Essential role of PI3-kinase pathway in p53-mediated transcription: Implications in cancer chemotherapy

ORIGINAL ARTICLE

Essential role of PI3-kinase pathway in p53-mediated transcription:

Implications in cancer chemotherapy

R Suvasini and K Somasundaram

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India

The PI3-kinase pathway is the target of inactivation inachieving better cancer chemotherapy. Here, we reportthat p53-mediated transcription is inhibited by pharma-cological inhibitors and a dominant-negative mutant ofPI3-kinase, and this inhibition was relieved by aconstitutively active mutant of PI3-kinase. Akt/PKBand mTOR, the downstream effectors of PI3-kinase,were also found to be essential. LY294002 (PI3-kinaseinhibitor) pre-treatment altered the post-translationalmodifications and the sub-cellular localization of p53.Although LY294002 increased the chemosensitivity ofcells to low concentrations of adriamycin (adriamycin-low),it protected the cells from cytotoxicity induced by highconcentrations of adriamycin (adriamycin-high) in a p53-dependent manner. Further, we found that LY294002completely abolished the activation of p53 target genes(particularly pro-apoptotic) under adriamycin-high condi-tions, whereas it only marginally repressed the p53 targetgenes under adriamycin-low conditions; in fact, it furtheractivated the transcription of NOXA, HRK, APAF1 andCASP5 genes. Thus, the differential effect of PI3-kinaseon p53 functions seems to be responsible for thedifferential regulation of DNA damage-induced cytotoxi-city and cell death by PI3-kinase. Our finding becomesrelevant in the light of ongoing combination chemotherapytrials with the PI3-kinase pathway inhibitors and under-scores the importance of p53 status in the carefulformulation of combination chemotherapies.Oncogene (2010) 29, 3605–3618; doi:10.1038/onc.2010.123;published online 26 April 2010

Keywords: p53; PI3-kinase; chemosensitivity; chemotherapy

Introduction

The PI3-kinase pathway is a prominent pro-survivalpathway deregulated in a wide spectrum of humancancers. Activating mutations and gene amplifications inClass IA-PI3K have been found in many humanmalignancies establishing this group of PI3Ks as potent

oncogenes (Vivanco and Sawyers, 2002). The PI3-kinasepathway and its downstream effectors (AKT andmTOR) are known to regulate various cellular processessuch as proliferation, growth, apoptosis and cytoskeletalrearrangement (Vivanco and Sawyers, 2002). Theattenuation of survival signals emanating downstreamto PI3-kinase is believed to be instrumental in increasingcancer cell death and this has made the PI3-kinasepathway a target for novel anti-cancer treatments alongwith conventional chemotherapy (LoPiccolo et al., 2008;Engelman, 2009).

Christened as ‘Guardian of the Genome’, the p53protein is one of the most potent tumour suppressorsknown, which integrates multiple stress signals toinitiate and execute the decisions between life and death(Lacroix et al., 2006). Our present understandingoutlines a vital function for p53 as a central regulatorof cell fate in response to various stresses—genotoxicstresses, hypoxia, nucleotide depletion, oncogene activa-tion, heat shock, telomere erosion and so on (Meek,2004; Lacroix et al., 2006; Levine and Oren, 2009;Vousden and Prives, 2009). Thus, p53 acts as a node formultiple stress signals and initiates appropriate res-ponses largely by virtue of its transcriptional activationfunctions (Meek, 1997). Keeping in mind the growthinhibitory effects of p53, its function is tightly regulatedin normal cells by multiple mechanisms largely throughalterations in the p53 protein, which regulate its activityunder non-stressed conditions (Kruse and Gu, 2009).The importance of p53 is also underlined by the fact thatit can contribute to efficient cell killing by the variousanti-neoplastic agents by virtue of its growth suppressivefunctions (Lowe et al., 1994).

The nodal functions of the PI3-kinase pathway andp53 have made them potent targets for the developmentof novel combination-based chemotherapeutic regimensaiming to inhibit the pro-survival effects of PI3-kinaseand to accentuate the growth suppressive effects of p53.We now report that the activation of p53 by DNA-damaging agents such as adriamycin requires thepresence of a functional PI3-kinase pathway, whichdifferentially regulates the resultant cytotoxic effects.Our findings become relevant in the light of the ongoingclinical trials for combination chemotherapy with thePI3-kinase pathway inhibitors and chemotherapeuticdrugs as it seems that by blocking the PI3-kinasepathway, one might be compromising p53 function,further leading to chemoresistance.

