RhoB Expression Research Articles

Simvastatin Inhibits Glucose‐Stimulated Vascular Smooth Muscle Cell Migration Involving Increased Expression of RhoB and a Block of Ras/Akt Signal

Diabetic patients are at high risk to develop atherosclerotic cardiovascular disease and have a higher restenotic rate after percutaneous coronary intervention (PCI). Statins improve cardiovascular outcome and reduce restenosis after PCI by inhibiting proliferation and migration of vascular smooth muscle cells (VSMCs). But the effect of statins on diabetes without dyslipidemia was still not fully understood. Our previous study has demonstrated that simvastatin inhibits VSMC proliferation in high glucose status without dyslipidemia, inducing a G0/G1 phase cell cycle growth arrest by acting on multiple steps upstream of pRb, including inhibition of CDK2/4 expression and upregulation of p53, p21, p16, and p27. Following our previous study, we investigated the mechanism of simvastatin inhibition of VSMC migration in a diabetes-like model (A7r5 cells under high glucose conditions without dyslipidemia). Under high glucose conditions, simvastatin dose-dependently inhibited VSMC migration, decreased PI3K/Akt pathway activity, reduced c-Raf and Ras expression, increased RhoB but not RhoA, Rac1, and Cdc2 expression, dose-dependently inhibited MMP-2, but not MMP-9, activity, and dose-dependently inhibited NF-κB activity. The inhibition of VSMC migration under high glucose conditions was via two different pathways. The first pathway is mevalonate-related but not RhoA protein-related and involves suppression of Ras and PI3K/Akt signals. The second pathway is not mevalonate-related and involves increasing RhoB expression directly.

Antitumor progression potential of caffeic acid phenethyl ester involving p75 NTR in C6 glioma cells

The previous data showed that caffeic acid phenethyl ester (CAPE), a component of propolis, possesses inducing cell cycle arrest and antiproliferation effect on C6 glioma cells in vitro and in vivo. In the present study, C6 glioma cells treated with CAPE resulted in morphological changes to an astrocytic phenotype and increased the expression of glial differentiation marker proteins including glial fibrillary acidic protein (GFAP) and S-100β. In addition, with scratch assay and Boyden chamber assay, CAPE exhibited inhibitory effects on the motility and invasion of C6 glioma cells. Furthermore, CAPE induced the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75 NTR), which were involved in neural cell differentiation. CAPE could also inhibit the activity of matrix metalloproteinases (MMPs) and induce the expression of RhoB, a tumor suppressor. To examine the involvement of p75 NTR in the anti-invasive property of CAPE, Western blotting and Boyden Chamber assay were performed by addition of an anti-p75 NTR antibody in C6 cells. The results showed that blocking p75 NTR could decrease the CAPE-induced expression of RhoB and the inactivation of MMP-2, -9 as well as the anti-invasion effect in C6 glioma cells. Furthermore, CAPE suppressed IκB-α phosphorylation which was down stream of p75 NTR. Finally, the effect of CAPE on metastasis by lung colonization of the tumor cell in nude mice was also evaluated. It was found that the groups of nude mice injected with CAPE-pretreated cells could decrease both lung size and weight as compared to the positive control group which did not receive CAPE treatment. In addition, histological examination of the mouse lung sections showed that the CAPE-treated group inhibited the metastasis of C6 glioma cells. These data suggest CAPE possesses antitumor progression potential.

JNK-mediated transcriptional upregulation of RhoB is critical for apoptosis of HCT-116 colon cancer cells by a novel diarylsulfonylurea derivative

Diarylsulfonylureas are potent antitumor agents that have been tested in clinical trials. However, detailed mechanisms of their apoptotic activity remain unclear. Here, we report a new diarylsulfonylurea derivative, LB2A, that upregulates RhoB, thereby inducing potent apoptosis in HCT-116 human colon cancer cells independently of p53 status. LB2A decreased procaspase-3, increased phospho-JNK, and cleaved PARP, leading to apoptosis of HCT-116 cells. Prior treatment of HCT-116 cells with the JNK inhibitor SP600125 and the RNA synthesis inhibitor DRB blocked apoptosis, implying that JNK activation and mRNA production are important for apoptosis by LB2A. Western blotting, RT-PCR, and RhoB-promoter luciferase reporter assays revealed that LB2A increased RhoB via JNK-mediated transcriptional activation. LB2A decreased HDAC1 and increased acetyl-H3, both of which activate the RhoB promoter and were blocked by SP600125. Ectopic expression of RhoB induced apoptosis of HCT-116 cells, suggesting that RhoB is critical for the anti-cancer activity of LB2A in human colon cancer cells. LB2A also exhibited potent tumor growth inhibition of HCT-116 cells in vivo using a mouse xenograft assay. Taken together, these results show that LB2A induces apoptosis of HCT-116 cells via JNK-mediated transcriptional upregulation of RhoB and may therefore provide a potential therapy for human colon cancer.

