A Novel KRAS Antibody Highlights a Regulation Mechanism of Post-Translational Modifications of KRAS during Tumorigenesis.

Mohamad Assi,Jean-Paul Thissen,Vincent Stroobant,Patrick Jacquemin,Boris Pirlot

doi:10.3390/ijms21176361

Abstract

KRAS is a powerful oncogene responsible for the development of many cancers. Despite the great progress in understanding its function during the last decade, the study of KRAS expression, subcellular localization, and post-translational modifications remains technically challenging. Accordingly, many facets of KRAS biology are still unknown. Antibodies could be an effective and easy-to-use tool for in vitro and in vivo research on KRAS. Here, we generated a novel rabbit polyclonal antibody that allows immunolabeling of cells and tissues overexpressing KRAS. Cell transfection experiments with expression vectors for the members of the RAS family revealed a preferential specificity of this antibody for KRAS. In addition, KRAS was sensitively detected in a mouse tissue electroporated with an expression vector. Interestingly, our antibody was able to detect endogenous forms of unprenylated (immature) and prenylated (mature) KRAS in mouse organs. We found that KRAS prenylation was increased ex vivo and in vivo in a model of KRASG12D-driven tumorigenesis, which was concomitant with an induction of expression of essential KRAS prenylation enzymes. Therefore, our tool helped us to put the light on new regulations of KRAS activation during cancer initiation. The use of this tool by the RAS community could contribute to discovering novel aspects of KRAS biology.

Highlights

The RAS family encompasses three genes that encode four related small GTPases, HRAS, NRAS, KRAS4A, and KRAS4B [1]
Three enzymes catalyze the C-terminal modifications of KRAS: farnesyltransferase (FTase), RAS-converting enzyme-1 (RCE1), and isoprenylcysteine carboxyl methyltransferase (ICMT)
Each peptide was coupled to keyhole limpet hemocyanin (KLH) and injected into four rabbits following a classical immunization protocol

Summary

Introduction

The RAS family encompasses three genes that encode four related small GTPases, HRAS, NRAS, KRAS4A, and KRAS4B [1]. The Kras gene undergoes an alternative splicing giving rise to two isoforms, KRAS4A and KRAS4B [2,3]. The latter is the predominant variant and is extensively studied in cancer [4]. Cell membrane localization of KRAS is essential for its signaling activity [7]. Targeting KRAS to the cell membrane requires post-translational prenylation of its C-terminal tail. Prenylation consists in the addition of branched unsaturated lipid groups (palmitate and farnesyl groups for KRAS4A, and farnesyl group for KRAS4B) essential for membrane localization [8]. Three enzymes catalyze the C-terminal modifications of KRAS: farnesyltransferase (FTase), RAS-converting enzyme-1 (RCE1), and isoprenylcysteine carboxyl methyltransferase (ICMT). Pharmacological inhibition of FTase leads to the addition of a geranylgeranyl group by the geranylgeranyltransferase I (GGTase-I) [9]

Methods

Results

Conclusion