Elucidating the Roles of Giant Obscurins in Breast Cancer Progression

Abstract

Obscurins are a family of giant cytoskeletal proteins expressed in striated muscle where they play roles in structural organization and contractility. The human OBSCN undergoes alternative splicing to give rise to two giant isoforms, obscurin A (720 kDa) and obscurin B (820 kDa), composed of tandem adhesion and signaling domains. Using a panel of obscurin-specific antibodies, I reveal the presence of giant and small isoforms ranging in size from 970-50 kDa in the skin and lung of rodent tissue. Obscurins were found to exhibit nuclear, cytosolic, and membrane localization within various cell types. Interestingly, some obscurin isoforms were ubiquitously expressed, while others were tissue-specific. In addition to identifying the presence of obscurins in skin and lung tissue, I have identified robust expression of obscurins in normal mammary epithelial tissue, where they exhibit membrane localization within the ducts and lobules, compared to matched breast cancer samples which exhibit decreased expression of obscurins. Stable clones of MCF10A cells depleted of giant obscurins fail to form adhesion junctions, undergo epithelial-to-mesenchymal transition, and generate >100 μm mammospheres bearing markers of cancer-initiating cells. Additionally, obscurin-deficient MCF10A cells display altered actin organization accompanied by increased actin dynamics, resulting in increased migration and invasion. MCF10A cells that stably express the K-Ras oncogene and obscurin shRNA, but not scramble control shRNA, exhibit increased primary tumor formation and lung colonization after subcutaneous and tail vein injections, respectively. Furthermore, my studies reveal loss of giant obscurins in MCF10A cells results in the upregulation of the PI3K signaling cascade, and blockade of the catalytic subunit of the PI3K protein resulted in a decrease in mammosphere formation in anchorage independent conditions, migration of both a monolayer and single cells, and in invasion through a matrigel coated chamber. Collectively, my findings reveal that obscurins are abundantly expressed in a variety of tissues including skin, lung, and breast. Moreover, loss of giant obscurins from breast epithelial cells results in alterations within the PI3K signaling pathway, which results in a more tumorigenic phenotype.