(622w) Integrin Alpha 6 and Calpain 2 Are Mechanosensitive Proteins in Breast Cancer

Cancer metastasis occurs when the primary tumor releases cells that travel through the vasculature and colonize distant tissues. Breast cancer spreads principally to the bone, brain and lung, however, it is not understood why there exists a clear preference to spread to these specific sites. Each secondary tissue has a distinct stiffness range and these differences alter gene expression, protein levels and activation, and cell phenotype, all of which impact the ability of cells to migrate, survive, and proliferate. We hypothesize that in breast cancer, mechanical forces could inform the tissue specificity of survival and growth at a secondary tissue site. To verify the role of tissue stiffness in metastatic cell fate, we used tunable biomaterials of varying stiffnesses and quantified genotypic and phenotypic characteristics of cells in each environment.

RNAseq implicated integrin α6 and calpain 2 as possible novel mechanosensitive genes and we examined their effects on cell adhesion and motility on synthetic PEG-PC hydrogel surfaces of varying stiffness. Integrin α6 is a surface receptor that binds specifically to laminin, which increases both the cell spreading rate and maximum area, as compared to collagen 1 alone, when on soft hydrogels. Gene expression levels show much higher expression of the B isoform at low stiffnesses. This variant of integrin alpha 6 has a splicing modification in the cytoplasmic domain, which is unique to a mesenchymal-like population and is a marker of cancer stem cells. Blocking integrin α6 function accentuates the stiffness dependence of cell spreading rate and area during adhesion on PEG-PC hydrogels. While integrin alpha 6 influences cell adhesion, calpain 2 is a protease that affects migration by controlling the disassembly rate of focal adhesions by cleaving structural proteins, such as talin. Calpain 2 inhibition decreases cell motility because cells are unable to release focal adhesions at the rear of the cell during migration. We are currently investigating the role of stiffness in mediating calpain 2 and we hypothesize that growth factor receptor activation (EGFR) accentuates the stiffness response due to receptor localization increasing the rate of focal adhesion turnover via cleavage of calpain 2. We have coupled genetic, protein and functional information to understand the mechanism by which stiffness cues initiate downstream signaling via integrin alpha 6B and calpain 2 to affect cell motility. We are working to connect the signaling roles of stiffness, EGF, and integrin binding to understand mechanosensitive cell adhesion and migration.