Essential role of PI3-kinase pathway in p53-mediated ... - Nature

Oncogene (2010) 29, 3605–3618

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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 in achieving better cancer chemotherapy. Here, we report that p53-mediated transcription is inhibited by pharmacological inhibitors and a dominant-negative mutant of PI3-kinase, and this inhibition was relieved by a constitutively active mutant of PI3-kinase. Akt/PKB and mTOR, the downstream effectors of PI3-kinase, were also found to be essential. LY294002 (PI3-kinase inhibitor) pre-treatment altered the post-translational modifications and the sub-cellular localization of p53. Although LY294002 increased the chemosensitivity of cells to low concentrations of adriamycin (adriamycin-low), it protected the cells from cytotoxicity induced by high concentrations of adriamycin (adriamycin-high) in a p53dependent manner. Further, we found that LY294002 completely abolished the activation of p53 target genes (particularly pro-apoptotic) under adriamycin-high conditions, whereas it only marginally repressed the p53 target genes under adriamycin-low conditions; in fact, it further activated the transcription of NOXA, HRK, APAF1 and CASP5 genes. Thus, the differential effect of PI3-kinase on p53 functions seems to be responsible for the differential regulation of DNA damage-induced cytotoxicity and cell death by PI3-kinase. Our finding becomes relevant in the light of ongoing combination chemotherapy trials with the PI3-kinase pathway inhibitors and underscores the importance of p53 status in the careful formulation 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-survival pathway deregulated in a wide spectrum of human cancers. Activating mutations and gene amplifications in Class IA-PI3K have been found in many human malignancies establishing this group of PI3Ks as potent Correspondence: Professor K Somasundaram, Department of Microbiology and Cell Biology, Indian Institute of Science, C V Raman Avenue, Bangalore, Karnataka 560012, India. E-mail: [email protected] Received 1 July 2009; revised 7 January 2010; accepted 28 February 2010; published online 26 April 2010

oncogenes (Vivanco and Sawyers, 2002). The PI3-kinase pathway and its downstream effectors (AKT and mTOR) are known to regulate various cellular processes such as proliferation, growth, apoptosis and cytoskeletal rearrangement (Vivanco and Sawyers, 2002). The attenuation of survival signals emanating downstream to PI3-kinase is believed to be instrumental in increasing cancer cell death and this has made the PI3-kinase pathway a target for novel anti-cancer treatments along with conventional chemotherapy (LoPiccolo et al., 2008; Engelman, 2009). Christened as ‘Guardian of the Genome’, the p53 protein is one of the most potent tumour suppressors known, which integrates multiple stress signals to initiate and execute the decisions between life and death (Lacroix et al., 2006). Our present understanding outlines a vital function for p53 as a central regulator of cell fate in response to various stresses—genotoxic stresses, hypoxia, nucleotide depletion, oncogene activation, 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 for multiple stress signals and initiates appropriate responses largely by virtue of its transcriptional activation functions (Meek, 1997). Keeping in mind the growth inhibitory effects of p53, its function is tightly regulated in normal cells by multiple mechanisms largely through alterations in the p53 protein, which regulate its activity under non-stressed conditions (Kruse and Gu, 2009). The importance of p53 is also underlined by the fact that it can contribute to efficient cell killing by the various anti-neoplastic agents by virtue of its growth suppressive functions (Lowe et al., 1994). The nodal functions of the PI3-kinase pathway and p53 have made them potent targets for the development of novel combination-based chemotherapeutic regimens aiming to inhibit the pro-survival effects of PI3-kinase and to accentuate the growth suppressive effects of p53. We now report that the activation of p53 by DNAdamaging agents such as adriamycin requires the presence of a functional PI3-kinase pathway, which differentially regulates the resultant cytotoxic effects. Our findings become relevant in the light of the ongoing clinical trials for combination chemotherapy with the PI3-kinase pathway inhibitors and chemotherapeutic drugs as it seems that by blocking the PI3-kinase pathway, one might be compromising p53 function, further leading to chemoresistance.

