Les GTPases de la sous famille RHOA participent à la régulation de nombreuses voies de signalisation qui contrôlent la dynamique du cytosquelette cellulaire et une grande diversité de fonctions telles que la prolifération, la division, la migration et la polarité cellulaires. Ce sont de véritables interrupteurs moléculaires qui, en réponse à un stimulus, changent de conformation tridimensionnelle pour activer leurs protéines effectrices cibles. Elles existent donc sous deux formes, une forme inactive liant le GDP et une forme active, liant le GTP. La proportion de forme active est extrêmement régulée au niveau spatial et temporel dans une cellule et représente moins de 10% de sa totalité. Depuis près de 20 ans, le seul outil disponible pour étudier leur activation est constitué par le domaine de liaison d'un effecteur, le RBD. Peu stable, faiblement soluble et peu adaptable, de nouveaux outils sont nécessaires afin de mieux comprendre la fine régulation de ces protéines. Les anticorps à simple domaine, VHH ou nanobodies, sont caractérisés par leur stabilité, solubilité, haut rendement de production et versatilité de fonctionnalisation. A partir d'une nouvelle banque d'anticorps à simple domaine optimisée pour la production d'intracorps, nous avons isolés différents clones capables de reconnaître in vitro et de bloquer in cellulo la forme active de ces protéines. L'un de ces clones permettra le développement d'un nouvel outil de mesure de l'activité de ces protéines in vitro tandis qu'un autre, in cellulo, permettra de mieux comprendre la régulation spatiale et temporelle des protéines endogènes. / RHOA small GTPase belongs to a subfamily acting as a molecular switch activating major signaling pathways that regulate cytoskeletal dynamics and a variety of cellular responses such as cell cycle progression, cytokinesis, migration and polarity. RHOA activity resides in a few percent of GTP loaded protein, which is finely tuned by a crosstalk between regulators of the GTPase cycle. Manipulating a single RHO at the expression level often induces imbalance in the activity of other RHO GTPases, suggesting that more specific tools targeting these active pools are needed to decipher RHOA functions in time and space. We decided to use single domain antibodies, also known as VHH or nanobodies, as a new tool for studying RHOA activation. We produced and screened a novel fully synthetic phage display library of humanized nanobodies (NaLi-H1) to develop conformational sensors of the GTP loaded active conformation of RHO subfamily. We obtained several high affinity nanobodies against RHOA's active form which we characterized as RHO active antibodies in vitro and RHO signaling blocking intrabodies in cellulo. These new tools will facilitate and improve our current knowledge of this peculiar protein subfamily and will be a paradigm for the study of other RHO related small GTPases.

RhoA is a small GTPase that acts as a molecular switch to control a variety of signal transduction pathways in eukaryotes. From an initial established role in the regulation of the actin cytoskeleton, RhoA has now been implicated in a range of functions that include gene transcription and regulation of cell morphology. In earlier studies from this laboratory that employed differential display and in situ hybridisation, RhoA was indicated as being up-regulated during the stages of early heart development in the developing chick embryo. Given the important effects of RhoA on both gene expression and morphology in other systems, it was hypothesised that RhoA plays a central role in the molecular mechanisms controlling cardiogenesis. This thesis describes investigations undertaken to elucidate the role of RhoA in these processes. As an initial approach to corroborate the earlier gene expression findings and provide further evidence for a role in tissue developmental mechanisms, RhoA proteins levels in the developing chick embryo were analysed using immunocytochemistry. These experiments demonstrated that RhoA is most abundant in heart-forming regions, findings compatible with the earlier gene expression studies and the proposed role of this protein in early heart development. Preliminary studies from this laboratory had also suggested that chick RhoA is expressed as different length mRNA transcripts that vary only in the 3' untranslated region (UTR). This thesis presents additional evidence for the existence of these different RhoA transcripts from experiments using Northern hybridisation and RT-PCR analyses. These analyses also serve to demonstrate that the second shortest RhoA transcript (designated RhoA2) is the most abundant transcript in developing heart tissue, in contrast to the situation in other embryonic tissues, findings that could be taken to suggest a possible role for this 3'UTR in developmental mechanisms