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Carcinogenesis Advance Access originally published online on March 19, 2008
Carcinogenesis 2008 29(5):991-998; doi:10.1093/carcin/bgn076
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Epigenetic inactivation of the secreted frizzled-related protein-5 (SFRP5) gene in human breast cancer is associated with unfavorable prognosis

Jürgen Veeck{dagger}, Cordelia Geisler{dagger}, Erik Noetzel, Sevim Alkaya, Arndt Hartmann1, Ruth Knüchel and Edgar Dahl*

Molecular Oncology Group, Institute of Pathology, University Hospital of the RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany
1 Department of Pathology, University of Erlangen, 91054 Erlangen, Germany

* To whom correspondence should be addressed. Tel: +49 241 8088431; Fax: +49 241 8082439; Email: edahl{at}ukaachen.de


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Disruption of the Wnt pathway is thought to be crucial in the development of human cancer. Pathway inhibitory members of the secreted frizzled-related protein (SFRP) family were found to be downregulated due to epigenetic inactivation in various malignancies. To date, only SFRP1 has been studied in human breast cancer and we questioned whether other SFRP genes may be implicated in the pathogenesis of this disease as well. An initial real-time polymerase chain reaction analysis of SFRP5 expression in normal human tissues (n = 9) revealed weak expression in most tissues, including breast. Malignant mammary cell lines showed further SFRP5 expression loss in five of six cases. Consistently, in matched pairs of primary breast tumor/normal breast tissue, this downregulation (>5-fold) could be confirmed (n = 8/13; 62%). We identified promoter methylation as the predominant mechanism of SFRP5 gene silencing since SFRP5 promoter methylation correlated significantly with loss of SFRP5 expression in cell lines (P = 0.040) and primary tumors (P = 0.003). Moreover, cancerous cell lines re-expressed SFRP5 messenger RNA following treatment with DNA-demethylating drugs. Of 168 primary breast carcinomas, 73% harbored a methylated SFRP5 promoter, whereas 27% were unaffected by epigenetic alteration. Most interestingly, SFRP5 methylation was associated with reduced overall survival (OS) (P = 0.045) and was an independent risk factor affecting OS in a multivariate Cox proportional hazard model (hazard ratio): 4.55; 95% confidence interval: 1.01–20.56; P = 0.049). In conclusion, SFRP5 is a target of epigenetic inactivation in human breast cancer, supporting the hypothesis of its role as tumor suppressor gene. SFRP5 methylation may be a novel DNA-based biomarker potentially useful in clinical breast cancer management.

Abbreviations: CI, confidence interval; DAC, 5-aza-2'-deoxycytidine; DFS, disease-free survival; EGF, epidermal growth factor; FC, fold change; GSK3-β, glycogen synthase kinase 3-β; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; HR, hazard ratio; mRNA, messenger RNA; MSP, methylation-specific polymerase chain reaction; OS, overall survival; PCR, polymerase chain reaction; SFRP, secreted frizzled-related protein; TCF/LEF, T-cell factor/lymphocyte enchancer factor; TSA, trichostatin A


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Secreted frizzled-related proteins (SFRPs) comprise a family of five secreted glycoproteins (SFRP1–5) that have been ascertained as modulators of the canonical Wnt-signaling pathway (1). SFRPs contain an N-terminal cysteine-rich domain homologous to the cysteine-rich domain of the Wnt receptor frizzled, but lack a transmembraneous region as well as a C-terminal domain required for intracellular signal transduction to stabilize β-catenin (2). This enables soluble SFRPs to compete with frizzled for Wnt binding by its cysteine-rich domain. In a resting state, β-catenin is phosphorylated by glycogen synthase kinase 3-β (GSK3-β) and immediately degraded via the ubiquitin proteasome. Binding of Wnt to frizzled activates the pathway by inhibition of GSK3-β, leading to cytoplasmic accumulation of dephosphorylated β-catenin, which is then translocated to the nucleus and interacts with transcription factors of the T-cell factor/lymphocyte enhancer factor (TCF/LEF) family (3). This complex promotes transcription of Wnt target genes like c-myc (4) that, in turn, activates a variety of genes involved in cell cycle regulation such as cyclin D1, cyclin D2, cyclin E and the phosphatase cdc25A (5,6).

