396

ARHGDIA

Rho GDP dissociation inhibitor (GDI) alpha;ARHGDIA;Rho GDP dissociation inhibitor (GDI) alpha

GDP-dissociation inhibitor, aplysia RAS-related 1|epididymis secretory sperm binding protein Li 47e|rho GDP-dissociation inhibitor 1

17q25.3

protein-coding

BACKGROUND: Estrogen receptor (ER) alpha is a successful therapeutic target in breast cancer, but patients eventually develop resistance to antiestrogens such as tamoxifen. METHODS: To identify genes whose expression was associated with the development of tamoxifen resistance and metastasis, we used microarrays to compare gene expression in four primary tumors from tamoxifen-treated patients whose breast cancers did not recur vs five metastatic tumors from patients whose cancers progressed during adjuvant tamoxifen treatment. Because Rho guanine dissociation inhibitor (GDI) alpha was underexpressed in the tamoxifen-resistant group, we stably transfected ERalpha-positive MCF-7 breast cancer cells with a plasmid encoding a short hairpin (sh) RNA to silence Rho GDIalpha expression. We used immunoblots and transcription assays to examine the role of Rho GDIalpha in ER-related signaling and growth of cells in vitro and as xenografts in treated nude mice (n = 8-9 per group) to examine the effects of Rho GDIalpha blockade on hormone responsiveness and metastatic behavior. The time to tumor tripling as the time in weeks from randomization to a threefold increase in total tumor volume over baseline was examined in treated mice. The associations of Rho GDIalpha and MTA2 levels with tamoxifen resistance were examined in microarray data from patients. All statistical tests were two-sided. RESULTS: Rho GDIalpha was expressed at lower levels in ERalpha-positive tumors that recurred during tamoxifen treatment than in ERalpha-positive tamoxifen-sensitive primary tumors. MCF-7 breast cancer cells in which Rho GDIalpha expression had been silenced were tamoxifen-resistant, had increased Rho GTPase and p21-activated kinase 1 activity, increased phosphorylation of ERalpha at serine 305, and enhanced tamoxifen-induced ERalpha transcriptional activity compared with control cells. MCF-7 cells in which Rho GDIalpha expression was silenced metastasized with high frequency when grown as tumor xenografts. When mice were treated with estrogen or estrogen withdrawal, tripling times for xenografts from cells with Rho GDIalpha silencing were similar to those from vector-containing control cells; however, tripling times were statistically significantly faster than control when mice were treated with tamoxifen (median tripling time for tumors with Rho GDIalpha small interfering RNA = 2.34 weeks; for control tumors = not reached, hazard ratio = 4.13, 95% confidence interval = 1.07 to 15.96, P = .040 [adjusted for multiple comparisons, P = .119]). Levels of the metastasis-associated protein MTA2 were also increased upon Rho GDIalpha silencing, and combined Rho GDIalpha and MTA2 levels were associated with recurrence in 250 tamoxifen-treated patients. CONCLUSION: Loss of Rho GDIalpha enhances metastasis and resistance to tamoxifen via effects on both ERalpha and MTA2 in models of ERalpha-positive breast cancer and in tumors of tamoxifen-treated patients.

The GDP dissociation inhibitors (GDIs) are pivotal regulators of Rho GTPases, which are essential for tumor progression, particularly in the area of metastasis. One member of GDIs was identified as RhoGDI (Rho GDP-dissociation inhibitor alpha, or RhoGDIalpha), but little is known about this protein in tumors. In this study, we used comparative proteomic analysis to show that RhoGDI is markedly up-regulated in metastatic colorectal cancer (CRC). The elevated level of RhoGDI protein in metastatic CRC was confirmed by Western blot at the tissue ( n = 24) and cell ( n = 6) levels. Further, we analyzed RhoGDI protein expression in 126 clinicopathologically characterized CRC cases by immunohistochemistry. Statistical analysis showed that there were significant differences of RhoGDI overexpression in patients categorized according to tumor invasion ( p = 0.018), lymph node metastasis ( p = 0.001) and clinical stage ( p = 0.009). A trend was also identified between high expression of RhoGDI and shorter overall survival ( p = 0.013). In the present work, we also analyzed the effect of RhoGDI on CRC cell line. Gene transfection-mediated overexpression of RhoGDI in HT29 cells, containing a low detectable level of endogenous RhoGDI, resulted in a significant increase in cell proliferation and motility in vitro. These data suggest that RhoGDI may promote CRC progression and metastasis by stimulating tumor cell growth and migration.

