Epigenomic Profiling Discovers Trans-Lineage SOX2 Partnerships Driving Tumor Heterogeneity in Lung Squamous Cell Carcinoma
Total Page：16
File Type：pdf, Size：1020Kb
Published OnlineFirst September 24, 2019; DOI: 10.1158/0008-5472.CAN-19-2132 Cancer Genome and Epigenome Research Epigenomic Profiling Discovers Trans-lineage SOX2 Partnerships Driving Tumor Heterogeneity in Lung Squamous Cell Carcinoma Takashi Sato1,2, Seungyeul Yoo3, Ranran Kong1,2,4, Abhilasha Sinha1,2, Prashanth Chandramani-Shivalingappa1, Ayushi Patel1,2, Maya Fridrikh1,2, Osamu Nagano5, Takashi Masuko6, Mary Beth Beasley7, Charles A. Powell1, Jun Zhu2,3,8, and Hideo Watanabe1,2,3 Abstract Molecular characterization of lung squamous Transcriptional programs cell carcinoma (LUSC), one of the major subtypes Super-enhancers Sox2 of lung cancer, has not sufficiently improved its p63 nonstratified treatment strategies over decades. Sox2 Sox2 p63 Accumulating evidence suggests that lineage- specific transcriptional regulators control differen- Genes involved in epithelial/ tiation states during cancer evolution and underlie p63 epidermal development Classical lineage their distinct biological behaviors. In this study, by Differential dependency Trans-lineage investigating the super-enhancer landscape of LUSC, we identified a previously undescribed Sox2 Brn2 fi "neural" subtype de ned by Sox2 and a neural Sox2 lineage factor Brn2, as well as the classical LUSC Lung squamous cell carcinoma Sox2 Brn2 fi subtype de ned by Sox2 and its classical squamous Neural lineage Brn2 Genes involved in neurogenesis partner p63. Robust protein–protein interaction and genomic cooccupancy of Sox2 and Brn2, in Transcriptional cooperation of Sox2 with p63 or Brn2 determines cancer lineage state in lung squamous cell carcinoma. place for p63 in the classical LUSC, indicated their © 2019 American Association for Cancer Research transcriptional cooperation imparting this unique lineage state in the "neural" LUSC. Forced expres- sion of p63 downregulated Brn2 in the "neural" LUSC cells and invoked the classical LUSC lineage with more squamous/ epithelial features, which were accompanied by increased activities of ErbB/Akt and MAPK–ERK pathways, suggesting differential dependency. Collectively, our data demonstrate heterogeneous cell lineage states of LUSC featured by Sox2 cooperation with Brn2 or p63, for which distinct therapeutic approaches may be warranted. Significance: Epigenomic profiling reveals a novel subtype of lung squamous cell carcinoma with neural differentiation. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/24/6084/F1.large.jpg. Introduction which provide a considerable survival benefit for patients with lung cancer, the overall 5-year survival remains less than adequate Lung cancer is the leading cause of cancer-related death world- at 19% (2). Lung squamous cell carcinoma (LUSC) is the second wide (1). Despite recent progress in diagnosis and treatment most common histologic subtype of lung cancer and is strongly including molecular targeted therapeutics and immunotherapy, associated with cigarette smoking. Although achievements in 1Division of Pulmonary, Critical Care and Sleep Medicine, Department of Med- Note: Supplementary data for this article are available at Cancer Research icine, Icahn School of Medicine at Mount Sinai, New York, New York. 2Tisch Online (http://cancerres.aacrjournals.org/). Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York. 3Department of Genetics and Genomic Sciences, Icahn School of Medicine at Corresponding Author: Hideo Watanabe, Icahn School of Medicine at Mount Mount Sinai, New York, New York. 4Department of Thoracic Surgery, The Second Sinai, One Gustave L. Levy Place, Box 1232, New York, NY 10029. Phone: 212-241- Affiliated Hospital of Medical School, Xi'an Jiaotong University, Xi'an, Shaanxi, 9242; Fax: 212-876-5519; E-mail: [email protected] China. 5Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan. 6Cell Biology Cancer Res 2019;79:1–17 Laboratory, School of Pharmacy, Kindai University, Higashiosaka, Osaka, Japan. 