DLC‐1 suppresses non‐small cell lung cancer growth and invasion by RhoGAP‐dependent and independent mechanisms
Kevin D. HealyLouis HodgsonTai Young KimAdam ShutesSavitri MaddiletiRudolph L. JulianoKlaus M. HahnT. Kendall HardenYung‐Jue BangChanning J. Der
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Abstract Expression of the tumor suppressor deleted in liver cancer‐1 (DLC‐1) is lost in non‐small cell lung (NSCLC) and other human carcinomas, and ectopic DLC‐1 expression dramatically reduces proliferation and tumorigenicity. DLC‐1 is a multi‐domain protein that includes a Rho GTPase activating protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC‐1 in tumor suppression, we performed biochemical and biological studies evaluating DLC‐1 in NSCLC. Full‐length DLC‐1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5‐ to 20‐fold enhanced activity for RhoA, RhoB, RhoC, and Cdc42. DLC‐1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC‐1 expression in DLC‐1‐deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC‐1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC‐1 expression in NSCLC cell lines impaired both anchorage‐dependent and ‐independent growth, as well as invasion in vitro. Surprisingly, we found that the anti‐tumor activity of DLC‐1 was due to both RhoGAP‐dependent and ‐independent activities. Unlike the rat homologue p122RhoGAP, DLC‐1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C‐δ1. Combined, these studies provide information on the mechanism of DLC‐1 function and regulation, and further support the role of DLC‐1 tumor suppression in NSCLC. © 2007 Wiley‐Liss, Inc.Keywords:
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Objective:To detect Rho family(RhoA,RhoB,RhoC) mRNA expression and study the role of Rho family in endometriosis.Method: Quantified RT-PCR technique was used in the detection.Result: RhoA and RhoC mRNA expression in endometriosis was lower than that in normal endometrium markedly(P0.05,vs normal).RhoB mRNA expression didn't show any distinction between the groups.Conclusion: Endometriosis may be related to the low expression of RhoA and RhoC.
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Objective To investigate changes of expressions of RhoA and RhoB and RhoC in lung cancertissues to explore the relationship of Rho subfamily with development,migration,and metastasis of lung carcinoma.MethodsThe expressions of RhoA,RhoB and RhoC mRNA and protein in four different lung cancer cell lines(LTEP-a-2,SK-MES-1,NCI-H446,and NCI-H292)were detected by real-time quantitative reverse transcriptionpolymerase chain reaction(QRT-PCR) and Western blotting, and were compared with MRC-5(control group). Theexpressions of RhoA,RhoB and RhoC protein in 45 cases of lung cancer tissue by immunocytochemistry.Results The expressions of RhoA and RhoB mRNA and protein in lung cancer cell line LTEP-a-2,SK-MES-1,NCI-H446,and NCI-H292 were significantly higher than those in control group(allP0.05),while no statisticaldifference was found among four lung cancer cell lines(allP0.05). The expressions of RhoA and RhoB protein inhuman lung cancer tissue were positive,but the levels of them were not statistically different between moderatelydifferentiated squamous carcinoma group and non-moderately differentiated squamous carcinoma group(allP0.05).The expression of RhoC protein was negative.ConclusionThe expressions of RhoA and RhoB were elevated inlung cancer,which indicates that Rho subfamily involve in the development,migration and metastasis of lungcancer.
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Although Rho family GTPases RhoA, RhoB and RhoC share more than 85% amino acid sequence identity, they may play distinct roles in tumor progression. RhoA and RhoC have been suggested to have positive effects on tumor progression, but the role of RhoB in cancer, particularly in gastric cancer, remains unclear. In our study, we have examined the expression levels of these three Rho GTPases in a large panel of specimens from gastric cancer patients by immunohistochemistry. We found that RhoA and RhoC expression were significantly elevated, while RhoB was reduced or absent, in surgically removed gastric cancer tissues when compared to normal gastric tissues. The significant reduction of RhoB expression was confirmed in another group of gastric cancer samples in comparison to the adjacent non-neoplastic tissues. Then we transfected the plasmids containing RhoA, RhoB or RhoC cDNA into two gastric cancer cell lines, SGC7901 and AGS cells, respectively. By overexpression experiments, we found that RhoA promoted the gastric cancer cell proliferation and RhoC stimulated migration and invasion of the cancer cell. RhoB expression, however, significantly inhibited the proliferation, migration and invasion of the gastric cancer cells and also enhanced the chemosensitivity of these cells to anticancer drugs. It appears that RhoB plays an opposing role from that of RhoA and/or RhoC in gastric cancer cells. Our work suggests that RhoB may play a tumor suppressor role and subsequently may have potential implications in future targeted therapy.
