Inman, Kristin S.

Protein kinase C iota (PKCι) functions as a bonafide human oncogene in lung and ovarian cancer and is required for KrasG12D-mediated lung cancer initiation and progression. PKCι expression is required for pancreatic cancer cell growth and maintenance of the transformed phenotype; however, nothing is known about the role of PKCι in pancreas development or pancreatic tumorigenesis. In this study, we investigated the effect of pancreas-specific ablation of PKCι expression on pancreatic cellular homeostasis, susceptibility to pancreatitis, and KrasG12D-mediated pancreatic cancer development. Knockout of pancreatic Prkci significantly increased pancreatic immune cell infiltration, acinar cell DNA damage, and apoptosis, but reduced sensitivity to caerulein-induced pancreatitis. Prkci-ablated pancreatic acinar cells exhibited P62 aggregation and a loss of autophagic vesicles. Loss of pancreatic Prkci promoted KrasG12D-mediated pancreatic intraepithelial neoplasia formation but blocked progression to adenocarcinoma, consistent with disruption of autophagy. Our results reveal a novel promotive role for PKCι in pancreatic epithelial cell autophagy and pancreatic cancer progression.

Abstract Background: The need to develop valid methods for sampling and analyzing fecal specimens for microbiome studies is increasingly important, especially for large population studies. Methods: Some of the most important attributes of any sampling method are reproducibility, stability, and accuracy. We compared seven fecal sampling methods [no additive, RNAlater, 70% ethanol, EDTA, dry swab, and pre/post development fecal occult blood test (FOBT)] using 16S rRNA microbiome profiling in two laboratories. We evaluated nine commonly used microbiome metrics: abundance of three phyla, two alpha-diversities, and four beta-diversities. We determined the technical reproducibility, stability at ambient temperature, and accuracy. Results: Although microbiome profiles showed systematic biases according to sample method and time at ambient temperature, the highest source of variation was between individuals. All collection methods showed high reproducibility. FOBT and RNAlater resulted in the highest stability without freezing for 4 days. In comparison with no-additive samples, swab, FOBT, and 70% ethanol exhibited the greatest accuracy when immediately frozen. Conclusions: Overall, optimal stability and reproducibility were achieved using FOBT, making this a reasonable sample collection method for 16S analysis. Impact: Having standardized method of collecting and storing stable fecal samples will allow future investigations into the role of gut microbiota in chronic disease etiology in large population studies. Cancer Epidemiol Biomarkers Prev; 25(2); 407–16. ©2015 AACR.

Abstract Pancreatic cancer is the fourth leading cause of cancer death in the United States with current 5-year survival rates at 6%. Greater than 90% of all pancreatic cancers harbor an activating mutation in codon 12 of the Kras gene (KrasG12D). Protein Kinase C iota (PKCι) has been shown to mediate KrasG12D downstream signaling and acinar-to-ductal metaplasia in an explant model of pancreatic cancer initiation. Additionally, inhibition of PKCι in human pancreatic cancer cells leads to decreased tumor size, angiogenesis, and metastasis in an orthotopic tumor model, suggesting that PKCι plays a role in maintaining the oncogenic phenotype of pancreatic cancer cells. We sought to test the hypothesis that PKCι plays a role development and maintenance of pancreatic cancer using a mouse model of KrasG12D-induced pancreatic cancer. Purpose: To identify the effect of PKCι loss on pancreas function and development of KrasG12D-induced pancreatic cancer. Procedures: Pancreata from mice with pancreas-specific deletion of PKCι, with and without pancreas-specific KrasG12D expression, were analyzed for histological and protein expression alterations. Acinar cells isolated from these transgenic mouse models were subjected to in vitro analyses to determine the role of PKCι in pancreatic cell homeostasis. Results: Mice lacking pancreatic PKCι survived and developed normally. Pancreatic knockout of PKCι expression resulted in a phenotype consistent with a defect in autophagic flux. In vitro analyses of isolated acinar cells supported the hypothesis that PKCι promotes autophagy in pancreatic cells. Autophagy exhibits a dual functionality in pancreatic cancer, acting as a tumor suppressor in normal cells and a tumor promoter in established tumor cells. We sought to determine the effect of PKCι knock-out on both of these cell types in a mouse model of pancreatic cancer. Using the LSL mouse model of K-ras induced pancreatic cancer, we demonstrated that pancreatic loss of PKCι lead to an increase in the number of Pancreatic Intraepithelial (PanIN) lesions formed in young mice. However, these lesions were protected from progressing past the early PanIN1/2 stage such that no mice lacking PKCι developed cancer, even after 1.5 years of age. By contrast, over 50% of mice with wild-type PKCι developed cancer; of those without cancer, many had progressed to the PanIN-3 stage, a stage representative of carcinoma in-situ. These data are similar to that seen in mouse models of pancreatic cancer with genetic inhibition of autophagy. Analysis of PanINs in these mice revealed that those lacking PKCι expressed significantly more P62 protein, a selective autophagy substrate, compared to PanINs with wild-type PKCι, supporting a block in the autophagic flux. These data suggest that PKCι plays an important role in both the initiation and progression of pancreatic cancer, possibly by regulating autophagic flux. Impact: Inhibition of pancreatic PKCι expression prevented progression of PanIN lesions past the early PanIN1/2 stage, resulting in a complete block in cancer development in mouse models of pancreatic cancer. Our preliminary data suggests that PKCι promotes cancer progression by regulating autophagic flux, a process that has been shown to be essential for KrasG12D-mediated pancreatic cancers. Pancreatic cancer is characterized by resistance to current therapies. Novel targets for new and more effective therapies are critical to improve outcomes for patients with this deadly disease. Inhibitors of PKCι are currently in clinical trials for ovarian and lung cancers. Our data supports the feasibility of using PKCι inhibitors for therapy in pancreatic cancer. Citation Format: Kristin S. Inman, Amanda A. Francis, Nicole R. Murray. PKCι in pancreatic cancer development: A possible role for autophagy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A02.