1. Field of the Invention
The invention relates to a method of blocking aberrant Ras signaling in a mammal while avoiding excessive cell toxicity.
2. Background Information
The mevalonic acid (MVA) pathway is responsible for the biosynthesis of cholesterol and isoprenoid intermediates such as geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP). Two sites in the MVA pathway have been cited to be of particular importance: the synthesis of MVA by 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), an early step thought to be the major point of regulation, and the so-called "branch-point" of FPP metabolism (Brown and Goldstein 1980; Sabine, 1983; reviewed in Grunler et al., 1994). FPP is the last common intermediate in the pathway and is the substrate for a number of different enzymes that catalyze committed steps in branching pathways leading to the biosynthesis of cholesterol, ubiquinone, dolichol, as well as isoprenylated proteins and hemes. GGPP synthase, one of the branch-point enzymes, catalyzes the condensation of FPP and isopentenyl pyrophosphate to form GGPP. GGPP and FPP are utilized by geranylgeranyl-transferases (GGTases) I and II, and farnesyltransferase (FTase), respectively, for posttranslational isoprenylation of proteins on carboxyl terminal cysteine residues (reviewed in Maltese, 1990; Casey, 1992; Grunler et al., 1994). FTase and GGTase I prenylate proteins with carboxyl termini that end with a CAAX box where C=cysteine, A=aliphatic, and X=any amino acid. FTase prefers X as a serine or methionine whereas GGTase I prefers X as a leucine or isoleucine. GGTase II prenylates proteins that end in XXCC and XCX where X is any amino acid. For several proteins, isoprenylation is essential for proper intracellular localization and biological function (Holtz et al., 1989; Fukada et al. 1990; Der and Cox 1991; Hori et al. 1991; Inglese et al. 1992). In contrast to FPP, GGPP is currently known to be utilized only for protein prenylation.
Geranylgeranylated proteins and farnesylated proteins appear to comprise distinct but overlapping, sets of proteins, with the former being greater in number than the latter (Farnsworth et al., 1990; Epstein et al., 1990). Many of these proteins have been shown to play essential roles in signal transduction pathways and some have been implicated in malignant transformation. For example, the geranylgeranylated low molecular weight (20-28 KDa) guanine nucleotide-binding proteins Rho and Rac have recently been shown to be critical players in regulating not only the organization of the actin cytoskeleton (Nobes and Hall 1995) but also the progression of the cell cycle through Gl (Olson et al. 1995). In addition Ras, another family of guanine nucleotide-binding proteins, control normal cell growth (Mulcahy et al., 1985) and differentiation (Bar-Sagi and Feramisco, 1985) and, when mutated, can produce malignant transformation (Reddy et al., 1982). The Ras family of proteins serve as transducers of extracellular signals from receptor tysosine kinases to the nucleus (McCormick 1993). Their stimulation by these receptors results in the activation of several growth-related pathways including a cascade of mitogen-activated protein (MAP) kinases such as Raf, MEK and ERK (McCormick 1993), the latter of which can translocate to the nucleus and regulate the activity of some transcription factors. In some human cancers, Ras is GTP-locked and constitutively activates the MAPK cascade. Such cancers include, but are not limited to, colorectal, pancreatic and lung carcinomas, and melanoma.
The ability of Ras to cause cancer requires its attachment to the plasma membrane which is mediated by prenylation (Der and Cox 1991; Kato et al., 1992). The prenylation of Ras in vitro exhibits a preference for farnesylation (James et al, 1995). Furthermore although the prenylation of K.sub.B -Ras in vivo is, at present, uncertain (Casey et al., 1989; Lerner et al., 1995b), H-Ras has been shown to require farnesylation for its cancer-causing activity (Hancock et al. 1989; Seabra et al., 1991; Lerner et al. 1995a). Moreover, in nude mice, inhibitors of FTase are effective at suppressing the growth of tumor cells possessing oncogenic H- or K-Ras (Sun et al., 1995).
Lovastatin is a potent competitive inhibitor of HMG-CoA reductase (Alberts 1988) and is used clinically as a cholesterol lowering agent. Following oral administration, the inactive lactone form is hydrolyzed to the .beta. hydroxy acid form, which inhibits HMG-CoA reductase. HMG-CoA reductase catalyzes the reduction of HMG-CoA to mevalonate, an early rate-limiting step in the biosynthesis of cholesterol.
Lovastatin significantly reduces not only the biosynthesis of the end-product cholesterol for which it is used clinically (Illingworth and Bacon 1989), but also depletes the intracellular pools of GGPP and FPP, resulting in the inhibition of protein geranylgeranylation and protein farnesylation (Leonard et al., 1990). It has been recently discovered that lovastatin disrupts early signaling events such as tyrosine phosphorylation levels of the PDGF receptor and its association with PI-3-kinase (McGuire et al., 1993). Moreover, lovastatin arrests cultured cells predominantly in the G.sub.1 phase of the cell cycle and produces a characteristic rounded morphology (Quesney-Hunecus et al., 1979; Sinensky and Logel, 1985; Fenton et al., 1992). Lovastatin has also been found to inhibit tumor growth of cells expressing oncogenic H-Ras in nude mice, but at doses that blocked tumor growth, the animals died (Sebti et al., 1991). It is not known whether inhibition of protein farnesylation and/or protein geranylgeranylation, or the reduction in levels of some other end-product of the MVA pathway, are responsible for lovastatin's toxicity.