Received 1 July 2009; revised 7 January 2010; accepted 28 February2010; published online 26 April 2010

Correspondence: Professor K Somasundaram, Department of Micro-biology and Cell Biology, Indian Institute of Science, C V RamanAvenue, Bangalore, Karnataka 560012, India.E-mail: [email protected]

Oncogene (2010) 29, 3605–3618& 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10

www.nature.com/onc

http://dx.doi.org/10.1038/onc.2010.123

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Results

PI3-kinase pathway is essential for p53-mediatedtranscriptionTo identify the cellular signalling pathways, whichregulate p53 activation on DNA damage, we determinedthe ability of adriamycin-induced p53 to activatetranscription from PG13-Luc, a synthetic p53-specificreporter, in cells treated with inhibitors of variouscellular signal transduction pathways (el-Deiry et al.,1993). Although adriamycin activated PG13-Luc effi-ciently in A549 cells, addition of PI3-kinase inhibitors,LY294002 and wortmannin, effectively repressed it(Figure 1a, compare lanes 3 and 4 with 2). A similarresult was also seen in HCT116 cells (data not shown).LY294002 also inhibited transcription by exogenouslyexpressed p53 (Figure 1b). Induction of transcript levelsof p53 targets, PUMA, p21WAF1/CIP1, GADD45a,NOXA, MDM2, BAX, CASP1 and APAF1 was alsoefficiently blocked by LY294002 (Figure 1c). Similarly,BIRC5 (Survivin), a transcriptionally repressed target ofp53, was efficiently down-regulated on adriamycinaddition, but this repression was relieved on LY294002pre-treatment (Figure 1d). Induction of p21WAF1/CIP1

protein by adriamycin was also abolished onLY294002 pre-treatment (Figure 1e, compare lanes 8–11 with 12–15). Similar loss of adriamycin-mediatedinduction was also seen for other p53 target proteinssuch as BAX and GADD45a on LY294002 pre-treatment (data not shown).

In an effort to determine whether the effect ofLY294002 on p53 is specific to PI3-kinase inhibition,we found that even 10 mM LY294002 (specific to PI3-kinase inhibition) abolished p53-mediated transcription(Supplementary Figure SF1A, compare lane 4 with 2).Further, we found that a dominant-negative constructof PI3-kinase (DNPI3K) also inhibited adriamycin-induced p53 activation (Figure 2a, compare lane 4 with2) and this inhibition was relieved by a constitutivelyactive mutant of PI3-kinase (CAPI3K) (Figure 2b,compare lane 5 with 4). In addition, small moleculeinhibitors of Akt/PKB and mTOR (downstream targetsof PI3-kinase) also inhibited p53 activation on DNAdamage (Figure 2c, compare lanes 5 and 6 with 2).Substantiating this finding, a dominant-negative con-struct of Akt (AKT K179M-T308A-S473A) also effec-tively inhibited adriamycin-induced p53 activation(Figure 2d, compare lane 4 with 2).

As adriamycin has been shown to activate the PI3-kinase pathway (Li et al., 2005), we monitored theSer473 phospho-Akt levels, as an indication of PI3-kinaseactivation, in our experimental conditions. We founda time-dependent increase in phosphorylated Aktlevels in adriamycin-treated cells with a maximumactivation at 8 h (Supplementary Figure SF1B). Ser473

phospho-Akt levels were completely abolished uponLY294002 treatment at all times tested (SupplementaryFigure SF1B), suggesting an efficient and immediateinhibition of PI3-kinase by LY294002. These resultstogether suggest an essential function for PI3-kinaseand its downstream effectors, Akt/PKB-mTOR, in

activating p53-mediated transcription during DNAdamage.

The existing paradigm about the nature of interactionsbetween the PI3-kinase and p53 pathways is of antag-onistic nature, wherein AKT inhibits p53 function in anMdm2-dependent manner (Mayo and Donner, 2001;Zhou et al., 2001; Gottlieb et al., 2002; Ogawara et al.,2002; Levine et al., 2006). Our results on the other handsuggest that a functional PI3-kinase pathway is essentialfor the transcriptional activation functions of p53.Investigation of Mdm2 phosphorylation status revealeda dramatic increase in the levels of Ser166 phospho-Mdm2upon adriamycin treatment, which was abrogated byLY294002 pre-treatment (Supplementary Figure SF1C).Thus, it seems that although the Akt-Mdm2-mediatedp53 degradative pathway is inactivated on LY294002treatment (as manifested by the loss of the activatingphosphorylation on Mdm2), p53 function is still severelycompromised. This adds another level of complexity tothe known interactions between PI3-kinase and p53,which merited further investigation.

Inhibition of PI3-kinase affects multiple aspectsof p53 activationTo determine the mechanism of inhibition of p53 byLY294002 in adriamycin-treated cells, we first analysedthe activation-associated post-translational modifica-tions of p53. Adriamycin addition resulted in a time-dependent increase in total p53 and Ser15, Ser20, Ser392

and Ser46 phosphorylations and Lys382 acetylation(Supplementary Figure SF2). However, on LY294002pre-treatment, adriamycin induced p53 protein lessefficiently (Figure 3A, compare lanes 11–14 with 7–10).Although Ser15 phosphorylation was not affectedsignificantly, Ser392 and Ser20 phosphorylations aresignificantly reduced (complete loss and 35%, respec-tively, at 24 h) in cells treated with LY294002 andadriamycin (Figure 3A, compare lanes 11–14 with 7–10).Lys382 acetylation, a measure of sequence-specific DNAbinding by p53, was also substantially reduced onLY294002 and adriamycin treatment (Figure 3A, com-pare lanes 11–14 with 7–10; 40% reduction at 24 h).