p53 requires PI3-kinase R Suvasini and K Somasundaram

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Results PI3-kinase pathway is essential for p53-mediated transcription To identify the cellular signalling pathways, which regulate p53 activation on DNA damage, we determined the ability of adriamycin-induced p53 to activate transcription from PG13-Luc, a synthetic p53-specific reporter, in cells treated with inhibitors of various cellular signal transduction pathways (el-Deiry et al., 1993). Although adriamycin activated PG13-Luc efficiently in A549 cells, addition of PI3-kinase inhibitors, LY294002 and wortmannin, effectively repressed it (Figure 1a, compare lanes 3 and 4 with 2). A similar result was also seen in HCT116 cells (data not shown). LY294002 also inhibited transcription by exogenously expressed p53 (Figure 1b). Induction of transcript levels of p53 targets, PUMA, p21WAF1/CIP1, GADD45a, NOXA, MDM2, BAX, CASP1 and APAF1 was also efficiently blocked by LY294002 (Figure 1c). Similarly, BIRC5 (Survivin), a transcriptionally repressed target of p53, was efficiently down-regulated on adriamycin addition, but this repression was relieved on LY294002 pre-treatment (Figure 1d). Induction of p21WAF1/CIP1 protein by adriamycin was also abolished on LY294002 pre-treatment (Figure 1e, compare lanes 8– 11 with 12–15). Similar loss of adriamycin-mediated induction was also seen for other p53 target proteins such as BAX and GADD45a on LY294002 pretreatment (data not shown). In an effort to determine whether the effect of LY294002 on p53 is specific to PI3-kinase inhibition, we found that even 10 mM LY294002 (specific to PI3kinase inhibition) abolished p53-mediated transcription (Supplementary Figure SF1A, compare lane 4 with 2). Further, we found that a dominant-negative construct of PI3-kinase (DNPI3K) also inhibited adriamycininduced p53 activation (Figure 2a, compare lane 4 with 2) and this inhibition was relieved by a constitutively active mutant of PI3-kinase (CAPI3K) (Figure 2b, compare lane 5 with 4). In addition, small molecule inhibitors of Akt/PKB and mTOR (downstream targets of PI3-kinase) also inhibited p53 activation on DNA damage (Figure 2c, compare lanes 5 and 6 with 2). Substantiating this finding, a dominant-negative construct of Akt (AKT K179M-T308A-S473A) also effectively inhibited adriamycin-induced p53 activation (Figure 2d, compare lane 4 with 2). As adriamycin has been shown to activate the PI3kinase pathway (Li et al., 2005), we monitored the Ser473 phospho-Akt levels, as an indication of PI3-kinase activation, in our experimental conditions. We found a time-dependent increase in phosphorylated Akt levels in adriamycin-treated cells with a maximum activation at 8 h (Supplementary Figure SF1B). Ser473 phospho-Akt levels were completely abolished upon LY294002 treatment at all times tested (Supplementary Figure SF1B), suggesting an efficient and immediate inhibition of PI3-kinase by LY294002. These results together suggest an essential function for PI3-kinase and its downstream effectors, Akt/PKB-mTOR, in Oncogene

