RhoE promotes metastasis in gastric cancer through a mechanism dependent on enhanced expression of CXCR4

Abstract

RhoE, a novel member of the Rho protein family, is a key regulator of the cytoskeleton and cell migration. Our group has previously shown that RhoE as a direct target for HIF-1α and mediates hypoxia-induced epithelial to mesenchymal transition in gastric cancer cells. Therefore, we assumed that RhoE might play an important role in gastric cancer metastasis. In the present study, we have explored the role of RhoE expression in gastric cancer, cell invasion and metastasis, and the influence of RhoE on regulating the potential expression of down-stream genes. RhoE expression was elevated in gastric cancer tissues as compared with normal gastric tissues. We also found a close correlation between the histological grade and the diagnosis of the patient. Up-regulation of RhoE significantly enhanced the migratory and invasive abilities of gastric cancer cells both in vitro and in vivo. Moreover, down-regulation of RhoE diminished the metastatic potential of cancer cells. PCR array and subsequent transwell assay showed that the regulation of gastric cancer metastasis by RhoE was partially mediated by CXCR4. This observation suggested that CXCR4 might be a downstream effector for RhoE. In summary, our study identified RhoE as a novel prognostic biomarker and metastatic-promoting gene of gastric cancer.

Figure 1. Expression of RhoE in gastric cancer tissues and cells, and its association with patient survival.

(A), Immunohistochemical analysis of RhoE in tissues; (a), normal gastric epithelium; and (b) well-differentiated, (c) moderately differentiated or (d) poorly-differentiated gastric cancer tissue; (e) metastatic site in the lymph node. Brown coloration represents positive RhoE staining. (B), Kaplan- Meier post-operative survival curve as a function of RhoE expression. (C), Western blot analysis of RhoE expression in different gastric cell-lines. β-actin was used as the internal control. The relative expression levels of RhoE in gastric cancer cell-lines were presented as bar charts. * P <0.05.

Figure 2. RhoE promotes the migratory and invasive abilities of gastric cancer cells in vitro.

(A), RhoE was down-regulated in SGC7901-M cells after treatment with siRNA while RhoE expression was up-regulated in SGC7901-NM cells after treatment with lentivirus. RhoE protein levels were confirmed by Western blot analysis. (B), The migratory ability of the cells was evaluated by a wound-healing assay. The wound widths of each sample were measured at different time-points by phase-contrast microscopy (Olympus, Tokyo, Japan), and the closure ratio was calculated in accord with the following formula: Wound Closure (%) = (width 0 h) - width 24 h) / width 0 h. *P <0.05. These results were then compared to those of the control cells. (C), Tumor cell invasion activities were measured by transwell assay. Representative image fields of invasive cells on the membrane are shown. Data are represented as normalized cellular invasion (invasion index) relative to control cells. *P <0.05. The images shown are representative of three experiments.

Figure 3. RhoE promotes the metastatic ability of gastric cancer cells in vivo.

H and E staining of both the lungs and livers were assessed from mice that had received intravenous tail injections of SGC7901-NM-RhoE and control cells respectively. Metastatic loci were identified and marked by arrows. The number of metastatic loci in the liver and lung were also counted (middle). *P <0.05.

Figure 4. Verification of the expression of downstream genes in different cell-lines by Western blot analysis of RhoE.

(A) RhoE, CXCR4, VEGFA, CD82 and CTSK expression of SGC7901-M-control, SGC7901-M- siRhoE, SGC7901-NM-control, and SGC7901-NM-RhoE cell-lines. The expression of β-actin was used as internal control. The relative expression levels of CXCR4 were shown as bar charts. *P <0.05. Values represent the arithmetic mean and standard error of the mean (SEM) as determined from at least three experiments. (B) Immunohistochemical staining of RhoE and CXCR4 in 60 pairs of gastric cancer tissues. Three representative cases showed that CXCR4 expression was well-correlated with that of RhoE.

Figure 5. RhoE induced metastasis of gastric cancer cells was mediated by CXCR4.

The influence of CXCR4 on RhoE induced metastasis was measured by a transwell assay. In SGC7901-M-siRhoE cells, CXCR4 was enhanced following lentivirus infection. In SGC7901-NM-RhoE cells, CXCR4 was knocked out by treatment with siRNA interference. The number of invading cells was determined and presented as bar charts. *P <0.05, in comparisons between the cell-lines exhibiting different levels of expression of RhoE. **P <0.05, in comparisons between cell-lines displaying differential levels of expression of CXCR4.