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Novel Transcriptional Targets of the SRY-HMG Box Transcription Factor SOX4 Link Its Expression to the Development of Small Cell Lung Cancer

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Novel Transcriptional Targets of the SRY-HMG Box Transcription Factor SOX4 Link Its Expression to the Development of Small Cell Lung Cancer Published OnlineFirst November 14, 2011; DOI: 10.1158/0008-5472.CAN-11-3506 Cancer Molecular and Cellular Pathobiology Research Novel Transcriptional Targets of the SRY-HMG Box Transcription Factor SOX4 Link Its Expression to the Development of Small Cell Lung Cancer Sandra D. Castillo1, Ander Matheu2, Niccolo Mariani1, Julian Carretero3, Fernando Lopez-Rios4, Robin Lovell-Badge2, and Montse Sanchez-Cespedes1 Abstract The HMG box transcription factor SOX4 involved in neuronal development is amplified and overexpressed in a subset of lung cancers, suggesting that it may be a driver oncogene. In this study, we sought to develop this hypothesis including by defining targets of SOX4 that may mediate its involvement in lung cancer. Ablating SOX4 expression in SOX4-amplified lung cancer cells revealed a gene expression signature that included genes involved in neuronal development such as PCDHB, MYB, RBP1, and TEAD2. Direct recruitment of SOX4 to gene promoters was associated with their upregulation upon ectopic overexpression of SOX4. We confirmed upregulation of the SOX4 expression signature in a panel of primary lung tumors, validating their specific response by a comparison using embryonic fibroblasts from Sox4-deficient mice. Interestingly, we found that small cell lung cancer (SCLC), a subtype of lung cancer with neuroendocrine characteristics, was generally characterized by high levels of SOX2, SOX4, and SOX11 along with the SOX4-specific gene expression signature identified. Taken together, our findings identify a functional role for SOX genes in SCLC, particularly for SOX4 and several novel targets defined in this study. Cancer Res; 72(1); 1–11. Ó2011 AACR. Introduction on chromosome 6p22, containing the SOX4 gene, which was expressed at very high levels and was the best candidate for an As with other types of cancer, lung cancer is undergoing a oncogene in the amplicon (2). therapeutic revolution characterized by the identification of The SOX4 gene belongs to the SOX family, which is divided novel driver oncogenes and the generation of drugs that inhibit into 8 groups, A to H, according to protein identity (3). Sox4, their activity in a very specific manner. The success of erloti- Sox11, and Sox12 form the Sox-C group, sharing a high degree of nib/gefitinib in epidermal growth factor receptor (EGFR)- identity in the high-mobility group domain, and in a group- mutant tumors and crizotinib in tumors carrying a transloca- specific transactivation domain (4). Sox4 is predominantly tion of the ALK oncogene are some of the current paradigms expressed during embryonic development in the heart, central (1). Because few tumors carry alterations of these genes, effort nervous system, lung and thymus (5–7). SOX4 protein levels are is required to identify additional targetable oncogenes. We increased in several types of carcinoma (8–11), and knocking previously carried out a wide-ranging DNA copy number down SOX4 induces apoptosis and growth suppression in analysis of lung cancer cell lines and identified an amplicon cancer cells (12–14). Providing further evidence of the onco- genic potential of SOX4, we reported that its overexpression in NIH3T3 cells increases the number of foci induced by the 1 Authors' Affiliations: Genes and Cancer Group, Cancer Epigenetics and mutant RHOA-Q63L (2). In addition, several independent Biology Program- (PEBC), Bellvitge Biomedical Research Institute-IDI- BELL, Barcelona, Spain; 2Division of Stem Cell Biology and Developmental studies have shown that Sox4 (also known as ecotropic viral Genetics, MRC National Institute for Medical Research, London, United integration site 16, Evi16) is a frequent target of retroviral Kingdom; 3Department of Physiology, Faculty of Medicine and Odontol- ogy, University of Valencia; and 4Hospital Universitario Madrid-Sanchi- insertional mutagenesis, leading to neoplastic transformation narro, Laboratorio Dianas Terapeuticas, Madrid, Spain in murine hematopoietic cells (15–18). In the particular case of Note: Supplementary data for this article are available at Cancer Research lung cancer, we previously reported the presence of a high level Online (http://cancerres.aacrjournals.org/). of SOX4 amplification in a subset of lung primary tumors and cancer cell lines (2). The relevance of SOX4 to lung cancer Array data deposited in the Gene Expression Omnibus (GEO) under accession number GSE31612. development has also been observed by others. A meta-analysis examining the transcriptional profiles of human tumors found Corresponding Author: Montse