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TP53 Mutants in the Tower of Babel of Cancer Progression

Alessandra Bisio,1 † Yari Ciribilli,1 † Gilberto Fronza,2 † Alberto Inga,1∗ † and Paola Monti2 †

1Laboratory of Transcriptional Networks, Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy ; 2Mutagenesis Unit, IRCSSAzienda Ospedaliera Universitaria San Martino-IST-Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy

For the TP53 Special IssueReceived 11 October 2013; accepted revised manuscript 6 January 2014.Published online 21 January 2014 in Wiley Online Library (www.wiley.com/humanmutation). DOI: 10.1002/humu.22514

ABSTRACT: Loss-of-function, partial-function, altered-function, dominant-negative, temperature sensitive, inter-fering, contact, structural, unfolded, misfolded, dimeric,monomeric, non-cooperative, unstable, supertrans, super-stable, intragenic suppressor. TP53 mutants are many,more than 2,000 in fact, and they can be very diverse.Sporadic; germline; gain-of-function (GoF); oncogenic;rebel-angel; yin and yang; prion-like; metastasis-inducer;mediator of chemo-resistance; modifier of stemness. TP53mutants can impact important cancer clinical variables, inmultiple, often subtle ways, as revealed by cell-based as-says as well as animal models. Here, we review studiesinvestigating TP53 mutants for their effect on sequence-specific transactivation function, and especially recentfindings on how TP53 mutants can exhibit GoF proper-ties. We also review reports on TP53 mutants’ impact oncancer cell transcriptomes and studies with Li–Fraumenipatients trying to classify and predict phenotypes in re-lation to experimentally determined transcription finger-prints. Finally, we provide an example of the complexityof correlating TP53 mutant functionality to clinical vari-ables in sporadic cancer patients. Conflicting results andlimitations of experimental approaches notwithstanding,the study of TP53 mutants has provided a rich body ofknowledge, mostly available in the public domain and ac-cessible through databases, which is beginning to impactcancer intervention strategies.Hum Mutat 35:689–701, 2014. C© 2014 Wiley Periodicals, Inc.

KEY WORDS: TP53; p53; cancer; Li–Fraumeni; transacti-vation; gain-of-function

IntroductionTP53 (MIM #191170) is a well-known tumor suppressor gene

whose protein product acts in the cell as tetrameric transcription

†All authors contributed equally and are listed alphabetically.

Contract grant sponsors: Compagnia di S. Paolo, Turin, Italy (Project 2012.1590 “Le

interazioni molecolari della proteina p53 mutata come bersaglio di nuove terapie anti-

tumorali personalizzate”); the Italian Association for Cancer Research, AIRC (IG#12869,

IG#5506). 5xmille donated to the IRCSS Azienda Ospedaliera Universitaria San Martino-

IST-Istituto Nazionale per la Ricerca sul Cancro.∗Correspondence to: Alberto Inga, Laboratory of Transcriptional Networks, Centre

for Integrative Biology (CIBIO), University of Trento, Via delle Regole 101, Trento 38123,

Italy. E-mail: inga@science.unitn.it

factor (TF) able to regulate target genes involved in cancer suppres-sion [Lane and Levine, 2010]. TP53 exerts its role primarily throughthe sequence-specific recognition of response elements (REs) at thepromoter level of regulated genes. The p53 RE consensus sequenceis represented by two repeats of 5′-RRRCWWGYYY-3′ sequence,where R stands for a purine, W for A/T, and Y indicates a pyrimidine[el-Deiry et al., 1992]. Noncanonical REs, consisting in½ sites or¾sites, have also been identified as capable to recruit TP53 at promoterregions and mediate responsiveness of target genes [Menendez et al.,2007; Jordan et al., 2008; Menendez et al., 2009; Menendez et al.,2013]. Unlike other tumor suppressor genes, TP53 is mostly alteredin human cancers by missense mutations (i.e., causing single amino-acid substitutions). As described in more detail in this special issue[Soussi, 2014], TP53 mutations in cancer affect mainly six majorhotspot residues within the DNA-binding domain (DBD) of theTP53 protein (R175, G245, R248, R249, R273, and R282). However,the spectrum of TP53 missense mutations is extremely broad withmore than 1,800 different amino-acid changes reported, althoughsome very infrequent in tumors [Soussi, 2011]. TP53 mutants havebeen primarily categorized as DNA contact mutants or structuralmutants, according to the effect of the amino-acid substitution onthe interaction with DNA (e.g., p.R273H) or on the structure of theTP53 protein itself (e.g., p.R175H) [Cho et al., 1994; Kitayner et al.,2006; Kitayner et al., 2010]. However, as discussed in many previ-ous articles and reviews, not all TP53 mutations affect equally TP53functions, potentially generating a wide range of phenotypic diver-sity that could impact important clinical variables, namely tumoraggressiveness, chemoresistance, and metastastic potential [Orenand Rotter, 2010; Muller et al., 2011; Walerych et al., 2012].

Over the last 15 years, a great amount of experimental data on thefunctional impact of TP53 missense mutations has been generatedand is freely available (p53.free.fr/) [Leroy et al., 2013]. Functionalassays have been performed by many laboratories, in yeast as wellas in human cells using reporter assays to measure different prop-erties of TP53 mutants including: (1) transactivation potential, (2)temperature sensitivity (TS), (3) dominant negative effect over thewild-type (WT) protein, (4) functional modulation of other mem-bers of the TP53 family (TP73 and TP63), usually reported as inter-ference, and (5) interactions with other TFs [Campomenosi et al.,2001; Monti et al., 2002; Kato et al., 2003; Monti et al., 2003; Resnickand Inga, 2003; Shiraishi et al., 2004; Ciribilli et al., 2010; Menendezet al., 2010]. A summary of the different properties of TP53 mutantsis presented in Figure 1. Some of these functional features may beincluded in the gain-of-function (GoF) effect reported for severalTP53 mutants (see the section on GoF properties, below) [Orenand Rotter, 2010]. Moreover, the recently identified propensity ofTP53 mutants to aggregate [Xu et al., 2011], exhibiting a prion-likebehavior, could also contribute to oncogenic functions, includingdominant negative (over WT TP53) and interfering (over TP63 andTP73) properties [Silva et al., 2013] (Fig. 1).

C© 2014 WILEY PERIODICALS, INC.

Figure 1. Functional classification of TP53 mutants. Presented is a graphical summary of different effects of TP53 mutations directly dependenton sequence-specific transactivation functions or due to protein–protein interactions either with WT TP53 or the TP53-related TP63 and TP73proteins. The potential to aggregate for TP53 mutants is also depicted. For each functional group, one or few TP53 mutations are listed as asignificant example. See text for details and references.

Although not the focus of this review, TP53 mutants pos-sess also biological activities that are cytosolic and transcription-independent with consequences on the induction of the intrinsicapoptosis pathway [Green and Kroemer, 2009].

At the Core of Sequence-SpecificTransactivation: Interactions of TP53 MutantProteins with p53 REs

Transactivation Activities of TP53 Mutants

Transactivation has been the most extensively studied feature ofTP53 mutants. Work of several groups, including our own, estab-lished that nearly all TP53 mutants at hotspot residues have lost orretain only a very weak transactivation function (defined as Loss-of-Function, LoF, or Severe Deficiency, SD, TP53 mutants) [Montiet al., 2007; 2011], with the exception of the p.R337H mutant thatexhibited a WT-like activity. Nevertheless, a significant fraction oftumor-associated TP53 mutants, that hit other positions of the TP53protein and are generally found at moderate to low frequency in can-cer [Soussi, 2011], retains some level of transactivation potential andcan discriminate between the p53 REs in gene reporter assays (con-sidered as Partial Function or Partial Deficiency, PD, TP53 mutants)[Campomenosi et al., 2001; Resnick and Inga, 2003; Jordan et al.,2010], among TP53 target genes in cell lines [Ludwig et al., 1996],and also in vivo, in mouse models [Lozano, 2010; Donehower, 2014].

