Fundam Clin Pharmacol (1990) 4, 401-422 0 Elsevier, Paris

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Oncogenic proteins new targets for chemotherapeutic agents against cancer I Rey, P Soubigou, T Cartwright, B Tocque‘ RhGne-Poulenc Santd, Centre de Recherche de Vitry, 94403 Vitry-sur-Seine, France (Received 12 July 1988; accepted 15 January 1990)

Summary - Over the past 10 years, more than 40 potentially oncogenic genes, termed protooncogenes, have been identified in the human genome. Little is known of the physiological role of the proteins encoded by these genes, but they seem to be involved in the reception and transmission of hormonal and other environmental information from the cell membrane to the nucleus. These proteins may acquire transforming properties when over-expressed or if structurally altered following partial deletions or point mutations. Cytogenetic analysis shows loss of genetic material from specific chromosomal loci in many human tumors, suggesting that the absence of a functional gene at these loci may permit tumor development. The genes involved have been termed “anti-oncogenes”. 1 Understanding the control mechanisms of cell proliferation is essential in order to understand how cancer cells escape from this control. To this end, numerous oncogenes have been cloned, permitting the production of modified forms of oncogenic proteins and identification of the regions essential for their biological activity. Availability of large amounts of protein also allows the production of specific antibody which can be used to verify whether blockage of a given protein results in reversion of the transformed phenotype. If it can be shown that the expression of an oncogenic protein is essential for transformation, it should be possible to search for molecules that inhibit its action or which mimic the effects of an anti-oncogene. This type of research is already well advanced for the oncogenic ras proteins, and models have been established that permit both screening for potential inhibitors and design of specific antagonists. oncogenes / therapeutic target / protein kinases / ras

The oncogene

- anti-oncogene concept

Cancer is a disease resulting from complex genetic changes leading to de-regulated cell proliferation. It is widely accepted that the development of human cancer is a multi-step process involving sequential modifications of several genes, rather ~

* Correspondence and reprints

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than a single step conversion of a normal cell to a neoplastic cell (Land et af, 1983 a, b). Thus, each additional gene alteration creates a further phenotypic aberration. This has major implications for possible pharmacological strategies since drugs acting at the beginning of this process would probably be more effective in avoiding tumor development than those acting late in the sequence of mutations.

Oncogenes and proto-oncogenes In the past 15 years, several genes responsible for cancer development have been discovered (Bishop, 1987). The first oncogene identified, named src (for sarcoma) was isolated in 1970 from the Rous sarcoma virus which causes tumors in chickens (Martin, 1970). Later, numerous other retroviruses were shown to be oncogenic (Bishop and Varmus, 1982, 1985). Stehelin et a/ (1976) then showed that src is not uniquely a retroviral gene, but rather an almost exact copy of a gene found in all chicken cells. This normal chicken gene (src proto-oncogene) was presumed to have been acquired by a retrovirus during infection and to have been modified (activated) in such a way as to cause tumor production when inserted into other cells by retroviral infection. Proto-oncogenes are very well conserved during evolution (Stehelin et af, 1976), and their presence in normal cells of all higher organisms suggests that they have a fundamental importance in cellular physiology. It is widely supposed that protooncogenes encode proteins that play a crucial role at different levels of the integration of the mitogenic signals carried by growth factors and hormones. Once modified at either the structural or the control level, proto-oncogenes may behave as oncogenes and favour tumor development. Thus, many oncogenes are the activated homologues of the proto-oncogenes that exist in normal cells. This view is supported by the fact that artificially induced mutation of proto-oncogenes produces deregulated cell growth which may result in immortalization and transformation in vitro (Land et af, 1983 a) and tumor development in vivo (Stewart et al, 1984). Equally, the tumorigenic phenotype may be conferred on cells by artificially enhancing the level of expression of a proto-oncogene by placing it under the control of more powerful transcriptional enhancer sequences (Bishop, 1987; Stern et uf, 1987). The high level of interspecific conservation of proto-oncogenes means that the identification of a new proto-oncogene in an animal permits identification of its human counterpart (Cooper, 1982), and that DNA from human tumor cells expressing an activated oncogene can be used to transform animal cells, (Perucho et af, 1981). These observations indicate that, in this context, the same mechanisms are involved in the generation of human and animal cancers. Thus, it is valid to establish animal cells of well defined genotype which are transformed by human oncogenes and to use these cell lines as models to look for drugs able to inhibit expression of the cancer phenotype. Later, more representative models using human oncogenes in transgenic animals will become available.

