The Role of RBM47 Inactivation in Colorectal Cancer Progression

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We have previously identified the RNA binding motif protein 47 (RBM47) as a putative inhibitor of colorectal cancer (CRC) metastasis: Low RBM47 expression was significantly associated with a mesenchymal-like cellular state, liver metastasis and poor survival. However, elevated RBM47 expression, which was found in normal intestinal epithelia and primary tumors, was associated with an epithelial-like state. siRNA-mediated suppression of RBM47 induced EMT, migration, and invasion. The transcription factors SNAIL and SLUG repressed RBM47 expression and siRNA-mediated down-regulation of RBM47 in xenografted CRC cells promoted metastasis formation. Here we propose to further study the role and functions of RBM47 in a genetic mouse model of metastatic CRC. Notably, our analysis of published RNA-Seq results obtained within this mouse model indicates that RBM47 expression is highest in normal colon, decreased in non-invasive CRCs, and lowest in invasive CRCs and in liver metastases. We plan to determine whether intestinal epithelial cell specific deletion of Rbm47 increases the incidence of distant metastasis in these mice. In addition, we will study an orthotopic transplantation model consisting of patient-derived metastatic CRCs injected into NOD/SCID/gamma (NSG) mice which recapitulates the entire adenoma/adenocarcinoma/liver-metastasis sequence of CRC progression. To study the role of RBM47 in this model, human CRC tumoroids with knockout or ectopic expression of RBM47 will be injected into the colon wall of NSG-mice using endoscopy, followed by monitoring of the formation of primary tumors and metastases. To identify downstream mediators of RBM47 functions in CRC metastasis, we will utilize tumoroids isolated from primary tumors and metastases of aforementioned mouse models. These will be subjected to Next-Generation-Sequencing in order to comprehensively analyze RNA editing and alternative splicing events, which are regulated by RBM47. In addition, we plan to identify and characterize additional transcriptional and post-transcriptional mechanism that maintain RBM47 expression in epithelial tissues. Our bioinformatics analysis identified the transcription factor FOXA1, several microRNAs, as well as CpG DNA methylation as potential regulators of RBM47 expression. These potential regulations will be validated and further studied in cell lines and CRC patient material by using multiplexed immunohistology and methylation-specific PCR. Finally, the association of RBM47 protein expression, its down-stream effectors and methylation of the RBM47 promoter with formation of distant metastases, patient survival and tumor progression will be analyzed in samples from three different CRC patient cohorts. The results obtained here should contribute to a better understanding of CRC progression and identify prognostic markers and therapeutic intervention points.