Aberrant nuclear and cytoplasmic localization of β-catenin in human breast cancer has been repeatedly observed (7–10), arguing for unscheduled Wnt signaling in this tumor type. Although β-catenin-stabilizing mutations in regulatory genes of the Wnt-signaling cascade (e.g. AXIN1, APC and CTNNB1) are associated with certain types of cancers (11,12), human breast tumors typically do not harbor mutations in any of these genes, yet there is evidence of active Wnt signaling in this disease (13,14). The most evident link so far is that Wnt antagonizing molecules, in particular SFRPs, were found to be downregulated in various human tumor entities (15–18). In line with this, loss of SFRP expression could be attributed to uncontrolled Wnt signaling (15,19), intriguingly leading to autonomous pathway activation in an autocrine manner (20). Altogether this indicates that suppression of Wnt inhibitors rather than mutational hits in downstream acting mediators most probably represents the mechanistic link to active Wnt signaling in human breast cancer.

Epigenetic inactivation of tumor suppressor genes that results in loss of their corresponding proteins is a well-established mechanism capable of driving human carcinogenesis (21,22). Enormous research efforts in this field have uncovered a variety of genes that become transcriptionally silenced during oncogenesis, mainly due to CpG hypermethylation within the gene promoter. SFRP5 promoter methylation was recently detected in several tumor entities, such as colon cancer (15), mesothelioma (16), bladder, lung (23,24), kidney (25), gastric cancer (26) and hepatocellular carcinoma (27). In colon cancer, Suzuki et al. (15) demonstrated that SFRP5 bears a potent capacity to suppress nuclear TCF/LEF activity, followed by downregulation of the Wnt target gene c-myc. Moreover, ectopic SFRP5 overexpression in colon cancer cells conferred susceptibility to apoptotic stimuli and decreased proliferation rates, altogether indicating a tumor-suppressive function of this gene.

To date, studies on differential expression and epigenetic regulation of SFRPs in human breast cancer have been rarely accomplished yet. So far, we and others have comprehensively characterized SFRP1 as an important tumor suppressor gene in breast cancer, which is epigenetically silenced in breast tumorigenesis (18,28–33). However, profound knowledge on the roles of other SFRPs in mammary tumorigenesis is missing yet.

Our aim was to perform an initial study on SFRP5 and to question a possible implication in human breast cancer development. In order to address this question, we assessed SFRP5 messenger RNA (mRNA) expression and SFRP5 promoter methylation in cancerous and non-cancerous breast cell lines as well as primary breast tissues and performed comprehensive statistical analyses to correlate these results with clinicopathological patient parameters including survival intervals. The presented findings strongly argue for further functional investigations in order to characterize SFRP5 as a novel tumor suppressor gene in human breast cancer.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Clinical specimens
Thirteen matched tumor/normal samples of breast cancer specimens and 155 unmatched breast carcinomas were obtained from patients treated by primary surgery for breast cancer at the Departments of Gynecology at the University Hospitals of Aachen, Jena, Regensburg and Düsseldorf, Germany. All patients gave informed consent for retention and analysis of their tissue for research purposes and the Institutional Review Boards of the participating centers approved the study. None of the patients had received neoadjuvant chemotherapy. For 133 patients, follow-up data were available with a median time of 64 months (range 1–174 months). Tumor material was snap frozen in liquid nitrogen immediately after surgery. Hematoxylin–eosin-stained sections were prepared for assessment of the percentage of tumor cells; only samples with >70% tumor cells were selected. Consecutive sections were dissolved in lysis buffer followed by DNA isolation using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA), both according to the protocols supplied by the manufacturers. For patient characteristics see supplementary Table 1 (available at Carcinogenesis Online).

Breast cell lines
The cancerous breast cell lines BT20, Hs578T, MCF7, MDA-MB231, MDA-MB468, SKBR3, T47D and ZR75-1 as well as the non-cancerous breast cell lines HMEC, MCF10A and MCF12A were obtained from the American Type Culture Collection, Rockville, MA and cultured as described previously (31).

Reverse transcription of RNA
Of the total RNA, 1 µg was reverse transcribed using the Reverse Transcription System (Promega, Madison, WI). In order to improve transcription rate, we mixed oligo-dT and pdN(6)-primers 1:2. Complementary DNA quality was checked for each preparation by standard reverse transcription–polymerase chain reaction (PCR) using glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) primers that yield an amplification product of 510 bp (see supplementary Table 2, available at Carcinogenesis Online). For normal human tissue expression screening, a set of commercially available polyA+ RNAs (Clontech, Heidelberg, Germany) was obtained and reverse transcribed using Superscript II RNaseH– and (dT)24 primers (Invitrogen).

