387

RHOA

epithelial to mesenchymal transition (EMT) is involved in embryological development, cancerous metastatic spread and organ fibrosis, including the kidney. This process is largely driven by transforming growth factor-beta and recent evidence has implicated Rho as a key intracellular signalling molecule. In this study we have used RNA interference to silence the genetically distinct Rho (A, B and C) isoforms to define their individual functions in human kidney epithelial cells undergoing EMT. We demonstrate that the downregulation of the epithelial cell marker E-cadherin is dependent upon the Rho effector, Rho-kinase. However, silencing RhoA or RhoC expression also results in E-cadherin loss, though each by different mechanisms. Loss of RhoA leads to an upregulation of Snail1 and a reduction in the transcription of E-cadherin whereas loss of RhoC upregulates its breakdown via proteasomal degradation. During EMT, the upregulation of alpha-smooth muscle actin can be blocked by inhibiting the expression of RhoA, but not by that of RhoB or RhoC. This effect is independent of Rho-kinase activity. RhoC is the isoform solely responsible for stress fibre formation and inhibiting its expression reduces EMT-induced migration by 50%. RhoB appears to play a role in cell survival as inhibiting its expression leads to >300% increase in cell apoptosis and a relocalization of focal adhesion kinase. We conclude that Rho is a key signalling molecule in the process of EMT but that each isoform has a distinct and specific role.

UNLABELLED: A high incidence of tumor recurrence and metastasis has been reported in hepatocellular carcinoma (HCC) patients; however, the underlying molecular mechanisms are largely unknown. In the present study a novel metastasis-related gene, eukaryotic initiation factor 5A2 (EIF5A2), was characterized for its role in HCC metastasis and underlying molecular mechanisms. Overexpression of EIF5A2 messenger RNA (mRNA) was detected in 50/81 (61.7%) of HCCs, which was significantly higher than those in nontumorous liver tissues. Compared with matched primary HCC, higher expression of EIF5A2 protein was observed in 25/47 (53.2%) of metastatic tumors. Functional studies found that ectopic expression of EIF5A2 could enhance cancer cell migration and invasion in vitro and tumor metastasis in vivo in an experimental mouse model. Moreover, inhibition of EIF5A by small interfering RNA (siRNA) or deoxyhypusine synthase (DHPS) inhibitor GC7, which inhibits EIF5A2 maturation, could effectively decrease cell motility. Further study found that EIF5A2 was able to induce epithelial-mesenchymal transition (EMT), a key event in tumor invasion and metastasis, characterized by down-regulation of epithelial markers (E-cadherin and beta-catenin) and up-regulation of mesenchymal markers (fibronectin, N-cadherin, alpha-SMA, and vimentin). In addition, EIF5A2 could also activate RhoA/Rac1 to stimulate the formation of stress fiber and lamellipodia. CONCLUSION: EIF5A2 plays an important role in HCC invasion and metastasis by inducing EMT, as well as stimulating cytoskeleton rearrangement through activation of RhoA and Rac1.

