Abstract 4024: Single-allele K-ras mutation mediates CXCR4 signaling in PanIN and pancreatic cancer cells

Abstract

Abstract Single-allele K-ras mutation mediates CXCR4 signaling in PanIN and pancreatic cancer cells Xiaoming Shen, Jianming Lu, Stefanie Christopher, Andrew M. Lowy, Joseph Kim Department of Oncologic Surgery, City of Hope Division of Surgical Oncology, Moores Comprehensive Cancer Center, UCSD Introduction: We previously demonstrated that the activated chemokine receptor CXCR4 signals through MAPK and PI-3K/AKT pathways in pancreatic intraepithelial neoplasia (PanIN) and invasive pancreatic cancer to enhance cell proliferation. It remains unclear whether mutant-derived constitutive K-Ras activity regulates CXCR4 signaling. Our aim was to characterize the role of mutant K-ras in CXCR4 signal transduction. Methods: Human pancreatic cancer cell lines with wild-type K-ras (BxPC-3 and Hs766T), single-allele mutant K-ras (PANC-1 and FG), and double-allele mutant K-ras (AsPC-1 and MIAPaCa-2) were evaluated. An established murine PanIN cell line with single allele mutant K-ras was also assessed. First, CXCR4 and K-Ras expression in all the cell lines were measured by Western blot assay. Then, changes in K-Ras activity induced by CXCL12, the selective CXCR4 ligand, were measured by a Raf-Ras binding domain ELISA assay. To further characterize the interaction between K-Ras and CXCR4, a K-ras knockdown assay was used to examine its effects on CXCL12-mediated activation of ERK1/2 and cell proliferation. Results: PCR and DNA sequencing verified K-ras mutation status in all the cell lines. Western blot assay showed that all cells expressed K-Ras and all cells expressed CXCR4, except MIAPaCa-2 (double-allele mutant K-ras). When CXCR4-expressing cells were exposed to CXCL12, we observed significantly increased K-Ras activity in single-allele mutant K-ras cells (what is the magnitude of change?111% for panIN and 18.6% for panc1), but not wild-type and double-allele mutant K-ras cells. Furthermore, the increased K-Ras activity corresponded with CXCL12-induced proliferation, which was only observed in the single-allele mutant K-ras cells. K-ras knockdown in the single allele mutant cells (PANC-1 and PanIN) blocked CXCL12-induced phosphorylation of ERK1/2 and abrogated cell proliferation. Because of concern that knockdown of dominant K-Ras signaling may block receptor-mediated signal transduction, we also exposed cells to epidermal growth factor, which induced phosphorylation of ERK1/2 despite K-ras knockdown. Conclusion: Our results verify that the activated chemokine receptor CXCR4 promotes the proliferation of PanIN and pancreatic cancer cells. We also show that while K-Ras conducts CXCR4 signals, only single-allele mutant K-ras mediates CXCR4-induced increases in K-Ras activity and the subsequent downstream enhancement in cell proliferation. 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 4024.