Transient transfection of Hs578T cells
Cells were transiently transfected using FuGENE 6 (Roche Diagnostics, Mannheim, Germany) applying a transfection ratio of 1:3. Briefly, 2.5 x 105 cells were seeded in six-well plates and after 24 h transfected with either 2 µg of pcDNA3.1/HisA (empty vector control; Invitrogen) or 2 µg pcDNA3.1/HisA-WNT1 expression vector. After 72 h allowing for protein expression, cells were harvested and subjected to RNA extraction.

Stimulation experiments
In six-well plates, 2.5 x 105 Hs578T cells were seeded and after 24 h exposed to 1 mM NaCl (Merck, Darmstadt, Germany), 1 mM LiCl (Merck), 10 mM NaCl, 10 mM LiCl, 2 ng/ml epidermal growth factor (EGF) (Sigma–Aldrich, Deisenheim, Germany) or 20 ng/ml EGF. After 72 h, cells were harvested and total RNA was extracted.

Quantitative SFRP5 mRNA real-time PCR
Expression levels of SFRP5 were determined using TaqMan predeveloped assay reagent sets for SFRP5 (Applied Biosystems, Foster City, CA). GAPDH TaqMan predeveloped assay reagent set (Applied Biosystems) was used as endogenous control to normalize results. Probes were labeled with 6-carboxyfluorescein. Each probe was quenched by 6-carboxy-tetramethylrhodamine at the 3'-end. TaqMan PCR assays were performed on an ABI® PRISM® 7000 Sequence Detection System (Applied Biosystems). The reaction mix contained 12.5 µl TaqMan Universal PCR Master Mix, 1.25 µl TaqMan Gene Expression Assay Mix, 10.25 µl of sterile water and 1 µl complementary DNA. Real-time PCR conditions applied for both genes were as follows: 50°C for 2 min, 95°C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min. To ensure experiment accuracy, all reactions were performed in triplicate. Gene expression was quantified by the comparative CT method, normalizing CT values to the housekeeping gene GAPDH and calculating relative expression values (34).

Semiquantitative real-time PCR
Semiquantitative SYBR Green I real-time PCR was carried out using a LightCycler device (Roche Diagnostics) as described elsewhere (31). Intron-spanning primer sequences and cycling conditions are listed in supplementary Table 2 (available at Carcinogenesis Online). In order to ensure experiment accuracy, all reactions were performed in triplicate.

Bisulfite modification and methylation-specific polymerase chain reaction
Approximately 1 µg DNA was bisulfite modified using the EZ DNA Methylation Kit (Zymo Research, Orange, CA) according to the manufacturer’s recommendations. Modified DNA was eluted in 20 µl Tris buffer (10 mM). Methylation-specific polymerase chain reaction (MSP) was performed according to Herman et al. (35). In short, 1 µl of modified DNA was amplified using MSP primers (see supplementary Table 2, available at Carcinogenesis Online) that specifically recognized either the unmethylated or methylated SFRP5 gene sequence after bisulfite conversion. Amplification products were visualized on 3% low-range ultra agarose gel (Bio-Rad Laboratories, Hercules, CA) containing ethidium bromide and illuminated under ultraviolet light.

Pharmacological DNA demethylation
Cells were seeded at a density of 3 x 104 cells/cm2 in a six-well plate on day 0. The demethylating agent 5-aza-2'-deoxycytidine (DAC; Sigma–Aldrich) was added to a final concentration of 2 µM in fresh medium on days 1, 2 and 3. Trichostatin A (TSA) was added at 300 nM on day 3. Cells were harvested on day 4 for RNA extraction. Control cells were incubated without the addition of DAC–TSA and fresh medium was also supplied on days 1, 2 and 3.