The interstitial chromosome (chr.) 1q21-q22 region is frequently amplified in human cancers, where it has been reported to carry prognostic significance for patients. We attempted to delineate chr. 1q21-q22 for affected gene(s) in hepatocellular carcinoma (HCC) by array-CGH and detected copy number gains of rho-guanine nucleotide exchange factor-H1 (GEF-H1) as most significant event. Gene expression evaluation in the HCC cohort indicated common up-regulations of GEF-H1 in 64% tumours compared to adjacent non-tumoural liver (64/100; paired t-test p < 0.0001). Moreover, GEF-H1 over-expressions correlated with microvascular invasion and advanced-stage tumours (p < 0.05). High GEF-H1 levels also predict shorter disease-free and overall survival of HCC patients (p < 0.03). Functional knock-down of GEF-H1 by RNAi indicated marked reduction in cell invasion through matrigel and an inhibition of cell migration (p < 0.035), but an effect on cell viability was not apparent. More interestingly, a mesenchymal-epithelial transition (MET) was readily observed in GEF-H1 knock-down cells, where a concomitant re-expression of epithelial markers (E-cadherin and cytokeratin 18) and cell adhesion proteins (alpha-catenin and gamma-catenin) was found but down-regulation of mesenchymal features (N-cadherin, vimentin and fibronectin). This phenotype was accompanied by reduced filamentous actin polymerizations and diminution of the stress fibre formation. In addition, reduced active form of GTP-RhoA, together with its downstream effectors, including cleaved ROCK1 and phosphorylated MLC2, were also detected in GEF-H1-depleted cells. Taken together, our findings underscore a potent oncogenic role for GEF-H1 in promoting the metastatic potentials of HCC, possibly through activation of RhoA signalling and the EMT phenomenon.CI - Copyright (c) 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Recently, an increasing number of studies have suggested that mTOR plays a critical role in the regulation of tumor cell motility, invasion and cancer metastasis. However, little is known about the signaling mechanisms in regulating epithelial-mesenchymal transition (EMT) of prostate cancer. In this study, we found that the expression levels of Raptor and Rictor in prostate cancer tissues were elevated, which may suggest that Raptor and Rictor signaling pathways are associated with prostate cancer progression and metastasis. Inhibition of mTORC1 or mTORC2 by knock down of Raptor or Rictor, respectively, migration and invasion of prostate cancer were attenuated. Furthermore, EMT, a characterized by the changed expression levels of various markers like E-cadherin, beta-catenin, N-cadherin, and vimentin emergend following inhibition of Raptor or Rictor. Finally, the small GTPases (RhoA and Rac1) which were crucial regulatory proteins in cell migration and invasion were inactivited after downregulating Raptor and Rictor. These results suggest that mTOR regulate EMT at least in part by down regulation of RhoA and Rac1 signaling pathways. Our findings provide novel very attractive target strategies that the inhibition of mTOR signaling pathways may retard prostate cancer migration and invasion at early stages.CI - (c) 2014 Wiley Periodicals, Inc.

F-box and WD repeat domain-containing7 (Fbxw7), a member of the F-box family of proteins, which are components of an E3 ubiquitin ligase complex, plays an important role as a general tumor suppressor in regulating the effects of various oncoproteins. Recently, accumulating studies have shown that Fbxw7 plays an important role in tumor cell motility, invasion and cancer metastasis. However, little is known about the signaling mechanisms that regulate tumor apoptosis, growth arrest and the epithelial-to-mesenchymal transition (EMT) in gastric cancer. In our study, we confirmed that Fbxw7 expression was decreased in gastric cancer tissues, and that Fbxw7 inhibited gastric cancer progression by inducing apoptosis and growth arrest. Furthermore, gastric cancer migration and invasion were decreased or increased following Fbxw7 overexpression or knockdown, respectively, and the expressions of various EMT markers, such as E-cadherin, N-cadherin and vimentin, were altered after Fbxw7 inhibition or overexpression. Furthermore, we demonstrated that Fbxw7 inhibits the EMT via the down-regulation of Snail 1 and ZEB 1, which are upstream transcription factors that promote this process. Additionally, RhoA showed higher expression in the same gastric cancer tissues than in normal tumor-adjacent samples. We found that Fbxw7 expression was negatively correlated with RhoA protein expression in gastric cancer tissues based on Pearson's correlation coefficient analysis. Moreover, we found that RhoA protein abundance was regulated by Fbxw7 via ubiquitination and proteasomal degradation in gastric cancer. We further demonstrated the effects of RhoA re-expression or inhibition on stable Fbxw7-overexpressing or Fbxw7-silenced cell lines in vitro and in vivo. These results suggest that Fbxw7 induces apoptosis and growth arrest and inhibits the EMT in part by down-regulating the RhoA signaling pathway.CI - Copyright (c) 2015 Elsevier Ireland Ltd. All rights reserved.