Expression of Ras homologous B protein in the human scalp skin and hair follicles: hair follicle cycle stages‐associated changes

RhoB belongs to the Ras homologous (Rho) subfamily which consists of low molecular weight mass GTP-binding proteins. Rho proteins are regulatory molecules that mediate changes in cell shape, contractility, motility and gene expression. To test the hypothesis that 'RhoB protein is expressed in the human skin and its expression undergoes hair follicle cycle dependent changes'. To test this hypothesis, we examined the expression of RhoB in the normal human skin and hair follicles (HFs) using immunohistochemical methods. A total of 50 normal human scalp skin specimens were obtained from 50 females (age: 53-57 years) undergoing elective cosmetic plastic surgery. The specimens were obtained from both frontal and temporal regions of the scalp. A total of 50 HF, (35 anagen, 10 catagen and 5 telogen) were examined in each case using immunohistologic staining methods. Semiquantitative analysis was done. RhoB protein was strongly expressed in the various elements of the human scalp skin and hair follicles. In the epidermis, a moderate RhoB immunoreactivity was found in all layers except stratum corneum where RhoB protein was completely absent. In sebaceous glands, a strong RhoB immunoreactivity was detected in all sebaceocytes. In the hair follicles, the expression of RhoB protein showed hair follicle cycle stages-associated changes, i.e. strong expression during anagen, but weak and completely absent expressions during catagen and telogen phases, respectively. Semiquantitative analysis revealed statistically significant high expression values (staining intensity, percentage of positive cells and immunoreactivity scores) in the anagen VI hair follicles compared to either cantagen or telogen ones (p < 0.05). Similarly, RhoB protein expression was significantly high in the stratum basale, stratum spinosum and sebaceous glands compared to stratum granulosum (p < 0.05). Here we report, for the first time, the distribution of RhoB protein in the human scalp skin and hair follicles. We also provide the first indication that there are variations in the expression of this protein in the different stages of the hair cycle.

Overexpression of miR-21 Promotes an In vitro Metastatic Phenotype by Targeting the Tumor Suppressor RHOB

Metastasis is a multistep process that involves the deregulation of oncogenes and tumor suppressors beyond changes required for primary tumor formation. RHOB is known to have tumor suppressor activity, and its knockdown is associated with more aggressive tumors as well as changes in cell shape, migration, and adhesion. This study shows that oncogenic microRNA, miR-21, represses RHOB expression by directly targeting the 3' untranslated region. Loss of miR-21 is associated with an elevation of RHOB in hepatocellular carcinoma cell lines Huh-7 and HepG2 and in the metastatic breast cancer cell line MDA-MB-231. Using in vitro models of distinct stages of metastasis, we showed that loss of miR-21 also causes a reduction in migration, invasion, and cell elongation. The reduction in migration and cell elongation can be mimicked by overexpression of RHOB. Furthermore, changes in miR-21 expression lead to alterations in matrix metalloproteinase-9 activity. Therefore, we conclude that miR-21 promotes multiple components of the metastatic phenotype in vitro by regulating several important tumor suppressors, including RHOB.

A distinct role of RhoB in gastric cancer suppression

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.

