Frequent Epigenetic Inactivation of DIRAS-1 and DIRAS-2 Contributes to Chemo-Resistance in Gliomas

SIMPLE SUMMARY: We investigated the genes DIRAS-1 and DIRAS-2 in terms of their regulation and functional relevance in brain tumors (gliomas). We found that in a majority of patients the expression of both genes is strongly downregulated on the mRNA level when comparing tumors with healthy brain tissue. We could show that epigenetic mechanisms account for this downregulation. Both promoter methylation and histone modifications are accountable. We performed experiments in tumor tissues (direct bisulfite sequencing and chromatin-immunoprecipitation) and we treated glioblastoma cell lines in a way to overcome epigenetic inactivation of both genes. When genes were re-expressed, the tumor cells turned out more sensitive to alkylating chemotherapeutic agents such as Lomustin. Changes in intracellular pathways related to p53-mediated DNA damage response may explain for this observation. ABSTRACT: We previously reported that DIRAS-3 is frequently inactivated in oligodendrogliomas due to promoter hypermethylation and loss of the chromosomal arm 1p. DIRAS-3 inactivation was associated with better overall survival. Consequently, we now investigated regulation and function of its family members DIRAS-1 and DIRAS-2. We found that DIRAS-1 was strongly downregulated in 65% and DIRAS-2 in 100% of analyzed glioma samples compared to non-neoplastic brain tissue (NNB). Moreover, a significant down-regulation of DIRAS-1 and -2 was detected in glioma data obtained from the TCGA database. Mutational analyses did not reveal any inactivating mutations in the DIRAS-1 and -2 coding regions. Analysis of the DIRAS-1 and -2 promoter methylation status showed significantly higher methylation in IDH-mutant astrocytic and IDH-mutant and 1p/19q-codeleted oligodendroglial tumors compared to NNB. Treatment of U251MG and Hs683 glioblastoma cells lines with 5-azacytidine led to significant re-expression of DIRAS-1 and -2. For IDH-wild-type primary gliomas, however, we did not observe significantly elevated DIRAS-1 and -2 promoter methylation levels, but still detected strong downregulation of both DIRAS family members. Additional analyses revealed that DIRAS-1 and -2 expression was also regulated by histone modifications. We observed a shift towards promoter heterochromatinization for DIRAS-1 and less promoter euchromatinization for DIRAS-2 in IDH-wild-type glioblastomas compared to controls. Treatment of the two glioblastoma cell lines with a histone deacetylase inhibitor led to significant re-expression of DIRAS-1 and -2. Functionally, overexpression of DIRAS-1 and -2 in glioblastoma cells translated into significantly higher sensitivity to lomustine treatment. Analyses of DNA damage markers revealed that DIRAS-1 and -2 may play a role in p53-dependent response to alkylating chemotherapy.