RAS is the most frequently mutated oncogene in human cancers. Despite decades of effort, anti-RAS therapies have remained elusive. Isoprenylcysteine carboxylmethyltransferase (ICMT) methylates RAS and other CaaX-containing proteins, but its potential as a target for cancer therapy has not been fully evaluated. We crossed a Pdx1-Cre;LSL-KrasG12D mouse, which is a model of pancreatic ductal adenocarcinoma (PDA), with a mouse harboring a floxed allele of Icmt. Surprisingly, we found that ICMT deficiency dramatically accelerated the development and progression of neoplasia. ICMT-deficient pancreatic ductal epithelial cells had a slight growth advantage and were resistant to premature senescence by a mechanism that involved suppression of cyclin-dependent kinase inhibitor 2A (p16INK4A) expression. ICMT deficiency precisely phenocopied Notch1 deficiency in the Pdx1-Cre;LSL-KrasG12D model by exacerbating pancreatic intraepithelial neoplasias, promoting facial papillomas, and derepressing Wnt signaling. Silencing ICMT in human osteosarcoma cells decreased Notch1 signaling in response to stimulation with cell-surface ligands. Additionally, targeted silencing of Ste14, the Drosophila homolog of Icmt, resulted in defects in wing development, consistent with Notch loss of function. Our data suggest that ICMT behaves like a tumor suppressor in PDA because it is required for Notch1 signaling.
Helen Court, Marc Amoyel, Michael Hackman, Kyoung Eun Lee, Ruliang Xu, George Miller, Dafna Bar-Sagi, Erika A. Bach, Martin O. Bergö, Mark R. Philips
(A) Phase contrast micrographs of primary PDECs harvested from the main pancreatic duct of animals of the indicated genotypes and grown as spheroids in 3D Matrigel. Scale bars: 200 μm. (B) Genotype analysis by PCR and agarose gel electrophoresis of DNA from PDECs of the indicated genotypes before and after initiation of LoxP recombination by infection with adenovirus-directed expression of Cre recombinase. (C and D) Proliferation as measured by MTS assay of PDECs of the indicated genotypes after switching from 3D to 2D culture on plastic. Values were normalized to the day-3 reading (value = 1). Data are plotted as the mean ± SEM; n = 4 for C and n = 3 for D. (E) Immunoblots for p16INK4A of lysates prepared from PDECs of the indicated genotypes on days 1 and 8 after switching from 3D to 2D culture. Blot shown is representative of three. (F) Phase contrast micrographs of PDECs of the indicated genotypes stained with X-gal for SAβgal 12 days after switching to 2D culture. Scale bars: 100 μm. Quantification of X-gal staining normalized to cell number is plotted as the mean ± SEM in the graph below (n = 4, P < 0.01).