that is yet to be elucidated. One potentially informative approach for testing the function of a protein in a biological system is to inhibit its expression and/or activity and observe the changes induced. The effects of inhibiting RhoA in early heart development and early organogenesis in the chick embryo model were investigated using small interfering RNAs (siRNA). Reduction in RhoA expression by siRNA treatment, as confirmed by real-time PCR, resulted in loss of heart tube fusion and abnormal head development, the former result providing further direct evidence of a role for RhoA in heart developmental processes. In order to investigate the function of RhoA specifically during the process of cardiomyocyte differentiation, an inducible model of cardiomyogenesis, P19CL6 cells, was used in combination with over-expression of different forms of mouse RhoA. The striking result from these investigations was that over-expression of the dominant negative mutant of mouse RhoA (mRhoAN19) prevented the differentiation of induced P19CL6 cells to the cardiomyocyte phenotype, results consistent with an essential role for RhoA in this cellular transition. The mechanism by which RhoA mediates its different cellular functions is unclear, however some studies have implicated RhoA in the regulation of transcription factors. To investigate such a mechanism as a possible explanation for the requirement of RhoA in cardiomyocyte differentiation, the P19CL6 inducible cell system over-expressing different forms of RhoA was analysed through real-time PCR to quantify the levels of transcription of genes known to play an important role in early heart development. These investigations indicated that RhoA inhibition causes an accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac-cx-actin. The expression of a protein is controlled by, among other factors, regulatory proteins that control transcription. To investigate factors in heart that potentially regulate RhoA expression at the molecular level, the chick RhoA gene organisation was analysed. The gene was shown to contain three introns that interrupt the protein coding sequence and at least one intron in the 5'UTR. Comparative RhoA gene studies indicated both an almost identical organisation and coding sequence of the chick, mouse and human RhoA genes, indicative of strict conservation of this gene during evolution. The putative promoter region of RhoA was predicted by computer analyses and tested for promoter activity using luciferase reporter analyses in non-differentiated and differentiated cardiomyocytes, using the inducible P19CL6 cell system. These investigations served to define a putative core promoter region that exhibited significantly higher promoter activity in differentiated cardiomyocytes than in non-differentiated cells, and other elements upstream of this core region that appear to be required for transcriptional regulation of RhoA. The majority of the consensus transcription factor sites identified in this putative promoter have been previously implicated in either heart development and/or organogenesis. These results therefore provide further, although indirect, evidence for an important role for RhoA in the molecular mechanisms controlling both cardiogenesis and embryogenesis in general. In summary, this thesis provides novel information on the role of RhoA in the processes of cardiogenesis and provides a firm foundation for continuing investigations aimed at elucidating the molecular basis of this contribution.

A fundamental yet unresolved issue in cell biology is how force regulates actin dynamics and how this biophysical regulation is modulated by biochemical signaling molecules. Here we show, by atomic force microscopy (AFM) force-clamp experiments, that tensile force regulates the kinetics of G-actin/G-actin and G-actin/F-actin interactions by decelerating dissociation at low forces (catch bonds) and accelerating dissociation at high forces (slip bonds). The catch bonds can be structurally explained by force-induced formation of new interactions between actin subunits (Steered molecular dynamics (SMD) simulations performed by Dr. Jizhong Lou, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China). K113S mutation on yeast actin suppressed the actin catch-slip bonds, supporting the structural mechanism proposed by SMD simulations. Moreover, formin controlled by a RhoA-mediated auto-inhibitory module can serve as a "molecular switch", converting the catch-slip bonds to slip-only. These results imply anisotropic stability of the actin network in cells subjected to directional forces, possibly explaining force-induced cell and actin fiber alignment controlled by RhoA and formin. Our study suggests a molecular level crosstalk mechanism bridging the actin-mediated mechanotransduction and biochemical signal transduction pathways.

