Oncogene (2010) 29, 1329–1338 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Mutant reactivation by PRIMA-1MET induces multiple signaling pathways converging on apoptosis

JMR Lambert1,2, A Moshfegh1, P Hainaut2, KG Wiman1 and VJN Bykov1

1Department of Oncology–Pathology, Cancer Center Karolinska, Karolinska Institutet, Stockholm, Sweden and 2Molecular Carcinogenesis and Biomarkers Group, International Agency for Research on Cancer (IARC), Cours Albert Thomas, Lyon, France

The low molecular weight compound PRIMA-1MET some anti-apoptotic (BCL2, hTERT) (Mirza reactivates mutant p53 and triggers mutant p53-dependent et al., 2003; Rahman et al., 2005). Furthermore, p53 apoptosis in human tumor cells. We investigated the effect has trancription-independent pro-apoptotic and anti- of PRIMA-1MET on global expression using micro- autophagic activity in the cytoplasm where it localizes to array analysis of Saos-2 cells expressing His273 mutant the mitochondria (reviewed by Green and Kroemer, p53 and parental p53 null Saos-2 cells. PRIMA-1MET 2009). p53 is also involved in processes such as DNA affected of a significantly larger number of repair (Livneh, 2006), cellular senescence (Xue et al., genes in the mutant p53-expressing cells compared to the 2007), and metabolism (Vousden and Ryan, 2009). p53 null cells. Genes affected by PRIMA-1MET in a Alterations in the p53 gene, mostly missense muta- mutant p53-dependent manner include the cell-cycle tions, are found in about half of all human tumors regulators GADD45B and 14-3-3c and the pro-apoptotic (Olivier et al., 2002). These mutations are clustered Noxa. Several of the affected genes are known p53 target within the DNA-binding domain and thus impair p53’s genes and/or contain p53 DNA-binding motifs. We also ability to function as a . The loss of found mutant p53-dependent disruption of the cytoskele- wild-type p53 tumor suppressor activity as well as the ton, as well as transcriptional activation of the XBP1 gene acquisition of novel functions (so called gain-of-func- and cleavage of its mRNA, a marker for endoplasmic tion) attributed to some p53 mutants, often counteracts reticulum stress. Our data show that PRIMA-1MET the effect of cancer treatment, as many anti-cancer drugs induces apoptosis through multiple transcription-depen- attack tumors by activating p53-dependent apoptosis dent and -independent pathways. Such integral engage- (Bunz et al., 1999; Andersson et al., 2005). Gain-of- ment of multiple pathways leading to apoptosis function effects include the ability of mutant p53 to is consistent with restoration of wild-type properties transactivate illegitimate targets such as MDR1, vascu- to mutant p53 and is likely to reduce the risk of drug lar endothelial growth factor (VEGF), and c-, and resistance development in clinical applications of inactivate other members of the p53 family by hetero- PRIMA-1MET. dimerization (Cadwell and Zambetti, 2001; Gaiddon Oncogene (2010) 29, 1329–1338; doi:10.1038/onc.2009.425; et al., 2001). published online 30 November 2009 Mutant p53 is often expressed at high levels in tumors. Therefore, therapeutic reactivation of mutant Keywords: cancer; mutant p53; apoptosis; novel therapy p53 should trigger massive apoptosis and allow efficient eradication of tumor cells in vivo. We have shown earlier that PRIMA-1 restores wild-type properties to mutant p53, that is transactivation of some of p53’s target genes Introduction (p21, MDM2, and Bax), and induces apoptosis in a mutant p53-dependent manner in vitro and in vivo The p53 tumor suppressor has a key role in the cellular (Bykov et al., 2002; Zache et al., 2008). We also showed response to diverse forms of stress (Vousden and Lu, that PRIMA-1 triggers apoptosis through the 2002). Under normal conditions, p53 is expressed at low mitochondrial pathway and activation of caspase-2, -3, levels because of negative feedback regulation by and -9 (Shen et al., 2008). Others have found that Bax- MDM2 (Haupt et al., 1997; Kubbutat et al., 1997). dependent apoptosis induced by PRIMA-1 is mutant Upon cellular stress, p53 is stabilized and transactivates p53-dependent but transcription-independent (Chipuk a number of genes that promote cell-cycle arrest et al., 2003). Recently, we have shown that PRIMA-1 is (GADD45, p21, 14-3-3s) and apoptosis (Bax, Noxa, converted to reactive products with Michael acceptor Puma) (Vousden and Lu, 2002). p53 can transrepress activity that bind covalently to the p53 core domain, and that such modification is critical for reactivation of Correspondence: Professor KG Wiman, Department of Oncology– mutant p53 (Lambert et al., 2009). Pathology, Cancer Center Karolinska, Karolinska Institutet, SE-171 Here, we have investigated how rescue of mutant p53 76 Stockholm, Sweden. MET E-mail: [email protected] by PRIMA-1 , an analog of PRIMA-1 (Bykov et al., Received 3 June 2009; revised 21 October 2009; accepted 22 October 2005), affects global gene transcription. We found that 2009; published online 30 November 2009 PRIMA-1MET affects transcription mainly in the mutant PRIMA-1 induces ER stress-mediated apoptosis JMR Lambert et al 1330 p53-expressing cells. Our results indicate that PRIMA-1 have observed earlier that PRIMA-1MET induces the induces cell death through multiple pathways encom- pro-apoptotic p53 target Bax at the level passing transcription-dependent and -independent sig- (Chipuk et al., 2003; Bykov et al., 2005), we investigated naling. These results provide significant novel insights Bax mRNA expression using a more sensitive method, into the mechanisms of PRIMA-1MET-induced cell real-time PCR. This showed a 2.43±0.92-fold upregula- death, and should facilitate the design of even more tion of Bax mRNA in Saos-2-His273 cells at 6 h, but not potent mutant p53-reactivating compounds. in the p53 null Saos-2 cells (0.80±0.24-fold change; data not shown). Importantly, PRIMA-1MET did not affect expression of any p53 target genes in Saos-2 cells according to the microarray data. Overall, we conclude Results that PRIMA-1MET significantly affects the transcriptome in a mutant p53-dependent manner, and induces MET PRIMA-1 affects the transcriptome in a mutant transcription of a subset of p53 target genes. p53-dependent manner The mutant p53-expressing Saos-2-His273 cells were treated with 37 mM PRIMA-1MET for 6 and 12 h. This PRIMA-1MET affects transcription of cell-cycle arrest and concentration was chosen based on our earlier studies, apoptosis-related genes in Saos-2-His273 cells as it gives a robust mutant p53-dependent apoptosis Next, we analyzed our data set using the Ingenuity (Figure 1a). Parental p53 null Saos-2 cells served as software to identify cellular pathways that were affected control. We used the Affymetrix GeneChip Human after exposure of Saos-2-His273 cells to PRIMA-1MET. Genome U133 Plus 2.0 arrays to monitor changes in At 6 h of treatment, the cell cycle and proliferation global after PRIMA-1MET treatment. pathways were the most affected, whereas genes involved After normalization and statistical analysis (ANOVA), in the cell death pathway were most affected at 12 h. we found that treatment with PRIMA-1MET yielded These findings are in full agreement with the observed changes in 305 genes in Saos-2 cells as compared to changes in cell-cycle distribution according to propi- untreated control cells, whereas 1318 genes were dium iodide staining and flow cytometry (Figure 1a). differentially expressed in Saos-2-His273 cells (P-value Although no cell-cycle changes in Saos-2 cells were o0.05) (Supplementary Table 1) on PRIMA-1MET observed after PRIMA-1MET treatment, a prominent G2 treatment. To focus on major changes in transcription, arrest and an increased sub-G1 population were we selected genes whose expression changed by factor of detected in Saos-2-His273 after 48 h exposure to 37 mM 1.3 or greater. This reduced the number of affected of PRIMA-1MET. All genes identified in our microarray genes to 185 in Saos-2-His273 cells and only 15 in Saos-2 analysis and genes involved in the cell cycle and cells (Supplementary Table 2). None of the identified proliferation or cell death pathways are listed in Table 1. genes were affected in both cell lines. We focused our GADD45B and 14-3-3g, both of which have roles in further analysis on the gene list generated from the Saos- regulation of cell-cycle progression and cell growth, 2-His273 cells. We selected significantly up- or down- were examined further. Upregulation of GADD45B regulated genes that are involved in different pathways, mRNA was detected by both the microarray analysis including cell-cycle regulation and apoptosis, and (3.05±0.66-fold induction) and real-time PCR analyzed their expression by real-time PCR. We con- (1.44±0.01-fold induction) (Figure 1b). For 14-3-3g, firmed the microarray data for 11 genes, including we observed a 1.80±0.17-fold induction of mRNA Noxa, GADD45B, Yin Yang 1 (YY1), PTCH1, and levels on the microarray but this could not be confirmed XBP1. by real-time PCR (Figure 1b). However, we did detect a Three genes induced in Saos-2-His273 cells, Scotin, significant induction at the protein level of up to five Noxa, and YY1, are known wild-type p53 targets. times in Saos-2-His273 cells by both western blotting Sequence analysis showed that 23 of the identified genes and immunostaining (Figure 1c; data not shown), contain putative p53-binding sites (Wang et al., 2001; whereas no such changes could be seen in Saos-2 cells. Hoh et al., 2002; Ceribelli et al., 2006; Table 2). Noxa is involved in apoptosis and is also known to be However, classical p53 transcription targets, such as p53 transcriptional target. Noxa showed 3.18±0.73-fold p21, MDM2, and Bax, were not affected by PRIMA- upregulation at the mRNA level at 12 h according to the 1MET in our microarray analysis. As we and others microarray. This was confirmed by real-time PCR

