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Cancer Therapy (2011) 18, 2–11 r 2011 Nature America, Inc. All rights reserved 0929-1903/11 www.nature.com/cgt

REVIEW Understanding wild-type and mutant activities in human cancer: new landmarks on the way to targeted therapies I Goldstein1,3, V Marcel2,3, M Olivier2, M Oren1, V Rotter1 and P Hainaut2 1Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovat, Israel and 2Molecular Group, International Agency for Research on Cancer, Lyon, France

Three decades of p53 research have led to many advances in understanding the basic biology of normal and cancer cells. Nonetheless, the detailed functions of p53 in normal cells, and even more so in cancer cells, remain obscure. A major breakthrough is the realization that mutant p53 has a life of its own: it contributes to cancer not only through loss of activity, but also through gain of specific ‘mutant functions’. This new focus on mutant p53 is the rationale behind the meeting series dedicated to advances on mutant p53 biology. This review provides an overview of results presented at the Fourth International Workshop on Mutant p53, held in Akko, Israel in March 2009. New roles and functions of p53 relevant for tumor suppressions were presented, including the regulation of microRNAs networks, the modulation of cell–stroma interactions and the induction of . A main focus of the meeting was the rapidly growing body of knowledge on autonomous properties of mutant p53 and on their oncogenic ‘gain of function’ impact. Importantly, the meeting highlighted that, 30 years after p53 discovery, research on mutant p53 is entering the clinical and translational era. Two major steps forward in this respect are a better understanding of the active mechanism of small drugs targeting mutant p53 in tumor cells and an improved definition of the prognostic and predictive value of mutant p53 in human cancer. Cancer Gene Therapy (2011) 18, 2–11; doi:10.1038/cgt.2010.63; published online 22 October 2010 Keywords: p53; tumor suppressor ; gain of function; p53 targeting drug; suppressive function

Introduction The TP53 gene is often mutated in cancer with a high proportion of missense mutations, leading to the produc- The p53 tumor suppressor protein, encoded by the TP53 tion of a protein expressed at high levels that differs from gene (OMIM 191170), lies at the center of an elaborated wild-type p53 by just one residue. This feature circuit controlling and interconnecting is a characteristic of p53, as many ‘classical’ suppressor with many cellular functions. In response to various , such as APC or BRCA1, are mainly altered forms of stress, p53 is activated and accumulates in the through nonsense or frameshift mutations leading to the nucleus, where it regulates the of numerous expression of inactive truncated . In the few years target genes using specific DNA response elements. From following the p53 discovery in 1979, it was reported that the p53 activation, several biological responses occur missense mutant p53 might exert some oncogenic function including , arrest, DNA repair, (or gain of function (GOF)). In particular, they can differentiation or senescence. The nature of the p53- cooperate with , such as Ras, to transform induced response depends on the type of stress and primary rodent fibroblasts. For more than a decade, the cell/tissue in which it occurs. Beyond its nuclear role, most convincing observation supporting a GOF has been p53 regulates some mitochondrial functions. Thus, p53 that mutant p53 is often expressed at high levels is the archetype of a truly multifunctional protein, throughout tumor progression stages, even in distant in which a small number of structural domains is metastases. Soon after the elucidation of the main specifically involved in vast arrays of complex interactions mechanisms controlling wild-type p53 in the 1990s, the with various species (DNA, RNA, proteins and cell hunt for mutant p53 functions became a major goal in metabolites). cancer research. Today, a large body of knowledge has accumulated on mutant p53 GOF, which appears as a major contender in human cancer. We summarize here some of the recent trophies of the hunt for mutant Correspondence: Dr P Hainaut, Molecular Carcinogenesis Group, International Agency for Research on Cancer, Lyon 69372, France. p53 functions, which were presented at the Fourth E-mail: [email protected] International Workshop on Mutant p53 (Akko, Israel (http:// 3These authors contributed equally to this work. www-p53.iarc.fr/P53meeting2009/P53meeting2009.html)). Received 24 February 2010; revised 22 June 2010; accepted 16 Lecture numbers available on following link: (http:// August 2010; published online 22 October 2010 www-p53.iarc.fr/p53meeting2009/Abstracts.html). Understanding wild-type and mutant p53 activities in human cancer I Goldstein et al 3 Modulation of p53 activity could be used in yeast and human cells, such as in the Importance of DNA sequence and structure to p53 VEGF and RAP80 genes. In the VEGF , the binding abilities second half site is constituted by an - binding element, while in RAP80, the half site becomes In response to stress, accumulation of p53 results in its 4,5 binding as a tetramer to DNA in a sequence-specific active only under exposure to UV. These observations manner. Initial in vitro studies identified a p53 consensus indicate that p53 transcriptional activity may be much response element (p53RE), which consists of a palindro- more complex than previously expected, with interplays mic sequence composed of two halves, each one consisting between p53 and other transcription factors favored by of a sequence matching the consensus RRRCWWGYYY the proximity of their DNA binding sites. (R: purine; W: A or T; Y: pyrimidine), separated by a The role of p53 acetylation with respect to DNA spacer (n) from 0 to 13 nucleotides of any type1 (Figure 1). binding has been highlighted by Prives (L7). Indeed, she This symmetric DNA motif is consistent with the binding reported that unacetylated p53 binds to its p53REs only of a tetramer: two monomers bind each side of the motif when these REs are present as naked DNA, for example forming a ‘grip’ over the double helix. However, several within the linker region between nucleosomes, although non-canonical p53REs have been described within the p53 acetylation may increase the scope and the avail- genome, indicating that p53 may have broader binding ability of sites within nucleosomal DNA. capacities. Using two different approaches, Shakked and Resnick demonstrated that p53 transcriptional activity Re-evaluation of p53 post-translational modifications was dependent on both the length and the nucleotide Post-translational modifications of p53 such as phosphor- sequence of the spacer (Figure 1). Shakked (L14) ylation, acetylation or sumoylation have been shown presented high resolution crystal structures of the p53 to be essential in determining and regulating p53 activity core domain bound to different p53RE oligonucleotides.2 in vitro. However, their effects in vivo remain difficult DNA with contiguous half sites (n ¼ 0) showed a local to assess. Sabapathy (S1) generated a ‘knock-in’ mouse change in the DNA double helix associated with a strain replacing the serine 312 residue, equivalent to the rearrangement of the four p53 monomers. This config- human serine 315, by alanine (S312A) to abolish uration resulted in increased protein–protein and protein– phosphorylation. This residue has been proposed to have a role in the regulation of p53 protein stability.6–9 DNA interactions and hence higher binding affinity as S312A/S312A compared with oligonucleotides carrying a spacer (nX1). p53 knock-in mice are viable, fertile and not – pre-disposed to spontaneous tumor formation. In addi- These data suggest that the presence of a spacer may be S312A associated with decreased affinity of p53 for its p53RE. tion, the p53 protein was found to be activated as Resnick (L9) made the same observation using a efficiently as wild-type p53 and its turnover rate was not diploid yeast system, expressing p53 under the control of affected, suggesting that despite in vitro evidence this a galactose-dependent promoter and a reporter gene phosphorylation event may not be critical for in vivo driven by p53RE.3 In this system, increasing the number suppressive functions. of spacer nucleotides resulted in a drastic decrease It is well established that wild-type p53 is extensively modified on several lysine residues and that these of both p53 transcriptional and DNA binding activities. 10 He also showed that conservation of the p53RE core modifications are relevant to its function. Prives (L7) motif (CWWG) is the main requirement for the definition has used the p53-null lung cancer cell line H1299 to of a half site, the CATG motif appearing to be the most generate clones expressing inducible p53 variants, in efficient one. In addition, half- and three-quarter-sites which the two lysines located within the p53 tetrameriza- tion domain, K351 and K357, were changed either to arginine (R, to conserve the negative charge), or to glutamine (Q, to neutralize the charge and possibly mimic acetylation). p53K351R/K357R cells displayed similar DNA binding, transcriptional abilities and cell cycle arrest capacities than wild-type p53 cells. On the other hand, p53K351Q/K357Q was selectively and transcriptionally impaired and could not induce cell cycle arrest even though its expression led to higher levels of than wild- type p53. Additionally, recent reports have shown that K357 can be acetylated and that K351 is mutated in cancer cells.11–13 These findings provide clues for a role of Figure 1 p53RE structure and p53 activity. The p53 acetylation in regulating p53 tetramerization. protein binds specifically to DNA within gene promoters or introns through p53REs, which are composed of two palindromic half sites Identification of a natural p53 antisense transcript (R ¼ purine; W ¼ A/T; P ¼ pyrimidine) separated by spacers composed of 0–13 nucleotides (n). Shakked and Resnick reported Recently, a new post-transcriptional mechanism has been that p53 DNA binding and transcriptional activities can be modulated implicated in the modulation of p53 function. A natural by the nature of the p53RE core motif (CWWG), as well as length of antisense transcript of p53, termed Wrap53, was identified spacer (n), the most efficient p53RE corresponding to the presence by Farnebo in Wiman’s group (L18) that may modulate 14 of the CATG core motif and the absence of any spacer (n ¼ 0). p53 activity. The WRAP53 gene is located on the