Aims Angiotensin II (Ang II) stimulates cardiac remodelling and fibrosis in the mechanically overloaded myocardium. Although Rho GTPases regulate several cellular processes, including myocardial remodelling, involvement in mediating mechanical stretch-induced regulation of angiotensinogen (Ao), the precursor to Ang II, remains to be determined. We, therefore, examined the role and associated signalling mechanisms of Rho GTPases (Rac1 and RhoA) in regulation of Ao gene expression in a stretch model of neonatal rat cardiac fibroblasts (CFs). Methods and resultsCFs were plated on deformable stretch membranes. Equiaxial mechanical stretch caused significant activation of both Rac1 and RhoA within 25 min. Rac1 activity returned to control levels after 4 h, whereas RhoA remained at a high level of activity until the end of the stretch period (24 h). Mechanical stretch initially caused a moderate decrease in Ao gene expression, but was significantly increased at 824 h. RhoA had a major role in mediating both the stretch-induced inhibition of Ao at 4 h and the subsequent upregulation of Ao expression at 24 h. β1 integrin receptor blockade by Tac β1 expression impaired acute (2 and 15 min) stretch-induced Rac1 activation, but increased RhoA activity. Molecular experiments revealed that Ao gene expression was inhibited by Rac1 through both JNK-dependent and independent mechanisms, and stimulated by RhoA through a p38-dependent mechanism. Conclusion These results indicate that stretch-induced activation of Rac1 and RhoA differentially regulates Ao gene expression by modulating p38 and JNK activation.

Indiana University-Purdue University Indianapolis (IUPUI) / The growth of mammary epithelial cells is regulated by interactions with neighboring cells and by exposure to soluble factors including hormones and growth factors. These cues are integrated within the cell, perpetuating changes onto the organization of the actin cytoskeleton, resulting in altered transcriptional programs. Rho family GTPases regulates actin dynamics that facilitate transcriptional reprogramming. In particular, RhoA induces the formation of actin stress fibers to promote the transcriptional co-activator YAP1 to translocate from the cytosol into the nucleus. There, it co-activates TEAD family transcription factors to drive the expression of pro-growth and survival genes. Rho family members are activated by guanine exchange factors (GEF) and inhibited by GTPase activating proteins (GAP). Here, we determined the relative effects of expression of 67 RhoGEFs and RhoGAPs on the activation of TEAD. This revealed that regulator of G-protein signaling (RGS) domain containing ArhGEF1, ArhGEF11 and ArhGEF12 all promoted YAP1 dependent activation of TEAD. These RhoGEFs mediate signaling from heptahelical receptors that are stimulated by lipid mitogens to activate the heterotrimeric G-proteins Gα12 and Gα13. Consistently, loss of expression of ArhGEF12 and to a lesser degree ArhGEF11 prevented actin stress fiber accumulation and activation of YAP1 mediated signaling by serum. Conversely, several complementary experiments revealed that ArhGEF1 dominantly limits Gα13 selective activation of YAP1 and the mitogen activated protein kinase (MAPK) cascades. Furthermore excessive Gα13 activity results in both high levels of filamentous actin and arrest cells in the G1/0 phase of the cell cycle. This is likely due to the systemic inhibition of cell cycle promoting signaling and a loss of protein translation. Further, YAP1 was found to be essential for the survival of ArhGEF1 silenced cells. Together, these studies define a circuit whereby the rgRhoGEFs regulate Gα 12/13-RhoA signaling flux to regulate cellular growth that is promoted by serum factors.

No description available.

Der spezifische Knockdown von RhoA in neonatalen kardialen Rattenfibroblasten führte auf molekularem Level zu einer Reduktion des Myofibroblastenmarkers α-Glattmuskelaktin und zu einem Anstieg im modifizierten acetylierten Tubulin. Auf subzellulärer Ebene konnte ein Verlust von Stressfasern, Aktinstrukturen höherer Ordnung und eine erhöhte Dichte des Golgi-Apparats beobachtet werden. Außerdem waren die Fokaladhäsionen kürzer und zufällig verteilt, was auf einen Verlust der Zellpolarität hinweist. Auf dem zellulären Level erhöhte der Knockdown von RhoA die Zellfläche aber nicht das Volumen. Diese Veränderungen führten zu einer schnelleren Adhäsion unabhängig vom Substrat, eine Reduktion der Migration in 2D und im Gegensatz dazu eine verbesserte Migration durch eine poröse Membran. Außerdem war die mitogene Antwort der Zellen auf einen Serumstimulus stark reduziert. Eine Veränderung in Zellviabilität konnte zudem nicht beobachtet werden. Die Expression und Sekretion des Fibrose-assoziierten Faktors CTGF war in gehungerten Zellen mit einer Reduktion in RhoA Expression signifikant vermindert, was jedoch in der Anwesenheit eines Serumstimulus aufgehoben werden konnte. Auf einer heterogenen multizellulären Ebene verringerte der Knockdown von RhoA die kontraktile Funktion von generierten künstlichen Herzgeweben unter Kalziumstimulation. Dies ging einher mit einer Reduktion der Expression von α-Glattmuskelaktin und Calsequestrin. Durch die Verwendung spezifischer Inhibitoren der Rho-assoziierten Kinase (ROCK) und HDAC6 konnten einige dieser zellulären Veränderungen imitiert und demensprechend einem Effektorprotein zugeordnet werden. Der ROCK Inhibitor Fasudil konnte die morphologischen Veränderungen und die reduzierte Migrationskapazität in Wildtyp-Fibroblasten abbilden, wobei eine Reduktion der Proliferation nach der Verwendung des HDAC6 Inhibitors Tubastatin A beobachtet wurde.

