II), and H3K4me3 binding to the CDK-6 promoter with and without AR-A014418 (GSK-3i) treatment (25 mmol/L for 12 hours). Means SD. D, qPCR illustrating reduced CDK-6 mRNA expression in PaTu8988t cells following GSK-3b inhibition with GSK-3i (25 mmol/L for 3 hours). E, lysates from PaTu8988t cells with stable expression of GSK-3b S9A or following transfection with GSK-3b siRNA were subjected to immunoblotting with indicated antibodies.

phosphorylation at Y705 in different pancreatic cancer cell lines expression (Fig. 4D and E). Consistent with the role of GSK-3b (Supplementary Fig. S3A–S3C), suggesting that GSK-3b stabilizes in promoting the transactivation of NFATc2–STAT3 complexes, NFATc2–STAT3 complexes by maintaining STAT3 phosphoryla- CDK-6 expression was decreased after upon GSK-3i treatment tion. Finally, proliferation assays revealed that the loss of STAT3 (Supplementary Fig. S3E). Taken together, our data strongly point expression reversed the NFATc2-dependent stimulation of cancer to a multilevel regulatory impact of GSK-3b on NFATc2, which is, cell proliferation, highlighting its importance for NFATc2- on one hand, mediated by SP2 phosphorylation-dependent sta- induced growth stimulation (Fig. 3G). Accordingly, cell prolifer- bilization of protein levels in the nucleus and on the other hand by ation of PaTu8988t was reduced after STAT3 siRNA transfection, recruitment of NFATc2–STAT3 complexes to target promoters and independent of SP2 phosphorylation (Supplementary Fig. S3D). subsequent transcription of cell-cycle promoters independent of In summary, p-STAT3 (Y705) efficiently binds to and transacti- SP2 phosphorylation. vates NFATc2 on responsive promoters under mitogenic and inflammatory conditions, and in turn, acts to sustain NFATc2- Targeting the NFATc2–STAT3–GSK-3b cooperativity interferes dependent cell growth in a GSK-3b–dependent manner. Collec- with inflammation-induced pancreatic cancer progression in G12D tively, GSK-3b maintains NFATc2 activation through stabilization Kras mice of NFATc2–STAT3 complexes independent of SP2 phosphoryla- To assess the therapeutic potential of targeting the identified tion, presumably through phosphorylation of STAT3 at Y705. NFATc2–STAT3–GSK-3b cooperativity in vivo, we utilized a well- We next analyzed the GSK-3b–dependent NFATc2/STAT3 inter- established model of pancreatitis-associated carcinogenesis to action on the previously described NFATc2 target promoter, CDK- ensure profound induction of NFATc2 and STAT3, as these tran- 6 (Fig. 4A and B). Importantly, GSK-3i treatment resulted not only scription factors were shown to be overexpressed or activated in the in the loss of NFATc2 binding to the CDK-6 promoter but also majority of Kras-mutated tumor specimen, especially upon inflam- reduced the binding of STAT3 (Fig. 4C). Furthermore, binding of matory conditions (37, 38). In line with previously published data, RNA-polymerase II and the activating histone modification we observed that cancer progression was massively accelerated H3K4-me3 was significantly decreased upon GSK-3i administra- when transgenic mice with pancreas-specific expression of onco- tion (Fig. 4C), followed by loss of CDK-6 gene and protein genic KrasG12D were challenged 3 weekly with the CCK analogue

