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. 2009 Oct 1;125(7):1613-21.
doi: 10.1002/ijc.24518.

Methylation-associated silencing of SFRP1 with an 8p11-12 amplification inhibits canonical and non-canonical WNT pathways in breast cancers

Zeng-Quan Yang  1 , Gang Liu, Aliccia Bollig-Fischer, Ramsi Haddad, Adi L Tarca, Stephen P Ethier
Affiliations

Methylation-associated silencing of SFRP1 with an 8p11-12 amplification inhibits canonical and non-canonical WNT pathways in breast cancers

Zeng-Quan Yang et al. Int J Cancer. .

Abstract

Recently, we analysed the 8p11-12 genomic region for copy number and gene expression changes in a panel of human breast cancer cell lines and primary specimens. We found that SFRP1 (Secreted frizzled related protein 1) is frequently under expressed even in breast tumours with copy number increases in this genomic region. SFRP1 encodes a WNT signalling antagonist, and plays a role in the development of multiple solid tumour types. In this study, we analysed methylation-associated silencing of the SFRP1 gene in breast cancer cells with the 8p11-12 amplicon, and investigated the tumour suppressor properties of SFRP1 in breast cancer cells. SFRP1 expression was markedly reduced in both the breast cancer cell lines and primary tumour specimens relative to normal primary human mammary epithelial cells even when SFRP1 is amplified. Suppression of SFRP1 expression in breast cancer cells with an SFRP1 gene amplification is associated with SFRP1 promoter methylation. Furthermore, restoration of SFRP1 expression suppressed the growth of breast cancer cells in monolayer, and inhibited anchorage independent growth. We also examined the relationship between the silencing of SFRP1 gene and WNT signalling in breast cancer. Ectopic SFRP1 expression in breast cancer cells suppressed both canonical and non-canonical WNT signalling pathways, and SFRP1 expression was negatively associated with the expression of a subset of WNT responsive genes including RET and MSX2. Thus, down-regulation of SFRP1 can be triggered by epigenetic and/or genetic events and may contribute to the tumourigenesis of human breast cancer through both canonical and non-canonical WNT signalling pathways.

