Abstract B69: Evaluation of Src-mediated signaling events in pancreatic cancer.

Abstract

The importance of the Src tyrosine kinase in pancreatic ductal adenocarcinoma (PDAC) has been validated by substantial evidence derived from preclinical genetic and pharmacological studies in PDAC cell lines and mouse models. In particular, a key role has been established for Src modulation of invasion and metastasis. Further, Src overexpression and activation has been correlated with poorer PDAC patient survival. Nevertheless, successful clinical application of Src inhibitors will requre a better understanding of the specific signaling events that contribute to Src-mediated transformation in PDAC and of appropriate biomarkers for its inhibition. Here we have applied Src-specific Rapamycin-regulated (RapR) allosteric activation of Src kinase activity, a novel technology that is highly controllable in a temporally and spatially regulated manner, to the analysis of Src-regulated functions. Most studies of Src-mediated events and Src substrates have utilized cells that have already been transformed by constitutively active Src, although mutational activation of Src is rare. We have engineered RapR-Src using the wildtype kinase that is still regulated by other signaling events. Our studies focus primarily on Src activation in the authentic aberrant signaling environment of PDAC cells that harbor the multitude of genetic alterations characteristic of patient tumors. In addition, essentially all PDAC tumors harbor mutationally active K-Ras. In model studies of K-Ras-driven pancreatic tumorigenesis, concurrent activation of Src dramatically facilitated formation of invasive PDAC and Src-mediated signaling was still required for tumor growth. Also, a recent study showed synergistic cooperation of K-Ras and Src activation in PDAC progression and growth. Therefore, to address how K-Ras may influence Src function, we also wished to evaluate RapR-Src in matched pair sets of control and KRAS-transformed human pancreatic ductal epithelial cells (HPDE). We first examined Src expression and activation by western blotting for total and phosphorylated Y416 Src, and determined that Src is strongly overexpressed and highly activated in a subset of our PDAC cell lines compared to HPDE cells. We then knocked down Src by using lentiviral delivery of short hairpin RNAs and examined the consequences to properties of transformed growth. Stable lentiviral knockdown of Src in PDAC cell lines robustly decreased cell motility and invasion, validating that endogenous Src is essential for these functions. In normal HPDE cells, we observed that activation of RapR-Src caused immediate cell spreading. In HPDE cells transformed by KRAS, activation of RapR-Src resulted not only in cell spreading but also in long filopodial protrusions terminating in highly dynamic ends. These results indicate that the application of RapR-Src technology to PDAC models can begin to provide a detailed characterization of cell behavior and identification of the signaling pathways that are specifically mediated by Src, required for the maintenance of pancreatic cancer, and inhibited by Src-directed therapeutics. CFPAC cells have been selected for further study, and results of our ongoing experiments to address these questions will be presented. Future studies will assess RapR-Src-mediated immediate and long-term phosphorylation events in Src-dependent PDAC cells and will assess resistance mechanisms in response to pharmacologic inhibition of Src. Citation Format: Leanna R. Gentry, Andrei V. Karginov, James J. Fiordalisi, Channing J. Der, Adrienne D. Cox. Evaluation of Src-mediated signaling events in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B69.