Abstract
BackgroundRASSF1A, a tumor suppressor gene, is frequently inactivated in lung cancer leading to a YAP-dependent epithelial-mesenchymal transition (EMT). Such effects are partly due to the inactivation of the anti-migratory RhoB GTPase via the inhibitory phosphorylation of GEF-H1, the GDP/GTP exchange factor for RhoB. However, the kinase responsible for RhoB/GEF-H1 inactivation in RASSF1A-depleted cells remained unknown.MethodsNDR1/2 inactivation by siRNA or shRNA effects on epithelial-mesenchymal transition, invasion, xenograft formation and growth in SCID−/− Beige mice, apoptosis, proliferation, cytokinesis, YAP/TAZ activation were investigated upon RASSF1A loss in human bronchial epithelial cells (HBEC).ResultsWe demonstrate here that depletion of the YAP-kinases NDR1/2 reverts migration and metastatic properties upon RASSF1A loss in HBEC. We show that NDR2 interacts directly with GEF-H1 (which contains the NDR phosphorylation consensus motif HXRXXS/T), leading to GEF-H1 phosphorylation. We further report that the RASSF1A/NDR2/GEF-H1/RhoB/YAP axis is involved in proper cytokinesis in human bronchial cells, since chromosome proper segregation are NDR-dependent upon RASSF1A or GEF-H1 loss in HBEC.ConclusionTo summarize, our data support a model in which, upon RASSF1A silencing, NDR2 gets activated, phosphorylates and inactivates GEF-H1, leading to RhoB inactivation. This cascade induced by RASSF1A loss in bronchial cells is responsible for metastasis properties, YAP activation and cytokinesis defects.
Highlights
RASSF1A, a tumor suppressor gene, is frequently inactivated in lung cancer leading to a YAP-dependent epithelial-mesenchymal transition (EMT)
Depletion of the NDR1/2 kinases reverts the migratory and metastatic phenotypes induced by RASSF1A loss in human bronchial epithelial cells (HBEC) Forty-eight hours after silencing RASSF1A or NDR1/2 in HBEC cells using siRNAs (Additional file 1: Table S2), we performed wound healing (Fig. 1a) and invasion (Fig. 1b) assays in the presence of mitomycin C, to inhibit the contribution of cell division in wound repair
The increase of migration and invasion induced by RASSF1A loss in HBEC-3 cells ([3], Fig. 1a and b) was inhibited by NDR1/2 depletion (Fig. 1a and b) without leading to cell death
Summary
RASSF1A, a tumor suppressor gene, is frequently inactivated in lung cancer leading to a YAP-dependent epithelial-mesenchymal transition (EMT) Such effects are partly due to the inactivation of the anti-migratory RhoB GTPase via the inhibitory phosphorylation of GEF-H1, the GDP/GTP exchange factor for RhoB. RASSF1A [Ras association (RalGDS/AF-6) domain family member 1], a tumor and metastatic suppressor gene, is frequently inactivated and an independent predictor of poor prognosis in resected early-stage non–small cell lung cancer (NSCLC) [1, 2]. RhoB inactivation leads to the cancer-associated phenotypes observed in RASSF1A-depleted HBEC
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