Inhibition Of Metastasis And Angiogenesis Of Hepatocellular Carcinoma Cells Via Targeting RhoC In Vitro And Vivo

Hepatocellular carcinoma (HCC) is one of the most common malignancies in Asia and Africa, especially in China. During the past decade, the hepatic resection for HCC has evolved into a safe procedure with low operative mortality. However, the long-term survival remains unsatisfactory because of a high incidence of recurrence and metastasis after the hepatic resection. Thus, the inhibition of invasion and metastasis is of great importance in the HCC therapies.The invasion and metastasis of HCC is a multi-step process that often involves many complex biological and pathological events. More recently, RhoC has attracted interest with its increased expression being linked to increased invasion in breast, melanoma, pancreatic, colon, bladder, hepatocellular, non small cell lung carcinoma and primary gastric tumor or cell lines. Our previous study revealed that RhoC was a prognostic marker with hepatocellular carcinoma and its expression was correlated with invasion and metastasis of HCC. However, the exact role of RhoC in HCC remains largely unknown. In this study, we used a stable retroviral small interfering RNA (siRNA) approach to selectively knockdown the expression of RhoC, which allowed us to confirm the correlation of RhoC overexpression and HCC metastasis. More importantly, we successfully established an in vivo RhoC retroviral siRNA gene delivery system to investigate whether inhibition of RhoC expression could effectively decrease the metastasis of HCC in vivo. 1. We first examined the mRNA expression of RhoGTPases, including Rac1, Cdc42, RhoA, RhoB and KhoC, in HepG₂, MHCC97-L and HCCLM3, three hepatocellular carcinoma cell lines with increasing spontaneous metastatic potential, to investigate the relationship between RhoGTPases expression and metastasis potential in HCC. The expression of RhoC in these cell lines was also determined at the protein levels using Western blot. the mRNA and protein expression of RhoC were significantly elevated in HCCLM3 and MHCC97L cell lines when compared with HepG2 cell line. Together, the results were in agreement with the correlation we previously reported that RhoC expression correlates with the metastatic potential in HCC.2. To define the correlation of RhoC expression and HCC metastasis, we employed siRNA approach to inhibit expression of RhoC. We identified three putative candidate sequences and one control sequence and cloned into the pSUPER.retro.neo retroviral expression vector. After three-week selection in media supplied with G418, HCCLM3 cell lines, stably expressed siRNA-expressing sequence and control sequences, were obtained and named as HCCLM3^RhoCRNAi+ and HCCLM3^RhoCRNAi- respectively. The expression of RhoC protein was decreased dramatically in HCCLM3^RhoCRNAi+ compared with HCCLM3^RhoCRNAi-, which showed a more than 70% inhibitory efficiency. To examine the specificity of RhoC siRNA, we analyzed the mRNA expression of Rac1, Cdc42, RhoA, RhoB and RhoC in HCCLM3^RhoCRNAi- and HCCLM3^RhoCRNAi- respectively. The expression of Rac1, Cdc42 and RhoB remained unchanged, which confirmed the specificity of RhoC siRNA, notwithstanding slight upregulated of RhoA in mRNA level was observed.3. The importance of Rho proteins in the regulation of cell motility in normal cells, and the association of upregulation of RhoC with metastasis of HCC suggest that RhoC may be involved in the invasive phenotype of HCC cell through promoting cell migration. To test this possibility, we first investigated the effect of RhoC on invasion and migration of HCCLM3 cell. Wound-healing assay was employed to determine the migration of HCCLM3 cell. HCCLM3^RhoCRNAi+ decreased 15% closure as compared with HCCLM3^RhoCRNAi-, suggesting a role for RhoC in the migration of HCCLM3 cell. Next, a matrigel invasion assay in transwell culture chambers was performed to determine the effect of RhoC on the in vitro invasion of HCCLM3 cell. The numbers of HCCLM3^RhoCRNAi+ cells that passed