Histone deacetylase inhibitors prime medulloblastoma cells for chemotherapy-induced apoptosis by enhancing p53-dependent Bax activation

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Abstract

Despite aggressive therapies, the prognosis of children with high-risk medulloblastoma is still poor, thus underscoring the need to develop novel treatment strategies. Here, we report that histone deacetylase inhibitors (HDACI), that is, MS-275, valproic acid or SAHA, provide a novel strategy for sensitization of medulloblastoma to DNA-damaging drugs such as Doxorubicin, VP16 and Cisplatin by promoting p53-dependent, mitochondrial apoptosis. Mechanistic studies reveal that single-agent treatment with MS-275 causes acetylation of the non-histone protein Ku70, an event reported to release Bax from Ku70, whereas DNA-damaging drugs trigger p53 acetylation and accumulation. Combined treatment with MS-275 and Doxorubicin or VP16 cooperates to promote binding of p53 to Bax and p53-dependent Bax activation, resulting in enhanced loss of mitochondrial membrane potential, cytochrome c release and caspase-dependent apoptosis. Overexpression of Bcl-2 almost completely abolishes the MS-275-mediated chemosensitization, underlining the importance of the mitochondrial pathway for inducing apoptosis. Also, MS-275 cooperates with chemotherapeutics to inhibit long-term clonogenic survival. Most importantly, MS-275 increases chemotherapeutic drug-induced apoptosis in primary medulloblastoma samples, and cooperates with Doxorubicin to suppress medulloblastoma growth in an in vivo model, which underscores the clinical relevance of the findings. Thus, HDACI such as MS-275 present a promising approach for chemosensitization of medulloblastoma by enhancing mitochondrial apoptosis in a p53-dependent manner. These findings have important clinical implications for the design of experimental treatment protocols for medulloblastoma.

Main

Medulloblastoma, an embryonic tumor of the cerebellum, constitutes the most frequent malignant brain tumor of childhood (Kleihues et al., 2002; Rossi et al., 2008). As conventional therapies and risk stratification have already been optimized in recent years (Rutkowski et al., 2007), experimental therapeutics are likely critical to improve the still poor prognosis of children with high-risk medulloblastoma.

Histone deacetylase (HDAC) inhibitors (HDACI) are considered as promising cancer therapeutics and exert their biological effects by at least two principal mechanisms. First, HDACI cause accumulation of acetylated histones, which favors an open state of the chromatin, thereby facilitating gene transcription (Bolden et al., 2006; Xu et al., 2007). More recently, HDACI have also been shown to trigger acetylation of a number of non-histone proteins, for example the DNA repair protein Ku70, which reduces its DNA repair capacity as well as its interaction with the proapoptotic protein Bax (Cohen et al., 2004; Subramanian et al., 2005; Chen et al., 2007). The anticancer effects of HDACI have been largely attributed to their ability to trigger programmed cell death (apoptosis) (Bolden et al., 2006). Permeabilization of mitochondrial membranes is considered as a central event in anticancer drug-induced apoptosis (Fulda and Debatin, 2006) and is controlled, for example, by two classes of Bcl-2 family proteins, that is, anti-apoptotic members such as Bcl-2 and proapoptotic proteins, that is, Bax (Adams and Cory, 2007). In preclinical studies, HDACI have shown promise as anticancer agents in a large number of hematological malignancies and solid tumors (Xu et al., 2007). In childhood, solid tumors including medulloblastoma, the benzamide-derivative MS-275 has been reported to display preclinical, in vitro and in vivo, anti-tumor activity (Jaboin et al., 2002), indicating that MS-275 presents a promising compound for pediatric cancers. Recently, we reported that re-expression of caspase-8 by HDACI and interfeon-γ was an essential event to prime medulloblastoma cells, with epigenetic silencing of caspase-8, for death receptor-triggered apoptosis (Hacker et al., 2009). In the present study, we investigated rational MS-275-based combinations with chemotherapy for the treatment of medulloblastoma.