epithelial-mesenchymal transition (EMT) is one of the decisive steps regulating cancer invasion and metastasis. However, the molecular mechanisms underlying this transition require further clarification. MDA-9/syntenin (SDCBP) expression is elevated in breast cancer patient samples as well as cultured breast cancer cells. Silencing expression of MDA-9 in mesenchymal metastatic breast cancer cells triggered a change in cell morphology in both 2D- and 3D-cultures to a more epithelial-like phenotype, along with changes in EMT markers, cytoskeletal rearrangement and decreased invasion. Conversely, over expressing MDA-9 in epithelial non-metastatic breast cancer cells instigated a change in morphology to a more mesenchymal phenotype with corresponding changes in EMT markers, cytoskeletal rearrangement and an increase in invasion. We also found that MDA-9 upregulated active levels of known modulators of EMT, the small GTPases RhoA and Cdc42, via TGFbeta1. Reintroducing TGFbeta1 in MDA-9 silenced cells restored active RhoA and cdc42 levels, modulated cytoskeletal rearrangement and increased invasion. We further determined that MDA-9 interacts with TGFbeta1 via its PDZ1 domain. Finally, in vivo studies demonstrated that silencing the expression of MDA-9 resulted in decreased lung metastasis and TGFbeta1 re-expression partially restored lung metastases. Our findings provide evidence for the relevance of MDA-9 in mediating EMT in breast cancer and support the potential of MDA-9 as a therapeutic target against metastatic disease.

BACKGROUND: Pancreatic cancer, one of the top two most fatal cancers, is characterized by a desmoplastic reaction that creates a dense microenvironment, promoting hypoxia and inducing the epithelial-to-mesenchymal transition (EMT) to facilitate invasion and metastasis. Recent evidence indicates that the long noncoding RNA NORAD may be a potential oncogenic gene and that this lncRNA is significantly upregulated during hypoxia. However, the overall biological role and clinical significance of NORAD remains largely unknown. METHODS: NORAD expression was measured in 33 paired cancerous and noncancerous tissue samples by real-time PCR. The effects of NORAD on pancreatic cancer cells were studied by overexpression and knockdown in vitro. Insights into the mechanism of competitive endogenous RNAs (ceRNAs) were gained from bioinformatics analyses and luciferase assays. In vivo, metastatic potential was identified using an orthotopic model of PDAC and quantified using bioluminescent signals. Alterations in RhoA expression and EMT levels were identified and verified by immunohistochemistry and Western blotting. RESULTS: NORAD is highly expressed in pancreatic cancer tissues and upregulated in hypoxic conditions. NORAD upregulation is correlated with shorter overall survival in pancreatic cancer patients. Furthermore, NORAD overexpression promoted the migration and invasion of pancreatic carcinoma cells, while NORAD depletion inhibited EMT and metastasis in vitro and in vivo. In particular, NORAD may function as a ceRNA to regulate the expression of the small GTP binding protein RhoA through competition for hsa-miR-125a-3p, thereby promoting EMT. CONCLUSIONS: Elevated expression of NORAD in pancreatic cancer tissues is linked to poor prognosis and may confer a malignant phenotype upon tumor cells. NORAD may function as a ceRNA to regulate the expression of the small GTP binding protein RhoA through competition for hsa-miR-125a-3p. This finding may contribute to a better understanding of the role played by lncRNAs in hypoxia-induced EMT and provide a potential novel diagnostic and therapeutic target for pancreatic cancer.

Upregulated gene 11 (URG11) represents a gene upregulated by hepatitis B virus X protein and is involved in the biological processes of multifarious tumors. The present study aimed to investigate the protective effects and regulatory mechanisms of URG11 in benign prostatic hyperplasia (BPH). URG11, Ras homolog family member A (RhoA) and Rhoassociated protein kinase 1 (ROCK1) expression was detected in patients with BPH using reverse transcriptionquantitative polymerase chain reaction (RTqPCR). Furthermore, URG11 expression was silenced using URG11targeting small interfering RNAs. In addition, cell viability was determined by performing a Cell Counting Kit8 assay, and the effect of URG11 on the cell cycle was investigated by flow cytometry. expression levels of cyclin D1, p27, Ecadherin, Ncadherin, vimentin, RhoA and ROCK1 were investigated by RTqPCR and western blotting. The results revealed that the expression levels of URG11, RhoA and ROCK1 were enhanced in patients with BPH1 cells compared with matched healthy controls. Furthermore, it was demonstrated that transforming growth factorbeta (TGFbeta) induced the proliferation of BPH1 cells in vitro, and silencing of URG11 inhibited the effects of TGFbeta on BPH1 cell proliferation and the cell cycle. In addition, silencing of URG11 altered the expression levels of cell cycleassociated genes, epithelialmesenchymal transitionassociated genes, and RhoA and ROCK1 protein levels. Thus, the results of the present study suggest that URG11 may be a potential therapeutic target, which may be important to inhibit the development and progression of prostatic hyperplasia.

