Rbm20 and Arhgap25 Expression Alteration and Roles in Breast Cancer in Vitro Models

Abstract

Breast cancer is the second leading type of cancer occurrence and mortality in women worldwide and studies are constantly attempting to find new treatment strategies, as well as better understand the mechanisms and pathways that control its initiation and progression. Alternative splicing is one of the major cellular processes that are deregulated in cancer, with more and more splicing factors being implicated in various steps of the tumorigenic process. RBM20 is a nuclear splicing factor mainly restricted in expression to the heart and skeletal muscle cells. Previous studies have implicated RBM20 in several heart defects and diseases that result from its altered splicing behavior due to mutations that are majorly loss of function. Several members of the RBM family have been implicated in various types of cancer, like RBM5, RBM6, RBM10 and others. However, RBM20 has not been shown to play any role in cancer. ARHGAP25 is a cytoplasmic RacGAP with known functions in cells of the hematopoietic lineage. Since it is part of the Rho/rac pathway, it has a major function in cell motility and migration. It also plays roles in immune system signaling pathways, as well as free radical production and phagocytosis in neutrophils. Importantly, it has been recently shown to have significant functions in each of rhabdomyosarcoma, colorectal cancer and lung cancer. In this study, we used western blotting, immunofluorescence staining, qRT-PCR, Co-IP and drug inhibition of AMPK to investigate the expression levels, subcellular localization and interactions between these two proteins, as well as potential pathways controlling the expression and activity of RBM20 in breast cancer. We showed that RBM20 expression, and more evidently, RBM20 isoform expression, is altered in two breast cancer in vitro models, MDA-MB-231 and MCF-7 cell lines, that are considered more and less aggressive models, respectively. Not only did we observe RBM20 isoform switching, but a novel and much smaller isoform was consistently seen exclusively in the MDA-MB-231 cells line which also had significantly higher levels of ARHGAP25 protein compared to MCF-7 cells. While ARHGAP25 subcellular localization was not altered in either cell line, that of RBM20 showed a significant shift towards the cytoplasm in the MCF-7 cell line. Phage display biopanning assays have shown that the C-terminal of RBM20 can potentially directly interact with a 7-amino acid peptide sequence that matches a sequence in the ARHGAP25 protein. Upon that, we aimed to validate this interaction in the breast cancer cell lines. Since the PI3K/mTOR pathway is implicated in RBM20 expression, we inhibited AMPK using the drug Dorsomorphin Dihydrochloride and showed that AMPK does not directly affect RBM20 protein expression levels but may potentially play a role in its phosphorylation levels. The results indicate that RBM20 and ARHGAP25 are both implicated in breast cancer and can potentially interact in a direct protein-protein manner. The functions each is involved in raises the possibilities that they affect and regulate several cancer hallmarks to be investigated.