Statistical evaluations
Statistical analyses were completed using SPSS version 14.0 (SPSS, Chicago, IL). Differences were considered significant when P-values were <0.05. A two-sided, non-parametrical Mann–Whitney U-test was performed to analyze differences in expression levels among distinct groups. Contingency table analysis and two-sided Fisher’s exact tests were used to study the statistical association between clinicopathological factors and promoter methylation status. Survival curves comparing patients with or without any of the factors were calculated using the Kaplan–Meier method with significance evaluated by two-sided log-rank statistics. Overall survival (OS) was measured from the day of surgery until tumor-related death and was censored for patients alive at last contact or in case of death unrelated to the tumor. Disease-free survival (DFS) was measured from surgery until local or distant relapse and censored for patients alive without evidence of relapse at the last follow-up. A multivariate Cox proportional hazard model was employed to assess relative risk of death and to test for independent prognostic relevance of clinical/investigational factors, respectively. Only patients for whom the status of all variables was known were included in the proportional hazard model. The limit for reverse selection procedures was P = 0.2. The proportionality assumption for all variables was assessed with log-negative–log survival distribution functions. Characteristics of all variables are summarized in supplementary Tables 3a– c (available at Carcinogenesis Online).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
SFRP5 mRNA is expressed in a tissue-specific manner
To start our analysis on SFRP5 and a possible implication in human breast cancer, we first asked whether SFRP5 mRNA is expressed in non-malignant breast epithelium. Since only few data on SFRP5 expression in normal human tissues were available, we determined its expression in breast, pancreas, placenta, colon, thyroid gland, lung, kidney, prostate and stomach by quantitative real-time PCR based on commercially available polyA+ RNA. Consistent with a previous study (36), we detected very abundant SFRP5 mRNA expression only in pancreas ({Delta}CT [GAPDH:SFRP5] = 0.9), weak SFRP5 expression in prostate, colon and lung and complete absence in placenta and kidney (Figure 1A). In addition, we detected weak SFRP5 expression in stomach and normal breast tissue ({Delta}CT = 9.0) and absence in the thyroid gland.


Figure 1
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  		Fig. 1. SFRP5 mRNA expression and SFRP5 promoter methylation analysis. (A) Quantitative real-time PCR of SFRP5 expression in normal primary human tissues based on polyA+ RNA. SFRP5 mRNA is abundantly expressed only in pancreas ({Delta}CT [GAPDH:SFRP5] = 0.9); lower transcript levels were detected in normal breast ({Delta}CT = 9.0). In prostate, colon, lung and stomach, SFRP5 mRNA is transcribed in comparable amounts as normal breast (mean {Delta}CT = 9.7), whereas very few mRNA transcripts were detected in placenta, thyroid gland and kidney (mean {Delta}CT = 14.7). (B) In breast cell lines, non-malignant HMEC cells and malignant Hs578T cells were found to express SFRP5 mRNA in detectable amounts, whereas in all other analyzed cell lines, SFRP5 expression was substantially diminished. Expression levels are standardized to normal breast. (C) MSP analysis of breast cell lines. Only HMEC and Hs578T cells showed absence of SFRP5 methylation indicated by exclusive amplification with primers specific to unmethylated promoter sequence. All other cell lines showed partial (MDA-MB468 and SKBR3) or complete SFRP5 promoter methylation, indicated by amplification with primers specific to methylated promoter sequence. NTC designates the ‘no template control’. (D) Breast cell lines were treated with 2 µM DAC and 300 nM TSA, and SFRP5 promoter methylation was assessed before (–) and after treatment (+). In MCF10A and MCF12A, no substantial gain of unmethylated DNA molecules could be detected after treatment, in contrast to BT20, MCF7, MDA-MB231, SKBR3 and T47D cells, which showed enrichment of unmethylated SFRP5 promoter sequence. (E) Real-time PCR analysis of SFRP5 mRNA expression after the demethylating treatment of breast cell lines. Scaling is standardized to SFRP5 expression in normal breast tissue. All cell lines originally bearing SFRP5 promoter methylation significantly enhanced SFRP5 mRNA expression after the treatment (P = 0.006), although induction levels were low in MCF10A and MCF12A cells. (F) RNA expression of cyclin D1, c-myc and β-actin of two malignant breast cell lines were assessed before (–) and after demethylating treatment (+). Cyclin D1 and c-myc expression was significantly suppressed after DNA demethylation (***P = 0.004) in contrast to expression of the housekeeping gene β-actin, which remained unaltered. Error bars in all graphics represent standard deviation from triplicate experiments.

 
Differential SFRP5 expression in breast cell lines
Next, we quantified SFRP5 mRNA expression in benign and cancerous breast cell lines by real-time PCR. Non-malignant HMEC cells exhibited abundant SFRP5 mRNA expression and Hs578T cells exhibited expression comparable with that found in benign primary epithelial cells (Figure 1B). In all other analyzed breast cancer cell lines (MCF7, MDA-MB231, T47D, BT20 and SKBR3), SFRP5 expression was substantially diminished. However, SFRP5 expression was also absent in the non-malignant cell lines MCF10A and MCF12A.

