Role Of RhoE In The Metastasis Of Gastric Carcinoma

Despite markedly reduced incidence and mortality, gastric cancer (GC) is the second most frequent cause of cancer death. Metastasis is a major obstacle to its successful treatment; more complete characterization of gastric cancer metastasis could lead to great improvements in therapeutic options.The three Rnd proteins, Rnd1, Rnd2, and RhoE (also known as Rnd3), compose a sub-group of the Rho family of small GTP-binding proteins. While typical Rho proteins cycle between a resting GDP-bound state and an active GTP-bound state, RhoE is always bound to GTP but does not hydrolysis GTP. This special feature gives RhoE some unusual functions, such as inhibiting actin cytoskeleton formation, blocking cell-cycle progression, inhibiting UVB-induced apoptosis, and enhancing cell motility and rounding. Extensive studies recently found RhoE to be a diagnostic or prognostic indicator in cancer. It is upregulated in several malignancies including pancreatic cancer, melanoma, and non-small cell lung cancer, but downregulated in prostate cancer.RhoE also promotes tumor migration and invasiveness by regulation of the actin cytoskeleton. Our group have found that RhoE has a positive effect on multi-drug resistance phenotypes in GC. but the data on the expression, function and underlying mechanisms of RhoE in human gastric cancer metastasis are very limited. Our previous study showed that the expression level of RhoE in metastatic gastric cancers were significantly higher than those in nonmetastatic tumorous tissue specimens, indicating that RhoE may play a important role in the invasion and metastasis of gastric carcinoma., but the underlying mechanisms need to be further investigated.Aims of this study:1. To further analyze the protein expression level of RhoE in human gastric cancer tissues with or without metastasis and the protein and mRNA expression level of RhoE in the human gastric cancer cell lines with different metastatic potentials.2. To study the effects of RhoE protein on the malignant behavior about metastasis (such as in vitro migratory and invasive abilities and in vivo metastatic abilitiy) of human gastric gastric cancer cells SGC7901.3. To investigate the underlying mechanisms of RhoE protein in regulating the metastasis of gastric cancer cells SGC7901. Methods:1. Immunohistochemistry and Western blot was used to analyze the protein expression level of RhoE in a panel of gastric cancer patient specimens including normal gastric mucosae, metastatic gastric cancer tissues, non-metastatic gastric cancer tissues and metastatic lymph nodes.2. Protein expression level of RhoE was examined by Western blot in two of human gastric cancer lines with different metastatic potentials including SGC7901-M and SGC7901-NM.3. Semi-guantitative PCR and Realtime PCR were used to investigate the difference of RhoE mRNA expression level between SGC7901-M and SGC7901-NM cell lines.4. Plasmid encoding RhoE and empty vector were transfected into SGC7901 cells through transient and stable transfection.5. Western blot was used to assure the transfection.6. Wound-healing assay was used to examine the in vitro migratory ability of the transfected cells and control cells.7. Cell invasion assay was performed using a Transwell plate pre-coated with Matrigel to examine the in vitro invasion ability of of the transfected cells and control cells.8. In vivo tail vein metastatic assay was used for analysis of the metastatic ability of transfected cells in vivo.9. Western blot analysis was performed to examine the expression changes of some MMPs. Results:1. We evaluated the relationship between the expression of RhoE and metastasis of gastric cancer. We first compared the expression of RhoE in gastric cancers with non-tumor gastric tissues from the same patients. In the 34 samples of non-tumor gastric tissues we studied, 25 had RhoE protein expression in the cytoplasm, with a 73.5% positive expression rate. However, in the 34 samples of gastric cancer tissue from the same patients, elevated RhoE protein expression was detected (positive rate, 23.5%). RhoE was predominantly located in the cytoplasm of gastric cancer cells and its expression level was significantly lower in gastric cancers than that in non-tumor tissues (P < 0.05). We then compared RhoE expression in GC tissues with and without metastasis. We found that in 17 metastatic GC samples, the positive rate of RhoE was 41.2% (7 of 17), which is markedly higher than that of non-metastatic GC (5.9%, 1 of 17) (P < 0.05). We further compared expression levels of RhoE between metastatic GCs and non-metastatic GCs. Data suggested that RhoE expression in GCs with metastasis was significantly higher than in non-metastatic GCs (P < 0.05). However, no significant difference in RhoE expression was found between the primary site and the metastatic site of lymph node in metastatic GCs.2. In 8 out of the 12 cases or 66.7% samples, RhoE was found overexpressed in metastatic cancerous tissues by Western blot, while poorly expressed in non-metastatic GC tissues (8.3%, 1 of 12), which consistent with the result from immunohistochemistry analysis.3. We further compared the RhoE mRNA and protein expression level in two gastric cancer cell lines with different potentials established in our lab. Both real-time PCR and semi-quantitative PCR revealed higher levels of RhoE mRNA in the SGC7901-M cell line, which has higher metastatic potential, than that in SGC7901-NM cell line, which has poor migratory and invasive abilities (P < 0.05). Western blot analysis showed that RhoE protein was upregulated in SGC7901-M cells compared to SGC7901-NM cells.4. Plasmids encoding RhoE, named pCMV-FLAG-RhoE and pcDNA3.1V5-His-B-RhoE, could specifically and significantly increase RhoE expression in AGS cells after transient and stable transfection, respectively.5. Upregulation of RhoE significantly enhanced cell migration from the edge of the wound, which indicates that overexpression of RhoE enhances in vitro migratory ability of SGC7901 cells.6. Upregulation of RhoE also notebly enhanced cell invasion to penetrate a Matrigel-coated Transwell, which indicates that overexpression of RhoE could also enhance in vitro invasive abilities of SGC7901 cells.7. Inoculation of cells stably transfected with pcDNA3.1-V5-HisB-RhoE led to significantly more visible lung-surface tumors compared to control cells (P < 0.05). Interestingly, neither SGC7901-RhoE cells nor control cells could induce liver metastasis in nude mice.8. Western blot revealed that overexpression of RhoE upregulated MMP9 rather than MMP2 (P < 0.05) in SGC7901 cells.Conclusion:1. RhoE was found significantly overexpressed in metastatic gastric cancer tissues and gastric cancer cells with high metastatic potentials compared with non-metastatic gastric cancer tissues and gastric cancer cells which have poor metastatic potential. RhoE expression was highly related to the metastasis of gastric caner; However, RhoE protein expression level was lower in gastric cancer tissues compared with non-tumor gastric tissues.2. Overexpression of RhoE produces a higher motile and invasive phenotype both in vitro and in vivo.3. Matrx metalloproteinases were involved in the regulation of metastasis of gastric cancer by RhoE protein.In a word, RhoE may play a critical role in the metastasis of gastric cancer. Therapeutic strategies targeting RhoE and RhoE-mediated pathways may be a novel avenue in gastric cancer intervention.