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To investigate the expression of RhoA, RhoB, RhoC, Rac1 and Cdc42 kinases in urothelial cell carcinoma (UCC) of the urinary bladder and determine the expression profile of 107 Rho-associated genes, including GTPases, GDIs, GAPs and GEFs.Rho expression was investigated using microarrays, qPCR and Western blotting in 77 UCC specimens with paired normal urothelium. Computational analysis was also performed on Gene Expression Omnibus datasets. Further microarray analysis was carried out for the expression profiling of the Rho-associated genes.RhoB mRNA and protein levels were significantly lower in UCC, suggesting a tumour-suppressor role. On the contrary, mRNA of RhoC and protein levels of RhoA, RhoC and Cdc42, respectively, were significantly higher in UCC vs. normal tissue. High Cdc42 mRNA levels correlated with worse overall survival (p=0.027), whereas high RhoB mRNA levels correlated both with better overall (p=0.0258) and cancer-specific (p=0.0272) survival. Computational analysis verified the expression profile of Rho kinases among superficial UCCs, muscle-invasive UCCs and normal tissues.The majority of the Rho-related genes showed over-expression in UCC vs. normal tissue. Alterations in RhoA, RhoB, RhoC, Rac1 and Cdc42 expression play a significant role in the genesis and progression of UCC of the urinary bladder.
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Summary Rho GTPases are well known to regulate cell motility through activation of a variety of downstream effector proteins, including enzymes, adaptor proteins and actin nucleators. The three closely related Rho GTPases RhoA, RhoB and RhoC all have the potential to interact with the same downstream effectors, yet they have substantially different effects on cell shape and migratory properties. Here I review the different ways in which RhoA, RhoB and RhoC expression is regulated in cancer and how they play distinct roles in cancer progression. I describe their main effectors known to contribute to cell motility. Recent results from our laboratory and others indicate that RhoA, RhoB and RhoC can be activated by specific stimuli and act through different effectors to control distinct aspects of cancer cell migration and invasion. This suggests that they each make unique contributions to cancer by participating in different protein complexes.
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The Ras homolog (Rho) family of small GTPases comprise several proteins with prominent roles in regulation of cell cycle transition, cell migration, and remodeling of actin cytoskeleton. Expression of these proteins is regulated by several factors among them are long non-coding RNAs (lncRNAs). The impact of lncRNAs on Rho GTPases signaling can be exerted through direct modulation of expression of these proteins or influencing expression of miRNAs that negatively regulate Rho GTPases. LINC00974/miR-122/RhoA, MALAT1/miR-429/RhoA, ZFAS1/miR-3924/RhoA/ROCK2, PCAT6/miR-326/RhoA/ROCK, SMILR/miR-141/RhoA/ROCK, DAPK1/miR-182/RhoA, GAS5/miR663a/RhoB, H19/miR-15b/CDC42/PAK1, TDRG1/miR-93/RhoC, TUG1/miR-498/CDC42, UCA1/miR-18a/Cdc42 and UCA1/miR-182/Cdc42 are examples of lncRNAs/miRNAs axes that regulate Rho GTPases. In the present manuscript, we describe the role of lncRNAs on Rho GTPases.
