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Inactivation of Icmt Inhibits Transformation by Oncogenic K-Ras

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Inactivation of Icmt Inhibits Transformation by Oncogenic K-Ras Inactivation of Icmt inhibits See the related Commentary beginning on page 513. transformation by oncogenic K-Ras and B-Raf Martin O. Bergo,1,2 Bryant J. Gavino,1 Christine Hong,1 Anne P. Beigneux,1,2 Martin McMahon,3 Patrick J. Casey,4 and Stephen G. Young1,2,5,6 1Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA 2Cardiovascular Research Institute, University of California, San Francisco, California, USA 3Cancer Research Institute, University of California, San Francisco Comprehensive Cancer Center, San Francisco, California, USA 4Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA 5Department of Medicine, University of California, San Francisco, California, USA 6The Medical Service, San Francisco General Hospital, San Francisco, California, USA Isoprenylcysteine carboxyl methyltransferase (Icmt) methylates the carboxyl-terminal isoprenylcys- teine of CAAX proteins (e.g., Ras and Rho proteins). In the case of the Ras proteins, carboxyl methy- lation 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-Icmt∆/∆ 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-Icmt∆/∆ fibroblasts to grow in soft agar. The effect of inactivating Icmt was not limited to the inhibition of K-Ras–induced transformation: inacti- vation 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. J. Clin. Invest. 113:539–550 (2004). doi:10.1172/JCI200418829. Introduction exposed isoprenylcysteine is methylated by an ER mem- Proteins that terminate with a carboxyl-terminal brane–bound methyltransferase, isoprenylcysteine car- “CAAX” motif, such as the Ras and Rho proteins, boxyl methyltransferase (Icmt) (3). These modifications undergo three sequential posttranslational processing render the C terminus of CAAX proteins more hydro- events. First, the cysteine (i.e., the C of the CAAX phobic, facilitating binding to membranes (4–6). sequence) is isoprenylated by protein farnesyltrans- The posttranslational processing of CAAX proteins ferase (FTase) or geranylgeranyltransferase type I has attracted interest because of the central role of (GGTase I) (1). Second, the last three amino acids of the mutationally activated Ras proteins in the develop- protein (i.e., the -AAX) are cleaved off by Rce1, an inte- ment of cancer (7, 8). The enzymes that carry out the gral membrane protein of the ER (2). Third, the newly posttranslational modifications of CAAX proteins (i.e., FTase, GGTase I, Rce1, and Icmt) have been considered as potential targets for modulating the activity of the Received for publication May 5, 2003, and accepted in revised form Ras proteins and for blocking the growth of Ras- November 25, 2003. induced malignancies. Farnesylation is critical for Ras Address correspondence to: Martin O. Bergo, activity (9), and farnesyltransferase inhibitors (FTIs) Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, California 94141-9100, USA. have shown promise in treating tumors, both in exper- Phone: (415) 695-3774; Fax: (415) 285-5632; imental animals (10, 11) and in humans (12–17). E-mail: [email protected]. A potential drawback of the clinical use of FTIs is that Conflict of interest: The authors have declared that no conflict of K-Ras and N-Ras—the isoforms most often mutated in interest exists. human tumors—can be efficiently geranylgeranylated Nonstandard abbreviations used: isoprenylcysteine carboxyl methyltransferase (Icmt); fibroblasts harboring a “floxed” Icmt in the setting of FTI therapy (18, 19). This alternate allele and expressing activated K-Ras (K-Ras-Icmtflx/flx); prenylation of the Ras proteins could limit the efficacy farnesyltransferase (FTase); geranylgeranyltransferase type I of FTIs in the treatment of Ras-induced tumors. The (GGTase I); farnesyltransferase inhibitor (FTI); Cre-adenovirus existence of an alternate means for prenylation has led (AdRSVCre); β-gal-adenovirus (AdRSVnlacZ); enhanced GFP (EGFP); mitogen-activated protein (MAP); polyinosinic- several groups to focus on the postisoprenylation steps polycytidylic ribonucleic acid (pI-pC). mediated by Rce1 and Icmt, since those steps are shared The Journal of Clinical Investigation | February 2004 | Volume 113 | Number 4 539 by farnesylated and geranylgeranylated CAAX proteins (G12V) and a