Abstract 3486: RBM3 causes differential expression lncRNA in colon cancer cell lines
Authors: Sayed Afreen Asif Ali , Subramaniam Dharmalingam , Umar Shahid , Thomas Sufi M. , Anant Shrikant 
Published: July 2017

Abstract
Introduction: Colon cancer is the third most commonly diagnosed cancer and leading cause of cancer-related deaths in the United States. It usually begins as a benign growth that transforms into a malignant adenocarcinoma through genetic changes in tumor suppressor genes and oncogenes. Cancer stem cells are a rare group of cells within a tumor that have self-renewal capacities and with high potency for tumor relapse and metastasis. Wnt/β-catenin is one of the major pathways deregulated in colon cancer and also important in stem cell self-renewal. Wnt/β-catenin signaling is important in maintaining intestinal homeostasis and the stem cell niche, but aberrant activation of the pathway increases the stem cell population leading to aggressive cancer. We determined that RBM3 induces stem-like characteristics in colon cancer cells in part, by enhancing Wnt/β-catenin-induced GSK3β inactivation. We are further evaluating the mechanism by which RBM3 increases Wnt/ β-catenin signaling leading to increased stemness.
Methods: HCT116 and DLD1 cell lines were used in this study. We used a PCR array to identify specific Wnt pathway proteins regulated by RBM3, in HCT116 RBM3 overexpressing cells and compared it to vector control cells. RNA-IP was performed to confirm if RBM3 bound to Wnt3a and LRP6. ChIP was performed for identify β-catenin target gene cMyc. RNA seq was performed to identify differential gene expression and also differential lncRNA expression. RNA-Immunoprecipitation seq was performed to identify RBM3 protein bound cellular proteins. Ingenuity pathway analysis was performed for all the sequencing data.
Results: Our data demonstrate that several Wnt pathway genes including Wnt3a and LRP6 are upregulated in RBM3 overexpressing cells. To identify mRNA regulated by RBM3, we performed RNA-IP for RBM3. We found that RBM3 was associated with the mRNA for Wnt3a and LRP6. The ingenuity pathway analysis for RNA seq shows expression of genes involved in cell growth, proliferation and metastasis like Jun, LAMC2, Myc, NR2F2, NT5E etc. when RBM3 is overexpressed in the cells, showing a role of RBM3 in cancer progression. The seq data also shows differential expression of lncRNA especially those involved in cancer like CCAT1 and SNHG16. The ingenuity pathway analysis for RNA-IP seq also shows increase in molecules involved in proliferation and cell movement. In-vivo studies were also performed to confirm role of RBM3 in tumorigenesis.
Conclusion: RBM3 increases stemness by activating β-catenin pathway possibly through Wnt3a and LRP6. RBM3 overexpression causes differential expression of lncRNA and also increases expression of genes involved in cell growth and proliferation possibly by interacting with its target mRNAs.
Citation Format: Afreen Asif Ali Sayed, Dharmalingam Subramaniam, Shahid Umar, Sufi M. Thomas, Shrikant Anant. RBM3 causes differential expression lncRNA in colon cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3486. doi:10.1158/1538-7445.AM2017-3486