Mobility and invasiveness of metastatic esophageal cancer are potentiated by shear stress in a ROCK- and Ras-dependent manner

Abstract

To metastasize, tumor cells must adopt different morphological responses to resist shear forces encountered in circulating blood and invade through basement membranes. The Rho and Ras GTPases play a critical role in regulating this dynamic behavior. Recently, we demonstrated shear-induced activation of adherent esophageal metastatic cells, characterized by formation of dynamic membrane blebs. Although membrane blebbing has only recently been characterized as a rounded mode of cellular invasion promoted through Rho kinase (ROCK), the role of shear forces in modulating membrane blebbing activity is unknown. To further characterize membrane blebbing in esophageal metastatic cells (OC-1 cell line), we investigated the role of shear in cytoskeletal remodeling and signaling through ROCK and Ras. Our results show that actin and tubulin colocalize to the cortical ring of the OC-1 cell under static conditions. However, under shear, actin acquires a punctuate distribution and tubulin localizes to the leading edge of the OC-1 cell. We show for the first time that dynamic bleb formation is induced by shear alone independent of integrin-mediated adhesion (P < 0.001, compared with OC-1 cells). Y-27632, a specific inhibitor of ROCK, causes a significant reduction in shear-induced bleb formation and inhibits integrin alpha(v)beta(3)-Ras colocalization at the leading edge of the cell. Direct measurement of Ras activation shows that the level of GTP-bound Ras is elevated in sheared OC-1 cells and that the shear-induced increase in Ras activity is inhibited by Y-27632. Finally, we show that shear stress significantly increases OC-1 cell invasion (P < 0.007), an effect negated by the presence of Y-27632. Together our findings suggest a novel physiological role for ROCK and Ras in metastatic cell behavior.

Shear stress induces internalization of E‐cadherin and invasiveness in metastatic oesophageal cancer cells by a Src‐dependent pathway

“…(16) In the current investigation we describe a novel role for shear in the internalization of E-cadherin. Specifically we show that: (1) venous shear induces the internalization of E-cadherin from cell-cell junctions into Rab5-positive endosomes; (2) Src inhibition disrupts shear-induced internalization of E-cadherin; and finally that (3) sheared OC-1 cells invade in a Src-dependent manner.…”

“…Previously, we (16) and others (4) have shown that exposure to shear causes activation, translocation, and clustering of integrins in tumor cells; however, it is unknown if shear induces rearrangement of adherens junctions, in particular, the cadherins in adherent metastatic cells. Because Fn supports maximal adhesion of OC-1 cells under venous fluid flow, (16) we used Fn as the adhesive matrix component in all parallel plate flow experiments. A period of 30 min enabled maximal interactions of OC-1 cells with Fn.…”

“…OC-1 tumor cell suspensions (5 × 10 4 cells/mL in the presence and absence of PP1 were sheared at 200/s for 15 min via cone plate viscometry and added to Matrigel invasion chambers as previously described. (16) In control experiments, non-sheared OC-1 cells pre-treated with and without PP1 or control (untreated) were washed in PBS, resuspended in serum-free DMEM, and added to the invasion chambers. Following the 22-h incubation period, OC-1 cells that had invaded the Matrigel layer were stained with crystal violet and quantified by light microscopy.…”

Growth Factors, Signal Transduction Pathways, and Tumor Suppressor Genes in Esophageal Cancer

“…Lawler et al [97] have also shown that mobility and invasiveness of metastatic esophageal cancer cells are potentiated by shear stress through the Rho kinase (ROCK) and Rassignaling pathways, suggesting a novel physiological role for Rock and Ras in metastatic behavior of cancer cells [97].…”

ATR-FTIR Spectroscopy of Membrane Bound Ras Protein

“…The proposed mechanism would thus be relating and combining the mechanical inputs to the biological outputs, in conjunction with the activity regulation of the Ras protein by the mechanical terms such as the shear force [260] and the plasma membrane viscosity [261]. The shear stress within the body, generated by the cardiac cycle and experienced by the endothelial cells, was given as a function of blood flow patterns throughout the vasculature [262].…”