through the matrigel was only 49% as compared with HCCLM3^RhoCRNAi- cells. Together, these results support a critical role for RhoC in the invasion of HCCLM3 cell. We also compared HCCLM3^RhoCRNAi+ and HCCLM3^RhoCRNAi- cells for proliferation rate using the MTT method, a very subtle inhibition of proliferation was observed in HCCLM3^RhoCRNAi+ cell, which counts a reduction of only 8% at 3th day and 11% at 5th day as compared with HCCLM3^RhoCRNAi- cell. At the same time, apoptosis in HCCLM3^RhoCRNAi+ and HCCLM3^RhoCRNAi- cells, as determined using FASC, showed no significant difference. These results suggest that RhoC contributes to metastasis without significantly effecting proliferation and apoptosis of HCCLM3 cell.4. Recent studies indicated that tumor cells secrete and respond to Autotaxin (ATX) in an autocrine fashion with increased cell motility, which contributes to metastasis of tumor cells. To examine a potential role for RhoC in ATX-induced metastasis, we first assessed whether HCCLM3 cell would respond to recombinant human ATX. A matrigel invasion assay in transwell culture chambers was performed to detect the response of HCCLM3 cell to ATX. A dose-dependent cell motility-stimulating activity of ATX was observed, with the cell motility reached saturation in the present of 50ng/mL ATX. To further confirm this response, GTPase pull-down assay was performed to analyze the GTP-bound form of RhoC GTPases in response to ATX. The results indicate an activation of RhoC by ATX. Active RhoC protein level was increased by 42% in response to ATX (50ng/mL) as compared with the control group (39% vs. 81% respectively). We next examined the pattern and morphology of F-actin to determine cytoskeletal and morphological changes induced by ATX. ATX at 50ng/mL concentration stimulated reorganization of actin leading to the formation of stress-fiber-like structures transversing in HCCLM3^RhoCRNAi- cells, whereas this process was not evident in HCCLM3^RhoCRNAi+ cell. Furthermore, we investigated whether the migration of HCCLM3 cells induced by ATX could be affected through inhibition of RhoC specially. Invasion and migration of HCCLM3^RhoCRNAi+ cells was dramatically reduced when compared with HCCLM3^RhoCRNAi- cells in response to ATX, supporting an important role for RhoC in mediating ATX induced invasion of HCCLM3 cells.5. To investigate whether RhoC gene could serve as a potential target to inhibit metastasis of HCC, we established an anti-RhoC retroviral gene delivery BALB/c nude mice model. Alter one week that HCCLM3 cells were subcutaneously transplanted to generate primary HCC tumor, PT67 cells (PT67^RhoCRNAi+ or PT67^RhoCRNAi-), with high title about 10⁵, were inoculated subcutaneously as a constant source of retrovirus, these nude mice model were named as Mice^RhoCRNAi+ and Mice^RhoCRNAi- respectively. Primary subcutaneous tumor of HCCLM3 was measured every five day and mice were sacrificed 45 days alter implantation to examine lung metastasis. Infection of HCCLM3 cells was confirmed firstly through histological examination of GFP expression. HCCLM3 tumor showed strong expression of GFP, indicative of a successful retroviral gene delivery. At the same time, the expression of RhoC in HCCLM3 tumor was also determined to evaluate the effect of siRNA against RhoC in vivo. As expected, expression of RhoC in primary HCCLM3 tumor was negative in Mice^RhoCRNAi+ whereas positive in Mice^RhoCRNAi-, which indicated the successful knockdown of RhoC in HCCLM3 cell in vivo. Next, the primary tumor size and the number of lung metastasis were assessed. Significantly, the numbers of lung metastasis was significantly fewer in Mice^RhoCRNAi+ than that in Mice^RhoCRNAi-. Unexpectedly, a difference in the primary tumor size was detected. The primary tumor was smaller in Mice^RhoCRNAi+ than in Mice^RhoCRNAi-, which suggested that knockdown of RhoC in vivo could inhibit growth of hepatocellular carcinoma. Finally, MVD in primary tumor was investigated. Primary tumor in Mice^RhoCRNAi+ showed significantly less MVD than Mice^RhoCRNAi-, suggesting