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Anti-oncogenes Cell division is stimulated by a variety of signals such as growth factors, hormones etc, but also appears to be subject to negative regulation. The most striking evidence for such negative control is that hybrids formed between normal and transformed cells generally revert to a normal phenotype with the morphology and growth parameters of untransformed cells (Noda et al, 1983). The reversion is sometimes only partial, with revertant cells keeping their abnormal morphology but losing their tumorigenicity (Geiser et al, 1986) or vice-versa (Schaefer et al, i 988). These experiments show unambiguously that oncogene expression is not always dominant, and that oncogenes can behave, in fact, as recessive genes. Moreover, revertant cells derived from v-Ki-ras transformed NIH 3T3 fibroblasts by hybridization with normal 3T3 cells acquire resistance to transformation by other oncogenes including v-src and v-fos (Noda et al, 1983). These observations led to the idea that genes exist (which have been termed “tumor suppressor genes”, “recessive oncogenes”, or more recently “antioncogenes”) which function by antagonizing the action of oncogenes. It has been proposed that, in some cases, it is the function loss of genes of this class that results in cancer development (Klein, 1987). The best documented example of the loss of an anti-oncogene resulting in human oncogenesis is that of the retinoblastoma gene (RB) where loss of both alleles at a single locus on chromosome 13 is necessary for tumor development. In the case of familial retinoblastoma, one copy of this gene is lost in the germ-line and the second is lost as a result of a subsequent somatic mutation. In sporadic retinoblastoma, both copies are lost in successive somatic mutations (Hansen and Cavenee, 1988). It is probable that the RB gene product, pl05 RB, regulates expression of specific cellular genes involved in mitogenesis (Lee et al, 1987 a, b). It has recently been shown that the neoplastic phenotype of retinoblastoma and osteosarcoma can be suppressed by transfection with the RB gene (Huang et al, 1988) confirming the key role of the RB gene in the control of carcinogenesis. Deletion of other genes has been shown to be involved in the development of several other human tumors suggesting that the anti-oncogene phenomenon may be widespread (Solomon et al, 1987; Law et al, 1988; Ponder, 1988).

Oncogenic DNA viruses Several DNA viruses are potentially oncogenic for humans (eg hepatitis B, Epstein-Barr, and papilloma viruses) but the oncogenic genes involved differ fundamentally from the cellular proto-oncogenes and their activated homologues. Genetic material from DNA viruses may integrate into the human genome and modify cell metabolism but does not represent a modified form of normal cellular genes. We now know that these viruses are oncogenic for several reasons:

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(i) expression of viral proteins can modify cellular proto-oncogene proteins with a similar result to that produced by proto-oncogene activation as described above ; for instance, it has been shown that the middle T protein of polyoma virus binds to src protein in the cytoplasm of infected cells (Courtneidge and Smith, 1983); the consequence is a dramatic increase in src tyrosine kinase activity following its phosphorylation at an unusual site (Bolen et al, 1984); (ii) it has recently been demonstrated that the transforming proteins of DNA viruses can act by binding to cellular nuclear proteins encoded by anti-oncogenes (De Caprio et al, 1988; Whyte et al, 1988i; Dyson et al, 1989), thus leading to neutralization of their activity in cell growth regulation. The biological functions of the different classes of oncogenic and antioncogenic proteins are still poorly understood. A major effort is required to identify or to design molecules able to interact specifically with oncoproteins and to modulate their oncogenic activity. Similarly, restoring or mimicking the deficient activities of anti-oncogenic proteins must be one of the goals of future cancer chemotherapy.

Mechanisms of proto-oncogene activation and anti-oncogene inactivation

Proto-oncogenes become transforming upon modification either of their nucleotide sequence or of their transcription and expression, so that the cell produces an abnormal protein with modified biochemical properties or expresses an abnormally high amount of the protein. Neoplastic transformation can also occur when an anti-oncogene is lost or its activity neutralized. In man there seems to be three main ways leading to oncogenic activation of proto-oncogenes : point mutations, chromosomal rearrangements and gene amplification (Land et al, 1983 a, b). Point mutations and chromosomal rearrangements may also be responsible for inactivation of anti-oncogenes.

