Bone marrow stromal antigen 2 (BST-2) DNA is demethylated in breast tumors and breast cancer cells.

BACKGROUND:Bone marrow stromal antigen 2 (BST-2) is a known anti-viral gene that has been recently identified to be overexpressed in many cancers, including breast cancer. BST-2 is critical for the invasiveness of breast cancer cells and the formation of metastasis in vivo. Although the regulation of BST-2 in immune cells is unraveling, it is unknown how BST-2 expression is regulated in breast cancer. We hypothesized that meta-analyses of BST-2 gene expression and BST-2 DNA methylation profiles would illuminate mechanisms regulating elevated BST-2 expression in breast tumor tissues and cells. MATERIALS AND METHODS:We performed comprehensive meta-analyses of BST-2 gene expression and BST-2 DNA methylation in The Cancer Genome Atlas (TCGA) and various Gene Expression Omnibus (GEO) datasets. BST-2 expression levels and BST-2 DNA methylation status at specific CpG sites on the BST-2 gene were compared for various breast tumor molecular subtypes and breast cancer cell lines. RESULTS:We show that BST-2 gene expression is inversely associated with the methylation status at specific CpG sites in primary breast cancer specimens and breast cancer cell lines. BST-2 demethylation is significantly more prevalent in primary tumors and cancer cells than in normal breast tissues or normal mammary epithelial cells. Demethylation of the BST-2 gene significantly correlates with its mRNA expression. These studies provide the initial evidence that significant differences exist in BST-2 DNA methylation patterns between breast tumors and normal breast tissues, and that BST-2 expression patterns in tumors and cancer cells correlate with hypomethylated BST-2 DNA. CONCLUSION:Our study suggests that the DNA methylation pattern and expression of BST-2 may play a role in disease pathogenesis and could serve as a biomarker for the diagnosis of breast cancer

BST-2 DNA in breast tumors is hypomethylated compared to normal tissues.

<p>(A) BST-2 expression versus methylation plot among all primary tumor (red) and normal (black) breast tissues from BRCA in the TCGA. Beta-value ranges from 0 to 1. Beta-values closer to one depict hypermethylation and closer to 0 depict hypomethylation. (B) Methylation status among different CpG sites in primary tumor (red) and normal (black) breast tissues from invasive breast cancer bearing patients (TCGA). Significance was taken at P<0.05 (*) and P<0.001 (****). Error bars correspond to SEM.</p

The effect of demethylating agents on BST-2 expression.

<p>(A) Meta-analysis of BST-2, Claudin-6 (CLDN6), and GAPDH expression levels in HB2, SK-BR-3, MDA-MB-231 cells (downloaded from GEO dataset GSE28976) and MCF-7 cells (downloaded from GEO dataset GSE36683) following treatment with 5-aza-2’-deoxycytidine (DAC). MCF-7 data was acquired from a different dataset, hence the difference in expression values. (B) BST-2, Claudin-6 (CLDN6), and GAPDH expression levels in HMLE, MCF-7, SK-BR-3, and MDA-MB-231 cells following treatment with DMSO (vehicle) or 1 uM of 5-azacytidine (5-AzaC) for 5 days. (C) BST-2 surface expression from HMLE, MCF-7, SK-BR-3 and MDA-MB-231 cells treated with DMSO (vehicle) or 1 uM of 5-azacytidine (5-AzaC) for 5 days as determined by flow cytometry. Numbers in parenthesis correspond to BST-2 expression presented as a percentage. Significance was taken at P<0.05 (*). Error bars correspond to SEM. n.s = not significant.</p

BST-2 expression inversely correlates with methylation density in neoplastic breast epithelial cell lines.

<p>(A) BST-2 and APOBEC3G (A3G) transcript levels in normal and tumor epithelial cells from patients with breast cancer. (B) BST-2 and A3G transcripts present in normal and tumor stromal cells of patients with breast cancer. Data were downloaded from GEO dataset GSE10797. (C) BST-2 expression levels among two luminal A cell lines (MCF-7 and T47D) and two triple-negative cell lines (MDA-MB-231 and SUM-159). Data were acquired from GEO dataset GSE41313 (bar graph, Log2 units) and GEO dataset GSE45732 (gray line graph, RPKM units). (D) Methylation status among different CpG sites from the breast cancer cell lines whose mRNA is presented in line graph in 5C. Data acquired from GEO dataset GSE49794. (E) BST-2 expression versus methylation plot among the four breast cancer cell lines presented in 5C (line graph) and 5D. Methylation beta-value is an average of probes 3 to 9 ± SEM. Methylation data was from GSE49794 (beta-value) and expression data was from GSE45732, a superseries from the same cells. Significance was taken at P<0.05 (*). n.s = not significant. Error bars correspond to standard error of the mean (SEM).</p

Profile of BST-2 DNA hypomethylation in breast cancer subtypes as ranked by significant difference compared to normal breast tissue.

<p>* Is degree of hyper- or hypo-methylation based on statistical significance relative to normalbreast tissues (unpaired t test with Welch's correction). P value (*), where:</p><p>* is p < 0.02,</p><p>** is p < 0.003,</p><p>*** is p < 0.0005 and</p><p>**** is p < 0.0001.</p><p>Profile of BST-2 DNA hypomethylation in breast cancer subtypes as ranked by significant difference compared to normal breast tissue.</p