NSC126188, a piperazine alkyl derivative, induces apoptosis via upregulation of RhoB in HeLa cells

We describe here a piperazine alkyl derivative, NSC126188, which induced apoptosis of HeLa cells by upregulating RhoB expression. NSC126188 caused multi-septation of fission yeast and hypersensitized a ∆rho3 mutant, which implicates the involvement of functional human homolog RhoB. The treatment of cells with NSC126188 induced apoptosis and a dramatic increase in RhoB expression. In addition, RhoB knockdown using siRNA rescued cells from apoptosis, indicating a crucial role of RhoB in NSC126188-induced apoptosis. In a reporter assay using luciferase and EGFP under control of the RhoB promoter, NSC126188 increased both luciferase activity and the expression of EGFP, implicating transcriptional activation of RhoB by NSC126188. Furthermore, NSC126188 demonstrated in vivo anti-tumor activity, inhibiting tumor growth by 66.8% in a nude mouse xenograft using PC-3 human prostate cancer cells. These results suggest that NSC126188 is a potential lead compound and that upregulation of RhoB is associated with NSC126188-induced apoptosis.

Abstract 2049: Post transcriptional regulation of RhoB expression

Abstract Rho GTPases contribute to most steps of cancer initiation and progression, including the acquisition of unlimited proliferation potential, survival, tissue invasion and the establishment of metastases. By contrast to RhoA and RhoC, RhoB has been proposed to act as a tumor suppressor as it is activated in response to various stresses including UV exposure, and since it has been reported to inhibit tumor growth, cell migration and invasion. Unlike RhoA and RhoC, RhoB is often down regulated in human tumour tissues including lung and head and neck cancer and its expression is inversely correlated with tumor aggressiveness. However, the epigenetic regulation of RhoB expression in cancer cells remains to be elucidated. Since several recent works demonstrate that post-transcriptional deregulation of gene expression is involved in carcinogenesis, we have investigated the post-transcriptional regulation of RhoB expression. The RhoB 3’ untranslated region is well conserved between species and has numerous putative binding sites for post-transcriptional regulators such as mRNA binding proteins and microRNAs. By using luciferase assays and polysome analysis, we have found that the translation of the RhoB mRNA is regulated upon UV exposure. One specific miRNA, miR-19b whose expression is regulated by UV, directly targets the RhoB mRNA to control RhoB expression and UV-induced apoptosis. Interestingly, other post-transcriptionnal regulators involved in RhoB expression, such as the mRNA binding proteins HuR and TiaR were also indentified by functional assays using RNA interference strategy. Immunohistochemical analysis on a cohort of human lung tissues ranging from normal to invasive carcinoma are underway to detect a possible correlation between the expression of these proteins and RhoB downregulation in tumor. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2049.

Abstract 1451: Lipid-based Akt inhibitors preferentially kill lung cancer cells with mesenchymal features

Abstract Most solid tumors are epithelial in origin. A loss of epithelial-cell markers and gain of mesenchymal-cell markers (epithelial mesenchymal transition, EMT) has been observed in patient tumor samples, particularly at the leading edge or invasive front of solid tumors such as non-small cell lung cancer (NSCLC). Pathological EMT is often associated with tumor metastasis and can confer a poor prognosis. EMT is promoted by Akt activation, which contributes to the formation, maintenance, and therapeutic resistance of cancer. In this study, we found that lipid-based Akt inhibitors (LBAI) such as phosphatidylinositol ether lipid analogues (PIAs) and alkylphospholipids (perifosine and miltefosine) preferentially kill tumor cells with mesenchymal features. NSCLC cell lines that exhibited epithelial, intermediate and mesenchymal phenotypes (H322, A549 and H157, respectively) were increasingly sensitive to the cytotoxic effects of LBAI. Prolonged treatment of A549 cells with TGFbeta induced a mesenchymal phenotype and increased sensitivity to LBAI. Because EMT can be controlled by the tumor suppressor RhoB, which we previously showed was transcriptionally activated by LBAI, we investigated the role of RhoB in these cell lines. An inverse relationship between Akt activation and RhoB expression was observed in the parental cell lines as well as A549 cells treated with TGFbeta, which correlated with LBAI cytotoxicity. Stable knockdown of RhoB in A549 cells induced features of EMT including loss of E-cadherin expression, induction of vimentin expression and Akt activation, and increased cellular migration. Stable knockdown of RhoB also increased sensitivity to LBAI. These studies show that lung cancer cells with a mesenchymal phenotype are preferentially sensitivity to LBAI, and that RhoB is a critical mediator of EMT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1451.

