BRAF and RAS oncogenes regulate Rho GTPase pathways to mediate migration and invasion properties in human colon cancer cells: a comparative study

Eleni Makrodouli,Senji Shirasawa,Michal Koc,Takehiko Sasazuki,Alexander Pintzas,Ladislav Andera,Eftychia Oikonomou

doi:10.1186/1476-4598-10-118

Abstract

BackgroundColorectal 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 BRAFV600E as compared to KRASG12V and HRASG12V oncoproteins. 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.MethodsColon adenocarcinoma cells with endogenous as well as ectopically expressed or silenced oncogenic mutations of BRAFV600E, KRASG12V and HRASG12V were 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.ResultsEvidence presented here indicate that BRAFV600E significantly induces cell migration and invasion properties in vitro in colon cancer cells, at least in part through activation of RhoA GTPase. The relationship established between BRAFV600E and RhoA activation is mediated by the MEK-ERK pathway. In parallel, KRASG12V enhances 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 HRASG12V in Caco-2 cells. Moreover, BRAF and KRAS oncogenes are shown to cooperate with the TGFβ-1 pathway to provide cells with additional transforming properties.ConclusionThis 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.

Highlights

Colorectal cancer is a common disease that involves genetic alterations, such as inactivation of tumour suppressor genes and activation of oncogenes
In CacoBR13 cells the formation of stress fibers was enhanced, whereas formation of filopodia-membrane protrusions enriched with actin- is evident in Caco-K15 cells (Figure 1A-ii, arrow)
Depletion of cell division cycle 42 (Cdc42) affected the filopodia formation, when Caco-2 cells overexpressing mutant KRASG12V (Caco-K) cells were treated with siRNA against Cdc42 acquired rounded cell membrane lacking filapodia protrusion suggesting that filopodia formation in Caco-K cells is Cdc42-dependent (Figure 7D). These findings suggest that KRASG12V regulates motility and invasiveness of colon cancer cells through the Cdc42 GTPase

Summary

Introduction

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. Colorectal cancer represents a complex disease that involves multiple steps of genetic alterations, like inactivation of tumour suppressor genes and activation of oncogenes, often associated with progression from premalignant lesion (adenoma) to invasive adenocarcinoma [1]. Mutant KRAS disrupts actin cytoskeleton and maintains motility in colon cancer cells [2]. BRAF, a major downstream effector of KRAS, is considered an oncogene whose activating mutations appear in 70% of human malignant melanomas and in about 12-18% of human colon cancers. Mutant BRAF may interfere with organization of cytoskeleton and affect cell migration and invasion ability [5]

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