Point mutations Molecular cloning and sequencing of ras proto-oncogenes has identified two main codons which can be modified by mutations to generate transforming alleles (Reddy et al, 1982;Tabin et al, 1982;Taparowski et al, 1982;Fasano et al, 1984; Newbold, 1984). There are at least three members of the ras gene family, each of which can become oncogenic when mutated at codons 12 and 61 (Barbacid, 1987). In vitro mutagenesis studies have demonstrated the existence of other sites potentially implicated in activation of the transforming properties of ras genes (Fasano et al, 1984). RB, the prototype anti-oncogene, is associated with the development of retinoblastomas in children, osteosarcomas and other types of human tumors (Friend et al, 1987). Several point mutations have been described which result in pl05 RB

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inactivation and these represent the initial event in the development of some tumors (Dunn et al, 1988i; Horowitz et al, 1989; Toguchida et al, 1989). Chromosomal rearrangements Of the chromosomal rearrangements, translocations are the best defined molecularly. In man, translocations involving c-myc proto-oncogene are very frequent in leukemias of both T and B cell lines (Croce, 1987). In Burkitt lymphoma, 80% of the neoplasms examined showed a characteristic translocation of the c-myc gene from chromosome 8 to chromosome 14 (Dalla-Favera et al, 1982). As a result of this event, the myc gene is moved to the immunoglobulin heavy chain locus and comes under the control of the powerful immunoglobulin transcriptional activating sequence. Thus, myc transcription is greatly enhanced in leukemic cells and is no longer responsive to its normal transcription control mechanisms (Croce, 1987). Increased c-myc transcription can also arise after the loss of the first exon of the gene during chromosomal translocation. A regulatory transcriptional role has been ascribed to this exon (Robertson, 1984), and moreover, its absence seems to protect mRNA from degradation (Piechaczyk et al, 1985), leading to an overproduction of the c-myc protein. Structural rearrangements may also be responsible for RB anti-oncogene inactivation. Thus, partial or total gene deletions and duplication of an exon have been shown in breast cancer cell lines (T’Ang et al, 1988). In each case, there was production of a truncated inactive RB protein or no protein at all. Similarly, rearrangement of the p53 gene has been involved in the formation of osteogenic sarcomas (Masuda et al, 1987). Gene amplication Proto-oncogene amplification has been described for N-myc in neuroblastomas (Seeger et al, 1985) and retinoblastomas, for L-myc, c-myc and N-myc in small cell lung carcinomas (Saksela et al, 1986) and for erb B2 in breast carcinomas (Slamon et al, 1987). In the case of ras proto-oncogenes, considerable amplification of Kirsten ras (Ki-ras) gene, over 40-fold, has been found in a primary bladder tumor (Fujita et al, 1985), and similar amplification (50-60-fold) has been detected in a lung carcinoma (Pulciani et al, 1985). Amplification of ras proto-oncogenes is a frequent phenomenon in established tumor cell lines (Nishimura and Sekiya, 1987).

Oncogenic proteins Proto-oncogenes are thought to regulate DNA replication and hormonal signal transduction processes via their different protein products. Known oncogene and

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proto-oncogene products fall into several functional classes : (i) Growth factors (sis, hst); (ii) Growth factor receptors (erb B, fms, kit, ros, met, neu, trk) and tyrosine kinases (src, fgr, fes, lck, yes); (iii) Membrane bound analogues of GTP binding proteins (ras) ; (iv) Cytoplasmic serine and threonine kinases (mil, mos, raf) ; (v) Nuclear proteins acting as transcriptional activators (myb, myc, fos, jun, c-erb A, sis, ets). The main functional groups of oncogenic proteins are summarized in figure 1 which also indicates their probable intracellular localization. Putative antioncogene products are indicated in large type. Members of each of these classes are over expressed in subsets of human cancers and their co-ordinated overproduction is thought to contribute to a variety of tumor properties including metastasis and invasion as well as cell proliferation (Weinberg, 1985). Figure 2 shows how oncogene encoded proteins from these different functional classes might fit into the cascade of events that regulates DNA replication in mammalian cells. Stimulation of GO arrested cells by growth factors or hormones leads to a rise in intracellular pH, increase in free internal calcium and activation of a considerable number of protein kinases (Rozengurt, 1986). This intense cellular activity results in part from phosphatidylinositol 4 3 bisphosphate (PIP2) breakdown (Berridge and Irvine, 1984) and opening of membrane Ca2+ channels induced by receptor -associated tyrosine kinase activity. This is always followed

ONCOGENES AND ANTI-ONCOGENES External signals

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Transduction of external signals

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Transmembrane receptors Tyrosine kinases

GTP-binding proteins

Nuclear events

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METABOLIC RESPONSE Fig 1. Oncogenes and anti-oncogenes : the main functional groups of oncogenic proteins and their probable intracellular localization. Putative anti-oncogenes are indicated in large type.

