Abstract IA6: Identification and characterization of factors required for KRAS-driven pancreatic tumorigenesis

Abstract

Abstract Activating mutations in the KRAS oncogene occur in the vast majority of pancreatic ductal adenocarcinomas (PDAC). These mutations are present in early precursor pancreatic intra-epithelial neoplasia (PanIN) lesions and are considered initiating driver mutations in this disease. Moreover, expression of an activated Kras allele potently initiates PanIN formation and PDAC development in the mouse. Therefore, inhibiting mutant KRAS-mediated signaling is potentially an effective therapeutic strategy for PDAC. While efforts directly targeting mutant KRAS are underway, an alternative approach is to target key signaling molecules that are stimulated downstream by activated KRAS. We hypothesized that factors required for the development of KRAS-driven pancreatic tumors will be similarly required during late stage disease and therefore represent potential therapeutic targets. We therefore utilize in vivo and in vitro systems to identify and characterize these factors. Mutated KRAS promotes the proliferation of primary pancreatic ductal epithelial cells (PDECs) as well as their survival following challenge with apoptotic stimuli. KRAS-induced survival in PDECs is dependent on signaling through the RAF/MEK/ERK and PI3K/AKT signaling cascades. Interestingly, we found that the insulin-like growth factor receptor (IGF1R) is required for KRASG12D-induced stimulation of the PI3K/AKT signaling axis. We determined that KRASG12D induces the expression of the IGF1R ligand IGF2 downstream of the MEK/ERK cascade and IGF2 stimulates IGF1R, and subsequently PI3K/AKT signaling, in an autocrine manner. Consistent with this, ectopic IGF2 expression restored KRASG12D-induced survival in cells treated with a MEK inhibitor but not a PI3K inhibitor. Critically, IGF1R is required for KRASG12D-induced survival after exposure to apoptotic stimuli, and inhibition of IGF1R impairs pancreatic tumor formation in an orthotopic mouse model. In PDAC cell lines, shRNA-mediated knockdown of IGF1R impairs their anchorage-independent growth and migration activity. Thus, IGF1R-mediated signaling is required for KRASG12D-driven pancreatic tumorigenesis in vivo and impacts the transformation of pancreatic cancer cells in culture. The mTOR signaling complex TORC2 promotes the phosphorylation of AKT on Ser473, an event required for full activation of AKT. Given the importance of IGF1R and PI3K/AKT signaling in promoting KRASG12D-induced survival and pancreatic tumor formation, we explored the requirement for TORC2 activity in KRAS-driven PDAC development in vivo. We inactivated TORC2 through the pancreas-specific genetic ablation of the essential subunit Rictor. Rictor deficient pancreata are architecturally normal but smaller than wild-type pancreata. Interestingly, Rictor deletion significantly impaired KRASG12D-driven PanIN formation. Moreover, the absence of Rictor also impaired KRASG12D-driven PanIN formation following caerulein-induced pancreatic injury. Together, these data indicate an important role for TORC2 signaling during PDAC initiation. We additionally characterized the role of TORC2 in PDAC cell lines. ShRNA-mediated inhibition of Rictor impaired PDAC cell proliferation and transformation as assessed by anchorage-independent growth, suggesting an important role for the TORC2 signaling complex in maintenance of the transformed phenotype of PDAC cells. Analysis of TORC substrate proteins identified reduced phosphorylation and activation of AKT and SGK1, suggesting these downstream molecules as potential mediators of TORC2-dependent phenotypes in pancreatic tumorigenesis. Together, these data suggest that TORC2 activity plays a critical role in KRAS-driven PDAC initiation and progression. Collectively, our studies have identified critical factors required for KRAS-driven pancreatic tumorigenesis, suggesting additional therapeutic strategies for targeting PDAC. Citation Format: Brian C. Lewis. Identification and characterization of factors required for KRAS-driven pancreatic tumorigenesis. [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 IA6.