To test whether altered localization could be anotherreason for reduced p53 activity, we measured p53 levelsin the nucleus and cytoplasm by confocal microscopyand sub-cellular fractionation. Although adriamycinaddition resulted in accumulation of p53 in the nucleus(Figure 3B, compare panel b with a), LY294002 pre-treatment resulted in 50% reduction in nuclear p53 withsubstantial amounts seen in the cytoplasm as well(Figures 3B, compare panel d with b, and C comparebar 4 with 3). This result was further confirmed by sub-cellular fractionation. We found a 50% reduction in thenuclear p53 in cells treated with LY294002 andadriamycin as against adriamycin alone (Figure 3D,compare lane 5 with 4). However, there was nocompensatory increase in cytoplasmic p53 (Figure 3D,compare lane 10 with 9), which could be due toproteasomal degradation as explained by the reductionin the total p53 levels on treatment with LY294002 and

p53 requires PI3-kinaseR Suvasini and K Somasundaram

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Figure 1 Effect of PI3-kinase inhibitors on p53-mediated transactivation. (a) A549 cells were transfected with 1mg of PG13-Luc. After 6hof transfection, cells were treated with LY294002 (50mM) or wortmannin (1mM) and 1h later, adriamycin (0.5mg/ml) was added. After 24hof adriamycin addition, lysates were prepared and assayed for luciferase activity. These experiments were repeated at least three times and arepresentative experiment result is shown. (b) A549 cells were transfected with 1mg of PG13-Luc and indicated amounts of p53. After 6h oftransfection, cells were treated with LY294002, and 24h post-transfection, lysates were prepared and assayed for luciferase activity. Theseexperiments were repeated at least three times and a representative experiment result is shown. (c, d) A549 cells were treated with LY294002(50mM) and 1h later, adriamycin (0.5mg/ml) was added. After 24h of adriamycin addition, total RNA was isolated and subjected to theRT–qPCR analysis for p21WAF1/CIP1, PUMA, MDM2, BAX, NOXA, GADD45a, CASP1 and APAF1 (c) and BIRC5 (d). (e) A549 cellswere treated with LY294002 (50mM) and 1h later, adriamycin (0.5mg/ml) was added. After 24h of adriamycin addition, total lysates wereprepared and subjected to western blot analysis for p21WAF1/CIP1 and PCNA (internal control) proteins.


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adriamycin. From these results we conclude that multi-ple aspects of p53 activation are affected on LY294002treatment.

Differential regulation of DNA damage-induced cellularcytotoxicity by PI3-kinaseOur finding that PI3-kinase pathway is required for theactivation of DNA damage-induced p53 would implythat inhibition of PI3-kinase pathway may lead tochemoresistance because of abrogation of p53-mediatedapoptosis. This was rather intriguing because inactiva-tion of the PI3-kinase pathway has in fact been reportedto induce apoptosis and sensitize the cells to chemother-apy (Fujiwara et al., 2006). With the hypothesis that theextent of DNA damage could potentially determine theeffect of the PI3-kinase pathway on p53 function andthereby on chemosensitivity, we carried out cytotoxicity

assays by treating the cells with varying adriamycinconcentrations with/without LY294002 pre-treatment.As expected, adriamycin treatment alone resulted ina concentration-dependent increase in cytotoxicity(Figure 4a, black bars). However, LY294002 pre-treatment resulted in varying effects on chemosensitiv-ity. On treatment with lower concentrations of adria-mycin (adriamycin-low; 0.05, 0.1, 0.2 and 0.4 mg/ml) andpresumably at lower levels of DNA damage, we found,as reported earlier (Fujiwara et al., 2006), thatLY294002 pre-treatment resulted in chemosensitization(Figure 4a, grey bars). However, as the concentration ofadriamycin and presumably the DNA damage wasincreased further (adriamycin-high, 0.8 and 1.0 mg/ml),this chemosensitization effect because of LY294002 waslost and, in fact, resulted in chemoresistance (Figure 4a,grey bars). The chemosensitivity index (cell viabilitywith adriamycin alone/cell viability with LY294002 and

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Figure 2 LY294002 inhibition of p53-mediated transcription is specific to PI3-kinase-Akt/PKB-mTOR pathway. (a) A549 cells weretransfected with 1 mg of PG13-Luc and 1 mg of DNPI3K (dominant negative) construct as indicated. After 6 h of transfection, cells weretreated with LY294002 (10 mM) and 1 h later, adriamycin (0.5mg/ml) was added. After 24 h of adriamycin addition, lysates wereprepared and assayed for luciferase activity. These experiments were repeated at least three times and a representative experiment resultis shown. (b) A549 cells were transfected with 1mg of PG13-Luc, 2mg of DNPI3K (dominant negative) and 2 mg of CAPI3K(constitutively active) constructs as indicated. After 6 h of transfection, cells were treated with adriamycin (0.5mg/ml). After 24 h ofadriamycin addition, lysates were prepared and assayed for luciferase activity. These experiments were repeated at least three times anda representative experiment result is shown. (c) A549 cells were transfected with 1mg of PG13-Luc. After 6 h of transfection, cells weretreated with LY294002 (50mM), wortmannin (1mM), rapamycin (100 nM) or AKT III-inhibitor (10mM) and 1 h later, adriamycin (0.5mg/ml) was added. After 24 h of adriamycin addition, lysates were prepared and assayed for luciferase activity. These experiments wererepeated at least three times and a representative experiment result is shown. (d) A549 cells were transfected with 1mg of PG13-Luc and5 mg of AKT K179M-T308A-S473A constructs as indicated. After 6 h of transfection, cells were treated with LY294002 (50mM) and 1 hlater, adriamycin (0.5mg/ml) was added. After 24 h of adriamycin addition, lysates were prepared and assayed for luciferase activity.These experiments were repeated at least three times and a representative experiment result is shown.


