Geranylgeraniol Potentiates Lovastatin Inhibition of Oncogenic H-Ras Processing and Signaling While Preventing Cytotoxicity
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Oncogene (1997) 14, 305 ± 312 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Geranylgeraniol potentiates lovastatin inhibition of oncogenic H-Ras processing and signaling while preventing cytotoxicity Terence F McGuire and SaõÈ d M Sebti* Department of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA Oncogenic H-Ras requires farnesylation for its trans- catalyzes the condensation of FPP and isopentenyl forming activity. Lovastatin inhibits both protein pyrophosphate to form GGPP. GGPP and FPP are farnesylation and geranylgeranylation by decreasing utilized by geranylgeranyltransferases (GGTases I and cellular pools of farnesylpyrophosphate (FPP) and II), and farnesyltransferase (FTase), respectively, for geranylgeranylpyrophosphate (GGPP), respectively. Use posttranslational isoprenylation of proteins on carbox- of lovastatin as a chemotherapeutic agent has been yl terminal cysteine residues (reviewed in GruÈ nler et al., precluded by its signi®cant cytotoxic eects. In this 1994; Maltese, 1990; Casey, 1992). FTase and GGTase report, we describe a novel approach utilizing a I prenylate proteins with carboxyl termini that end combination of lovastatin and geranylgeraniol (GGOH) with a CAAX box where C=cysteine, A=aliphatic, to potentiate the ability of lovastatin to block oncogenic and X=any amino acid. FTase prefers X as a serine or H-Ras signaling and concomitantly rescue lovastatin methionine whereas GGTase I prefers X as a leucine or toxicity. GGOH co-treatment with lovastatin enhances isoleucine. GGTase II prenylates proteins that end in inhibition of oncogenic H-Ras processing and constitutive XXCC and XCX where X is any amino acid. For activation of mitogen-activated protein kinase (MAPK), several proteins, isoprenylation is essential for proper and preserves the processing of geranylgeranyltransfer- intracellular localization and biological function (Holtz ase (GGTase) I and GGTase II protein substrates. et al., 1989; Fukada et al., 1990; Der and Cox, 1991; Moreover, co-treatment with GGOH signi®cantly (15- Hori et al., 1991; Inglese et al., 1992). In contrast to fold) attenuates the cytotoxic eects of lovastatin as well FPP, GGPP is currently known to be utilized only for as prevents lovastatin-induced cell rounding. These protein geranylgeranylation and possibly ubiquinone results demonstrate that GGOH potentiates the anti- biosynthesis (ViganoÁ et al., 1995). oncogenic/anti-signaling activity of lovastatin while Geranylgeranylated proteins and farnesylated pro- antagonizing its cytotoxicity. These opposing eects teins appear to comprise distinct, but overlapping sets are due to a GGOH metabolite that serves simulta- of proteins, with the former being greater in number neously as a potent inhibitor for farneslyltransferase as than the latter (Epstein et al., 1990; Farnsworth et al., well as a substrate for GGTases I and II. 1990). Many of these proteins have been shown to play essential roles in signal transduction pathways Keywords: Ras inhibition; lovastatin; geranylgeraniol and some have been implicated in malignant transformation. For example, the geranylgeranylated guanine nucleotide-binding proteins Rho and Rac have recently been shown to be critical players in Introduction regulating not only the organization of the actin cytoskeleton (Nobes and Hall, 1995) but also the The mevalonic acid (MVA) pathway is responsible for progression of the cell cycle through G1 (Olson et al., the biosynthesis of cholesterol and isoprenoid inter- 1995). In addition, Ras, another family of guanine mediates such as geranylgeranylpyrophosphate nucleotide-binding proteins, control normal cell (GGPP) and farnesylpyrophosphate (FPP). Two sites growth (Mulcahy et al., 1985) and dierentiation in the MVA pathway have been cited to be of (Bar-Sagi and Feramisco, 1985) and, when mutated, particular importance: the synthesis of MVA by can produce malignant transformation (Reddy et al., hydroxymethylglutaryl-coenzyme A (HMG-CoA) re- 1982). The Ras family of proteins serve as transducers ductase, an early step thought to be the major point of of extracellular signals from receptor tyrosine kinases regulation, and the so-called `branch-point' of FPP to the nucleus (McCormick, 1993). Their stimulation metabolism (Brown and Goldstein, 1980; Sabine, 1983; by these receptors results in the activation of several reviewed in GruÈ nler et al., 1994). FPP is the last growth-related pathways including a cascade of common intermediate in the pathway and is the mitogen-activated protein kinases (MAPKs) such as substrate for a number of dierent enzymes that Raf, MEK and ERK (McCormick, 1993), the latter of catalyze committed steps in branching pathways which can translocate to the nucleus and regulate the leading to the biosynthesis of cholesterol, ubiquinone, activity of some transcription factors. In some human dolichol, as well as isoprenylated proteins and hemes. cancers, Ras is GTP-locked and constitutively GGPP synthase, one of the branch-point