Several attempts have been made to understand the mechanismunderlying the capacity of TP53 mutants to discriminate betweendifferent target promoters. Growth suppression of tumor cells byTP53 proteins might result from the activation of both apoptosis andcell cycle arrest pathways. Specific amino-acid changes in the TP53DBD can lead to a reduction in DNA binding potential that wouldparticularly affect the recognition of weaker target sites, more fre-quent among apoptotic target genes [Friedlander et al., 1996]. How-ever, researchers demonstrated that also some mutants outside theDBD, for example in the TP53 hinge domain—a small linker regionbetween the DBD and the tetramerization domain—differentiallyactivated the promoters of TP53 target genes and showed an im-paired apoptotic function [Kong et al., 2001]. Those results sug-

gested that conformational changes, potentially impacting on TP53tetramer assembly, might play an important role in differentiallyregulating TP53 cell cycle arrest and apoptotic functions. Morerecently, the importance of cooperative interactions at the levelof TP53 DBDs has been revealed to contribute to transactivationspecificity and to be a target for mutation in cancer [Schlereth et al.,2010].

Overall, data on transactivation activity have led to the develop-ment of functional classifications of TP53 mutants with the attemptto explore their prognostic relevance in somatic cancers [Petitjeanet al., 2007]. The most comprehensive functional study examined2.314 mutants, virtually all possible missense mutations that resultfrom single nucleotide substitutions in the TP53 coding sequence.The transactivation activity of TP53 mutants was characterized to-ward eight different p53 REs using yeast reporter cells [Kato et al.,2003]. However, for a large cohort of sporadic breast cancer patients,that functional classification of TP53 mutants did not provide ad-ditional prognostic value compared with the information on TP53mutation status [Olivier et al., 2006]. A more recent study, on thecontrary, reported a trend for TP53 mutant function to stratify lo-cally advanced breast cancers for different clinical outcome [Jordanet al., 2010].

Other categories of TP53 mutants were identified that are virtu-ally absent from the TP53 mutation spectrum in cancer (Fig. 1).These mutations have been defined as “altered specificity,” “super-trans,” or “intragenic-suppressor” and proven to be useful toolsto study mechanisms underlying transactivation specificity [Brach-mann et al., 1998; Inga et al., 2001; Inga and Resnick, 2001; Resnickand Inga, 2003]. Interestingly, the majority of these TP53 mutants,which can also exhibit enhanced DNA binding affinity and transac-tivation activity with respect to WT TP53, lie in the L1 loop, a highlyconserved portion of the TP53 DBD that is a “cold spot” for muta-tion in cancer. An example of this group of TP53 mutants is p.S121Fthat has been shown in several cell-based assays to have lost the abil-ity to induced the CDKN1A gene (P21; MIM ∗116899), but to havegained a higher capability with respect to WT TP53 to transactivateother target genes including apoptotic targets such as BAX (MIM∗600040), BBC3 (PUMA, MIM ∗605854), and TNFRSF10B (MIM∗605854) (KILLER), resulting in higher potential to induce apopto-sis in cancer cells [Saller et al., 1999; Menendez et al., 2006]. In

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addition, p.S121F could induce apoptosis in a transcription-independent manner [Yasuda et al., 2012]. Consistently, these mu-tants were also found to possess a higher DNA binding affinity invitro compared with WT TP53, and the capacity to act as secondsite suppressor of LoF cancer mutations [Brachmann et al., 1998].Interestingly, the described changes in transactivation specificitieswere also observed for L1 loop mutants of the TP53-related pro-tein TP73 (MIM #601990), where structural studies indicated animpact of the mutations also on tetramer assembly on target REs[Ethayathulla et al., 2012; Ciribilli et al., 2013].

Taken collectively, the studies on transactivation potential have re-vealed considerable differences among the effects of TP53 mutants.Combined with the growing number of target genes and biologicalpathways modulated by WT TP53 (recently reviewed in [Sullivanet al., 2012]), the variable impact of TP53 mutations on transacti-vation led Resnick and Inga (2003) to propose a model for TP53mutant-associated phenotypic diversity that employs, as analogy,chords produced from the piano musical instrument. Each ‘‘key’’ ofthe piano corresponds to a gene whose cellular expression is modu-lated by the interaction of a sequence-specific TF (e.g., TP53) witha target RE. A master regulator such as TP53 can be considered asthe ‘‘hand’’ that plays many keys with variable intensity (being thepressure of the fingers on the keys influenced, among other factors,by differences in binding affinity toward target REs). Changes in thefeatures of the hand because of specific amino-acid substitutionsmay determine a variety of differences in the sound level arisingfrom each key and in the chords; in the analogy, new chords corre-spond to potentially different biological phenotypes [Resnick andInga, 2003; Menendez et al., 2007].

TS of TP53 Mutants

TS reflects the thermodynamic impact of introduced singleamino-acid changes and it is a common feature of many TP53 mu-tant proteins [Jagosova et al., 2012] (Fig. 1). Usually TP53 mutantsloose ability to activate transcription at higher temperature (heatsensitivity). Although rare, cold sensitivity of specific TP53 mu-tants has been also reported [Campomenosi et al., 2001]. Cell lineswith temperature-sensitive TP53 mutants have provided an elegantsystem to study TP53 functions [Zhang et al., 1994; Friedlanderet al., 1996]. A systematic study [Shiraishi et al., 2004] identified142 temperature-sensitive TP53 mutants using a functional assayin yeast. These TP53 mutants clustered in the β-strands withinthe DBD, particularly in one of the two β-sheets, and 15 residues(T155, R158, M160, A161, V172, H214, S215, P223, T231, T253,I254, T256, S269, E271, and E285) were susceptible to amino-acidchanges resulting in TS. Some of those TP53 mutants were con-firmed as temperature sensitive in the Saos-2 human osteosarcomaTP53 null cell line. Temperature-sensitive TP53 mutants have beenproposed as candidate for structure-dependent restoration of TP53function [Liu et al., 2013].

Dominant-Negative Potential of TP53 Mutants

It is established that cancer-associated TP53 mutants can abro-gate transactivation by WT TP53 (dominant negative potential)(Fig. 1). TP53 is active in a tetrameric form and the formation ofheterotetramers (mutant and WT) with defined stoichiometry hasbeen shown in heterozygous cells as well as in vitro [Natan et al.,2009]. Studying the biogenesis of TP53 tetramers in vitro, it has beendemonstrated that TP53 dimers can be formed cotranslationally onthe polysomes, whereas tetramers are generated posttranslationally,

through the dimerization of dimers in solution or on DNA bindingsites [Nicholls et al., 2002]. In order to better understand the natureof the tumorigenic activity of TP53 mutants, many groups, includingours, have tried to uncover the mechanisms by which TP53 mutantscan have a dominant negative effect. Biochemical data suggestedthat the C-terminal tetramerization domain within TP53 is abso-lutely required for the dominant negative effect, since TP53 mutantswithout a functional tetramerization domain lost the dominant neg-ative potential and were not oncogenic [Chene and Bechter, 1999].Willis et al. (2004) demonstrated that TP53 mutants remarkablyreduced WT TP53 binding to p53 REs in the CDKN1A, MDM2, andTP53I3 (PIG3; MIM ∗605171) promoters. Those findings were alsocorrelated to the decreased ability of WT TP53 to induce growthsuppression and cell cycle arrest in the presence of TP53 mutant[Willis et al., 2004]. Furthermore, the dominant negative potentialof two TP53 mutants (p.R270H and p.P275S, corresponding in hu-mans to p.R273H and p.P278S, respectively) was also demonstratedin vivo, in mouse models [de Vries et al., 2002].

