Aberrant CBFA2T3B gene promoter methylation in breast tumors

Abstract

Background: The CBFA2T3 locus located on the human chromosome region 16q24.3 is frequently deleted in breast tumors. CBFA2T3 gene expression levels are aberrant in breast tumor cell lines and the CBFA2T3B isoform is a potential tumor suppressor gene. In the absence of identified mutations to further support a role for this gene in tumorigenesis, we explored whether the CBFA2T3B promoter region is aberrantly methylated and whether this correlates with expression.

Results: Aberrant hypo and hypermethylation of the CBFA2T3B promoter was detected in breast tumor cell lines and primary breast tumor samples relative to methylation index interquartile ranges in normal breast counterpart and normal whole blood samples. A statistically significant inverse correlation between aberrant CBFA2T3B promoter methylation and gene expression was established.

Conclusion: CBFA2T3B is a potential breast tumor suppressor gene affected by aberrant promoter methylation and gene expression. The methylation levels were quantitated using a second-round real-time methylation-specific PCR assay. The detection of both hypo and hypermethylation is a technicality regarding the methylation methodology.

Figure 1

CBFA2T3 gene structure and promoter methylation patterns. (A) CBFA2T3 encodes two alternative transcripts, CBFA2T3A and CBFA2T3B. CBFA2T3A is encoded by exons 1A and 2–12. CBFA2T3B is encoded by exons 1B-12 splicing out exon 3. Relative exon sizes are shown. The exon 1A start site contains no CpG island. The black box marks the location of a high-density CpG island located five prime to the exon 1B start site. The black arrowheads mark the primers used for real-time RT-PCR. (B) CBFA2T3B contains a CpG island of approximately 160 CpG sites spanning 1-kb of sequence. The single black bars represent CpG sites scaled relative to each other. CAT ELISA promoter constructs and primers used for MSP, real-time MSP and bisulfite sequencing are shown. The asterisk marks the location of the amplicon and internal primers used for second-round real-time MSP. (C) CBFA2T3B promoter methylation patterns were examined in hypermethylated cell lines using sodium bisulfite sequencing. A characteristic sinusoidal pattern of approximately six high to low frequency methylation levels every 40–150 bp was detected. The high-frequency cytosine methylation levels residing within a consensus Sp1 binding site located approximately minus 450 bp from the transcriptional start of exon 1B are shown.

Figure 2

CBFA2T3 gene expression levels, 5-Aza-dC re-expression and promoter activity. (A) CBFA2T3 gene expression levels were assayed using real-time RT-PCR. Several breast tumor cell line and normal tissue sample expression levels are shown. The y-axis represents mRNA mlcls expressed per 10⁴ cells shown on a log scale (mean ± SD, n = 6). Fold changes in expression relative to normal breast expression are shown above each sample. The white diamonds and white squares represent expression levels of the housekeeping genes cyclophilin A (CYPA) and ATPase coupling factor 6 subunit (ATP5A), respectively. CBFA2T3B and CBFA2T3 expressed at endogenously low yet aberrant levels in breast tumor cell lines. Using normal breast as a reference, CBFA2T3B (600 mRNA mlcls per 10⁴ cells) and CBFA2T3 (1,800) were low compared to ATP5A (600,000) and CYPA (1,600,000). CBFA2T3 expression ranged 30,000-fold from 4 to 120,000 mRNA mlcls per 10⁴cells in MDA-MB-231 and BT-483, respectively. In contrast, CYPA and ATP5A expression ranged 2-fold and 20-fold, respectively (100–200 and 15–300 mRNA mlcls per cell). Expression levels were also examined in several primary breast tumor samples for which total RNA was available (see Figure 6). (B) CBFA2T3 re-expression levels were examined in MDA-MB-231 cells using 5-Aza-dC. Fold changes in CBFA2T3B, CBFA2T3 and SYK expression levels are shown upon exposure to 5-Aza-dC relative to control untreated cells. > 100-fold re-expression was detected in CBFA2T3 and CBFA2T3B. 5-Aza-dC had no affect on CBFA2T3A expression (data not shown). > 1,000-fold re-expression was also detected in SYK, a control gene known to be hypermethylated and down-regulated in MDA-MB-231 cells. (C) CBFA2T3B promoter activity was assayed using CAT ELISA. Promoter constructs labeled A to D (see Figure 1B) were inserted upstream of the CAT reporter in pBLCAT3 (Boehringer Mannheim). pBLCAT2 driven by the tyrosine kinase promoter was used as a positive control. 2.0 × 10⁶ 293T cells were transfected in triplicate using Lipofectamine 2000 (Invitrogen) with 1.5 μg of construct and control vector and 0.3 μg of the internal pSVβ-galactosidase control vector (Stratagene). Cells were lysed after 24 h and CAT concentrations determined using ELISA. Of the four constructs labeled A to D, the 1-kb B construct promoted a 30-fold increase in CAT expression (mean ± SD are triplicates, n = 3).

