Quantitation of RhoA activation: differential binding to downstream effectors.

The small GTPase RhoA controls many important cellular processes through its ability to activate multiple downstream effector pathways. Most RhoA effectors contain a Rho-binding domain (RBD), and interaction between active RhoA and the RBD typically induces a conformational change in effectors that stimulates their recruitment or activity. Isolated GTPase binding domains fused to GST have been widely used in so-called pulldown assays to measure the activation state of other GTPases in cell lysates. Similarly, GST fusions containing the RBD of the RhoA effector Rhotekin have been widely adopted as a standardized tool for the measurement of RhoA activation. RBDs have also been used to generate fluorescent reporter constructs to localize sites of GTPase activation in intact cells. In this report, we demonstrate that not all forms of active RhoA are capable of interacting with the Rhotekin RBD. A constitutively active RhoA-G14V mutant, which interacted with the RBDs of ROCK2 and mDIA1, was unable to bind the Rhotekin RBD as evidenced by both conventional GST pulldown assay and our newly established BRET assay. Furthermore, active RhoA induced by different stimuli in cells also displayed binding preference for its diverse effectors. Our data demonstrate that RhoA may undergo effector-specific activation for differential regulation of its downstream pathways, and that RhoA activation should not be defined solely by its interaction with Rhotekin.

The complex of Fas-associated factor 1 with Hsp70 stabilizes the adherens junction integrity by suppressing RhoA activation.

Fas-associated factor 1 (FAF1) is a scaffolding protein that plays multiple functions, and dysregulation of FAF1 is associated with many types of diseases such as cancers. FAF1 contains multiple ubiquitin-related domains (UBA, UBL1, UBL2, UAS, and UBX), each domain interacting with a specific partner. In particular, the interaction of UBL1 with heat shock protein 70 (Hsp70) is associated with tumor formation, although the molecular understanding remains unknown. In this study, the structural analysis revealed that His160 of FAF1 is important for its interaction with Hsp70. The association of Hsp70 with FAF1 is required for the interaction with IQGAP1. FAF1 negatively regulates RhoA activation by FAF1-Hsp70 complex formation, which then interacts with IQGAP1. These steps play a key role in maintaining the stability of cell-to-cell junction. We conclude that FAF1 plays a critical role in the structure and function of adherens junction during tissue homeostasis and morphogenesis by suppressing RhoA activation, which induces the activation of Rho-associated protein kinase, phosphorylation of myosin light chain, formation of actin stress fiber, and disruption of adherens junction. In addition, depletion of FAF1 increased collective invasion in a 3D spheroid cell culture. These results provide insight into how the FAF1-Hsp70 complex acts as a novel regulator of the adherens junction integrity. The complex can be a potential therapeutic target to inhibit tumorigenesis and metastasis.

MYBL2 disrupts the Hippo-YAP pathway and confers castration resistance and metastatic potential in prostate cancer.

Rationale: Resistance to androgen-deprivation therapy (ADT) associated with metastatic progression remains a challenging clinical task in prostate cancer (PCa) treatment. Current targeted therapies for castration-resistant prostate cancer (CRPC) are not durable. The exact molecular mechanisms mediating resistance to castration therapy that lead to CRPC progression remain obscure. Methods: The expression of MYB proto-oncogene like 2 (MYBL2) was evaluated in PCa samples. The effect of MYBL2 on the response to ADT was determined by in vitro and in vivo experiments. The survival of patients with PCa was analyzed using clinical specimens (n = 132) and data from The Cancer Genome Atlas (n = 450). The mechanistic model of MYBL2 in regulating gene expression was further detected by subcellular fractionation, western blotting, quantitative real-time PCR, chromatin immunoprecipitation, and luciferase reporter assays. Results: MYBL2 expression was significantly upregulated in CRPC tissues and cell lines. Overexpression of MYBL2 could facilitate castration-resistant growth and metastatic capacity in androgen-dependent PCa cells by promoting YAP1 transcriptional activity via modulating the activity of the Rho GTPases RhoA and LATS1 kinase. Importantly, targeting MYBL2, or treatment with either the YAP/TAZ inhibitor Verteporfin or the RhoA inhibitor Simvastatin, reversed the resistance to ADT and blocked bone metastasis in CRPC cells. Finally, high MYBL2 levels were positively associated with TNM stage, total PSA level, and Gleason score and predicted a higher risk of metastatic relapse and poor prognosis in patients with PCa. Conclusions: Our results reveal a novel molecular mechanism conferring resistance to ADT and provide a strong rationale for potential therapeutic strategies against CRPC.

CREPT Promotes Melanoma Progression Through Accelerated Proliferation and Enhanced Migration by RhoA-Mediated Actin Filaments and Focal Adhesion Formation.

