Abstract
Simple SummaryWe 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. 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.
Highlights
DIRAS-1 and DIRAS-2 are members of the distinct subfamily of small Ras GTPases and were first described by Kontani et al [1]
Downregulation of DIRAS-1 expression was reported to be partly due to aberrant promoter methylation in esophageal squamous cell carcinoma, renal cell carcinoma, and colorectal cancer [4,5,7] and increased DIRAS-1 expression was shown in one renal cell carcinoma cell line after treatment with a histone deacetylase inhibitor [8]
When looking at phosphorylation of p53 tion of p53, we found a strong increase after lomustine treatme, we found a strong increase after lomustine treatment in DIRAS-1 and in DIRAS-1 and in DIRAS-2 transfected cells compared to 6B)
Summary
DIRAS-1 and DIRAS-2 are members of the distinct subfamily of small Ras GTPases and were first described by Kontani et al [1]. DIRAS-1, in contrast to common oncogenic small GTPases like Ras or Rho family members [3], has been reported as a potential tumor suppressor in human glioblastoma [2], colorectal cancer [4], renal cell carcinoma [5], and ovarian cancer [6], and reduced expression of DIRAS-1 predicts poor prognosis in esophageal squamous cell carcinoma [7]. SmgGDS showed a stronger binding affinity for DIRAS-1 than for other small GTPases and DIRAS-1, prevented binding of SmgGDS to pro-oncogenic. Since DIRAS-1 expression is frequently reduced or lost in malignant tissues, more SmgGDS is available to interact with and activate pro-oncogenic GTPases [3]. Downregulation of DIRAS-1 expression was reported to be partly due to aberrant promoter methylation in esophageal squamous cell carcinoma, renal cell carcinoma, and colorectal cancer [4,5,7] and increased DIRAS-1 expression was shown in one renal cell carcinoma cell line after treatment with a histone deacetylase inhibitor [8]
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