activating p53-mediated transcription during DNA damage. The existing paradigm about the nature of interactions between the PI3-kinase and p53 pathways is of antagonistic nature, wherein AKT inhibits p53 function in an Mdm2-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 hand suggest that a functional PI3-kinase pathway is essential for the transcriptional activation functions of p53. Investigation of Mdm2 phosphorylation status revealed a dramatic increase in the levels of Ser166 phospho-Mdm2 upon adriamycin treatment, which was abrogated by LY294002 pre-treatment (Supplementary Figure SF1C). Thus, it seems that although the Akt-Mdm2-mediated p53 degradative pathway is inactivated on LY294002 treatment (as manifested by the loss of the activating phosphorylation on Mdm2), p53 function is still severely compromised. This adds another level of complexity to the known interactions between PI3-kinase and p53, which merited further investigation. Inhibition of PI3-kinase affects multiple aspects of p53 activation To determine the mechanism of inhibition of p53 by LY294002 in adriamycin-treated cells, we first analysed the activation-associated post-translational modifications of p53. Adriamycin addition resulted in a timedependent increase in total p53 and Ser15, Ser20, Ser392 and Ser46 phosphorylations and Lys382 acetylation (Supplementary Figure SF2). However, on LY294002 pre-treatment, adriamycin induced p53 protein less efficiently (Figure 3A, compare lanes 11–14 with 7–10). Although Ser15 phosphorylation was not affected significantly, Ser392 and Ser20 phosphorylations are significantly reduced (complete loss and 35%, respectively, at 24 h) in cells treated with LY294002 and adriamycin (Figure 3A, compare lanes 11–14 with 7–10). Lys382 acetylation, a measure of sequence-specific DNA binding by p53, was also substantially reduced on LY294002 and adriamycin treatment (Figure 3A, compare lanes 11–14 with 7–10; 40% reduction at 24 h). To test whether altered localization could be another reason for reduced p53 activity, we measured p53 levels in the nucleus and cytoplasm by confocal microscopy and sub-cellular fractionation. Although adriamycin addition resulted in accumulation of p53 in the nucleus (Figure 3B, compare panel b with a), LY294002 pretreatment resulted in 50% reduction in nuclear p53 with substantial amounts seen in the cytoplasm as well (Figures 3B, compare panel d with b, and C compare bar 4 with 3). This result was further confirmed by subcellular fractionation. We found a 50% reduction in the nuclear p53 in cells treated with LY294002 and adriamycin as against adriamycin alone (Figure 3D, compare lane 5 with 4). However, there was no compensatory increase in cytoplasmic p53 (Figure 3D, compare lane 10 with 9), which could be due to proteasomal degradation as explained by the reduction in the total p53 levels on treatment with LY294002 and


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


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Figure 2 LY294002 inhibition of p53-mediated transcription is specific to PI3-kinase-Akt/PKB-mTOR pathway. (a) A549 cells were transfected with 1 mg of PG13-Luc and 1 mg of DNPI3K (dominant negative) construct as indicated. After 6 h of transfection, cells were treated with LY294002 (10 mM) and 1 h later, adriamycin (0.5 mg/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. (b) A549 cells were transfected with 1 mg of PG13-Luc, 2 mg of DNPI3K (dominant negative) and 2 mg of CAPI3K (constitutively active) constructs as indicated. After 6 h of transfection, cells were treated with adriamycin (0.5 mg/ml). 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. (c) A549 cells were transfected with 1 mg of PG13-Luc. After 6 h of transfection, cells were treated with LY294002 (50 mM), wortmannin (1 mM), rapamycin (100 nM) or AKT III-inhibitor (10 mM) and 1 h later, adriamycin (0.5 mg/ 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. (d) A549 cells were transfected with 1 mg of PG13-Luc and 5 mg of AKT K179M-T308A-S473A constructs as indicated. After 6 h of transfection, cells were treated with LY294002 (50 mM) and 1 h later, adriamycin (0.5 mg/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.

adriamycin. From these results we conclude that multiple aspects of p53 activation are affected on LY294002 treatment. Differential regulation of DNA damage-induced cellular cytotoxicity by PI3-kinase Our finding that PI3-kinase pathway is required for the activation of DNA damage-induced p53 would imply that inhibition of PI3-kinase pathway may lead to chemoresistance because of abrogation of p53-mediated apoptosis. This was rather intriguing because inactivation of the PI3-kinase pathway has in fact been reported to induce apoptosis and sensitize the cells to chemotherapy (Fujiwara et al., 2006). With the hypothesis that the extent of DNA damage could potentially determine the effect of the PI3-kinase pathway on p53 function and thereby on chemosensitivity, we carried out cytotoxicity Oncogene

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


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adriamycin) was measured as effectiveness of the combination treatment. A high chemosensitivity index was evident at adriamycin-low conditions, but was lost at adriamycin-high conditions implying the development 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 cells after combination therapy by allowing the cells to grow in drug-free medium for 3 days after 48 h of combination Oncogene