Methylation of the SFRP5 promoter in breast cell lines
Aberrant promoter methylation of tumor suppressor genes is a major oncogenic mechanism during carcinogenesis resulting in downregulation and functional inactivation of these genes (22). Knowing that SFRP5 mRNA expression was downregulated in malignant breast cell lines, we performed promoter methylation analysis in these cells by use of MSP on bisulfite-treated DNA (35). We found a methylated SFRP5 promoter sequence in all cell lines showing reduced SFRP5 expression, i.e. MCF10A, MCF12A, BT20, MCF7, MDA-MB231, SKBR3, T47D and in addition in ZR75-1 and MDA-MB468 (Figure 1C), indicated by amplification with primers specific for the methylated DNA sequence. In contrast, the SFRP5-expressing cell lines HMEC and Hs578T lacked SFRP5 promoter methylation in the analyzed region, indicated by exclusive amplification with primers specific for the unmethylated DNA sequence. A two-sided U-test comparing expression levels of SFRP5-unmethylated versus SFRP5-methylated cell lines revealed a significant difference in their transcript level (P = 0.040).

In vitro demethylation of the SFRP5 promoter
To prove a direct association of SFRP5 promoter methylation with loss of SFRP5 mRNA expression, we treated seven breast cell lines with both 2 µM of the DNA methyltransferase inhibitor DAC and 300 nM of the histone deacetylase inhibitor TSA and determined SFRP5 promoter methylation and SFRP5 expression before and after the drug treatment, respectively. Drug concentrations had been adjusted in advance to warrant viability and replication of all cell lines. MSP analyses after drug treatment (Figure 1D) confirmed that SFRP5 promoter demethylation had occurred in some of the originally methylated cell lines by the appearance/enhancement of signals indicative of an unmethylated SFRP5 promoter. Those cell lines gaining unmethylated promoter sequence after treatment consistently showed elevated SFRP5 mRNA expression (MCF10A, MCF12A, BT20, MCF7, MDA-MB231, SKBR3 and T47D; Figure 1E). SFRP5 mRNA induction as determined by real-time PCR ranged from 2-fold (MCF12A) to 576-fold (BT20) in originally methylated cells. The median relative SFRP5 expression before treatment was 0.2 and after treatment 9.7, which equals a median induction of 49-fold in these cells (P = 0.006). To exclude the possibility that DAC–TSA treatment resulted in unspecific upregulation of gene expression, we coassessed expression of the known Wnt target genes cyclin D1 and c-myc, as well as β-actin in two representative breast cell lines (MCF7 and T47D; Figure 1F). In both cell lines, mRNA expression of cyclin D1 and c-myc was found to be significantly reduced after DNA demethylation (P = 0.004), in contrast to β-actin expression that remained unaltered.

SFRP5 methylation in primary breast carcinomas
Since cell lines may acquire additional genetic and epigenetic alterations during in vitro cultivation (37,38), it is mandatory to investigate these aberrations in primary tissues as well. To this end, we analyzed 168 primary mammary tumor samples by MSP. For 13 tumors, corresponding normal breast tissue samples were available. In total, 122 of 168 tumors (72.6%) showed SFRP5 promoter methylation (e.g. #1 in Figure 2A), whereas 46 of 168 tumors (27.4%) beared no SFRP5 methylation in the analyzed promoter region. In these cases, MSP signals were achieved exclusively with primers specific for unmethylated promoter (e.g. #4 in Figure 2A). Of the normal breast tissues, all samples showed only unmethylated promoter sequence. Tumor samples generally showed also unmethylated promoter sequence due to possible contamination with small parts of stromal and endothelial cells, as has also been described by Suzuki et al. (15).


Figure 2
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  		Fig. 2. SFRP5 promoter methylation and SFRP5 mRNA expression in primary breast cancer. (A) MSP results from four matched pairs of tumorous (T)/corresponding normal breast tissue (N) and six additional breast tumors are shown. Normal tissues are not affected by SFRP5 promoter methylation, in contrast to primary breast tumors that frequently harbor a methylated SFRP5 promoter (e.g. #1). In addition, human placenta (Plac) and peripheral blood lymphocytes (PBL) exhibited unmethylated SFRP5 promoter sequence. (B) Real-time PCR analysis of SFRP5 mRNA expression in 13 matched pairs of normal (N, gray bars) and tumorous (T, black bars) breast tissue. As indicated by arrows, in eight of these pairs, a substantial downregulation of SFRP5 mRNA >FC5 could be detected in the tumor tissue. In these eight tumors, we detected SFRP5 promoter methylation, whereas the five tumors without expression loss harbored an unmethylated SFRP5 promoter. Error bars represent standard deviation from triplicate experiments. (C) Correlation of SFRP5 mRNA expression with SFRP5 promoter methylation in breast cancer. In five unmethylated mammary tumors (TU), a median expression FC of 0.833 (plotted as 1/FC) was detected in contrast to eight methylated tumors (TM), which revealed a median expression FC (plotted as 1/FC) of 0.006 (P = 0.003).