Cancer Gene Therapy Understanding wild-type and mutant p53 activities in human cancer I Goldstein et al 4 of stem and committed progenitor cells. Stem/progenitor cells can be identified on the basis of their high expression of aldehyde dehydrogenase. It turned out that ALDE þ cells also contain high p53 levels, suggesting a role for p53 in controlling stemness. One mechanism by which p53 may influence this process, is the regulation of asymmetric cell division, a hallmark of stemness. Stem/progenitor cells devoid of tight control over cell fate regulation are prone to Figure 2 Wrap53, a natural antisense transcript of p53. Within the expansion via symmetric cell divisions. These results support recent studies showing a role for p53 in embryonic 17.1.13 locus, two genes transcribed in opposite direction have been 19–22 described: TP53 expressing 11 and WRAP53. Wrap53 and differentiation and apoptosis. p53 transcripts anneal each other owing to the presence on both MdmX, also termed Mdm4, is a Mdm2 homologue that molecules of the proximal part of TP53 gene, including the non- has been shown to inhibit p53 activity during embryonic coding 1 and part of the intron 1. This annealing stabilizes p53 development. Its uncontrolled overexpression may drive mRNA and thus, increases p53 protein levels in cells. oncogenic transformation.23,24 A study presented by de Lange (S3) indicated that MdmX is highly expressed in a subset of uveal melanoma cell lines. As TP53 mutations 17p13.1 locus and is transcribed in the opposite direction are uncommon in uveal melanoma the hypothesis arose to TP53, the proximal part of Wrap53 mRNA over- that MdmX may be overexpressed in this cancer to lapping with the non-coding exon 1 and part of the long inactivate the p53 pathway. Uveal melanoma cell lines intron 1 of p53 mRNA (Figure 2). Thus, Wrap53 with high MdmX expression were used. Knock down of transcripts anneal with p53 transcripts leading to p53 MdmX significantly impaired cell growth and induced accumulation at both mRNA and protein levels in basal apoptosis. These effects appeared to be at least partially conditions, although Wrap53 silencing resulted in dec- p53-independent. This study, together with previous reased accumulation of p53 upon DNA damage. These reports,25,26 suggests a role of MdmX that stretches observations suggest that this antisense transcript may beyond p53 inhibition. represent a novel and important regulator of p53 function, although its effect on mutant p53 activities p53, an important key in the carcinogenic process remains to be investigated. The process of carcinogenesis involves sequential accu- mulation of genetic alterations causing loss of tumor suppressor genes and overactivation of oncogenes. To overcome genetic variations that epitomize cancer- The p53 pathway: from normal to tumor cells derived cell lines, Rotter (L8) has established an in vitro Expanding the range of tumor suppressive functions transformation model, in which diploid human fibro- of the p53 pathway blasts WI-38 were immortalized by the introduction (1) of Normal cells have a limited dividing capacity in vitro, the telomerase catalytic subunit hTERT, (2) of the p53 reaching a state of senescence in which the capability to dominant negative peptide GSE56, which inactivates replicate is irreversibly lost. activation or p53 and (3) of the oncogenic H-RasV12. In this protocol, genotoxic stress may also induce a senescent state, which it appeared that, only INK4A-deficient cells expressing has a role in tumor suppression and is regulated by H-RasV12 and GSE56 peptide became fully transformed p53.15,16 p53-induced senescence involves the activation of and could form tumors in nude mice. Genome-wide specific microRNA targets, such as members of the miR- microarray analysis revealed several stable genetic signa- 34 family (a-c). Harris (L1) showed that Nutlin-induced tures associated with the carcinogenic process. A ‘Cancer- cellular senescence in normal human fibroblasts is related Gene Signature’ (CGS), which mainly consisted of p53-dependent and correlates with elevated miR-34s inflammatory chemokines, cytokines, interleukins and expression.17 When fibroblasts were treated with antisense extracellular matrix related proteins,27,28 was upregulated miR-34a, they extended their replicative capacity by three when both H-RasV12 and the p53 dominant-negative population doublings. Additionally, Nutlin-induced peptide GSE56 were concomitantly expressed in the cells. senescence was significantly impaired by oligonucleo- Wild-type p53 repressed CGS expression by mechanisms tide-mediated knock down of endogenous miR-34a.18 involving the p53 targets BTG2 and ATF3, previously With the emergence of cancer stem cells as the critical described as involved in Ras-induced transformation.29,30 source of cell populations during carcinogenesis, it is ATF3 directly binds to the CGS promoters and represses essential to determine how p53 functions participate in the their expression, while BTG2 interacts with H-Ras and control and maintenance of normal and cancer stem cell represses its activity.31 This effect may represent a novel status. Tolstonog (L4) presented new results exploring the suppressive function of wild-type p53. These findings are role of p53 in controlling the proliferation and differ- supported by reports showing that pro-malignant genes entiation of stem/progenitor cells. Mouse F9 teratocarci- are upregulated following p53 loss and acquisition of noma cells were used, a well-described model system for activated Ras.32,33 embryo-derived cancer cell differentiation. In culture, Crosstalk between tumor cells and their microenviron- F9 cells are heterogeneous and contain mixed populations ment are critical for tumor formation and progression.