The serum response factor (SRF) regulates the transcription of target genes by binding to serum response elements in dimeric form and by interacting with ternary complex factors. In this study, we have analyzed the role of the serum response factor and mechanisms that regulate its activity in tumor progression utilizing a multistage model of mouse skin carcinogenesis. We demonstrate elevated SRF DNA binding activity only in the cell lines that have undergone an epithelial to mesenchymal transition and have increased actin stress fiber formation. Transient transfection experiments of activated or dominant negative forms of RhoA showed that the high activity of SRF and the induced formation of actin stress fibers in cells with spindle morphology were mediated by RhoA signaling. A dominant negative form of SRF inhibited RhoA-induced actin polymerization and stress fiber formation. The DNA binding activity of SRF in mesenchymal tumor cells was also correlated with elevated expression of SRF target genes, similar to SRF itself, actin, and vinculin. These observations suggest for the first time that SRF may play an important role in tumor progression, specifically at the transition to an invasive metastatic stage of carcinogenesis.

Molecular and cellular mechanisms of epithelial-mesenchymal transition (EMT), crucial in development and pathogenesis, are still poorly understood. Here we provide evidence that distinct cellular steps of EMT occur sequentially during gastrulation. Basement membrane (BM) breakdown is the first recognizable step and is controlled by loss of basally localized RhoA activity and its activator neuroepithelial-transforming-protein-1 (Net1). Failure of RhoA downregulation during EMT leads to BM retention and reduction of its activity in normal epithelium leads to BM breakdown. We also show that this is in part mediated by RhoA-regulated basal microtubule stability. Microtubule disruption causes BM breakdown and its stabilization results in BM retention. We propose that loss of Net1 before EMT reduces basal RhoA activity and destabilizes basal microtubules, causing disruption of epithelial cell-BM interaction and subsequently, breakdown of the BM.

epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) is a key process of peritoneal fibrosis. Rapamycin has been previously shown to inhibit EMT of PMCs and prevent peritoneal fibrosis. In this study, we investigated the undefined molecular mechanisms by which rapamycin inhibits EMT of PMCs. To define the protective effect of rapamycin, we initially used a rat PD model which was daily infused with 20 mL of 4.25% high glucose (HG) dialysis solution for 6 weeks to induce fibrosis. The HG rats showed decreased ultrafiltration volume and obvious fibroproliferative response, with markedly increased peritoneal thickness and higher expression of alpha-smooth muscle actin (alpha-SMA) and transforming growth factor-beta1. Rapamycin significantly ameliorated those pathological changes. Next, we treated rat PMCs with HG to induce EMT and/or rapamycin for indicated time. Rapamycin significantly inhibited HG-induced EMT, which manifests as increased expression of alpha-SMA, fibronectin, and collagen I, decreased expression of E-cadherin, and increased mobility. HG increased the phosphorylation of PI3K, Akt, and mTOR. Importantly, rapamycin inhibits the RhoA, Rac1, and Cdc42 activated by HG. Moreover, rapamycin repaired the pattern of F-actin distribution induced by HG, reducing the formation of stress fiber, focal adhesion, lamellipodia, and filopodia. Thus, rapamycin shows an obvious protective effect on HG-induced EMT, by inhibiting the activation of Rho GTPases (RhoA, Rac1, and Cdc42).CI - (c) 2016 Federation of European Biochemical Societies.