Cells in a Stiff Matrix: ROBO1 is Regulated by Extra-Cellular Matrix Stiffness to Modulate Cellular Contractility Through the Maturation of Focal Adhesions
Breast tumors have the ability to remodel the extracellular matrix (ECM) surrounding it to induce a stiffened matrix, which then activates mechanically sensitive signaling cascades that promote tumor progression. In this thesis, I sought to identify mechanosignaling pathways that are turned on by exposing cells to artificially stiffened, rigid matrices. By doing so I aimed to identify stiffness induced cellular responses of normal mammary cells. Previous work from the lab established SLIT2/ROBO1 as a regulator of pubertal branching morphogenesis by controlling proliferation. We show here that this signaling pathway is also responsive to mechanical stimuli from the ECM, where stiffness induces ROBO1 expression as a necessary cellular response to maintain the normal phenotype of adult mammary cells. We began our investigation by dissecting the functional roles of SLIT2/ROBO1 signaling within mammary cells and observed an increase in Rac1 activation that led to significant increases in focal adhesion (FA) size and cellular contractility. The contractile behavior is necessary for cells to sense the mechanical state of their environment and make appropriate behavioral decisions. To determine the consequence of the loss of Robo1 in stiff 3D primary culture, we cultured Robo1+/+ and Robo1-/- primary murine mammary epithelial cells (MMEC) in 3D collagen matrices of low and high stiffness. We found that Robo1+/+ organoids constrained protrusions of cells in high stiffness as compared to organoids in low stiffness collagen gels. In contrast, Robo1-/- organoids created protrusions in both conditions. This data suggests that ROBO1 is necessary for mammary cells to regulate organoid morphology in response to ECM stiffness. We next questioned how this important signaling response is regulated by ECM stiffness by investigating mechanisms of Robo1 mRNA regulation. We identified miR203 to be a mechanically sensitive repressor of Robo1 expression where increased stiffness inhibits miR203 expression. By overexpressing the precursor pre-miR203 in mammary cells and culturing them in 3D collagen matrices, we found that ROBO1 expression is indeed tuned through stiffness regulated miR203 expression. To determine the clinical significance and prevalence of this expression profile, we queried publicly available clinical datasets for repressed miR203 and increased Robo1 expression indicative of a normal cellular response to a stiffened ECM. Normal tissues maintain this expression pattern in 75% of all samples as compared to basal breast tumors that have this profile in only 23% of samples. We further assessed for long-term survivability of patients with the basal breast tumors that contain this expression profile. We observed a significant increase of long-term survival in patients with tumors that have low miR203 and elevated Robo1 expression. Taken together, these results suggest SLIT2/ROBO1 signaling is employed by the cell as a protective response against ECM stiffness induced tumorigenesis.