The dominant negative effect of TP53 mutants appears to im-pact the development and clinical manifestations of at least somesporadic tumors. Among 40 patients with sporadic glioblastomas,the average age at diagnosis was significantly lower in patients withtumors harboring dominant negative alleles than in those with re-cessive alleles mutations or in those without mutations, suggest-ing that dominant negative mutations can accelerate the develop-ment of glioblastomas [Marutani et al., 1999]. In another studyon squamous cell carcinoma (SCC), patients with dominant neg-ative TP53 mutants presented a significantly shorter disease-freesurvival than those with recessive or with WT alleles, suggestingthat the presence of a dominant negative TP53 mutant may providea predictor of early recurrence in oral SCC patients [Hassan et al.,2008].

GoF Properties of Cancer-Associated TP53Mutants

The high preponderance of TP53 missense mutations in humancancers led to develop the concept that TP53 mutant protein mightpossess activities of its own not present in the WT (i.e., GoF) thatcan confer a selective advantage, contributing to various aspects oftumor progression [Oren and Rotter, 2010; Goh et al., 2011; Freed-Pastor and Prives, 2012; Walerych et al., 2012].

The concept of GoF dates back to 1993 when it was formallyshown that some TP53 mutants, but not the WT protein, couldtransform TP53-null cells, enabling them to form colonies in softagar in vitro and tumors in mice [Dittmer et al., 1993]. To demon-strate GoF of TP53 mutants, two experimental approaches are cur-rently used: (1) overexpression of the studied TP53 mutant in aTP53-null cell and (2) depletion of endogenous TP53 mutant incancer cells that have already lost the WT TP53 allele, using RNA in-terference. However, the most compelling evidence for GoF activityof TP53 mutants comes from knock-in mice engineered to har-bor tumor-associated hotspot TP53 mutations [Donehower, 2014].Knock-in mouse model expressing the hotspot mutant p.R172H orp.R270H (corresponding to human p.R175H and p.R273H, respec-tively) exhibited a broader tumor spectrum with a more invasiveand metastatic phenotype compared with TP53–/– or TP53+/– mice[Lang et al., 2004; Olive et al., 2004]. Moreover, the generation ofHUPKI (HUmanized TP53 Knock-In) mouse models harboringdifferent TP53 hotspot mutations (p.R175H, p.G245S, p.R273H,p.R248Q, and p.R248W) provided also an in vivo proof for the

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differential oncogenic potential among human TP53 mutants [Songet al., 2007; Liu et al., 2010; Hanel et al., 2013].

Multiple mechanisms can account for TP53 mutant GoF, notbeing necessarily mutually exclusive, rather coexisting and cooper-ating based on the cellular background. Experiments using 10(3)mouse fibroblasts, cells stably expressing the tumor-derived TP53p.D281G GoF mutant with additional mutations in cis at aminoacids 22 and 23 (p.L22Q/p.W23S) in the activation domain 1, wereno longer tumorigenic when injected into nude mice [Lin et al.,1995; Scian et al., 2004]. That result demonstrated the requirementof transactivation function for the execution of TP53 mutant GoFproperties. In a more recent study, the activation domain 2 and theproline-rich domain also appeared to be necessary for GoF, whereasthe C-terminal basic domain had a negative impact on TP53 mutantfunctions [Yan and Chen, 2010].

Recently, the impact of posttranslational modifications on TP53mutant GoF was proposed in several studies [Nguyen et al., 2014].One of them presented findings for a provocative novel oncogenicfunction of the tumor suppressor protein PTEN (MIM +601728).PTEN expression appeared in fact capable of stabilizing thep.R175H mutant via inhibition of MDM2-dependent degradationand possibly through a direct protein binding [Li et al., 2008].Related to this finding, TP53 mutant mice also lacking MDM2(MIM ∗164785) or CDKN2A (MIM ∗600160) (p16INK4a) developedtumors earlier in life and more aggressive in phenotype [Terzianet al., 2008]. Another study identified an oncogenic autoregulatoryfeedback loop: the polo-like kinase-2 (PLK2; MIM ∗607023) bindsand phosphorylates TP53 mutant, fostering its oncogenic potentialthrough enhanced recruitment of p300 and NF-Y on promotersof genes involved in proliferation, including PLK2 [Valenti et al.,2011]. Posttranslational modifications can also counteract TP53mutant GoF. For example, acetylation of some TP53 mutantproteins by PCAF acetyl-transferase could result in the partialrestoration of WT DNA-binding activity associated with growthsuppression [Perez et al., 2010].

A graphical summary of GoF phenotypes is presented in Figure 2and described in the following sections, where the emphasis is givento recently reported findings.

GoF TP53 Mutants can Affect TP63 or TP73 Functions

The TP63 (MIM ∗603273) and TP73 genes express, by alternativepromoter usage and splicing, multiple transactivation competent(TA) and truncated N-terminal (�N) variants [Kaghad et al., 1997;Yang et al., 1998]. Although evolutionary conservation is higherfor the DBD, it is still apparent in the amino-terminal transactiva-tion domain and in the carboxy-terminal tetramerization domain[Dotsch et al., 2010]. The homology between tetramerization do-mains initially suggested that these proteins could form heterote-tramers. Davison et al. (1999) showed that TP73 and TP63 formhomotetramers capable of weak heterotypic interactions with eachother but not with TP53. This finding was independently confirmed,and the divergence between oligomerization domains is presentlyconsidered an important determinant in the functional divergencebetween TP53 and TP63/TP73 proteins [Joerger et al., 2009; Ethay-athulla et al., 2012]. However, it became also apparent that sometumor-derived TP53 mutants could inhibit TP63 and TP73 trans-activation from a p53-responsive promoter in eukaryotic cells, aphenotype defined as “interference” [Di Como et al., 1999; Gaid-don et al., 2001; Monti et al., 2003]. The mechanisms by whichTP53 mutants, but not WT TP53, can affect transactivation can bedue to a direct interaction at the level of the DBDs [Gaiddon et al.,

2001; Melino, 2011; Tucci et al., 2012], rather than a competition atthe DNA binding sites among homotetramers of the different P53family proteins.

Binding to TP63 and TP73 has been associated with the GoF activ-ity of certain tumor-derived TP53 mutants to promote chemoresis-tance, migration, invasion, and metastasis [Bergamaschi et al., 2003;Adorno et al., 2009; Muller et al., 2009]. Specifically, TP53 mutantproteins (e.g., p.R175H and p.R273H) play an important role instimulating cell migration and invasion via inhibition of TA-P63α

isoform that instead can act as a metastasis suppressor of mam-mary tumors by upregulating CCNG2 (cyclinG2; MIM #603203)and BHLHE41 genes (SHARP1; MIM #606200) [Adorno et al., 2009;Montagner et al., 2012]. In fact, in concert with oncogenic RAS(MIM #190020), TP53 mutants and transforming growth factor β

(TGFβ)-activated SMAD2/3 are engaged in a ternary complex withTA-TP63 isoform, compromising TP63 functions [Adorno et al.,2009].