Downregulation of RhoB GTPase confers resistance to cisplatin in human laryngeal carcinoma cells

Acquired resistance to cisplatin represents a major obstacle to an efficient chemotherapy. We found downregulation of RhoB expression in cisplatin-resistant tumor cell lines from different origin. In cisplatin-resistant laryngeal carcinoma subline overexpression of farnesylated or geranylgeranylated RhoB increased cisplatin-induced cell death, while silencing of RhoB expression diminished sensitivity of parental HEp-2 cells via decreased cellular accumulation of cisplatin. However, since RhoB silencing in additional tumor cell lines did not alter their sensitivity to cisplatin, we can assume that RhoB downregulation does not provide general protective role in cell response to cisplatin. Nevertheless, gene therapy involving restoration of RhoB expression might improve the efficiency of cisplatin treatment, especially in patients with laryngeal carcinoma that acquired resistance to this chemotherapeutic drug.

Correlation between RhoB and Estrogen Receptor α (ERα) Expressions: From Experimental Data to Human Breast Tumors.

Abstract Introduction: Hormonotherapy (HT) of breast cancers (BC) is strongly limitated by the resistances. Because Rho proteins are key elements in the cross-talks between ERα and growth factors signaling, they may be involved in the response to HT. RhoB could play a negative role in oncogenesis but there is no valid data regarding RhoB expression in BC. Experimental results: Our data suggest that RhoB participates in the balance of expression of ERα. The inhibition of the expression of RhoB using two sequences of interfering RNA in hormonodependent cell lines (MCF-7, T47D, ZR75) and in anti-estrogen resistant cell lines (LCC2, LCC9), decreases the expression of ERα both at the protein and mRNA levels. Moreover, in Mouse Embryonic Fibroblasts and uteri collected from RhoB knock-out mice, a dramatic decrease of ERα expression is observed. We therefore investigated the expression level of RhoB and ERα in BC tumors. Patients and methods: A tissue microarray (TMA) was constructed from a cohort of 113 patients (pts) enrolled in a randomized trial for adjuvant tamoxifen (median follow-up: 249.9 months). RhoB, ERα, and PR expressions were measured by immunochemistry. Cut-off used for ERα and PR + was 10% of stained cells. The expression of RhoB was calculated with the IRS score. Correlation of RhoB expression score with clinically diagnostic and prognostic variables was assessed using Man Whitney and Spearman's rank tests. Univariate survival analysis was performed for disease free survival (DFS) by applying the log-rank test to RhoB expression levels stratified by median value. TMA results: 65 (58.6%) were grade I-II; 74(66.1%) were ERα+ and 59(52.7%) PR+; 23 (22.1%) presented lymphovascular (LV) invasion. 39 pts (34.8%) had lymph nodes (LN) +. Pts under tamoxifen (n=62) had a less favorable pathological profile regarding + LN (p=0.0039), pathological tumour size (p=0.0486) and number of mitoses (p=0.0556). Age, ERα/PR status, histological grade or type and LV invasion status were similar in the two groups. We found less RhoB IRS expression in pts with tumor grade III (median 8 [1;12]) than in grade I-II (median 9 [3;12], p=0.0142). RhoB IRS expression was much higher in ERa + tumors (median:10.5 [3;12]) than in ERa negative tumors (median=8 [1;12], p=0.0023). RhoB IRS score was not correlated with the presence or not of LV invasion (p=0.26), neither with the presence of LN invasion (p= 0.74). Furthermore, RhoB expression is i) strongly correlated with the % of expression of ERα (Spearman'sρ=0.3659; p = 0.001) and PR (Spearman's ρ=0.2544 ; p=0.0076) ii) inversely correlated with histologic size (Spearman's ρ= -0.2344 ; p = 0.0166) and with number of mitose (Spearman's ρ=-0.2009 ; p=0.0362). The DFS of pts with ERα + tumors under tamoxifen or not was not affected by the level of RhoB expression. Conclusion: The analysis of 113 human breast tumors allowed to confirm experimental results, demonstrating that RhoB expression is strongly correlated with ERα expression. The role of RhoB as a potential suppressor gene of tumours in BC and its role in the response to HT have then to be investigated further. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5146.

