RhoA-ROCK competes with YAP to regulate amoeboid breast cancer cell migration in response to lymphatic-like flow

Lymphatic drainage generates force that induces prostate cancer cell motility via activation of Yes-associated protein (YAP), but whether this response to fluid force is conserved across cancer types is unclear. Here, we show that shear stress corresponding to fluid flow in the initial lymphatics modifies taxis in breast cancer. Whereas some cell lines employ rapid amoeboid migration behavior in response to fluid flow, a separate subset decrease movement. Positive responders displayed transcriptional profiles typical of an amoeboid cell state. Regulation of the HIPPO tumor suppressor pathway and YAP activity also differed between breast subsets and prostate cancer. Although subcellular localization of YAP to the nucleus positively correlated with overall velocity of locomotion, YAP gain- and loss-of-function demonstrates that YAP inhibits breast cancer motility but is outcompeted by other pro-taxis mediators in the context of flow. Specifically, we show that RhoA dictates response to flow. GTPase activity of RhoA, but not Rac1 or Cdc42 Rho family GTPases, is elevated in cells that positively respond to flow and is unchanged in cells that decelerate under flow. Disruption of RhoA or the RhoA effector, Rho-associated kinase (ROCK), blocked shear stress-induced motility. Collectively, these findings demonstrate stratification of breast cancer subsets by flow-sensing mechanotransduction pathways and point to a role for biophysical force in regulation of an amoeboid cell state. Breast cancer cell lines were cultured under static conditions or fluid wall shear stress (WSS) of 0.05 dyne cm-2 for 6 hr in collagen-coated microfluidic channels. Breast cancer cell lines examined included MCF-7, HCC1187, EFM-19, and MDA-MB-231. Three independent biological replicates were included for each cell line. MCF-7 and HCC1187 were classified as negatively responding to WSS by decreased migration; whereas, EFM-19 and MDA-MB-231 were classified as positive responders associated with induction of rapid amoeboid migration in response to fluid flow.