Figure 1 PRIMA-1MET-induced cell-cycle arrest and apoptosis and upregulation of cell cycle and apoptosis-related genes. (a) Cell- cycle analysis showing G2 arrest and an increased sub-G1 cell population after PRIMA-1MET treatment with 37 mM PRIMA-1MET in mutant p53-expressing Saos-2 cells but not in parental p53 null Saos-2 cells. (b) Expression of GADD45B, YWHAG/14-3-3g, and PMAIP1/Noxa on treatment with 37 mM PRIMA-1MET as assessed by microarray analysis and real-time PCR. (c) Western blot analysis showing increased levels of 14-3-3 in Saos-2-His273 cells after treatment with the indicated concentrations of PRIMA-1MET. b-actin was used as loading control. (d) Western blot analysis of Noxa expression in Saos-2, SW480, H1299, and HCT116 cells with different p53 status as indicated. b-actin was used as loading control. (e) Western blot analysis of Noxa, 14-3-3, and p53 protein expression in p53 null Saos-2 cells transfected with increasing amounts of wild-type p53 expression plasmid. Mutant p53 levels in Saos-2-His273 cells are shown for comparison. b-actin was used as loading control. (f) Induction of GADD45, 14-3-3g, and Noxa mRNA in SW480 cells treated with PRIMA-1MET at 25 or 50 mM, as assessed by real-time PCR.

Oncogene PRIMA-1 induces ER stress-mediated apoptosis JMR Lambert et al 1331 (10.41±3.83-fold) (Figure 1b) and at the protein level manner in several other cell lines (Figure 1d). For by western blotting (Figure 1d). Furthermore, we comparison, we transfected parental Saos-2 cells with observed Noxa upregulation in a mutant p53-dependent increasing amounts of wild-type p53 vector and assessed

256 256 256 256 Cells

0 0 0 0 100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103 FL3-H FL3-H FL3-H FL3-H Untreated PRIMA-1MET Untreated PRIMA-1MET Saos-2 Saos-2-His273

GADD45B YWHAG/14-3-3γ PMAIP1/Noxa 16 2.0 4.0 14 3.5 1.8 1.6 12 3.0 1.4 10 2.5 1.2 8 2.0 1.0 0.8 6 1.5 4 Fold change 0.6 1.0 0.4 2 0.5 0.2 0 0.0 0.0 Ctrl 6h 12h Ctrl 6h 12h Ctrl 6h 12h Ctrl 6h 12h Ctrl 6h 12h Ctrl 6h 12h Microarray RT-PCR Microarray RT-PCR Microarray RT-PCR Saos-2 Saos-2-His273

Saos-2 Saos-2-His273 Saos-2 Saos-2-His273

14-3-3 Noxa β-actin β-actin HCT116 p53+/+ HCT116 p53-/- - 10 25 50 - 10 25 50 PRIMA-1MET μ ( M) Noxa Saos-2 His273 Saos-2 β-actin

Noxa HCT116 p53-/248 SW480

14-3-3 Noxa

p53 β-actin

β-actin H1299 H1299-His175 μ 0 0.5 1 2 3 g of wt p53 Noxa

β-actin - 25 37 50 - 25 37 50 PRIMA-1MET (μM) SW480 12

10

8

6 Untreated 25μM PRIMA-1MET 50μM PRIMA-1MET

Fold change 4

2

0 DNAJB2GADD45B Noxa XBP1 YY1 14-3-3γ VEGF

Oncogene PRIMA-1 induces ER stress-mediated apoptosis JMR Lambert et al 1332 Table 1 Genes related to apoptosis or cell-cycle arrest whose expression is up- or downregulated by 1.3-fold or more upon PRIMA-1MET treatment in Saos-2-His273 cells Affymetrix Gene title/symbol P-value Fold change probe set ID 6 h 12 h