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To correlate the above findings with the extent of apoptosis, we measured the proportion of the sub-G1 population under similar conditions (Supplementary Figures SF5A and B). At 24 h after adriamycin treatment, a concentration-dependent increase in apoptosis was observed (Figure 4e, grey line). At adriamycinlow conditions (0.25 and 0.5 mg/ml), LY294002 pretreatment resulted in a dramatic increase in apoptosis compared with adriamycin treatment alone (Figure 4e, 78.65% from 9.45% (8.32-fold) and 65.18% from 15.00% (4.35-fold)). However, LY294002 pre-treatment at adriamycin-high conditions (1.00 mg/ml) resulted in only 1.24-fold increase in apoptosis (Figure 4e, 36.93% from 29.90%). However, at 48 h, LY294002 pre-treatment actually showed a protective effect under adriamycin-high conditions (Figure 4f, 76.21% from 93.83%). We also measured the extent of apoptosis based on caspase-3/7 activity and the presence of fragmented DNA in the apoptotic cells. As can be seen by these more accurate indicators of apoptosis, there is indeed a remarkable decrease in cell death on treatment with high dosages of adriamycin in LY294002 pretreated cells at 24 h (Figures 4g, compare lanes 11 and 12 with 7 and 8; i compare lanes 9 and 10 with 5 and 6) and 48 h (Figure 4h, compare lanes 11 and 12 with 7 and 8). We have also investigated this phenomenon in cells treated with other DNA-damaging agents such as cisplatin and etoposide. PG13-Luc activity induced by both these drugs was efficiently inhibited by LY294002 pre-treatment (Supplementary Figure SF6A, compare lanes 2 and 3 with 4 and 5, and B compare lanes 2 and 3 with 4 and 5). Further, LY294002 enhanced the cytotoxicity of cisplatin at low concentrations, but conferred resistance to high concentrations of cisplatin (Supplementary Figures SF6C and D). These results

together suggest a differential role for the PI3-kinase pathway in modulating DNA damage-induced cytotoxicity and cell fate decisions, which is most likely mediated by its regulation of p53 functions. WT p53 is required for differential regulation of DNA damage-induced cytotoxicity by PI3-kinase To correlate the regulation of p53 functions by PI3kinase with differential cytotoxicity as seen above, we investigated the effect of combination therapy in isogenic cells with different p53 status. HCT116 p53WT and HCT116 p53 / cells were subjected to a similar experiment as described in Figure 4a and the chemosensitivity index was measured. As can be seen in Figure 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 chemosensitivity, which was lost on combination treatment at adriamycin-high conditions (0.5, 0.75, 1, 2 and 5 mg/ml). However, in HCT116 p53 / cells, increased chemosensitivity on combination treatment was seen in both adriamycin-low and -high conditions (Figure 5, grey bars). We also found similar results in HeLa cells, in which p53 is degraded by HPV18 E6 (Supplementary Figures SF7A and B). These results suggest an essential function for p53 in mediating differential regulation of DNA damage-induced cytotoxicity by PI3-kinase. Differential requirement of PI3-kinase for p53 activation Above results suggest a possibility for differential requirement of PI3-kinase in regulating p53 activation. To address this, we investigated the effect of LY294002 and DNPI3K on p53-mediated PG13-Luc activation across 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 (50 mM), and 1 h later, indicated concentrations of adriamycin were added. After 48 h of adriamycin addition, proportion of live cells was quantified by MTT assay. (b) The chemosensitivity index (the ratio of cell viability on treatment with adriamycin alone and on treatment with LY294002 and adriamycin) from (a) was calculated and shown in log scale. (Please note that chemosensitivity index is high and low in adriamycin-low and -high conditions, respectively.) (c) HCT116 cells were treated with LY294002 (50 mM), and 1 h later, indicated concentrations of adriamycin were added. After 48 h of adriamycin addition, proportion of live cells was quantified by MTT assay. (d) The chemosensitivity index (the ratio of cell viability on treatment with adriamycin alone and on treatment with LY294002 and adriamycin) from (a) was calculated and shown in log scale. (Please note that chemosensitivity index is high and low in adriamycin-low and -high conditions, respectively.) (e) A549 cells were either untreated or treated with LY294002 (50 mM), and 1 h later, indicated concentrations of adriamycin were added. After 24 h of adriamycin addition, the cells were harvested and subjected to flow cytometry analysis. The proportion of apoptotic cell population measured as sub-G1 phase is shown. The grey and black lines represent 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 0.25 mg/ml concentration of adriamycin, they come very close at 1 mg/ml concentration of adriamycin, suggesting the loss of chemosensitization at adriamycin-high conditions.) (f) A549 cells were either untreated or treated with LY294002 (50 mM), and 1 h later, indicated concentrations of adriamycin were added. After 48 h of adriamycin addition, the cells were 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-treated samples, respectively. (Note: Although grey and black lines are far from each other at 0.25 mg/ml concentration of adriamycin, they cross 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 (50 mM), 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 the cleavage of the fluorescent substrate have been duly indicated. (h) A549 cells were either treated with DMSO or LY294002 (50 mM), and 1 h later, indicated concentrations of adriamycin were added. After 48 h of adriamycin addition, the Apo-one caspase-3/7 cleavage assay (Promega) was carried out. The fluorescent units from the cleavage of the fluorescent substrate have been duly indicated. (i) A549 cells were either treated with DMSO or LY294002 (50 mM), and 1 h later, indicated concentrations of adriamycin were added. After 48 h of 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 apoptotic nuclei in each sample was calculated. Oncogene


