Background: Colorectal cancer is a common disease that involves genetic alterations, such as inactivation of tumour suppressor genes and activation of oncogenes. Among them are RAS and BRAF mutations, which rarely coexist in the same tumour. Individual members of the Rho (Ras homology) GTPases contribute with distinct roles in tumour cell morphology, invasion and metastasis. The aim of this study is to dissect cell migration and invasion pathways that are utilised by BRAF^V600Eas compared to KRAS^G12Vand HRAS^G12Voncoproteins. In particular, the role of RhoA (Ras homolog gene family, member A), Rac1 (Ras-related C3 botulinum toxin substrate 1) and Cdc42 (cell division cycle 42) in cancer progression induced by each of the three oncogenes is described.Methods: Colon adenocarcinoma cells with endogenous as well as ectopically expressed or silenced oncogenic mutations of BRAF^V600E, KRAS^G12Vand HRAS^G12Vwere employed. Signalling pathways and Rho GTPases were inhibited with specific kinase inhibitors and siRNAs. Cell motility and invasion properties were correlated with cytoskeletal properties and Rho GTPase activities.Results: Evidence presented here indicate that BRAF^V600Esignificantly induces cell migration and invasion propertiesin vitroin colon cancer cells, at least in part through activation of RhoA GTPase. The relationship established between BRAF^V600Eand RhoA activation is mediated by the MEK-ERK pathway. In parallel, KRAS^G12Venhances the ability of colon adenocarcinoma cells Caco-2 to migrate and invade through filopodia formation and PI3K-dependent Cdc42 activation. Ultimately increased cell migration and invasion, mediated by Rac1, along with the mesenchymal morphology obtained through the Epithelial-Mesenchymal Transition (EMT) were the main characteristics rendered by HRAS^G12Vin Caco-2 cells. Moreover, BRAF and KRAS oncogenes are shown to cooperate with the TGFβ-1 pathway to provide cells with additional transforming properties.Conclusion: This study discriminates oncogene-specific cell migration and invasion pathways mediated by Rho GTPases in colon cancer cells and reveals potential new oncogene-specific characteristics for targeted therapeutics.

Several food polyphenols act as chemopreventers by reducing the incidence of many types of cancer, especially in colon epithelia. In this study, we have investigated whether the flavonoid quercetin can modulate cell proliferation and survival by targeting key molecules and/or biological processes responsible for tumor cell properties. The effect of quercetin on the expression of Ras oncoproteins was specifically studied using systems of either constitutive or conditional expression of oncogenic RAS in human epithelial cells. Our findings suggest that quercetin inhibits cell viability as well as cancer cell properties like anchorage-independent growth. These findings were further supported at the molecular level, since quercetin treatment resulted in a preferential reduction of Ras protein levels in cell lines expressing oncogenic Ras proteins. Notably, in cells that only express wild-type Ras or in those where the oncogenic Ras allele was knocked out, quercetin had no evident effects upon Ras levels. We have shown that quercetin drastically reduces half-life of oncogenic Ras but has no effect when the cells are treated with a proteasome inhibitor. Moreover, in Ha-RAS-transformed cells, quercetin induces autophagic processes. Since quercetin downregulates the levels of oncogenic Ras in cancer cells, we propose that this flavonoid could act as a chemopreventive agent for cancers with frequent mutations of RAS genes.

Previous studies have uncovered several transcription factors that determine biological alterations in tumor cells to execute the invasion-metastasis cascade, including the epithelial-mesenchymal transition (EMT). We sought to investigate the role of miR-21 in colorectal cancer regulation. For this purpose, miR-21 expression was quantified in a panel of colorectal cancer cell lines and clinical specimens. High expression was found in cell lines with EMT properties and in the vast majority of human tumor specimens. We demonstrate in a cell-specific manner the occupancy of MIR-21 gene promoter by AP-1 and ETS1 transcription factors and, for the first time, the pattern of histone posttranslational modifications necessary for miR-21 overexpression. We also show that Integrin-β4 (ITGβ4), exclusively expressed in polarized epithelial cells, is a novel miR-21 target gene and plays a role in the regulation of EMT, since it is remarkably de-repressed after transient miR-21 silencing and downregulated after miR-21 overexpression. miR-21-dependent change of ITGβ4 expression significantly affects cell migration properties of colon cancer cells. Finally, in a subgroup of tumor specimens, ROC curve analysis performed on quantitative PCR data sets for miR-21, ITGβ4, and PDCD4 shows that the combination of high miR-21 with low ITGβ4 and PDCD4 expression is able to predict presence of metastasis. In conclusion, miR-21 is a key player in oncogenic EMT, its overexpression is controlled by the cooperation of genetic and epigenetic alterations, and its levels, along with ITGβ4 and PDCD4 expression, could be exploited as a prognostic tool for CRC metastasis.

BRAF vs RAS oncogenes: Are mutations of the same pathway equal? Differential signalling and therapeutic implications BRAF vs RAS oncogenes: are mutations of the same pathway equal? differential signalling and therapeutic implications

Current clinical problems in colorectal cancer (CRC) diagnostics and therapeutics include the disease complexity, tumor heterogeneity, and resistance to targeted therapeutics. In the present study, we examined 171 CRC adenocarcinomas from Greek patients undergoing surgery for CRC to determine the frequency of KRAS, BRAF, and PIK3CA point mutations from different areas of tumors in heterogeneous specimens. Ninety two out of 171 (53.8%) patients were found to bear a KRAS mutation in codons 12/13. Of the 126 mutations found, 57.9% (73/126) were c.38G>A mutations (p.G13D) and 22.2% (28/126) were c.35G>T (p.G12V). Remarkably, RAS mutations in both codons 12 and 13 were recorded in the same tumor by pyrosequencing. Moreover, differences in KRAS mutations between tumor center and periphery revealed tumor heterogeneity in 50.7% of the specimens. BRAF c.1799T>A (V600E) mutations were moderately detected in 4/171 (2.3%) specimens, whereas most PIK3CA mutations were revealed by pyrosequencing 6/171 (3.5%). Remarkable tumor heterogeneity is revealed, where double mutations of KRAS in the same tumor and different KRAS mutation status between tumor core and margin are detected with high frequency. It is expected that these findings will have a major impact in cancer diagnosis and personalized therapies.