TP63 inactivation by different TP53 mutants (p.R175H, p.R273H,and p.D281G) has been also associated with enhanced chemokineexpression (CXCL-5, CXCL-8, and CXCL-12), leading to the promo-tion of cell migration [Yeudall et al., 2012]. Concomitantly, repres-sion of TA-TP63 activity by TP53 mutants results in enhanced Rabcoupling protein (RCP)-mediated recycling to plasma membraneof α5β1 integrin, Epidermal Growth Factor receptor (EGFR; MIM#131550), and Mesenchymal–Epithelial Transition factor (MET,i.e., the receptor for hepatocyte growth factor), contributing onceagain to metastatic migration of cancer cells [Muller et al., 2009;Muller et al., 2013]. However, the effect of TP53 mutants on MET-dependent cell invasion can be also TP63 independent. The TP63transcriptional targets involved in the stimulation of the endocyticrecycling of adhesion molecules are currently unknown, but a pos-sible role of microRNAs (miRs) and the machinery that processesthem has been hypothesized.

The involvement of miRs in TP53 mutant-driven invasionthrough the inhibition of P53 family members has been highlightedin breast cancer cell lines. In fact, miR-155 (MIM #609337) expres-sion was directly repressed by TP63, and TP53 mutants, throughtheir interaction with and inhibition of TP63, could therefore stim-ulate miR-155 expression [Neilsen et al., 2013]. The transcrip-tional repressor ZNF652 (MIM #613907) has been identified asa miR-155 target. As ZNF652 is involved in suppression of inva-sion and metastasis through constitutive repression of key drivers ofepithelial–mesenchymal transition (EMT), such as TGFB1, TGFB2,TGFBR2, EGFR, SMAD2, TP53 mutants would indirectly repress itsfunctions.

Recently, Liu et al. (2011) demonstrated that the formation ofcomplexes between TP53 mutant and TP63/TP73 is facilitated by P1(MIM #607760), a protein involved in DNA replication, thereby pre-venting promoter occupancy by TP63/TP73 and consequently in-hibiting their transcriptional activities. Through this action, TopBP1may facilitate the chemoresistance phenotype by decreasing theexpression of apoptosis-related genes (e.g., BBC3, BAX, PMAIP1AIP1). Conversely, TP53 mutant-TP63/TP73 complexes have beenshown to be the target of ANKRD11 (MIM ∗611192) representingthe first evidence of an endogenous protein that can restore na-tive conformation to TP53 mutant, causing its dissociation fromTP63/TP73. Stable expression of ANKRD11 in cells lines of differ-ent origin and harboring different TP53 mutants (e.g., p.R175Hand p.R273H) was shown to inhibit the ability of TP53 mutantsin driving invasion, thus reducing the tumorigenic properties [Nollet al., 2012].

The reduction of TP73-dependent MDM2 expression by TP53mutant represents another mechanism of GoF activity, aimed at

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Figure 2. Mechanisms of TP53 mutant GoF. Overview of the processes that can be influenced by TP53 mutant GoF activities. Effectors, dividedas protein interactors (solid lines with a big dot), induced gene targets (arrows) or repressed targets (Stop L connectors) are indicated, as well asthe potential outcomes associated with those interactions. Solid lines indicate genes modulated by direct or indirect TP53 mutant effect, whereasbroken lines stand for upstream activators’ stimuli on TP53 mutant. When multiple independent interactions have been reported, the gene namesare listed in column. See text for details and references.

increasing cell invasion [Wang et al., 2009]. Specifically, p.R248Wmutant interferes with TP73 on the expression of MDM2, increasingthe level of SNAIL (MIM #602150) (SLUG), a target for MDM2-dependent degradation. SNAIL acts as a transcriptional repressor ofcell adhesion molecule E-cadherin, which is a known inhibitor ofinvasion and metastasis formation [Wang et al., 2009].

Based on the previous observations, the inhibition of TP63 andTP73 constitutes a key GoF mechanism adopted by TP53 mutants.

Interactions of TP53 Mutants with WT TP53 BindingPartners

GoF activity of TP53 mutants can be mediated by the formation ofprotein complexes with various proteins that physiologically interactwith WT TP53 [Haupt et al., 2009], including the promyelocyticleukemia (PML; MIM #102578) protein and the peptidyl-prolylcis/trans isomerase (PIN1; MIM #601052) [Girardini et al., 2011].The PML protein interacts and enhances the transactivation abilityof TP53 mutants [Haupt et al., 2009] with a mechanism not yetwell defined. PIN1 docks with phosphorylated serine or threonineresidues that are followed by proline and catalyzes the switch ofthe proline residue from the cis to trans configuration, resultingin a conformational change that stimulates the activity of TP53mutant protein and can lead to the activation of a proaggressivenesstranscriptional program. The existence of the PIN1/TP53 mutantaxis was revealed in a knock-in TP53 mutant mouse model, wherethe lack of PIN1 reduced tumorigenesis. Lack of PIN1 also impairedthe ability of TP53 mutants to promote anchorage-independentgrowth and tumorigenicity of H-RASV12 transformed fibroblasts,thus indicating that oncogenic signaling is not sufficient to activateGoF activity of TP53 mutants [Girardini et al., 2011]. PIN1 was alsoshown to enhance TP53 mutant interaction with P63.

MDM2, as well as heat shock protein 70 (HSP70), can impactTP53 mutant GoF by influencing aggregation [Xu et al., 2011].Interaction of HSP70 with TP53 mutant causes the transient expo-

sition of aggregation-prone domains of TP53 mutant protein, in anATP-dependent manner. Moreover, aggregation propensity can behigher for specific TP53 mutants. Subsequent aggregation of TP53mutant is favored by the MDM2–TP53 interaction, and can leadalso to the sequestration of other tumor suppressors (e.g., TP73)[Wiech et al., 2012].

Interestingly, all described mechanisms may converge also on theinhibition of P53 family proteins by TP53 mutant.

Interactions of TP53 Mutants with Binding Partners notShared with WT TP53

Recently, Coffill et al. (2012) compared the interaction partners ofp.R273H and WT TP53 using the triple-label SILAC immunoprecip-itation assay, and identified nine proteins that exclusively interactedwith p.R273H. Among them Nardilysin (NRD1; MIM ∗602651)was confirmed as a key binding partner of p.R273H, promoting aninvasive phenotype in the presence of heparin binding-epidermalgrowth factor-like protein (HB-EGF; MIM ∗126150) in H1299 cellline. Another protein that was shown to cooperate with TP53 mutantto mediate cell invasion is periostin, a protein of the cellular matrixeven though periostin–TP53 direct interaction was not explored.Interestingly, it drives invasion to a greater extent when coexpressedwith a TP53 conformational mutant (p.R175H) compared with aTP53 DNA-contact mutant (p.R273H) [Wong et al., 2013].

Conformational TP53 mutants showed different GoF activitieswith respect to contact TP53 mutants also in the cooperation withthe H-RAS oncogene to induce cellular transformation [Solomonet al., 2012]. Specifically, the conformational TP53 mutants p.R175Hand p.H179R modulate H-RAS-mediated signaling differently fromthe contact mutants p.R248Q and p.R273H. p.R175H and p.H179R,by strongly binding to BTG2 (MIM ∗601597), a protein that inter-acts with H-RAS and represses its activity, squelch BTG2 activity andconsequentially stimulate H-RAS, promoting tumor cell prolifera-tion through the activation of genes like CXCL-1 (MIM ∗155730),

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MMP3 (MIM ∗185250), and IL-1b (MIM ∗147720). Conversely,the contact TP53 mutants p.R248Q and p.R273H weakly interactwith BTG2, exerting their proproliferative GoF by binding to theNF-kB/relA TF. The p.G245S mutant, which shows an altered con-formation of L3 loop, does not affect any of the above describedmechanisms [Solomon et al., 2012].

