Department of Surgery, University of Virginia, Charlottesville, VA, USA.
J Pathol. 2010 Jun;221(2):229-37. doi: 10.1002/path.2707.
Breast cancer metastasis suppressor gene-1 (BRMS1) mRNA and protein expression are significantly decreased in non-small cell lung cancer (NSCLC) and this is a poor prognostic indicator. Given that the BRMS1 promoter region contains a promoter-associated CpG island (CGI) that encompasses the transcriptional start site, we hypothesized that decreased BRMS1 mRNA and protein levels in NSCLC was secondary to increased BRMS1 promoter methylation. Methylation-specific PCR (MSP) of the two known CGIs (-3477 to - 2214 and - 531 to + 608) in the BRMS1 genome was performed in NSCLC cells. This demonstrated a robust increase in methylation of the promoter-associated CGI (-531 to + 608) but not of the upstream CGI (-3477 to - 2214). To experimentally verify that methylation contributes to BRMS1 transcriptional repression, we cloned the BRMS1 promoter region, including the promoter-associated CGI, into a luciferase reporter gene and found that BRMS1 promoter activity was dramatically inhibited under methylated conditions. We then assessed the BRMS1 methylation profile with MSP and bisulphite-sequencing PCR in human NSCLC adenocarcinoma (n = 20) and squamous cell carcinoma (n = 20) relative to adjacent non-cancerous bronchial epithelium. There was a significant increase in BRMS1 promoter methylation in all NSCLC specimens relative to non-cancerous tissues, with the most dramatic difference in squamous cell cancer histology. Subsequent immunostaining demonstrated that nuclear BRMS1 expression is reduced in lung cancer specimens compared to normal bronchial epithelium. The association between BRMS1 promoter methylation and specific clinical and histopathological variables was examined using a general linear model. Pathological tumour stage was associated with increased BRMS1 methylation in squamous cell cancers. These observations demonstrate that methylation of the promoter-associated CGI in BRMS1 results in its transcriptional repression, and highlight the potential clinical relevance of this methylation event with respect to NSCLC tumour histology and pathological stage.
乳腺癌转移抑制基因 1（BRMS1）mRNA 和蛋白表达在非小细胞肺癌（NSCLC）中显著降低，这是预后不良的指标。鉴于 BRMS1 启动子区域包含一个与启动子相关的 CpG 岛（CGI），该 CGI 包含转录起始位点，我们假设 NSCLC 中 BRMS1 mRNA 和蛋白水平降低是由于 BRMS1 启动子甲基化增加所致。对 BRMS1 基因组中的两个已知 CGI（-3477 至-2214 和-531 至+608）进行了甲基化特异性 PCR（MSP）。结果显示，与上游 CGI（-3477 至-2214）相比，启动子相关 CGI（-531 至+608）的甲基化显著增加。为了实验验证甲基化导致 BRMS1 转录抑制，我们将 BRMS1 启动子区域（包括启动子相关 CGI）克隆到荧光素酶报告基因中，发现 BRMS1 启动子活性在甲基化条件下受到显著抑制。然后，我们通过 MSP 和亚硫酸氢盐测序 PCR 评估了 20 例 NSCLC 腺癌和 20 例鳞癌与相邻非癌性支气管上皮组织中 BRMS1 的甲基化谱。与非癌组织相比，所有 NSCLC 标本中的 BRMS1 启动子甲基化均显著增加，其中鳞癌组织的差异最为显著。随后的免疫染色显示，与正常支气管上皮相比，肺癌标本中核 BRMS1 表达减少。使用广义线性模型检查 BRMS1 启动子甲基化与特定临床和组织病理学变量之间的关系。鳞癌中病理肿瘤分期与 BRMS1 甲基化增加相关。这些观察结果表明，BRMS1 启动子相关 CGI 的甲基化导致其转录抑制，并强调了这种甲基化事件与 NSCLC 肿瘤组织学和病理分期的潜在临床相关性。
