Abstract 1091: The role for retinoblastoma-like p130 (RBL2) and DNA damage response in human pancreatic cancer cells

Abstract

Abstract Background: Pancreatic cancer is the fourth leading cause of cancer death in North America. Infiltrating pancreatic cancer putatively evolves from histologically-defined Pancreatic Intraepithelial Neoplasias (PanINs) precursor lesions. In pancreatic duct adenocarcinoma (PDA), KRAS oncogene activation occurs in 80-95% of cases, while the inactivation of tumor suppressor genes (TSGs) including SMAD4, p53, and p16 occurs in 55%, 75%, and 100% of cases, respectively. Our laboratory has previously established a near normal HPV-16E6E7-immortalized human pancreatic ductal epithelial (H6c7) cells to investigate the mechanism of duct cell carcinogenesis. Despite the abrogation of p53 and RB pathway, H6c7 cells are not tumorigenic in scid mice. The expression of mutant KRASG12V in H6c7 cells resulted in weakly tumorigenic transformation. This implies that KRAS oncogene is insufficient for the full malignant transformation of H6c7 cells, consistent with the mouse models of pancreatic carcinogenesis. Recent studies demonstrate that DNA damage response (DDR) is highly elevated in PanIN lesions and the p53 inactivation in late-stage PanIN and PDA is associated with bypass of the DDR checkpoint. RBL2 has recently been implicated to play a vital role in DDR through stable maintenance of G2-M arrest. We hypothesized that the loss of RBL2 in H6c7 cells would compromise DDR and enhance the transforming activity of Kras oncogene. Methods: The RBL2 expression in H6c7 cells is downregulated using the retroviral shRNA expression system. Cell invasion and migration are assayed using matrigel and collagen type IV coated inserts. Radiation or drug effects are evaluated by flow cytometry measurement of mitochondrial-membrane potential. Results: Adriamycin treatment of RBL2 down-regulated H6c7 cells resulted in an increased pool of mitotic genes and increased apoptosis, consistent with compromised DDR. RBL2 knockdown also sensitized H6c7 cells to γ-irradiation following inhibition of Chk1 by UCN-01. Expression of KRASG12V oncogene in RBL2 down-regulated H6c7 cells resulted in increased anchorage-independent growth and enhanced migration and invasion, suggesting an interaction between RBL2 and KRAS oncogene. Direct sequencing of exons 19, 20, 21 and 22 of the RBL2 gene in 19 PDAC cell lines revealed no evidence of mutations. However, the RNA and protein expression of RBL2 was significantly decreased in number of PDAC cell lines compared to the H6c7 cells suggesting that RBL2 might be functionally inactivated in PDAC. PDAC cell lines with low RBL2 expression and mutated p53 showed increased radiosensitivity following treatment with Chk1 inhibitor UCN-01. Conclusion: Our results suggest that RBL2 plays an important role in pancreatic duct cell carcinogenesis and DDR response of PDA cells to radiation treatment. (Supported by the CIHR grant MOP-49585 and the Vanier Scholarship) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1091.