Promoter hypermethylation of the SFRP2 gene is a high-frequent alteration and tumor-specific epigenetic marker in human breast cancer.
BACKGROUND
We have previously reported that expression of the Wnt antagonist genes SFRP1 and SFRP5 is frequently silenced by promoter hypermethylation in breast cancer. SFRP2 is a further Wnt inhibitor whose expression was recently found being downregulated in various malignancies. Here we investigated whether SFRP2 is also implicated in human breast cancer, and if so whether SFRP2 promoter methylation might serve as a potential tumor biomarker.


METHODS
We analyzed SFRP2 mRNA expression and SFRP2 promoter methylation in 10 breast cell lines, 199 primary breast carcinomas, 20 matched normal breast tissues and 17 cancer-unrelated normal breast tissues using RT-PCR, realtime PCR, methylation-specific PCR and Pyrosequencing, respectively. SFRP2 protein expression was assessed by immunohistochemistry on a tissue microarray. Proliferation assays after transfection with an SFRP2 expression vector were performed with mammary MCF10A cells. Statistical evaluations were accomplished with SPSS 14.0 software.


RESULTS
Of the cancerous breast cell lines, 7/8 (88%) lacked SFRP2 mRNA expression due to SFRP2 promoter methylation (P < 0.001). SFRP2 expression was substantially restored in most breast cell lines after treatment with 5-aza-2'-deoxycytidine and trichostatin A. In primary breast carcinomas SFRP2 protein expression was strongly reduced in 93 of 125 specimens (74%). SFRP2 promoter methylation was detected in 165/199 primary carcinomas (83%) whereas all cancer-related and unrelated normal breast tissues were not affected by SFRP2 methylation. SFRP2 methylation was not associated with clinicopathological factors or clinical patient outcome. However, loss of SFRP2 protein expression showed a weak association with unfavorable patient overall survival (P = 0.071). Forced expression of SFRP2 in mammary MCF10A cells substantially inhibited proliferation rates (P = 0.045).


CONCLUSIONS
The SFRP2 gene is a high-frequent target of epigenetic inactivation in human breast cancer. Its methylation leads to abrogation of SFRP2 expression, conferring a growth advantage to epithelial mammary cells. This altogether supports a tumor suppressive function of SFRP2. Although clinical patient outcome was not associated with SFRP2 methylation, the high frequency of this epimutation and its putative specificity to neoplastic cells may qualify SFRP2 promoter methylation as a potential candidate screening marker helping to improve early breast cancer detection.
Veeck
Jürgen
J
Molecular Oncology Group, Institute of Pathology, University Hospital of the RWTH Aachen, Aachen, Germany. juergen.veeck@rwth-aachen.de
Noetzel
Erik
E
Bektas
Nuran
N
Jost
Edgar
E
Hartmann
Arndt
A
Knüchel
Ruth
R
Dahl
Edgar
E
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics*; D001940:Breast / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D049109:Cell Proliferation; D019175:DNA Methylation; D044127:Epigenesis, Genetic / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006262:Health; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D015996:Survival Rate; D000094742:Secreted Frizzled-Related Proteins
D014408:Biomarkers, Tumor; D008565:Membrane Proteins; D012333:RNA, Messenger; C109618:SFRP2 protein, human; D000094742:Secreted Frizzled-Related Proteins