Apotosis-related genes 210025_s_at Caspase recruitment domain family, member 10 CARD10 0.0193 1.38 1.81 212140_at KIAA0648 protein, SCC-112 protein SCC-112 0.0091 1.47 1.84 224621_at Mitogen-activated protein kinase 1 MAPK1 0.0309 1.43 1.74 200797_s_at qw03a03.x1 NCI_CGAP_Ut3 Homo sapiens cDNA clone MCL1 0.0364 1.44 1.68 IMAGE:1989964 3’, mRNA sequence 204286_s_at Phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1 or NOXA) NOXA 0.0308 1.79 3.18 222986_s_at Scotin SCOTIN 0.0237 1.43 1.97

Cell-cycle arrest-related genes 209165_at Apoptosis antagonizing transcription factor AATF 0.0083 2.05 2.65 209304_x_at Growth arrest and DNA-damage-inducible, b GADD45B 0.0416 2.00 3.05 200788_s_at Phosphoprotein enriched in astrocytes 15 PEA15 0.0186 1.58 2.23 204292_x_at Serine/threonine kinase 11, Peutz-Jeghers syndrome STK11 0.0175 1.65 1.98 222985_at Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, 14-3-3 g 0.0176 1.42 1.80 g polypeptide (YWHAG or 14-3-3 g ) 201901_s_at YY1 transcription factor YY1 0.047 1.75 2.45

Abbreviation: YY1, Yin Yang 1.

expression of 14-3-3 and Noxa. As shown in Figure 1e, ton rearrangements, which are hallmarks of the acti- expression of exogenous wild-type p53 in these cells led vated cell death program. to upregulation of both 14-3-3 and Noxa to levels comparable to those observed in PRIMA-1MET-treated Saos-2-His273 cells, although Noxa was induced more PRIMA-1MET treatment induces endoplasmic efficiently by wild-type p53. Next, we examined the reticulum stress expression of 14-3-3 and Noxa in SW480 colon One of the genes that were strongly upregulated in Saos- carcinoma cells that carry endogenous His273 mutant 2-His273 cells on PRIMA-1MET treatment is XBP1. We p53, and confirmed upregulation of Noxa, 14-3-3g,and observed a 2.36±0.39-fold increase at 12 h. These GADD45 mRNA after PRIMA-1MET treatment results were confirmed by real-time PCR, which showed (Figure 1f). Altogether these results show that the a 2.36±0.29-fold induction at 12 h (Figure 3a). XBP1 is reactivation of mutant p53 by PRIMA-1MET leads to associated with endoplasmic reticulum (ER) stress enhanced expression of genes involved in cell-cycle (Yoshida et al., 2001). Its mRNA is cleaved on ER regulation and apoptosis. stress, giving rise to a spliced form, (S) XBP1, that activates the unfolded protein response. Figure 3b shows that PRIMA-1MET treatment induced cleavage of XBP1 Disruption of the after PRIMA-1MET mRNA more prominently in the mutant p53-expressing treatment cells. Thapsigargin, which blocks the ER Ca2 þ pump by We observed that PRIMA-1MET induces changes in inhibiting the Ca2 þ -ATPase and thus elicits ER stress expression of several genes involved in the rearrange- (Inesi and Sagara, 1992), was used as a positive control ment of the cytoskeleton in a mutant p53-dependent to induce XBP1 cleavage. Moreover, we detected XBP1 manner, including WIRE, CNN2, Vinculin, and Super- cleavage in SW480 colon carcinoma cells carrying villin. PRIMA-1MET induced characteristic apoptosis- mutant p53 (Figure 3b). Interestingly, we found that related cytoskeleton rearrangements. The degradation DNAJB2, an Hsp40-like downstream protein target of of actin filaments in Saos-2-His273 cells was evident XBP1, was upregulated after PRIMA-1MET treatment only after 24 h and was concentration dependent (Figure 3c), although this occurred in both Saos-2 and (Figure 2). At 25 mM, an actin ring was formed at the Saos-2-His273 cells. Finally, we tested whether PRIMA- cell borders, accompanied by the formation of mem- 1MET treatment would lead to an accumulation of brane blebs and clumps. At 50 mM, at a later stage of unfolded , the fundamental characteristic of apoptosis, the actin cytoskeleton was fully disrupted, ER stress (Lai et al., 2007). GRP78, another protein with only some actin clumps remaining (Figure 2). No involved in ER stress, is known to bind unfolded such distortion of the cytoskeleton was observed in proteins accumulating in the ER (Bertolotti et al., 2000). parental p53 null Saos-2 cells (Figure 2). We observed We treated cells with PRIMA-1MET, immunoprecipitated the reduction of filaments in Saos-2 cells only with GRP78, and resolved proteins by PAGE followed by substantially higher concentrations of PRIMA-1MET (not silver staining. Densitometry showed a 20% increase in shown). These data suggest that mutant p53-dependent total proteins co-immunoprecipitated with GRP78 in apoptosis induced by PRIMA-1MET involves cytoskele- Saos-2-His273 treated with PRIMA-1MET, whereas

Oncogene PRIMA-1 induces ER stress-mediated apoptosis JMR Lambert et al 1333 Phalloidin DAPI et al., 2002, 2005). We have also shown that PRIMA-1 induces apoptosis through activation of caspase-2 and the intrinsic mitochondrial cell death pathway (Shen et al., 2008). Here, we have investigated the effects of mutant p53 reactivation on global transcription. Treat- Control ment with PRIMA-1MET led to mutant p53-dependent induction of genes involved in regulation of cell growth and survival, including 14-3-3g (YWHAG) and GADD45B that inhibit cell-cycle progression, and Noxa M μ that promotes apoptosis. These results are fully con- 40x 25 sistent with the documented ability of PRIMA-1MET to inhibit growth and induce apoptosis in human and mouse tumor cells in a mutant p53-dependent manner. A significant fraction, 23 out of 185 (12.4%), of M

μ the identified genes carry putative p53-binding sites 50 (Table 2). This is in agreement with our earlier findings that PRIMA-1MET restores wild-type conformation and DNA binding to mutant p53. Nonetheless, we did not detect any major changes in expression of classical p53 target genes such as p21 and MDM2, although upregulation of both genes was observed earlier at the MET

Control protein level in PRIMA-1 or PRIMA-1 -treated cells (Bykov et al., 2002, 2005). A plausible explanation for this discrepancy is that mRNAs for these genes are only transiently upregulated between the 6 and 12 h time