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Figure 5. G12D Targeting GSK-3 prolongs pancreatic cancer progression in KrasG12D mice. A, representative H&E staining in Pdx1;Kras mice after treatment with PBS (n ¼ 10) caerulein (n ¼ 7) or caerulein along with the GSK-3 inhibitor 9-ING-41 (designated GSK-3i; n ¼ 8), showing retarded tumor progression upon GSK-3 inhibition. Scale bars, 100 mmol/L (top) and 200 mmol/L (bottom). B, quantification of normal and preneoplastic ducts in Pdx1;KrasG12D mice upon indicated treatment. Means SD (n 6). , P values are related to untreated Pdx1;KrasG12D cohorts and D values indicate caerulein versus caerulein and GSK-3i treatment. and D, P < 0.05; and DD, P < 0.01; and DDD, P < 0.001. C, immunohistologic p-STAT3, p-NFATc2, and Ki67 staining in Pdx1; G12D G12D Kras mice after treatment as indicated. Scale bars, 100 mmol/L. D, tissues from Pdx1;Kras mice after treatment as indicated were assayed for p-STAT3, G12D STAT3, p-NFATc2, and NFATc2 expression. E, proliferation index was measured in Ki67-stained pancreatic sections (n 3) of Pdx1;Kras mice after indicated treatment. Means SE. , P < 0.05; , P < 0.001. caerulein to induce a persistent organ inflammation (9, 10). Mice Discussion developed the full spectrum of precursor lesions ranging from Pancreatic cancer exhibits a high therapeutic resistance against noninvasive PanIN1 to PanIN2/3 and carcinoma in situ lesions cytotoxic and apoptosis-inducing agents (39), and although (Fig. 5A and B). This was accompanied by induced p-NFATc2 (SP2) genetic approaches targeting Kras in mice have shown encourag- and p-STAT3 (Y705) levels (Fig. 5C and D). When mice were ing results (40, 41), the oncogene is still deemed "undruggable" in additionally treated with a GSK-3i, however, cancer progression mice and man. Persistent inflammation contributes to tumor was significantly blocked in terms of proliferation, neoplastic progression and therapeutic resistance in pancreatic cancer, and transformation, and tumor incidence, as evidenced by restored the activation of proinflammatory cytokines and transcription normal ducts, decreased precursor lesions (Fig. 5B), and signifi- factors is a molecular feature of this disease. Although GSK-3b is cantly reduced Ki67 expression levels (Fig. 5E). Moreover, we not induced by inflammation itself, it governs a variety of inflam- observed a reduced tendency to develop tumors upon GSK-3i matory responses such as enhanced cytokine production driven treatment (30% after caerulein; n ¼ 7 vs. 0% after caerulein and by NF-kB and STAT3 pathways (21–24). Interestingly, the anti- GSK-3i; n ¼ 8). Finally, induction of p-NFATc2 and p-STAT3 was inflammatory potential of GSK-3 inhibition was evidenced in our antagonized upon treatment with GSK-3i (Fig. 5C and D), clearly study by a clear reduction of immune cell infiltrations in GEMM of highlighting the regulatory impact of GSK-3 on NFATc2/STAT3– pancreas cancer. mediated cancer progression and growth. Interestingly, increased NFAT proteins represent another class of major proinflam- immune cell infiltrations upon caerulein treatment were markedly matory transcription factors that were originally described as reduced upon GSK-3 inhibition (Supplementary Fig. S4). inducible factors binding to the IL2 promoter in activated T

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Figure 6. Proposed model of GSK-3b–mediated regulation of the NFATc2 metabolism in pancreatic cancer cells. Activation of the phosphatase calcineurin following an intracellular calcium inﬂux dephosphorylates NFAT proteins and stimulates their nuclear import. There, GSK-3b–mediated SP2 phosphorylation protects NFATc2 from degradation and further stimulates DNA binding and transcriptional activation of target genes (left). In addition, GSK-3b stabilizes NFATc2/ STAT3 complex formation independent of and dominant to SP2 phosphorylation resulting in target gene expression (right).

