Abstract 2347: Inhibition of metastasis: A novel role for RNA binding protein CUGBP2

Abstract

Abstract Introduction: Cancer metastasis is the key determinant of a patient's death. Interestingly, cells have an effective system to inhibit the ability to metastasize, termed metastasis suppression, and loss of this regulatory mechanism has been associated with cancer progression. In the present study, we have extended our previous observation that RNA binding protein CUGBP2 is not only downregulated in various cancers, but also that there is a clear-cut correlation with CUGBP2 expression and the metastatic state of a cancer cell. Methods: Human colon epithelial cell line SW620, SW480 and mouse fibroblast cell line NIH3T3 were cultured under recommended conditions. Migration assay was performed using a 24-well transwell plate, and fetal bovine serum was used as a chemoattractant. We have developed a metastatic animal model where NIH-3T3 cells overexpressing RBM3 induce tumors in nude mice and contain malignant DCAMKL-1 (a bonafide stem cell marker) positive cells. When these cells were sorted and injected into the flanks of athymic nude mice, tumors were observed after 4 weeks not only in the flanks, but also in the lungs and liver. Total RNA and protein were isolated for Real Time RT-PCR and western blot analyses, respectively. Tumors were formalin fixed and used for immunohistochemistry. Results: Lower levels of CUGBP2 were present in highly metastatic SW620 when compared to the poorly metastatic SW480 cells. Furthermore, CUGBP2 levels were significantly decreased in lung and liver metastasis when compared to the primary tumors in the flanks of the nude mice. Similarly, CUGBP2 levels were reduced in SW620 cells that had migrated through the collagen in the in vitro cell invasion assay. Forced overexpression of CUGBP2 in SW620 cells however, significantly reduced cell migration through the collagen. Moreover, CUGBP2 overexpression reduced cell migration in the in vitro scratch plate/migration assay. Conversely, siRNA mediated knockdown of CUGBP2 in SW480 cells increased the migration of cells in the scratch plate assay. In previous studies, we have demonstrated that CUGBP2 inhibit the translation of mRNAs that contain AU-rich sequences in the 3′ untranslated region of transcripts such as cyclooxygenase-2 (COX-2). Immunoprecipitation coupled with RT-PCR assay demonstrated the binding of CUGBP2 protein with MMP-9 and COX-2 mRNAs. To confirm that the CUGBP2 is playing a role in regulating MMP-9 expression, we also determined the effect of CUGBP2 downregulation in the cells. CUGBP2 was downregulated with the specific siRNA in HCT-116 cells. There was significant increase in MMP-9 and COX-2 protein levels in the cells with reduced CUGBP2, which was not affected when cells were also transfected with the scrambled siRNA. Conclusion: Taken together, these data suggest that RNA binding protein CUGBP2 is a critical player in modulating tumor metastasis. 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 2347.