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adriamycin) was measured as effectiveness of thecombination treatment. A high chemosensitivity indexwas evident at adriamycin-low conditions, but was lostat adriamycin-high conditions implying the develop-ment of chemoresistance (Figure 4b). In principle,

similar results were obtained in HCT116 cells also(Figures 4c and d).

We next determined the fate of these surviving cellsafter combination therapy by allowing the cells to grow indrug-free medium for 3 days after 48 h of combination

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Figure 3 Effect of LY294002 on post-translational modification and nuclear localization of p53 in adriamycin-treated cells. (A) A549cells were treated with LY294002 (50mM), and 1 h later, adriamycin (0.5mg/ml) was added as indicated. The total lysates were preparedat indicated time points after adriamycin addition and subjected to western blot analysis for Ser15 phospho p53, Ser392 phospho p53,Ser20 phospho p53, Lys382Ac p53, total p53 and actin (internal control) proteins using specific antibodies. (B) A549 cells were untreated(panels a and b) or treated with LY294002 (50mM) (panels c and d), and 1 h later, adriamycin (0.5mg/ml) was added (panels b and d).After 24 h of adriamycin addition, the cells were fixed and stained with p53 antibody followed by FITC-conjugated anti-mousesecondary antibody. The cells were counterstained with propidium iodide and analysed by confocal microscopy. (C) Theco-localization index for panels a, b, c and d of Figure 3B was calculated and shown as a bar diagram. (D) A549 cells were treated withLY294002 (50 mM), and 1 h later, adriamycin (0.5mg/ml) was added as indicated. The cells were collected after 24 h of adriamycinaddition and separated into nuclear and cytoplasmic fractions and subjected to western blot analysis for total p53, actin (cytoplasmcontrol) and lamin B1 (nucleus control) proteins using specific antibodies.


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treatment. The loss of viable cells was seen on adriamycintreatment in a concentration-dependent manner withcomplete loss seen at 1mg/ml (Supplementary FigureSF3, adriamycin only). The observed chemoresistancewith LY294002 and adriamycin-high conditions corre-lated with the presence of adherent viable-appearing cells,which were absent on LY294002 treatment in adriamycin-

low conditions (Supplementary Figure SF3, adriamycinand LY294002 combination). In a more rigorous experi-mental setup, the adriamycin and LY294002 treatmentwas renewed every 48h, and similar to the earlier scenario,viable cells were seen only at adriamycin-high conditionseven after 7 and 10 days of the combination treatment(Supplementary Figures SF4A and B).

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To correlate the above findings with the extent ofapoptosis, we measured the proportion of the sub-G1population under similar conditions (SupplementaryFigures SF5A and B). At 24 h after adriamycintreatment, a concentration-dependent increase in apop-tosis was observed (Figure 4e, grey line). At adriamycin-low conditions (0.25 and 0.5 mg/ml), LY294002 pre-treatment resulted in a dramatic increase in apoptosiscompared with adriamycin treatment alone (Figure 4e,78.65% from 9.45% (8.32-fold) and 65.18% from15.00% (4.35-fold)). However, LY294002 pre-treatmentat adriamycin-high conditions (1.00 mg/ml) resulted inonly 1.24-fold increase in apoptosis (Figure 4e, 36.93%from 29.90%). However, at 48 h, LY294002 pre-treat-ment actually showed a protective effect under adria-mycin-high conditions (Figure 4f, 76.21% from93.83%). We also measured the extent of apoptosisbased on caspase-3/7 activity and the presence offragmented DNA in the apoptotic cells. As can be seenby these more accurate indicators of apoptosis, there isindeed a remarkable decrease in cell death on treatmentwith high dosages of adriamycin in LY294002 pre-treated cells at 24 h (Figures 4g, compare lanes 11 and 12with 7 and 8; i compare lanes 9 and 10 with 5 and 6) and48 h (Figure 4h, compare lanes 11 and 12 with 7 and 8).

We have also investigated this phenomenon in cellstreated with other DNA-damaging agents such ascisplatin and etoposide. PG13-Luc activity induced byboth these drugs was efficiently inhibited by LY294002pre-treatment (Supplementary Figure SF6A, comparelanes 2 and 3 with 4 and 5, and B compare lanes 2 and 3with 4 and 5). Further, LY294002 enhanced thecytotoxicity of cisplatin at low concentrations, butconferred resistance to high concentrations of cisplatin(Supplementary Figures SF6C and D). These results

together suggest a differential role for the PI3-kinasepathway in modulating DNA damage-induced cytotoxi-city and cell fate decisions, which is most likely mediatedby its regulation of p53 functions.