To explore the potential of HDACI to increase chemosensitivity of medulloblastoma cells, we used the benzamide-derivative MS-275 because of its potent in vitro anti-tumor activity against pediatric solid tumor cell lines including medulloblastoma (Jaboin et al., 2002). Control experiments showed that treatment of medulloblastoma cells with MS-275 caused acetylation of histone H3 (Supplementary Figure 1a). Analysis of the administration schedule revealed that pretreatment with MS-275 for 24 h was more effective to enhance Doxorubicin-induced apoptosis compared with simultaneous administration of MS-275 and Doxorubicin, treatment with Doxorubicin for 24 h followed by treatment with MS-275 for 24 h or, alternatively, another length of preincubation (Supplementary Figure 1b). Therefore, a 24 h pretreatment period was subsequently used in all experiments. We selected anticancer drugs of different pharmacological classes, including topoisomerase I and II inhibitors (Topotecan, Doxorubicin, VP16), platin analogs (Cisplatin), anti-metabolites (Methotrexate) and microtubule-interfering agents (Taxol, Vincristine). Importantly, pre-exposure to sub-toxic concentrations of MS-275 significantly enhanced apoptosis induced by Doxorubicin, VP16 or Cisplatin in several medulloblastoma cell lines, and by Topotecan in D458 cells in a dose-dependent manner (Figure 1a, Supplementary Figure 1c, Supplementary Table 1). In comparison, no cooperative effect of MS-275 was observed for Methotrexate, Taxol or Vincristine (Supplementary Table 1). Also, MS-275 acted in concert with Doxorubicin or VP16 to suppress clonogenic growth compared with treatment with either agent alone (Figure 1b), demonstrating that MS-275 has an effect on long-term survival. To exclude that our findings are restricted to one particular HDACI, we extended our studies to additional HDACI. Similarly, valproic acid and SAHA significantly augmented Doxorubicin- or VP16-induced apoptosis in a dose-dependent manner at a concentration that caused histone H3 acetylation (Supplementary Figure 1d). In contrast with malignant cells, MS-275 did not enhance Doxorubicin-induced apoptosis in normal fibroblasts (data not shown). Together, these findings demonstrate that HDACI enhance the sensitivity of medulloblastoma cells for chemotherapy-induced apoptosis and also act in concert with chemotherapeutic agents to suppress clonogenic survival.

Figure 1
figure1

MS-275 sensitizes medulloblastoma cells to chemotherapy-induced apoptosis. (a) D458 medulloblastoma cells were cultured as described previously (Fulda and Debatin, 2006) and treated for 24 h with 0.6 μM MS-275 (kindly provided by Bayer Schering Pharma AG, Berlin, Germany) before the indicated concentrations of Doxorubicin (Doxo), VP16 or Cisplatin were added for 24 h. Apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei as described previously (Vogler et al., 2008) and percentage of specific apoptosis is shown. White bars, no pretreatment, black bars, pretreatment with MS-275. (b) D458 cells were treated for 24 h with 0.6 μM MS-275 before 0.1 μg/ml Doxorubicin (Doxo) or 0.3 μg/ml VP16 was added for 3 h. Afterwards cells were seeded at 0.63 × 103 cells/cm2 in a 1% methylcellulose medium (StemCell Technologies, Vancouver, Canada) consisting of a 2.6% solution of methylcellulose in Iscove's MDM (40%), IMEMZO medium with 40% FCS (49%) and 1% L-glutamine. After ten days, colonies with more than twenty cells per colony were counted under the microscope. (c) D458 cells were treated with 0.6 μM MS-275 for 24 h before 0.3 μg/ml Doxorubicin (Doxo) or 1 μg/ml VP16 was added for additional 24 h. Apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei in the presence or absence of 50 μM of the broad-range caspase inhibitor z-VAD-fmk (Bachem, Heidelberg, Germany) and percentage of specific apoptosis is shown. (d) D458 cells were treated with 0.6 μM MS-275 for 24 h before 0.3 μg/ml Doxorubicin (Doxo) or 1 μg/ml VP16 was added for the indicated times. Caspase activity was determined in living cells as described previously (Giagkousiklidis et al., 2005) by using substrates specific for caspase-3 (zDEVD-R110) or caspase-9 (zLEHD-R110). In ad, data represent mean+s.e.m. of three independent experiments performed in triplicate. Statistical significance was assessed by Student's t-test (two-tailed distribution, two-sample unequal variance); *P<0.05; #P<0.001.

To test whether apoptosis in this setting occurs in a caspase-dependent manner, we used the broad-range caspase inhibitor z-VAD-fmk. Notably, addition of z-VAD-fmk almost completely blocked apoptosis upon treatment with either Doxorubicin or VP16 in combination with MS-275 (Figure 1c), demonstrating that caspase activity is necessary for apoptosis induction. Monitoring of caspase activation by enzymatic caspase assay revealed that MS-275 significantly enhanced caspase-9 and -3 activities upon treatment with Doxorubicin or VP16 (Figure 1d). Similarly, MS-275 substantially enhanced Doxorubicin- or VP16-induced cleavage of caspase-9 into p37/35 fragments and cleavage of caspase-3 into p17/12 active fragments (Supplementary Figure 2a, for example, at 12 h), resulting in decreased caspase-3 proenzyme levels as a marker of its proteolytic turnover in cells at 24 h. This demonstrates that MS-275 cooperates with chemotherapeutic drugs to induce caspase activation and caspase-dependent apoptosis in medulloblastoma cells.

