Histone deacetylase inhibitors reverse resistance to methylating agents : mechanisms in malignant melanoma and glioblastoma cells
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Abstract
The aggressive characteristics of metastatic melanoma and glioblastoma necessitate DNA-damaging therapies, in particular with methylating agents such as Temozolomide (TMZ) or Dacarbazine (DTIC). However, the efficacy of therapy is limited by resistance factors such as DNA repair, DNA damage signaling and impaired apoptosis execution. Histone deacetylase (HDAC) inhibitors have proven antineoplastic as they are associated with cell cycle and apoptosis regulation as well as with DNA repair. Therefore, this study investigated whether HDAC inhibition enhances the efficacy of DNA damaging therapies in malignant melanoma and glioblastoma cell lines as well as its influence on DNA damage-resistance pathways. First, it was shown that malignant melanoma cells express high levels of class I HDACs HDAC1, HDAC2 and HDAC3 in comparison to non-cancerous cells. Pre-treatment inhibition of these HDACs by valproic acid or Entinostat enhanced the apoptotic response of melanoma cells to the methylating agent TMZ, the chloroethylating agent Fotemustine and to ionizing radiation without sensitizing primary melanocytes. This work proposes that HDAC inhibition sensitizes melanoma cells to DNA damaging insults by downregulating proteins involved in homologous recombination (HR) repair of replication-dependently formed DNA double-strand breaks (DSBs), which leads to an increase in apoptosis. This is substantiated by the following observations obtained in this study: Inhibition of class I HDACs reduced the expression of the HR factors RAD51 and FANCD2 on transcript and protein level. The resulting functional impairment of HR decreased the repair of DSBs by this pathway and increased the sensitivity to PARP1 inhibition, which relies on HR derogation. Consequently, HDAC inhibition increased replication dependent DSB formation following TMZ exposure. The protective role of HR in melanoma TMZ toxicity was confirmed by RAD51 knockdown experiments. RAD51 knockdown cells were more susceptible to TMZ-induced apoptosis than the corresponding control lines.
In glioblastoma cells, half of the tested cell lines exhibited increased TMZ-induced cell death in combination with the HDAC inhibitor Entinostat. The findings support a possible mechanism whereby HDAC inhibition disrupts DNA damage signaling in glioblastoma cells and alters the cell cycle regulation in response to TMZ, leading to apoptosis. TMZ treatment activated the DNA damage signaling kinases ATM/ATR, whereas in combination with Entinostat, ATM/ATR activation was abolished. Similarly, TMZ-induced downstream signaling to checkpoint kinases (CHK1/CHK2) as well as the following p53 stabilization and expression of the cell cycle regulator p21Cip1/Waf1 were abrogated in the presence of Entinostat. Further, HDAC inhibition protected from endoreplication upon TMZ-treatment and instead induced apoptosis in glioblastoma cells. In conclusion, this work demonstrates that class I HDAC inhibitors are DNA damage sensitizers in cancer cells. This HDAC inhibitor-mediated sensitization is due to the impairment of RAD51 and FANCD2 dependent HR repair in melanoma cells, or due to the premature shutdown of DNA damage signaling in glioblastoma cells. As inhibitors like valproic acid and Entinostat are well-tolerated in patients, the combination of adjuvant HDAC inhibition with methylating agents is a reasonable approach for improving therapeutic efficacy. Since acting on general DNA damage resistance pathways, HDAC inhibition might further provide a way for combination with genotoxic agents other than the common O6-alkylating, thereby circumventing the major resistance factor O6-methylguanine-DNA methyltransferase.