Genomic and epigenomic EBF1 alterations modulate TERT expression in gastric cancer
Manjie Xing, … , Bin Tean Teh, Patrick Tan
Manjie Xing, … , Bin Tean Teh, Patrick Tan
Published May 4, 2020
Citation Information: J Clin Invest. 2020;130(6):3005-3020. https://doi.org/10.1172/JCI126726.
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Research Article Gastroenterology Oncology

Genomic and epigenomic EBF1 alterations modulate TERT expression in gastric cancer

Abstract

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA–binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.

Authors

Manjie Xing, Wen Fong Ooi, Jing Tan, Aditi Qamra, Po-Hsien Lee, Zhimei Li, Chang Xu, Nisha Padmanabhan, Jing Quan Lim, Yu Amanda Guo, Xiaosai Yao, Mandoli Amit, Ley Moy Ng, Taotao Sheng, Jing Wang, Kie Kyon Huang, Chukwuemeka George Anene-Nzelu, Shamaine Wei Ting Ho, Mohana Ray, Lijia Ma, Gregorio Fazzi, Kevin Junliang Lim, Giovani Claresta Wijaya, Shenli Zhang, Tannistha Nandi, Tingdong Yan, Mei Mei Chang, Kakoli Das, Zul Fazreen Adam Isa, Jeanie Wu, Polly Suk Yean Poon, Yue Ning Lam, Joyce Suling Lin, Su Ting Tay, Ming Hui Lee, Angie Lay Keng Tan, Xuewen Ong, Kevin White, Steven George Rozen, Michael Beer, Roger Sik Yin Foo, Heike Irmgard Grabsch, Anders Jacobsen Skanderup, Shang Li, Bin Tean Teh, Patrick Tan

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Figure 4

EBF1 exhibits dominant-negative somatic mutations in GC.

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EBF1 exhibits dominant-negative somatic mutations in GC. (A) Distributi...
(A) Distribution of EBF1 coding mutations in 14 GC patients with annotated functional domains. Each dot represents 1 patient. (B) Western blotting revealed equal levels of EBF1 protein abundance after overexpression of WT and mutant EBF1 in AGS cells. (C and D) Proliferation assays (C) and monolayer colony formation assays (D) showed cell proliferation capacity after overexpression of WT and mutant EBF1 in GC cells. Error bars indicate SD. (E) 3D structural analysis of EBF1 and missense mutations. The x-ray crystal structure reveals that 2 EBF1 protomers (green and cyan ribbons) are predicted to dimerize and bind to DNA (orange ribbon with blue ladders). Purple sticks and labels indicate the positions of GC-associated missense mutations on 3 functional domains. HLH, helix-loop-helix. (F and G) Alterations of DNA-binding loop flexibility by EBF1 mutations. (F) EBF1 DBDs are represented by green ribbons. Purple sticks indicate the position of 4 missense mutations. The 3 orange beads mark Cα atoms of R63, N172, and P205. Double helix shows the relative orientation of DNA in the complex. (G) Distance distributions of specified Cα atoms obtained from MD simulations of unbound EBF1 DBD. Vertical dashed lines indicate the distances measured from x-ray crystal structures. (H) EMSAs showed that Q196P or R209Q mutated recombinant EBF1 protein did not have DNA binding to probes containing EBF1-binding motifs. (I) Western blots showed similar protein levels of WT EBF1, Q196P-mutant EBF1, and combined WT plus Q196P-mutant EBF1 after overexpression. Proliferation assay (J) and monolayer colony formation assay (K) showed cell proliferation capacity after overexpression. Error bars indicate SD. (L) Gene expression levels (RNA-Seq) of AGS cells after retroviral infection with either empty vector (black), WT EBF1 (blue), Q196P-mutant EBF1 (red), or combined WT plus Q196P-mutant EBF1 (purple). Expression levels of EBF1 and TERT are shown.