Variability in functional p53 reactivation by PRIMA-1Met|[sol]|APR-246 in Ewing sarcoma

P53 suppresses tumour growth via its diverse cellular activities, including induction of apoptosis, cell cycle arrest, differentiation and senescence (Vousden and Lu, 2002). Most of these effects reflect the transactivation of a number of genes by p53 acting as a transcription factor, but p53 also activates mitochondrial-dependent apoptotic pathways that are independent of p53 transcriptional activity (Green and Kroemer, 2009). Activation of p53 leads to apoptosis through either the death receptor pathway or the mitochondrial pathway (Selivanova, 2004). In the mitochondrial apoptotic pathway, p53 induces several genes including Bax, APAF-1, Puma and Noxa (Miyashita and Reed, 1995; Burns and El-Deiry, 1999; Lowe and Lin, 2000; Nakano and Vousden, 2001; Robles et al, 2001). TP53 mutations may be associated with an aggressive phenotype and poor prognosis, and some p53 mutants counteract the effects of anticancer agents that attack tumours (Bunz et al, 1999; Poeta et al, 2007). Given the high frequency of p53 mutations in human tumours, reactivation of the p53 pathway has been widely proposed as beneficial for cancer therapy (Selivanova, 2010). In addition, several reported structural studies have shown that mutant p53 core domain unfolding is reversible and, as mutant p53 is expressed at high levels in many tumours, it therefore serves as a potential target for novel cancer therapy (Lambert et al, 2010). Its reactivation will restore p53-dependent apoptosis, among others, resulting in efficient eradication of tumour cells (Tovar et al, 2006). Wild-type p53 can be induced by small organic molecules such as nutlin-3 inhibiting the p53/MDM2 interaction (Vassilev et al, 2004). PRIMA-1 (p53 reactivation and induction of massive apoptosis) and its potent methylated analog, APR-246/PRIMA-1Met, are small molecules that have the ability to convert mt-p53 to an active conformation, thereby restoring its sequence-specific DNA binding and transcriptional activation (Bykov et al, 2002b). It was reported that PRIMA-1 induces cell death through multiple pathways encompassing transcription-dependent and -independent signaling (Chipuk et al, 2003). In vitro and in vivo studies have shown that p53 reactivating small molecules are less toxic to normal cells than to cancer cells and have no significant adverse or genotoxic effects (Stuhmer et al, 2005; Tovar et al, 2006). Although about half of all human malignancies harbour dysfunctional, mutated p53 proteins, approximately 90% of all ES retain wild-type p53, and the downstream DNA damage cell cycle checkpoints and p53 pathways remain functionally intact (de Kovar et al, 1993; Alava et al, 2000; Kovar et al, 2003b; Huang et al, 2005). Exposure of ES cells to Nutlin-3a, a small organic molecule known to reactivate wild-type 53, resulted in a robust apoptotic phenotype that required the presence of wild-type p53 but did not affect the growth of mutant p53 expressing cells (Pishas et al, 2011). TP53 mutation alone rates high among variables, including p16/p14ARF alteration and tumour stage, predicting poorer overall survival in Ewing sarcoma (de Alava et al, 2000; Lopez-Guerrero et al, 2011). Multivariate analysis identified alterations of TP53 as an adverse prognostic factor defining a subset of ES with highly aggressive behaviour and poor chemoresponse (Huang et al, 2005). Therefore, novel treatment options specifically targeting mutant p53 are highly warranted. There is a prospective COG (Children’s Oncology Group, USA) study ongoing to validate a retrospective study which strongly suggested that mutant p53 ES constitutes a particularly bad prognostic group (ClinicalTrials.gov identifier: NCT00898053). If data from the retrospective study is confirmed, this would strongly support the need for novel mutant p53 targeting therapeutic strategies in ES. In this study, we investigated whether the small pharmacological molecule APR-246 is able to reactivate mutant p53 in Ewing sarcoma cells in order to drive tumour cells into apoptosis. Using Ewing sarcoma patients’ derived cell lines, we show that APR-246 is able to induce apoptosis to variable degrees independent of the p53 status. We observed that cell lines with similar p53 mutations as well as cell lines established from the same patient at different stages of the disease all exhibited variable responses to the drug. To interrogate the molecular basis for the differential responses to APR-246, we performed comparative transcriptomic analysis on the three STA-ET-7 cell lines established