Cancer Gene Therapy Understanding wild-type and mutant p53 activities in human cancer I Goldstein et al 5 Recent results show that p53 could modulate these gain new oncogenic activities exerted by mutant p53 interactions through the expression of secreted factors.34 (Figure 3).39 In a recent report, Oren has shown that cancer cells, or their conditioned medium, reduced p53 response Diversity of mutant p53 targets to cisplatin treatment in fibroblasts, as reflected by a 35 As mentioned above, studies by Rotter and colleagues attenuated accumulation of both p53 and p21. In (L8) identified a CGS regulated by p53 and Ras. The role addition, the suppression by cancer cells of p53 response of mutant p53 in regulating CGS was examined. in adjacent fibroblasts was required for neoplastic Interestingly, different mutants exhibited different prop- progression. In his presentation, Oren (L2) presented erties with respect to CGS regulation. A common new data on the impact of p53 expression in stromal conformational mutation within the L3 loop (p53G245S) cells on the growth of epithelial cancer cells. In mice, affected neither CGS expression nor activity of H-Ras. introduction of human PC3 prostate cancer cells in The drastic conformational mutants with substitutions combination with p53-null mouse embryonic fibroblasts within the p53 Zn2 þ binding domain (p53R175H, (MEFs) resulted in the development of tumors four fold p53H179R) showed comparable CGS expression levels larger than with wild-type p53 MEFs. Additionally, and H-Ras activity as in p53 knock down cells, suggesting culturing of cancer cells in conditioned medium of a complete loss of function with dominant negative effect p53-null MEFs increased their migration and invasion of the mutant allele over the remaining wild-type allele. capabilities. Oren showed that these biological events However, DNA contact mutants (p53R248Q and p53R273H) depended upon stromal cell-derived factor 1, a chemokine 36 showed significantly higher expression of the CGS, repressed by p53 in MEFs. These findings are in suggesting a GOF mechanism. This GOF effect was agreement with a previous study demonstrating a selec- found to be mediated by NFkB, in agreement with tion of stromal cells towards p53 loss in response 37 previous reports showing that mutant p53 can exert GOF to epithelial tumorigenesis, thus suggesting that cancer activities by stimulating NFkB activity.40,41 Finally, CGS cells may evolve to inhibit p53 suppressive activities in the expression was examined in lung, and head and neck tumor microenvironment, facilitating their growth and tumors, where high CGS expression correlated with progression. mutant p53 bearing tumors. This study suggests different abilities of the different p53 mutants to control the CGS Mutant p53 GOF and thus affect tumor development. TP53 mutations are very common in cancer (www-p53. The transforming growth factor b (TGFb) signaling iarc.fr38) and tend to cluster at some hot-spot codons, cascade has a dual role in carcinogenesis. Although in leading to point mutations rather than deletions. This led early stages TGFb acts to antagonize cancer progression, to the assumption that TP53 mutation not only abrogates in later stages TGFb has pro-metastatic effects.42 tumor suppression functions of wild-type p53, but also Although there is evidence that mutant p53 reduces the

Figure 3 Properties of mutant p53 proteins. Missense mutations at hot-spot codons located in the DNA binding domain of p53 are the most frequent alterations in human cancers. Three types of p53 mutants have been described: dominant negative mutants (DN) inhibit the suppressive function of the residual wild-type p53 protein; loss of function mutants (LOH) lack suppressive function; while gain of function mutants (GOF) exhibit oncogenic properties through an array of mutant specific activities including aberrant protein interactions or gene regulation. TADI/II: transactivation domain I/II; PXXP: proline-rich domain; DBD: DNA binding domain; OD: oligomerization domain; BD: basic domain.