 
Differential SFRP5 expression in primary breast tissues
Next, we examined SFRP5 mRNA expression in primary breast tissues by real-time PCR. Thirteen pairs of tumor and corresponding normal breast tissue were analyzed by quantitative real-time PCR. A substantial downregulation of SFRP5 in tumor compared with its adjacent normal tissue was detected in 8 of 13 pairs (62%; Figure 2B) as defined by an expression fold change (FC) of >5. Since SFRP5 promoter methylation had been assessed in those tumors in parallel, we compared SFRP5 methylation with SFRP5 mRNA expression. All eight tumors exhibiting SFRP5 expression loss >FC5 were found being hypermethylated in the SFRP5 promoter, whereas the five tumors without expression loss harbored an unmethylated SFRP5 promoter. Figure 2C illustrates the median SFRP5 expression FC of tumor versus corresponding normal tissue (inversely plotted) among the groups of unmethylated and methylated tumors. The median FC of unmethylated tumors (1/FC = 0.833) was significantly different to the median FC of methylated tumors (1/FC = 0.006) (P = 0.003).

Correlation of SFRP5 promoter methylation with clinicopathological factors
For descriptive data analysis, clinicopathological patient characteristics were correlated with SFRP5 methylation status. In bivariate analysis, a methylated gene promoter was associated with advanced patient age ≥60 years (P = 0.003) and histological type of breast cancer, i.e. ductal and lobular carcinomas showed higher prevalence of methylation (P = 0.032) than other histological types (group consisting of four medullary, three mucinous, two scirrhous, two papillar and one tubular carcinoma). SFRP5 methylation was not associated with tumor size, lymph node status, histological grade or estrogen receptor/progesterone receptor status of invasive breast cancers (Table I). OS and DFS were compared between methylated versus unmethylated SFRP5 promoter by univariate log-rank statistics. SFRP5 methylation was not associated with DFS (P = 0.525) but significantly associated with reduced OS (P = 0.045; 5-year survival: 82% for methylated promoter versus 88% for unmethylated promoter; 10-year survival: 40 versus 88%; see Table II) as also illustrated by Kaplan–Meier survival curves (Figure 3). Based on a mean OS of 128 months in our tumor cohort, mean OS was 145 months [95% confidence interval (CI): 131–159 months] for patients retaining an unmethylated SFRP5 promoter, in contrast to 117 months (95% CI: 98–136 months) for patients harboring a methylated SFRP5 promoter. A multivariate Cox regression analysis was employed to test for independency of SFRP5 methylation as a prognostic factor in patient’s OS (supplementary Table 3a is available at Carcinogenesis Online). Lymph node status, histological grade, estrogen receptor status and SFRP5 methylation, all of which were significant factors in univariate analysis, and tumor size and age were included in the model. After reverse selection, SFRP5 methylation, histological grade and estrogen receptor status remained significant in the analysis (SFRP5 HR: 4.554; 95% CI: 1.008–20.560; P = 0.049).


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  		Table I. Correlation analysis of SFRP5 promoter methylation with clinicopathological and immunohistochemical patient characteristics

 


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  		Table II. Univariate survival analysis of clinicopathological and immunohistochemical parameters and SFRP5 promoter methylation

 


Figure 3
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  		Fig. 3. Kaplan–Meier survival distribution curves in relation to SFRP5 promoter methylation. (A) Breast cancer patients bearing a methylated SFRP5 promoter show reduced OS compared with patients unaffected by SFRP5 methylation. (B) Recurrence in breast cancer patients after complete remission is not associated with SFRP5 methylation.

 
Survival analysis of a combined SFRP5–SFRP1 methylation marker
In a previous study, we have demonstrated that promoter methylation of the well-characterized tumor suppressor gene SFRP1 is associated with adverse patient survival in breast cancer (31). Since we now assessed SFRP5 promoter methylation in the same tumor cohort, we were able to accomplish a combined statistical analysis to answer whether this may add supplementary information considering SFRP biomarker performance in breast cancer. For n = 168 patients with methylation results on both genes, SFRP1 was found being methylated in 104 cases (62%), thus displaying a methylation frequency quite comparable with SFRP5. SFRP5 methylation was significantly associated with SFRP1 methylation (P = 0.03), suggesting that simultaneous epigenetic inactivation of both genes is not a random occurrence during breast carcinogenesis. OS characteristics for SFRP1 methylation were as follows: patients with unmethylated SFRP1 in tumor tissue revealed mean OS of 149 months (95% CI: 136–161) compared with 114 months for patients harboring SFRP1 methylation (95% CI: 95–133); P = 0.002. Interestingly, combination of SFRP1 and SFRP5 methylation indeed adds complementary information to the prediction of patient OS. Cases harboring methylation in both gene promoters displayed reduced OS of 108 months (95% CI: 88–130) compared with patients with any/no marker positive (146 months; 95% CI: 135–158); P < 0.001. This prognostic improvement could be confirmed in a multivariate Cox proportional hazard model, in which a combinatory marker of SFRP1/SFRP5 methylation performed better than either marker alone (SFRP1/5 HR: 4.605; 95% CI: 1.308–16.219; P = 0.017) (supplementary Tables 3b and c are available at Carcinogenesis Online).