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by bursts of CAMP-dependent protein kinase activity. A result of the activation of this cascade is the transient expression, within a few minutes, of some nuclear proto-oncogene products such as myc, fos and jun which exert direct effects on transcription. The bypassing or the subversion of the receptor-controlled Ca+ phospholipid hydrolysis/protein kinase C pathway is probably behind the escape from regulatory control of cell multiplication which characterizes all neoplastic tranformations. Confirmation of this view is provided by the recent demonstration that the disruption of CAMP production in human pituitary tumors results from a single point mutation in the well documented Gs protein. (Landis et al, 1989). This is one of the first reports that establishes a direct connection between a known second messenger, a point mutation in a well characterized gene and the development of a human tumor.

Use of antisense mRNA and oligonucleotides to decrease expression or to regulate the activity of oncogenic proteins Recently, naturally occurring regulator genes have been discovered that govern the synthesis of species of RNA capable of directly controlling the expression of other

CELLULAR SIGNALLING = RELATIONSHIP BETWEEN THE DIFFERENT ONCOGENES I

PIP 2 signalling pathway alterations

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Tyrosino kinases

Transcription

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METABOLIC RESPONSE Fig 2. Cellular Signalling : functional relationships between the different classes of oncogenic proteins and their probable position in the cascade of events leading to the proliferation of growth arrested cells.

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genes (Green et af, 1986). These RNA repressors are highly specific inhibitors of gene expression which act as anti-sense RNA by hybridization with the sense mRNA from the gene to be controlled. Such RNA/RNA complexes have been shown to inhibit mRNA translation, but the intranuclear localization of duplexes suggests that transport or processing of RNA might also be affected (Kim and Wold, 1985). These observations led to the development of strategies to artificially control the expression of harmful genes such as oncogenes. Several recent reports suggest that the suppression of specific gene products can indeed be achieved by the expression of antisense RNA (Weintraub et af, 1985). This approach has been used to directly demonstrate the role of c-fos protooncogene in cell proliferation (Holt et af, 1986). In this study, antisense RNA complementary to c-fos mRNA was produced in mouse 3T3 cells under the control of a steroid inducible promoter. Induction of the antisense RNA was shown to inhibit cell proliferation to an extent proportional to the degree of inhibition of production of the c-fos protein. It was also shown that some expression of the c-fos protein is essential for normal cell division. The use of such gene transfer techniques in human therapy remains far in the future. However, it is possible, using existing technology, to produce candidate oligonucleotides, and, using an in vitro translation system, to select those capable of effective inhibition of a given gene. Thus, oligonucleotides complementary to

MODEL FOR GAP PROTEINS AS DOWNSTREAM EFFECTORS FOR RAS AND RAP

METABOLIC RESPONSE cell divbion dlffermtiatbn development

Fig 3. GAP proteins as downstream effectors for ras and rap: a model for the interaction of ras and rap proteins with their respective GAP proteins in the positive and negative regulation of cellular proliferation.

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the region of the c-myc gene including the initiation codon, have been shown to inhibit mitogen-induced c-myc expression in human T lymphocytes (Heikkila et al, 1987) and HL-60 promyelocytic cells (Holt et af, 1988). This very promising approach has howewer encountered major practical difficulties due to problems of stability, bioavailability and toxicity of oligonucleotides. The use of double stranded oligonucleotides which mimic binding sites on DNA may also prove useful by reducing by way of competition, the quantity of transcription factors available for binding to the specific DNA sequences involved in oncogene expression. In addition, oligonucleotides have been produced that bind directly to duplex DNA in a site specific manner. The resulting formation of a triple helix was shown to inhibit the binding of transcriptional activating factors, thus suggesting another approach to specific suppression of harmful genes (Maher et al, 1989). Development of in vitro transcription systems might facilitate identification of transcription factors specific for an oncogene. For instance, the expression of ras genes is developmentally regulated in Dictyostelium discoideum (Pawson et al, 1985). The level of both ras transcripts and ras proteins is highest in vegetative amoeba and declines with the onset of differentiation. This finding indicates that differentiated cells either lack an inducer or gain a repressor of ras gene expression. Identification of the putative ras repressor from differentiated Dictyostefium cells could provide a strategy for therapy of ras-induced cancers.

