Abstract 5409: Ron silencing by shRNA inhibits growth, induces apoptosis and sensitizes tumor response to c-Met inhibitor treatment through down-regulating MAPK signaling in B-Raf mutant colon carcinomas

Abstract

Abstract RON, the receptor for macrophage-stimulating protein, belongs to the MET receptor tyrosine kinase family and shares many common features with MET. Inappropriate activation of RON signaling plays an important roll in colon carcinoma cell proliferation, survival, migration and invasive growth. Ron and Met are overexpressed in patient colon tumors, where co-overexpression of both Ron and Met correlates with the worst patient survival compared to the overexpression of Ron or Met alone. BRaf-activating mutations occur in about 10% of colorectal carcinomas. Mutant BRaf has constitutive kinase activity and causes hyperactivation of the mitogen-activated protein kinase (MAPK) pathway, and BRaf mutant cancers are generally resistant to standard therapies. Here we report that knockdown of the Ron gene by Ron shRNA in BRafV600E mutant HT29 colon carcinoma cells induced HT29 tumor apoptosis and resulted in significant tumor growth inhibition (TGI) by 59%, which is comparable to that observed with the selective EGFR inhibitor-Erlotinib treatment (57% TGI), but more potent than that of the selective Met inhibitor-PF-04217903 treatment (38% TGI). When Ron shRNA knockdown and treatment with the selective Met inhibitor were combined, enhanced tumor growth inhibition (76%) was observed. Comparable experiments using the BRaf mutant Colo205 model yielded similar results. Western Blot analysis showed that combination of Ron shRNA and Met inhibition in HT29 cells decreased phosho-ERK, elevated cleaved caspase-3 and, surprisingly, increased total Ras protein levels. No significant changes in total and phospho-AKT levels were observed. In vitro, the stable HT29-shRon cells initially grew more slowly compared to HT29-vector cells, but the growth rate gradually increased, approaching that observed in normal cell culture conditions. Interestingly, when co-culturing stable HT29-shRon or HT29-vector cells with bone marrow derived mesenchymal stem cells, the selective Met inhibitor-PF-04217903 induced significant cell death only in the HT29-shRon cells under hypoxic conditions (1% oxygen). No cell death was observed with either HT29-shRon cells under normoxia or HT29-vector cells under hypoxia or normoxia conditions, indicating the collaborative roll of Ron and Met in colon cancer cell survival during hypoxia. In summary, inhibiting both Ron and Met pathways in BRaf mutant colon cancer cells down- regulated ERK activity, induced apoptosis and led to enhanced antitumor efficacy in vivo. 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 5409.