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

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.

KRAS protein expression becomes progressively restricted during embryogenesis and in adulthood

“…Therefore, our Kras Cit mouse model represents an interesting tool to investigate the subcellular location of KRAS in tissues in a physiological or pathophysiological setting. In that context, the induction of enzymes that catalyze post-translational modifications of KRAS necessary for its membrane localization was shown to be highly context-dependent ( Assi et al, 2020 ). Moreover, cytoplasmic expression of KRAS was associated with resistance of pancreatic acinar cells to tumor-promoting KRAS mutations: KRAS mutants were expressed, but not properly located for exerting their function ( Assi et al, 2020 ).…”

“…In that context, the induction of enzymes that catalyze post-translational modifications of KRAS necessary for its membrane localization was shown to be highly context-dependent ( Assi et al, 2020 ). Moreover, cytoplasmic expression of KRAS was associated with resistance of pancreatic acinar cells to tumor-promoting KRAS mutations: KRAS mutants were expressed, but not properly located for exerting their function ( Assi et al, 2020 ). We therefore speculate that our present data showing KRAS predominantly in the cytoplasm of pancreatic endocrine cells provides an explanation to the insulin-producing β cells’ refractoriness to KRAS-mediated transformation ( Gidekel Friedlander et al, 2009 ).…”

“…PVDF membranes (ISEQ00010, Millipore) were blocked with a solution of 5% low-fat milk diluted in Tris-buffered saline (TBS)/0.1% Tween-20 (Sigma-Aldrich) for 1 h at room temperature. Membranes were incubated overnight at 4 °C with primary antibodies against KRAS (own antibody) ( Assi et al, 2020 ) or HSC70 (Santa Cruz sc-7298) (dilution 1:1000 for all antibodies). Then, membranes were washed with TBS/0.1% Tween-20 and incubated with secondary antibodies for 1 h at RT.…”

Genetic Inactivation of Peroxiredoxin-I Impairs the Growth of Human Pancreatic Cancer Cells

“…Whole-tissue quantification for PRX-I staining was performed using the HALO software (Indica Labs, Albuquerque, NM, USA). For cell labeling, cells cultured on coverslips were washed once with PBS and then fixed with 4% PFA for 30 min at RT, as described previously [ 22 ]. Then cells were permeabilized, blocked with solution 1, and labeled with Microtubule-associated protein 1A/1B-light chain (LC3) (1/100; 12741S, Cell Signaling Technology, Leiden, Netherlands) and Lysosomal-associated membrane protein 1 (LAMP1) antibodies (1/100; 1D4B, DSHB, Iowa, IA, USA) at 4 °C overnight.…”

Olfactory Receptor OR7A17 Expression Correlates with All-Trans Retinoic Acid (ATRA)-Induced Suppression of Proliferation in Human Keratinocyte Cells

“…Interestingly, as shown in Figures 3B,C, we observed two or three bands of OR7A17 protein in the Western blot. Although further research is needed, the appearance of several bands for OR7A17 might be attributed to post-translational modifications such as glycosylation, acetylation, and methylation [21][22][23]. Furthermore, to establish a connection between ATRA-regulated OR7A17 expression and ATRA antiproliferative effects, we subjected the cells treated with ATRA and RAR antagonists to EdU incorporation assays (Figure 3D).…”