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conditions (Figure 6i, compare lane 3 with 4 and 7 with 8 and j). Addition of LY294002, thus, selectively abrogates the activation of the pro-apoptotic genes by p53 under conditions of extensive DNA damage, and thereby possibly keeps the cells viable. However, with low concentrations of adriamycin (and presumably less DNA damage), addition of LY294002 failed to inhibit p53 function efficiently and thereby leads the cells towards apoptotic death. These results together suggest an important function for the PI3-kinase pathway in regulating p53 functions thereby mediating differential chemosensitivity.

0.1 Figure 5 p53 status and differential regulation of DNA damageinduced cytotoxicity by PI3-kinase HCT116 p53 WT and HCT116 p53 / cells were treated with LY294002 (50 mM), and 1 h later, indicated concentrations of adriamycin were added. After 48 h of adriamycin addition, proportion of live cells was quantified by MTT assay. The chemosensitivity index (the ratio of cell viability on treatment with adriamycin alone and on treatment with LY294002 and adriamycin) was calculated and shown.

LY294002 and DNPI3K inhibited PG13-Luc activation at all adriamycin concentrations used (Supplementary Figures SF7C and D), suggesting an absolute requirement of PI3-kinase for p53-mediated transcription. Altered drug retention could also be responsible for the differential cytotoxicity observed above. Our investigation revealed that while intra-cellular adriamycin (as measured by its fluorescence; see Supplementary Information) increased in a concentration-dependent manner (Supplementary Figures SF8A and B), LY294002 pre-treatment did not reduce adriamycin retention (Supplementary Figures SF8C, D and E). Corroboratively, the transcript levels of the major transporters (ABCC1 and ABCG2), known to be involved in adriamycin efflux, did not increase in cells treated with varying concentrations of adriamycin in the presence and absence of LY294002 (data not shown). Next, we monitored the transcript levels of various pro-apoptotic targets of p53 under conditions of differential DNA damage and the effect of LY294002 on this regulation. On adriamycin treatment, PUMA, NOXA, HRK, CASP1, APAF1 and CASP5 transcript levels increased dramatically in a concentration-dependent manner (Figures 6a–f). Interestingly, although LY294002 addition completely inhibited the activation of these genes at adriamycin-high conditions (1.0 mg/ml), it only repressed them marginally at adriamycin-low conditions (0.25 mg/ml) (Figures 6a–f). In fact, adriamycin-low conditions activated NOXA, HRK, APAF1 and CASP5 more efficiently in the presence of LY294002 (2.5–4.0-fold) than alone (Figures 6b, c, e and f). We also found differential activation of p21WAF1/CIP1 and GADD45a by varied concentrations of adriamycin in the presence of LY294002 (Figures 6g and h). Corroboratively, although adriamycin caused an increase in Ser46 phospho-p53 (needed for activation of proapoptotic targets), LY294002 pre-treatment inhibited Ser46 phosphorylation selectively at adriamycin-high Oncogene

