DNA methylation profiles of HOXB13 in breast cancer cell lines. Bisulfite-converted DNA samples were analyzed by qMSP with HOXB13 methylation-specific primers and reference COL2A1 primers. Copy numbers were determined by absolute standard curves of PCR products. Results are presented as a percentage of Sss I-treated control DNA, % methylated reference. A PMR of >4% classifies a significant DNA methylation event. Columns, mean (n = 3); bars, standard deviation. (A) Levels of HOXB13 methylation in 17 ERα-positive cell lines. (B) Levels of HOXB13 methylation in 21 ERα-negative cell lines. Published Affymetrix U133A gene expression data for breast cancer cell lines analyzed for HOXB13 DNA methylation were obtained from Neve et al. (18). (C) Box plot of HOXB13 expression array data. The 38 cell lines were categorized by HOXB13 methylation status (4% PMR threshold) and centered RMA HOXB13 expression values were plotted. Box represents interquartile range, inset horizontal line represents median, vertical lines represent 95% CIs.

Confirmation of HOXB13 DNA methylation in breast cancer cells. (A) High resolution mapping of DNA methylation at the HOXB13 locus in breast cancer and normal cells. Bisulfite sequence analysis of a 319 bp region located within the promoter and first exon of the HOXB13 locus, encompassing 25 CpG dinucleotides. (A) Methylation in cancer cell lines MCF7^T and T47D, as well as disease-free non-immortalized mammary-derived epithelial cells (normal). Clear circles, non-methylated CpGs; black circles, methylated CpGs; stems, unclassifiable sites and bent arrow, transcription start site. (B) HOXB13 qMSP analysis of MCF7^T cells after 5 days of treatment with 5 μM 5-aza-2′-deoxycytidine (DAC) or vehicle control [dimethyl sulfoxide (DMSO)]. qMSP performed as described in Figure 1. (C) Comparison of HOXB13 expression in MCF7^T cells treated with 5 μM 5-aza-2′-deoxycytidine or vehicle control. Absolute expression levels of HOXB13 were assessed by qRT–PCR and normalized to the reference gene TATA binding protein. Copy numbers were determined by absolute standard curves of cloned PCR products. Columns, mean (n = 3); bars, standard deviation. Results presented relative to MCF7^H cells (data not shown). (D) Effect of HOXB13 hypermethylation on gene expression in 10 breast cancer cell lines. Linear regression analysis was performed on arcsine-transformed qMSP and qRT–PCR values for each cell line (circles). Horizontal and vertical bars represent standard deviations of DNA methylation and expression values, respectively. Solid line, regression line. Dotted lines, 95% CIs of regression line. Solid arrows indicate results for T47D and MCF7^H cells.

Expression of HOXB13 is differentially regulated via estrogen signaling in breast cancer cells. (A–B) Absolute expression levels of HOXB13 were assessed by qRT–PCR in ER-positive MCF7^H and T47D cells as described in Figure 2. Cells were hormone deprived for 72 h followed by treatment with 10 nM E2 or vehicle (dimethyl sulfoxide) for the indicated times. Data presented relative to 0 h time points. Results are the mean ± SD of two independent experiments. Significance of messenger RNA (mRNA) level changes relative to 0 time point were measured by paired t-test, *P < 0.05 and **P < 0.005. (A) Effect of estrogen on HOXB13 expression in MCF7^H cells. (B) Effect of estrogen on HOXB13 expression in T47D cells. (C) Effect of estrogen on HOXB13 protein expression in T47D cells. Hormone-deprived T47D cells were treated with 10 nM E2 or dimethyl sulfoxide for the indicated times. HOXB13 protein levels in whole-cell lysates were assayed by western blotting. Beta-actin protein was used as a loading control. (D) Effect of 4-OHT and estrogen on HOXB13 expression in T47D cells. Hormone-deprived T47D cells were treated with vehicle or 1 μM 4-OHT for 48 h prior to E2 treatment. Cells were then coadministered with 1 μM 4-OHT and 10 nM E2 for the indicated times. Absolute expression levels of HOXB13 were assessed by qRT–PCR as described in Figure 2. Results are presented relative to vehicle (data not shown). Columns, mean (n = 3); bars, standard deviation.

DNA methylation profiles of HOXB13 in primary tumors and adjacent normal tissues. Bisulfite-converted DNA samples were analyzed by qMSP as described in Figure 1. (A) Analysis of HOXB13 methylation in 78 ERα-positive and 42 ERα-negative primary breast cancers. The solid horizontal line represents 4% PMR, the threshold level for classifying a significant DNA methylation event. (B) HOXB13 methylation analysis of paired tumor (T) and tumor adjacent normal (N) tissues from seven breast cancer patients. Columns, mean (n = 3); bars, standard deviation. (C) Relationship between HOXB13 methylation status and DFS curves analyzed in 57 ERα-positive breast cancer patients. Kaplan–Meier curves were plotted based on methylation status (PMR >4%).