Malgré les avancées récentes, le cancer reste la première cause de mortalité en France avec ~ 150000 décès recensés par an (INCa 2012). Notamment, le cancer broncho-pulmonaire est l’un des plus agressifs, avec 29100 décès en 2011. La survie à cinq ans de seulement 10% des patients atteints des cancers du poumon non à petites cellules (NSCLC, 80% des cancers bronchiques), pose un problème d’ordre scientifique, médical et de santé publique. Il est communément admis qu’une sous-population de la tumeur acquiert en raison d’une instabilité génétique de nouvelles propriétés agressives nécessaires à sa dissémination, sa prolifération et une plus grande résistance aux chimiothérapies. Une meilleure compréhension de ces propriétés tumorales constitue un enjeu majeur pour prévenir à terme cette progression maligne. Nous avons axé notre recherche sur la macro-autophagie, un processus catabolique lysosomal essentiel à l’homéostasie cellulaire et sur un de ses substrats SQSTM1 (séquestosome ou p62). Au moment où j’ai initié ma thèse, une relation étroite entre l’autophagie, SQSTM1 et la progression tumorale venait d’être mise en lumière. Plusieurs membres de la machinerie autophagique sont fréquemment mutés/déletés dans les cancers. De façon paradoxale, l’autophagie peut également permettre la survie des cellules tumorales face à l’hypoxie, la carence nutritionnelle ou les chimiothérapies. / Despite recent advances, cancer remains the leading cause of death in France with~150,000 annual deaths (INCA, 2012). Notably, lung cancer is one of the most aggressive, with 29 100 deaths in 2011. The five-year survival is only 10 % of patients with lung cancer non-small cell (NSCLC, 80% of lung cancers), so it’s a medical/scientific challenge, and public health problem. Due to genetic instability, it’s commonly accepted that a subpopulation of tumor acquires new aggressive properties for its dissemination, its proliferation and greater resistance to chemotherapy. A better understanding of these tumor properties is a major issue to prevent this malignant progression. We focused our research on macro-autophagy, a lysosomal catabolic process essential for cellular homeostasis, and alos on one of its substrates SQSTM1 (Sequestosome or p62). When I started my PhD, a close relationship between autophagy, SQSTM1 and tumor progression had been highlighted. Several members of the autophagic machinery are frequently mutated/deleted in cancers. Paradoxically, autophagy can also allow the survival of tumor cells under hypoxia, nutritional deficiency or chemotherapy. How could we understand these conflicting functions, considering that the only autophagy substrates known are long half-lives proteins, damaged organelles and scaffold SQSTM1? Thus, SQSTM1 is essential for RasV12 driven oncogenesis (A Duran et al, Cancer Cell 2008).