Cancer Gene Therapy Understanding wild-type and mutant p53 activities in human cancer I Goldstein et al 6 expression of TGFb receptor type II,43 data presented by conformational changes that regulate protein activity Cordenonsi (S7) revealed an interplay between TGFb, and stability.52 Studies by Del Sal (L10) revealed that mutant p53 and Ras during . TGFb, in concert Pin1 formed physical complexes with mutant p53 with oncogenic Ras, induced the assembly of a mutant (as with wild-type p5352,53) and was required for mutant p53–p63 protein complex, which favors metastasis.44 p53 to cooperate with oncogene-induced transformation These data imply for a role for mutant p53 in late stage, in primary mouse fibroblasts. Moreover, in human breast pro-metastatic TGFb signaling. cancer cell lines, Pin1 cooperated with mutant p53 to Another role of mutant p53 in growth signaling was enhance tumor cell aggressiveness. These data indicate described by Bossi (S2). Building up on previous evidence that Pin1 is required for activation of mutant p53 GOF. of interactions between p53 and the mitogen-activated protein kinase pathway, he showed that mitogen-acti- Mouse models to explore mutant p53 functions vated protein kinase-kinase 3, a specific activator of p38 Until recently, it was frequently assumed that mutant p53 mitogen-activating protein kinase, was transcriptionally was systematically stabilized in tumors owing to impaired regulated by p53. mitogen-activated protein kinase-kinase Mdm2-mediated degradation. A study by Lozano (L20) 3-depleted cells showed attenuated growth, although challenged this concept. In a knock-in mice harboring the overexpression enhanced cell growth.45 These findings p53R172H mutant (p53H), equivalent to the human hot-spot suggest upregulation of mitogen-activated protein kinase- mutant p53R175H, no p53 accumulation was found in the kinase 3 expression represent a mechanism for mutant normal tissues of p53H/H homozygote mice, whereas p53 GOF. increased expression was observed in p53H/H Mdm2À/À Two novel genes targeted by mutant p53 may partici- and p53H/H p16À/À mice. Furthermore, only 21% of tumors pate in p53 GOF activities as described by Blandino developed in these mice exhibited detectable p53 expression, (L12). These genes, inhibitor of DNA-binding 4 (ID4) and underscoring that accumulation is not an intrinsic property polo-like kinase-2 (Plk2), emerged from transcriptome of the mutant and that deregulation of Mdm2 was required analysis of H1299 cells expressing an inducible mutant for mutant p53 stabilization. This stabilization led to a p53R175H. Inhibitor of DNA binding 4 is a member of a metastatic phenotype, which was not observed in p53H/H protein family which function as dominant-negative mice. Otherwise, the tumor spectrum of p53H/H and p53H/H regulators of basic helix-loop-helix transcription factors Mdm2À/À or p53H/H p16À/À mice was similar.54 These involved in cell proliferation, differentiation and tumori- findings underline the need for careful assessment of the genesis.46 siRNA-mediated depletion of p53R175H severely drug effects that stabilize wild-type p53, as these drugs may impaired inhibitor of DNA binding 4 expression in proli- also stabilize mutant p53 and thus, promote carcinogenesis ferating tumor cells. Mutant p53 formed a complex with through mutant GOF activities. on a specific region of inhibitor of DNA-binding 4 Hollstein (L19) has generated over 200 immortalized (ID4) promoter to upregulate expression, resulting in MEF cell lines harboring mutant p53 using senescence an increased angiogenic potential of mutant p53-expressing bypass of embryonic fibroblasts from mice carrying a cancer cells.47 The other target gene, Plk2 was targeted by humanized p53 gene (human p53 knock-in).55–57 As mutant p53 following DNA damage. Plk2 interacted with replicative senescence in hupki mouse cells exerts a and phosphorylated mutant p53, leading to a transcriptional selection pressure for outgrowth of cells with a mutated feedback loop involving mutant p53 and Plk2 in response to TP53 gene, these cells can be used to analyze the role of DNA damage. Knocking down Plk2 increased cell sensiti- p53 modifications in escaping spontaneous or DNA vity to DNA damaging, cytotoxic treatments. damage-induced senescence. Although hupki embryonic fibroblasts (HUFs) immortalized at a similar rate as Is GOF a consequence of unusual protein interactions? normal MEFs, hupki embryonic fibroblasts with S46A The tumor suppressive promyelocytic leukemia protein mutation escaped senescence more readily than their wild- (PML) is the major component of PML nuclear bodies type counterpart. Furthermore, when pre-senescent hupki implicated in apoptosis, cell proliferation and senes- embryonic fibroblasts were exposed to aristolochic acid, a cence.48,49 In humans, complete or partial loss of PML mutagenic compound present in extracts of Aristolochia has been observed in many types of cancers, including used in some traditional medications, emerging immorta- breast, colon and prostate.50 Although interconnections lized hupki embryonic fibroblast showed a high rate of between wild-type p53 and PML have been described,48 A–T transversion mutations in TP53.58 These mutations recent results of Haupt (L11) demonstrated that PML showed a remarkable correspondence with the mutations physically interacts with mutant p53 and enhances the found in tumor of patients with Balkan endemic nephro- transcriptional activity of mutant p53.51 Cancer cells with pathy, supporting the claim that this plant carcinogen has mutant p53 presented attenuated growth following PML a direct role in the etiology of this regional disease.59 knock down. Furthermore, mutant p53 GOF activities, Another mouse model of mutant p53 GOF was such as enhanced proliferation and colony formation, presented by Deppert (L3). In this model, transgenic required PML. These finding suggest that the presence of mice carrying the SV40 early region were crossed with both mutant p53 and PML may promote oncogenesis. mice carrying mutant p53.60 The use of the whey acidic The prolyl isomerase Pin1 protein has been shown to protein promoter allowed for the targeted expression of bind phosphorylated S/T-P sites on protein substrates and both transgenes in the mammary .61,62 In this to isomerize the intervening peptide bond leading to double transgenic model, binding of SV40-LT to p53

Cancer Gene Therapy Understanding wild-type and mutant p53 activities in human cancer I Goldstein et al 7 abrogates wild-type p530s function. Crossing these mice Nutlin-3 and RITA may be owing to their different with mice expressing mutant p53R270H enhanced the effects on Mdm2-dependent degradation of homeo- formation of mammary tumors with more aggressive domain-interacting protein kinase 2.66 features and increased their metastatic potential. Loss of Screening of drugs and peptides libraries has proved a p53 led to activation of c-MET, an event that appeared useful approach to identify drugs that specifically target essential for initiation and progression of mammary mutant p53 but not wild-type form (Table 1). Bisso (S5) . Consistent with these observations, a recent isolated five peptide aptamers (PAs) interacting specifi- study has shown that miR-34a, a microRNA induced by cally with p53 mutants. He demonstrated that PAs had a p53, inhibited uveal melanoma cell proliferation and higher affinity for unfolded conformational mutant migration through downregulation of c-MET.63 p53 than for wild-type p53 or contact mutants, which conserve the overall protein architecture. Interaction with PAs inhibited mutant p53 transcriptional activity and Towards clinically effective p53 targeting drugs induced apoptosis in tumor cell lines expressing mutant p53.67 Three-dimensional models of mutant p53–PAs In recent years, two drugs have been developed to activate complexes indicated that PAs interacted with the prox- wild-type p53 protein in tumors (Table 1). Nutlin-3 binds imal part of the p53 core domain via three important Mdm2 and induces accumulation of both p53 and Mdm2 residues (T102, L130 and N131). Another approach was leading to mitotic arrest, whereas RITA binds and presented by Joerger, from the group of A. Fersht (L13), stabilizes p53 while reducing Mdm2 expression, leading to identify compounds which target the hot-spot mutant to apoptosis. However, the mechanisms by which these p53Y220C and restore its wild-type activity by stabilizing two drugs induce different responses remain unknown. the protein structure. Mutation at codon 220 is one of the Selivanova (L17) showed that RITA affected the trans- most common mutation outside the DNA binding surface activation of p53-dependent pro-apoptotic genes but not of p53 and it has the particularity to affect a crevice cell cycle arrest genes.64 She presented two molecular amenable to the binding of designed small chemicals. mechanisms explaining this particular transactivation Using in silico analyses, Fersht group identified a family pattern (Figure 4a). First, RITA inhibited p21 expression of drugs, PhiKan, that specifically interact with mutant by increasing its proteasomal degradation and by down- p53Y220C and restored wild-type conformation.68 regulating hnRNP K, a cofactor of p53 transactivation, Several small drugs have been shown to restore wild- which is degraded in a Mdm2-dependent manner. In type p53 activity leading to apoptosis, among which is contrast to RITA, Nutlin-3 did not affect p21 and hnRNP PRIMA-1 (Table 1). However, the mechanisms respon- K expression nor did it induce the transactivation of pro- sible for this restoration are still unknown. Wiman (L18) apoptotic genes. Second, at high levels, RITA induced reported that intracellular metabolization of PRIMA-1 both expression of pro-apoptotic genes and downregula- generates a thiol reactive intermediate that induces tion of oncogenes and pro-survival factors.64 Soddu (L16) alkylation of cysteines located in the p53 core domain. reported another molecular mechanism that may explain This chemical modification, in turn, appears to facilitate the different response induced by RITA and Nutlin-3. the restoration of wild-type p53 conformation and DNA She demonstrated that apoptosis-inducing doses of binding activity.69 The affinity of PRIMA-1 appeared to adriamycin resulted in the accumulation of homeo- depend on the degree of p53 unfolding, explaining the domain-interacting protein kinase 2, which is responsible selectivity of PRIMA towards mutant p53. Re-introduc- for the phosphorylation of S46 of p53.65 In contrast, tion of PRIMA-1-treated p53R175H in p53-null cells was doses that induced mitotic arrest but not apoptosis were correlated with upregulation of BAX and caspase associated with a decrease of both homeodomain-inter- activation leading to apoptosis (Figure 4b). acting protein kinase 2 expression and S46 phosphory- These studies support the notion that mutant p53 is a lation. The decrease in homeodomain-interacting protein ‘druggable’ target in order to restore wild-type function. kinase 2 levels was owing to its degradation mediated by However, these compounds still need to be assessed for Mdm2, suggesting the different responses elicited by their functional and biological effects.

