Investigating the Regulation and Metastasis Suppressor Function of Obscurin in Breast Cancer

Abstract

Mounting evidence has implicated the giant cytoskeletal protein obscurin (720-870 kDa), encoded by the OBSCN gene, in the predisposition and development of breast cancer. Accordingly, prior work from our group has shown that downregulation of OBSCN in immortalized normal breast epithelial cells promotes survival and chemoresistance, induces cytoskeletal alterations, and enhances their migratory, invasive, and metastatic potentials. Consistent with these observations, analysis of Kaplan-Meier-Plotter data sets reveals that low OBSCN levels correlate with significantly reduced survival and relapse-free survival in breast cancer patients. Despite the compelling evidence linking OBSCN loss in breast tumorigenesis and progression, its regulation has remained elusive, limiting any efforts to target and potentially restore its expression a major challenge given its molecular complexity and gigantic size (~170 kb). Our studies revealed that OBSCN-AS1, a novel nuclear long noncoding RNA (lncRNA) gene, and OBSCN display positively correlated expression and are downregulated in breast cancer. OBSCN-AS1 regulates OBSCN expression in a cis-fashion through chromatin remodeling involving H3 lysine 4-trimethylation enrichment, associated with an open chromatin conformation, and RNA polymerase-II recruitment. CRISPR-activation of OBSCN-AS1 in triple negative breast cancer cells effectively and specifically restores OBSCN expression, and markedly suppresses cell migration, invasion, and dissemination from three-dimensional spheroids in vitro and metastasis in vivo. Furthermore, given the associations of OBSCN dysregulation and silencing in cancer formation and progression, we aimed to decipher the downstream molecular consequences of obscurin loss in breast epithelial cells. Our findings revealed that OBSCN knockout in MCF10A breast epithelial cells leads to upregulation of the PI3K/AKT/NFkB signaling cascade and alters the expression of genes involved in important biological processes including extracellular matrix organization, angiogenesis, and cell adhesion. Importantly, loss of obscurin expression promotes malignant transformation of immortalized normal breast epithelial cells. Taken together, our studies reveal the previously unknown regulation of OBSCN by an antisense lncRNA, the metastasis suppressor function of the OBSCN-AS1/OBSCN gene pair, and the downstream activation of the PI3K/AKT/NFkB signaling pathway with OBSCN deletion. These discoveries expose fundamental insights into the regulation and function of obscurin in breast cancer and pave the way for future research with significant implications in cancer.