To better understand the mechanism underlying colorectal carcinoma (CRC) genesis or metastasis, and to search for potential markers for CRC prognosis, a comparative proteomic analysis was performed on CRC tissue. Proteins were extracted from normal colorectal mucosa, non-metastatic CRC (nmCRC) and metastatic CRC (mCRC) tissue samples. Protein profiling of each sample was performed by two-dimensional electrophoresis coupled with MALDI-TOF MS, followed by confirmation by Western blotting. Thirty-one proteins were found to be differentially expressed between normal mucosa, nmCRC and mCRC tissue. In 126 paraffin-embedded CRC samples, three differentially expressed proteins, identified as LASP-1, S100A9 and RhoGDI by proteomic analysis, were detected by immunohistochemical staining to determine the clinicopathological characteristics of these proteins in CRC. Increased expression levels of these proteins were found in CRC, especially mCRC, compared with normal mucosa. The results provide the basis for searching for potential markers for CRC genesis and metastasis, and also provide clues for elucidating the mechanism of CRC progression. The pattern changes identified have the potential to be used for the design of marker panels for assistance in diagnostic and therapeutic strategies in CRC.

The Rho GDP dissociation inhibitor (RhoGDI) can bind to small GTPases and keep them in a biologically inactive state in cytoplasm, through which it affects actin polymerization and cell motility. However, mechanisms underlying how RhoGDI regulates Rho GTPase complex formation/membrane extraction/GTPase dissociation remain largely unexplored. Our previous studies reported that X-linked inhibitor of apoptosis protein (XIAP) interacted with RhoGDI via its RING domain and negatively modulated RhoGDI SUMOylation and HCT116 cancer cell migration. Here, we identified that RhoGDI SUMOylation specifically occurred at Lys-138, which was inhibited by XIAP domain. We further demonstrated that RhoGDI SUMOylation at Lys-138 was crucial for inhibiting actin polymerization and cytoskeleton formation as well as cancer cell motility. Moreover, SUMO-RhoGDI had a much higher binding affinity to small Rho GTPase compared with the un-SUMOylated form of RhoGDI. Taken together, our study demonstrated a novel modification of RhoGDI, SUMOylation at Lys-138, which played a key role in regulating Rho GTPase activation in cancer cells. The physiological regulation of RhoGDI SUMOylation by the RING domain of XIAP may account for modulation of cancer cell invasion and metastasis by XIAP.

Since metastasis is one of the most important prognostic factors in colorectal cancer, development of new methods to diagnose and prevent metastasis is highly desirable. However, the molecular mechanisms leading to the metastatic phenotype have not been well elucidated. In this study, a proteomics-based search was carried out for metastasis-related proteins in colorectal cancer by analyzing the differential expression of proteins in primary versus metastasis focus-derived colorectal tumor cells. Protein expression profiles were determined using a tissue microarray (TMA), and the results identified Rho GDP-dissociation inhibitor alpha (Rho GDI) as a metastasis-related protein in colon and prostate cancer patients. Consequently, Rho GDI may be useful as a diagnostic biomarker and/or a therapeutic to prevent colon and prostate cancer metastasis.

Recurrent chromosomal aberrations are often observed in hepatocellular carcinoma (HCC), but little is known about the functional non-coding sequences, particularly microRNAs (miRNAs), at the chromosomal breakpoints in HCC. Here we show that 22 miRNAs are often amplified or deleted in HCC. MicroRNA-151 (miR-151), a frequently amplified miRNA on 8q24.3, is correlated with intrahepatic metastasis of HCC. We further show that miR-151, which is often expressed together with its host gene FAK, encoding focal adhesion kinase, significantly increases HCC cell migration and invasion in vitro and in vivo, mainly through miR-151-5p, but not through miR-151-3p. Moreover, miR-151 exerts this function by directly targeting RhoGDIA, a putative metastasis suppressor in HCC, thus leading to the activation of Rac1, Cdc42 and Rho GTPases. In addition, miR-151 can function synergistically with FAK to enhance HCC cell motility and spreading. Thus, our findings indicate that chromosome gain of miR-151 is a crucial stimulus for tumour invasion and metastasis of HCC.