Protein acylation Post-translational specific fatty acylation is required for the anchoring of certain cellular proteins to the cell membrane (Sefton and Buss, 1987). Thus, myristylation of p60 src, p65 gag or palmitoylation of p21 ras stabilize the interaction of these proteins with membranes. This protein membrane interaction is, important biologically since point mutations that prevent acylation prevent membrane interaction, and abolish biological activity (Willumsen et al, 1985; Buss et al, 1986) p21 ras is acylated at a cysteine residue 4 amino acids from the C-terminus which is followed in the sequence by 2 aliphatic residues. These residues are conserved in yeast (Powers et al, 1984) and in Dictyostelium (Reymond et af, 1984) ras proteins, and also in other partially homologous proteins (Madaule et af, 1987). It has recently been shown by Hancock et al(l989) that the acylation process is complex and first involves a polyisoprenylation of the C-terminal cysteine followed by palmitoylation at the cysteine 4 residues upstream. All p21 ras molecules ares polyisoprenylated, but not all are necessarily palmitoylated. Molecules that are only polyisoprenylated are still localized at the membrane and can still produce transformation, however, palmitoylation increases both the avidity of membrane binding and transforming activity (Hancock.et af, 1989). THe importance of the nature of the membrane binding group has also been demonstrated in the work of Buss et a1 (1989), who showed that substitution of myristate

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for palmitate in actived p21 ras still permitted membrane localization and transformation. Surprisingly however, myristylated forms of the normal cellular ras product were also transforming, showing that activation of a cellular protooncogene can be obtained simply by modification of its interaction with the cell membrane. The enzymes responsible for the acylation of proteins belonging to the ras family have not yet been identified. However, once purified and characterized, these enzymes could be used in screening or in drug design studies to discover specific inhibitors of ras protein acylation. Non-acylated ras p21 produced in E coli could be used as substrate in these studies. It should thus be possible to modulate the activity of some oncogenic proteins at the post-translational level, perhaps by using drugs that interfere with the enzymes of isoprenoid synthesis. Isoprenoids are derived from mevalonate and are precursors in the synthesis. of cholesterol and other complex lipids. In this context, it is interesting to note that drugs like mevinolin or lovastatin, which are inhibitors of cholesterol synthesis and which deplete cellular isoprenoid pools (Doyle and Kandutsch, 1988 ; Repko and Maltese, 1989) also exhibit anti-ras activity (Schafer et al, 1989).

Growth factor antagonists

Involvement of growth factor-like molecules It is well known that the development and maintenance of certain kinds of human tumors are strictly dependent on the presence of specific growth factors. One obvious anti-tumor strategy is thus to identify any growth factor involved in expression of the transformed phenotype and to search for antagonists. This approach was tested using anti-growth factor antibodies. Thus, small cell lung carcinoma (SCLC), with is an aggressive form of lung cancer, is characterized by secretion of the neuropeptide bombesin which is not secreted by other lung cancers (Moody et al, 1981). Very elaborate experiments were designed to demonstrate that anti-bombesin antibodies block the binding of bombesin to cellular receptors, and inhibit the clonal growth of SCLC cells in vifro and of SCLC xenografts in vivo (Cuttita et al, 1985). Peptide antagonists of bombesin that inhibit the growth of SCLC cell lines have also been described (Woll and Rozengurt, 1988). Human melanoma cell lines express a platelet derived growth factor (PDGF) like protein that can stimulate 3H thymidine incorporation in human fibroblasts (Westermark et al, 1986). Similar factors have been found in human glioma cells (Nister el al, 1984). The synthesis of PDGF or a closely related polypeptide by human sarcomas indicates an abnormal expression of the c-sis gene (encoding the B chain of PDGF) which may contribute to the transformation of these cells. Other growth factor-like molecules with possible transforming activity include

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transforming growth factor (Y (TGFcu) (Todaro et al, 1980), TGFP (Tucker et al, 1984) and basic fibroblast growth factor (bFGF) (Rogelj et al, 1987).