from the same patient. Data analysed revealed genes annotating to p53 and apoptosis pathways whose expression varies between the more APR-246-sensitive and the less APR-246-sensitive cell lines. We propose, therefore, that APR-246 will not be a suitable candidate to consider for targeting p53 mutant Ewing sarcoma. As shown in Figure 1A, the expression level of mutant p53 in the ES cell lines varied. However, in all the mt-p53 harbouring cell lines, the levels of the protein was higher than in the TC252 (wt-p53) cell line, which was only visualised after longer exposure, and there was no measurable expression of full-length p53 in the p53-null (A673) and p53-truncated (SK-N-MC) cell lines. Treatment of cells for 24 h with 20 μM APR-246 increased Annexin-V-positive (apoptotic) cells by up to 40% in some ES cell lines (Figure 1B). Although the breast carcinoma cell line MDA-MB-468, used as a control, already exhibited a significant induction of apoptosis at 10 μM APR-246, significant apoptosis induction was only achieved with 20 μM APR-246 in the ES cell lines indicated (Figure 1B). A slight induction of apoptosis was observed in the wild-type p53 cell line, TC252, whereas no measurable induction was seen in the p53-null cell line A673. PARP cleavage, an indicator of apoptosis induction, is shown in both the wild-type p53 cell line TC252 and the mutant p53 cell line RM82 in a dose-dependent manner on APR-246 treatment (Figure 1C). This is consistent with the apoptosis induction by APR-246 of the TC252 cell line as shown in Figure 1B and indicates that APR-246 also influences wild-type p53 function. ES is a very aggressive disease and though TP53 mutations are rare in ES, with the majority of tumours expressing wild-type p53 (Kovar et al, 1993; de Alava et al, 2000; Huang et al, 2005), patients with point mutation of TP53 are associated with a dismal prognosis (Huang et al, 2005). However, even in the absence of mutation, there is evidence that wild-type p53 may be functionally disabled in Ewing sarcoma as a consequence of the EWS-FLI1 oncogene (Ban et al, 2008; Li et al, 2012). A study of 308 ES cases established that mutant p53 expression was more frequent in disseminated disease than in primary localised tumours, indicating a role in the progression and metastasis of ES (Lopez-Guerrero et al, 2011). This makes functional restoration of the p53 pathway an attractive therapeutic option in this tumour entity. In the current study, we addressed the possibility of using the small-molecule compound, APR-246/PRIMA-1Met, capable of reactivating mutant p53 and inducing apoptosis in several different cancer types, to induce cell death in ES cells harbouring different p53 mutants. In this study, we have shown that ES cells exhibit different sensitivities to APR-246 exposure. For instance, three ES cell lines established from the same patient at different stages of the disease (with identical p53 mutation) all reacted variably to APR-246 treatment (Figure 2A). We also asked whether tumour cell lines with the same mutation will show similar cellular response to APR-246. To address this question, we took three approaches: (1) we used the breast cancer cell line MDA-MB-468 (with the R273H p53 mutation) as a positive control to investigate an ES cell line with identical p53 mutation, RM82, (2) we investigated the response of two ES cell lines, established from different ES patients, with identical p53 mutations and, (3) we studied three cell lines from different tumour materials of the same patient to the same concentrations of APR-246. We observed that response of the cells was unrelated to the mutation type, alluding to the cellular context dependency of the response to APR-246 (Figure 1B). To investigate whether induction of apoptosis was mediated via p53 upon APR-246 exposure, we used RNAi to knockdown p53 in mt-p53 cell lines before treatment with APR-246. We found apoptosis induction after APR-246 treatment of the STA-ET-7.2 cell line transfected with p53 siRNA was reduced compared with scrambled siRNA-transfected control cells (Figure 2B). We now report that treatment with APR-246 evoked apoptosis to variable extents in mt-p53 ES cell lines and also on the p53 wild-type cell line (TC252) but no measurable effect on a p53-null cell line (A673). It has recently been shown that APR-246 can bind to unfolded wt-p53 and activate it by inducing correct folding (Lambert et al, 2009). As the actual mechanism of action of APR-246 is not completely clear (Zandi et al, 2011), our data corroborate reports showing that APR-246 also affects wt-p53-containing cells. We