WT p53 is required for differential regulation of DNAdamage-induced cytotoxicity by PI3-kinaseTo correlate the regulation of p53 functions by PI3-kinase with differential cytotoxicity as seen above, weinvestigated the effect of combination therapy inisogenic cells with different p53 status. HCT116p53WT and HCT116 p53�/� cells were subjected to asimilar experiment as described in Figure 4a and thechemosensitivity index was measured. As can be seen inFigure 5, in HCT116 p53WT cells (black bars),LY294002 pre-treatment at adriamycin-low conditions(0.05, 0.1 and 0.25 mg/ml) resulted in increased chemo-sensitivity, which was lost on combination treatment atadriamycin-high conditions (0.5, 0.75, 1, 2 and 5 mg/ml).However, in HCT116 p53�/� cells, increased chemosen-sitivity on combination treatment was seen in bothadriamycin-low and -high conditions (Figure 5, grey bars).We also found similar results in HeLa cells, in which p53is degraded by HPV18 E6 (Supplementary FiguresSF7A and B). These results suggest an essential functionfor p53 in mediating differential regulation of DNAdamage-induced cytotoxicity by PI3-kinase.

Differential requirement of PI3-kinase for p53 activationAbove results suggest a possibility for differentialrequirement of PI3-kinase in regulating p53 activation.To address this, we investigated the effect of LY294002and DNPI3K on p53-mediated PG13-Luc activationacross a varied concentration range of adriamycin. Both

Figure 4 Effect of LY294002 on adriamycin-induced cytotoxicity and apoptosis. (a) A549 cells were treated with LY294002 (50mM),and 1 h later, indicated concentrations of adriamycin were added. After 48 h of adriamycin addition, proportion of live cells wasquantified by MTT assay. (b) The chemosensitivity index (the ratio of cell viability on treatment with adriamycin alone and ontreatment with LY294002 and adriamycin) from (a) was calculated and shown in log scale. (Please note that chemosensitivity index ishigh and low in adriamycin-low and -high conditions, respectively.) (c) HCT116 cells were treated with LY294002 (50mM), and 1 hlater, indicated concentrations of adriamycin were added. After 48 h of adriamycin addition, proportion of live cells was quantified byMTT assay. (d) The chemosensitivity index (the ratio of cell viability on treatment with adriamycin alone and on treatment withLY294002 and adriamycin) from (a) was calculated and shown in log scale. (Please note that chemosensitivity index is high and low inadriamycin-low and -high conditions, respectively.) (e) A549 cells were either untreated or treated with LY294002 (50mM), and 1 hlater, indicated concentrations of adriamycin were added. After 24 h of adriamycin addition, the cells were harvested and subjected toflow cytometry analysis. The proportion of apoptotic cell population measured as sub-G1 phase is shown. The grey and black linesrepresent per cent apoptosis in adriamycin alone and adriamycin plus LY294002-treated samples, respectively. (Note: Although(grey and black lines) are far from each other at 0.25mg/ml concentration of adriamycin, they come very close at 1mg/ml concentrationof adriamycin, suggesting the loss of chemosensitization at adriamycin-high conditions.) (f) A549 cells were either untreated or treatedwith LY294002 (50 mM), and 1 h later, indicated concentrations of adriamycin were added. After 48 h of adriamycin addition, the cellswere harvested and subjected to flow cytometry analysis. The proportion of apoptotic cell population, measured as sub-G1 phase, from(g) is shown. The grey and black lines represent per cent apoptosis in adriamycin alone and adriamycin plus LY294002-treatedsamples, respectively. (Note: Although grey and black lines are far from each other at 0.25 mg/ml concentration of adriamycin, theycross each other at 1 mg/ml concentration of adriamycin, suggesting the loss of chemosensitization at adriamycin-high conditions.)(g) A549 cells were either treated with DMSO or LY294002 (50mM), and 1 h later, indicated concentrations of adriamycin were added.After 24 h of adriamycin addition, the Apo-one caspase-3/7 cleavage assay (Promega) was carried out. The fluorescent units from thecleavage of the fluorescent substrate have been duly indicated. (h) A549 cells were either treated with DMSO or LY294002 (50mM), and1 h later, indicated concentrations of adriamycin were added. After 48 h of adriamycin addition, the Apo-one caspase-3/7 cleavageassay (Promega) was carried out. The fluorescent units from the cleavage of the fluorescent substrate have been duly indicated. (i) A549cells were either treated with DMSO or LY294002 (50mM), and 1 h later, indicated concentrations of adriamycin were added. After 48 hof adriamycin addition, the Fragel assay (Calbiochem) was carried out to selectively label the apoptotic nuclei with fragmented DNA.The number of positive nuclei and the total number of nuclei based on DAPI staining were counted and the percentage of apoptoticnuclei in each sample was calculated.


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LY294002 and DNPI3K inhibited PG13-Luc activationat all adriamycin concentrations used (SupplementaryFigures SF7C and D), suggesting an absolute require-ment of PI3-kinase for p53-mediated transcription.Altered drug retention could also be responsible forthe differential cytotoxicity observed above. Our in-vestigation revealed that while intra-cellular adriamycin(as measured by its fluorescence; see SupplementaryInformation) increased in a concentration-dependentmanner (Supplementary Figures SF8A and B),LY294002 pre-treatment did not reduce adriamycinretention (Supplementary Figures SF8C, D and E).Corroboratively, the transcript levels of the majortransporters (ABCC1 and ABCG2), known to beinvolved in adriamycin efflux, did not increase in cellstreated with varying concentrations of adriamycin in thepresence and absence of LY294002 (data not shown).

