A High-Dimensional, Deep-Sequencing Study of Lung Adenocarcinoma in Female Never-Smokers

Deep-Sequencing Study of Lung Adenocarcinoma in Female Never-Smokers. PLoS ONE 8(2): e55596. doi:10.1371/journal.pone.0055596 A High-Dimensional, Deep-Sequencing Study of Lung Adenocarcinoma in Female Never-Smokers Sang Cheol Kim 0 Yeonjoo Jung 0 Jinah Park 0 Sooyoung Cho 0 Chaehwa Seo 0 Jaesang Kim 0 Pora Kim 0 Jehwan Park 0 Jihae Seo 0 Jiwoong Kim 0 Seongjin Park 0 Insu Jang 0 Namshin Kim 0 Jin Ok Yang 0 Byungwook Lee 0 Kyoohyoung Rho 0 Yeonhwa Jung 0 Juhee Keum 0 Jinseon Lee 0 Jungho Han 0 Sangeun Kang 0 Sujin Bae 0 So-Jung Choi 0 Sujin Kim 0 Jong-Eun Lee 0 Wankyu Kim 0 Jhingook Kim 0 Sanghyuk Lee 0 William C S Cho, Queen Elizabeth Hospital, Hong Kong 0 1 Korean Bioinformation Center (KOBIC), Korea Research Institute of Bioscience and Biotechnology , Daejeon , Korea , 2 Ewha Research Center for Systems Biology (ERCSB), Ewha Womans University , Seoul , Korea , 3 Division of Life and Pharmaceutical Sciences and the Center for Cell Signaling and Drug Discovery Research, Ewha Womans University , Seoul , Korea , 4 Samsung Biomedical Research Institute (SBRI) and Cancer Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine , Seoul , Korea , 5 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine , Seoul , Korea , 6 DNA Link Inc., Seoul, Korea, 7 Department of Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine , Seoul , Korea Background: Deep sequencing techniques provide a remarkable opportunity for comprehensive understanding of tumorigenesis at the molecular level. As omics studies become popular, integrative approaches need to be developed to move from a simple cataloguing of mutations and changes in gene expression to dissecting the molecular nature of carcinogenesis at the systemic level and understanding the complex networks that lead to cancer development. Results: Here, we describe a high-throughput, multi-dimensional sequencing study of primary lung adenocarcinoma tumors and adjacent normal tissues of six Korean female never-smoker patients. Our data encompass results from exome-seq, RNAseq, small RNA-seq, and MeDIP-seq. We identified and validated novel genetic aberrations, including 47 somatic mutations and 19 fusion transcripts. One of the fusions involves the c-RET gene, which was recently reported to form fusion genes that may function as drivers of carcinogenesis in lung cancer patients. We also characterized gene expression profiles, which we integrated with genomic aberrations and gene regulations into functional networks. The most prominent gene network module that emerged indicates that disturbances in G2/M transition and mitotic progression are causally linked to tumorigenesis in these patients. Also, results from the analysis strongly suggest that several novel microRNA-target interactions represent key regulatory elements of the gene network. Conclusions: Our study not only provides an overview of the alterations occurring in lung adenocarcinoma at multiple levels from genome to transcriptome and epigenome, but also offers a model for integrative genomics analysis and proposes potential target pathways for the control of lung adenocarcinoma. - Funding: This work was funded by grants from the KRIBB Research Initiative Program (S.L.), National Research Foundation of Korea (grant numbers 20110019747 and 20110002321 to S.L. and grant number 20110014992 to WK), GIST Systems Biology Infrastructure Establishment Grant through ERCSB (SL and JK), and RPGrant 2011 of Ewha Womans University (SC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors acknowledge that two of the authors, Sujin Kim and Jong-Eun Lee belong to a commercial company, DNA Link Inc. The authors duly state here that no competing interests, financial or non-financial, are incurred by this affiliation. Furthermore, this does not alter the authors adherence to the PLOS ONE policies on sharing data and materials. . These authors contributed equally to this work. Recent advances in DNA sequencing technology have revolutionized genomics and biomedical research, especially in the field of cancer research [1]. Various types of mutations as well as large scale chromosomal aberrations are being reported and cataloged, and the rate of data accumulation will likely accelerate for the foreseeable future. This should certainly apply to lung cancer which is currently the second most common cancer and the primary cause of mortality among cancer-related death in the United States [2]. The first complete sequence of a lung adenocarcinoma genome revealed about 50 000 single nucleotide variations in the tumor relative to normal lung [3]. This was followed by the sequencing study of a small-cell lung cancer genome which highlighted the role of tobacco carcinogens such as polycyclic aromatic hydrocarbons in shaping mutational patterns in lung cancers from smokers [4]. Transcriptome analysis of multiple lung adenocarciFigure 1. Circos plot of somatic mutations, copy number variations, transcriptome expression, and structural variations. From inside to out, structural variations (purple and orange), copy number variations (gain in dark red, loss in dark blue, mRNA expression (up in gold, down in olive), differentially expressed microRNAs (up in red, down in green), DNA methylation with sky-blue background (up in dark orange, down in chartreuse), somatic mutations with a gene symbols, and chromosomal cytobands. doi:10.1371/journal.pone.0055596.g001 noma patients using next-generation sequencing (NGS) recently showed the existence of a fusion gene containing the tyrosine kinase domain of the c-RET oncogene in 1%2% of patients; this fusion leads to aberrant activation of RET kinase and is considered to be a new driver mutation of lung adenocarcinoma [5]. This finding was further confirmed through an independent study using a combination of targeted sequencing with an integrated molecular- and histopathology-based screening system [6]. Given that patients with c-RET fusions do not harbor mutations or fusions in EGFR, KRAS or ALK oncogenes, it is likely that c-RET fusion genes represent lung adenocarcinoma drivers and will lead to the definition of a new subclass of lung cancer [5]. Identifying mutations with high probabilities of being drivers, mutations that confer genes with oncogenic activity, is clearly a prototypical and certainly already a productive application of NGS, but the greater challenge is moving beyond the simple cataloging of mutations and establishing means for integrating diverse high-throughput data generated by NGS [7] to understand cancer at the multiple levels of gene networks and signaling pathways [8]. In this report, we describe a high-dimensional, highthroughput sequencing study of primary lung tumors and matched normal tissues isolated from 6 Korean female never-smoker pat (...truncated)