M points used in our study. It should also be noted that μ

40x although our microarray analysis failed to detect 25 increased expression of Bax, real-time PCR showed a marked mutant p53-dependent induction (data not shown). The correlation between Affymetrix and real-

M time PCR data was reported to be about 85%; however, μ Saos-2-His273 Saos-2 weakly expressed mRNA showed a lower correlation 50 (Rogojina et al., 2003). This may explain some of the observed differences between our Affymetrix and real- time PCR data. Moreover, YY1 has been reported to bind to p53-target sites in a sequence-dependent manner and inhibit transcription of some p53 target genes, such M μ as p21 but not Bax (Gronroos et al., 2004; Yakovleva 100x 25 et al., 2004). Thus, the increased expression of YY1 favors apoptosis. Figure 2 Phalloidin Texas-Red staining of actin filaments showing We observed a marked mutant p53-dependent change major rearrangement of the cytoskeleton in Saos-2-His273 cells in expression of genes that are functionally related to the after 24 h of PRIMA-1MET treatment. Treatment with 25 mM cytoskeleton, including upregulation of WIRE and PRIMA-1MET induced the formation of an F-actin ring at the cell downregulation of Supervillin. Ectopic expression of edges and membrane blebs as shown at higher magnification ( Â 100). No cytoskeleton changes were observed in PRIMA-1MET- the WASP-binding protein, WIRE, causes actin treated Saos-2 cells at this concentration range. cytoskeleton redistribution and actin depolymerization (Hossain et al., 2003). Overexpression of supervillin, an F-actin-binding protein, results in increased levels of no increase was observed in the p53 null Saos-2 cells F-actin (Wulfkuhle et al., 1999). Upregulation of WIRE (data not shown). Thus, the observed cleavage of XBP1 and downregulation of Supervillin may account for the and accumulation of unfolded proteins, along with observed reduction of filamentous actin or depolymer- the upregulation of Noxa, which is involved in ER ization in the PRIMA-1MET-treated mutant p53-expres- stress-induced cell death, indicate that PRIMA-1MET sing cells. triggers ER stress. It is particularly noteworthy that several genes associated with ER stress were induced by PRIMA- 1MET, such as XBP1, Noxa, and DNAJB2 (Abcouwer Discussion et al., 2002; Westhoff et al., 2005; Li et al., 2006). This indicates that PRIMA-1MET somehow affects protein PRIMA-1MET has been shown to restore wild-type folding in the cell, leading to activtion of the ER stress conformation to mutant p53, resulting in upregulation pathway. This notion is further supported by the of p53 targets such as p21, MDM2, and Bax (Bykov observed cleavage of XBP1 mRNA. Moreover, an

Oncogene PRIMA-1 induces ER stress-mediated apoptosis JMR Lambert et al 1334 XBP1 Recently, we have shown that PRIMA-1, through its 2.8 conversion products, modifies mutant p53 protein by 2.4 binding to thiol groups in the core domain (Lambert et al., 2009). Such modification of p53 per se was 2.0 sufficient to induce apoptosis in tumor cells. However, 1.6 our data indicate that reactive conversion products of PRIMA-1 could potentially modify a number of 1.2

Fold change other proteins in cells, including other members of 0.8 the p53 family, such as p63 and . Mutant p53 has 0.4 been shown to bind and inactivate p63 and p73 (Di Como et al., 1999; Strano et al., 2002). It is conceivable 0.0 Ctrl 6h 12h Ctrl 6h 12h that modifications of p63 or p73 could disrupt mutant p53 binding and thus trigger p63/p73-dependent Microarray RT-PCR apoptosis. PRIMA-1-modified p63 or p73 may also Saos-2 Saos-2-His273 have its own set of target genes. Further studies of the effect of PRIMA-1 on p53 family proteins should 500 (U) XBP1 answer this question and may open new possibilities 400 (S) XBP1 for the use of PRIMA-1 in pathological conditions linked to p63 or p73. Modifications of other proteins 300 - + ++ - - + ++ - PRIMA-1MET might explain the observed induction of ER stress, as - - - + - - - + Thapsigargin thiol alkylation of multiple proteins should trigger the 3.1 4.5 5.0 16.4 3.3 7.6 9.1 20.7 (S)XBP1 / (U)XBP1 % unfolded protein response and eventually ER stress. MET SW480 Surprisingly, PRIMA-1 -induced ER stress depends on the expression of mutant p53 (Figure 4). We (U) XBP1 (S) XBP1 have observed mutant p53-dependent induction of reactive oxygen species (ROS) by PRIMA-1MET - + ++ - PRIMA-1MET (Lambert et al., 2009), consistent with restoration of - - - + Thapsigargin wild-type properties to mutant p53 because wild-type DNAJB2 (Hsp40-like) p53 can promote ROS on severe stress, leading to 4.0 apoptosis (Johnson et al., 1996; Polyak et al., 1997; 3.5 Sablina et al., 2005). As ROS are known inducers 3.0 of ER stress (He et al., 2008), our results suggest that PRIMA-1MET triggers the ER stress pathway at least 2.5 partially through mutant p53-dependent induction of 2.0 ROS. Our data also suggest that modification of many 1.5 proteins in PRIMA-1-treated cells has a relatively small Fold change impact on cell survival, in agreement with our earlier 1.0 findings that PRIMA-1-modified albumin does not 0.5 induce apoptosis when introduced in tumor cells 0.0 (Lambert et al., 2009). Therefore, it is likely that Ctrl 6h 12h Ctrl 6h 12h PRIMA-1 targets only a limited number of cellular Microarray RT-PCR proteins that are capable of triggering cell death Saos-2 Saos-2-His273 signaling pathways, and that one of these targets is Figure 3 PRIMA-1MET induces genes involved in ER stress. (a) mutant p53. XBP1 mRNA is induced by PRIMA-1MET at 37 mM in mutant p53- Furthermore, Bax and Bak are known to interact and expressing cells according to both microarray analysis and real- activate IRE-1, leading to increased ER stress signaling time PCR. (b) Retrotranscriptase PCR (RT–PCR) showed the MET (Hetz et al., 2006). Therefore, we suggest that PRIMA- presence of a spliced form of XBP1, (S)XBP1 after PRIMA-1 MET treatment. The intensity of (S)XBP1 was quantified and is shown as 1 activates the ER stress signaling cascade through percentage of (U)XBP1, that is ratio between (S)XBP1 and both ROS induction and Bax-mediated IRE-1 activa- (U)XBP1. (c) Expression of DNAJB2 mRNA in Saos-2-His273 tion. ER stress can trigger apoptosis through two main cells after PRIMA-1MET treatment and to a lesser extent in parental pathways, either through the mitochondrial pathway Saos-2 cells. involving Bcl-2 family proteins (Li et al., 2006) or through JNK through its activation by the IRE-1 kinase earlier study showed that PRIMA-1MET increases the (Urano et al., 2000). This may explain why earlier level of HSP70, a chaperone involved in protein folding studies have shown PRIMA-1MET induced apoptosis (Rokaeus et al., 2007), suggesting an increase in the through Bax and the mitochondrial pathway (Chipuk unfolded protein fraction on PRIMA-1MET treatment. et al., 2003; Wang et al., 2007; Shen et al., 2008) or Interestingly, XBP1 contains a putative p53 response the JNK pathway in a mutant p53-dependent manner element, consistent with the involvement of p53 in ER (Li et al., 2005). stress (Ceribelli et al., 2006) and the upregulation of VEGF expression is known to be downregulated by XBP1 by PRIMA-1MET in this study. wild-type p53 (Mukhopadhyay et al., 1995). We found a