Les lymphocytes T (LT) perçoivent et intègrent en permanence des signaux solubles et cellulaires, conditionnant leur comportement et leur devenir. A l’état de repos, les propriétés des LT s’appuient sur un réseau moléculaire caractéristique, au sein duquel les facteurs de transcription FoxOs jouent un rôle majeur. En effet, ces derniers sont impliqués dans le maintien de la quiescence et de la capacité circulatoire des LT, de par le profil transcriptionnel qu’ils induisent. Nous avons identifié Fam65b comme une nouvelle cible transcriptionnelle de FOXO1. D’un point de vue fonctionnel, nous avons démontré que Fam65b régule négativement le seuil de prolifération des LT en réponse à une stimulation du récepteur à l’antigène (TCR) ou du récepteur aux chimiokines CCR7. In vivo, dans un modèle de souris transgénique pour le TCR, ces caractéristiques fonctionnelles se traduisent par une réponse secondaire plus efficace en absence de Fam65b. Physiologiquement, la moindre expression de Fam65b que nous avons observé dans les LT mémoires par comparaison aux LT naïfs corrèle avec leur plus grande réactivité. L’ensemble de ces résultats suggère que Fam65b pourrait être un marqueur fonctionnel des LT mémoires. Enfin, nous avons pu démontrer que les effets fonctionnels de Fam65b résultent d’une inhibition de l’activité de RhoA.Fam65b est donc un régulateur de la quiescence et de la migration des LT. De par son rôle de régulateur de l’activité de RhoA, Fam65b constitue un nouveau lien fonctionnel entre deux familles majeures, contrôlant la physiologie des LT : les Rho-GTPases et les FoxOs. / T cells continually sense numerous soluble and cellular signals, which determine their behavior and differentiation pattern. At the steady state, lymphocytes properties rely on a specific network, in which FoxOs transcription factors play a central role. Indeed, FoxOs-induced transcriptome is involved in the maintenance of T cell quiescence and circulation.We identified a new transcriptional target of FoxO1 named Fam65b. We show that Fam65b acts as a brake on T lymphocyte activation downstream of the T cell receptor. Functionally, we demonstrate that Fam65b negatively regulates the threshold of T cell activation downstream of TCR or CCR7 stimulation. These characteristics allow a more efficient secondary response as Fam65b expression is inhibited. Physiologically, the lower expression of Fam65b in memory T cells compared to naïve T cells takes part in their enhanced reactivity. This suggests that Fam65b could be a functional marker of memory T cells. Finally, we demonstrated that the role of Fam65b is mediated by an inhibition of RhoA activity. Therefore, Fam65b is a regulator of T cell quiescence and migration. Because he regulates the activity of RhoA, Fam65b constitutes a functional link between two major families of proteins which control T cell physiology: Rho GTPases and FoxOs.

Le cancer de la prostate est la principale cause de décès chez les hommes des pays occidentaux. Traitement du cancer de la prostate résistant à la castration (CRPC) métastatique est encore limité. RhoA/ROCK sont les régulateurs principaux du cytosquelette et sont impliqués dans l'angiogenèse et l'invasion tumorale. Dans la première étude (partie 1), nous avons étudié les effets anti-angiogéniques de fasudil ( inhibiteur de ROCK ) in vitro. Des cellules endothéliales de la veine ombilicale sont stimulé par les cellules du cancer de la prostate. La prolifération est détectée par la Bromodéoxyuridine (BrdU). La migration est détectée par la cicatrisation des plaies. Les conditions de l'angiogenèse in vitro sont évaluées par la formation des tubes et un ellipsoïde test de germination. Fasudil inhibe la prolifération et la migration des cellules endothéliales induite par le cancer de la prostate. Dans les tests in vitro de l'angiogenèse, les formations des tubes et des germes sphériques sont inhibées significativement par fasudil dans d'une manière dépendante de la dose. Selon les résultats de Western Blot, l'expression de MYPT-1 a été significativement réduite après le traitement de Fasudil, confirmé l'inhibition de l'activité de ROCK par fasudil. Dans la deuxière étude (partie 2 et 3), l'expression et l'activité de RhoA sont évaluées chez 34 tissues paraffinées et 20 échantillons congelés de la prostate, obteni de 45 malades du cancer de la prostate qui à été reçu une prostatectomie radicale. Les motifs de l'expression de RhoA sont detecté par la coloration immunomarquage et western blot. Les différence entre le centre, l'avant et plus loin autour de la tumeur sont évalué. L’activité de RhoA sont évalué par G-LISA. Le gradient d'augmentation de l'expression de RhoA a été trouvé du centre a la region autour de la tumeur. L'expression de RhoA est supérieure significativement dans les cellules de l’avant de la tumeur par rapport au centre de la tumeur par Immunohistochimie (p = 0001). Gleason score était significativement supérieur chez des patients avec l'expression de RhoA élevée dans