A New Understanding of Obscurins: A Ubiquitously-Expressed Family of Cytoskeletal Regulators With Important Roles in Breast Cancer Development

Abstract

The obscurins comprise a family of proteins that regulate the assembly, contractile function, and membrane organization of striated muscles. Expressed from the OBSCN gene, located on human chromosome 1, the giant isoforms (720-870 kDa) are modular in structure and include approximately sixty adhesion domains followed by several domains involved in signal transduction, including a RhoGEF domain and a pair of serine/threonine kinases. Although their expression and function was thought to be limited to striated muscle, whole exome sequencing of various types of tumors revealed that mutation of OBSCN could drive the development of cancer. We sought to elucidate i) the expression pattern of obscurins in normal and cancerous breast epithelial cells, ii) the signaling pathways in which obscurins participated, and iii) the expression and localization of obscurins in a broad range of tissues. We first showed that the giant isoforms of obscurin are readily expressed in normal breast, skin, and colon epithelial cells, but not in their cancerous counterparts. Abrogation of the expression of obscurin giant isoforms using shRNA in MCF10A normal breast epithelial cells resulted in increased anchorage-independent growth and reduced apoptosis in response to DNA damage or substrate detachment, properties associated with the development and metastasis of breast cancer. Furthermore, loss of giant obscurins enhances microtentacle formation and reattachment, even in the presence of the chemotherapeutic, paclitaxel. These properties can be traced to downregulated RhoA activity upon loss of the obscurin RhoGEF, and are rescued by overexpression of the obscurin RhoGEF domain. Lastly, we employed immunoblotting to demonstrate the wide range of isoforms, ranging from ~50-900 kDa, in an array of murine organs, including brain, skin, lung, liver, spleen, and kidney. Immunohistochemistry revealed obscurins to be present at diverse subcellular locations, including the nucleus and sites of cell-cell contact. Together, these results illuminate the previously unsuspected complexity of the expression and function of obscurins in non-muscle tissues, particularly their role in the development and metastasis of breast cancer.