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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE191142 | GEO | 2022/03/27

REPOSITORIES: GEO

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Project description:Diaphanous-related formin-3 (DIAPH3), a cytoskeletal regulator of the formin family, is lost at high frequency in metastatic breast and prostate cancers and has characteristics of a non-canonical metastasis suppressor. We previously demonstrated that DIAPH3 silencing evokes transition to an “amoeboid” phenotype, characterized by rapid motility, RhoA/ROCK and MEK/ERK pathway activation, dynamic membrane blebbing, and increased shedding of microvesicles. Here we report that low DIAPH3 mRNA expression correlates with reduced survival in multiple cancer patient cohorts. In line with induction of amoeboid behavior, traction force microscopy (TFM) revealed that polarized force generation, contractility, and response to substrate stiffness are reduced by DIAPH3 loss. Proteomic analyses revealed that DIAPH3 precipitates with tubulins, and reciprocal co-immunoprecipitation analyses revealed greater binding of DIAPH3 to MT polymers than soluble tubulin. DIAPH3 also bound to acetylated-tubulin (Ac-tubulin), suggesting association with a stable MT population. DIAPH3 silencing reduced Ac-tubulin and increased the cellular content of dynamic MT, as indicated by TFM and live cell imaging. DIAPH3 silencing also increased responsiveness to MT-disrupting agents. Treatment with the taxanes paclitaxel and docetaxel, and the non-taxane epothilone B, induced a greater change in Ac-tubulin when cells lacked DIAPH3. Intracellular accumulation of Oregon Green 488-paclitaxel was also increased by DIAPH3 loss, and accordingly, DIAPH3 silencing enhanced cytotoxicity by paclitaxel, docetaxel, or epothilone B. Gene expression profiles of breast cancer patients treated with chemotherapeutic regimens containing taxanes revealed an association of low DIAPH3 expression with improved relapse-free survival. Collectively, these results suggest that regulation of MT stability/dynamics by DIAPH3 enhances amoeboid tumor cell susceptibility to microtubule poisons. These findings have potential therapeutic implications.

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

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