Rothhammer-Hampl Tanja, Liesenberg Franziska, Hansen Natalie, Hoja Sabine, Delic Sabit, Reifenberger Guido, Riemenschneider Markus J
Department of Neuropathology, Regensburg University Hospital, 93053 Regensburg, Germany.
Institute of Neuropathology, Medical Faculty, University Hospital Düsseldorf, Heinrich Heine University, 40225 Düsseldorf, Germany.
Cancers (Basel). 2021 Oct 12;13(20):5113. doi: 10.3390/cancers13205113.
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 and coding regions. Analysis of the and promoter methylation status showed significantly higher methylation in -mutant astrocytic and -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 -wild-type primary gliomas, however, we did not observe significantly elevated and 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 and less promoter euchromatinization for in -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.
我们之前报道过，由于启动子高甲基化和染色体臂1p缺失，DIRAS - 3在少突胶质细胞瘤中经常失活。DIRAS - 3失活与更好的总生存期相关。因此，我们现在研究了其家族成员DIRAS - 1和DIRAS - 2的调控及功能。我们发现，与非肿瘤性脑组织（NNB）相比，在65%的分析胶质瘤样本中DIRAS - 1强烈下调，在100%的样本中DIRAS - 2下调。此外，在从TCGA数据库获得的胶质瘤数据中检测到DIRAS - 1和 - 2显著下调。突变分析未在 和 编码区发现任何失活突变。 和 启动子甲基化状态分析显示，与NNB相比， - 突变型星形细胞瘤、 - 突变型和1p/19q共缺失少突胶质细胞瘤中的甲基化显著更高。用5 - 氮杂胞苷处理U251MG和Hs683胶质母细胞瘤细胞系导致DIRAS - 1和 - 2显著重新表达。然而，对于 - 野生型原发性胶质瘤，我们未观察到 和 启动子甲基化水平显著升高，但仍检测到DIRAS家族两个成员的强烈下调。进一步分析表明，DIRAS - 1和 - 2的表达也受组蛋白修饰调控。与对照相比，我们观察到在 - 野生型胶质母细胞瘤中 向启动子异染色质化转变， 启动子常染色质化减少。用组蛋白去乙酰化酶抑制剂处理这两种胶质母细胞瘤细胞系导致DIRAS - 1和 - 2显著重新表达。在功能上，胶质母细胞瘤细胞中DIRAS - 1和 - 2的过表达转化为对洛莫司汀治疗的显著更高敏感性。DNA损伤标志物分析表明，DIRAS - 1和 - 2可能在p53依赖的烷化化疗反应中起作用。