Recurrent chromosomal aberrations are often observed in hepatocellular carcinoma (HCC), but little is known about the functional non-coding sequences, particularly microRNAs (miRNAs), at the chromosomal breakpoints in HCC. Here we show that 22 miRNAs are often amplified or deleted in HCC. MicroRNA-151 (miR-151), a frequently amplified miRNA on 8q24.3, is correlated with intrahepatic metastasis of HCC. We further show that miR-151, which is often expressed together with its host gene FAK, encoding focal adhesion kinase, significantly increases HCC cell migration and invasion in vitro and in vivo, mainly through miR-151-5p, but not through miR-151-3p. Moreover, miR-151 exerts this function by directly targeting RhoGDIA, a putative metastasis suppressor in HCC, thus leading to the activation of Rac1, Cdc42 and Rho GTPases. In addition, miR-151 can function synergistically with FAK to enhance HCC cell motility and spreading. Thus, our findings indicate that chromosome gain of miR-151 is a crucial stimulus for tumour invasion and metastasis of HCC.

RhoGDI (Rho GDP-dissociation inhibitor alpha, or RhoGDIalpha) was identified as a regulator of Rho GTPases, but its role in cancer remains controversial. In this study, increased expression of RhoGDI was detected in hepatocellular carcinoma (HCC) cell lines and tissues with highly metastatic potential. RhoGDI overexpression correlated with postoperative distant metastasis. Enforced expression of RhoGDI in HCC cells significantly enhanced cell proliferation and migration. Conversely, knockdown of RhoGDI caused an inhibition of the aggressive phenotypes of HCC cells. Furthermore, RhoGDI up-regulated Rho, but not Rac, and enhanced PI3K/AKT and MAPK pathway activity. Our findings suggest that RhoGDI overexpression is a predictor of distant metastasis and plays an important role in the progression of HCC.

Rho GTPases control a wide range of cellular processes and contribute to tumor invasion and metastasis. As a regulator of Rho activity, ARHGDIA is aberrantly expressed in several types of tumors and plays different roles in the tumor process. To elucidate the role of ARHGDIA in HCC, we investigated the patterns of its expression, prognosis and clinical profiles in HCC. Functional assays were performed to investigate whether the alteration of ARHGDIA has an effect on cell growth, migration and invasion, as well as the status of Rho GTPases. We found that ARHGDIA was frequently downregulated in HCC and associated with tumor invasion and metastasis. Moreover, ARHGDIA was significantly associated with OS and TTR of HCC patients. Low level of ARHGDIA exhibited a decreased postoperative OS and a shorter TTR compared those with high levels. Functional assays showed that loss of ARHGDIA promoted HCC cell migration and invasion in vitro and lung metastasis formation in vivo. We found that loss of ARHGDIA significantly induced Rac1 and RhoA activation which may contribute to invasion and metastasis of HCC. In conclusion, the present study has identified loss of ARHGDIA contributed to the processes of hepatic tumorigenesis, in particular invasion and metastasis which may provide a potential therapeutic target for HCC.

The Rho GDP dissociation inhibitor (RhoGDI) can bind to small GTPases and keep them in a biologically inactive state in cytoplasm, through which it affects actin polymerization and cell motility. However, mechanisms underlying how RhoGDI regulates Rho GTPase complex formation/membrane extraction/GTPase dissociation remain largely unexplored. Our previous studies reported that X-linked inhibitor of apoptosis protein (XIAP) interacted with RhoGDI via its RING domain and negatively modulated RhoGDI SUMOylation and HCT116 cancer cell migration. Here, we identified that RhoGDI SUMOylation specifically occurred at Lys-138, which was inhibited by XIAP domain. We further demonstrated that RhoGDI SUMOylation at Lys-138 was crucial for inhibiting actin polymerization and cytoskeleton formation as well as cancer cell motility. Moreover, SUMO-RhoGDI had a much higher binding affinity to small Rho GTPase compared with the un-SUMOylated form of RhoGDI. Taken together, our study demonstrated a novel modification of RhoGDI, SUMOylation at Lys-138, which played a key role in regulating Rho GTPase activation in cancer cells. The physiological regulation of RhoGDI SUMOylation by the RING domain of XIAP may account for modulation of cancer cell invasion and metastasis by XIAP.

Since metastasis is one of the most important prognostic factors in colorectal cancer, development of new methods to diagnose and prevent metastasis is highly desirable. However, the molecular mechanisms leading to the metastatic phenotype have not been well elucidated. In this study, a proteomics-based search was carried out for metastasis-related proteins in colorectal cancer by analyzing the differential expression of proteins in primary versus metastasis focus-derived colorectal tumor cells. Protein expression profiles were determined using a tissue microarray (TMA), and the results identified Rho GDP-dissociation inhibitor alpha (Rho GDI) as a metastasis-related protein in colon and prostate cancer patients. Consequently, Rho GDI may be useful as a diagnostic biomarker and/or a therapeutic to prevent colon and prostate cancer metastasis.