Table 1 Diversity of p53 targeting drugs Category Name Target Effect on p53 Biological Reference response

Peptide aptamers Pas mut p53 Inhibition of mut p53 transcriptional activity Apoptosis S5, Grinkevich et al.64 Small molecule PhiKan mut p53Y220C Stabilization and restoration of wt p53 activity ? L13, Rinaldo et al.65 PRIMA-1 mut p53 Restoration of wt p53 conformation and DNA Apoptosis L18, Rinaldo et al.66 binding activity RITA wt p53 wt p53 accumulation Apoptosis L17, Schulze-Garg et al.61 Nutlin-3 Mdm2 wt p53 accumulation Mitotic arrest L16, Krepulat et al.62 Abbreviations: Mut, mutant; ?, unknown; wt, wild type.

Cancer Gene Therapy Understanding wild-type and mutant p53 activities in human cancer I Goldstein et al 8

Figure 4 Molecular and biological events resulting from p53 targeting drugs. (a) Nutlin binds Mdm2 protein leading to homeodomain-interacting protein kinase 2 degradation that may result in the inhibition of p53-induced apoptosis and the induction of cell cycle arrest. In contrast, RITA dissociates the p53-Mdm2 complex resulting in Mdm2-dependent degradation of p21 and hnRNPK, as well as in p53-dependent inhibition of oncogenes and pro-survival factors with the end result being apoptosis. (b) PRIMA1-derived compounds bind to mutant p53 and restore its wild-type abilities.

Genotype/phenotype correlations of TP53 gene To investigate this hypothesis, Olivier and colleagues variations (L23) analyzed the crosstalks between p53 and pathways in cell responses to , anti- TP53 mutations have generally been associated with poor hormone or chemotherapy treatments. They showed that prognosis and poor response to treatment in several types estrogens levels could modulate both wild-type and of cancers. In squamous cell carcinomas of the head and mutant p53 protein levels. Estrogen deprivation compro- neck, TP53 mutations are found in the majority of the mised p53 response to DNA damage by reducing p53 cases and are associated with poor survival after surgery. protein steady-state levels.73 These results are in line with Myers (L22) described the squamous cell carcinomas of previous observations that were made in a clinical series, the head and neck cell line Tu138, which carries the where a larger effect of TP53 mutation status was found p53P151S mutation that made these cells resistant to cell for patient survival in cases with death induced by loss of anchorage (anoikis). He positive status, a marker of a functional estrogen receptor demonstrated that expression of this mutant in anoikis- pathway. How estrogens may affect p53 mutant activities sensitive cells led to downregulation of PUMA and remain to be addressed. Additionally, it was shown that NOXA, inducing resistance to anoikis and enhanced in TP53 mutated cells, the antiestrogen tamoxifen had tumorigenicity in an orthotopic nude mouse model of proliferative effects, although it had expected antiprolifera- oral cancer. tive effects in TP53 wild-type cells (Ferna´ndez-Cuesta 2010, In breast cancers, TP53 mutations are found in about Int J Cancer, in press). It remains to be determined which 25% of the cases and are associated with poor prognosis activities of mutant p53 may be responsible for this effect. and resistance to doxorubicin treatment.70 Expression TP53 mutations are very frequent is lung cancer, in signature of mutant TP53 status have been described that particular in smokers. Hainaut (L24) reported that, in a include genes involved in the estrogen receptor pathway large randomized therapeutic clinical trial, International and in the control of cell proliferation,71,72 suggesting a Adjuvant Lung cancer Trial, n ¼ 783 aimed at assessing close link between p53 and estrogen receptor pathways. the benefits of cisplatin-based adjuvant therapy in

Cancer Gene Therapy Understanding wild-type and mutant p53 activities in human cancer I Goldstein et al 9 completely resected non-small cell lung is now swiftly moving towards translation in the clinics. (NSCLC) patients, TP53 status had no prognostic value It is now technically possible to assess TP53 mutation at 8 years of follow-up. However, TP53 status was a status in large clinical studies and to combine mutation significant predictor of response to therapy, in particular identification with expression studies of gene and micro- in squamous cell carcinoma. Although, wild-type TP53 RNA, paving the way to the definition of ‘mutant p53 did not predict a better response to therapy, mutant TP53 molecular profiles’. Such profiles may provide extremely predicted a decreased survival and disease-free interval in precise predictors of disease outcomes and of responses to patients receiving chemotherapy. Thus, the presence of therapy. Furthermore, a whole range of small drugs is mutant p53, although having no significant effect on the available, which act on different aspects of mutant p53 course of the disease in the absence of adjuvant therapy, activities. These drugs are now ready to move into clinical significantly reduced the survival of patients upon trials, either alone or in combination with classical treatment. These results are the first evidence for GOF therapeutic approaches. In the next 10 years, such in a clinical setting. molecules are expected to contribute in an important Polymorphisms in genes of the TP53 pathway, such as way to the large panel of specific drugs that will be MDM2 SNP309 and p53R72P have been shown to have required to deliver the promises of evidence-based and modifier effects on the penetrance of TP53 germline personalized medicine. mutations in Li-Fraumeni’s syndrome.74,75 Marcel (S4) analyzed another common TP53 polymorphism, TP53 PIN3, a 16 duplication located in intron 3 (rs17878362; A1, non-duplicated allele; A2, duplicated Conflict of interest allele) that is present at an allele frequency of 0.2 in Caucasians. In a cohort of Brazilian LFS, she found that The authors declare no conflict of interest. TP53 PIN3 was associated with a difference of 19.0 years in the mean age at first diagnosis in TP53 mutation carriers (n ¼ 25; A1A1: 28.0 years; n ¼ 7; A1A2: 47.0 Acknowledgements years; P ¼ 0.01). As a result, cancer occurrence before age of 35 years was exclusively detected in homozygotes The Fourth International Workshop on Mutant p53 was of the more frequent allele (A1).76 These results highlight supported by the EU FP6 program on ‘Mutant p53’ and the modifier effect of TP53 intronic polymorphisms by the International Agency for Research on Cancer. and further suggest that not only the type and effect of Collaborations between IARC and The Weizmann mutation, but also the nature of the affected haplotype Institute on ‘Mutant p53’ are supported by a grant and of the remaining wild-type haplotype, are critical to of Cance´ropoˆle Rhoˆne-Alpes Auvergne, France. The understand the full impact of mutant p53 in cancer. authors gratefully thank the different lecturers for their contribution to the Workshop and to this manuscript.

