Brother Of The Regulator Of Imprinted Sites Expression Research Articles

MP52-03 DISRUPTION OF CHD8-CTCF CHROMATIN COMPLEX IN PROSTATE CANCER ALTERS DNA METHYLATION PATTERNS

INTRODUCTION AND OBJECTIVES: Epigenetic features drive cancer progression, but little is known regarding the targeting of DNA methylation. CCCTC-binding factor (CTCF), Chromodomain helicase DNA-binding protein 8 (CHD8), and Brother of the regulator of imprinted sites (BORIS) are interrelated chromatin proteins involved in the regulation of epigenetic marks. BORIS and CTCF are paralogues which compete for CTCF binding sites, exhibit opposing functions, and require CHD8. We evaluated the role that this complex plays in protecting methylated regions, and investigated their expression in prostate cancer (PCa). METHODS: Doxocycline-inducible CTCF targeting shRNAs were stably introduced into PCa lines PPC1, LNCaP and HPV16 immortalized prostate epithelial cells. Proliferation and cell cycle was assessed using flow cytometry. DNA methylation was tested at CTCF binding sites using quantitative pyrosequencing. To evaluate gene expression in PCa, tissue microarrays consisting of benign (N1⁄496), localized (N1⁄4146), metastatic PCa (N1⁄444) and HGPIN (N1⁄450) were chromogenically stained for CTCF, CHD8, and BORIS. A novel, automated quantitative imaging system VECTRA was used to evaluate epithelial staining in both the nucleus and cytoplasm. RESULTS: Knockdown of CTCF was performed to disrupt this chromatin complex. It had no effect on cell proliferation or apoptosis. However, CTCF loss leads to an increase in methylation across CTCF binding sites within the Cav1 gene and the Igf2-H19 imprinted region. Quantitative protein expression in clinical samples demonstrated a marked downregulation of CHD8 expression in HGPIN (P<0.01), localized (P < 0.001), and metastatic PCa (P < 0.0001) compared to benign. CHD8 expression was downregulated in 52% of primary cancer samples indicating a common alteration. Decreased nuclear CHD8 expression was associated with positive margins (P 1⁄4 0.047), extracapsular extension (P<0.01), and presence of metastases (P1⁄40.025). BORIS expression, as was the BORIS/CTCF ratio, was increased in localized PCa (P1⁄40.03 and 0.03) compared to benign. Heterogeneity of staining was assessed using the Simpson’s coefficient which revealed CTCF expression to be more heterogeneous in cancerous tissue (both P < 0.001), especially with higher Gleason grade (p<0.01). CONCLUSIONS: In the first detailed analysis in cancer, a marked loss of CHD8 expression and increased BORIS/CTCF ratio indicate disruption of this chromatin complex. Loss of this complex results in DNA hypermethylation potentially explaining the targeting of DNA methylation changes in cancer.

Abstract P6-05-26: BORIS: A potential novel breast cancer biomarker

Abstract Background: Breast cancer is the most common cancer in the UK excluding non-melanoma skin cancers. Worldwide 1.38 million women were diagnosed with breast cancer and caused 458,503 deaths (13.7% of cancer deaths in women) in 2008. Presently there are no early predictors for detection of breast cancer. There is a pressing need to identify “biomarkers” that complement or replace the currently available diagnostic tools. BORIS (Brother of the Regulator of Imprinted Sites) is a protein belonging to the cancer testis antigen gene family. It is present in early spermatocytes and is absent in women. It is aberrantly expressed in various malignancies. Our discovery of the appearance of BORIS in leukocytes and in cancer tissue of breast cancer patients offers a promise. As the patients who received neoadjuvant chemotherapy showed lower BORIS expression, BORIS may be an indicator of patient's response to treatment. We reasoned that the appearance of BORIS in the “high risk” women may predict the presence of the condition and hence BORIS may be considered as a potential early breast cancer biomarker. We also assessed the potential of BORIS as a marker of treatment monitoring in metastatic breast cancer (MBC). Methods: 1) Familial breast cancer study The aim is to recruit 300 women between 40 and 50 years of age who have high familial risk of breast cancer. Blood samples (10 ml) are collected from each participant every year for 5 consecutive years at the time of their annual mammogram. BORIS levels in leukocytes were assessed by Immunocytochemistry (measured as immunoreactivity score (IRS)) and Western Blot Analysis and were correlated with their respective annual mammograms. If a woman shows an increase in BORIS level and her corresponding mammogram is negative, she is offered MRI scan to confirm the presence or absence of the disease. 2) MBC study We recruited 64 patients who are either on chemotherapy or endocrine therapy. BORIS expression in the leukocytes of MBC patients was assessed in pre- and post- treatment samples by Immunocytochemistry and Western Blot Analysis. Results: 1) In the first year, 129 women have been recruited. Protein and RNA samples were obtained from the blood leukocytes and BORIS expression assessed by different methods. The BORIS expression measured as immunoreactivity score (IRS) was found to be higher in the family history group (IRS 0.36 ± 0.004) than in healthy donors (IRS 0.17±0.068). Positive BORIS expression was observed in 8% of participants on Western blot analysis. In one participant, BORIS was detected in leukocytes by both methods. This woman had a positive mammogram and had been histologically diagnosed of Invasive Ductal Carcinoma of breast. Clinically, mammograms of all other participants were negative. 2) BORIS was not found in the leukocytes of patients with advanced breast cancer. Conclusions: Presence of BORIS expression in the leukocytes of one woman correlated with the mammographic and histological evidence of breast cancer. Likewise, the absence of BORIS in remaining participants correlated with the negative results of their mammograms. These findings support the hypothesis that BORIS may be an early biomarker of breast cancer. Its absence in MBC patients rules out its use as biomarker for treatment monitoring. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-26.