Melanoma is one of the most aggressive cancers, and patients with distant metastases have dire outcomes. We observed previously that melanoma progression is driven by a high migratory potential of melanoma cells, which survive and proliferate under harsh environmental conditions. In this study, we report that CREPT (cell-cycle related and expression-elevated protein in tumor), an oncoprotein highly expressed in other cancers, is overexpressed in melanoma cells but not melanocytes. Overexpression of CREPT stimulates cell proliferation, migration, and invasion in several melanoma cell lines. Further, we show that CREPT enhances melanoma progression through upregulating and activating Ras homolog family member A (RhoA)-induced actin organization and focal adhesion assembly. Our study reveals a novel role of CREPT in promoting melanoma progression. Targeting CREPT may be a promising strategy for melanoma treatment.

The V protein of human parainfluenza virus type 2 promotes RhoA-induced filamentous actin formation.

We previously demonstrated that human parainfluenza virus type 2 (hPIV-2) induces RhoA activation, which promotes its growth. RhoA controls the equilibrium between globular and filamentous actin (F-actin). We found that F-actin formation is induced by wild type (wt) hPIV-2 infection, and that inhibition of F-actin formation by cytochalasin D decreases hPIV-2 growth. In wt RhoA-expressing cells, F-actin formation occurs and hPIV-2 growth is promoted. Overexpression of T19N RhoA, a dominant negative (DN) form of RhoA, inhibits hPIV-2-induced F-actin formation, and suppresses hPIV-2 growth. Immunoprecipitation assays reveal that hPIV-2 V protein binds only to DN RhoA, and this interaction requires its C-terminal Trp residues. F-actin formation is not observed during infection of recombinant hPIV-2 expressing Trp-mutated V protein (VW178H/W182E/W192A). Overexpression of V protein, but not that of VW178H/W182E/W192A, causes F-actin formation. Our results suggest that hPIV-2 V protein enhances hPIV2 growth through RhoA-induced F-actin formation, by selectively binding to inactive RhoA.

Cell type-dependent HIF1 α-mediated effects of hypoxia on proliferation, migration and metastatic potential of human tumor cells.

Tumor hypoxia promotes neoangiogenesis and contributes to the radio- and chemotherapy resistant and aggressive phenotype of cancer cells. However, the migratory response of tumor cells and the role of small GTPases regulating the organization of cytoskeleton under hypoxic conditions have yet to be established. Accordingly, we measured the proliferation, migration, RhoA activation, the mRNA and protein levels of hypoxia inducible factor-1alpha (HIF-1α) and three small G-proteins, Rac1, cdc42 and RhoA in a panel of five human tumor cell lines under normoxic and hypoxic conditions. Importantly, HT168-M1 human melanoma cells with high baseline migration capacity showed increased HIF-1α and small GTPases expression, RhoA activation and migration under hypoxia. These activities were blocked by anti- HIF-1α shRNA. Moreover, the in vivo metastatic potential was promoted by hypoxia mimicking CoCl2 treatment and reduced upon inhibition of HIF-1α in a spleen to liver colonization experiment. In contrast, HT29 human colon cancer cells with low migration capacity showed limited response to in vitro hypoxia. The expression of the small G-proteins decreased both at mRNA and protein levels and the RhoA activation was reduced. Nevertheless, the number of lung or liver metastatic colonies disseminating from orthotopic HT29 grafts did not change upon CoCl2 or chetomin treatment. Our data demonstrates that the hypoxic environment induces cell-type dependent changes in the levels and activation of small GTPases and results in varying migratory and metastasis promoting responses in different human tumor cell lines.

Epigallocatechin-3 gallate inhibits cancer invasion by repressing functional invadopodia formation in oral squamous cell carcinoma.

Although the polyphenol EGCG has various beneficial biological effects, its effect on cytoskeletal activities during cancer invasion is not well defined, and the precise molecular mechanisms are largely unknown. Here, we provide molecular evidence on the anti-invasion effect of EGCG in OSCC cells using an in vitro 3-D culture system and in vivo athymic mouse model. Briefly, EGCG exerted an inhibitory effect on the Matrigel-based Transwell invasion and migration of OSCC cells. These effects were not due to decreased cell viability or adhesion capacity to ECM. EGCG-treated OSCC cells possessed fully extended actin fibers without invadopodia, indicating a loss of ECM degradation capacity. Decreased phosphorylation of Src, CTTN, and FAK also followed EGCG treatment. Additionally, EGCG reduced activation of RhoA in dominant-negative RhoA N19 and constitutively active RhoA Q63E cells, and inhibited the invasive capability of these cells in the 3-D cell growth model. Furthermore, the administration of EGCG led to substantial inhibition of tumor growth and activation of invadopodial proteins in the tumor tissues of mice inoculated with OSCC cells. The data indicate the potential value of EGCG as an invadopodia-targeted anti-invasive agent in cancer therapy.