PI3-kinase inhibition before p53 activation is needed for modulation of chemosensitivity As the inhibition of PI3-kinase rendered cells chemoresistant at high concentrations of adriamycin (higher DNA damage), it was of our interest to further investigate the effect of varied timings of PI3-kinase inhibition and chemotherapy administration with respect to chemoresistance. Adriamycin cytotoxicity assays were carried out as before with LY294002 addition at different time intervals either before or after adriamycin addition. Efficient chemoresistance at adriamycin-high conditions was seen when LY294002 was added 1 h before adriamycin, simultaneously or up to 2 h after adriamycin addition (Figures 7a and i, b and j, c and k, d and l). Under these conditions, we also observed increased chemosensitization on LY294002 pre-treatment with adriamycin-low conditions. However, the observed chemoresistance at adriamycin-high conditions was lost when the LY294002 was added at 6 h or later after adriamycin addition (Figures 7e and m, f and n, g and o, h and p). It is also interesting to note that the effective chemosensitization by LY294002 in cells treated with low concentrations of adriamycin was minimized if LY294002 was added beyond 12 h of adriamycin addition (Figures 7g and o, h and p). This may be due to the fact that cell fate decisions have already been made by this time and blocking PI3-kinase at this point fails to provide any additional benefit. Thus, it seems that effective PI3-kinase inhibition before p53 activation is required for both chemoresistance and chemosensitization seen under adriamycin-high and low conditions, respectively.

Discussion Activated PI3-kinase pathway, through Akt/PKBmTOR, has been found to have an important function during oncogenesis and chemoresistance (Wendel et al., 2004). As genetic alterations that activate the PI3K–Akt pathway are common in human cancers (Sakai et al., 1998; Min et al., 2003), this pathway has been the target for inactivation to achieve better chemotherapy (Hennessy et al., 2005; Guillard et al., 2009). P53, which gets induced by genotoxic stress, has a major function in preventing genomic instability and thereby tumour development (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 (50 mM), and 1 h later, indicated concentrations of adriamycin were added. After 24 h of adriamycin addition, total RNA was isolated and subjected to the 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 (50 mM), and 1 h later, indicated concentrations of adriamycin were added. After 24 h of adriamycin 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 after normalizing with actin levels from (i) and shown.

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Further, p53 has an important function in apoptosis induced by DNA-damaging anti-cancer drugs (Johnstone et al., 2002). The current understanding of the interplay between these two pathways is that the PI3kinase pathway inhibits p53 function and thereby promotes survival (Vivanco and Sawyers, 2002). In contrast, we show in this work that the DNA damageactivated PI3-kinase pathway has an essential function in p53-mediated transcription. Although there are reports of PI3-kinase inhibitors repressing p53-mediated transcription, these were inconclusive and led to many unanswered questions (Price and Youmell, 1996; Bar

et al., 2005). In this work, we provide definitive evidences that it is indeed the specific inhibition of PI3-kinase by LY294002 that represses p53-mediated transcription. Besides the fact that lower concentrations of LY294002 (10 mM), which specifically inhibit PI3kinase, were able to abolish p53-mediated transcription, we also show that the expression of a dominant-negative form of PI3-kinase inhibited p53-mediated transcription and this was efficiently overcome by a constitutively active form of PI3-kinase. The downstream effectors of the PI3-kinase-mediated activation of p53 functions were not defined clearly in

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Figure 7 Correlation of chemoresistance with PI3-kinase inhibition and p53 activation (a–h) A549 cells were treated with indicated concentrations of adriamycin. LY294002 (50 mM) was added 1 h before (a), simultaneously (b), 1 h after (c), 2 h after (d), 6 h after (e), 12 h after (f), 24 h after (g) and 36 h after (h) of adriamycin addition. After 48 h of adriamycin addition, proportion of live cells was quantified by MTT assay. (i–p) The chemosensitivity index (the ratio of cell viability on treatment with adriamycin alone and on treatment with LY294002 and adriamycin) from (a–h) was calculated and shown in (i–p), respectively. Oncogene


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Figure 7 Continued.

the earlier studies (Price and Youmell, 1996; Bar et al., 2005). In addition, present understanding of the field suggests an Mdm2-mediated repression of p53 functions by the PI3-kinase pathway (Ogawara et al., 2002). In our work, we show that PI3-kinase uses the Akt/PKBmTOR pathway to activate p53 functions. This was shown by the increase in Ser473 phosphorylated form of Akt on adriamycin treatment. Inhibition of both Akt and mTOR abrogated transcription by DNA damageinduced p53 in adriamycin-treated cells. Further, a dominant-negative mutant of Akt carrying mutations in the active sites was able to inhibit adriamycin-induced p53-mediated transcription. We also establish that the known interplay between the PI3K-Akt-Mdm2 is inconsequential in the above shown regulation of p53 functions by PI3-kinase. Our results, thus, indicate the existence of another layer of complexity in the interactions between PI3-kinase and p53. Phosphorylation of p300 at Ser1834 by Akt is shown to be essential for its histone acetyl-transferase activity, which is indispensible