Immunohistological Profile of the Ras Homologous B Protein (RhoB) in Human Testes Showing Normal Spermatogenesis, Spermatogenic Arrest and Sertoli Cell Only Syndrome

Ras homologous B protein (RhoB) belongs to the Ras homologous subfamily which consists of low molecular weight (21kDa) GTP-binding proteins. Rho proteins are regulatory molecules associated with various kinases and as such they mediate changes in cell shape, contractility, motility and gene expression. To date, no data are available about the expression pattern of RhoB protein in the human testis showing normal and abnormal spermatogenesis. The present study addresses these issues. Human testicular biopsy specimens were obtained from patients suffering from post-testicular infertility (testis showing normal spermatogenesis, 10 cases) and testicular infertility (testis showing Sertoli cell only syndrome and spermatogenic arrest, 10 patients each). The expression of RhoB was examined using in situ immunofluorescent staining methods. In testes showing normal spermatogenesis, RhoB had a strong expression in the seminiferous epithelium (cytoplasm of Sertoli-cells, spermatogonia and spermatocytes) and in the interstitium (Leydig cells). RhoB expression was weak in the myofibroblasts and absent in the spermatids and sperms. In the testes showing abnormal spermatogenesis, RhoB expression was moderate in the seminiferous epithelium (cytoplasm of Sertoli cells, spermatogonia and spermatocytes) and was completely absent in the Leydig cells, myofibroblasts, spermatids and sperms. To the best of our knowledge, this study provides the first morphological indication that RhoB protein is expressed in human testis and its expression undergoes testicular infertility associated changes. These findings suggest the involvement of RhoB in the process of spermatogenesis in human and their possible therapeutic ramifications in testicular infertility are open for further investigations.

Increase of RhoB in γ-radiation-induced apoptosis is regulated by c-Jun N-terminal kinase in Jurkat T cells

The Ras-related small GTP-binding protein RhoB is known to be a pro-apoptotic protein and immediate-early inducible by genotoxic stresses. In addition, JNK activation is known to function in γ-radiation-induced apoptosis. However, it is unclear how JNK activation and γ-radiation-dependent RhoB induction are related. Here we verified the relationship between JNK activation and RhoB induction. RhoB induction by γ-radiation occurred at the transcriptional level and transcriptional activation of RhoB was concomitant with an increase in RhoB protein. γ-Radiation-induced RhoB expression was markedly attenuated by pretreatment with a JNK-specific inhibitor, SP600125, but not by a p38 MAPK inhibitor, SB203580. Inhibition of JNK caused a decrease in early apoptotic cell death that correlated with RhoB expression. However, PI3K inhibition had no significant effects, indicating that the AKT survival pathway was not involved. The siRNA knockdown of JNK resulted in a decrease in RhoB expression and the siRNA knockdown of RhoB restored cell growth even in the γ-irradiated cells. These results suggest that RhoB regulation involves the JNK pathway and contributes to the early apoptotic response of Jurkat T cells to γ-radiation.

Loss of RhoB Expression Enhances the Myelodysplastic Phenotype of Mammalian Diaphanous-Related Formin mDia1 Knockout Mice

Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis and hyperplastic bone marrow. Complete loss or interstitial deletions of the long arm of chromosome 5 occur frequently in MDS. One candidate tumor suppressor on 5q is the mammalian Diaphanous (mDia)-related formin mDia1, encoded by DIAPH1 (5q31.3). mDia-family formins act as effectors for Rho-family small GTP-binding proteins including RhoB, which has also been shown to possess tumor suppressor activity. Mice lacking the Drf1 gene that encodes mDia1 develop age-dependent myelodysplastic features. We crossed mDia1 and RhoB knockout mice to test whether the additional loss of RhoB expression would compound the myelodysplastic phenotype. Drf1 −/− RhoB −/− mice are fertile and develop normally. Relative to age-matched Drf1 −/− RhoB +/− mice, the age of myelodysplasia onset was earlier in Drf1 −/− RhoB −/− animals—including abnormally shaped erythrocytes, splenomegaly, and extramedullary hematopoiesis. In addition, we observed a statistically significant increase in the number of activated monocytes/macrophages in both the spleen and bone marrow of Drf1 −/− RhoB −/− mice relative to Drf1 −/− RhoB +/− mice. These data suggest a role for RhoB-regulated mDia1 in the regulation of hematopoietic progenitor cells.