To decipher the molecular mechanisms underlying the HDACI-mediated chemosensitization, we initially performed a survey of pro- and anti-apoptotic proteins following exposure to MS-275. Although caspase-8 expression was undetectable in untreated cells, consistent with its epigenetic silencing in medulloblastoma (Grotzer et al., 2000; Fulda et al., 2001), treatment with MS-275 substantially upregulated caspase-8 (Supplementary Figure 2b). Silencing of caspase-8 did not interfere with the MS-275-mediated sensitization for Doxorubicin- or VP16-induced apoptosis (data not shown), whereas caspase-8 knockdown blocked TRAIL-induced apoptosis in medulloblastoma cells (Hacker et al., 2009), indicating that upregulation of caspase-8 is not required for the sensitization to DNA-damaging drugs by MS-275.

Next, we assessed the effect of MS-275 on activation of the DNA damage checkpoint. Doxorubicin caused phosphorylation of ATM and Chk2, as well as accumulation of p53 and Puma, with no further increase upon the addition of MS-275 (Supplementary Figure 2c). Further, exposure to MS-275 did not cause detectable changes in expression of proteins involved in non-homologous end joining, such as DNA-PK, Ku70 and Ku80, or in homologous recombination, such as BRCA1, BRCA2, RAD51 and NBS1 (data not shown).

As there is increasing evidence that HDACI alter the acetylation status of non-histone proteins besides histones (Bolden et al., 2006; Xu et al., 2007), we assessed the acetylation status of Ku70. Indeed, MS-275 markedly increased acetylation of Ku70, and significantly increased Doxorubicin-induced DNA strand breaks in a time-dependent manner (Figure 2a). This increase in DNA strand breaks was not simply secondary to enhanced apoptotic DNA fragmentation, as it similarly occurred in the presence of the caspase inhibitor z-VAD-fmk (data not shown), which blocked apoptosis (Figure 1c). Together, this set of experiments demonstrates that MS-275 triggers Ku70 acetylation and enhances Doxorubicin-induced DNA strand breaks.

Figure 2
figure2

MS-275 causes Ku70 acetylation, and enhances Doxorubicin-induced DNA strand breaks and chemotherapy-induced mitochondrial perturbations. (a) D458 cells were treated with 0.6 μM MS-275 for 24 h. In the left panel, acetylation of Ku70 was assessed by lysing cells in a hypotonic buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl), followed by immunoprecipitation of Ku70 from protein lysates (2 mg) using 4 μg mouse anti-Ku70 antibody (Abcam, Cambridge, UK) and finally immunoblotting using an antibody against acetyl–lysine residues (Upstate, Lake Placid, NY, USA). In the right panel, DNA strand breaks following the addition of 0.3 μg/ml Doxorubicin (Doxo) for 3, 6 and 12 h were determined by alkaline comet assay as described previously (Karl et al., 2009) using an alkaline denaturation time of 20 min and an electrophoresis time of 15 min. DNA damage is expressed as Olive Tail Moment. In panels b and c, D458 cells were treated for 24 h with 0.6 μM MS-275 before 0.3 μg/ml Doxorubicin (Doxo) or 1 μg/ml VP16 was added for 9 h (b) or for the indicated times (c). In b, Bax activation was analyzed by immunoprecipitation of protein lysates using the active conformation specific anti-Bax antibody 6A7 as previously described (Hacker et al., 2009), and Bax expression was analyzed by western blotting. In c, mitochondrial membrane potential was assessed by flow cytometry using the fluorescent dye CMXRos (1 μM, Molecular Probes, Karlsruhe, Germany). In d, D458 cells were stably transduced with pMSCV vector containing murine Bcl-2 (Bcl-2) or empty vector (EV) using the packaging cell line PT67, and were treated for 24 h with 0.6 μM MS-275. Expression of Bcl-2 and β-actin was assessed by western blotting (upper panel). Apoptosis after addition of 0.3 μg/ml Doxorubicin (Doxo) or 0.3 μg/ml VP16 for 24 h was determined by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei and percentage of specific apoptosis is shown (lower panel). In a (left panel), b and d (upper panel), a representative of three independent experiments is shown. In a (right panel), c and d (lower panel), mean+s.e.m. of three independent experiments performed in triplicate are shown; *P<0.05; #P<0.001.

