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

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.

M6A modification regulates tumor suppressor DIRAS1 expression in cervical cancer cells

“…The same trend was also found in other types of solid tumor tissues. Similar downregulation of DIRAS1 expression was found in renal cell carcinoma, 6 ovarian cancer, 7 colorectal cancer, 8 glioma 9 and esophageal squamous cell carcinoma 10 tissues. The low-level expression of the DIRAS1 protein in tumor tissues may be the main reason for its lack of nuclear localization.…”

“…Current mutation analysis did not identify inactivating mutations on the DIRAS1 coding region. 9 Heterozygous deletion of the DIRAS1 gene is present in esophageal squamous cell carcinoma tissues. 10 The present study was not equipped to detect mutations in DIRAS1 occurring in cervical cancer tissues and mainly focused on epigenetic regulation.…”

“…In addition, experimental data have shown that 5-aza-dC treatment of renal cell carcinoma cell lines (ACHN, 786-O, and Caki-1) (10 µM for 96 h), 6 colorectal cancer cell lines (2 µM for 96 h, with fluid changes every 24 h), 8 glioblastoma cell lines (U251MG and Hs683) (1 µM for 72 h) 9 and esophageal squamous cell carcinoma cell lines (KYSE30 and KYSE510) 10 result in a significant elevation of DIRAS1 mRNA levels. Treatment of both glioblastoma cell lines 9 and renal cell carcinoma cell line (UOK146) 19 with histone deacetylase inhibitors also result in a significant increase in DIRAS1 mRNA levels. These findings seem to suggest that DIRAS1 expression is regulated by DNA methylation and histone modifications.…”

“… 5 The DIRAS1 gene, located on chromosome 19p13.3, consists of 2 exons and encodes a protein of 198 amino acids. Unlike common oncogenic small GTPases (e.g., Ras or Rho family members), DIRAS1 has been reported as a potential tumor suppressor in human renal cell carcinoma, 6 ovarian cancer, 7 colorectal cancer, 8 gliomas, 9 and esophageal squamous cell carcinoma. 10 However, its expression pattern and role in cervical cancer remain unknown.…”

The function of histone methylation and acetylation regulators in GBM pathophysiology

“…Trichostatin A, a class I and II HDAC inhibitor, has shown a similar ability to radiosensitize U87 and U373 cells in vitro and triggers similar transcriptional shifts away from TCGA proneural and classical expressional signatures in patient-derived cell lines in vitro ( 76 , 77 ). Trichostatin A treatment of U251 and Hs683 cell lines in vitro has also been shown to upregulate mRNA expression of DIRAS-1, a small Ras GTPase and potential tumor suppressor in various solid tumors ( 84 ). Panobinostat, a nonselective HDAC inhibitor that has been explored as a potential therapeutic agent in a variety of cancers, has been shown to impact GBM cells in a manner similar to other HDAC inhibitors.…”