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Inflammatory breast carcinoma (IBC) is a highly aggressive form of locally advanced breast cancer that has the ability to invade and block the dermal lymphatics of the skin overlying the breast. In a previous series of studies, our laboratory identified overexpression of RhoC GTPase in >90% of IBCs (K. L. van Golen et al., Clin. Cancer Res., 5: 2511-2519, 1999) and defined RhoC as a mammary oncogene involved in conferring the metastatic phenotype (K. L. van Golen et al., Cancer Res., 60: 5832-5838, 2000). RhoC GTPase is involved in cytoskeletal reorganization during cellular motility. Farnesyl transferase inhibitors (FTIs) were previously shown to be effective in modulating tumor growth in Ras-transformed tumor cells. Recently, studies have focused on RhoB as a putative non-Ras target of FTI action. In the present study, we assessed the effect of the FTI L-744,832 on RhoC-overexpressing IBC and RhoC-transfected human mammary epithelial (HME-RhoC) cells. Treatment of the SUM149 IBC cell line and HME-RhoC transfectants with the FTI L-744,832 led to reversion of the RhoC-induced phenotype, manifested by a significant decrease in anchorage-independent growth, motility, and invasion. Although RhoC expression and activation were not affected, RhoB levels were increased by FTI treatment. Transient transfection of geranylgeranylated RhoB (RhoB-GG) into the same cells reproduced the effects of the FTI, thus suggesting that FTI-induced reversion of the RhoC phenotype may be mediated by an increase in RhoB-GG levels. These data provide direct evidence that FTIs may find use in the clinic when directed against RhoC-overexpressing tumors and suggest appropriate biological markers to evaluate during FTI treatment.
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The three Rho subfamily members, RhoA, RhoB, and RhoC, share 85% sequence identity, yet display opposite phenotypes. Whereas RhoA and RhoC proteins promote cellular proliferation, invasion, metastasis, and human cancers, RhoB hinders oncogenesis. How such similar proteins can have such opposing functions is not clearly defined. HMG-CoA reductase inhibitors (statins), typically prescribed for their cholesterol-lowering properties, were additionally found to lower rates of various cancers. Rho proteins were hypothesized to contribute to this anti- tumorigenic effect given that one of the pleiotropic effects of statins is inhibition of Rho prenylation. In our experiments, we found that RhoA and RhoC were more sensitive to the statin-induced decline in prenylation than RhoB, likely due to the ability of RhoB to be additionally farnesylated. In addition, we found that RhoA, RhoB, and RhoC activation and expression were greatly increased in lovastatin-treated cells. An increase in activated, GTP-bound Rho occurred even when RhoA and RhoC were completely unprenylated. This increase in Rho activation was at least partly explained by the decreased ability of unprenylated Rho to associate with its Rho- dissociation inhibitor, RhoGDI\[alpha\]. An increase in activated, yet unprenylated, Rho in response to statins could lead to a potential gain-of-function or a loss-of- function for Rho, and we hypothesized that this might explain how statins provide their anti-oncogenic properties. However, many statin-induced effects on proliferation, transcription, apoptosis, and polyploidy could not be explained by a change in Rho function and we found no evidence that the lovastatin-induced increase in RhoB contributed significantly to the anti-proliferative and pro-apoptotic effects of lovastatin in human leukemia cells. We used a proteomics approach to search for Rho interacting factors with differential binding affinity for RhoA/C and RhoB. Through the use of affinity chromatography and mass spectrometry, we identified a novel RhoA and RhoC isoform-specific interacting protein, IQGAP1. The binding to IQGAP1 was found to be dependent on the activation and prenylation state of Rho. Like RhoA and RhoC, IQGAP1 has many pro-proliferative, pro-invasion, and pro-oncogenesis properties. The lack of RhoB association with IQGAP1 may explain in part how this Rho isoform differs so dramatically in its function from RhoA and C.
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To clarify the role of the Rho small GTP-binding protein (Rho) and its major downstream target, ROCK (Rho-associated serine-threonine protein kinase), in progression of renal cell carcinoma (RCC), we examined mRNA expression for Rho and ROCK genes in surgical specimen of RCC tissues from 78 Japanese patients and in the corresponding non-tumor tissues originating from the same patient using a real-time reverse transcription polymerase chain reaction (RT-PCR). Expression of mRNA for RhoA did not differ between tumor and non-tumor tissues. RhoB mRNA expression was higher in the tumor (P < 0.05), but expression was not associated with tumor grade, stage, or prognosis. However, degree of RhoC and ROCK mRNA expression was related to tumor grade (P < 0.05) and stage (P < 0.0001). A positive relationship was seen between expression of mRNA for RhoC and that for ROCK in tumor tissues (P < 0.0001). Kaplan-Meier plots showed high RhoC and ROCK mRNA expression to be negatively associated with overall survival (P < 0.0001). Multivariate analysis showed mRNA expression of RhoC and ROCK to be independent poor prognostic factors concerning overall survival. Our findings implicate the RhoC/ROCK pathway in carcinogenesis and progression of RCC, indicating that RhoC/ROCK may be a useful prognostic marker and a possible molecular target for treatment of the disease.
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