As the acetylation status of Ku70 has been reported to also affect its interaction with Bax (Cohen et al., 2004), we next assessed Bax activation. Interestingly, MS-275 and Doxorubicin or VP-16 cooperated to trigger Bax conformational change (Figure 2b), which was also detected in the presence of the caspase inhibitor z-VAD-fmk (Supplementary Figure 3), demonstrating that it occurred before caspase activation. Additionally, MS-275 significantly enhanced Doxorubicin- or VP16-induced loss of mitochondrial membrane potential and cytochrome c release (Figure 2c, Supplementary Figure 3b). Importantly, Bcl-2 overexpression significantly reduced apoptosis upon combined treatment with either MS-275 and Doxorubicin or VP16, and also upon treatment with Doxorubicin or VP16 alone (Figure 2d). This demonstrates that the MS-275-induced sensitization for chemotherapeutic drug-induced apoptosis depends on the mitochondrial pathway of apoptosis.

Monitoring of p53 as another non-histone substrate of HDACI revealed a marked increase in acetylated p53 and in total p53 protein levels upon Doxorubicin treatment, with no further increase by MS-275 (Figure 3a). Of note, Doxorubicin and MS-275 cooperated to stimulate the binding of p53 to Bax without changes in Bax expression levels (Figure 3a), in line with the concerted action of Doxorubicin and MS-275 to trigger Bax activation (Figure 2b). Importantly, knockdown of p53 profoundly inhibited activation of Bax, and significantly reduced apoptosis upon treatment with MS-275 and Doxorubicin or VP16 (Figures 3b and c, Supplementary Figure 4). This set of experiments demonstrates that Doxorubicin as a single agent causes p53 acetylation and accumulation, whereas the combination of Doxorubicin and MS-275 acts in concert to trigger binding of p53 to Bax, p53-dependent Bax activation and apoptosis.

Figure 3
figure3

Requirement of p53 for Bax activation and mitochondrial apoptosis. (a) D458 cells were treated for 24 h with 0.6 μM MS-275 before 0.3 μg/ml Doxorubicin (Doxo) was added for 9 h. Acetylation of p53 on lysine residues 373 and 382 and interaction of p53 and Bax was analyzed by lysing cells in CHAPS buffer (10 mM HEPES pH7.4; 150 mM NaCl; 1% CHAPS), followed by an immunoprecipitation of Ku70 from protein lysates (2 mg) using 5 μg mouse anti-p53 antibody (DO-7, BD Biosciences, Heidelberg, Germany), and immunoblotting using antibodies against acetylated p53, p53 and Bax. Panels b and c, D458 cells were stably transduced with a vector expressing shRNA against p53 (Brummelkamp et al., 2002) or control shRNA using pRETRO-SUPER vector as previously described (Vogler et al., 2008) and stable clones were selected. In b, cells were treated for 24 h with 0.6 μM MS-275 before 0.1 μg/ml Doxorubicin (Doxo) or 0.3 μg/ml VP16 was added for 9 h, and Bax conformational change was determined by immunoprecipitation (IP), Bax expression in lysates served as controls. In c, cells were treated for 24 h with 0.6 μM MS-275 before 0.1 μg/ml Doxorubicin (Doxo) or 0.3 μg/ml VP16 were added for 24 h. Apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei and percentage of specific apoptosis is shown. In panels a and b, a representative of three independent experiments is shown. In panel c, mean+s.e.m. of three independent experiments performed in triplicate are shown; *P<0.05; #P<0.001.

To investigate the clinical relevance of our combination approach, we extended our studies to primary medulloblastoma cells that were freshly isolated from surgical specimens of children with medulloblastoma. Importantly, pre-exposure to MS-275-sensitized primary medulloblastoma cells to Doxorubicin- or VP16-induced apoptosis in a dose-dependent manner (Figure 4a). Finally, we evaluated the anti-tumor activity of the combination treatment in vivo using the chorioallantoic membrane model, an established in vivo model of cancer including medulloblastoma (Kuefer et al., 2004; Vogler et al., 2008; Hacker et al., 2009). Intriguingly, the combination of MS-275 and Doxorubicin was significantly more effective to suppress medulloblastoma growth in vivo compared with treatment with either Doxorubicin or MS-275 alone (Figure 4b).