Growth factor receptors The modification of growth factor receptors represents another way in which oncogenes may become activated. Thus v-erb-B is a truncated form of the EGF receptor and v-fms is an altered macrophage CSF-1 receptor (Ullrich et al, 1984; Sherr et al, 1985). The neu gene is frequently activated in chemically induced neuro- and glioblastomas of the rat. The protein encoded by the neu gene, p185, has a domain structure consistent with a function as a membrane receptor. Although p185 shows about 50% homology with the EGF receptor, the neu gene and the erb-B gene are distinct and located on different chromosomes. The human counterpart of neu has been identified and designated c-erb-B 2 (Yamamoto et al, 1986) or HER2 (Coussens el al, 1985), and may be involved in the pathogenesis of some human breast cancer (Slamon et al, 1989). Unlike the major structural changes involved in erb-B activation, a single base mutation in the rat neu gene, producing a single amino acid change in the transmembrane domain, appears to be sufficient to create tranforming potential (Bargmann et al, 1986). It was suggested that this mutation (Val to glu) might stabilize the receptor protein in a conformation normally occupied transiently in response to ligand binding and thus permit the receptor to continue to transmit a proliferation signal even in the absence of ligand. In human breast cancer it appears that the HER2 gene is not mutated but is over-expressed (Slamon el al, 1989). The ligand for the neu encoded protein is still unknown. Its identification and the synthesis of antagonists and inhibitors of receptor activation may offer new possibilities in cancer therapy (Yarden and Weinberg, 1989).

Anti-proliferative molecules Endogenous anti-proliferative molecules represent area for anti-cancer research. One interesting example is TGF P, the prototype of a large family of dimeric proteins that control cell growth and differentiation in a wide range of cell types. TGF /3 can either have growth promoting or growth inhibitory activity depending on cell type and conditions. Thus, TGF P stimulates proliferation of cells of mesenchymal origin, apparently by inducing the expression of the c-sis protooncogene which codes for a PGDF-like protein (Leof et al, 1986). TGF P also inhibits proliferation of cells of epithelial origin, endothelial cells, B lymphocytes, and thymocytes, and induces differentiation of several cell types (Sporn et al, 1986). Studies of the molecular mechanism of TGF 8 , inhibition of endothelial cell growth have shown that the expression of high affinity EGF receptor is reduced in TGF3!, treated cells, and that such cells become incapable of responding to the

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mitogenic signal of EGF (Takehara et a/, 1987). Furthermore, it has recently been shown that whereas retinal cells are normally responsive to the anti-mitogenic effects of TGF p, retinoblastoma cells may cease to express TGF ,8 receptor and this may represent one mechanism by which these cells escape from negative control of proliferation (Kimchi et al, 1988). Other endogenous anti-proliferative proteins include TNF CY and interferon y which both inhibit, apparently by different mechanisms, the expression of c-myc which is essential for entry into cell division (Yarden and Kimchi, 1986). Both of these factors have shown to exert anti-tumor effects.

Oncogenes and protein kinase activity Nearly half of all known oncogene products are protein kinases. The proteins encoded by several oncogenes such as yes, fgr, abl, fps, fes, ros and src show similarities in their catalytic domains, suggesting the existence of a possible common ancestor (Hunter, 1984). These genes may become activated either by overexpression or by point mutations. Over-expression seems to occur at the transcriptional level as examplified by the c-erb-B oncogene in breast cancer (Davidson et al, 1987). In the case of the src gene, a point mutation results in production of an altered protein with increased and uncontrolled tyrosine protein kinase activity (Hunter, 1984). In addition to the level of activity of the different kinases, availability of specific substrates also contributes to the complexity of control by protein phosphorylation. At present, little is known about the substrates of the oncogenic protein kinases, but it appears that these enzymes are essential elements in intracellular regulatory circuits. Several observations support the idea that the kinase activity associated with oncogenic proteins is important for the induction of transformation. (i) Transformation of cells with this class of oncogenes leads to increased levels of phosphotyrosine in proteins (Sefton et al, 1980). (ii) Transformation capacity can be reduced or abolished if protein kinase activity is impaired (Erikson et a/, 1979; Cooper et a/, 1983), however this does not prove that tyrosine phosphorylation is the key event in this transformation. (iii) Vanadate inhibits phosphatases that remove phosphate groups from phosphotyrosine, and accordingly produces a major (40-fold) increase in protein phosphotyrosine when added to cell culture medium; in parallel, vanadate induces expression of the transformed phenotype in several different kinds of cell (Klarlund, 1985), thus providing a further indication that protein phosphorylation is indeed critical for transformation. (iv) The erb B2/HER-2 (neu) gene product, an analogue of the EGF receptor, possesses intrinsic tyrosine kinase activity. In several chemically-induced rat neuroblastomas the neu gene product is expressed in a structurally altered form that probably exhibits constitutive tyrosine kinase activity (Bargmann et al, 1986).