Next, we monitored the transcript levels of variouspro-apoptotic targets of p53 under conditions ofdifferential DNA damage and the effect of LY294002on this regulation. On adriamycin treatment, PUMA,NOXA, HRK, CASP1, APAF1 and CASP5 transcriptlevels increased dramatically in a concentration-depen-dent manner (Figures 6a–f). Interestingly, althoughLY294002 addition completely inhibited the activationof these genes at adriamycin-high conditions (1.0 mg/ml),it only repressed them marginally at adriamycin-lowconditions (0.25 mg/ml) (Figures 6a–f). In fact, adriamy-cin-low conditions activated NOXA, HRK, APAF1 andCASP5 more efficiently in the presence of LY294002(2.5–4.0-fold) than alone (Figures 6b, c, e and f). Wealso found differential activation of p21WAF1/CIP1 andGADD45a by varied concentrations of adriamycin inthe presence of LY294002 (Figures 6g and h). Corro-boratively, although adriamycin caused an increase inSer46 phospho-p53 (needed for activation of pro-apoptotic targets), LY294002 pre-treatment inhibitedSer46 phosphorylation selectively at adriamycin-high

conditions (Figure 6i, compare lane 3 with 4 and 7 with8 and j). Addition of LY294002, thus, selectivelyabrogates the activation of the pro-apoptotic genes byp53 under conditions of extensive DNA damage, andthereby possibly keeps the cells viable. However, withlow concentrations of adriamycin (and presumably lessDNA damage), addition of LY294002 failed to inhibitp53 function efficiently and thereby leads the cellstowards apoptotic death. These results together suggestan important function for the PI3-kinase pathway inregulating p53 functions thereby mediating differentialchemosensitivity.

PI3-kinase inhibition before p53 activation is neededfor modulation of chemosensitivityAs the inhibition of PI3-kinase rendered cells chemore-sistant at high concentrations of adriamycin (higherDNA damage), it was of our interest to furtherinvestigate the effect of varied timings of PI3-kinaseinhibition and chemotherapy administration with re-spect to chemoresistance. Adriamycin cytotoxicityassays were carried out as before with LY294002addition at different time intervals either before or afteradriamycin addition. Efficient chemoresistance at adria-mycin-high conditions was seen when LY294002 wasadded 1 h before adriamycin, simultaneously or up to2 h after adriamycin addition (Figures 7a and i, b and j,c and k, d and l). Under these conditions, we alsoobserved increased chemosensitization on LY294002pre-treatment with adriamycin-low conditions. How-ever, the observed chemoresistance at adriamycin-highconditions was lost when the LY294002 was added at6 h or later after adriamycin addition (Figures 7e and m,f and n, g and o, h and p). It is also interesting to notethat the effective chemosensitization by LY294002 incells treated with low concentrations of adriamycin wasminimized if LY294002 was added beyond 12 h ofadriamycin addition (Figures 7g and o, h and p). Thismay be due to the fact that cell fate decisions havealready been made by this time and blocking PI3-kinaseat this point fails to provide any additional benefit.Thus, it seems that effective PI3-kinase inhibition beforep53 activation is required for both chemoresistance andchemosensitization seen under adriamycin-high and lowconditions, respectively.

Discussion

Activated PI3-kinase pathway, through Akt/PKB-mTOR, has been found to have an important functionduring oncogenesis and chemoresistance (Wendel et al.,2004). As genetic alterations that activate the PI3K–Aktpathway are common in human cancers (Sakai et al.,1998; Min et al., 2003), this pathway has been the targetfor inactivation to achieve better chemotherapy (Hen-nessy et al., 2005; Guillard et al., 2009). P53, which getsinduced by genotoxic stress, has a major function inpreventing genomic instability and thereby tumourdevelopment (Somasundaram, 2000; Vousden, 2006).

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Figure 6 Effect of LY294002 on DNA damage-induced p53 activation. (a–h) A549 cells were treated with LY294002 (50mM), and 1 hlater, indicated concentrations of adriamycin were added. After 24 h of adriamycin addition, total RNA was isolated and subjected tothe RT–PCR analysis for PUMA (a), NOXA (b), HRK (c), CASP1 (d), APAF1 (e), CASP6 (f), GADD45a (g) and p21WAF1/CIP1 (h).(i) A549 cells were treated with LY294002 (50mM), and 1 h later, indicated concentrations of adriamycin were added. After 24 h ofadriamycin addition, total lysates were prepared and subjected to western blot analysis for Ser46 p53 and actin (internal control)proteins using specific antibodies. (j) The densitometric estimation of Ser46 phosphorylated form of p53 was calculated afternormalizing with actin levels from (i) and shown.


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Further, p53 has an important function in apoptosisinduced by DNA-damaging anti-cancer drugs (John-stone et al., 2002). The current understanding of theinterplay between these two pathways is that the PI3-kinase pathway inhibits p53 function and therebypromotes survival (Vivanco and Sawyers, 2002). Incontrast, we show in this work that the DNA damage-activated PI3-kinase pathway has an essential functionin p53-mediated transcription. Although there arereports of PI3-kinase inhibitors repressing p53-mediatedtranscription, these were inconclusive and led to manyunanswered questions (Price and Youmell, 1996; Bar

et al., 2005). In this work, we provide definitiveevidences that it is indeed the specific inhibition ofPI3-kinase by LY294002 that represses p53-mediatedtranscription. Besides the fact that lower concentrationsof LY294002 (10 mM), which specifically inhibit PI3-kinase, were able to abolish p53-mediated transcription,we also show that the expression of a dominant-negativeform of PI3-kinase inhibited p53-mediated transcriptionand this was efficiently overcome by a constitutivelyactive form of PI3-kinase.