Oncogene PRIMA-1 induces ER stress-mediated apoptosis JMR Lambert et al 1335 Table 2 Genes containing putative p53-binding site whose expression is up- or downregulated by PRIMA-1MET treatment in Saos-2-His273 cells with fold change greater than 1.3 Affymetrix probe set ID Gene title/symbol P-value Fold change

6 h 12 h

209165_at Apoptosis antogonizing transcription factor AATF 0.0083 2.05 2.65 215483_at A kinase anchor protein 9 AKAP9 0.0309 0.84 0.74 210025_s_at Caspase recruitment domain family, member 10 CARD10 0.0193 1.38 1.81 200675_at CD81 antigen CD81 0.0211 1.41 2.62 202500_at DnaJ (Hsp40) homolog, subfamily B, member 2 DNAJB2 0.0075 1.67 2.80 208708_x_at 208290_s_at Eukariotic translation initiation factor 5 EIF5 0.0496 1.55 2.09 0.0178 2.41 3.99 203706_s_at Frizzled homolog 7 (Drosophila) FZD7 0.0051 1.33 2.01 209304_x_at Growth arrest and DNA-damage-inducible, b GADD45B 0.0416 2.00 3.05 209769_s_at Peanut-like 1(Drosophila) GP1BB 0.0435 1.48 2.23 201162_at Insulin-like growth factor-binding protein 7 IGFBP7 0.0498 1.07 2.529 210582_s_at LIM domain kinase 2 LIMK2 0.0245 1.22 1.84 212566_at -associated protein 4 MAP4 0.0325 1.34 1.66 224621_at Mitogen-activated protein kinase 1 MAPK1 0.0309 1.43 1.74 200797_s_at qw03a03.x1 NCI_CGAP_Ut3 Homo sapiens cDNA clone MCL1 0.0364 1.44 1.68 IMAGE:1989964 3’ mRNA sequence 200788_s_at Phosphoprotein enriched in astrocytes 15 PEA15 0.0186 1.58 2.23 204286_s_at Phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1 or NOXA) NOXA 0.0308 1.79 3.18 1555520_at Patched homolog (Drosophila) PTCH 0.0451 0.80 0.64 212217_at Ran GTPase activating protein 1 RANGAP1 0.0108 1.57 2.76 217264_s_at SCNN1A 0.0382 1.47 1.78 222986_s_at Scotin SCOTIN 0.0237 1.43 1.97 201195_s_at Solute carrier family 7, member 5 SLC7A5 0.0136 1.66 3.27 204292_x_at Serine/threonine kinase 11, Peutz-Jeghers syndrome STK11 0.0175 1.65 1.98 200931_s_at Vinculin VCL 0.0301 1.56 1.96 210512_s_at Vascular endothelial growth factor VEGF 0.0246 2.01 3.45 200670_at X-box-binding protein 1 XBP1 0.0268 1.54 2.36 201901_s_at YY1 transcription factor YY1 0.047 1.75 2.45

Abbreviation: YY1, Yin Yang 1. strong upregulation of this gene after reactivation of Materials and methods mutant p53 by PRIMA-1MET. However, VEGF mRNA expression is rapidly increased after exposure to Cell lines and reagents chemical inducers of ER stress (Abcouwer et al., The human Saos-2 osteosarcoma and H1299 lung adenocarci- 2002). Thus, the increase observed here may not be noma cell lines are p53 null. Their sublines Saos-2-His273 and directly p53 dependent but rather a response to H1299-His175 are stably transfected with the tetracycline- MET regulated His273 or His175 mutant p53 expression constructs PRIMA-1 -induced ER stress. (Tet-Off), respectively. The human SW480 colon adenocarci- In conclusion, our microarray analysis demonstrates noma line expresses endogenous His273/Ser309 mutant p53. MET that PRIMA-1 has profound impact on global gene The human HCT116 p53 þ / þ colon carcinoma line harbors expression in a mutant p53-dependent manner. PRIMA- wild-type p53. Its sublines HCT116 p53À/À and HCT116 p53À/248 1MET treatment leads to induction of series of p53 target lack p53 or express Trp248 mutant p53, respectively. All cells genes that promote cell-cycle arrest and apoptosis. We were grown at 37 1C under 5% CO2 in Iscove’s-modified have uncovered an ER stress signaling pathway Dulbecco’s medium containing 10% FBS, 2 mML-glutamine triggered by PRIMA-1MET in mutant p53-expressing and 40 mg/ml gentamycin. Wild-type p53 plasmid carrying the cells. Interestingly, ER stress-induced apoptosis can be full-length p53 cDNA sequence inserted into the pCMV achieved through either the JNK pathway or the expression vector was generously provided by Dr Bert Vogel- stein. The transient transfection experiments were performed mitochondrial pathway. Thus, we have shown that with Lipofectamine 2000 according to the manufacturer’s PRIMA-1 restores both transcription-dependent and recommendations (Invitrogen Life Technologies, Breda, the transcription-independent pro-apoptotic activity to mu- Netherlands). For all experiments, cells were plated at a density tant p53. This is in agreement with the notion that p53 of 15 000 cells/cm2 and were incubated overnight before treat- can induce apoptosis also in a transcription-independent ment. manner (Caelles et al., 1994). Activation of multiple apoptotic pathways not only allows a robust apoptotic Microarrays response, but also reduces the risk of emerging drug- Cells were treated with PRIMA-1MET for 6 and 12 h. RNA resistant variants. Thus, our results have identified novel was extracted using RNeasy Mini Kit (Qiagen, Solna, Sweden) molecular mechanisms for PRIMA-1-induced cell death according to manufacturer’s instructions. The optical density and may facilitate the design for novel anti-cancer at 260/280 nm was between 1.9–2.1. Double-stranded cDNA drugs, and ultimately more efficient cancer therapy. was synthesized with 1 mg of total RNA using the SuperScript