Figure 4
figure4

MS-275 cooperates with chemotherapeutic drugs to induce apoptosis in primary medulloblastoma cells and to suppress medulloblastoma growth in vivo. (a) Primary medulloblastoma cells were isolated and cultured as described previously (Hacker et al., 2009), and treated with 1 μM MS-275 for 24 h before the indicated concentrations of Doxorubicin (Doxo) or VP16 were added. Apoptosis was determined by FACS analysis of DNA fragmentation of propidium iodide-stained nuclei and percentage of specific apoptosis is shown. Data from one experiment performed in duplicate is shown. White bars: no pretreatment, black bars: pretreatment with MS-275. (b) Approximately 2 × 106 D458 cells were seeded with matrigel on the chorioallantoic membrane of chicken embryos as described previously (Vogler et al., 2008) and tumors were allowed to form for 24 h. For next three days, tumors were treated with 1.4 μg MS-275 and/or 1.9 μg Doxorubicin and four days after seeding, tumors were sampled with the surrounding chorioallantoic membrane. Tumor growth was analyzed using hematoxylin and eosin-stained paraffin sections of the chorioallantoic membrane. Representative pictures of hematoxylin and eosin-stained sections (Bars=1000 μm) and tumor area are shown (AU, arbitrary units). Error bars indicate mean+s.e.m. of eight samples per group; *P<0.05; #P<0.001. Similar results were obtained in three independent experiments.

In the present study, we provide first evidence that HDACI, for example, MS-275, valproic acid or SAHA, cooperate with chemotherapeutics to induce apoptosis in medulloblastoma cells by triggering Bax conformational change, leading to mitochondrial outer membrane permeabilization, caspase activation and apoptosis (Supplementary Figure 5). Treatment with MS-275 alone causes Ku70 acetylation and Bax activation, consistent with previous reports that acetylation of Ku70 disrupts its interaction with Bax (Cohen et al., 2004; Subramanian et al., 2005), whereas monotherapy with Doxorubicin triggers p53 acetylation and accumulation. Both compounds cooperate to trigger the binding of p53 to Bax and p53-dependent Bax activation, resulting in loss of mitochondrial membrane potential, cytochrome c release and caspase-dependent apoptotic cell death. The critical role of the mitochondrial pathway in MS-275- and chemotherapy-induced apoptosis is demonstrated by overexpression of the anti-apoptotic protein Bcl-2, which inhibits the cooperative interaction of MS-275 and chemotherapeutics. In addition, reduced DNA repair activity of acetylated Ku70 (Chen et al., 2007) may contribute to sensitization to DNA-damaging drugs, as we found increased Doxorubicin-induced DNA strand breaks in the presence of MS-275. MS-275 and Doxorubicin likely trigger Bax activation independent of the transcriptional activity of p53, as Bax was rapidly activated without changes in Bax levels. There is mounting evidence that acetylated p53 can enhance apoptosis independently of transcription, for example, by disrupting the binding of Mcl-1 to Bak (Sykes et al., 2009), or by destroying the Ku70-Bax complex (Yamaguchi et al., 2009). As TP53 mutations have recently been shown to correlate with resistance to conventional therapies in medulloblastoma (Tabori et al., 2010), it will be interesting to explore whether the combination of HDACI and chemotherapy can restore chemosensitivity in TP53-mutated cases.

The clinical relevance is underscored by our data obtained in primary patients-derived medulloblastoma cells and in an in vivo model, which similarly show the cooperative interaction of MS-275 and chemotherapeutics. As HDACI are currently under evaluation in early clinical trials (Hess-Stumpp et al., 2007; Lee et al., 2008), it appears feasible that this combination approach of HDACI for chemosensitization of medulloblastoma can be translated into a clinical application.

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Acknowledgements

We thank R Agami (The Netherlands Cancer Institute, Amsterdam, The Netherlands) for kindly providing pRETRO-SUPER vector, CA Schmitt (Berlin, Germany) for providing mouse Bcl-2 vector and A Dittrich for expert technical assistance. This work has been partially supported by grants from the Deutsche Forschungsgemeinschaft, Else Kröner-Fresenius-Stiftung, IAP6/18 and the European Community (ApopTrain, APO-SYS) (to SF).

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Correspondence to S Fulda.

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Häcker, S., Karl, S., Mader, I. et al. Histone deacetylase inhibitors prime medulloblastoma cells for chemotherapy-induced apoptosis by enhancing p53-dependent Bax activation. Oncogene 30, 2275–2281 (2011) doi:10.1038/onc.2010.599

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Keywords

  • HDACI
  • apoptosis
  • medulloblastoma
  • mitochondria
  • chemotherapy

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