puromycin-resistance cassette. Experi- (6). We previously generated Rce1-knockout mice and ments were performed with two different K-Ras–trans- found that they died quite late in gestation, after all of fected Icmtflx/flx cell lines (A and B), unless otherwise the major organ systems were formed (20). The Ras indicated. To produce B-Raf–transformed cell lines, proteins in Rce1-deficient fibroblasts were mislocalized spontaneously immortalized Icmtflx/flx fibroblasts were away from the plasma membrane, and the cells grew transfected with a retrovirus encoding an oncogenic slightly slower than wild-type cells (20). More recently, form of human B-Raf (V599E) and a blasticidin-resist- we showed that the inactivation of Rce1 partially ance cassette. Control cells were infected with a retro- blocked transformation of cells by an activated form of virus encoding wild-type B-Raf. To inactivate Icmt (to H-Ras or K-Ras and sensitized transformed cells to the produce Icmt∆/∆ cells), Icmtflx/flx cells were infected with antiproliferative effects of an FTI (21). 108 PFU/ml of Cre-adenovirus (AdRSVCre). In paral- The phenotype of Icmt deficiency in mice was more lel, control Icmtflx/flx cells were treated with 108 PFU/ml severe than Rce1 deficiency; an Icmt knockout caused of a β-gal adenovirus (AdRSVnlacZ). The efficiency of grossly retarded growth during embryonic develop- Cre-mediated recombination was assessed with South- ment and death at embryonic day 10.5–11.5 (22), pos- ern blotting. All experiments with Icmt∆/∆ cells were ini- sibly due to agenesis of the liver (23). Icmt deficiency tiated three to four passages after the AdRSVCre infec- causes mislocalization of the Ras proteins within cells, tion. To produce mixed populations of Icmtflx/flx and but virtually nothing is known about the effects of Icmt Icmt∆/∆ cells, Icmtflx/flx cells were infected with low titers deficiency on cell growth and oncogenic transforma- of AdRSVCre (106 PFU/ml) and high titers (108 PFU/ tion. To address these issues, we created a conditional ml) of AdRSVnlacZ (so that all of the cells in the dish (“floxed”) Icmt allele, generated fibroblast cell lines, and would be infected with adenovirus). then analyzed the consequences of inactivating Icmt. Icmt activity assay. Icmt activity was measured with base- hydrolysis assays with S-adenosyl-L-[methyl-14C]methio- Methods nine as the methyl donor and either N-acetyl-S-geranyl- Production of a conditional Icmt allele. To generate a con- geranyl-L-cysteine or farnesyl-K-Ras as the substrate. The ditional Icmt allele, exon 1 of Icmt along with upstream accumulation of “methylatable substrates” in cell ex- promoter sequences and parts of intron 1 were flanked tracts was quantified by measuring the methylation of with loxP sites. A 5′ arm of the gene-targeting vector (4 proteins in cell extracts in the presence of recombinant kb in length) was amplified from bacterial artificial Ste14p (the yeast ortholog of Icmt) and S-adenosyl-L- chromosome DNA (24) with primers 5′-CTCTGTGCG- [methyl-14C]methionine (24). GCCGCCTGTGTATAACTGTTTCCTTAGGTATG-3′ and 5′- Analyzing fibroblast growth. Anchorage-dependent ACGACGGCGGCCGCCCGGCGACGCCGGCTCGGGA- growth was assessed with a tetrazolium compound AGGGC-3′ and cloned into the NotI site of pKSloxPNT (MTS)–based cell proliferation assay (Cell Titer 96 (25). A 2.1-kb 3′ arm was amplified with primers 5′- AQueous One Solution Cell Proliferation Assay; ACGACGGCGGCCGCAGGGTAGGTGCACCAGGTA- Promega Corp., Madison, Wisconsin, USA). Icmtflx/flx CATTAGAACCG-3′ and 5′-ACGACGGAATTCCTTCAGTT- and Icmt∆/∆ cells were seeded on 96-well plates (500 or CTGGCCAGAAGATGTTGTCGAGCG-3′ and cloned into 1,000 cells/well, n = 12 wells/cell line, 1 plate per time the polylinker EcoRI site. A 717-bp fragment contain- point) and incubated at 37°C. At various time points, ing promoter sequences, exon 1, and parts of intron 1 20 µl of the MTS reagent ([3-(4,5-dimethylthiazol-2- was amplified with 5′-ACGACTATCGATTTGAATGCG- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)]- CAGGCCGGCCTTCGGGTTTCCC-3′ and 5′-ACGACG- 2H-tetrazolium, inner salt) was added to each well GCGGCCGCATAACTTCGTATAATGTATGCTATAC- and incubated for 2 hours at 37°C. Cell density was GAAGTTATGCCCCATCCCTGACTGATCGGTCCCTG-3′, quantified by analyzing absorbance at 490 nm. The which contained a loxP site. That fragment was insert- relative growth rates
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