cells (42). Strongly induced upon inflammatory stimuli, NFAT fine-tuning DNA binding and specificity to target promoters. This proteins have been shown to be overexpressed in the majority cooperation with partner proteins serves to stabilize and modu- of invasively growing cancers, presumably in those with per- late DNA binding of NFAT proteins, thus generating a sophisti- sistent inflammation, and to dramatically accelerate Kras-driv- cated axis of interdependencies and feedback loops. STAT3 pro- en carcinogenesis in the pancreas (27, 28, 30). However, as teins are aberrantly activated in most cancers, and recently therapeutic targeting of transcription factors is difficult, it is emerged as potent mediators of pancreatic malignant transfor- critical to identify upstream regulatory pathways that may be mation and cancer progression, especially under inflammatory targetable for cancer therapeutics. conditions (43, 44). In line with these observations, we have Here, we investigated the role of GSK-3b on the regulation previously shown that NFATc1 induces STAT3 expression with and activation of NFATc2 in pancreatic cancer progression and subsequent formation of NFATc1/STAT3 nucleoprotein com- growth. We propose the following model: in contrast to results plexes in pancreatic cancer cells. Likewise, STAT3 affected NFATc1 in immune cells, where GSK-3b phosphorylates three serine transcriptional activity by maintaining its DNA binding on distant residues in the N-terminal SP2 domain and thus terminates enhancers and hence stimulated enhancer–promoter communi- DNA binding of NFAT (34–36), we show that specificphos- cation leading to the expression of oncogenes such as EGFR, cyclin phorylation of the SP2 region of NFATc2 by GSK-3b induces D3, or Wnt proteins (27, 28, 30). NFATc2 protein stability in the nucleus of pancreatic cancer From a therapeutic point of view, targeting of central upstream cells (Fig. 6). Thus, we extend our existing knowledge of the regulators, and subsequently the complete proinflammatory GSK-3b–NFATc2 stabilization pathway by uncovering nuclear oncogenic network, appears feasible and may help overcome activation of p-NFATc2 as a potentially specific diagnostic tool drug resistance and apoptosis inhibition. Notably, caution has and therapeutic target, as targeting p-NFATc2 would suppos- been raised in the therapeutic field owing to the putative tumor edly not harm NFAT signaling in immune cells. However, suppressive functions of GSK-3b as inhibition may result in the protein stability itself is not the exclusive mechanism governing activation of the Wnt pathway and subsequent tumorigenesis. robust protumorigenic effects driven by the GSK-3b–NFATc2 Reassuringly, however, Wnt signaling and b-catenin nuclear accu- axis. In fact, our data highlight that GSK-3b orchestrates an SP2 mulation are not perturbed in GSK-3b–deficient mice (14). Also, phosphorylation–independent mechanism involving NFATc2– lithium, a known inhibitor of GSK-3, causes a reduced cancer STAT3complexformationthatisrequiredtostimulateeuchro- morbidity in psychiatric patients compared with the general matin formation of NFAT target promoters, such as CDK-6. This population and inhibits proliferation in hepatocellular carcino- is likely dominant over SP2-dependent NFATc2 protein stabi- ma cell lines (45, 46). lization, as it can be disrupted through GSK-3b inhibition, even Collectively, our data suggest that GSK-3b fine-tunes NFATc2 in the presence of p-NFATc2. and STAT3 transcriptional networks to integrate upstream In addition, our observations suggest that the activity of NFAT signaling events to eventually govern pancreatic cancer pro- proteins is not only executed by a variety of phosphorylation gression and growth. Furthermore, the therapeutic potential of events (some GSK-3b–mediated) but to a great extent by dynamic GSK-3b to delay inflammation-induced malignant transforma- and complex protein–protein interactions influencing and tion is demonstrated for the first time in a relevant GEMM for

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Inﬂammatory Signaling in Pancreatic Cancer

pancreatic cancer. In a broader sense, our data add to the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, molecular understanding of GSK-3b–mediated tumorigenesis computational analysis): S. Baumgart, N.-M. Chen, S.K. Singh, D. Fink, € and may thus open new avenues for individually tailored P. Strobel, L. Zhang, W.R. Bamlet, A. Koenig, E. Hessmann, A. Neesse Writing, review, and/or revision of the manuscript: S. Baumgart, N.-M. Chen, therapies. For instance, one can imagine that patients with J.-S. Zhang, D.D. Billadeau, A.P. Kozikowski, P. Strobel,€ A. Koenig, E. Hessmann, high expression of NFATc2 may beneﬁtthemostfromGSK- A. Neesse 3b inhibitor-based therapy. Our results are particularly ger- Administrative, technical, or material support (i.e., reporting or organizing mane given the fact that a large number of clinical GSK-3b data, constructing databases): N.-M. Chen, T.M. Gress inhibitors are available and currently being tested in several Study supervision: N.-M. Chen, S.K. Singh, V. Ellenrieder, A. Neesse disease entities, including pancreatic cancer patients.

Disclosure of Potential Conﬂicts of Interest Grant Support A.P. Kozikowski is the owner and founder of, has ownership interest This work was supported in part by the NCI Pancreatic Cancer SPORE grant (including patents) in, and is a consultant/advisory board member for P50CA102701 (to D.D. Billadeau), by a Max Eder Fellowship (110972) Actuate Therapeutics. No potential conﬂicts of interest were disclosed by Deutsche Krebshilfe (to A. Neesse), and by the National Natural Science the other authors. Foundation of China (grant number 81472601, to J.S. Zhang). The costs of publication of this article were defrayed in part by the Authors' Contributions payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Conception and design: S. Baumgart, N.-M. Chen, J.-S. Zhang, S.K Singh, this fact. V. Ellenrieder, A. Neesse Development of methodology: N.-M. Chen, J.-S. Zhang, A.P. Kozikowski Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Baumgart, N.-M. Chen, J.-S. Zhang, I.N. Gaisina, Received April 17, 2015; revised November 13, 2015; accepted November 28, S.K. Singh, D. Fink, C. Klindt, E. Hessmann, T.M. Gress, A. Neesse 2015; published OnlineFirst January 28, 2016.

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GSK-3β Governs Inflammation-Induced NFATc2 Signaling Hubs to Promote Pancreatic Cancer Progression

Sandra Baumgart, Nai-Ming Chen, Jin-San Zhang, et al.

Mol Cancer Ther Published OnlineFirst January 28, 2016.

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