TP53 GoF mutants can promote tumorigenesis also by a mecha-nism involving the interaction with the nuclease MRE11 and thesuppression of the binding of the MRE11–RAD50–NBS1 com-plex to DNA double-strand breaks, leading to impaired Ataxia-Telangiectasia Mutated activation. This interaction confers a GoFactivity to p.R248W and p.R273H mutants by promoting geneticinstability in tumors as well as pretumorigenic lesions in the HUPKIknock-in mouse models [Song et al., 2007].

Interactions of TP53 Mutants with TFs

The cooperation of TP53 mutants with different TFs is an impor-tant route to execute GoF activity. TP53 mutants can interact withdifferent TFs (NF-Y, NF-kB, NF-kB2, E2F1, VDR, NFR2, SREBPs,ZEB1, ETS2, STAT2) and can be recruited to their respective cog-nate binding sites and either stimulate or repress transcription [Scianet al., 2005; Di Agostino et al., 2006; Fontemaggi et al., 2009; Gurtneret al., 2010; Schneider et al., 2010; Stambolsky et al., 2010; Do et al.,2012; Freed-Pastor et al., 2012; Kalo et al., 2012; Masciarelli et al.,2013]. TP53 mutants can regulate the expression of target genes alsoby causing chromatin reorganization on the promoters; chromatinmodifications are brought into action by histone acetylases and/orhistone deacetylases in cooperation with transcriptional coactiva-tors (p300, CREB) that interact with TP53 mutants and modulatethe level of histone acetylation [Vaughan et al., 2012]. A novel mech-anism has been described where the PTEN protein is able to enhancea transcriptional complex containing TP53 mutant, CBP, and NF-Yin glioblastoma cells, revealing a context-dependent oncogenic po-tential of PTEN [Huang et al., 2013]. The recruitment of the TP53mutant–NF-Y complex together with the p300 coactivator can bealso mediated by the TopBP1 protein [Liu et al., 2011]. Moreover,the assembly of selective protein complexes containing TP53 mu-tant, sequence-specific TFs along with coactivators or corepressorscan influence the output of gene expression ranging from activa-tion to repression. As an example with opposite outcomes, althougha report by Yan et al. (2008) showed that the ID2 target gene isrepressed by TP53 mutants in proliferating SW480 colon adenocar-cinoma cell line, Fontemaggi et al. (2010) demonstrated that underDNA damage condition, the TP53 mutant/E2F1 complex activatesID2 expression.

The target genes of TP53 mutant/TFs complexes are quite diversein terms of their biological effects comprising genes that stimulateproliferation, inhibit apoptosis, promote chemoresistance, and in-fluence EMT transition [Brosh and Rotter, 2009; Liu et al., 2011;Kogan-Sakin et al., 2011; Freed-Pastor and Prives, 2012 and ref-erence therein; Kalo et al., 2012; Noll et al., 2012; Vaughan et al.,2012; Campion et al., 2013; Garritano et al., 2013]. These genesinclude, among others, c-MYC [Frazier et al., 1998], PCNA (Prolif-erating Cell Nuclear Antigen), hTERT (human Telomerase ReverseTranscriptase)[Scian et al., 2004], ASNS (Asparagine Synthetase)[Scian et al., 2004], EGFR, EGR1 (Early Growth Response 1) [Weiszet al., 2004], and ABCB1 (Multi Drug Resistance-1), the first genediscovered as TP53 mutant target [Chin et al., 1992].

An important role of TP53 mutants in regulating metabolic path-ways linked to tumor progression and metastasis, particularly in themodulation of genes involved in lipid metabolism, has been dis-

covered in high-grade serous ovarian cancer [Hu et al., 2013] andbreast cancers [Freed-Pastor et al., 2012]. TP53 mutant can interactwith the steroid regulatory element binding proteins (SREBPs) thatactivate the genes encoding key enzymes of the fatty acid and sterolbiosynthetic pathways. Consistent with this view, WT TP53 appearsto have the opposite impact in the modulation of SREBP1/2 [Huet al., 2013] and to modulate lipid biosynthesis in multiple ways[Goldstein et al., 2012]. Interestingly, cell culture models in 3D havebeen instrumental in revealing the involvement of TP53 mutant inthe mevalonate pathway, through the upregulation of many impor-tant genes involved in that cascade (HMGCS1, MVK, and SQLEamong others) [Freed-Pastor et al., 2012].

Crosstalk between cancer cells and surrounding stroma is also acrucial factor affecting tumor outcome and can be shaped by TP53mutant GoF effects on gene expression. It was recently shown thatTP53 mutant counteracts the response to interferon β secreted by fi-broblasts, attenuating its antitumorigenic activity. Specifically, TP53mutant is involved in prevention of STAT1 phosphorylation by overactivating SOCS1 gene, an inhibitor of JAK1 kinase. The inhibitionof STAT1 activity was associated with a moderation of interferon β

pathway and the consequent stimulation of cell migration [Madaret al., 2013].

miRs can Contribute to TP53 Mutant GoF

Recently, the role of miRs in TP53 mutant GoF is emerging, as acounterpoint to the role of miRs in supporting WT TP53 function[Jones and Lal, 2012]; moreover, all members of the P53 familyhave been shown to regulate miR gene expression and maturation[He et al., 2007; Su et al., 2010; Huang et al., 2011; Knouf et al.,2012]. miR-128–2 is the first miR whose expression was determinedto be dependent on p.R175H mutant expression and associatedwith chemoresistance of lung cancer cells [Donzelli et al., 2012].However, the TP53 mutant-containing transcriptional complexesrecruited on the promoter region of miR-128–2 have not been yetidentified. At the molecular level, miR-128–2 is induced by TP53mutant and targets the transcriptional repressor E2F5, leading toabrogation of its repressive activity toward CDKN1A transcription.The observed accumulation of P21 protein in the cytoplasm canprotect from apoptosis cells exposed to anticancer drugs.

Another recent study [Masciarelli et al., 2013] provided molecularevidences of the transcriptional complexes containing TP53 mutantthat can modulate the expression of miRs. By screening a groupof miRs deregulated in lung cancer, a decrease in the expression ofmiR-223 in H1299 cell line upon p.R175H protein induction wasobserved. By expanding this observation to different cell lines, itwas shown that TP53 mutant, together with the ZEB1 TF (MIM#189909), inhibited miR-223 transcription. This resulted in reliev-ing miR-223-dependent translational inhibition of STMN-1 (MIM#151442), a key microtubule-regulatory oncoprotein. The conse-quential accumulation of STMN-1 and of other, yet unidentified,miR-223 targets, was associated with the chemoresistance of cul-tured cells [Masciarelli et al., 2013].

miR-dependent upregulation of ZEB1 and subsequent activationof downstream signaling pathways that led to EMT transition, is alsoinvolved in the GoF activity of different TP53 mutants (p.C135Y,p.R175H, and p.R273H) in endometrial cancer cells. Specifically,TP53 mutants can bind directly to the promoter and inhibit tran-scription of miR-130b, a negative regulator of ZEB1 [Dong et al.,2013].

