Abstract PR04: p120 catenin loss drives pancreatic cancer EMT and metastasis through activation of calcium signaling

Abstract

Abstract Purpose: We have generated a mouse model to delete the gene Ctnnd1, whose gene product p120 catenin (p120ctn) is necessary for E-CADHERIN stability, resulting in enhanced metastasis in the conventional KPC pancreatic tumor mouse model. An unbiased screen of tumor cells isolated from these mice identified misregulated calcium signaling as a previously unappreciated contributor to epithelial-to-mesenchymal transition (EMT) and metastasis. Thus, our overarching hypothesis is that p120ctn loss in pancreatic cancer drives EMT and metastasis through functional upregulation of the calcium signaling component PTHLH. Background: Pancreatic ductal adenocarcinoma (PDAC) is a major health issue, with only 7% of patients surviving beyond 5 years, and increases in PDAC-associated deaths project this disease to be the second leading cause of cancer deaths by 2020. An unbiased approach to discover candidate cancer genes in PDAC identified the p120 catenin gene as one of the top 20 PDAC cancer genes, and further analysis revealed that p120ctn loss was associated with reduced PDAC patient survival. Recent work from our lab further demonstrated that conditional p120ctn/Ctnnd1 deletion in the esophagus was sufficient to drive invasive squamous cell carcinoma, establishing p120ctn/Ctnnd1 as a bona fide tumor-suppressor gene. Results presented herein determined the in vivo role of p120ctn loss in PDAC tumorigenesis and metastasis through genetic mouse models and unbiased RNA-seq analysis. Methods: A PDAC mouse model was established to study the role of p120ctn in pancreatic carcinogenesis and metastasis. Specifically, LSL-KrasG12D/+; p53fl/+; Pdx1cre; Rosa26LSL-YFP; Ctnnd1fl/wt mice (herein KPCY-p120CKO) were generated to determine the effect of conditional p120ctn loss on pancreatic cancer. Furthermore, RNA-seq was performed on p120-intact or p120-null pancreatic tumor cells isolated from these mice to identify novel mechanisms of pancreatic tumorigenesis and metastasis. Results: We demonstrate p120ctn loss as a catastrophic event for tumor epithelial cell identity in vivo, leading to enhanced EMT and metastasis. Specifically, we show that KPCY-p120CKO mice have an enhanced metastatic phenotype relative to KPCY controls. Our data therefore suggest that p120ctn is a critical factor in metastatic cell dissemination, and that p120ctn loss results in tumor cells being “locked” in a mesenchymal phenotype by failing to stabilize E-cadherin at the metastatic site. To determine the mechanism of enhanced EMT and metastasis, RNA-seq analysis of p120ctn-null tumor cells was performed, which surprisingly revealed aberrant activation of calcium signaling. Unexpectedly, two of the top five most upregulated genes in p120ctn-null cells were the secreted factor Pthlh and the kinase Camk2b, both of which are key signaling molecules involved in calcium signaling. We demonstrate that PTHLH binding to its cognate receptor leads to cytosolic calcium ion (Ca2+) release, resulting in phosphorylation and activation of CaMKII. We further establish that genetic deletion or pharmacologic inhibition of Pthlh results in proliferation and migration defects. Moreover, orthotopic implantation of KPC-PthlhNULL tumor cell lines reduced tumor growth and metastasis in vivo. Finally, we show that PDAC patients with high expression of Pthlh have significantly decreased survival, suggesting that calcium signaling may be a potent oncogenic pathway in pancreatic cancer and that blocking this pathway may be of therapeutic benefit. Conclusions: This work has demonstrated the importance of the previously unappreciated role of calcium signaling in pancreatic cancer progression and metastasis, and future studies will look to determine the efficacy of calcium-modulating therapeutics in preclinical models of pancreatic cancer. This abstract is also being presented as Poster A42. Citation Format: Jason R. Pitarresi, Maximilian Reichert, Basil Bakir, Leticia Moreira, Lauren Simon, Anil K. Rustgi. p120 catenin loss drives pancreatic cancer EMT and metastasis through activation of calcium signaling [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr PR04.