Transcription Factor BORIS (Brother of the Regulator of Imprinted Sites) Directly Induces Expression of a Cancer-Testis Antigen, TSP50, through Regulated Binding of BORIS to the Promoter

Cancer-testis antigens (CTAs) are normally expressed in testis but are aberrantly expressed in a variety of cancers with varying frequency. More than 100 proteins have been identified as CTA including testes-specific protease 50 (TSP50) and the testis-specific paralogue of CCCTC-binding factor, BORIS (brother of the regulator of imprinted sites). Because many CTAs are considered as excellent targets for tumor immunotherapy, understanding the regulatory mechanisms governing their expression is important. In this study we demonstrate that BORIS is directly responsible for the transcriptional activation of TSP50. We found two BORIS binding sites in the TSP50 promoter that are highly conserved between mouse and human. Mutations of the binding sites resulted in loss of BORIS binding and the ability of BORIS to activate the promoter. However, although expression of BORIS was essential, it was not sufficient for high expression of TSP50 in cancer cells. Further studies showed that binding of BORIS to the target sites was methylation-independent but was diminished by nucleosomal occupancy consistent with the findings that high expression of TSP50 was associated with increased DNase I sensitivity and high BORIS occupancy of the promoter. These findings indicate that BORIS-induced expression of TSP50 is governed by accessibility and binding of BORIS to the promoter. To our knowledge this is the first report of regulated expression of one CTA by another to be validated in a physiological context.

Abstract 5001: BORIS and MageA expression correlate positively in melanoma

Abstract Cancer Testis Antigens (CTAs) are proteins that are normally expressed only in the male germ cells. These proteins are not expressed in other normal somatic tissues but are aberrantly upregulated in a variety of cancers like lung and melanoma. The expression of CTA genes is known to be regulated by promoter methylation. Using an integrative epigenetic screening approach, we have previously shown that CTAs are upregulated in non-small cell lung cancer and head and neck squamous cell carcinoma by promoter hypomethylation. Although it is now well accepted that CTAs are regulated by promoter methylation, the regulatory mechanisms of these genes remains unclear. Transcription factor BORIS (Brother Of the Regulator of Imprinted Sites) or CTCFL has been implicated in the regulation of two CTAs, mageA1 and NY-ESO-1. In the present study, we analyzed the expression of BORIS in a cohort of 16 melanoma tissues using quantitative reverse-transcription PCR. BORIS expression was detected in ∼70% of the tissues analyzed. Taking into account the evidence that BORIS regulates the expression of at least two CTAs, we also analyzed the expression of four CTAs, mageA1, mageA2, mageA3 and mageA4, in these tissues to assess the correlation between BORIS and CTA gene expression. We found that expression of mageA1, A2 and A3 correlated significantly with that of BORIS. We also analyzed two publicly available melanoma gene expression datasets and found that BORIS expression correlated with all four mageA genes analyzed. In our cohort as well as in the published gene expression datasets, the expression of the mageA genes tightly correlate with each other. Analysis of the methylation status of the mageA genes in our cohort showed that the methylation levels of mageA1, A2 and A3 genes also correlated with each other significantly. These observations support the phenomenon of coordinated CTA expression in solid tumors. We also found inverse correlation between the methylation and expression levels of these genes in some of the tissues analyzed. These results indicate that coordinated transcriptional regulation of the mageA genes may occur via promoter methylation dependent as well as independent mechanisms. Given the role of BORIS in the derepression of mageA1 gene via promoter demethylation, we also evaluated the correlation between BORIS expression and CTA promoter demethylation. We found that BORIS expression and mageA gene demethylation correlated in a subset of the tissues analyzed. The association between expression of BORIS and the mageA genes supports a role for BORIS as an effector in the upregulation of these genes. Also, the coordinated expression of the mageA genes suggests a common transcriptional mechanism for their regulation and our study presents evidence that implicates BORIS as a common regulator of the CTAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5001. doi:10.1158/1538-7445.AM2011-5001

Selective apoptosis of breast cancer cells by siRNA targeting of BORIS

Brother of the regulator of imprinted sites (BORIS) is an epigenetically acting transcription factor which represses the tumor inhibitor functions of the tumor suppressor protein CTCF. BORIS expression has not been documented in adult females, making it an exciting molecular target for drug development in breast cancer. Previously, we demonstrated that vaccination of mice with zing-finger (ZF)-deleted non-functional BORIS results in regression of breast cancer and generation of potent anti-tumor immune responses. RNAi induction can be used as an alternative approach for selective tumor cell killing. Short interfering RNA (siRNA) molecules targeting BORIS were generated and their efficacy was tested in MDA-MB-231 breast cancer and non-malignant epithelial cell lines. Treatment with BORIS-specific siRNA, but not control siRNA led to a concentration-dependent reduction in BORIS expression and proportional apoptotic death of the cancer but not control cells. To our knowledge this is first report demonstrating a critical role of BORIS in maintaining tumor cell viability.