Conclusion and perspectives References In all, 10 years of research focusing on mutant p53 have yielded remarkable progress in understanding their 1 el-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, specific functions. It is now clear that GOF mutant p53 Vogelstein B. Definition of a consensus binding site for is not a single and generic property. In a manner remi- p53. Nat Genet 1992; 1: 45–49. niscent of wild-type p53, mutant p53 is a multifunctional 2 Kitayner M, Rozenberg H, Kessler N, Rabinovich D, factor, of which the effects may differ according to the Shaulov L, Haran TE et al. Structural basis of DNA cancer cells in which the mutation occurs. Two main recognition by p53 tetramers. Mol Cell 2006; 22: 741–753. avenues for further research may be identified. First, 3 Jordan JJ, Menendez D, Inga A, Noureddine M, Bell DA, Resnick MA. Noncanonical DNA motifs as transactivation accumulation of mutant p53 at higher levels than those targets by wild type and mutant p53. PLoS Genet 2008; 4: normally found in healthy cells, may allow mutant e1000104. proteins to interact with a larger panel of DNA and 4 Menendez D, Inga A, Snipe J, Krysiak O, Schonfelder G, proteins than wild-type p53. Second, it should be Resnick MA. A single-nucleotide polymorphism in a half- considered that mutant p53 may exert particular effects binding site creates p53 and estrogen receptor control of depending on its structure and its affinity for defined vascular endothelial growth factor receptor 1. Mol Cell Biol DNA or protein targets. The respective importance of this 2007; 27: 2590–2600. dosage effect and these specific structural effects induced 5 Yan J, Menendez D, Yang XP, Resnick MA, Jetten AM. A by the mutation should be further investigated, but a regulatory loop composed of rap80-HDM2-p53 provides combination of these two properties may account for the rap80 enhanced p53 degradation by HDM2 in response to DNA damage. J Biol Chem 2009; 284: 19280–19289. wide diversity of mutant p53 effects. 6 Bischoff JR, Friedman PN, Marshak DR, Prives C, Beach It is becoming clear that all cancers have a defective p53 D. Human p53 is phosphorylated by p60-cdc2 and cyclin pathway, either by TP53 mutation or by deregulation of B-cdc2. Proc Natl Acad Sci USA 1990; 87: 4766–4770. another determinant of the p53 pathway. Despite this 7 Katayama H, Sasai K, Kawai H, Yuan ZM, Bondaruk J, increased complexity, the field of mutant p53 research Suzuki F et al. Phosphorylation by aurora kinase A induces