also observed that APR-246 induced expression of variable sets of classical p53 target genes in ES cells harbouring mutant and wild-type p53 (Figures 3A and B). By real-time quantitative PCR assay in the STA-ET-7.2 cell line after APR-246 treatment, we found p21 mRNA to be significantly induced (Figure 3A). APR-246 also activated transcription of the pro-apoptotic genes Noxa (PMAIP1) in RM82 and Puma in STA-ET-7.2. The changes in Noxa and Puma mRNA expression observed support the potential activation of the p53-dependent apoptotic pathway by APR-246. In the wild-type p53 cell line, TC252, both Puma and p21 mRNAs were induced by APR-246 treatments, whereas no effect on p53 target genes was seen in the p53-null cell line A673 (Figure 3B). Genome-wide gene expression analysis performed in the STA-ET-7 cell line triplet revealed, among others, differential expression of genes that annotated to p53 and apoptosis pathways. Among them, overexpression of the gene encoding for apolipoprotein 6 (APOL6), as was observed in STA-ET-7.2, has been shown to induce mitochondrial-mediated apoptosis in colorectal cancer cells (DLD-1) (Liu et al, 2005). Another gene, PENK, which is highly upregulated in the STA-ET-7.2 cell line relative to the other two cell lines, has been shown to assist stress-activated apoptosis through transcriptional repression of NF-kappaB- and p53-regulated gene targets (McTavish et al, 2007). Also, some anti-apoptotic genes such as PCDH7 (Zhang and DuBois, 2001) and MST4 (Sung et al, 2003) are highly suppressed in the more APR-246-sensitive STA-ET-7.2 cell line in comparison to the less sensitive STA-ET-7.1 and STA-ET-7.3 cell lines (Figure 4). The relative expression levels of these genes among the cell lines could at least be partially responsible for their disparate sensitivities to APR-246 treatment. These results are consistent with the reported capability of APR-246 to restore transcriptional activity to mutant p53 and trigger mutant p53-dependent apoptosis (Bykov et al, 2002a). This is also in line with the suggestion by Lambert et al (2009, 2010) that adducts of the APR-246 conversion product methylene quinuclidinone could create novel protein–DNA contacts, which could affect the choice of target genes. It is also reported that APR-246 could target other proteins in the cell, which might lead to synergistic effects promoting apoptosis rather than growth arrest (Rokaeus et al, 2010). This notion reflects our finding of the induction of different p53 target genes in different mutant p53 cell lines after APR-246 treatments, notwithstanding the cellular context dependency exhibited by ES cells in response to APR-246. In our real-time quantitative PCR data, we did not observe high induction of Bax mRNA by APR-246 treatment. This corroborates reported results by Chipuk et al (2003), where they found that Bax-dependent apoptosis induced by APR-246 is mutant p53 dependent but transcription independent. In this study, we also show that there is no association between the type of p53 mutation and the response to APR-246 treatment. It is also speculated that APR-246 can induce ER stress that may cause p53-independent responses (Lambert et al, 2010). Therefore, cellular responses to APR-246 may not be directly p53 dependent but rather a response to its induced ER stress. In conclusion, our results suggest that there is variability in functional p53 reactivation by APR-246 in ES cells independent of the p53 status. We also observed that APR-246 variably enhanced the apoptosis-inducing capacity of the chemotherapeutic agent Etoposide, which is currently used in the treatment of patients with ES (data not shown). Although in vivo studies would be required to validate the results, the in vitro data do not support APR-246 as a prime drug to advance into animal and clinical studies. Those genes identified in our gene expression profiling to be differentially expressed between the APR-246-sensitive cell line (STA-ET-7.2) and the less sensitive cell lines (STA-ET-7.1 and STA-ET-7.3) should stimulate further studies to investigate the relevance of these genes as potential biomarkers for stratification for APR-246 treatment. Together, our data downplay the prospect of considering APR-246 as a candidate for the future development of novel treatment modalities for ES patients. We are grateful to David Herrero-Martin and Eleni Tomazou-Bock for critical reading of the manuscript. We thank Argyro Fourtouna and Dieter Prinz for help with the FACS analyses. This work was supported by grant 14205 from the ‘Jubilaeumsfonds’ of the Austrian National Bank to DNTA.