ABSTRACT
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 ρ-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 (α-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.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Rho Guanine Nucleotide Exchange Factors/genetics , Signal Transduction/physiology , rhoA GTP-Binding Protein/metabolism , Adult , Aged , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/secondary , Cell Movement/genetics , Epithelial Cells/metabolism , Epithelial Cells/pathology , Epithelial-Mesenchymal Transition/physiology , Female , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Middle Aged , Neoplasm Invasiveness , Neoplasm Staging , Phenotype , RNA, Small Interfering/genetics , Rho Guanine Nucleotide Exchange Factors/metabolismABSTRACT
We have previously described the physical localization of a constitutional t(5;6)(q21;q21) in a patient (tumor cell sample designated as MA214) with bilateral Wilms tumor (WT). We have now physically refined the breakpoints and identified putative gene targets within this region. The translocation breakpoints are contained within a 2.5-Mbp region on 5q21 containing four candidate genes and a 1.3-Mbp region on 6q21 that contains three candidate genes. To explore the role of this region in WT genesis, we have performed loss of heterozygosity (LOH) analysis with markers flanking the translocation breakpoints in tumor from MA214 and a panel of sporadic WT. Alleles were retained for all informative markers used in the MA214 tumor. In sporadic tumors LOH was found in 6 of 63 (9.5%) and 5 of 62 (8%) informative cases for flanking markers D6S301 and D6S1592 on 6q21. LOH was found in 3 of 58 (5.2%) and 2 of 54 (3.6%) for flanking markers D5S495 and D5S409 on 5q21. These preliminary data suggest LOH at the t(5;6)(q21;q21) region is unlikely to be a mechanism for tumor development in MA214, but may be important for a subgroup of sporadic WT.