Loss of RhoB Expression Promotes Migration and Invasion of Human Bronchial Cells Via Activation of AKT1

Lung cancer is the leading cause of cancer-related death worldwide, mainly due to its highly metastatic properties. Previously, we reported an inverse correlation between RhoB expression and the progression of the lung cancer, occurring between preinvasive and invasive tumors. Herein, we mimicked the loss of RhoB observed throughout lung oncogenesis with RNA interference in nontumoral bronchial cell lines and analyzed the consequences on both cell transformation and invasion. Down-regulation of RhoB did not modify the cell growth properties but did promote migration and invasiveness. Furthermore, RhoB depletion was accompanied by modifications of actin and cell adhesion. The specific activation of the Akt1 isoform and Rac1 was found to be critical for this RhoB-mediated regulation of migration. Lastly, we showed that RhoB down-regulation consecutive to K-RasV12 cell transformation is critical for cell motility but not for cell proliferation. We propose that RhoB loss during lung cancer progression relates to the acquisition of invasiveness mediated by the phosphatidylinositol 3-kinase (PI3K)/AKT and Rac1 pathways rather than to tumor initiation.

RhoB induces apoptosis via direct interaction with TNFAIP1 in HeLa cells

RhoB, a tumor suppressor, has emerged as an interesting cancer target, and extensive studies aimed at understanding its role in apoptosis have been performed. In our study, we investigated the involvement of RhoB-interacting molecules in apoptosis. To identify RhoB-interacting proteins, we performed yeast-two hybrid screening assays using RhoB as a bait and isolated TNFAIP1, a TNFalpha-induced protein containing the BTB/POZ domain. The interaction between RhoB and TNFAIP1 was demonstrated in vivo through coimmunoprecipitation studies and in vitro binding assays. RFP-TNFAIP1 was found to be partially colocalized with EGFP-RhoB. The partial colocalization of RhoB and TNFAIP1 in endosomes suggests that RhoB-TNFAIP1 interactions may have a functional role in apoptosis. TNFAIP1 elicited proapoptotic activity, while simultaneous expression of RhoB and TNFAIP1 resulted in a dramatic increase in apoptosis in HeLa cells. Furthermore, knockdown of RhoB using siRNA clearly rescued cells from apoptosis induced by TNFAIP1. This finding suggests that interactions between RhoB and TNFAIP1 are crucial for induction of apoptosis in HeLa cells. The observation of increased SAPK/JNK phosphorylation in apoptotic cells and the finding that a JNK inhibitor suppressed apoptosis indicates that SAPK/JNK signaling may be involved in apoptosis induced by RhoB-TNFAIP1 interactions. In conclusion, we found that RhoB interacts with TNFAIP1 to regulate apoptosis via a SAPK/JNK-mediated signal transduction mechanism.

Reactivation of Suppressed RhoB is a Critical Step for the Inhibition of Anaplastic Thyroid Cancer Growth

Anaplastic thyroid carcinoma (ATC) is a highly aggressive form of the disease for which new therapeutic options are desperately needed. Previously, we showed that the high-affinity peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, RS5444, inhibits cell proliferation of ATC cells via induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21). We show here that up-regulation of RhoB is a critical step in PPARgamma-mediated activation of p21-induced cell stasis. Using multiple independently derived ATC cell lines, we found that treatment with RS5444 leads to the up-regulation of RhoB and subsequent activation of p21, and that silencing of RhoB by RNAi blocks the ability of RS5444 to induce p21 and to inhibit cell proliferation. Our results show that transcriptional regulation of RhoB by the nuclear transcription factor PPARgamma is responsible for the induction of p21 mRNA and protein. We further implicate RhoB as a key signaling effector for the growth inhibition of ATC, as treatment with a histone deacetylase inhibitor shown to increase RhoB expression in lung cancer cells caused the up-regulation of RhoB in ATC cells accompanied by increased expression of p21 and inhibition of cell proliferation; this effect occurred even in ATC cells that were unresponsive to RS5444 due to a lack of expression of PPARgamma. Our results implicate RhoB as a novel intermediate in critical signaling pathways and as an additional target for therapeutic intervention in ATC.

