Isoprenylcysteine carboxylmethyltransferase deficiency exacerbates KRAS-driven pancreatic neoplasia via Notch suppression
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
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
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Research Article Oncology

Isoprenylcysteine carboxylmethyltransferase deficiency exacerbates KRAS-driven pancreatic neoplasia via Notch suppression

Abstract

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.

Authors

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

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Figure 4

ICMT deficiency mislocalizes KRAS in pancreatic cells but does not inhibit ERK activation or proliferation.

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ICMT deficiency mislocalizes KRAS in pancreatic cells but does not inhib...
(A) Membrane association of endogenous KRAS in subcellular fractionations of homogenized pancreas from 3-month-old Icmtflx/+;Pdx1-Cre (Icmtflx/+) and Icmtflx/flx;Pdx1-Cre (Icmtflx/flx) mice. A representative immunoblot of KRAS in cytosolic (S100) and membrane (P100) fractions is shown, as is a quantification of three such experiments, the results of which are shown as the mean ± SEM of the ratios (Icmtflx/flx/Icmtflx/+) of immunodetected KRAS in P100 and S100 fractions normalized to the amount of β-tubulin present in each fraction. Na+K+-ATPase and RhoGDI were blotted to assess the purity of the membrane versus cytosolic fractions. (B) Immunohistochemical staining for pERK in paraffin sections of pancreata from 3-month-old mice of the indicated genotypes. Whereas normal ducts (asterisk) and acinar cells (arrows) were negative for pERK, early PanIN lesions showed strong nuclear and cytoplasmic staining (arrowheads) in pancreata from both genotypes, indicating that MAPK activation is unaffected by ICMT deficiency. (C) Immunohistochemical staining for Ki67 in paraffin sections of pancreata from 2-month-old mice of the indicated genotypes. Upper panels show areas of PanIN lesions, and lower panels show normal ducts. The nuclei of ductal cells in PanINs stained for Ki67 in both genotypes (arrows). Scale bars: 100 μm (B and C). Percentage of nuclei positive for pERK or Ki67 in 500 PanIN-associated cells examined (mean ± SEM, n = 3, NS).