The downstream effectors of the PI3-kinase-mediatedactivation of p53 functions were not defined clearly in




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the earlier studies (Price and Youmell, 1996; Bar et al.,2005). In addition, present understanding of the fieldsuggests an Mdm2-mediated repression of p53 functionsby the PI3-kinase pathway (Ogawara et al., 2002). Inour work, we show that PI3-kinase uses the Akt/PKB-mTOR pathway to activate p53 functions. This wasshown by the increase in Ser473 phosphorylated form ofAkt on adriamycin treatment. Inhibition of both Aktand mTOR abrogated transcription by DNA damage-induced p53 in adriamycin-treated cells. Further, adominant-negative mutant of Akt carrying mutations inthe active sites was able to inhibit adriamycin-inducedp53-mediated transcription. We also establish that theknown interplay between the PI3K-Akt-Mdm2 isinconsequential in the above shown regulation of p53functions by PI3-kinase. Our results, thus, indicate theexistence of another layer of complexity in the interac-tions between PI3-kinase and p53. Phosphorylation ofp300 at Ser1834 by Akt is shown to be essential for itshistone acetyl-transferase activity, which is indispensible

for the activation of p53 functions (Huang and Chen,2005). Similarly, constitutive mTOR activity amplifiesp53 activation by stimulating p53 translation (Lee et al.,2007). Thus, it is clear that Akt/PKB-mTOR have animportant function in p53 activation downstream ofPI3-kinase during DNA damage.

The observed inhibition of p53 function by LY294002could occur by multiple means as the regulation of p53function is extremely complex (Kruse and Gu, 2009). Inaddition to a significant reduction in total p53, we foundfunctionally important modifications such as Ser392,Ser20, Ser46 phosphorylations and Lys382 acetylation tobe significantly affected. The mechanism by which theDNA damage-induced PI3-kinase pathway alters p53phosphorylations at important residues is not clear atpresent. However, the reduction in Lys382 acetylation inLY294002-treated cells could be explained by the factthat p300, which acetylates p53, is activated by Akt-mediated phosphorylation (Huang and Chen, 2005). Wealso found substantial reduction in nuclear p53 as





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another reason for p53 inhibition. In good correlation,treatment of cells with Leptomycin-B, a nuclear exportinhibitor, could partially abrogate the inhibition of p53-mediated transcription by LY294002 (data not shown).Thus, it seems that PI3-kinase inhibition affects multipleaspects of p53 activation.

Although the relationship between p53 activation andcell fate regulation by itself is very complex (Meek,2004), the interplay between the classical ‘pro-survival’PI3-kinase pathway and the ‘pro-apoptotic’ p53 path-way, as established in this study, adds another layer ofcomplexity to the outcome on cell fate. Our hypothesisto begin with was that PI3-kinase pathway inhibitionmay not always result in apoptosis induction orincreased chemosensitivity. Indeed, we found that PI3-kinase inhibition in cells treated with higher concentra-tions of adriamycin led to complete loss of chemosensi-tization and to chemoresistance in some cell types. Atlower concentrations of adriamycin, the combinationtreatment with LY294002 led to effective chemosensiti-zation in almost all cell lines tested as reported byothers. We also show that the differential effect ofLY294002 on DNA damage-induced cytotoxicity is dueto differential induction of apoptosis under thoseconditions. In good correlation, activation of p53 targetgenes (particularly pro-apoptotic genes) was severelycompromised on LY294002 pre-treatment in adriamy-cin-high conditions than in adriamycin-low conditions.In addition, this chemoresistance with LY294002 andhigh-adriamycin treatment was lost in a p53-null back-ground, suggesting that the differential regulation ofDNA damage-induced cytotoxicity by PI3-kinase isindeed mediated through WT p53. Although chemo-sensitization on LY294002 pre-treatment was seen at allconcentrations of adriamycin tested in cells lackingfunctional p53, there was still a significant reduction inchemosensitivity at adriamycin-high conditions, whichcould be attributed to similar inhibition of resident p73functions. Indeed, we found p73-mediated transcriptionto be inhibited by LY294002 (data not shown), whichsuggests that the PI3-kinase pathway may also have animportant function in regulating the activation of p53family members such as p73. This suggests that theregulation of DNA damage-induced cytotoxicity by PI3-kinase may be operative in the p53 mutant cancers aswell, through p73, albeit at a more restricted level.Taken together, these results imply a differential functionfor the PI3-kinase pathway in modulating DNA damage-induced cytotoxicity and cell fate decisions by regulatingthe functions of p53 and its family members.