In vitro induction of SFRP5 transcription by Wnt-1, LiCl and EGF
So far, we have shown that SFRP5 bears some typical features of a tumor suppressor gene in breast cancer. Despite, we wondered whether loss of a secreted protein that is weakly expressed in normal tissue may confer benefits to tumor development or growth. To test whether SFRP5 expression might be inducible by distinct stimuli, we transfected Hs578T cells with pcDNA3.1/HisA-WNT1 expression vector, or cells were treated with either LiCl or EGF. Control cells were either transfected with empty vector (Mock) or treated with NaCl. After 72 h of stimulation, we prepared total RNA and assessed mRNA expression of SFRP1, SFRP2 and SFRP5 by real-time PCR. Figure 4 shows that ectopic Wnt-1 overexpression leads to no substantial induction of SFRP1 and SFRP2 but a 4.9-fold induction of SFRP5 expression in these cells. Likewise, stimulation with LiCl, an inhibitor of GSK3-β activity and thus downstream promoter of Wnt pathway activation, results in strong induction of SFRP5 expression in a dose-dependent manner (11.2- and 27.6-fold, respectively), in contrast to SFRP1 and SFRP2 expression, which did not exceed a FC>2. In addition to LiCl, EGF had a comparable dose-dependent effect on SFRP5 expression (14.0 and 20.9, respectively), whereas no effect (SFRP2) or only marginal upregulation <FC2 (SFRP1) could be detected with other SFRPs.


Figure 4
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  		Fig. 4. Induction of SFRP5 mRNA transcription in a breast cancer cell line. Hs578T cells were either transiently transfected (Mock or pCMV-WNT1) or treated with NaCl, LiCl or EGF and SFRP1, SFRP2 and SFRP5 mRNA expressions were determined after 72 h by real-time PCR, respectively. Numbers indicate fold induction of mRNAs; only FC of ≥2 are listed. All graphs are related to SFRP1 expression in mock-transfected cells (assigned to 1). SFRP1 and SFRP2 are much stronger expressed than SFRP5, but SFRP5 expression is significantly inducible by the distinct stimuli. Error bars represent standard deviation from triplicate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
In human breast cancer, numerous genes have been identified with abolished expression due to 5'cytosine methylation within a CpG-rich region of their gene promoter (41). Typically, those genes affect important aspects of normal growth control, like cell cycle regulation (p16INK4a) (42), cell adhesion (E-cadherin) (43), extracellular matrix integrity (ITIH5) (44) or steroid receptor biology (ESR1) (45). Recent studies have shown that expression of Wnt antagonist genes is commonly silenced due to promoter hypermethylation in human carcinogenesis (15,16,23,25–27,31,46,47), occurring even in those organs in which pathway activating mutations in downstream mediators simultaneously arise (12,15). Of the SFRP gene family, only SFRP1 has been identified as target of epigenetic inactivation in breast cancer to date (31,32), and we asked whether additional members of the SFRP family might be implicated in human breast tumorigenesis as well. SFRP5, the most recently discovered SFRP gene, was first isolated in a study aiming at identifying human genes associated with apoptosis (36) and was later on characterized as inhibitor of Wnt signaling (48). However, a role in human breast tissue has not been investigated so far.