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Monoclonal antibodies directed against neu protein have been shown to suppress the neoplastic phenotype of cells Carrying mutated alleles of this gene (Drebin et al, 1985). (v) Riedel et a1 (1987) constructed a chimeric protein composed of the extracellular ligand-binding and the transmembrane domains of the human EGF receptor with the cytoplasmic domain of v-erbB which carries abnormal, constitutively activated, tyrosine kinase activity. This hybrid molecule retained transforming capacity, showing that the addition of the ligand binding domain was not sufficient to restore the transforming protein to a normal, ligand-sensitive condition, and that the abnormal, unregulated enzyme is responsible for its oncogenic potential. (vi) Several specific tyrosine kinase inhibitors were identified. One of them, herbimycin, has been shown to reverse expression of the transformed phenotype by cells expressing the src oncogene (Murakami et al, 1988). Another antibiotic protein kinase inhibitor, erbstatin, inhibits in vitro growth of human epidermoid carcinoma A431 cells (which are very rich in EGF receptors) together with EGFstimulated receptor autophosphorylation (Umezawa et al, 1986). Several analogues of erbstatin have been synthetized that block EGF-dependent proliferation of A431 cells in vitro, apparently by competing with substrate for the EGF receptor kinase (Yaish et al, 1988). No in vivo antiproliferative activity of these molecules has yet been reported.

Ras oncogenes: targets for antitumor drugs? Because ras oncogenes activated by single nucleotide substitutions are associated with a significant percentage of human cancers of diverse histological origin (Barbacid, 1987; Bos, 1988), there is intense interest in elucidating the biological functions of the ras gene products. The ras gene family encodes 21 kDa membrane associated proteins, and antibodies to these proteins can block transformation by activated ras oncogenes (Feramisco et al, 1985; Kung et al, 1986). These oncogene products thus represent important targets for therapeutic intervention. The p21 proteins show significant structural similarity with the G proteins which behave as molecular switches in intracellular signal transduction, and also share with G proteins several key biochemical properties including GTP binding and GTPase activity (Gibbs et al, 1984; Sweet et al, 1984). Oncogenic mutations at position 12 or at position 61 specifically impair intrinsic GTPase activity so that activated p21 proteins have an in vitro GTPase activity 10-fold less than that of normal p21 (Gibbs et a/, 1984; Sweet et al, 1984). Furthermore, oncogenic mutants of p21 lose the ability to be stimulated by endogenous GAP (GTPase activating protein) which significantly increases the GTPase activity of normal ras p21s (Trahey and Mc Cormick, 1987). It is estimated that the combination of these 2 effects reduces GTPase activity of the oncogenic p21 proteins by a factor of lo00 (Trahey and Mc Cormick, 1987). By analogy with other G proteins, it is probable that this

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impaired hydrolysis of GTP results in either permanent or less regulated signal transmission by p21s that are unable to return normally to their resting state (figure 2). The fact that ras proteins require GTP for transformation is consistent with this view (Lacal and Aaronson, 1986). It follows from the above that one pharmacological approach might be to look for drugs that restore the wild-type levels of intrinsic GTPase activity to mutated ras p21, and thus to cause reversion of the ras transformed cells (Sigal et al, 1987). The recently putli;:led crystal structure of the Ha-ras/GDP complex (De Vos et al, 1988; Tong et al, 1989) and of the Ha-ras GTP complex (Pai ef al, 1989) showing that oncogenic mutations are near the GTPase catalytic site, supports this approach. It might be more useful, however, to look for compounds able to restore the stimulation of ras GTPase by GAP, since, in addition to enhancing GTPase activity, GAP interacts with ras p21 at a site previously identified as the ras effector region (Sigal et al, 1986), strongly suggesting that GAP represents the natural biological target for regulation by p21 (Adari et al, 1988; Cales et al, 1988). Recently this view was strengthened by the work of Vogel et a1 (1988) in which the GA P gene was cloned and shown to contain sequences homologous to those of known effector molecules such as non-receptor tyrosine kinases and phospholipase C. Thus, the displacement of oncogenic p21 from GAP could provide another target for therapeutic action (Sigal et al, 198). In fact the discovery of drugs that specifically block the ras-dependent mitogenic cascade at any level would probably be of use in cancer therapy.