We made another interesting finding when wecompared the chemoresistance in cells receiving combi-nation therapy with the kinetics of activation of PI3-kinase and p53 on DNA damage. We found an efficientPI3-kinase activation (as read by Ser473 phospho-Akt) by4 h with maximum activation seen at 8 h after adriamy-cin addition (Supplementary Figure SF1B). With respectto p53, the DNA damage-induced activation, as read bythe various post-translational modifications, althoughseen by 2 h, reached its maximum levels by 12 h ofadriamycin addition (Supplementary Figure SF2). Thus,

it seems that an efficient PI3-kinase activation is a pre-requisite for DNA damage-induced p53 activation.Indeed our results show that addition of LY294002even up to 2 h after adriamycin addition could modulatep53-activation and thereby the resultant cytotoxicity.However, when LY294002 was added after 6 h ofadriamycin addition, there was a loss of chemoresistanceat adriamycin-high conditions and a progressive lossof chemosensitization at adriamycin-low conditions.These results suggest that inhibition of PI3-kinasebefore p53 activation is required for modulation ofchemosensitivity.

Although most of our work has been carried out withadriamycin as a DNA-damaging agent, we also foundthat similar observations are seen with other cytotoxicagents such as cisplatin and etoposide. Thus, it appearsthat the PI3-kinase pathway plays an essential role inthe transcriptional activation functions of p53 andthereby modulating cell fate decisions. Our findingsbecome very important in view of many ongoingcombination cancer chemotherapy trials with PI3-kinasepathway inhibitors. Thus, this study basically suggeststhat a careful consideration of chemotherapy dosages,timing of PI3-kinase inhibition and p53 status is veryimportant for a successful outcome of combinationchemotherapy regimens.

Materials and methods

Plasmids and reporter constructsPlasmids PG13-Luc and pCEP4/p53 were described before(el-Deiry et al., 1993) (Somasundaram and El-Deiry, 1997).pCMV-LacZ was used to normalize for the transfectionefficiency in the various reporter assays. CAPI3K (pCDNA3-CD2p110myc) and DNPI3K (pSG5-rCD2p85) constructs asdescribed before were kindly provided by Dr A Rangarajan(Reif et al., 1996). A dominant-negative mutant of Akt,pCDNA3AKT1-K179M-T308A-S473A, with three point mu-tations at the active sites was obtained from Dr WR Sellersthrough Addgene (plasmid 9031) (Ramaswamy et al., 1999).

Drugs and inhibitorsLY294002, wortmannin and rapamycin (Alomone Bio-sciences, Jerusalem, Israel) were used at a final concentrationof 50 mM, 1mM and 100 nM, respectively. AKT inhibitor-III andleptomycin-B (Calbiochem, EMD4Biosciences, Gibbstown,NJ, USA) were used at a final concentration of 10mM and10 ng/ml, respectively. Adriamycin/doxorubicin was purchasedfrom Sigma (Sigma-Aldrich, St Louis, MO, USA).

Cell lines, transfections and reporter assaysA549 (p53 WT), HCT116 (p53 WT), HCT116 p53�/� (p53 null;kindly provided by Dr Bert Vogelstein) and HeLa (HPV16positive) were cultured in DMEM with 10% Fetal calf serum.Transfection and reporter assays were performed as describedbefore (Das et al., 2003).

RNA isolation, cDNA synthesis and real time PCRTotal RNA isolation, cDNA synthesis and real time PCRwere performed as described (Reddy et al., 2008). GAPDH(glyceraldehyde-3-phosphate dehydrogenase) ACTB (actin),18 s rRNA (18s ribosomal RNA) and RPL35a (ribosomal


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protein L35a) were used as internal reference. The primersequences and conditions used for RT–PCR will be providedon request.

Western blot analysis, flow cytometry, MTT assay, caspase-3/7cleavage assay, fragel DNA fragmentation assay and ELISAWestern blot analysis, FACS analysis and MTT assays wereperformed as described (Mungamuri et al., 2006; see alsoSupplementary Information). Caspase 3/7 cleavage assay(Promega, Madison, WI, USA) Fragel DNA fragmentationassay (Calbiochem, EMD4 Biosciences) and ELISA (CellSignaling Technologies, MA, USA) were performed as permanufacturer’s instructions.

Confocal analysis and sub-cellular fractionationA549 cells were seeded onto chamber slides (BD Biosciences,San Jose, CA, USA) and treated as indicated. The cells werefixed with 70% ethanol for 10min at room temperature. Afterblocking with goat serum, the samples were incubated with theprimary antibody for 2 h followed by three washes withphosphate-buffered saline. Samples were subsequently incu-bated with the FITC-conjugated secondary antibody in darkfor 2 h and then washed three times with phosphate-bufferedsaline. After mounting with an anti-fade agent, confocalimages were taken on Zeiss LSM 510 Meta confocal laserscanning microscope using plan-Apochromat 63X/1.4 oil DICobjective. For sub-cellular fractionation, appropriately treated

cells were harvested and nuclear and cytoplasmic fractionswere isolated as described before (Kemler et al., 1989). Thenuclear and cytoplasmic fractions from equal number of cellswere then used for western blotting and probed for p53, laminB1 and actin as described earlier.

Abbreviations

PI3-kinase, phosphoinositide 3-kinase; PKB/Akt, proteinkinase B/Akt; mTOR, mammalian target of rapamycin.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

KS is a Wellcome Trust International Senior Research Fellow.Infrastructural support by funding from ICMR, DBT, DSTand UGC to MCB is acknowledged. RS gratefully acknowl-edges SRF from CSIR.

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