Oncogene PRIMA-1 induces ER stress-mediated apoptosis JMR Lambert et al 1336

ER GRP78 (Affymetrix Inc.). The scanned images were inspected and GRP78 ATF-6 analyzed using established quality control criteria. IRE-1

GRP78 Data analysis PERK Comparative analysis between expression profiles for Affyme- trix experiments was carried out using GeneSpring software version 7.3 (Silicon Genetics, Redwood City, CA, USA). The ‘Cross gene error model for deviation from 1.0’ was active. ER PRIMA-1 Gene expression data were normalized in two ways: ‘per chip GRP normalization’ and ‘per gene normalization’. For ‘per chip ROS GRP78 78 normalization’, all expression data on a chip are normalized to GRP78 the 50th percentile of all values on that chip. For ‘per gene P normalization’, the data for a given gene are normalized to the p53 mutp53 IRE-1 E-1 median expression level of that gene across all samples. The IR P Bax data sets were then assigned to six groups for either experiment VEGF Saos-2 (0, 6, and 12 h) or Saos-2-His273 (0, 6, and 12 h). The PUMA NOXA expression profiles from three independent experiments were XBP1 JNK pathway compared using ANOVA (parametric test, assume variances equal) to identify genes that were differentially expressed BAX/BAK pathway Chaperone proteins between the six groups. For sample clustering, standard correlation was applied to measure the similarity of the expression pattern between different samples. Gene lists were made with genes that were either induced or suppressed >1.3- fold between treatments and time points. The genes were Apoptosis categorized according to their biological functions as described Figure 4 Putative model for induction of ER stress by PRIMA- in Gene Spring, Ingenuity and GeneCards. data accesion 1MET.(a) In the absence of cellular stress, GRP78 is localized in the number online: GSE15658 (http://www.ncbi.nlm.nih.gov/geo/ ER and bound to transcription factors including IRE-1. (b) query/acc.cgi?acc ¼ GSE15658) PRIMA-1MET reactivates mutant p53, which triggers the mitochon- drial apoptosis pathway through Puma and Bax. Reactivation of mutant p53 is followed by the production of ROS that can induce Real-time PCR ER stress, and as a consequence, the accumulation of unfolded Cells were treated with PRIMA-1MET and were collected after 6 proteins in the ER. During stress, GRP78 binds unfolded proteins and 12 h, RNA was extracted and cDNA was synthesized, as and releases IRE-1, which is dimerized. Activated IRE-1 leads to described above. cDNA of 12 ng was added to 50 ml of Taqman the cleavage of XBP1 mRNA and the activation of the JNK Universal PCR Master Mix (Applied Biosystems, Foster City, pathway and apoptosis. Bax, which is upregulated after PRIMA- CA, USA) and total volume was adjusted to 100 mlwithRNase- MET 1 treatment, can directly activate IRE-1. During sustained ER free water. Samples were loaded into the 384-wells Taqman Low stress, Noxa is induced in a mutant p53-dependent manner. We do Density Array (Applied Biosystems). The following probes not exclude that PRIMA-1 may have direct effects on protein folding/ER stress. The downstream effects of ER stress are were available on the array: Hs00820472_m1 (EIF5), highlighted in blue. Hs00177066_m1 (MAPK1), Hs00192541_m1 (RAD23A), Hs00169587_m1 (GADD45B), Hs00370193_m1 (WIPF2/ WIRE), Hs00560402_m1 (PMAIP1/Noxa), Hs00176092_m1 (STK11), Hs01579127_m1 (Scotin) Hs01113553_mH Choice system (Invitrogen, Stockholm, Sweden). T7-(dT24) (YWHAG/14-3-3g), Hs00159048_m1 (MAP4), Hs00701790_s1 oligomer was used for priming the first-strand cDNA (UBE2D3), Hs00231936_m1 (XBP-1), Hs00231533_m1 (YY1), synthesis. The resulting cDNA was purified using the sample Hs00367225_m1 (CARD10), Hs00204044_m1 (ASF1A), clean up kit (Affymetrix Inc., Santa Clara, CA, USA). The Hs00198807_m1 (DNAJB2), Hs00266026_m1 (IGFBP7), cDNA pellet was collected and dissolved in an appropriate Hs00854264_s1 (CNN2), Hs00269428_m1 (PEA15), volume. Using cDNA as the template, cRNA was synthesized Hs00173626_m1 (VEGFA), Hs00181117_m1 (PTCH1), using an in vitro transcription kit (Affymetrix Inc.). Biotiny- and Hs99999901_s1 (18S) (Applied Biosystems). lated-11-CTP and 16-UTP ribonucleotides (Affymetrix Inc.) Amplification started with 2 min at 50 1C and 10 min at were added to the reaction as labeling reagents. in vitro 95 1C, and was followed by 40 cycles with 15 s denaturation at transcription reactions were carried out at 37 1C for 16 h and, 95 1C and 1 min annealing/extension at 60 1C. Reactions were the labeled cRNA was purified using the sample clean up kit run on the ABI PRISM 7900HT Fast real-time PCR System (Affymetrix Inc.). The cRNA was fragmented in a fragmenta- (Applied Biosystems). Results were analyzed with the com- tion buffer (40 mmol/l Tris-acetate, pH 8.1, 100 mmol/l KOAc, parative Ct method using 18S as gene of reference. Real time 30 mmol/l MgOAc) for 35 min at 94 1C. Fragmented cRNA for Bax was run separately using the standard protocol and the (15 mg/probe array) was hybridized with the human U133 2.0 Taqman Gene Expression Assay Hs00414514_m1 (BAX) and plus GeneChip array at 45 1C for 18 h in a hybridization oven Hs99999905_m1 (GAPDH) (Applied Biosystems). with constant rotation (60 r.p.m.). The chips were washed and stained using the Affymetrix fluidics stations. Staining was Retrotranscriptase PCR for XBP1 performed using streptavidin phycoerythrin conjugate (SAPE; Cells were treated with PRIMA-1MET for 12 h. Then cells were Molecular Probes, Eugene, OR, USA) followed by the collected and RNA was extracted as described above. Samples addition of a biotinylated anti-streptavidin antibody (Vector were incubated with recombinant DNase (Roche Applied Laboratories, Burlingame, CA, USA), and finally with Science, Bromma, Sweden). cDNA was synthesized as streptavidin phycoerythrin conjugate. Probe arrays were described above but by using random primers instead of scanned using GeneChip Scanner 3000 with AutoLoader OligodT. cDNA of 50 ng was amplified with TopTaq