Anti-ras bnti-oncogenes Recently, two independent groups have cloned a human gene which can abolish the transformed phenotype when transfected into cells transformed by the v-Ki-ras oncogene. This gene has been named K-rev 1 (Kitayama el al, 1989; Noda et al, 1989) or rap 1 (Pizon et al, 1988). The protein encoded by this gene is a 21 kDa GTP binding protein which shows about 50% homology with Ha-ras p21 (Pizon et al, 1988; Kitayama et al, 1989). This homology is especially strong at sites corresponding to the presumed functionally important regions of Ha-ras p21 including the phosphoryl group binding domain (residues 5-22), the guanine binding domain (residues 116-120 and 145-147) and the C-terminal membrane localization sequence. In particular, the putative effector region of Ha-ras p21 (residues 32-44) is identical with the corresponding region in the rap 1/K-rev 1 protein. This suggests that rap 1 might have opposing effects to ras p21 on a common or analogous cellular target, or that, in contrast to the presumed growth promoting role of the ras proteins, rap 1 could act as a G protein involved in the transduction of a growth inhibitory signal. Kikuchi et al (1989) have also isolated a GTP binding protein termed smg p21 which was shown to be identical to the rap 1 and K-rev 1 gene products. Bovine brain contains two different GTPase activating proteins, GAP-1 and GAP-2,

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which stimulate the GTPase activity of smg-p21 but not that of c-Ha-ras or of several other GTP binding proteins. GAP-1 and GAP-2 are chromatographically distinct from the c-Ha-ras p21 GAP and from other GAP proteins, raising the possibility that each 21 kDa G protein may operate through its own specific GAP (s) (Kikuchi et al, 1989). A possible model for the intersection of GAP proteins with ras and rap is shown in figure 3. On the basis of these experiments it is tempting to propose that molecules which inhibit specific p21-GAP interactions associated with cell proliferation or which promote or mimic the p21-GAP interactions associated with growth inhibitory signals might be potentially useful as novel anti-cancer drugs. It must, however, be remembered that unequivocal evidence that GAP is the ras protein target has not yet been obtained. Recent studies by Gibbs et al (1989) support the notion that GAP may be the cytosolic factor required for ras activity. In these experiments, microinjection of a truncated, non membrane-bound RAS 1 protein into Xenopus oocytes inhibited germinal vesicle breakdown stimulated by either insulin-like growth factor 1 or by microinjection of [vaI**]Ras. This inhibition could be reversed by co-injection of purified mammalian GAP, suggesting that the cytosolic factor involved is either GAP or a factor with which GAP competes for binding to the truncated RAS protein. Although the intracellular targets of Ras and the control circuits involved are not perfectly understood, the information currently available shows that the ras system is important in the control of cell proliferation and is sufficiently accessible at the molecular level to represent a major target for the identification of new anti-cancer drugs by screening and drug design.

Conclusion Many different cellular events may give rise to cancer, but damage to DNA remains as a unifying theme. Studies of the different proto-oncogenes discussed here show how genetic lesions of various types may result in the expression of aberrant growth signals, or in the case of the anti-oncogenes, in suppression of growth inhibitory signals. As the molecular mechanisms of signal transduction between the extracellular and intracellular domains of receptors, the molecular events by which receptor occupancy is coupled to the production of early growth signals, and the pathways by which these signals induce the expression of regulatory genes become unravelled, it is clear that each step in the proliferation signal cascade can be modified and may contribute to the expression of the transformed phenotype. Thus each of these steps represents a potential target for therapeutic intervention. Further research on the molecular mechanisms involved, particularly on the control of transduction by tyrosine phosphorylation and by GTPase activity is therefore of fundamental importance. It is also important to determine whether transformation induced by an oncogene can be repressed at any time, or whether cells gradually acquire a higher degree of transformation which becomes independent of the earliest oncogenic stimuli.

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Anti-oncogenes represent a particularly promising approach for the restoration of normal control of cellular proliferation. Since oncogene expression is not a dominant character and tumor development results from both proto-oncogene activation and anti-oncogene inactivation (Bishop, 1987), a major aim in future cancer chemotherapy will be to sustain the expression of anti-oncogenes or to substitute or replace their defective function. This should permit a more specific and less traumatic treatment of cancer.

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