that knockdown of RhoC in vivo may inhibit growth of hepatocellular carcinoma through inhibition of angiogenesis.Angiogenesis, also known as neovascularization, is the formation of new blood vessels through endothelial cell (EC) migration and stem cell recruitment in combination with morphogenesis. Recent research revealed that Rho activity critically and selectively regulates endothelial cell organization during angiogenesis. Our previous study indicated the possibility of critical role of RhoC in angiogenesis of hepatocellular carcinoma. More importantly, our results suggested that knockdown of RhoC in vivo can inhibit growth of hepatocellular carcinoma through inhibition of angiogenesis, we hypothesized that it is knockdown of RhoC in endothelial cell inhibited the migration of endothelial cell, which may be responsible for inhibition of angiogenesis. To address this hypothesis, we employed HMEC-1 cell, an immortalized human microvascular endothelial cell line, and angiogenesis model in vitro and in vivo, to investigate the role of RhoC during angiogenesis of hepatocellular carcinoma.1. To define the role of RhoC in angiogenesis, we employed siRNA approach (as described in Part one)combined multiple rounds of infection to inhibit expression of RhoC in HMEC-1 cells. This approach resulted in 80%～90% transfection efficiency and successful inhibition of RhoC in HMEC-1 cells.2. As we hypothesized that it is RhoC taken critical role in migration of endothelial cell, which may be responsible for influence of angiogenesis. We first investigated the effect of RhoC on invasion and migration of HMEC-1 cell. Wound-healing assay was employed to determine the migration of HMEC-1 cell. HMEC-1^RhoCRNAi+ decreased 23% closure as compared with HMEC-1^RhoCRNAi-, suggesting a role for RhoC in the migration of HMEC-1 cell. Next, a matrigel invasion assay in transwell culture chambers was performed to determine the effect of RhoC on the in vitro invasion of HMEC-1 cell. The numbers of HMEC-1^RhoCRNAi+ cells that passed through the matrigel was 49±6 as compared with HMEC-1^RhoCRNAi- cells was 70±3. Together, these results support a critical role for RhoC in the invasion of HMEC-1 cell. We also compared HMEC-1^RhoCRNAi+ and HMEC-1^RhoCRNAi- cells for proliferation rate using the MTT method and for apoptosis using FACS, no significant difference were observed both in proliferation and apoptosis. These results suggest that RhoC contributes to metastasis without significantly effecting proliferation and apoptosis of HMEC-1 cell.3. Next, we investigated the effects of RhoC for HMEC-1 organization behavior in Matrigel. Our results showed inhibition of RhoC in HMEC-1 could result in an inhibition of capillary network formation of HMEC-1 in matrigel. Using rhodamine-conjugated phalloidin, F-actin filaments of HMEC-1 cells in Matrigel were visualized, which indicated that knockdown expression of RhoC could inhibit reorganization of the F-actin filaments. Meanwhile, using a three dimensions angiogenesis model, we demonstrated that knockdown of RhoC could inhibit tube formation of HMEC-1 in three dimensions matrigel. Together, these results demonstrated that knockdown of RhoC inhibits angiogenesis in vitro.4. To investigate whether RhoC gene could serve as a potential target to inhibit angiogenesis in vivo, we established an anti-RhoC retroviral gene delivery BALB/c nude mice model, in which PT67 packing cells with high title about 10⁵, were inoculated subcutaneously as a constant source of retrovirus and Matrigel mixed with VEGF was inoculated subcutaneously as derivation of angiogenesis. Our results showed that neovascularization was markedly reduced in RhoC-deficient mice, which indicated that RhoC critically regulates angiogenesis driven by VEGF in vivo.In summary, the present study provides evidence indicating a critical and specific role of RhoC in metastasis and angiogenesis of HCC. Furthermore, data from our present study implicate a new cancer therapy that blocks HCC metastasis and angiogenesis by specifically inhibiting RhoC.