Cancer Gene Therapy Understanding wild-type and mutant p53 activities in human cancer I Goldstein et al 10 Mdm2-mediated destabilization and inhibition of p53. tumorigenesis in p53-deficient cells and mice. Mol Cell Biol Nat Genet 2004; 36: 55–62. 2008; 28: 1265–1273. 8 Price BD, Hughes-Davies L, Park SJ. Cdk2 kinase phos- 27 Milyavsky M, Shats I, Erez N, Tang X, Senderovich S, phorylates serine 315 of human p53 in vitro. Oncogene 1995; Meerson A et al. Prolonged culture of telomerase-immorta- 11: 73–80. lized human fibroblasts leads to a premalignant phenotype. 9 Radhakrishnan SK, Gartel AL. CDK9 phosphorylates p53 on Cancer Res 2003; 63: 7147–7157. serine residues 33, 315 and 392. Cell Cycle 2006; 5: 519–521. 28 Milyavsky M, Tabach Y, Shats I, Erez N, Cohen Y, 10 Kruse JP, Gu W. Modes of p53 regulation. Cell 2009; 137: Tang X et al. Transcriptional programs following genetic 609–622. alterations in p53, INK4A, and H-Ras genes along defined 11 Goldstein I, Rotter V. Mutations in the tetramerization stages of malignant transformation. Cancer Res 2005; 65: domain of p53: more than just keeping monomers apart. 4530–4543. Cell Cycle 2009; 8: 3259–3260. 29 Lu D, Wolfgang CD, Hai T. Activating 12 Joubel A, Chalkley RJ, Medzihradszky KF, Hondermarck 3, a stress-inducible gene, suppresses Ras-stimulated tumor- H, Burlingame AL. Identification of new p53 acetylation igenesis. J Biol Chem 2006; 281: 10473–10481. sites in COS-1 cells. Mol Cell Proteomics 2009; 8: 1167–1173. 30 Boiko AD, Porteous S, Razorenova OV, Krivokrysenko VI, 13 Muscolini M, Montagni E, Caristi S, Nomura T, Kamada R, Williams BR, Gudkov AV. A systematic search for down- Di Agostino S et al. Characterization of a new cancer-associated stream mediators of tumor suppressor function of p53 mutant of p53 with a missense mutation (K351N) in the reveals a major role of BTG2 in suppression of Ras-induced tetramerization domain. Cell Cycle 2009; 8: 3396–3405. transformation. Genes Dev 2006; 20: 236–252. 14 Mahmoudi S, Henriksson S, Corcoran M, Mendez-Vidal C, 31 Buganim Y, Solomon H, Rais Y, Kistner D, Nachmany I, Wiman KG, Farnebo M. Wrap53, a natural p53 antisense Brait M et al. p53 Regulates the Ras circuit to inhibit the transcript required for p53 induction upon DNA damage. expression of a cancer-related gene signature by various Mol Cell 2009; 33: 462–471. molecular pathways. Cancer Res 2010; 70: 2274–2284. 15 Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, 32 Coppe JP, Patil CK, Rodier F, Sun Y, Munoz DP, Goldstein Issaeva N et al. Oncogene-induced senescence is part of the J et al. Senescence-associated secretory phenotypes reveal tumorigenesis barrier imposed by DNA damage checkpoints. cell-nonautonomous functions of oncogenic RAS and the Nature 2006; 444: 633–637. p53 tumor suppressor. PLoS Biol 2008; 6: 2853–2868. 16 Halazonetis TD, Gorgoulis VG, Bartek J. An oncogene- 33 McMurray HR, Sampson ER, Compitello G, Kinsey C, induced DNA damage model for cancer development. Newman L, Smith B et al. Synergistic response to oncogenic Science 2008; 319: 1352–1355. mutations defines gene class critical to cancer phenotype. 17 Kumamoto K, Spillare EA, Fujita K, Horikawa I, Nature 2008; 453: 1112–1116. Yamashita T, Appella E et al. Nutlin-3a activates p53 to 34 Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev both down-regulate inhibitor of growth 2 and up-regulate Cancer 2006; 6: 392–401. mir-34a, mir-34b, and mir-34c expression, and induce 35 Bar J, Feniger-Barish R, Lukashchuk N, Shaham H, senescence. Cancer Res 2008; 68: 3193–3203. Moskovits N, Goldfinger N et al. Cancer cells suppress 18 Fujita K, Mondal AM, Horikawa I, Nguyen GH, p53 in adjacent fibroblasts. Oncogene 2009; 28: 933–936. Kumamoto K, Sohn JJ et al. p53 isoforms Delta133p53 36 Moskovits N, Kalinkovich A, Bar J, Lapidot T, Oren M. and p53beta are endogenous regulators of replicative cellular p53 Attenuates cancer cell migration and invasion through senescence. Nat Cell Biol 2009; 11: 1135–1142. repression of SDF-1/CXCL12 expression in stromal fibro- 19 Han MK, Song EK, Guo Y, Ou X, Mantel C, Broxmeyer HE. blasts. Cancer Res 2006; 66: 10671–10676. SIRT1 regulates apoptosis and Nanog expression in 37 Hill R, Song Y, Cardiff RD, Dyke TV. Selective evolution of mouse embryonic stem cells by controlling p53 subcellular stromal mesenchyme with p53 loss in response to epithelial localization. Cell Stem Cell 2008; 2: 241–251. tumorigenesis. Cell 2005; 123: 1001–1011. 20 Lin T, Chao C, Saito S, Mazur SJ, Murphy ME, Appella E et al. 38 Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, p53 induces differentiation of mouse embryonic stem cells by Hainaut P et al. Impact of mutant p53 functional properties suppressing Nanog expression. Nat Cell Biol 2005; 7: 165–171. on TP53 mutation patterns and tumor phenotype: lessons 21 Maimets T, Neganova I, Armstrong L, Lako M. Activation from recent developments in the IARC TP53 database. of p53 by nutlin leads to rapid differentiation of human Hum Mutat 2007; 28: 622–629. embryonic stem cells. Oncogene 2008; 27: 5277–5287. 39 Brosh R, Rotter V. When mutants gain new powers: news 22 Qin H, Yu T, Qing T, Liu Y, Zhao Y, Cai J et al. Regulation from the mutant p53 field. Nat Rev Cancer 2009; 9: 701–713. of apoptosis and differentiation by p53 in human embryonic 40 Weisz L, Damalas A, Liontos M, Karakaidos P, Fontemaggi stem cells. J Biol Chem 2007; 282: 5842–5852. G, Maor-Aloni R et al. Mutant p53 enhances nuclear factor 23 Danovi D, Meulmeester E, Pasini D, Migliorini D, Capra M, kappaB activation by tumor necrosis factor alpha in cancer Frenk R et al. Amplification of Mdmx (or Mdm4) directly cells. Cancer Res 2007; 67: 2396–2401. contributes to tumor formation by inhibiting p53 tumor 41 Scian MJ, Stagliano KE, Anderson MA, Hassan S, Bowman suppressor activity. Mol Cell Biol 2004; 24: 5835–5843. M, Miles MF et al. Tumor-derived p53 mutants induce 24 Migliorini D, Lazzerini Denchi E, Danovi D, Jochemsen A, NF-kappaB2 . Mol Cell Biol 2005; 25: Capillo M, Gobbi A et al. Mdm4 (Mdmx) regulates p53-induced 10097–10110. growth arrest and neuronal cell death during early embryonic 42 Derynck R, Akhurst RJ, Balmain A. TGF-beta signaling in mouse development. Mol Cell Biol 2002; 22: 5527–5538. tumor suppression and cancer progression. Nat Genet 2001; 25 Matijasevic Z, Krzywicka-Racka A, Sluder G, Jones SN. 29: 117–129. MdmX regulates transformation and chromosomal stability 43 Gerwin BI, Spillare E, Forrester K, Lehman TA, Kispert J, in p53-deficient cells. Cell Cycle 2008; 7: 2967–2973. Welsh JA et al. Mutant p53 can induce tumorigenic 26 Matijasevic Z, Steinman HA, Hoover K, Jones SN. MdmX conversion of human bronchial epithelial cells and promotes bipolar mitosis to suppress transformation and reduce their responsiveness to a negative growth factor,

