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Original article
Integrated paired-end enhancer profiling and whole-genome sequencing reveals recurrent CCNE1 and IGF2 enhancer hijacking in primary gastric adenocarcinoma
  1. Wen Fong Ooi1,
  2. Amrita M Nargund2,
  3. Kevin Junliang Lim2,3,
  4. Shenli Zhang2,
  5. Manjie Xing1,
  6. Amit Mandoli2,
  7. Jing Quan Lim4,
  8. Shamaine Wei Ting Ho5,
  9. Yu Guo6,
  10. Xiaosai Yao1,
  11. Suling Joyce Lin1,
  12. Tannistha Nandi1,
  13. Chang Xu2,
  14. Xuewen Ong2,
  15. Minghui Lee2,
  16. Angie Lay-Keng Tan2,
  17. Yue Ning Lam1,
  18. Jing Xian Teo7,
  19. Atsushi Kaneda8,
  20. Kevin P White9,10,
  21. Weng Khong Lim2,7,
  22. Steven G Rozen2,3,7,
  23. Bin Tean Teh2,5,7,11,
  24. Shang Li2,
  25. Anders J Skanderup6,
  26. Patrick Tan1,2,5,7
  1. 1Cancer Therapeutics and Stratified Oncology, Genome Institute of Singapore, Singapore, Singapore
  2. 2Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore, Singapore
  3. 3Centre for Computational Biology, Duke-NUS Medical School, Singapore, Singapore
  4. 4Lymphoma Genomic Translational Laboratory, National Cancer Centre Singapore, Singapore, Singapore
  5. 5Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore
  6. 6Computational and Systems Biology, Genome Institute of Singapore, Singapore, Singapore
  7. 7SingHealth/Duke-NUS Institute of Precision Medicine, National Heart Centre, Singapore, Singapore
  8. 8Department of Molecular Oncology, Chiba University, Chiba, Japan
  9. 9Institute for Genomics and Systems Biology, University of Chicago and Argonne National Laboratory, Chicago, Illinois, USA
  10. 10Tempus Labs, Chicago, Illinois, USA
  11. 11Laboratory of Cancer Epigenome, National Cancer Centre Singapore, Singapore, Singapore
  1. Correspondence to Dr Patrick Tan, Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore 169857, Singapore; gmstanp{at}duke-nus.edu.sg

Abstract

Objective Genomic structural variations (SVs) causing rewiring of cis-regulatory elements remain largely unexplored in gastric cancer (GC). To identify SVs affecting enhancer elements in GC (enhancer-based SVs), we integrated epigenomic enhancer profiles revealed by paired-end H3K27ac ChIP-sequencing from primary GCs with tumour whole-genome sequencing (WGS) data (PeNChIP-seq/WGS).

Design We applied PeNChIP-seq to 11 primary GCs and matched normal tissues combined with WGS profiles of >200 GCs. Epigenome profiles were analysed alongside matched RNA-seq data to identify tumour-associated enhancer-based SVs with altered cancer transcription. Functional validation of candidate enhancer-based SVs was performed using CRISPR/Cas9 genome editing, chromosome conformation capture assays (4C-seq, Capture-C) and Hi-C analysis of primary GCs.

Results PeNChIP-seq/WGS revealed ~150 enhancer-based SVs in GC. The majority (63%) of SVs linked to target gene deregulation were associated with increased tumour expression. Enhancer-based SVs targeting CCNE1, a key driver of therapy resistance, occurred in 8% of patients frequently juxtaposing diverse distal enhancers to CCNE1 proximal regions. CCNE1-rearranged GCs were associated with high CCNE1 expression, disrupted CCNE1 topologically associating domain (TAD) boundaries, and novel TAD interactions in CCNE1-rearranged primary tumours. We also observed IGF2 enhancer-based SVs, previously noted in colorectal cancer, highlighting a common non-coding genetic driver alteration in gastric and colorectal malignancies.

Conclusion Integrated paired-end NanoChIP-seq and WGS of gastric tumours reveals tumour-associated regulatory SV in regions associated with both simple and complex genomic rearrangements. Genomic rearrangements may thus exploit enhancer-hijacking as a common mechanism to drive oncogene expression in GC.

  • gastric cancer
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Footnotes

  • WFO and AMN contributed equally.

  • Contributors PT led the study and was involved in its conception, design, data collection and assembly, and manuscript writing. WFO, AMN and KJL were involved in study conception, design, data analysis and manuscript writing. JQL, YG, SJL, TN, JXT and WKL were involved in data analysis. AMN, SZ, MX, AM, SWTH, XY, CX, XO and YNL were involved in performing experiments. ML and AL-KT provided genomic profiling (sequencing and microarray) and data preprocessing services. AK, KPW, SR, BTT, SL, AJS provided facilities, reagents and intellectual input. WFO and AMN contributed equally to this article. All authors were involved in proof-reading and gave final approval of the manuscript.

  • Funding This work was supported by the National Research Foundation Singapore under its Translational and Clinical Research (TCR) Flagship Programme and administered by the Singapore Ministry of Health’s National Medical Research Council (TCR/009-NUHS/2013) and grant NMRC/STaR/0026/2015. Other sources of support include A*STAR A*ccelerate GAP fund (ETPL/15-R15 GAP-0021), core funding from Duke-NUS Medical School, and the Cancer Science Institute of Singapore, NUS, under the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centres of Excellence initiative.

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Ethics approval Primary patient samples were obtained from the SingHealth tissue repository with Institutional Review Board approval and signed patient informed consent.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement Data are available in a public, open access repository. All data relevant to the study are included in the article or uploaded as supplementary information.

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