Activation of P53 by Oncogenes

Endocrine-Related Cancer (1999) 6 45-48 Activation of p53 by oncogenes

S W Lowe

Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA

Abstract p53 is activated by a variety of cellular stresses, including DNA damage, hypoxia, and mitogenic oncogenes, but the extent to which each signal engages p53 as a tumour suppressor remains unknown. In non-immortal cells, the adenovirus E1A oncogene activates p53 to promote apoptosis, whereas oncogenic ras activates p53 to promote cellular senescence. Inactivation of p53 prevents E1A-induced apoptosis or Ras-induced senescence, allowing proliferation to continue unabated. In each instance, the ability of the oncogene to activate p53 involves the same functions as are required for their transforming potential, implying that p53 activation acts as a fail-safe mechanism to counter hyperproliferative signals. Furthermore, p19ARF is strictly required for oncogene signalling to p53. The fact that ARF - itself a tumour suppressor - acts as an intermediary in this response argues that the tumour suppressor activity of p53 can arise from its ability to eliminate oncogene-expressing cells.

Endocrine-Related Cancer (1999) 6 45-48

Introduction remarkable ability of E1A to enhance radio- and chemosensitivity (Lowe et al. 1993). Although E1A is a The high frequency of p53 mutations in human cancer mitogenic oncogene, p53 acts to limit its oncogenic implies a central role for p53 in tumorigenesis, but the potential. Thus, p53-deficient primary fibroblasts express- signals that trigger p53 in suppressing tumour growth ing E1A are resistant to apoptosis and become oncogeni- remain poorly defined. p53 is a sequence-specific DNA cally transformed (Lowe et al. 1994b). The ability of E1A binding protein that promotes cell-cycle arrest or to activate p53 is not unique, as c-Myc activates p53 to apoptosis in response to a variety of cellular stresses promote apoptosis (Hermeking & Eick 1994) and (Kastan et al. 1991, Graeber et al. 1994, Linke et al. 1996; oncogenic ras induces p53, leading to premature reviewed by Giaccia & Kastan 1998, Prives 1998). For senescence (Serrano et al. 1997). How oncogenic signals example, p53 levels and activity increase after DNA activate p53 is unknown, and it is conceivable that they damage, in part as a result of de novo phosphorylation and induce p53 by inadvertently damaging DNA. Never- the accompanying conformational changes (Shieh et al. theless, the general involvement of p53 in the cellular 1997, Siliciano et al. 1997). Phosphorylation at serine 15 response to oncogenes raises the possibility that these prevents the interaction of p53 with Mdm2 (Shieh et al. stimuli are relevant triggers of the tumour suppressor 1997), a protein that can downregulate p53 via ubiquitin- activity of p53. mediated proteolysis (Haupt et al. 1997, Kubbutat et al. The INK4a/ARF locus is second only to p53 in the 1997). In principle, failure of p53 to suppress proliferation frequency of its disruption in human cancer (reviewed by after DNA damage might indirectly promote tumour Haber 1997). This locus encodes p16INK4a, a cyclin- development by allowing the growth and survival of cells dependent kinase inhibitor that acts upstream of with mutations (Livingstone et al. 1992, Yin et al. 1992, retinoblastoma protein (RB) to promote cell-cycle arrest Griffiths et al. 1997), but whether this provides the (Serrano et al. 1993). Whereas compelling evidence primary driving force for p53 mutation in tumours is indicates that p16INK4a is an important tumour suppressor, unclear. the INK4a/ARF locus encodes a second protein translated Oncogenes can also induce p53, leading to increased in an alternate reading frame, designated p19ARF (Quelle apoptosis or premature senescence (Lowe & Ruley 1993, et al. 1995). p19ARF and p16INK4a are often co-deleted in Hermeking & Eick 1994, Serrano et al. 1997). For tumour cells, but mice lacking p19ARF alone are highly example, the adenovirus E1A oncogene induces p53 and susceptible to cancer (Kamijo et al. 1997; reviewed by promotes apoptosis in primary cells (Debbas & White Haber 1997). p19ARF promotes cell-cycle arrest (Quelle et 1993, Lowe & Ruley 1993, Querido et al. 1997, al. 1995), whereas ARF-null primary mouse embryo Samuelson & Lowe 1997), which is reflected by the fibroblasts (MEFs) do not undergo replicative senescence