Oncogene PRIMA-1 induces ER stress-mediated apoptosis JMR Lambert et al 1337 polymerase (Qiagen) using a pairs of primers corresponding to Immunoprecipitation nucleotides 412–431 (CCTTGTAGTTGAGAACCAGG) and Cells were treated with PRIMA-1MET for 6 h, and then 834–853 (GGGGCTTGGTATATATGTGG) of XBP1 cDNA. collected and incubated for 45 min on ice in lysis buffer. Amplification started with a first step of 2 min at 94 1C, Samples containing equal amount of proteins were precleared continued with a second step made of 40 cycles 94 1C for 30 s, by incubating 2 h at 4 1C with Dynabeads protein A (Invitro- 55 1C for 30 s, and 72 1C for 60 s, and finished with a last step of gen). Supernatants were transferred into new tubes and 10 min at 72 1C. PCR products were run on a 3% agarose gel. incubated overnight at 4 1C with anti-BiP/GRP78. Antibodies were then captured by incubating with 10 ml Dynabeads 1 Western blotting protein A for 1 h on a rotation wheel at 4 C. Proteins were Cells were treated with PRIMA-1MET for 16–24 h and collected eluted with 0.1 M citrate pH 3.1 and neutralized with 25 mM and lysed. Equal amount of total proteins were separated by NaOH. Eluted samples were separated on a SDS–polyacyla- electrophoresis on an SDS–polyacrylamide gel, and then mide gel, which were then stained with the Silver Quest kit transferred on a nitrocellulose membrane using the iBlot Dry (Invitrogen) according to the manufacturer’s instructions. Blotting System (Invitrogen). Anti-b-actin (AC-15, Sigma, Stockholm, Sweden), anti-p53 (FL393, Santa Cruz Biotech- nology, Santa Cruz, CA, USA), anti-14-3-3 (H8, Santa Cruz Biotechnology), anti-Noxa (OP180, Calbiochem, Darmstadt, Conflict of interest Germany), and anti-BiP/GRP78 (610978, BD Biosciences, San Jose, CA, USA) antibodies were used. Protein bands were Klas Wiman and Vladimir Bykov are co-founders and share- visualized using SuperSignal West Femto Maximum Sensitiv- holders of Aprea AB, a company that develops novel p53-based ity Substrate (Pierce, Rockford, IL, USA). cancer therapy. Klas Wiman is a member of its board.

Cell staining MET Cells were treated with PRIMA-1 for 8 or 24 h at final Acknowledgements concentrations ranging from 10 to 75 mM. Cells were fixed in 4% formaldehyde and permeabilized in 0.2% Triton-X. To We thank Bert Vogelstein, Johns Hopkins Oncology Center, visualize F-actin, cells were stained with TexasRed-X phalloi- for HCT116 cells. This work was supported by the Swedish din (Invitrogen). All samples were mounted with Vectashied Cancer Society (Cancerfonden), Cancerfo¨reningen, Karolinska mounting medium with DAPI (Vector laboratories). Images Institutet and the EU 6th framework program. This publica- were captured on a Zeiss Axioplan 2 microscope. tion reflects the author’s views and not necessarily those of the EC. The information in this document is provided as is and no Flow cytometry guarantee or warranty is given that the information is fit for Cells were treated with PRIMA-1MET for 48 h, collected, fixed any particular purpose. The user thereof uses the information in 70% ethanol, treated with RNase A, stained with propidium at his/her sole risk and liability. The Community is not liable iodide and analyzed with a FACScan flow cytometer (Becton for any use that may be made of the information contained Dickinson, Franklin Lakes, CA, USA). herein.

References

Abcouwer SF, Marjon PL, Loper RK, Vander Jagt DL. (2002). Ceribelli M, Alcalay M, Vigano MA, Mantovani R. (2006). Repression Response of VEGF expression to amino acid deprivation and of new p53 targets revealed by ChIP on chip experiments. Cell Cycle inducers of endoplasmic reticulum stress. Invest Ophthalmol Vis Sci 5: 1102–1110. 43: 2791–2798. Chipuk JE, Maurer U, Green DR, Schuler M. (2003). Pharmacologic Andersson J, Larsson L, Klaar S, Holmberg L, Nilsson J, Inganas M activation of p53 elicits Bax-dependent apoptosis in the absence of et al. (2005). Worse survival for TP53 (p53)-mutated breast cancer transcription. Cancer Cell 4: 371–381. patients receiving adjuvant CMF. Ann Oncol 16: 743–748. Di Como CJ, Gaiddon C, Prives C. (1999). p73 function is inhibited by Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D. (2000). tumor-derived p53 mutants in mammalian cells. Mol Cell Biol 19: Dynamic interaction of BiP and ER stress transducers in the 1438–1449. unfolded-protein response. Nat Cell Biol 2: 326–332. Gaiddon C, Lokshin M, Ahn J, Zhang T, Prives C. (2001). A subset of Bunz F, Hwang PM, Torrance C, Waldman T, Zhang Y, Dillehay L tumor-derived mutant forms of p53 down-regulate p63 and p73 et al. (1999). Disruption of p53 in human cancer cells alters the through a direct interaction with the p53 core domain. Mol Cell Biol responses to therapeutic agents. J Clin Invest 104: 263–269. 21: 1874–1887. Bykov VJ, Issaeva N, Shilov A, Hultcrantz M, Pugacheva E, Green DR, Kroemer G. (2009). Cytoplasmic functions of the tumour Chumakov P et al. (2002). Restoration of the tumor suppressor suppressor p53. Nature 458: 1127–1130. function to mutant p53 by a low-molecular-weight compound. Nat Gronroos E, Terentiev AA, Punga T, Ericsson J. (2004). YY1 inhibits Med 8: 282–288. the activation of the p53 tumor suppressor in response to genotoxic Bykov VJ, Zache N, Stridh H, Westman J, Bergman J, Selivanova G stress. Proc Natl Acad Sci USA 101: 12165–12170. et al. (2005). PRIMA-1(MET) synergizes with cisplatin to induce Haupt Y, Maya R, Kazaz A, Oren M. (1997). Mdm2 promotes the tumor cell apoptosis. Oncogene 24: 3484–3491. rapid degradation of p53. Nature 387: 296–299. Cadwell C, Zambetti GP. (2001). The effects of wild-type p53 tumor He S, Yaung J, Kim YH, Barron E, Ryan SJ, Hinton DR. (2008). suppressor activity and mutant p53 gain-of-function on cell growth. Endoplasmic reticulum stress induced by oxidative stress in retinal Gene 277: 15–30. pigment epithelial cells. Graefes Arch Clin Exp Ophthalmol 246: Caelles C, Helmberg A, Karin M. (1994). p53-dependent apoptosis in 677–683. the absence of transcriptional activation of p53-target genes. Nature Hetz C, Bernasconi P, Fisher J, Lee AH, Bassik MC, Antonsson B 370: 220–223. et al. (2006). Proapoptotic BAX and BAK modulate the unfolded

