Veeck Jürgen, Noetzel Erik, Bektas Nuran, Jost Edgar, Hartmann Arndt, Knüchel Ruth, Dahl Edgar
Molecular Oncology Group, Institute of Pathology, University Hospital of the RWTH Aachen, Aachen, Germany.
Mol Cancer. 2008 Nov 6;7:83. doi: 10.1186/1476-4598-7-83.
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.
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.
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).
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.
我们之前报道过，Wnt拮抗剂基因SFRP1和SFRP5的表达在乳腺癌中常因启动子高甲基化而沉默。SFRP2是另一种Wnt抑制剂，最近发现其在多种恶性肿瘤中表达下调。在此，我们研究了SFRP2是否也与人类乳腺癌有关，以及SFRP2启动子甲基化是否可作为一种潜在的肿瘤生物标志物。
我们分别使用逆转录聚合酶链反应（RT-PCR）、实时定量聚合酶链反应（real-time PCR）、甲基化特异性聚合酶链反应（methylation-specific PCR）和焦磷酸测序法，分析了10种乳腺癌细胞系、199例原发性乳腺癌、20例配对的正常乳腺组织和17例癌旁正常乳腺组织中的SFRP2 mRNA表达和SFRP2启动子甲基化情况。通过组织芯片免疫组化评估SFRP2蛋白表达。用SFRP2表达载体转染乳腺MCF10A细胞后进行增殖试验。使用SPSS 14.0软件进行统计评估。
在癌性乳腺癌细胞系中，7/8（88%）因SFRP2启动子甲基化而缺乏SFRP2 mRNA表达（P < 0.001）。用5-氮杂-2'-脱氧胞苷和曲古抑菌素A处理后，大多数乳腺癌细胞系中SFRP2表达得到显著恢复。在原发性乳腺癌中，125例标本中有93例（74%）SFRP2蛋白表达强烈降低。199例原发性癌中有165例（83%）检测到SFRP2启动子甲基化，而所有癌旁和癌无关的正常乳腺组织均未受SFRP2甲基化影响。SFRP2甲基化与临床病理因素或临床患者预后无关。然而，SFRP2蛋白表达缺失与患者总体生存不良呈弱相关（P = 0.071）。在乳腺MCF10A细胞中强制表达SFRP2可显著抑制增殖率（P = 0.045）。
SFRP2基因是人类乳腺癌中表观遗传失活的高频靶点。其甲基化导致SFRP2表达缺失，赋予乳腺上皮细胞生长优势。这完全支持SFRP2的肿瘤抑制功能。虽然临床患者预后与SFRP2甲基化无关，但这种表观突变的高频率及其对肿瘤细胞的推定特异性可能使SFRP2启动子甲基化有资格作为一种潜在的候选筛查标志物，有助于改善早期乳腺癌的检测。