Lastly, p.R273H mutant expressed under an inducible system inH1299 cells was shown to bind miR-27a promoter and suppress

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its expression, thereby increasing the level of a newly identifiedmiR-27a target, that is, the EGFR. In response to EGF stimulation,p.R273H promotes the activation of EGFR/ERK1/2 pathways andthus increases cell proliferation [Wang et al., 2013c].

Up to now, other types of noncoding RNAs, particularly lincR-NAs, have not been associated with GoF activities of TP53 mutants.

TP53 Mutants Binding with Non-B-DNA

Recruitment of TP53 mutants to promoters through protein–protein interactions (e.g., with TP63, TP73, NF-Y, VDR) is a well-documented mechanism of the regulation of gene expression asdescribed above. However, a new direct recruitment of TP53 mu-tants to DNA was recently proposed. It was shown that p.R273H canbind to G/C-rich regions in proximity of transcription start sites inU251 cells [Quante et al., 2012]. Previously, it was shown that TP53mutant proteins could strongly bind also to genomic DNA elementsrepresenting matrix attachment regions that exhibit a high propen-sity to form non-B-DNA structures [Will et al., 1998]. Influenceof DNA topology has been addressed recently by Brazdova et al.(2013), who demonstrated that hotspot TP53 mutant proteins pref-erentially bind to supercoiled DNA both in vitro and in culturedcells. DNA supercoiling is associated with the level of repression byTP53 mutant of BAX and MSP/MST1 expression. Moreover, bindingis strictly dependent on the presence of an intact TP53 C-terminalDNA binding site (aa 363–382) that has been previously shown topossess the ability to interact with DNA in non-sequence-specificmanner [Kim and Deppert, 2003; Shakked, 2007; Leith et al., 2012].

TP53 Mutants and Li–Fraumeni SyndromeGermline mutations of TP53 gene result in cancer proneness syn-

dromes ranging from the more severe known as Li–Fraumeni (LFS),Li-Fraumeni-like, to the less severe nonsyndromic predispositionswith (FH) or without Family History [Malkin, 2011]. The spectrumof TP53 germline mutations comprises many different alleles butthe vast majority yields single amino-acid changes in the DBD ofthe protein [Petitjean et al., 2007].

The wide variety of adult-onset and childhood cancers and thedistribution of TP53 mutations were deeply examined, allowing anunderstanding of cancer genotype–phenotype correlations. Never-theless, the LFS phenotype is complex and cannot be readily ex-plained by the simple identification of TP53 germline mutations inaffected individuals [Malkin, 2011].

Recent works have identified genetic events that modulate theLFS phenotype. These include intragenic polymorphisms, muta-tions/polymorphisms of genes in the TP53 regulatory pathway, aswell as more global events such as aberrant copy number variations(CNVs) and telomere shortening. Shlien et al. (2008) reported thatnot only mutation carriers have higher frequencies of CNVs thanpeople with WT TP53 gene, but also the carriers with the highestCNVs frequencies were more likely to have family histories of can-cer. Thus, CNVs frequency could prove to be useful in assigningTP53 mutation carriers into risk groups, thereby providing criteriato be used in screening and genetic counseling.

Interestingly, the telomere length in peripheral blood cells ofTP53 germline mutation carriers was found to be shorter than thatof normal individuals of corresponding age [Trkova et al., 2007].Furthermore, it was found that earlier age of cancer initiation wasassociated with shorter telomere length in LFS patients and there wasconvincing evidence of increased telomere attrition in succeedinggenerations [Tabori et al., 2007].

Transactivation and Dominant-Negative Features ofGermline TP53 Mutants

We previously explored genotype/phenotype associations bycomparing a functional classification of all TP53 germlinemutant alleles to clinical data from the IARC database(http://www-p53.iarc.fr/Germline.html). Our analyses revealed thatSD alleles were associated with more severe cancer proneness syn-dromes (e.g., LFS), whereas PD alleles were associated with less se-vere cancer proneness conditions (e.g., FH), indicating that the lossof transactivation ability influences clinical manifestations in pa-tients who inherited TP53 mutations and developed cancer [Montiet al., 2007].

As discussed above, mutations in TP53 can affect the tumori-genic process through at least three different mechanisms: LoF, GoF[Bossi et al., 2006; Di Agostino et al., 2006] or dominant-negative ef-fects. Given the heterozygous state of germline TP53 mutations, theexistence of a correlation between dominant-negative features andclinical manifestations in patients who inherited TP53 mutationsand developed cancer was explored. Clinical data from the IARCgermline database were combined with functional data (transacti-vation and dominant-negative) for 104 of the 106 (98%) germlinemutant TP53 alleles with a single amino-acid substitution [Montiet al., 2011]. Although a classification based on transactivation alonewas confirmed to identify familial cancer cases with more severeclinical features, classification based on dominant-negative effectshighlighted similar associations but did not reveal distinct clinicalsubclasses of SD alleles. We concluded that in carriers of germlineTP53 mutations, the transactivation-based classification of TP53 al-leles appears more important for genotype–phenotype correlationsthan dominant-negative effects and that haploinsufficiency of theTP53 gene is an important factor in cancer proneness in humans[Monti et al., 2011].

GoF Properties of Germline TP53 Mutants

To our knowledge, no attempt to correlate clinical features andGoF other than dominance was performed. This is also becauseof the potential mechanistic heterogeneity of GoF properties. Veryrecently, however, new observations and hypothesis were proposedfor understanding LFS.

Cells from LFS sufferers are known to exhibit telomere dys-function, genomic instability, and spontaneous immortalization.Pantziarka (2013) hypothesized that these features are evidencesthat the host microenvironment is “primed” for carcinogenesisover and above the lack of TP53 tumor suppressor function. Fur-ther, the “two-compartment tumor metabolism” hypothesis, a newparadigm that describes a metabolic shuttle between autophagiccells in the tumor stroma and tumor cells, was proposed. The hy-pothesis suggests that cancer cells induce oxidative stress in thestroma by secreting hydrogen peroxide. Cancer-associated fibrob-lasts respond to this environmental challenge by entering into anautophagic state and undergoing mitophagy, resulting in mitochon-drial dysfunction and a shift of metabolism toward aerobic glycol-ysis. This issue brings in the spotlight the role of TP53 in cellularhomeostasis, metabolism, and in how cells respond to nutrient de-ficiency, hypoxia, and other stresses. TP53 can upregulate oxidativephosphorylation by inducing the synthesis of cytochrome c oxidase(SCO2; MIM #604272) and downregulate glycolysis through activa-tion of TP53-induced glycolysis regulator (TIGAR; MIM #610775)[Reinhardt and Schumacher, 2012]. This role of TP53 as metabolismmodulator, initially considered “one among the many,” is presently

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getting more and more attention as it was very recently shown thattumor suppression was observed in the absence of TP53-mediatedcell-cycle arrest, apoptosis, and senescence, but in the presence of theability of TP53 to regulate energy metabolism and reactive oxygenspecies production [Li et al., 2012]. These findings suggest thatunconventional activities of TP53, such as metabolic regulationand antioxidant function, are critical for suppression of early-onsetspontaneous tumorigenesis.

Analysis of the redox parameters in blood samples of healthyLFS individuals (harboring the p.R337H mutant) compared withhealthy noncarriers of TP53 mutations indicated that the for-mer group had significantly elevated indicators of protein oxida-tive damage, including a fourfold increase in plasma malondi-aldehyde levels, indicating increased lipid peroxidation [Macedoet al., 2012]. These findings provide the first evidence that ox-idative damage occurs in carriers of a TP53 germline mutationwith important implications regarding the understanding of themechanisms responsible for germline p.R337H mutant-associatedcarcinogenesis.

