Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf
Martin O. Bergo, … , Patrick J. Casey, Stephen G. Young
Martin O. Bergo, … , Patrick J. Casey, Stephen G. Young
Published February 15, 2004
Citation Information: J Clin Invest. 2004;113(4):539-550. https://doi.org/10.1172/JCI18829.
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Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf

Abstract

Isoprenylcysteine carboxyl methyltransferase (Icmt) methylates the carboxyl-terminal isoprenylcysteine of CAAX proteins (e.g., Ras and Rho proteins). In the case of the Ras proteins, carboxyl methylation is important for targeting of the proteins to the plasma membrane. We hypothesized that a knockout of Icmt would reduce the ability of cells to be transformed by K-Ras. Fibroblasts harboring a floxed Icmt allele and expressing activated K-Ras (K-Ras-Icmtflx/flx) were treated with Cre-adenovirus, producing K-Ras-IcmtΔ/Δ fibroblasts. Inactivation of Icmt inhibited cell growth and K-Ras–induced oncogenic transformation, both in soft agar assays and in a nude mice model. The inactivation of Icmt did not affect growth factor–stimulated phosphorylation of Erk1/2 or Akt1. However, levels of RhoA were greatly reduced as a consequence of accelerated protein turnover. In addition, there was a large Ras/Erk1/2-dependent increase in p21Cip1, which was probably a consequence of the reduced levels of RhoA. Deletion of p21Cip1 restored the ability of K-Ras-IcmtΔ/Δ fibroblasts to grow in soft agar. The effect of inactivating Icmt was not limited to the inhibition of K-Ras–induced transformation: inactivation of Icmt blocked transformation by an oncogenic form of B-Raf (V599E). These studies identify Icmt as a potential target for reducing the growth of K-Ras– and B-Raf–induced malignancies.

Authors

Martin O. Bergo, Bryant J. Gavino, Christine Hong, Anne P. Beigneux, Martin McMahon, Patrick J. Casey, Stephen G. Young

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Figure 3

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Reduced capacity of K-Ras–transfected Icmt-deficient fibroblasts to form...
Reduced capacity of K-Ras–transfected Icmt-deficient fibroblasts to form colonies in soft agar. (a) K-Ras-Icmtflx/flx and derivative K-Ras-IcmtΔ/Δ fibroblasts (2,000 cells of each) were mixed with 0.35% agarose and poured onto plates containing a 0.70% agarose base. Colonies were stained and photographed 21 days later. Nontransfected cells (i.e., no activated K-Ras) did not form colonies in soft agar. (b) Bar graph illustrating the number of colonies formed in soft agar in four independent experiments; data in each experiment were normalized to the number of colonies that formed with the parental K-Ras-Icmtflx/flx fibroblasts. Inactivation of Icmt significantly reduced the number of colonies that formed in soft agar (*P < 0.0001). For experiments involving K-Ras-Icmtflx/flx:ICMT cells (expressing a human ICMT cDNA) and the derivative K-Ras-IcmtΔ/Δ:ICMT cells, data show the results from three independent experiments. In the cells expressing human ICMT, inactivation of mouse Icmt did not affect colony formation (P = 0.63). (c) Western blot showing higher K-Ras expression levels in K–Ras-transfected cells (+) compared with nontransfected cells (–). The blot was stripped and incubated with an anti-Erk1/2 antibody as a loading control. IB, immunoblot.