Abstract LB-289: Hypermethylation enforces promoter switch in glioblastoma multiforme tumors
Authors: Jin Guangxu , Liu Liang , Zhou Xiaobo 
Published: July 2016

Complete understanding why a patient is sensitive or resistant to a treatment needs comprehensive whole-genome characterization of the patient's tumor to address underlying molecular mechanism. The developed consortiums, such as The Cancer Genome Atlas (TCGA) and the Encyclopedia of DNA Elements (ENCODE), provide such genomic data at different dimensions (e.g., DNA methylation, RNA gene expression, DNA copy number, and DNA mutation). However, there lacks a patient-specific approach to integration of the multi-dimensional genomic data, which is helpful to provide mechanistic insights for primary or recurrent tumor of an individual patient. Here, we developed a new patient-specific analysis for the genomic data integration and its application to Glioblastoma Multiforme (GBM) tumors discovered a mechanism of epigenetic regulation on drug response of Temozolomide (TMZ).

We applied the new patient-specific analysis to 13 recurrent tumors of GBM patients in the TCGA and identified a top gene (#1), CYP19A1, with alternative promoters and intragenic hypermethylation. We detected the gene isoforms of CYP19A1 in the different brain tissues. The whole gene (Exons 1-10) is seldomly expressed in both of the normal brain tissues and low-grade gliomas tumors whereas new gene isoforms are specifically expressed in the primary GBM tumors and recurrent GBM tumors. Among 175 primary GBM tumors, 166 (95%) expressed the isoforms of CYP19A1. In the primary and recurrent GBM tumors, there are 78% (129/166) and 83% (10/12) expressing the isoforms starting from the TSS near to Exon 4 (Ex-4).

CYP19A1 has (i) hypermethylated CpGs at the regions of original promoter and the introns upstream of Ex-4, and (ii) alternative exon usage: Exons 4-10 are highly used whereas exons 1-3 are seldomly used. We hypothesize that the hypermethylated CpGs located in the region covering from the promoter (near to TSS) to the intron close to Ex-4 may prevent the start of the TF complex binding to the original promoter and enforce the switch to alternative promoter proximal to Ex-4. The Pol-II ChIP-Seq data of U87 cell line from the ENCODE indicate that the Pol-II alternatively binds to the upstream of Ex-4 because the CpGs close to the original promoters, I.f and I.3, are hypermethylated. We further treated the U87 cell line with 5-AZA to induce demethylation of CpGs. Strikingly, we observed that Ex-2 is highly expressed after demethylation. The results confirmed that the region near to Ex-4 is an alternative promoter for expression of the isoforms of CYP19A1 and the hypermethylation of CpGs controls the usage of Exons 1-10.

We tested if the gene expression and new transcript isoforms of CYP19A1 are related to patients’ survival. We checked the gene expression of CYP19A1 in 161 primary tumors of GBM from TCGA, who received radiotherapy, surgery, and chemotherapy of TMZ. The primary tumors of GBM patients with lower CYP19A1 tend to benefit more and survive longer. The statistical significance for the survival analysis based on Cox regression is P &lt; 0.007. We analyzed that the survival statuses of these patients with different gene isoforms. The new isoform starting from Exon 4 to Exon 8 benefits patients’ survival more (628±453), compared to those terminated proximal to Exon 9 (317±152) and those proximal to Exon 10 (367±251). The statistical significance is P = 0.008, Wilcoxon rank sum test.

Citation Format: Guangxu Jin, Liang Liu, Xiaobo Zhou. Hypermethylation enforces promoter switch in glioblastoma multiforme tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-289.