Abstract #LB-287: Protooncogene RBM3 mediated upregulation of miR210 induces and is a potential target for anti-angiogenic compound marmelin
Authors: Satish Ramalingam, Dharmalingam Subramaniam, Cindy Simpson-Durand, Aarthi Varman, Michael Ihnat, Vibhudutta Awasthi, Rama Ramanujam, Courtney Houchen, Shrikant Anant
Journal: Cancer Research (2009)

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

RNA binding protein RBM3 is a novel protooncogene that transforms primary cells when overexpressed. RBM3 is upregulated in many cancers including colon, breast, and lung. Forced RBM3 overexpression in SW480 colon cells (SW480-RBM3) resulted in significantly bigger tumors in xenografts when compared to controls (SW480-Ctrl). Moreover, CD31 immunohistochemistry showed that SW480-RBM3 tumors had more microvasculature suggesting that RBM3 induces angiogenesis. This was confirmed by the chicken chorioallantoic membrane assay, where the SW480-RBM3 increased capillary vascular density. We recently identified marmelin, a natural compound from the medicinal plant Aegle marmelos and demonstrated that it potently inhibits cancer growth, in part through inhibiting angiogenesis. Marmelin administration (ip)significantly inhibited growth of SW480-RBM3 xenografts in nude mice and angiogenesis in a dose dependent manner (0-200 mcg/Kg bw). A critical proangiogenic factor is vascular endothelial growth factor (VEGF). VEGF-A levels were increased 3-fold in the SW480-RBM3 cells but was reduced to control levels following marmelin treatment. VEGF expression is regulated by transcription factor HIF-1\#945;, while it is downregulated by HIF3\#945;. In SW480-RBM3 cells, there was a 4-fold increase in HIF1\#945; levels while HIF3\#945; was reduced by 3-fold. Marmelin treatment on the other hand, resulted in suppression of HIF1\#945; and induction of HIF3\#945;. To confirm that RBM3 induced HIF1\#945; expression, J82-Ctrl and J82-RBM3 bladder cancer cells were stably transfected with a luciferase reporter plasmid under the control of hypoxia response element. HRE activity was 8-fold higher in J82-RBM3 cells when compared to Ctrl cells. MicroRNAs (miRNAs) are small, non-coding RNAs that recognize target sequences in cognate mRNAs and inhibit protein translation. To examine the potential involvement of miRNAs in RBM3 mediated regulation of angiogenesis, we performed a miRNA microarray analysis with HCT-116-RBM3 cells, and subsequently confirmed the expression in HCT-116-RBM3, SW480-RBM3 and J82-RBM3 cells by real time RT-PCR. In all three cell lines, expression of miR-210 was upregulated. Promoter analysis indicated the presence of HRE upstream of miR-210. Activation of HIF1\#945; induced the expression of miR-210, which was suppressed by marmelin. Using target prediction algorithms, we identified that HIF3\#945; is a target of miR-210. We are currently confirming this using luciferase reporters that express miR210 binding sites of HIF3\#945;. We are also confirming the effect of marmelin on HIF1\#945;, HIF3\#945;, and miR-210 expression in the tumor xenografts. These data taken together, suggest that protooncogene RBM3 increases miR210 expression by inducing HIF1\#945;, and miR210 in turn suppresses HIF3\#945; expression. Furthermore, the anti-cancer compound marmelin downregulates RBM3 and miR210 expression to suppress tumor angiogenesis.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-287.