Protein Kinase C Iota Regulates Pancreatic Acinar-to-Ductal Metaplasia

et al. (2012) Protein Kinase C Iota Regulates Pancreatic Acinar-to-Ductal Metaplasia. PLoS ONE 7(2): e30509. doi:10.1371/journal.pone.0030509 Protein Kinase C Iota Regulates Pancreatic Acinar-to- Ductal Metaplasia Michele L. Scotti 0 Kristin E. Smith 0 Amanda M. Butler 0 Shelly R. Calcagno 0 Howard C. Crawford 0 Michael Leitges 0 Alan P. Fields 0 Nicole R. Murray 0 Guenter Schneider, Technische Universitat M unchen, Germany 0 1 Department of Cancer Biology, Mayo Clinic , Jacksonville , Florida, United States of America, 2 Department of Pharmacological Sciences, Stony Brook University , Stony Brook , New York, United States of America, 3 Biotechnology Centre of Oslo, University of Oslo , Oslo , Norway Pancreatic acinar-to-ductal metaplasia (ADM) is associated with an increased risk of pancreatic cancer and is considered a precursor of pancreatic ductal adenocarcinoma. Transgenic expression of transforming growth factor alpha (TGF-a) or KrasG12D in mouse pancreatic epithelium induces ADM in vivo. Protein kinase C iota (PKCi) is highly expressed in human pancreatic cancer and is required for the transformed growth and tumorigenesis of pancreatic cancer cells. In this study, PKCi expression was assessed in a mouse model of K-rasG12D-induced pancreatic ADM and pancreatic cancer. The ability of K-rasG12D to induce pancreatic ADM in explant culture, and the requirement for PKCi, was investigated. PKCi is elevated in human and mouse pancreatic ADM and intraepithelial neoplastic lesions in vivo. We demonstrate that K-rasG12D is sufficient to induce pancreatic ADM in explant culture, exhibiting many of the same morphologic and biochemical alterations observed in TGF-a-induced ADM, including a dependence on Notch activation. PKCi is highly expressed in both TGF-a- and K-rasG12D-induced pancreatic ADM and inhibition of PKCi significantly reduces TGF-a- and K-rasG12D-mediated ADM. Inhibition of PKCi suppresses K-rasG12D-induced MMP-7 expression and Notch activation, and exogenous MMP-7 restores KrasG12D-mediated ADM in PKCi-depleted cells, implicating a K-rasG12D-PKCi-MMP-7 signaling axis that likely induces ADM through Notch activation. Our results indicate that PKCi is an early marker of pancreatic neoplasia and suggest that PKCi is a potential downstream target of K-rasG12D in pancreatic ductal metaplasia in vivo. - Funding: Mayo Clinic SPORE in Pancreatic Cancer Career Development award P50 CA102701 (N.R. Murray), NIH/NCI R01CA140290-1 (N.R. Murray), NIH/NCI R01CA081436-13 (A.P. Fields), Fraternal Order of Eagles Cancer Research Fund Pilot Program (N.R. Murray), Daniel Foundation of Alabama Postdoctoral Fellowship (M.L. Scotti) and The Mayo Clinic Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: A provisional patent related to this research has been filed (MLS, APF, NRM). This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. All other authors have nothing to disclose. Patent Application Title: Methods and Materials for Treating Pancreatic Cancer, Patent Application No.: 12/981,114, Docket No.: 07039.0972001, Mayo Case No.: 2009-364. Oncogenic KRAS mutations are found in .90% of pancreatic ductal adenocarcinomas (PDACs). [1] Mutational activation of KRAS is thought to occur early in PDAC development, as KRAS mutations are observed in ,30% of PDAC precursor lesions, pancreatic intraepithelial neoplasia (PanIN). [1] A mouse model for conditional expression of an activated Kras (KrasG12D) allele in the pancreas from its physiological promoter has been utilized to investigate the role of oncogenic K-ras in initiation and progression of PDAC. [2,3,4] Expression of oncogenic K-ras induces formation of preneoplastic lesions in mice that are histologically similar to human PanINs (mouse PanINs, mPanINs). [2,4] K-rasG12Dinduced mPanINs become increasingly dysplastic, with a small percent progressing to invasive and metastatic adenocarcinomas, strongly suggesting that acquisition of an oncogenic Kras mutation can be an initiating event in pancreatic cancer. [2,4] Acinar-to-ductal metaplasia (ADM), the replacement of acinar cells with metaplastic ductal cells, is thought to be a source of neoplasia in the initiation of human PDAC. [4,5,6] Dysplastic features often arise in areas of ductal metaplasia, and metaplastic ductal cells exhibit many properties of embryonic progenitor cells, including Nestin expression. [4,7,8] The K-rasG12D-initiated mouse model of PDAC exhibits morphological, molecular and biochemical features indicative of ADM as early as 4 weeks of age, prior to the development of mPanINs. [2,4] Aberrant activation of EGFR signaling in mouse pancreas also induces ADM and subsequent formation of PDAC. [7,9,10] EGFR-mediated ADM has been further characterized in an explant model. [11,12] TGFa induces primary mouse pancreatic acinar cells to transition through a de-differentiated, Nestin-positive intermediate to form metaplastic ductal structures. [7,11,12] Additional studies revealed that Notch signaling is both necessary and sufficient for acinar cell de-differentiation, Nestin expression and ADM in explant culture. [2,12] MMP-7, which is also upregulated in human and mouse PanINs and PDAC, promotes activation of Notch signaling and ADM. [13,14] MMP-7 is required for ADM in explant culture, and expression of a constitutively active Notch construct reconstitutes ADM in MMP-7depleted acinar cells, indicating that MMP-7-dependent Notch activity is required for ADM. [14] These studies demonstrate the utility of the pancreatic acinar cell explant model for characterization of ADM, and strengthen the link between pancreatic metaplasia, neoplasia and initiation of PDAC. We have identified PKCi as an important effector in oncogenic K-ras-induced transformation of lung and intestinal epithelial cells. [15,16] We have also demonstrated that PKCi expression is elevated in a large percent of primary pancreatic adenocarcinomas, and high PKCi expression predicts poor patient survival. [17] In the current study, we demonstrate that PKCi is elevated in pancreatic metaplasia associated with human PDAC tumors and in K-rasG12D-mediated pancreatic metaplasia in vivo. To further characterize the molecular mechanism of K-rasG12D-mediated pancreatic ADM we employed a well-characterized mouse pancreatic acinar cell explant model. In this context, we evaluated the role of PKCi in K-rasG12D-mediated pancreatic ADM. Expression of oncogenic K-ras, the most frequently mutated oncogene in PDAC, is sufficient to induce pancreatic ADM in explant culture. PKCi expression is elevated in K-rasG12D- and TGFa-induced ADM. Inhibition of PKCi significantly reduces both K-rasG12D- and TGFa-induced ADM and also significantly reduces K-rasG12D-mediated Nestin expression, Notch activation and MMP-7 expression. Exogenous MMP-7 partially but significantl (...truncated)