Oncogene PRIMA-1 induces ER stress-mediated apoptosis JMR Lambert et al 1338 protein response by a direct interaction with IRE1alpha. Science Rokaeus N, Klein G, Wiman KG, Szekely L, Mattsson K. (2007). 312: 572–576. PRIMA-1(MET) induces nucleolar accumulation of mutant Hoh J, Jin S, Parrado T, Edington J, Levine AJ, Ott J. (2002). The p53 and PML nuclear body-associated proteins. Oncogene 26: p53MH algorithm and its application in detecting p53-responsive 982–992. genes. Proc Natl Acad Sci USA 99: 8467–8472. Sablina AA, Budanov AV, Ilyinskaya GV, Agapova LS, Kravchenko Hossain MM, Hwang DY, Huang QQ, Sasaki Y, Jin JP. (2003). JE, Chumakov PM. (2005). The antioxidant function of the p53 Developmentally regulated expression of calponin isoforms and the tumor suppressor. Nat Med 11: 1306–1313. effect of h2-calponin on cell proliferation. Am J Physiol Cell Physiol Shen J, Vakifahmetoglu H, Stridh H, Zhivotovsky B, Wiman KG. 284: C156–C167. (2008). PRIMA-1MET induces mitochondrial apoptosis through Inesi G, Sagara Y. (1992). Thapsigargin, a high affinity and global activation of caspase-2. Oncogene 27: 6571–6580. inhibitor of intracellular Ca2+ transport ATPases. Arch Biochem Strano S, Fontemaggi G, Costanzo A, Rizzo MG, Monti O, Baccarini Biophys 298: 313–317. A et al. (2002). Physical interaction with human tumor-derived p53 Johnson TM, Yu ZX, Ferrans VJ, Lowenstein RA, Finkel T. (1996). mutants inhibits p63 activities. J Biol Chem 277: 18817–18826. Reactive oxygen species are downstream mediators of p53- Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, Harding HP et al. dependent apoptosis. Proc Natl Acad Sci USA 93: 11848–11852. (2000). Coupling of stress in the ER to activation of JNK Kubbutat MH, Jones SN, Vousden KH. (1997). Regulation of p53 protein kinases by transmembrane protein kinase IRE1. Science stability by Mdm2. Nature 387: 299–303. 287: 664–666. Lai E, Teodoro T, Volchuk A. (2007). Endoplasmic reticulum stress: Vousden KH, Lu X. (2002). Live or let die: the cell’s response to p53. signaling the unfolded protein response. Physiology (Bethesda) 22: Nat Rev Cancer 2: 594–604. 193–201. Vousden KH, Ryan KM. (2009). p53 and metabolism. Nat Rev Cancer Lambert JM, Gorzov P, Veprintsev DB, Soderqvist M, Segerback D, 9: 691–700. Bergman J et al. (2009). PRIMA-1 reactivates mutant p53 by Wang L, Wu Q, Qiu P, Mirza A, McGuirk M, Kirschmeier P et al. covalent binding to the core domain. Cancer Cell 15: 376–388. (2001). Analyses of p53 target genes in the Li J, Lee B, Lee AS. (2006). Endoplasmic reticulum stress-induced by bioinformatic and microarray approaches. J Biol Chem 276: apoptosis: multiple pathways and activation of p53-up-regulated 43604–43610. modulator of apoptosis (PUMA) and NOXA by p53. J Biol Chem Wang T, Lee K, Rehman A, Daoud SS. (2007). PRIMA-1 281: 7260–7270. induces apoptosis by inhibiting JNK signaling but promoting Li Y, Mao Y, Brandt-Rauf PW, Williams AC, Fine RL. (2005). the activation of Bax. Biochem Biophys Res Commun 352: Selective induction of apoptosis in mutant p53 premalignant and 203–212. malignant cancer cells by PRIMA-1 through the c-Jun-NH2-kinase Westhoff B, Chapple JP, van der Spuy J, Hohfeld J, Cheetham ME. pathway. Mol Cancer Ther 4: 901–909. (2005). HSJ1 is a neuronal shuttling factor for the sorting of Livneh Z. (2006). Keeping mammalian mutation load in check: chaperone clients to the proteasome. Curr Biol 15: 1058–1064. regulation of the activity of error-prone DNA polymerases by p53 Wulfkuhle JD, Donina IE, Stark NH, Pope RK, Pestonjamasp KN, and p21. Cell Cycle 5: 1918–1922. Niswonger ML et al. (1999). Domain analysis of supervillin, an F- MirzaA,WuQ,WangL,McClanahanT,BishopWR,GheyasFet al. actin bundling plasma membrane protein with functional nuclear (2003). Global transcriptional program of p53 target genes during the localization signals. J Cell Sci 112: 2125–2136. process of apoptosis and cell cycle progression. Oncogene 22: 3645–3654. Xue W, Zender L, Miething C, Dickins RA, Hernando E, Mukhopadhyay D, Tsiokas L, Sukhatme VP. (1995). Wild-type p53 Krizhanovsky V et al. (2007). Senescence and tumour clearance is and v-Src exert opposing influences on human vascular endothelial triggered by p53 restoration in murine liver carcinomas. Nature 445: growth factor gene expression. Cancer Res 55: 6161–6165. 656–660. Olivier M, Eeles R, Hollstein M, Khan MA, Harris CC, Hainaut P. Yakovleva T, Kolesnikova L, Vukojevic V, Gileva I, Tan-No K, (2002). The IARC TP53 database: new online mutation analysis and Austen M et al. (2004). YY1 binding to a subset of p53 DNA-target recommendations to users. Hum Mutat 19: 607–614. sites regulates p53-dependent transcription. Biochem Biophys Res Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B. (1997). A Commun 318: 615–624. model for p53-induced apoptosis. Nature 389: 300–305. Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K. (2001). XBP1 Rahman R, Latonen L, Wiman KG. (2005). hTERT antagonizes p53- mRNA is induced by ATF6 and spliced by IRE1 in response to induced apoptosis independently of telomerase activity. Oncogene ER stress to produce a highly active transcription factor. Cell 107: 24: 1320–1327. 881–891. Rogojina AT, Orr WE, Song BK, Geisert Jr EE. (2003). Comparing Zache N, Lambert JM, Wiman KG, Bykov VJ. (2008). PRIMA- the use of Affymetrix to spotted oligonucleotide microarrays using 1(MET) inhibits growth of mouse tumors carrying mutant p53. Cell two retinal pigment epithelium cell lines. Mol Vis 9: 482–496. Oncol 30: 411–418.

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

Oncogene