Downloaded from Bioscientifica.com at 09/30/2021 05:13:27PM via free access Lowe: Activation of p53 by oncogenes and are transformed by oncogenic ras alone (Kamijo et al. B), c-Jun-N-terminal kinase, Rac, the Rho proteins, and 1997). Thus ARF is a bona fide tumour suppressor. NF-κB (Ridley et al. 1992, Finco & Baldwin 1993, Van Several observations suggest that p19ARF may Aelst et al. 1993, Derijard et al. 1994, Franke et al. 1997, function in a genetic and biochemical pathway that Robinson & Cobb 1997, Rodriguez-Viciana et al. 1997). involves p53. At the level of the organism, the In normal cells, Ras induces premature senescence consequences of deleting p53 and ARF are remarkably through activation of the MAPK cascade - the same similar (Donehower et al. 1992, Kamijo et al. 1997). In pathway important for Ras-induced mitogenesis in either case, the mutant mouse develops normally, but is immortal cells (Lin et al. 1998; see also Zhu et al. 1998). highly predisposed to malignant tumours of a similar In human fibroblasts, Ras ‘effector loop’ mutants that overall pattern and latency. At the cellular level, enforced retain their ability to bind Raf-1 promote premature expression of p19ARF can induce cell-cycle arrest in cells senescence and, among a series of Ras downstream harbouring wild-type, but not mutant, p53 (Kamijo et al. components examined, only activated Raf-1 and MEK 1997). In turn, p19ARF can physically associate with p53 induced p53, p16, and features of senescence. Moreover, itself, with Mdm2, or with both, to alter p53 levels and a MEK inhibitor (PD98059) prevents Ras-induced cell- activity (Kamijo et al. 1998, Pomerantz et al. 1998, Stott cycle arrest and senescence. In primary murine fibroblasts, et al. 1998, Zhang et al. 1998). Nevertheless, ARF is not activated MEK arrested wild-type MEFs, but forced required for the p53 response following DNA damage, uncontrolled mitogenesis and transformation when ex- because radiation induces G1 arrest in ARF-deficient pressed at comparable levels in p53-/- or INK4aex2-/- fibroblasts and apoptosis in ARF-deficient thymocytes MEFs. The precisely opposite response of normal and (Kamijo et al. 1997, 1998). Thus an understanding of the functionally immortal cells to constitutive mitogen- signals that activate p19ARF may help to explain its role as activated protein kinase (MAPK) activation implies that a tumour suppressor, in addition to that of p53. premature senescence acts as a fail-safe mechanism to limit the transforming potential of excessive Ras mitogenic signalling. Thus constitutive MAPK signalling E1A functions that activate p53 activates p53 and p16 as tumour suppressors. To determine how E1A induces p53 and promotes apoptosis, a series of E1A mutants were introduced into Oncogene signalling to p53 primary human and mouse fibroblasts using high-titre ARF recombinant retroviruses, allowing analysis of E1A in involves p19 genetically normal cells outside the context of adenovirus Using MEFs derived from wild-type, ARF -/-, and p53-/- infection (Samuelson & Lowe 1997). In primary human mice, we investigated whether oncogene signalling to p53 and mouse fibroblasts, E1A mutations that prevented p53 involves p19ARF. E1A induces p19ARF mRNA and pro- accumulation and apoptosis separated into two tein, and the ability of E1A to induce p53 and its trans- complementation groups, which correlated precisely with criptional targets is severely compromised in ARF-null the ability of E1A to associate with either the p300/CRE cells (de Stanchina et al. 1998). ARF -/- cells expressing binding protein (CBP) or RB-related proteins. Furthe- E1A are resistant to apoptosis, albeit to a lesser extent then rmore, E1A mutants incapable of binding RB, p107, and their p53-null counterparts. Re-introduction of p19ARF p130 induce p53 and promote apoptosis in fibroblasts restores p53 accumulation and resensitizes ARF-null cells derived from RB-deficient mice, but not in fibroblasts to apoptotic signals. Like E1A, oncogenic ras induces from mice lacking p107 or p130. Hence, inactivation of p19ARF in wild-type cells. Moreover, Ras is unable to RB, but not p107 or p130, is required for p53 arrest ARF -/- cells, which continue to grow at rates similar accumulation and apoptosis induced by E1A. to p53-/- and INK4aex2-/- MEFs. Ras-induced p53 trans- Interestingly, the regions of E1A that promote apoptosis criptional targets are induced in wild-type, but not ARF -/- are precisely those required for the oncogenic potential of cells (unpublished observations). Thus ARF is required for E1A (Whyte et al. 1988), suggesting that apoptosis and p53 activation and cell-cycle arrest induced by oncogenic p53 accumulation are a response to oncogenic ‘stress’ Ras. Our data, together with those of others (Bates et al. rather than a direct effect of E1A. 1998, Palmero et al. 1998, de Stanchina et al. 1998, Zindy et al. 1998) argue that ARF mediates p53 activation by oncogenes. Ras functions that activate p53 We also examined the Ras signalling pathway(s) respon- sible for p53 activation and premature senescence of non- The ARF-p53 tumour immortal human and mouse fibroblasts. Known Ras suppressor pathway effectors include Raf-1 and components of the MAPK Although p53 integrates signals from multiple stress- cascade, phosphoinositide-3-kinase, Akt (protein kinase induced pathways, its action can, in turn, lead to several

Downloaded from Bioscientifica.com at 09/30/2021 05:13:27PM via free access Endocrine-Related Cancer (1999) 6 45-48 possible anti-proliferative responses, depending upon importance in suppressing proliferation of oncogene- cellular context. For example, enforced ARF expression in expressing cells. MEFs induces cell-cycle arrest, but cells overexpressing ARF p19 together with E1A or Myc undergo apoptosis (de Acknowledgements Stanchina et al. 1998, Zindy et al. 1998), which is potentiated by withdrawal of serum survival factors (Evan The author is a Kimmel Scholar and supported by grant et al. 1992, Lowe & Ruley 1993, Lowe et al. 1994a). ARF- CA13106 from the National Cancer Institute (USA). null MEFs are resistant to both E1A- and Myc-induced apoptosis and escape Ras-induced senescence, bypassing References the p53-dependent fail-safe mechanism that normally Bates S, Phillips AC, Clark PA, Stott F, Peters G, Ludwig RL & protects them from these oncogenic signals, and thereby Vousden KH 1998 p14ARF links the tumour suppressors RB enabling mitogenic oncogenes to function as pure growth and p53 [letter]. Nature 395 124-125. promoters. Debbas M & White E 1993 Wild-type p53 mediates apoptosis by Like p53, ARF has no overt role in normal cell cycle E1A, which is inhibited by E1B. Genes and Development 7 control or development; hence, the physiological 546-554. circumstances in which it might become activated to DeGregori J, Leone G, Miron A, Jakoi L & Nevins JR 1997 inhibit proliferation or suppress tumour growth are not Distinct roles for E2F proteins in cell growth control and apoptosis. 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