for the activation of p53 functions (Huang and Chen, 2005). Similarly, constitutive mTOR activity amplifies p53 activation by stimulating p53 translation (Lee et al., 2007). Thus, it is clear that Akt/PKB-mTOR have an important function in p53 activation downstream of PI3-kinase during DNA damage. The observed inhibition of p53 function by LY294002 could occur by multiple means as the regulation of p53 function is extremely complex (Kruse and Gu, 2009). In addition to a significant reduction in total p53, we found functionally important modifications such as Ser392, Ser20, Ser46 phosphorylations and Lys382 acetylation to be significantly affected. The mechanism by which the DNA damage-induced PI3-kinase pathway alters p53 phosphorylations at important residues is not clear at present. However, the reduction in Lys382 acetylation in LY294002-treated cells could be explained by the fact that p300, which acetylates p53, is activated by Aktmediated phosphorylation (Huang and Chen, 2005). We also found substantial reduction in nuclear p53 as Oncogene


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another reason for p53 inhibition. In good correlation, treatment of cells with Leptomycin-B, a nuclear export inhibitor, could partially abrogate the inhibition of p53mediated transcription by LY294002 (data not shown). Thus, it seems that PI3-kinase inhibition affects multiple aspects of p53 activation. Although the relationship between p53 activation and cell fate regulation by itself is very complex (Meek, 2004), the interplay between the classical ‘pro-survival’ PI3-kinase pathway and the ‘pro-apoptotic’ p53 pathway, as established in this study, adds another layer of complexity to the outcome on cell fate. Our hypothesis to begin with was that PI3-kinase pathway inhibition may not always result in apoptosis induction or increased chemosensitivity. Indeed, we found that PI3kinase inhibition in cells treated with higher concentrations of adriamycin led to complete loss of chemosensitization and to chemoresistance in some cell types. At lower concentrations of adriamycin, the combination treatment with LY294002 led to effective chemosensitization in almost all cell lines tested as reported by others. We also show that the differential effect of LY294002 on DNA damage-induced cytotoxicity is due to differential induction of apoptosis under those conditions. In good correlation, activation of p53 target genes (particularly pro-apoptotic genes) was severely compromised on LY294002 pre-treatment in adriamycin-high conditions than in adriamycin-low conditions. In addition, this chemoresistance with LY294002 and high-adriamycin treatment was lost in a p53-null background, suggesting that the differential regulation of DNA damage-induced cytotoxicity by PI3-kinase is indeed mediated through WT p53. Although chemosensitization on LY294002 pre-treatment was seen at all concentrations of adriamycin tested in cells lacking functional p53, there was still a significant reduction in chemosensitivity at adriamycin-high conditions, which could be attributed to similar inhibition of resident p73 functions. Indeed, we found p73-mediated transcription to be inhibited by LY294002 (data not shown), which suggests that the PI3-kinase pathway may also have an important function in regulating the activation of p53 family members such as p73. This suggests that the regulation of DNA damage-induced cytotoxicity by PI3kinase may be operative in the p53 mutant cancers as well, through p73, albeit at a more restricted level. Taken together, these results imply a differential function for the PI3-kinase pathway in modulating DNA damageinduced cytotoxicity and cell fate decisions by regulating the functions of p53 and its family members. We made another interesting finding when we compared the chemoresistance in cells receiving combination therapy with the kinetics of activation of PI3kinase and p53 on DNA damage. We found an efficient PI3-kinase activation (as read by Ser473 phospho-Akt) by 4 h with maximum activation seen at 8 h after adriamycin addition (Supplementary Figure SF1B). With respect to p53, the DNA damage-induced activation, as read by the various post-translational modifications, although seen by 2 h, reached its maximum levels by 12 h of adriamycin addition (Supplementary Figure SF2). Thus, Oncogene