First, we screened various non-malignant human tissues for SFRP5 mRNA expression and consistent with a previous study (36), we found abundant SFRP5 expression only in pancreas, indicating that SFRP5 is not as ubiquitously expressed as other SFRPs in differentiated epithelial tissues (49,50). However, we detected weak SFRP5 mRNA transcription in normal mammary tissue, providing initial evidence for a potentially functional role of SFRP5 in the human breast. In contrast to normal breast tissue, most cancerous breast cell lines showed diminished SFRP5 expression that was significantly associated with hypermethylation of cytosine residues within the SFRP5 promoter. Moreover, SFRP5 was re-expressed after DNA-demethylating treatment with DAC, demonstrating that SFRP5 is transcriptionally silenced by epigenetic inactivation in breast cancer cell lines. In line with this, we detected SFRP5 promoter hypermethylation also in a large fraction of primary breast carcinomas in close association with SFRP5 expression loss, whereas corresponding normal breast tissues were not affected by SFRP5 methylation. Importantly, SFRP5 methylation in our study was equally prevalent in small-sized (pT1) and in large-sized (pT2–4) tumors, suggesting that this epigenetic aberration is not only an early event in breast tumor initiation, but moreover is stably inherited during tumor growth suggesting a continued role in tumor progression. In summary, we conclude that epigenetic suppression of SFRP5 is a frequent tumorigenic event in human breast carcinogenesis.

To date, hypermethylation of the SFRP5 gene has been demonstrated in various human cancer entities such as colon cancer (15), mesothelioma (16), bladder, lung (23,24), kidney (25), gastric cancer (26) and hepatocellular carcinoma (27), implying that SFRP5 might represent a general tumor suppressor gene in human tissues. However, only some of these studies investigated in parallel SFRP5 expression in normal tissues, e.g. in colonic mucosa or normal pleural tissue (15,16), whereas in normal gastric, liver, bladder, kidney and lung tissue, SFRP5 expression remained undetermined, leaving open the question whether SFRP5 methylation indeed leads to a shift of SFRP5 expression in tumorous tissues. Since we found that expression of SFRP5 was weak in some of these tissues, we speculated that SFRP5, in contrast to SFRP1 and SFRP2, may represent an inducible ad hoc response under certain circumstances, e.g. responding to Wnt stimuli. In fact, SFRP5 expression was significantly induced by Wnt-1 overexpression, whereas SFRP1 and SFRP2 expression was not. Similarly, LiCl, a selective inhibitor of GSK3-β able to mimic Wnt signaling (51), leads to significant upregulation of SFRP5 expression. EGF, which was recently shown to mediate β-catenin–TCF/LEF transcriptional activity in tumor cells in a GSK3-β-independent manner (52), also induced SFRP5 expression stronger than expression of any other SFRP. In conclusion, these data imply that SFRP5 may be a direct target of β-catenin-mediated transcription. More importantly, it reveals that genes, despite lacking abundant expression in normal tissues, may nevertheless bear important tumor-suppressive functions, possibly due to protective properties that only become effective under certain tumorigenic circumstances.

Interestingly, supporting a tumor suppressive role of SFRP5 in human tissue, we found an association of SFRP5 promoter methylation with adverse patient survival in breast cancer patients, both by univariate and multivariate analysis. This is consistent with a recent study in kidney cancer (25) showing that methylation of Wnt antagonist genes correlates with poor patient survival. Since this study employed a marker panel of several Wnt antagonists, we performed a combined statistical analysis of SFRP1/SFRP5 promoter methylation in breast cancer as well. Recently, we had shown that SFRP1 promoter methylation is a potent biomarker in breast cancer prognosis (31). Indeed, the combinatory SFRP1/SFRP5 methylation marker panel in our study performed better than either marker alone, raising expectations toward defining the most potent methylation marker panel consisting of Wnt antagonists in human breast cancer, of which we will report in a future study.

In summary, our data demonstrate for the first time that epigenetic inactivation of SFRP5 is a frequent alteration in human breast cancer associated with poor patient outcome. SFRP5 methylation as a single marker or in combination with other established methylation biomarkers may provide valuable information to the clinical oncologist in breast cancer management.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Tables 1– 3 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
This work is a research project within the German Human Genome Project and has been supported with a grant from the Bundesministerium für Bildung und Forschung (BMBF) to Edgar Dahl (01KW040-1).


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
The expert technical help of Sonja von Serényi and Inge Losen is greatly appreciated. We thank Dr Dieter Niederacher (Department of Gynecology and Obstetrics, Heinrich-Heine-University, Düsseldorf, Germany) and Prof. Matthias Dürst (Friedrich-Schiller University, Jena, Germany) for kindly providing patient samples. We are thankful to Dr Monika Klinkhammer-Schalke and Armin Pauer from the Tumor Registry Regensburg for continuous help in obtaining clinical follow-up data. The WNT1 expression vector was a kind gift from Dr Hiromu Suzuki (Sapporo Medical University, Sapporo, Japan).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received January 14, 2008; revised February 22, 2008; accepted March 11, 2008.


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