Consistent with the growing evidence that alterations inmetabolism may contribute to tumorigenesis, Wang et al. (2013b)demonstrated that members of LFS families who carry germline mu-tations have increased oxidative phosphorylation of skeletal mus-cle compared with the family members who are not carriers andto healthy volunteers. Basic experimental studies of tissue samplesfrom patients with the LFS and a mouse model of the syndromesupport this in vivo finding of increased mitochondrial function.Whether this metabolic phenotype contributes to tumorigenesisin LFS patients remains still to be determined. Thus, a change in“oxidative metabolism” resulting from GoF properties appears tobe one of the earliest events associated with the presence of TP53mutations even in the heterozygous state. This finding raises thequestion of seeking a signature of metabolic changes in the geneexpression of LFS specimens with specific TP53 mutant alleles.

TP53 Mutant-Associated Transcriptomes in CellLines as Well as in Cancer Samples

The previous sections dealt with all possible mechanisms by whichTP53 mutants may change some properties (biochemical, biologi-cal, metabolic) of the cell with respect to the WT allele. It is possiblethat such features are “recorded” in changes of the expression pro-gramme of the cell. Are there genes involved in the plethora ofmechanisms described above (Fig. 2) whose changes in expressionlevels are recognizable through transcriptome analysis? More gener-ally: is an allele-specific gene expression profile (GEP) recognizablein cells expressing a specific TP53 mutant allele?

In fact, the possibility to use genome-scale approaches, mainlytranscriptome analyses, but also promoter occupancy and pro-teomics, has offered the opportunity to explore more globally theimpact of TP53 mutant expression in cancer cell lines or even in pri-mary cells derived from individuals who inherited TP53 germlinemutations. Omics approaches on cancer samples along with muta-tion analysis also led to the possibility to explore correlations withTP53 status, presence of specific TP53 mutations, transcription fin-gerprints, and clinical outcomes [Donzelli et al., 2008]. As noted in arecent review, the correlation between TP53 status and patients’ out-come has produced controversial results [Robles and Harris, 2010].Here, we review recent attempts to profile TP53 mutant functionsusing transcriptome analysis, at three different levels: (1) in cell linesoverexpressing a specific TP53 mutant, (2) in EBV immortalized LFSlymphocytes, and (3) in cancer biopsies.

Table 1. TP53 Mutant Alleles Studied by Gene Expression Profiles(GEPs) and Considered in Garritano et al. (2013)

AlleleOccurrencein tumorsa

Numberof GEPb

Transactivationcapabilitiesc

p.A138P 33 1 PDp.V157F 238 1 PDp.R175H 1347 7 SDp.M237I 211 1 PDp.G245S 517 1 SDp.R248Q 1043 1 SDp.R248W 838 2 SDp.R249S 612 1 PDp.R273H 926 5 SDp.R273C 816 1 SDp.R280K 94 1 SDp.R281G 18 4 PD

aAccording to the “Curated TP53 database,” which contains a total num-ber of 30.280 mutations (23.997 exonic missense mutations, 2.612 exonic non-sense mutation, 414 Splice mutations, 3.257 Frameshift mutations) (http://p53.fr/TP53_database_download/TP53_tumor_database/tumor_database.html).bFor each allele, number of independent gene expression profiling (GEP) obtained indifferent cell lines.cTransactivation capacities according to (http://p53.fr/TP53Mutload/database_access/search.php) considering Severe Deficiency (SD) allele, an allele that showedan activity <25% with respect to WT on every transactivation assay tested and PartialDeficiency (PD) allele, an allele that showed an activity >25% with respect to WT on atleast one transactivation assay tested (Monti et al., 2007).

TP53 Mutants’-Dependent GEP in Cancer Cell Lines

Recently, Garritano et al. (2013) reviewed studies that investi-gated transcriptional reprogramming upon ectopic expression of aTP53 mutant as a means to identify deregulated targets and path-ways. Twenty-six GEPs obtained with 12 different alleles and de-riving from 10 studies were compared. Most of the studied TP53mutant proteins are classified as SD, based on the TP53 mutant asses-sor tool (http://p53.fr/TP53Mutload/database_access/search.php)(Table 1). The comparison among differentially expressed genesthat were identified upon the overexpression of TP53 mutants ledto define cellular pathways potentially affected. Interestingly, nearly600 genes were found to be commonly up- or downregulated uponectopic expression of at least three different TP53 mutants. Com-paring these genes with the list of well-established TP53 target genes[Riley et al., 2008] and ChIP-seq occupancy data for WT TP53[Nikulenkov et al., 2012; Menendez et al., 2013] the authors con-cluded that it is conceivable that a majority of genes differentiallyexpressed by TP53 mutants are a consequence of LoF, that is, lossof WT TP53-mediated transactivation or repression. However, GoFproperties of TP53 mutants could not be excluded. Interestingly,pathway analysis indicated that TP53 mutants led to differential ex-pression of genes involved in several metabolic pathways, includingthe metabolism/catabolism of amino acids such as aspartate, gluta-mate, arginine, and proline (Table 5 in [Garritano et al., 2013]).

A recent systematic study used clones of TP53-null H1299 cellline, with inducible expression of six TP53 LoF hotspot mutants(p.R175H, p.R248Q, p.R248W, p.R249S, p.R273H, and p.R282W)and examined global transcriptional changes upon induction ofTP53 mutant [Neilsen et al., 2011]. Of the 59 activated target genescommon to all TP53 mutants, only five were found upregulated byall TP53 mutants but not by WT TP53. Among the 59 genes, en-richment for secreted factors influencing cell invasion was observedand a new mechanism involving corecruitment of TP53 mutantstogether with P63 at promoter sites was proposed.

A very recent article established a link between TP53 func-tions and innate immunity [Wang et al., 2013a]. Included in the

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Figure 3. Analysis of transcriptome changes by exons arrays fol-lowing TP53 mutant ectopic expression in the TP53-null HCT116 cellline. Raw data from the experiments [Wang et al., 2013a] were re-trieved and compared as described in the text. Analyses of gene expres-sion changes common to the five mutants (p.R175H, p.G245D, p.R273C,p.R280T, and p.R282W) compared with WT TP53 identified 62 repressedand 85 induced genes. Ontology pathways significantly enriched for up-or downregulated genes are shown.

manuscript was an analysis of transcriptome changes by exonarrays obtained for five SD TP53 mutants (p.R175H, p.G245D,p.R273C, p.R280T, and p.R282W) ectopically expressed in theTP53 null HCT116 cell line, with WT TP53 included as control.Raw data from the experiments (GSE49046) were retrieved andcomparison between TP53 mutant-driven transcription changeswas examined using GEO2R (Gene Expression Omnibus Webtool; http://www.ncbi.nlm.nih.gov/geo/geo2r/) and DAVID (TheDatabase for Annotation, Visualization and Integrated Discovery;http://david.abcc.ncifcrf.gov/home.jsp). Analysis of gene expressionchanges common to the five mutants compared with WT TP53identified 62 repressed, and 85 induced genes (log2FC > 1). Theonly ontology pathway enriched with the 62 genes group was “TP53signaling,” consistent with the LoF of the TP53 mutants studied;other enriched terms were glycosylation and signal peptide. For thegroup of 85 induced genes by TP53 mutants, “olfactory transduc-tion” was the only enriched pathway with enriched terms including“olfaction” and “sensory perception” (Fig. 3).