Sanchez-Cespedes, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute- SOX4 to be one of 64 genes uniquely upregulated in cancer IDIBELL 08908, Hospitalet de Llobregat, Barcelona 08907, Spain. Phone: thereby making it part of a general gene expression signature of 34-932-607132; Fax: 34-932-607219; E-mail: [email protected] cancer (19). Lung cancer was among the tumors with the doi: 10.1158/0008-5472.CAN-11-3506 greatest levels of SOX4 expression. In addition, Sox4 was among Ó2011 American Association for Cancer Research. the set of genes overexpressed in the lungs of c-myc transgenic www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 5, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst November 14, 2011; DOI: 10.1158/0008-5472.CAN-11-3506 Castillo et al. mice, and c-myc was also found to be one of the transcription Gene expression microarrays and real-time quantitative factors with overrepresented Sox4-binding sites among the set PCRs of overexpressed genes (20). The mRNA was extracted using conventional methods, and 1 SOX4 is involved in neural development and the main- mg of it was amplified from each sample and used for gene tenance of some stem cell types (21, 22). Recent studies expression microarray analysis. Universal Human Reference have reported SOX4 to be a direct TGF-beta target that RNA (P/N 740000; Stratagene), was used as reference for activates SOX2 transcription while retaining the stem cell hybridization and analysis. For labeling we used the commer- properties of glioma-initiating cells (21). In addition, in cial Two-Color Microarray-Based Gene Expression Analysis Kit normal hair follicles, Sox4 is expressed in the developing (version 5.5). MMLV-RT retrotranscription of sample from a T7 hair germ (23). promoter primer was followed by a T7 RNA polymerase- In spite of this, and although SOX4 was one of the first catalyzed in vitro transcription reaction in the presence of members of the SOX family to be isolated and characterized, either Cy3-CTP or Cy5-CTP fluorophores. Cy3 labeling was including the demonstration that it has separable DNA-bind- used for the reference sample. Hybridization was carried out on ing and transactivation domains, our present knowledge of the the Whole Human Genome Microarray (4 Â 44 K), scanned genes controlled by SOX4 activity is scarce. This paper reports with a G2505B DNA microarray scanner and quantified using on the role of SOX4 in human lung carcinogenesis, focusing Agilent Feature Extraction Software (version 9.5; Agilent). especially on identifying novel targets of SOX4 transcriptional Fluorescence intensity from each array element was subtracted activity. from the local background and data normalized as previously described (24). We further selected transcripts that fulfilled the Materials and Methods following criteria: (i) repressed at least 2 times in the H522Tr- shSOX4-1 at 48 and 96 hours after induction of shSOX4 relative Cancer cell lines and primary tumors to the level at 0 hour and (ii) no changes in gene expression Cancer cell lines were obtained from the American Type between the parental H522 and the H522Tr-shSOX4-1. The Culture Collection and grown under recommended condi- mRNA and genomic DNA were measured by real-time quan- tions.DNAandRNAfromadditionallungcancercelllines titative PCR. DNase-treated RNA was reverse-transcribed. The used for real-time quantitative reverse transcriptase (RT) cDNA and genomic DNA were amplified using an ABI Prism PCR were kindly provided by Luis M. Montuenga and Ruben 7900 Sequence detector (Applied Biosystems), and levels of Pio of the Centro de Investigacion Medica Aplicada (CIMA), genes were measured by SYBR green real-time PCR. Reactions University of Navarra, Spain, and Jun Yokota, National were carried out in triplicate. As controls we used the human Cancer Center Research Institute, Tokyo, Japan. Fresh frozen GAPDH, B-ACTIN, and TATA box–binding protein (TBP)to lung primary tumors were provided by the CNIO Tumour correct for inter-individual/tumor variations. The primer Bank Network, CNIO, Spain, and were selected as previously sequences used are included in Supplementary Table S2. described (24). Antibodies, Western blot analysis, and immunostaining Expression plasmids and reporters For Western blot analysis, cells were scraped from the dishes We stably transfected the H522 cell line with a Tet repressor into the lysis buffer. A total of 25 mg of total protein was (TetR) expression construct, pCMB1b-TrS, kindly provided by separated by SDS-PAGE and blotted with rabbit anti-SOX4 M.V. de Wetering (Hubrecht Institute, Utrecht, The Nether- (A574) 1:5,000 (CS-129-100, Diagenode), mouse anti-SOX2 lands) using hygromycin selection. We examined TetR activity 1:2,500 (R&D Systems); rabbit anti-NSE 1:1,000 (Abcam), or by transfecting the cells with the pcDNA4/TO/Luc construct mouse anti-GAPDH. The secondary goat anti-mouse-IgG:HRP that expressed the firefly luciferase
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