Abstract
BackgroundCell migration is essential for development and tissue repair, but it also contributes to disease. Rho GTPases regulate cell migration, but a comprehensive analysis of how each Rho signalling component affects migration has not been carried out.ResultsThrough an RNA interference screen, and using a prostate cancer cell line, we find that approximately 25% of Rho network components alter migration. Some genes enhance migration while others decrease basal and/or hepatocyte growth factor-stimulated migration. Surprisingly, we identify RhoH as a screen hit. RhoH expression is normally restricted to haematopoietic cells, but we find it is expressed in multiple epithelial cancer cell lines. High RhoH expression in samples from prostate cancer patients correlates with earlier relapse. RhoH depletion reduces cell speed and persistence and decreases migratory polarity. Rac1 activity normally localizes to the front of migrating cells at areas of dynamic membrane movement, but in RhoH-depleted cells active Rac1 is localised around the whole cell periphery and associated with membrane regions that are not extending or retracting. RhoH interacts with Rac1 and with several p21-activated kinases (PAKs), which are Rac effectors. Similar to RhoH depletion, PAK2 depletion increases cell spread area and reduces cell migration. In addition, RhoH depletion reduces lamellipodium extension induced by PAK2 overexpression.ConclusionsWe describe a novel role for RhoH in prostate cancer cell migration. We propose that RhoH promotes cell migration by coupling Rac1 activity and PAK2 to membrane protrusion. Our results also suggest that RhoH expression levels correlate with prostate cancer progression.
Highlights
Cell migration is essential for development and tissue repair, but it contributes to disease
An RNA interference (RNAi) screen of the Rho network identifies groups of genes with different effects on cancer cell migration To investigate which Rho GTPase network components affect cell migration, we used a small interfering RNA library containing pools of four siRNAs targeting each of 202 genes encoding Rho GTPases, RhoGEFs, RhoGAPs and Rho targets, together with four control siRNA pools (Additional file 1: Table S1)
After 24 h, control siRNA-transfected cells covered approximately 50–60% of the wound area (Fig. 1b). This was established as the optimal time point for the screening, because it allowed the identification of genes that inhibited and enhanced migration
Summary
Cell migration is essential for development and tissue repair, but it contributes to disease. Many cell-surface receptors and intracellular signalling proteins contribute to cell migration, and inhibitors of some of these molecules are being developed for the treatment of human diseases [4, 5]. Most Rho GTPases cycle between an active GTP-bound and an inactive GDP-bound form. They are activated by guanine nucleotide exchange factors (GEFs), which stimulate exchange of GDP for GTP, and inactivated by GTPase-activating proteins (GAPs), which stimulate their intrinsic GTPase activity, hydrolysing GTP to GDP [6]. Some Rho GTPases are regulated by guanine nucleotide dissociation inhibitors (GDIs), which extract them from membranes by binding to the lipid group as well as other regions of the protein [7]. When bound to GTP, Rho GTPases interact with a wide range of downstream effectors to induce signalling, including
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