Seniski Gerusa G, Camargo Anamaria A, Ierardi Daniela F, Ramos Edneia A S, Grochoski Mariana, Ribeiro Enilze S F, Cavalli Iglenir J, Pedrosa Fabio O, de Souza Emanuel M, Zanata Silvio M, Costa Fabrício F, Klassen Giseli
Department of Basic Pathology, Federal University of Parana, PR, Brazil.
BMC Cancer. 2009 Mar 6;9:80. doi: 10.1186/1471-2407-9-80.
ADAM33 protein is a member of the family of transmembrane glycoproteins composed of multidomains. ADAM family members have different activities, such as proteolysis and adhesion, making them good candidates to mediate the extracellular matrix remodelling and changes in cellular adhesion that characterise certain pathologies and cancer development. It was reported that one family member, ADAM23, is down-regulated by promoter hypermethylation. This seems to correlate with tumour progression and metastasis in breast cancer. In this study, we explored the involvement of ADAM33, another ADAM family member, in breast cancer.
First, we analysed ADAM33 expression in breast tumour cell lines by RT-PCR and western blotting. We also used 5-aza-2'-deoxycytidine (5azadCR) treatment and DNA bisulphite sequencing to study the promoter methylation of ADAM33 in breast tumour cell lines. We evaluated ADAM33 methylation in primary tumour samples by methylation specific PCR (MSP). Finally, ADAM33 promoter hypermethylation was correlated with clinicopathological data using the chi-square test and Fisher's exact test.
The expression analysis of ADAM33 in breast tumour cell lines by RT-PCR revealed gene silencing in 65% of tumour cell lines. The corresponding lack of ADAM33 protein was confirmed by western blotting. We also used 5-aza-2'-deoxycytidine (5-aza-dCR) demethylation and bisulphite sequencing methodologies to confirm that gene silencing is due to ADAM33 promoter hypermethylation. Using MSP, we detected ADAM33 promoter hypermethylation in 40% of primary breast tumour samples. The correlation between methylation pattern and patient's clinicopathological data was not significantly associated with histological grade; tumour stage (TNM); tumour size; ER, PR or ERBB2 status; lymph node status; metastasis or recurrence. Methylation frequency in invasive lobular carcinoma (ILC) was 76.2% compared with 25.5% in invasive ductal carcinoma (IDC), and this difference was statistically significant (p = 0.0002).
ADAM33 gene silencing may be related to the discohesive histological appearance of ILCs. We suggest that ADAM33 promoter methylation may be a useful molecular marker for differentiating ILC and IDC.
ADAM33蛋白是由多个结构域组成的跨膜糖蛋白家族的成员。ADAM家族成员具有不同的活性，如蛋白水解和黏附，这使其成为介导细胞外基质重塑和细胞黏附变化的良好候选者，而这些变化是某些病理学和癌症发展的特征。据报道，该家族成员之一ADAM23因启动子高甲基化而下调。这似乎与乳腺癌的肿瘤进展和转移相关。在本研究中，我们探究了ADAM家族的另一个成员ADAM33在乳腺癌中的作用。
首先，我们通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法分析了ADAM33在乳腺肿瘤细胞系中的表达。我们还使用5-氮杂-2'-脱氧胞苷（5azadCR）处理和DNA亚硫酸氢盐测序来研究ADAM33在乳腺肿瘤细胞系中的启动子甲基化。我们通过甲基化特异性聚合酶链反应（MSP）评估原发性肿瘤样本中ADAM33的甲基化情况。最后，使用卡方检验和Fisher精确检验将ADAM33启动子高甲基化与临床病理数据相关联。
通过RT-PCR对乳腺肿瘤细胞系中ADAM33的表达分析显示，65%的肿瘤细胞系中该基因沉默。蛋白质免疫印迹法证实了相应的ADAM33蛋白缺失。我们还使用5-氮杂-2'-脱氧胞苷（5-aza-dCR）去甲基化和亚硫酸氢盐测序方法来证实基因沉默是由于ADAM33启动子高甲基化所致。使用MSP，我们在40%的原发性乳腺肿瘤样本中检测到ADAM33启动子高甲基化。甲基化模式与患者临床病理数据之间的相关性与组织学分级、肿瘤分期（TNM）、肿瘤大小、雌激素受体（ER）、孕激素受体（PR）或人表皮生长因子受体2（ERBB2）状态、淋巴结状态、转移或复发均无显著关联。浸润性小叶癌（ILC）中的甲基化频率为76.2%，而浸润性导管癌（IDC）中的甲基化频率为25.5%，且这种差异具有统计学意义（p = 0.0002）。
ADAM33基因沉默可能与ILC的分散性组织学表现有关。我们认为ADAM33启动子甲基化可能是区分ILC和IDC的有用分子标志物。
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