(723i) Loss of Giant Obscurins Promotes a Metastatic Phenotype in Breast Epithelium

Introduction: Obscurins, encoded by the single OBSCN gene, are a family of giant proteins that are expressed in striated muscle cells where they play key roles in their structural organization and contractile activity. In a recent systematic study of 13,023 genes in breast and colorectal cancers, OBSCN was identified as one of 189 “candidate cancer genes” due to its high mutational frequency (1). Indeed, findings from our laboratory indicate that loss of giant obscurins provides breast epithelial cells with a survival and growth advantage after exposure to DNA damaging agents (2). Here, we explore the role of obscurins in other processes related to cancer development, including epithelial-to-mesenchymal transition, cell invasion and migration, and cytoskeletal dynamics.

Materials and Methods: Stable clones of MCF10A breast epithelial cells depleted of giant obscurins were created using small hairpin RNA (shRNA) technology (2). A combination of immunoblotting, semi-quantitative RT-PCR and immunostaining was used to quantify the expression levels and localization of adherens junctions proteins, as well as proteins and transcriptional regulators associated with an epithelial-to-mesenchymal transition (EMT) in cells depleted of obscurins. Furthermore, a novel microfluidic chemotaxis-based device was engineered and used to quantify cell migration velocity and persistence within narrow and wide (3-50mm) channels. Finally, fluorescence recovery after photobleaching (FRAP) was used to measure actin cytoskeletal dynamics upon depletion of obscurins.

Results and Discussion: We first demonstrated that giant obscurins are down-regulated in human breast cancer biopsies, in comparison with their matched normal samples. Furthermore, depletion of obscurins promoted epithelial-to-mesenchymal transition, as evidenced by (1) disruption of the localization and reduction of the amounts of the adherens junctions proteins E-cadherin, β-catenin, and α-catenin, (2) increased expression of the mesenchymal proteins N-cadherin and vimentin and of the transcriptional regulators Slug and Twist, (3) decreased expression of the junctional epithelial proteins claudin-1, ZO-1, connexin-43, and plakoglobin. Loss of obscurins also promoted a more migratory phenotype, as demonstrated using our microfluidic chemotaxis-based device. Cell migration velocity and chemotactic index (a measure of persistence) were both elevated in obscurin-depleted cells compared to controls (Fig. 1A-B). This trend held for cell migrating in channels of width 3, 6, 10, 20 and 50 μm. Furthermore, FRAP experiments measuring Lifeact-GFP dynamics at cell-cell junctions revealed an increase in the mobile actin fraction and recovery time in cells depleted of obscurin, in comparison with control cells (Fig. 1C-D). This increase in actin cytoskeletal dynamics likely contributes to the enhanced ability of obscurin-depleted cells to transition from an epithelial to mesenchymal phenotype.

Conclusions: Loss of obscurins leads to epithelial-to-mesenchymal transition, enhanced actin dynamics at cell-cell borders, and increased cell migration and invasion into confined spaces. Collectively, our results suggest that obscurins play a role in preventing breast epithelial cells from progressing to a metastatic phenotype. In future work, we plan to explore the possible role of obscurins in cell mechanosensing of matrix mechanical properties.

References: (1) Sjoblom T. et al, Science 314: 268-274 (2006). (2) Perry N.A. et al, FASEB J 26(7): 2764-2775 (2012).