Figure 3

CBFA2T3B promoter methylation levels examined using MSP. The full results of this analysis in breast tumor cell lines, primary breast tumors, normal breast counterparts and normal whole blood samples are summarized in Additional file 2. (A) An example of the characteristic basal methylation levels detected in all samples is shown for normal blood samples examined at region 2. (B) An example of the complex high to low methylation levels is shown for 20 primary breast tumor samples with adjacent normal breast counterparts. The pUC19 DNA/MspI marker concentrations reflect an approximate concentration of 100 to 1 unmethylated to methylated mlcls. The asterisks indicate the samples examined by bisulfite sequencing.

Figure 4

CBFA2T3B promoter methylation patterns examined using bisulfite sequencing. The methylation patterns in breast tumor cell lines, primary breast tumors, normal breast counterparts and normal whole blood samples are shown. The y-axis represents protected 5-methylcytosines scored as percent cytosines methylated per 5–10 clones. The complete methylation maps displaying 160 CpG sites spanning 1-kb of sequence are shown for breast tumor cell lines only. The white bars indicate the Sp1 sites targeted by second-round real-time MSP. Bisulfite sequencing of the CBFA2T3B promoter region in hypermethylated breast tumor samples MDA-MB-231, MDA-MB-468 and 14T revealed a characteristic sinusoidal methylation pattern. This sinusoidal pattern was also detected in samples SK-BR-3, 14N and 17T at levels approximately 1 tenth of the hypermethylated samples. No methylation was detected in the normal blood samples 4B and 5B or the breast tumor samples 3T and MCF-7.

Figure 5

CBFA2T3B promoter methylation levels assayed using second-round real-time MSP. The methylation levels in normal whole blood samples (nwb), normal breast counterparts (nbc), primary breast tumors (pbt) and breast tumor cell lines (btcl) were assayed at the Sp1 site shown in Figure 1C using real-time MSP. The y-axis represents methylation levels plotted as methylation indices [mi = m/(m + u)] on a log scale. Each white circle represents a different sample. The breast tumor cell lines examined are shown in descending order from high to low methylation. The horizontal bars mark the median methylation indices calculated for each group. The asterisks mark the interquartile ranges for normal groups. The median methylation indices and (interquartile ranges) were 0.02 (0.02–0.04) for nwb, 0.01 (0.008–0.03) for nbc, 0.03 (0.009–0.08) for pbt and 0.02 (0.002–0.3) for btcl. The median methylation index variance in each tumor group was statistically significantly different than the normal groups (P = .001); nwb/btcl (P < .0001), nwb/pbt (P = .009), nbc/btcl (P = .01), nbc/pbt (P = .05). The normal group median methylation index variances were not significantly different; nwb/nbc (P = 0.6).

Figure 6

CBFA2T3B promoter methylation levels versus gene expression. The data for 24 breast tumor cell lines and 20 primary breast tumor samples are shown. The y-axis represents methylation levels assayed using real-time MSP and plotted as methylation indices [mi = m/(m + u)] on a log scale. The x-axis represents total expression levels assayed using real-time RT-PCR and plotted as mRNA mlcls per 10⁴ cells on a log scale. Each white circle represents a different primary breast tumor sample and the black circles represent the breast tumor cell lines. The breast tumor cell lines examined are shown in descending order from high to low methylation. A statistically significant inverse correlation was established between promoter hypermethylation (mi = 0.9) and reduced expression (4 mRNA mlcls per 10⁴ cells) versus hypomethylation (0.0001) and elevated expression (120,000) (r² = 0.72; r = -0.9, P < .0001). A power regression (y = cx^b) describes the relationship between methylation and expression.