Abstract 5287: RNA binding protein RBM3 promotes metastasis through induction of hemangiogenesis and lymphangiogenesis
Authors: Ramalingam Satish , Subramaniam Dharmalingam , Standing David , Neradugomma Naveen K. , Umar Shahid , Anant Shrikant 
Published: August 2012

Introduction: Tumor metastasis, rather than primary tumors is the main cause of cancer-related deaths. A critical aspect of tumor metastasis, driven by vascular endothelial growth factors (VEGF) is the development of new vessels through hemangiogenesis and lymphangiogenesis. RNA binding protein RBM3 is a novel protooncogene that transforms normal cells when overexpressed. Here, we report that RBM3 mediates both hemangiogenesis and lymphangiogenesis. Results: Immunohistochemistry analysis of a human tissue microarray demonstrated increased RBM3 expression in tumors when compared to normal tissue, with even higher levels in metastatic tissues. Furthermore, RBM3-transformed NIH3T3 cells were injected into flanks of athymic nude mice metastasized to the liver and lungs. Immunohistochemistry analysis of the primary NIH3T3-RBM3 xenograft demonstrated increased number of microvessels that were lined by either CD31 or LYVE1 positive cells suggesting the increase in hem- and lymphangiogenesis. To demonstrate that RBM3 induces hemangiogenesis, tube formation assay using human umbilical vein endothelial (HUVEC) cells was performed. RBM3 overexpression increased, while RBM3 knockdown inhibited tube formation. Furthermore, NIH3T3-RBM3 cells significantly induced de novo capillary formation in the Chorio-Allantoic Membrane assay. Similarly, RBM3 overexpression enhanced, while knockdown suppressed tube formation by human lymphatic endothelial (HLEC) cells suggesting that it also affects lymphangiogenesis. To confirm the RBM3 role in hem- and lymphangiogenesis, HCT116 conditioned media was used in the tube formation assays. Tube formation by both HUVEC and HLEC cells was significantly increased in media from HCT116-RBM3 overexpressing cells. Moreover, both HUVEC and HLEC cells showed significant migration through collagen in transwell invasion assay. We further determined that this was due to the increased expression of VEGF A, VEGF C and VEGF D because siRNA mediated suppression of either one of these factors significantly reduced RBM3-mediated enhanced HLEC tube formation and migration through collagen. We next determined the mechanism of RBM3 mediated upregulation of the three VEGFs. HCT116 cells overexpressing RBM3 showed increased steady state levels of VEGF A, C and D mRNAs when compared to control. In addition, actinomycin D-stability assays demonstrated that there was increased stability of the three VEGF mRNAs in HCT116-RBM3 cells when compared to HCT116 control cells. Conclusion: Taken together these data suggests that RBM3 is a key regulator of hemangiogenesis and lymphangiogenesis, thereby inducing a highly metastatic phenotype through upregulation of VEGF A, C and D at the posttranscriptional levels of mRNA stability and translation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5287. doi:1538-7445.AM2012-5287