l’avant et centre de la tumeur (p =0044 et 0039, respectivement). Après un suivi médian de 52 mois, le taux de récidive est plus élevé chez des patients avec l'expression de RhoA élevée dans l’avant de la tumeur (62,5% contre 35%), une tendance évidente a la différence significative (P = 0089). Il n'y a pas de corrélation entre l'expression de RhoA, PSA et la stadification pathologique. On a aussi découvert que l'expression de le ROCK2, mais pas l'expression de le ROCK1, est eleve significativement dans l’avant de la tumeur du cancer de la prostate. En conclusion, nous avons trouvé que fasudil inhiber la prolifération, la migration, la formation de capillaires et de la sphère de la germination des cellules endothéliales vasculaires d’'une manière dépendante de la dose. Ces effets peut-être grâce à l'inhibition de l'activité de la ROCK résultant de la sécrétion de cellules du cancer de la prostate. Nous avons aussi trouvé que l'expression de la RhoA et ROCK2 dans l’avant de la tumeur de la prostate sont plus élevés. La corrélation de l'expression de RhoA avec Gleason score et récidive est identifiée. Cela montre l’association entre la voie RhoA/ROCK et l’agressive du cancer de la prostate. L'étude décrite ici peut fournir de nouveaux traitements cible la voie RhoA/ROCK contre l’angiogenèse et agressive du cancer de la prostate. Fasudil peut être utile comme agent anti - angiogénique, doit être étudiée pour leur rôle potentiel dans le traitement du cancer de la prostate. / Prostate cancer remains a major cause of mortality among males in western countries. Treatment options for metastatic castration-resistant disease remain limited. There is a continuing unmet need for new systemic interventions in patients with progressive prostate cancer. RhoA/Rho-associated protein kinases (ROCK) are key regulators of the cytoskeleton and have been implicated in PCa angiogenesis and tumour invasion. In the first study (Part I), we investigated the anti-angiogenic effects of fasudil, a ROCK inhibitor, on PCa-induced angiogenesis in vitro. Proliferation of PCa-conditioned human umbilical vein endothelial cells (HUVECs) was assessed using a bromodeoxyuridine (BrdU) assay, and migration was assessed with a wound healing assay. In vitro angiogenesis of PCa-conditioned HUVECs was evaluated by tube formation and a spheroid sprouting assay. Fasudil inhibited PCa-induced endothelial cell proliferation, and also decreased PCa-induced endothelial cell migration. In the in vitro angiogenesis assay, tube formation and spheroid sprouts were significantly inhibited at fasudil in a dose dependent manner. Western blotting results showed that expression of phosphorylated myosin phosphatase target subunit 1 (MYPT-1) was significantly lower after fasudil treatment, confirming that fasudil inhibited ROCK activity in these model systems. In the second study (Part II & III), we evaluated RhoA expression and activity in a total of 34 paraffin embedded and 20 frozen prostate specimens, respectively, obtained from 45 patients treated with radical prostatectomy for clinically localized cancer. The expression patterns of RhoA were tested by immunohistochemical staining and Western blotting, and further compared between the tumour centre, tumour front and distant peritumoral tissue. RhoA activity was assessed by G-LISA. Our results showed an increasing gradient of expression from the centre to the periphery of index tumour foci. RhoA expression was indeed significantly higher at the tumour front as compared to tumour centre, using immunohistochemistry (p=0.001). Gleason score was significantly higher in the patients with higher RhoA expression in both the tumour front and tumour centre (p=0.044 and 0.039, respectively). After a median follow-up of 52 months, the rate of PSA relapse was higher in patients with a higher RhoA expression at the tumour front (62.5% vs 35%), although the difference was not significant (p=0.089). There was no association between RhoA expression and PSA, pathological stage. We also found ROCK2 expression, but not ROCK1 expression, was significantly higher in the prostate cancer tumor front. In conclusion, we found fasudil significantly inhibits the key steps of endothelial cell angiogenesis, including proliferation, migration, capillary tube formation and spheroid sprouting, in a dose-dependent manner. These effects may due to inhibition of ROCK activity induced by PCa cell secretions. We also identified higher RhoA and ROCK2 expression in human prostate tumour front. The correlation of higher RhoA expression with higher Gleason score and higher rate of cancer relapse. This indicated the association of RhoA/ROCK2 pathway with aggressiveness of prostate cancer. The insights described here may provide the foundation for novel therapeutic approaches targeting RhoA/ROCK pathway to inhibit angiogenesis and clinically aggressiveness of PCa. Fasudil may be a useful anti-angiogenic agent and should be investigated further for its potential role in PCa treatment.