Regulation of temozolomide-induced death of glioblastoma cells: dose responses and role of HIPK2
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Abstract
Glioblastoma multiforme is the most frequent and aggressive form of high-grade malignant glioma. Due to the dismal prognosis faced by patients suffering from this disease, there is a need for identifying new targets that might improve its therapy. O6-methylguanine (O6-MeG) is the most toxic DNA adduct caused by temozolomide (TMZ). The damage induces DNA double-strand breaks (DSBs) that trigger apoptosis. Since TMZ is used for the treatment of glioblastoma and the dual function of p53 regulates both cell death and cell survival mechanisms, the question arises as to the possible threshold to start apoptosis that is evoked by TMZ. To determine whether there is a threshold for the TMZ-induced DNA damage response and exploring the factors regulating the switch in p53 dependent death or survival in glioblastoma cells, the glioblastoma lines LN229, LN308 and LN229MGMT were exposed to different doses of TMZ. p53 protein expression, phosphorylation levels of p53ser15 and p53ser46 were determined by Western blotting. Apoptosis, senescence and autophagy levels were checked after different doses of TMZ. The results showed that pro-survival (p53ser15) and pro-death (p53ser46) factors are induced by O6-MeG in a dose and time dependent manner. We observed that O6-MeG triggers apoptosis at later time points. A survival shoulder was observed in p53 deficient LN308 cells, but not in p53 proficient LN229 cells after TMZ treatment. LN308 also shows more resistance in senescence and autophagy than LN229 after TMZ treatment, while the two cell lines have the same level of DSBs induced by TMZ. These data suggest that the p53 status may influence the threshold of TMZ-induced damage responses in glioblastoma cells, and the phosphorylation level of p53ser46 can be used as an index of O6-MeG triggered apoptosis.
The DNA damage-activated kinase HIPK2 serves as a potent cell death activator through engaging tumor suppressor p53. Whether HIPK2 plays a role in therapy-induced glioblastoma cell death is unknown. In this work, evidence is provided that HIPK2 and its negative regulatory E3 ubiquitin ligase SIAH1 are critical factors controlling TMZ- induced glioblastoma cell death. We show that HIPK2 downregulation (HIPK2kd) significantly reduced the level of TMZ-induced apoptosis. This was not the case in cells expressing the DNA repair enzyme MGMT, which removes TMZ-induced O6-alkylations, suggesting that the primary DNA lesion responsible for triggering HIPK2-mediated death is O6-methylguanine. Following TMZ treatment p53 was phosphorylated at Ser46, and HIPK2kd had an impact exclusively on p53Ser46 while left Ser15 phosphorylation unaffected. TMZ-induced apoptosis in p53 wildtype glioblastoma cells is driven by activation of the death receptor FAS (alias CD95/APO1), which becomes upregulated following TMZ. Accordingly, the expression level of FAS was clearly attenuated upon HIPK2 downregulation, supporting the conclusion that HIPK2 regulates TMZ-induced apoptosis via p53Ser46 driven FAS expression. Using chromatin-immunoprecipitation studies we found that binding of p53 to the FAS promotor is positively regulated by HIPK2. Remarkably, other proapoptotic proteins like PUMA, NOXA, BAX and PTEN were not affected in HIPK2kd. Finally, we show that downregulation of the E3 ubiquitin ligase SIAH1, but not SIAH2, significantly ameliorates TMZ-induced apoptosis, suggesting that the ATM/ATR target SIAH1 plays a key role in TMZ-induced apoptotic death. Since our database analysis revealed that SIAH1 is frequently overexpressed in gliomas, the results bear important implications for TMZ-based malignant brain cancer therapy and suggest the HIPK2-SIAH1 module as a potential target in glioma therapy.