GEF-H1 over-expression in hepatocellular carcinoma promotes cell motility via activation of RhoA signalling

The interstitial chromosome (chr.) 1q21-q22 region is frequently amplified in human cancers, where it has been reported to carry prognostic significance for patients. We attempted to delineate chr. 1q21-q22 for affected gene(s) in hepatocellular carcinoma (HCC) by array-CGH and detected copy number gains of θ-guanine nucleotide exchange factor-H1 (GEF-H1) as most significant event. Gene expression evaluation in the HCC cohort indicated common up-regulations of GEF-H1 in 64% tumours compared to adjacent non-tumoural liver (64/100; paired t - test p < 0.0001). Moreover, GEF-H1 over-expressions correlated with microvascular invasion and advanced-stage tumours (p < 0.05). High GEF-H1 levels also predict shorter disease-free and overall survival of HCC patients (p < 0.03). Functional knock-down of GEF-H1 by RNAi indicated marked reduction in cell invasion through matrigel and an inhibition of cell migration (p < 0.035), but an effect on cell viability was not apparent. More interestingly, a mesenchymal-epithelial transition (MET) was readily observed in GEF-H1 knock-down cells, where a concomitant re-expression of epithelial markers (E-cadherin and cytokeratin 18) and cell adhesion proteins (a-catenin and γ-catenin) was found but down-regulation of mesenchymal features (N-cadherin, vimentin and fibronectin). This phenotype was accompanied by reduced filamentous actin polymerizations and diminution of the stress fibre formation. In addition, reduced active form of GTP-RhoA, together with its downstream effectors, including cleaved ROCK1 and phosphorylated MLC2, were also detected in GEF-H1-depleted cells. Taken together, our findings underscore a potent oncogenic role for GEF-H1 in promoting the metastatic potentials of HCC, possibly through activation of RhoA signalling and the EMT phenomenon.