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

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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. Ó2016 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 initia- tion 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- Ó2016 American Association for Cancer Research. stitutive active and multifunctional Ser/Thr kinase, which, due to www.aacrjournals.org OF1 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst January 28, 2016; DOI: 10.1158/1535-7163.MCT-15-0309 Baumgart et al. 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 Â 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
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