7Department of Pathology and Laboratory Medicine, Icahn School of Medicine at doi: 10.1158/0008-5472.CAN-19-2132 Mount Sinai, New York, New York. 8Sema4, a Mount Sinai venture, Stamford, Connecticut. Ó2019 American Association for Cancer Research. www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst September 24, 2019; DOI: 10.1158/0008-5472.CAN-19-2132 Sato et al. tobacco control in developed countries have contributed to a and that forced expression of p63 leads to classical squamous cell decline in the mortality rate of lung cancer, LUSC still remains a differentiation and suppression of Brn2 in this novel LUSC major cause of cancer-related death worldwide and shows even subtype. higher incidence than lung adenocarcinoma in several coun- tries (3, 4). Comprehensive genomic characterization of LUSC conducted by The Cancer Genome Atlas (TCGA) project revealed Materials and Methods its heterogeneous features with complex genomic alterations (5). Cell lines Unfortunately, these findings have not resulted in the successful Lung cancer cell lines (HCC95, KNS62, HCC2814, LK2, development of clinically approved targeted drugs for LUSC. NCI-H520, SQ-1, EBC-1, HCC2279, NCI-H2887, HCC2450, Compared with patients with lung adenocarcinoma, where mul- Calu-1, HARA, LC-1/SQSF) were maintained in RPMI1640 tiple genome-based targeted therapeutics have been approved, (Gibco) with 10% FBS and 1% penicillin–streptomycin (Gibco). patients with LUSC have very limited therapeutic options. More HEK293T cells were maintained in DMEM with 10% FBS and 1% recently, immune checkpoint inhibitors have emerged as key penicillin–streptomycin (Gibco). Original sources for these cell therapeutic options for solid tumors including LUSC (6, 7); lines are shown in Supplementary Table S1. HCC95, HCC2814, however, the response rate for unselected LUSC population HCC2279, NCI-H2887, and HCC2450 cells were a gift from remains only approximately 20% and initially responded tumors Dr. John Minna (University of Texas Southwestern Medical eventually progress in most cases. Therefore, innovative strategies Center, Dallas, TX). Cells were authenticated by copy number to better characterize and classify LUSC for a better patient variation analyses and regularly tested for Mycoplasma using the stratification for current and future therapeutic options are des- MycoAlert Detection Kit (Lonza). perately needed. We previously identified SOX2 as the most commonly ampli- Chromatin immunoprecipitation sequencing fied oncogene in LUSC (8). We proposed SOX2 as a lineage- For H3K27ac, Sox2, and p63 chromatin immunoprecipitation survival oncogene in squamous cell cancers for its essential role (ChIP), cells were performed as described previously with mod- during the development in the specification of the squamous cell ifications (10). Cells were crosslinked with 1% formaldehyde in lineages by opposing the role of Nkx2-1 in the dividing foregut PBS for 10 minutes at room temperature, washed in 5 mg/mL BSA and its essentiality for LUSC cell survival (9). In a following study, in PBS and then in just cold PBS, resuspended in lysis buffer we identified another squamous lineage factor, p63, as an impor- [50 mmol/L Tris-HCl pH 8.1, 10 mmol/L EDTA, 1% SDS, 1 Â tant cooperative partner of Sox2 in LUSC (10). SOX2 amplifica- protease inhibitor cocktail (Thermo Fisher Scientific)] and son- tion on chromosome 3q in LUSC often extends to its telomeric icated with the Covaris M220 sonicator or the Diagenode side to include the locus of TP63, which encodes p63. While focal Bioruptor sonicator to obtain chromatin fragment lengths of amplification and/or overexpression of both SOX2 and TP63 100-to-1,000 bp judged by Bioanalyzer DNA High Sensitivity Kit genes is found in only 7% of LUSC tumors, broader copy number (Agilent). Fragmented chromatin was diluted in immunoprecip- gains on 3q telomeric ends are observed in the vast majority of itation (IP) buffer (20 mmol/L Tris-HCl pH 8.1, 150 mmol/L LUSCs (5). Studies on expression profiles classified LUSCs into NaCl, 2 mmol/L EDTA, 1% Triton X-100) and incubated over- four expression subtypes (primitive, classical, secretory, and bas- night at 4C with protein G magnetic beads (Dynabeads, Thermo al), suggesting the heterogeneity of transcriptional programs Fisher Scientific) that had been preincubated with anti-H3K27ac within LUSCs (5, 11, 12). On the basis of this classification, (Abcam, ab4729) or anti-Sox2 (R&D Systems, AF2018) or anti- coamplification of SOX2 and TP63 is enriched in the classical p63 (Santa Cruz Biotechnology, sc-8431) antibodies. subtype of LUSC, whereas TP63 expression is relatively low in the For Brn2 ChIP, cells were crosslinked with 2 mmol/L disucci- primitive and secretory subtypes. However, it remains largely nimidyl glutarate in PBS with 1 mmol/L MgCl2 for 45 minutes at unknown what mechanisms are involved in controlling transcrip- room temperature and 1% formaldehyde in PBS for 11 minutes at tional programs in the heterogeneous group of LUSCs. room temperature, washed in 5 mg/mL BSA in PBS and then A series of recent genome-wide histone modification analyses in just cold PBS, re-suspended in nuclear extraction buffer have demonstrated the presence of large clusters of putative (10 mmol/L Tris-HCl pH 7.5, 10 mmol/L NaCl, 3 mmol/L MgCl2, enhancers in close genomic proximity, coined super- 0.1 % IGEPAL CA-630, 1 Â protease inhibitor cocktail), and enhancers (13,