Role of RHOB in the antiproliferative effect of glucocorticoid receptor on macrophage RAW264.7 cells

Although glucocorticoid (GC) has been reported to inhibit macrophage killing activity and cytokine production in response to proinflammatory stimuli, the effect of GC on macrophage proliferation is controversial. In our previous study, we found that inhibition of glucocorticoid receptor (GR) expression in murine macrophage cell line RAW264.7 cells (RAW-GR(-) cells) by RNAi significantly promoted cell proliferation. In the present study, we provide the evidence that the expression of Rhob, a member of Rho GTPases with anti-cancer character, remarkably decreased in RAW-GR(-) and RAW264.7 cells transiently transfected with GR-RNAi vector. Overexpression or constitutive activation of Rhob in RAW-GR(-) and RAW264.7 cells by transfection with wild-type Rhob expression vector (Rhob-wt) or constitutively activated Rhob plasmid (Rhob-V14) resulted in decreased proliferation of the two cell lines. Oppositely, the proliferation of RAW264.7 cells was significantly increased when the expression of Rhob by RNA interference technique or the activity of Rhob by transfection with dominant negative Rhob mutant that is defective in nucleotide binding (Rhob-N19) was inhibited. In addition, enhanced activity of Akt, but not MAPK3/1 or MAPK14, was found in RAW-GR(-) cells. Blocking the pathway of phosphatidylinositol 3-kinase (PI3K)/Akt with the specific inhibitor LY294002 decreased the proliferation and elevated RHOB protein level, indicating that PI3K/Akt signal plays its role of proliferation modulation upstream of RHOB protein. In conclusion, these results demonstrate that Rhob plays an important role in the antiproliferative effect of GR on RAW264.7 cells by GR-->Akt-->Rhob signaling and Rhob negatively regulates the proliferation of RAW264.7 cells.

In vivo restoration of RhoB expression leads to ovarian tumor regression

Ovarian cancers are very aggressive cancers most often diagnosed when metastasis has already occurred in the entire peritoneal cavity. Ovarian adenocarcinoma cells present an undetectable level of RhoB GTPase. Using preclinical ovarian cancer models, we aimed to evaluate the potential use of RhoB cDNA as a tumor suppressor gene in gene therapy. RhoB restoration in vitro, through recombinant adenovirus transduction, resulted in the apoptosis of endogenous RhoB protein low-expressing cell lines (OVCAR-3 and IGROV-1) through the activation of the intrinsic apoptotic caspase cascade. We showed that a single injection of 10(8) p.f.u. of adenoviral vector encoding a reporter gene into the peritoneal cavity of ovarian tumor bearing mice can induce the gene modification of a large quantity of cells throughout the cavity. We thereby tested the effect of AdRhoB injections to treat ovarian cancer-bearing mice. The ectopic expression of RhoB, following its introduction via viral transduction into nude mice in vivo, was highly effective in suppressing tumor growth of ovarian cancer xenografts. Therapeutic agents designed to correct defects of RhoB at the molecular level may thereby provide innovative treatment options for patients not responding to standard therapies.

MAP1A Light Chain-2 Interacts with GTP-RhoB to Control Epidermal Growth Factor (EGF)-dependent EGF Receptor Signaling

Rho GTPases have been implicated in the control of several cellular functions, including regulation of the actin cytoskeleton, cell proliferation, and oncogenesis. Unlike RhoA and RhoC, RhoB localizes in part to endosomes and controls endocytic trafficking. Using a yeast two-hybrid screen and a glutathione S-transferase pulldown assay, we identified LC2, the light chain of the microtubule-associated protein MAP1A, as a novel binding partner for RhoB. GTP binding and the 18-amino acid C-terminal hypervariable domain of RhoB are critical for its binding to MAP1A/LC2. Coimmunoprecipitation and immunofluorescence experiments showed that this interaction occurs in U87 cells. Down-regulation of MAP1A/LC2 expression decreased epidermal growth factor (EGF) receptor expression and modified the signaling response to EGF treatment. We concluded that MAP1A/LC2 is critical for RhoB function in EGF-induced EGF receptor regulation. Because MAP1A/LC2 is thought to function as an adaptor between microtubules and other molecules, we postulate that the RhoB and MAP1A/LC2 interactions facilitate endocytic vesicle trafficking and regulate the trafficking of signaling molecules.