Overall, global gene expression studies with overexpressed TP53mutants in cancer cell lines are not revealing a consistent pattern oftranscriptional changes, a result possibly suggestive of cell-specificas well as allele-specific effects, but potentially biased by the experi-mental approaches.

GEP of Germline TP53 Mutants

Very recently, Zerdoumi et al. (2013) showed a highly significantlower mean age of tumor onset in LFS patients harboring dominantnegative missense mutations compared with those harboring TP53deletions, hence resulting in a null phenotype (22.6 and 37.5 years,respectively). These authors immortalized peripheral blood lym-phocytes by Epstein–Barr virus (EBV) infection from three healthycontrol subjects and from six TP53 mutation carriers belonging tosix unrelated LFS families. They showed that missense TP53 mu-tations do not alter TP53 mRNA expression in EBV-immortalizedlymphocytes. Next, they developed an ex vivo functional assay for

the TP53 pathway. The EBV-immortalized lymphocytes were ex-posed to doxorubicin and whole GEP in WT versus TP53 mu-tants were characterized and compared. In TP53 WT immortalizedlymphocytes, a group of 173 genes, whose expression was inducedmore than twofold, defined a core response to DNA damage. Thiscore included 46 genes that had previously been characterized asP53 target genes, such as CDKN1A, MDM2, BBC3, TP53I3, DDR1(MIM #600408), and GLS2. In LFS immortalized lymphocytes withp.R175H, p.R248W, and p.R273H, the number of induced TP53 tar-get genes and the level of induction were strongly reduced as com-pared both with controls and with LFS lymphocytes with mutationsthat produced no full length protein. The authors concluded thatthe TP53 response to DNA damage was particularly compromisedin LFS EBV-immortalized lymphocytes cells with known dominantnegative TP53 germline missense mutations. Interestingly, of the15 known TP53 responsive genes showing altered expression inLFS versus WT EBV-immortalized lymphocytes six ([GLS2; MIM#606365], [SULF2; MIM #610013], TP53I3, SLC52A1 [GPR172B;MIM #607883], [SESN1; MIM #606103], [FDXR; MIM #103270])are related to metabolism.

GEP in Tumors. The Elusive Quest for TP53 Mutant-SpecificGEPs in Tumor Samples: An Example from Breast Cancer

Perturbations of the TP53 pathway are associated with more ag-gressive and therapeutically refractory tumors. However, molecularassessment of TP53 status, by using sequence analysis and immuno-histochemistry, is an incomplete assessor of TP53 functional effects.Miller et al. (2005) proposed that a transcriptional fingerprint wouldbe a more definitive downstream indicator of TP53 functions. Theyanalyzed gene transcription profiles of 251 TP53-sequenced primarybreast tumors, 58 of which showed TP53 mutations. To look moreclosely at these results also in the light of TP53 mutant functionalclassifications, 21 mutations by virtue of nonsense mutations, dele-tion or insertion are expected to produce a truncated protein (i.e.,they are Obligate SD alleles), whereas the remaining 37 containeda mutation of the missense type, resulting in 29 different amino-acid changes. Of these 37 tumors, 21 expressed SD TP53 mutants(Table 2). Hence, in the whole cohort of breast tumor patients themajority of GEPs (42/58) tumors with TP53 mutations harbor SDalleles and are expected to have lost ability to transactivate canonicalWT TP53 target genes.

The authors identified a signature consisting of 32 genes that wasable to distinguish TP53 mutant and WT breast tumors of differenthistology, and more importantly, outperformed sequence-based as-sessments of TP53 in predicting prognosis and therapeutic response[Miller et al., 2005]. Provocatively, the authors noted that none ofthe signature genes were known TP53 targets nor had been previ-ously implicated in the TP53 pathway. Moreover, promoter analysisrevealed evidence of p53-binding sites at �15% (5/32) of these genes([CYBRD1; MIM ∗605745], [AFF3; MIM ∗601464], [DHRS2; MIM∗615194], [STC2; MIM ∗603665], [LRP2; MIM ∗600073]), a re-sults that holds globally true also considering very recent ChIP-seqdata [Nikulenkov et al., 2012; Menendez et al., 2013]. However, the32 genes identified may represent either specific targets of TP53 mu-tants through a GoF activity or reflect gene expression programs as-sociated with tumors where the TP53 pathway is inactivated throughtranscription independent mechanisms, hence they would not beexpected to be direct targets of WT TP53.

Overall, the current state of the art indicates that it is very difficultto recognize in cancer biopsies specific GEPs dependent from thepresence of individual sporadic TP53 mutants.

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Table 2. TP53 Mutant Alleles Studied by GEPs and Considered inMiller et al. (2005)

AlleleOccurrencein tumorsa

Number ofGEPb

Transactivationcapabilitiesc GoFd

p.G28A 1 1 PD N.A.p.P36L 2 1 PD N.A.p.P152L 99 1 PD N.A.p.V157G 19 1 PD N.A.p.R158P 25 1 PD N.A.p.A159V 55 1 PD N.A.p.V173M 103 1 SD YESp.R175H 1347 2 SD YESp.H179Q 29 1 PD N.A.p.R181H 34 1 PD N.A.p.H193R 123 2 SD N.A.p.H193L 81 1 SD N.A.p.Y205C 147 2 PD YESp.T211A 7 1 PD N.A.p.Y220C 486 2 SD N.A.p.Y236C 102 1 SD N.A.p.M237I 211 1 SD YESp.M246T 9 1 SD N.A.p.R248Q 1043 3 SD YESp.R248W 838 2 SD YESp.R249S 612 1 PD YESp.I255F 52 1 PD N.A.p.G266V 71 1 SD N.A.p.R273C 816 2 SD YESp.R273L 162 1 SD YESp.A276G 8 1 PD N.A.p.R280G 53 1 PD N.A.p.R282P 29 1 SD N.A.p.E285K 99 1 PD YES

aAccording to the “Curated TP53 database.”bFor each allele, number of independent GEPs obtained in different cell lines.cTransactivation capacities according to (http://p53.fr/TP53Mutload/database_access/search.php) and considering the definition described in Monti et al. (2007).dExistence of information about Gain-of-Function (GoF) properties of the differentalleles according to (http://p53.fr/TP53Mutload/database_access/search.php).N.A., data not available.

Gene signatures that can serve as biomarkers of TP53 pathwayfunctionality or else of TP53 mutant GoF can be, however, an achiev-able task (Fig. 4).

Figure 4. Variables limiting the possibility to identify TP53 mutant-dependent GEPs in tumors. A GEP is the final result of a complex processin which the specific impact of TP53 mutant proteins is one among manyvariable influencing gene expression networks.

ConclusionsThe amount of information available for TP53 mutants expressed

in cancer cell lines and tumors, and the variability and complexity ofresults obtained are staggering. Remarkably, despite nearly 30 yearsof intense research after recognizing the tumor suppressor functionof WT TP53, it is not yet entirely clear what the critical TP53 path-way for tumor suppression is, among cell cycle control, inductionof apoptosis, or modulation of metabolic pathways, or others [Liet al., 2012]. Is there a common “language” of TP53 mutants thatcould be learned and exploited in the clinical settings? The fact thattranscription signatures can be shaped by various levels of indirecteffects and by interindividual variability, limits the possibility toascertain TP53 dependence of gene expression in human specimen[Nikulenkov et al., 2012]. The gain of knowledge of recent years hasbeen vast. The challenge for the near future is to sift through thevariability and noise in the data, and translate the diversity of TP53mutant driven effects into personalized intervention strategies.

Acknowledgment

Disclosure statement: The authors declare no conflict of interest.

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