RhoA and RhoC Are Both Required for the ROCK II-Dependent Promotion of Centrosome Duplication

Molecular Oncology Program, H Lee Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USA.
Oncogene (Impact Factor: 8.46). 11/2010; 29(45):6040-50. DOI: 10.1038/onc.2010.328
Source: PubMed


CDK2-cyclin E triggers centrosome duplication, and nucleophosmin (NPM/B23) is found to be one of its targets. NPM/B23 phosphorylated by CDK2-cyclin E acquires a high binding affinity to Rho-associated kinase (ROCK II), and physically associates with ROCK II. The NPM/B23-binding results in superactivation of ROCK II, which is a critical event for initiation of centrosome duplication. The activation of ROCK II also requires the binding of Rho small GTPase to the auto-inhibitory region; hence the availability of the active Rho protein is an important aspect of the centrosomally localized ROCK II to properly initiate centrosome duplication. There are three isoforms of Rho (RhoA, B and C), all of which are capable of binding to and priming the activation of ROCK II. Here, we investigated which Rho isoform(s) are involved in the activation of ROCK II in respect to the initiation of centrosome duplication. We found that both RhoA and RhoC, but not RhoB, were required for initiation of centrosome duplication, and overactivation of RhoA, as well as RhoC, but not RhoB, promoted centrosome duplication and centrosome amplification.

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    • "The RhoA-like sub-family molecules of small GTPases (RhoA, RhoB and RhoC) share nearly 84% amino acid sequence homology, differing predominantly in their C-terminus domain [1]. Until recently, this sequence homology has prevented specific causality being attributed to each Rho GTPase for their explicit roles in distinct biological functions [2–4]. Increased expression of RhoC has been implicated in the metastatic process in pathologically distinct human cancers [5–7]. "
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    ABSTRACT: Human pancreatic ductal adenocarcinoma (PDAC) is characterized by early systemic dissemination. Although RhoC has been implicated in cancer cell migration, the relevant underlying molecular mechanisms remain unknown. RhoC has been implicated in the enhancement of cancer cell migration and invasion, with actions which are distinct from RhoA (84% homology), and are possibly attributed to the divergent C-terminus domain. Here, we confirm that RhoC significantly enhances the migratory and invasive properties of pancreatic carcinoma cells. In addition, we show that RhoC over-expression decreases cancer cell adhesion and, in turn, accelerates cellular body movement and focal adhesion turnover, especially, on fibronectin-coated surfaces. Whilst RhoC over-expression did not alter integrin expression patterns, we show that it enhanced integrin α5β1 internalization and re-cycling (trafficking), an effect that was dependent specifically on the C-terminus (180-193 amino acids) of RhoC protein. We also report that RhoC and integrin α5β1 co-localize within the peri-nuclear region of pancreatic tumor cells, and by masking the CAAX motif at the C-terminal of RhoC protein, we were able to abolish this interaction in vitro and in vivo. Co-localization of integrin α5β1 and RhoC was demonstrable in invading cancer cells in 3D-organotypic cultures, and further mimicked in vivo analyses of, spontaneous human, (two distinct sources: operated patients and rapid autopsy programme) and transgenic murine (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre), pancreatic cancers. In both cases, co-localization of integrin α5β1 and RhoC correlated with poor differentiation status and metastatic potential. We propose that RhoC facilitates tumor cell invasion and promotes subsequent metastasis, in part, by enhancing integrin α5β1 trafficking. Thus, RhoC may serve as a biomarker and a therapeutic target.
    PLoS ONE 12/2013; 8(12):e81575. DOI:10.1371/journal.pone.0081575 · 3.23 Impact Factor
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    • "Aneuploidy and genomic instability are features of most solid tumors [5], and disruption of mitosis and cytokinesis facilitates these processes. A number of studies have implicated Rho GTPases and their regulators and effectors in centrosome duplication, mitotic spindle formation, kinetochore-microtubule attachments, and cytokinesis [6,7,8,9,10]. Perturbation of any of these processes can lead to abnormal chromosome segregation and aneuploidy. "
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    ABSTRACT: Rho GTPases are overexpressed and hyperactivated in many cancers, including breast cancer. Rho proteins, as well as their regulators and effectors, have been implicated in mitosis, and their altered expression promotes mitotic defects and aneuploidy. Previously, we demonstrated that p190B Rho GTPase activating protein (RhoGAP) deficiency inhibits ErbB2-induced mammary tumor formation in mice. Here we describe a novel role for p190B as a regulator of mitosis. We found that p190B localized to centrosomes during interphase and mitosis, and that it is differentially phosphorylated during mitosis. Knockdown of p190B expression in MCF-7 and Hela cells increased the incidence of aberrant microtubule-kinetochore attachments at metaphase, lagging chromosomes at anaphase, and micronucleation, all of which are indicative of aneuploidy. Cell cycle analysis of p190B deficient MCF-7 cells revealed a significant increase in apoptotic cells with a concomitant decrease in cells in G1 and S phase, suggesting that p190B deficient cells die at the G1 to S transition. Chemical inhibition of the Rac GTPase during mitosis reduced the incidence of lagging chromosomes in p190B knockdown cells to levels detected in control cells, suggesting that aberrant Rac activity in the absence of p190B promotes chromosome segregation defects. Taken together, these data suggest that p190B regulates chromosome segregation and apoptosis in cancer cells. We propose that disruption of mitosis may be one mechanism by which p190B deficiency inhibits tumorigenesis.
    Cancers 06/2012; 4(2):475-489. DOI:10.3390/cancers4020475
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    • "After NPM phosphorylation by CDK2–cyclin E, the binding between NPM and ROCK2 increases and ROCK2 is activated at centrosomes, leading to centrosome duplication (Ma et al., 2006b). In ROCK2 activation, the binding of Rho small GTPase to the auto-inhibitory region is also required (Kanai et al., 2010). Among three isoforms of Rho, both RhoA and RhoC, but not RhoB, promoted centrosome duplication and centrosome amplification. "

    Lung Diseases - Selected State of the Art Reviews, 03/2012; , ISBN: 978-953-51-0180-2
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