enzymes, activates the MAPK cascade. The ability of Ras to cause cancer requires its attachment to the plasma Correspondence: SM Sebti membrane which is mediated by prenylation (Der and *Present address: H. Lee Mott Cancer Center & Dept. Biochem. Cox, 1991; Kato et al., 1992). The prenylation of Ras Mol. Biol. Univ. South Florida 12902 Magnolia Dr, Tampa, FL in vitro exhibits a preference for farnesylation (James 33612, USA This work was supported by NIH grant CA-55823. et al., 1995). Furthermore, although the prenylation of Received 13 May 1996; revised 12 September 1996; accepted KB-Ras in vivo is, at present, uncertain (Casey et al., 12 September 1996 1989; Lerner et al., 1995), H-Ras has been shown to GGOH/lovastatin: selective inhibition of H-Ras signaling TF McGuire and SM Sebti 306 require farnesylation for its cancer-causing activity a (Hancock et al., 1989; Seabra et al., 1991; Lerner et 12345678 al., 1995). Moreover, in nude mice, inhibitors of U Ras P FTase are eective at suppressing the growth of tumor MAPK–P cells possessing oncogenic H- or K-Ras (Sun et al., MAPK 1995). Lovastatin, a potent competitive inhibitor of HMG- CoA reductase (Alberts, 1988), signi®cantly reduces not b 1234 5678 91011121314 1516 only the biosynthesis of the end-product cholesterol, for which it is used clinically (Illingworth and Bacon, Ras 1989), but also depletes the intracellular pools of Rap1A GGPP and FPP, resulting in the inhibition of protein geranylgeranylation and protein farnesylation (Leonard Figure 1 GGOH potentiates lovastatin-induced inhibition of H- et al., 1990). We have recently shown that lovastatin Ras-CVLS processing and signaling while rescuing that of H-Ras- disrupts early signaling events such as tyrosine CVLL. (a), NIH3T3 cells transformed with either H-Ras- CVLS(61L) (lanes 1 ± 4) or H-Ras-CVLL(61L) (lanes 5 ± 8) were phosphorylation levels of the PDGF receptor and its treated two consecutive days with vehicle (lanes 1 and 5), association with PI-3-kinase (McGuire et al., 1993). lovastatin (20 mM on day 1 and 10 mM on day 2) (lanes 2, 4, 6 Moreover, lovastatin arrests cultured cells predomi- and 8), and 20 mM GGOH (lanes 3, 4, 7 and 8). Cells were lysed and equivalent amounts of protein electrophoresed and immuno- nantly in the G1 phase of the cell cycle and produces a characteristic rounded morphology (Quesney-Huneeus blotted with either anti-Ras antibody (Y13-238) or anti-MAPK as described under Materials and methods. The slower-migrating, et al., 1979; Sinensky and Logel, 1985; Fenton et al., unprocessed form (U) and the processed form (P) of Ras as well 1992). Lovastatin has also been found to inhibit tumor as the inactive and activated forms of MAPK (MAPK and growth of cells expressing oncogenic H-Ras in nude MAPK-P, respectively) are indicated. (b) NIH3T3 cells trans- mice, but at doses that blocked tumor growth, formed with H-Ras-CVLS(61L) were treated two consecutive lovastatin was extremely toxic and some animals died days with vehicle (lanes 1 ± 4) or with lovastatin at 1 mM (lanes 5 ± 8), 5 mM (lanes 9 ± 12), or 15 mM (lanes 13 ± 16) in conjuction with (Sebti et al., 1991). It is not known whether inhibition GGOH at either 0 mM (lanes 1, 5, 9 and 13), 7.5 mM (lanes 2, 6, 10 of protein farnesylation and/or protein geranylgerany- and 14), 15 mM (lanes 3, 7, 11 and 15), or 30 mM (lanes 4, 8, 12 lation, or the reduction in levels of some other end- and 16). Cells were lysed and equivalent amounts of protein product of the MVA pathway, are responsible for electrophoresed and immunoblotted with anti-Ras antibody (Y13- 238). The identical membane was then reprobed with anti-Rap1A lovastatin's toxicity. antibody. Data are representative of three and four independent In this report, we describe the use of lovastatin experiments for a and b, respectively treatment of cells in conjunction with geranylgeraniol (GGOH) as a novel approach for selectively inhibiting oncogenic H-Ras processing and signaling while rescuing cells from lovastatin toxicity. Our mechanistic processed form of Ras. Oncogenic H-Ras constitutively investigations lead to a model where GGOH is activates MAPK and, therefore, both the active converted to a metabolite which concomitantly (hyperphos-phorylated) and inactive (hypophosphory- synergizes with lovastatin to inhibit protein farnesyla- lated) forms of MAPK are observed in control cells tion and which serves as a substrate for geranylger- (Figure 1a, lanes 1 and 5). Treatment of cells with anyltransferases to reverse inhibition of protein GGOH alone had no eect on the processing or signaling geranylgeranylation. of either H-Ras-CVLS or H-Ras-CVLL (Figure 1a, lanes 3 and 7). Lovastatin inhibited the processing of both H- Ras-CVLS and H-Ras-CVLL and inhibited the activa- tion of MAPK slightly in H-Ras-CVLS-transformed Results cells and completely in H-Ras-CVLL-transformed cells (Figure 1a, lanes 2 and 6). GGOH enhanced greatly the GGOH potentiates the ability of lovastatin to inhibit ability of lovastatin to inhibit Ras-CVLS farnesylation oncogenic H-Ras processing and constitutive activation while it restored Ras-CVLL geranylgeranylation (Figure of MAPK 1a, lanes 4 and 8).