Cancer Gene Therapy Understanding wild-type and mutant p53 activities in human cancer I Goldstein et al 11 transforming growth factor beta 1. Proc Natl Acad Sci USA 61 Schulze-Garg C, Lohler J, Gocht A, Deppert W. A transgenic 1992; 89: 2759–2763. mouse model for the ductal carcinoma in situ (DCIS) of the 44 Adorno M, Cordenonsi M, Montagner M, Dupont S, Wong C, . Oncogene 2000; 19: 1028–1037. Hann B et al. A Mutant-p53/Smad complex opposes p63 to 62 Krepulat F, Lohler J, Heinlein C, Hermannstadter A, empower TGFbeta-induced metastasis. Cell 2009; 137:87–98. Tolstonog GV, Deppert W. Epigenetic mechanisms affect 45 Gurtner A, Starace G, Norelli G, Piaggio G, Sacchi A, Bossi mutant p53 transgene expression in WAP-mutp53 transgenic G. Mutant p53-induced up-regulation of mitogen-activated mice. Oncogene 2005; 24: 4645–4659. protein kinase kinase 3 contributes to gain of function. 63 Yan D, Zhou X, Chen X, Hu DN, Dong XD, Wang J et al. J Biol Chem 2010; 285: 14160–14169. MicroRNA-34a inhibits uveal melanoma cell proliferation 46 Lasorella A, Uo T, Iavarone A. Id proteins at the cross-road and migration through downregulation of c-Met. of development and cancer. Oncogene 2001; 20: 8326–8333. Invest Ophthalmol Vis Sci 2009; 50: 1559–1565. 47 Fontemaggi G, Dell’Orso S, Trisciuoglio D, Shay T, Melucci 64 Grinkevich VV, Nikulenkov F, Shi Y, Enge M, Bao W, E, Fazi F et al. The execution of the transcriptional axis Maljukova A et al. Ablation of key oncogenic pathways by mutant p53, E2F1 and ID4 promotes tumor neo-angiogen- RITA-reactivated p53 is required for efficient apoptosis. esis. Nat Struct Mol Biol 2009; 16: 1086–1093. Cancer Cell 2009; 15: 441–453. 48 Zimber A, Nguyen QD, Gespach C. Nuclear bodies and 65 Rinaldo C, Prodosmo A, Mancini F, Iacovelli S, Sacchi A, compartments: functional roles and cellular signalling Moretti F et al. MDM2-regulated degradation of HIPK2 in health and disease. Cell Signal 2004; 16: 1085–1104. prevents p53Ser46 phosphorylation and DNA damage- 49 Trotman LC, Alimonti A, Scaglioni PP, Koutcher JA, induced apoptosis. Mol Cell 2007; 25: 739–750. Cordon-Cardo C, Pandolfi PP. Identification of a tumour 66 Rinaldo C, Prodosmo A, Siepi F, Moncada A, Sacchi A, suppressor network opposing nuclear Akt function. Selivanova G et al. HIPK2 regulation by MDM2 determines Nature 2006; 441: 523–527. tumor cell response to the p53-reactivating drugs nutlin-3 50 Gurrieri C, Capodieci P, Bernardi R, Scaglioni PP, Nafa K, and RITA. Cancer Res 2009; 69: 6241–6248. Rush LJ et al. Loss of the tumor suppressor PML in human 67 Guida E, Bisso A, Fenollar-Ferrer C, Napoli M, Anselmi C, cancers of multiple histologic origins. J Natl Cancer Inst Girardini JE et al. Peptide aptamers targeting mutant p53 induce 2004; 96: 269–279. apoptosis in tumor cells. Cancer Res 2008; 68: 6550–6558. 51 Haupt S, di Agostino S, Mizrahi I, Alsheich-Bartok O, 68 Boeckler FM, Joerger AC, Jaggi G, Rutherford TJ, Voorhoeve M, Damalas A et al. Promyelocytic leukemia Veprintsev DB, Fersht AR. Targeted rescue of a destabilized mutant of p53 by an in silico screened drug. Proc Natl Acad protein is required for gain of function by mutant p53. Sci USA 2008; 105: 10360–10365. Cancer Res 2009; 69: 4818–4826. 69 Lambert JM, Gorzov P, Veprintsev DB, Soderqvist M, 52 Zacchi P, Gostissa M, Uchida T, Salvagno C, Avolio F, Segerback D, Bergman J et al. PRIMA-1 reactivates mutant Volinia S et al. The prolyl isomerase Pin1 reveals a p53 by covalent binding to the core domain. Cancer Cell mechanism to control p53 functions after genotoxic insults. 2009; 15: 376–388. Nature 2002; 419: 853–857. 70 Olivier M, Petitjean A, Marcel V, Petre A, Mounawar M, 53 Zheng H, You H, Zhou XZ, Murray SA, Uchida T, Wulf G Plymoth A et al. Recent advances in p53 research: an et al. The prolyl isomerase Pin1 is a regulator of p53 in interdisciplinary perspective. Cancer Gene Ther 2009; 16: 1–12. genotoxic response. Nature 2002; 419: 849–853. 71 Langerod A, Zhao H, Borgan O, Nesland JM, Bukholm IR, 54 Terzian T, Suh YA, Iwakuma T, Post SM, Neumann M, Lang Ikdahl T et al. TP53 mutation status and gene expression GA et al. The inherent instability of mutant p53 is alleviated by profiles are powerful prognostic markers of . Mdm2 or p16INK4a loss. Genes Dev 2008; 22: 1337–1344. Breast Cancer Res 2007; 9: R30. 55 Feng L, Hollstein M, Xu Y. Ser46 phosphorylation regulates 72 Miller LD, Smeds J, George J, Vega VB, Vergara L, Ploner p53-dependent apoptosis and replicative senescence. A et al. An expression signature for p53 status in human Cell Cycle 2006; 5: 2812–2819. breast cancer predicts mutation status, transcriptional 56 Luo JL, Yang Q, Tong WM, Hergenhahn M, Wang ZQ, effects, and patient survival. Proc Natl Acad Sci USA 2005; Hollstein M. Knock-in mice with a chimeric human/murine 102: 13550–13555. p53 gene develop normally and show wild-type p53 responses 73 Fernandez-Cuesta L, Anaganti S, Hainaut P, Olivier M. to DNA damaging agents: a new biomedical research tool. Estrogen levels act as a rheostat on p53 levels and modulate Oncogene 2001; 20: 320–328. p53-dependent responses in breast cancer cell lines. 57 Song H, Hollstein M, Xu Y. p53 gain-of-function cancer Breast Cancer Res Treat 2010; e-pub ahead of print 11 mutants induce genetic instability by inactivating ATM. March 2010; doi:10.1007/s10549-010-0819-x. Nat Cell Biol 2007; 9: 573–580. 74 Bond GL, Hirshfield KM, Kirchhoff T, Alexe G, Bond EE, 58 Nedelko T, Arlt VM, Phillips DH, Hollstein M. TP53 Robins H et al. MDM2 SNP309 accelerates tumor formation mutation signature supports involvement of aristolochic acid in a gender-specific and hormone-dependent manner. in the aetiology of endemic nephropathy-associated tumours. Cancer Res 2006; 66: 5104–5110. Int J Cancer 2009; 124: 987–990. 75 Bougeard G, Baert-Desurmont S, Tournier I, Vasseur S, 59 Slade N, Moll UM, Brdar B, Zoric A, Jelakovic B. p53 Martin C, Brugieres L et al. Impact of the MDM2 SNP309 mutations as fingerprints for aristolochic acid: an environ- and p53 Arg72Pro polymorphism on age of tumour onset in mental carcinogen in endemic (Balkan) nephropathy. Li-Fraumeni syndrome. J Med Genet 2006; 43: 531–533. Mutat Res 2009; 663: 1–6. 76 Marcel V, Palmero EI, Falagan-Lotsch P, Martel-Planche G, 60 Heinlein C, Krepulat F, Lohler J, Speidel D, Deppert W, Ashton-Prolla P, Olivier M et al. TP53PIN3 and MDM2 Tolstonog GV. Mutant p53(R270H) gain of function SNP309 polymorphisms as genetic modifiers in the Li- phenotype in a mouse model for oncogene-induced mam- Fraumeni syndrome: impact on age at first diagnosis. J Med mary carcinogenesis. Int J Cancer 2008; 122: 1701–1709. Genet 2009; 46: 766–772.

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