Taijun Hana1,2,3, Akitake Mukasa4, Masashi Nomura1, Genta Nagae2, Shogo Yamamoto2, Kenji Tatsuno2, Hiroki Ueda2,5, Shiro Fukuda2, Takayoshi Umeda2, Shota Tanaka1, Takahide Nejo1,6, Yosuke Kitagawa1, Erika Yamazawa1,2, Satoshi Takahashi1, Tsukasa Koike1, Yoshihiro Kushihara1, Hirokazu Takami1, Shunsaku Takayanagi1, Hiroyuki Aburatani2, Nobuhito Saito1
1Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan;
2Genome Science & Medicine Laboratory, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan;
3Department of Neurosurgery, Saitama Medical Center, Saitama Medical University, Kawagoe, Japan;
4Department of Neurosurgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan;
5Advanced Data Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan;
6Department of Neurological Surgery, University of California, San Francisco, CA, USA
Correspondence to: Taijun Hana, MD, PhD. Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan; Genome Science & Medicine Laboratory, Research Center for Advanced Science and Technology, The University of Tokyo, Meguro-ku, Tokyo, Japan; Department of Neurosurgery, Saitama Medical Center, Saitama Medical University, Kawagoe, Saitama, Japan. Email: hanat_ns@saitama-med.ac.jp.
Background: Gliomas vary in prognosis with World Health Organization (WHO) grade. Low-grade gliomas can undergo malignant progression (MP), becoming aggressive high-grade tumors, worsening prognosis. This is prevalent in isocitrate dehydrogenase-mutant (IDH-mt) gliomas like astrocytoma and oligodendroglioma, but the mechanism of MP is still not fully understood. High-grade IDH-mt gliomas have been reported to exhibit TET-mediated DNA hydroxymethylation, which is suggested to potentially influence gene expression. We hypothesized that hydroxymethylation in specific regions could be implicated in triggering MP.
Methods: We collected glioma tumor samples over a decade, using WHO 2021 classification to study IDH-mt astrocytoma grade 2 progression to grades 3 or 4, indicating MP. Samples from five patients, demonstrating MP, were analyzed for DNA hydroxymethylation status across more than 850,000 genomic locations using the oxidative bisulfite process and Infinium EPIC methylation array. This was complemented by RNA sequencing for gene expression analysis and its correlation with hydroxymethylation, and motif-enrichment analysis to infer transcription factor involvement in hydroxymethylation-based gene regulation. Additionally, to delve into the fundamental causes of hydroxymethylation, we exposed an IDH-mt glioma cell line to hypoxic conditions and systematically explored the genomic locations where hydroxymethylation occurred.
Results: Our comprehensive analysis identified a significant overlap of hydroxymethylated genomic regions across samples during MP, with a notable enrichment in open sea and intergenic regions (P<0.001). These regions were significantly associated with cancer-related signalling pathways. Integration with RNA sequencing data revealed 91 genes with significant correlations between hydroxymethylation and gene expression, implying roles in cell cycle regulation and antineoplastic functions. Furthermore, motif-enrichment analysis suggested the potential regulatory role of KLF4 in these processes. The cell culture results revealed that a certain similarity exists between the hydroxymethylation patterns observed during MP and those in glioma cells cultured under hypoxic conditions.
Conclusions: This study elucidates the importance of region-specific DNA hydroxymethylation in the MP of IDH-mt astrocytomas, suggesting its potential impact on gene expression relevant to cancer malignancy. Our findings propose a complex interplay between hydroxymethylation and gene regulation, which may offer new insights into the mechanisms driving glioma progression and highlight potential targets for therapeutic intervention.
Keywords: Glioma; hydroxymethylation; isocitrate dehydrogenase (IDH); malignant progression (MP)
