Published OnlineFirst May 10, 2011; DOI: 10.1158/1078-0432.CCR-11-0653 Clinical Cancer Human Cancer Biology Research BORIS Binding to the Promoters of Cancer Testis Antigens, MAGEA2, MAGEA3, and MAGEA4, Is Associated with Their Transcriptional Activation in Lung Cancer Sheetal Bhan1, Sandeep S. Negi2, Chunbo Shao1, Chad A. Glazer1, Alice Chuang2, Daria A. Gaykalova1, Wenyue Sun1, David Sidransky1,2, Patrick K. Ha1,3, and Joseph A. Califano1,2,3 Abstract Purpose: Aim of this study was to determine whether BORIS (Brother of the Regulator of Imprinted Sites) is a regulator of MAGEA2, MAGEA3, and MAGEA4 genes in lung cancer. Experimental Design: Changes in expression of MAGEA genes upon BORIS induction/knockdown were studied. Recruitment of BORIS and changes in histone modifications at their promoters upon BORIS induction were analyzed. Luciferase assays were used to study their activation by BORIS. Changes in methylation at these promoters upon BORIS induction were evaluated. Results: Alteration of BORIS expression by induction/knockdown directly correlated with expression of MAGEA genes. BORIS was enriched at their promoters in H1299 cells, which show high expression of these cancer testis antigens (CTA), compared with normal human bronchial epithelial (NHBE) cells which show low expression of the target CTAs. BORIS induction in A549 cells resulted in increased amounts of BORIS and activating histone modifications at their promoters along with a corresponding increase in their expression. Similarly, BORIS binding at these promoters in H1299 correlates with enrichment of activating modifications, whereas absence of BORIS binding in NHBE is associated with enrichment of repressive marks. BORIS induction of MAGEA3 was associated with promoter demethylation, but no methylation changes were noted with activation of MAGEA2 and MAGEA4. Conclusions: These data suggest that BORIS positively regulates these CTAs by binding and inducing a shift to a more open chromatin conformation with promoter demethylation for MAGEA3 or independent of promoter demethylation in case of MAGEA2 and MAGEA4 and may be a key effector involved in their derepression in lung cancer. Clin Cancer Res; 17(13); 4267–76. Ó2011 AACR. Introduction promoter hypomethylation is the cancer testis antigens (CTA), so named because they are expressed only in male Genomes of tumors are frequently characterized by germ cells and are also aberrantly upregulated in a variety of alterations in the DNA methylation patterns (1–3). Hyper- cancers including lung and melanoma (11–14). The fre- and hypomethylation of individual gene promoters (4–10) quency of expression of the CTAs is variable in different as well as global hypomethylation are commonly found in a tumor types and has been found to correlate with tumor variety of tumors. Hypermethylation of tumor suppressor grade. Melanomas, lung cancers, and ovarian cancers have genes leads to their inactivation, whereas demethylation the highest frequency of CTA expression, whereas leukemia, may cause activation of proto-oncogenes. One class of genes lymphomas, renal, colon, and pancreatic cancers have a that is upregulated in many types of human tumors by lower frequency of CTA expression. An expression array study conducted in lung cancer cell lines showed that 30% of the overexpressed genes (6 of 20) were CTAs (5 MAGEA 1 and NY-ESO-1; ref. 15). The results were validated by Authors' Affiliations: Departments of Otolaryngology-Head and Neck Surgery and 2Oncology, Johns Hopkins Medical Institutions; and 3Milton immunohistochemical testing for protein using a tissue J. Dance Head and Neck Center, Greater Baltimore Medical Center, microarray containing 187 non–small cell lung cancer Baltimore, Maryland (NSCLC) clinical samples. Derepression of these genes in Note: Supplementary data for this article are available at Clinical Cancer cultured cells after treatment with DNA methyltransferase Research Online (http://clincancerres.aacrjournals.org/). inhibitors like 5-aza-20-deoxycytidine shows that demethy- Corresponding Author: Joseph A. Califano, Department of Otolaryngol- ogy-Head and Neck Surgery, Johns Hopkins Medical Institutions, 1550 lation is a key factor governing the regulation of these genes Orleans Street, Room 5N.04, Baltimore, MD 21231. Phone: 410-955-6420; (16–19). Using an integrative epigenetic screening Fax: 410-614-1411; E-mail: [email protected] approach, we have previously shown that CTAs are upre- doi: 10.1158/1078-0432.CCR-11-0653 gulated in NSCLC and head and neck squamous cell carci- Ó2011 American Association for Cancer Research. noma (HNSCC) by promoter hypomethylation (18, 19). www.aacrjournals.org 4267 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst May 10, 2011; DOI: 10.1158/1078-0432.CCR-11-0653 Bhan et al. Translational Relevance Materials and Methods Cancer testis genes are potential oncogenes that are Cell lines, plasmids, and transfections activated in a variety of cancers. These genes are known Normal human bronchial epithelial cells (NHBE) were to be coordinately regulated and promoter hypomethy- obtained from Lonza and grown according to man- lation plays a major role in their derepression in cancers. ufacturer’s instructions. Lung cancer cell lines H1299 and Here, we have shown that Brother of the Regulator of A549 were obtained from American Type Cell Culture. The Imprinted Sites (BORIS) is a key common factor lung cancer cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomy- involved in the derepression of these genes in lung cancer cell lines. Cancer testis antigens (CTA) are pro- cin and incubated at 37 C and 5% CO2. Small hairpin RNA mising targets for cancer immunotherapy because they (shRNA) plasmids for BORIS knockdown were purchased are exclusively expressed in cancers and elicit cellular from Origene. H1299 cells were transiently transfected with m and humoral immune responses. If BORIS functions as 4 g shRNA plasmid carrying BORIS-specific shRNA cassette a master activator of these genes, its induction might or the plasmid carrying a noneffective scrambled shRNA help in their upregulation that may in turn help the host cassette using Lipofectamine 2000 (Invitrogen). They were in mounting an enhanced immune response. Also, harvested in QIAzol (Qiagen) for total RNA extraction 48 CTAs have been shown to have growth stimulatory hours posttransfection. For ectopic BORIS expression, properties and the involvement of a common regulatory BORIS expression plasmid (pBIG2i-BORIS) and the control control of CTA expression provides an opportunity for empty vector were used (19, 25). A549 cells were transfected m development of therapeutic agents for CTA expressing with 1 g of the plasmids using FuGENE HD (Roche). cancers. Twenty-four hours post transfection, cells were induced with 0.125 mg/mL doxycycline and were allowed to grow for 48 hours before harvesting for RNA/DNA extraction. For chromatin immunoprecipitation (ChIP) analysis, cells were Although it is now well accepted that CTAs are epigen- grown in 150 cm2 dishes and transfected with 16 mg of the etically controlled, the regulatory mechanisms of these BORIS expression plasmid or the control empty vector. They genes remain unclear. The transcription factor Brother of were then induced with doxycycline as described above and the Regulator of Imprinted Sites (BORIS) has emerged as harvested for ChIP analysis. a plausible candidate for the role of a dominant regulator of these genes. BORIS is a transcription factor that is RNA extraction and quantitative reverse-transcription paralogous to the well-characterized, highly conserved, PCR multivalent 11 Zn finger factor CTCF (20, 21). The Total RNA was extracted from cultured cells using QIAzol combinatorial use of its 11 Zn fingers allows CTCF to and RNeasy mini kit (Qiagen). Cells were harvested in bind to distinct DNA sequences and makes it functionally QIAzol and RNA was extracted using the RNeasy Kit versatile (22, 23). It functions as a transcriptional regu- according to the manufacturer’s instructions. RNA was lator and forms methylation-dependent insulators that reverse transcribed to cDNA using qScript cDNA mix regulate genomic imprinting and X chromosome inacti- (Quanta Biosciences). Real-time PCR was carried out using vation (23). BORIS is the paralogue of CTCF with an the Fast SYBR Green Master Mix on the ABI 7900HT Real- identical 11 Zn finger domain but distinct N- and C- Time PCR Machine (Applied Biosystems). Primers used are termini. It plays a crucial role in spermatogenesis by listed in Supplementary Table S1. regulating the expression of testis-specific form of CST, a gene that is essential for spermatogenesis (24). Thus far, ChIP assay 2 reports have suggested a direct role for BORIS in the Exponentially growing H1299 and NHBE cells were used regulation of the CTA gene expression. Vatolin and col- for ChIP assay. ChIP was carried out using the Magna ChIP leagues have shown that overexpression of BORIS in G Chromatin Immunoprecipitation Kit (Millipore) accord- normal cells leads to MAGEA1 promoter demethylation ing to manufacturer’s instructions. We used 2 different resulting in derepression of MAGEA1 expression (25). BORIS antibodies for this assay: commercially available Also, reciprocal binding of CTCF and BORIS to the NY- antibody from Abcam (Cambridge) and an antibody ESO-1 promoter in lung cancer cells regulates the expres- kindly provided