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Figures

Figure 1
Figure 1
(A) Cartoon of the 8p11-12 amplified region in 2 breast cancer cell lines and 3 primary samples. Localization of the 8p11-12 genes including SFRP1 are represented to the right of the chromosome 8 ideogram. The lines at far right represent the amplified region of each sample based on the array-CGH data. (B) Down-regulation of SFRP1 in primary breast cancer specimens. SFRP1 mRNA levels were analyzed by using gene expression data sets from the cancer gene microarray meta-analysis public database, Oncomine. The Y-axis represents normalized expression units. Shaded boxes represent the interquartile range between the 25th and 75th percentile; whiskers represent the 10th to 90th percentile range; horizontal lines represent the median. The P-value was calculated using Student’s t-test. The following analyses were performed: (1) normal breast vs. breast carcinoma, P = 1.1E-8 (Richardon et. al. 31); (2) normal breast vs. breast carcinoma, P = 1.8E-5 (Radvanyi et. al. 32); (3) normal breast vs. breast ductal carcinoma, P = 2.7E-5 (Perou et al. 33); (4) normal breast vs. breast lobular carcinoma, P= 0.001 (Perou et al. 33); (5) benign breast vs. breast carcinoma, P = 0.007 (Sorlie et. al. 34); (6) adjacent normal ductal vs. invasive ductal carcinoma, P = 0.015 (Turashvili et. al. 35); (7) adjacent normal lobular vs. invasive lobular carcinoma, P = 0.04 (Turashvili et. al. 35).
Figure 2
Figure 2
Methylation analysis of SFRP1 in breast cancer cells. Methylation-specific PCR was performed on bisulphite-treated DNA from (A) 10 SUM breast cancer cell lines and (B) 9 primary breast cancer samples. DNA bands in lanes labeled M indicate PCR products amplified with primers recognizing the methylated promoter sequence. DNA bands in lanes labeled U represent amplified products with an unmethylated-specific primer. The immortalized, nontransformed mammary epithelial cell line, MCF10A, served as the positive control for the U reaction. Water was used instead of template in the negative control (DDW). The stars indicate samples that had SFRP1 gene amplification and arrows indicate samples that had methylated DNA molecules. (C) Re-expression of SFRP1 by 5-aza-dC treatment was analyzed by Q-RT-PCR. SUM-44 and SUM-52, and the control cell line MCF10A were treated with 1 μM 5-aza-dC (AZA) or DMSO for 3 days. SFRP1 expression level (fold change) in AZA-treated cells relative to DMSO-treated control cells were measured by Q-RT-PCR and normalized to GAPDH expression. For DMSO-treated control cells, the baseline was set to one.
Figure 3
Figure 3
(A) Expression of SFRP1 mRNA in SUM-44 and SUM-52 cells after being transfected with the control vector or a SFRP1 construct. SUM-44 and SUM-52 cells were transfected with 2 μg/well of the pcDNA-GW-SFRP1 expression vector (pc-SFRP1) or pcDNA-GW-CAT control vector using FuGENE HD transfection reagent and cultured in 400 μg/ml (SUM-44) or 200 μg/ml(SUM-52) G418 for 4 weeks. SFRP1 mRNA expression levels were measured by RT-PCR and normalized to GAPDH expression. PCR product from 25 cycles are shown. (B) Colony formation assays of the SUM-44 and SUM-52 cells transfected with the SFRP1 expression vector. The graphs indicate the number of colonies in the SUM-44 and SUM-52 cells transfected with the pcDNA-GW-SFRP1 expression vector (pc-SFRP1) relative to the number of colonies formed after control vector transfection, which was set to 100%. (C) Representative images of colonies in SUM-52 cells are shown. (D) SUM-44 and SUM-52 cell lines were infected with a control lentivirus (pLenti6-LacZ) and a lentivirus encoding SFRP1 (pL-SFRP1=pLenti6-SFRP1) to generate the cell lines indicated. Exogenous expression of SFRP1 in SUM-44-pL-SFRP1 and SUM-52-pL-SFRP1 cells was detected by western blot analysis with anti-V5 antibody. (E) Ectopic SFRP1 expression inhibited anchorage-independent growth in vitro. Ectopic SFRP1 expression in SUM-44 and SUM-52 breast cancer cells with the pLenti-SFRP1 lentivirus (pL-SFRP1) reduced colony formation in soft agar compared with cells transduced with the control vector virus. The graph indicates the number of colonies formed by exogenous SFRP1 over expressing cells relative to the number of colonies formed by control cells in soft agar, which was set to 100%.
Figure 4
Figure 4
Luciferase reporter gene assays were done to compare the activities of TCF, NFAT and AP1 transcription factors in A) SUM-44-SFRP1 and B) SUM-52-SFRP1, which stably express SFRP1. All of the luciferase reporter constructs were identical, including a minimal promoter, except that each contained tandem repeats of a response element for TCF/LEF, NFAT, or AP1. Resulting luciferase activities (in light units) were normalized to protein per assay and shown as a percentage relative to the corresponding lacz expressing control. Error bars are the standard deviation of each representative assay in triplicate. A similar transfection efficiency and minimal promoter activity for the cell lines were observed by transfection of a GFP expressing vector driven by a minimal promoter (data not shown).
Figure 5
Figure 5
(A) Unsupervised hierarchical clustering of SFRP1 and 4 representative Wnt/β-catenin target genes in 153 breast cancer specimens. Affymetrix-based gene expression profiles of 130 primary breast cancer samples were obtained from published data . Each row represents a probe, and each column represents a sample. Green indicates low expression level of the transcript; black indicates intermediate and red indicates high. (B) Up-regulation of RET and MSX2 in SUM-44 and SUM-52 breast cancer cell lines. The mRNA expression level of RET and MSX2 in SUM-44 and SUM-52 breast cancer cells and control MCF10A cells were measured by Q-RT-PCR and normalized to GAPDH expression. The number of cycles to threshold values for RET and MSX2 in SUM-44 and SUM-52 were normalized to values for GAPDH and then compared to RET and MSX2 expression in MCF10A cells to determine their fold change. (C) Down-regulation of RET and MSX2 in SFRP1 over expressing SUM-44 breast cancer cells. The mRNA expression levels of RET and MSX2 in SFRP1 over expressing SUM-44 cancer cells and control SUM44-LacZ cells were measured by Q-RT-PCR and normalized to GAPDH expression.
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