it seems that an efficient PI3-kinase activation is a prerequisite for DNA damage-induced p53 activation. Indeed our results show that addition of LY294002 even up to 2 h after adriamycin addition could modulate p53-activation and thereby the resultant cytotoxicity. However, when LY294002 was added after 6 h of adriamycin addition, there was a loss of chemoresistance at adriamycin-high conditions and a progressive loss of chemosensitization at adriamycin-low conditions. These results suggest that inhibition of PI3-kinase before p53 activation is required for modulation of chemosensitivity. Although most of our work has been carried out with adriamycin as a DNA-damaging agent, we also found that similar observations are seen with other cytotoxic agents such as cisplatin and etoposide. Thus, it appears that the PI3-kinase pathway plays an essential role in the transcriptional activation functions of p53 and thereby modulating cell fate decisions. Our findings become very important in view of many ongoing combination cancer chemotherapy trials with PI3-kinase pathway inhibitors. Thus, this study basically suggests that a careful consideration of chemotherapy dosages, timing of PI3-kinase inhibition and p53 status is very important for a successful outcome of combination chemotherapy regimens. Materials and methods Plasmids and reporter constructs Plasmids 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 transfection efficiency in the various reporter assays. CAPI3K (pCDNA3CD2p110myc) and DNPI3K (pSG5-rCD2p85) constructs as described 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 mutations at the active sites was obtained from Dr WR Sellers through Addgene (plasmid 9031) (Ramaswamy et al., 1999). Drugs and inhibitors LY294002, wortmannin and rapamycin (Alomone Biosciences, Jerusalem, Israel) were used at a final concentration of 50 mM, 1 mM and 100 nM, respectively. AKT inhibitor-III and leptomycin-B (Calbiochem, EMD4Biosciences, Gibbstown, NJ, USA) were used at a final concentration of 10 mM and 10 ng/ml, respectively. Adriamycin/doxorubicin was purchased from Sigma (Sigma-Aldrich, St Louis, MO, USA). Cell lines, transfections and reporter assays A549 (p53 WT), HCT116 (p53 WT), HCT116 p53 / (p53 null; kindly provided by Dr Bert Vogelstein) and HeLa (HPV16 positive) were cultured in DMEM with 10% Fetal calf serum. Transfection and reporter assays were performed as described before (Das et al., 2003). RNA isolation, cDNA synthesis and real time PCR Total RNA isolation, cDNA synthesis and real time PCR were performed as described (Reddy et al., 2008). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) ACTB (actin), 18 s rRNA (18s ribosomal RNA) and RPL35a (ribosomal


3617 protein L35a) were used as internal reference. The primer sequences and conditions used for RT–PCR will be provided on request. Western blot analysis, flow cytometry, MTT assay, caspase-3/7 cleavage assay, fragel DNA fragmentation assay and ELISA Western blot analysis, FACS analysis and MTT assays were performed as described (Mungamuri et al., 2006; see also Supplementary Information). Caspase 3/7 cleavage assay (Promega, Madison, WI, USA) Fragel DNA fragmentation assay (Calbiochem, EMD4 Biosciences) and ELISA (Cell Signaling Technologies, MA, USA) were performed as per manufacturer’s instructions. Confocal analysis and sub-cellular fractionation A549 cells were seeded onto chamber slides (BD Biosciences, San Jose, CA, USA) and treated as indicated. The cells were fixed with 70% ethanol for 10 min at room temperature. After blocking with goat serum, the samples were incubated with the primary antibody for 2 h followed by three washes with phosphate-buffered saline. Samples were subsequently incubated with the FITC-conjugated secondary antibody in dark for 2 h and then washed three times with phosphate-buffered saline. After mounting with an anti-fade agent, confocal images were taken on Zeiss LSM 510 Meta confocal laser scanning microscope using plan-Apochromat 63X/1.4 oil DIC objective. For sub-cellular fractionation, appropriately treated

cells were harvested and nuclear and cytoplasmic fractions were isolated as described before (Kemler et al., 1989). The nuclear and cytoplasmic fractions from equal number of cells were then used for western blotting and probed for p53, lamin B1 and actin as described earlier.

Abbreviations PI3-kinase, phosphoinositide 3-kinase; PKB/Akt, protein kinase 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, DST and UGC to MCB is acknowledged. RS gratefully acknowledges SRF from CSIR.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene