Abstract 3901: RBM3 drives tumor angiogenesis by modulating miR-210.

Abstract

Abstract Background: Colorectal cancer is the second leading cause of cancer related deaths in the United States. While primary tumors can be resected out to inhibit progression, it is tumor metastasis that is deadly. A critical feature of metastatic spread of cells is tumor angiogenesis, wherein a network of blood vessels penetrates into the tumor and supplies nutrients and oxygen. Recently, we demonstrated that RNA binding protein RBM3 is a novel protooncogene that transforms normal cells to cancerous cells that can form xenografts in athymic nude mice. Here, we report that RBM3 induces angiogenesis through regulation of microRNA expression. Methods: Colon cancer cells HCT116, SW480 (Human) and CT26 (mouse), endothelial cells HUVEC, and the mouse fibroblast cells NIH3T3 were used in the study. RBM3 overexpressing cells were plated in soft agar and allowed to grow as spheroids, which were then injected into flanks of athymic nude mice. Total RNA and protein were isolated for Real Time RT-PCR and western blot analyses, respectively. Tumors were formalin fixed and used for immunohistochemistry. Microarray analyses were performed with total RNA from HCT116 cells overexpressing RBM3 to identify microRNAs that were differentially regulated. Results: Both HCT116 and SW480 cells demonstrated increased migration in scratch assay and in invasion assay when RBM3 is overexpressed. In addition, NIH3T3 and CT26 cells overexpressing RBM3 injected into the flanks of nude mice demonstrated increased metastasis to liver and lungs. There were also a significantly higher number of microvessels marked by CD31 endothelial cells within the primary tumor of the mice, within the tumor xenografts. In contrast, siRNA mediated knockdown of RBM3 expression in HCT116 tumor xenografts resulted in reduced tumor growth coupled with significant reduction in tumor microvasculature. RBM3 overexpression increased tube formation of HUVEC cells in matrigel tube formation assays. In contrast, RBM3 knockdown reduced tube formation. We next determined the effect of RBM3 expression on microRNA expression. Microarray studies showed that RBM3 overexpression in HCT116 cells resulted in increased levels of several microRNAs. miR210, a hypoxia-induced miRNA, is a critical angiogenesis regulator and endothelial cell survival factor whose overexpression stimulates angiogenesis and VEGF-induced cell migration. Microarray studies showed that miR210 was upregulated 3-fold in RBM3 overexpressing cells, which was validated by Real Time PCR analyses. miR210 was also upregulated in HUVEC cells overexpressing RBM3. Furthermore, the microRNA was reduced in HCT116 cells in culture and in tumor xenografts when RBM3 levels were downregulated by specific siRNA. Antagomir-mediated silencing of miR-210 significantly suppressed RBM3 mediated angiogenesis. Conclusion: RBM3 overexpression promotes tumor angiogenesis by upregulating miR-210 expression. Citation Format: Satish Ramalingam, dharmalingam Subramaniam, Shrikant Anant. RBM3 drives tumor angiogenesis by modulating miR-210. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3901. doi:10.1158/1538-7445.AM2013-3901