Epigenetic regulation of the ras effector/tumour suppressor RASSF2 in breast and lung cancer.
Epigenetic regulation of the ras effector/tumour suppressor RASSF2 in breast and lung cancer.
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DOI：
10.1038/sj.onc.1210805
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发表时间：
2008-03-13
期刊：
影响因子：
8
通讯作者：
Latif, F.
中科院分区：
文献类型：
--
作者：
Cooper, W. N.;Dickinson, R. E.;Dallol, A.;Grigorieva, E. V.;Pavlova, T. V.;Hesson, L. B.;Bieche, I.;Broggini, M.;Maher, E. R.;Zabarovsky, E. R.;Clark, G. J.;Latif, F.
RASSF2是最近发现的一类新的肿瘤抑制基因的成员，它们都含有ras相关结构域。我们先前的研究表明，在大肠肿瘤和腺瘤中，RASSF2的A亚型经常被启动子区域的高甲基化灭活，甲基化是肿瘤特异性的，在甲基化的肿瘤株中的表达可以通过5-aza-2dc的处理而重新激活。RASSF2位于20p13，该区域在人类肿瘤中经常缺失。在这份报告中，我们研究了一系列乳腺癌、卵巢癌和非小细胞肺癌(NSCLC)中RASSF2A启动子CpG岛的甲基化状态。RASSF2A在乳腺肿瘤细胞系中的甲基化频率为65%(13/20)，在原发性乳腺肿瘤中的甲基化频率为38%(15/40)。经5-aza-2dC处理后，甲基化的乳腺癌细胞株中RASSF2A基因的表达可以恢复，而未甲基化的乳腺癌细胞株在5-aza-2dC处理前后的表达水平没有差异。44%(22/50)的非小细胞肺癌组织中RASSF2A基因甲基化频率较高。乳腺肿瘤和非小细胞肺癌中的甲基化是肿瘤特异性的。我们在卵巢肿瘤中未检测到RASSF2A甲基化(0/17)。此外，在这些卵巢肿瘤中未发现RASSF2A编码区的突变。RASSF2A在体外(通过集落形成和软琼脂实验)和体内抑制乳腺肿瘤细胞的生长。我们在RASSF2A序列的151和167位氨基酸之间发现了一个高度保守的两部分核定位信号(NLS)，并证明了内源RASSF2A定位于细胞核。假定的核定位信号的突变取消了核定位，因此RASSF2A成为主要的细胞质。我们的数据表明，RASSF2A在结直肠癌、乳腺癌和非小细胞肺癌中经常发生甲基化，而且这种甲基化是肿瘤特有的。因此，我们已确定RASSF2A是一种新的多种恶性肿瘤的甲基化标志物，它有可能发展成为利用启动子超甲基化图谱同时筛查几种癌症的有价值的标志物。我们还证明了RASSF2具有功能性的NLS信号。此外，这是第一个表明RASSF2在体内抑制癌细胞生长的报告。从而为其作为位于20p13的肿瘤抑制基因的作用提供了进一步的证据。
RASSF2 is a recently identified member of a class of novel tumour suppressor genes, all containing a ras association domain. We previously demonstrated that the A isoform of RASSF2, is frequently inactivated by promoter region hypermethylation in colorectal tumours and adenomas, methylation was tumour specific and that expression in methylated tumour lines could be reactivated by treatment with 5-aza-2dc. RASSF2 resides at 20p13, this region has been demonstrated to be frequently lost in human cancers. In this report we investigated methylation status of the RASSF2A promoter CpG island in a series of breast, ovarian and non-small cell lung cancers (NSCLC). RASSF2A was frequently methylated in breast tumour cell lines 65% (13/20) and in primary breast tumours 38% (15/40). RASSF2A gene expression could be switched back on in methylated breast tumour cell lines after treatment with 5-aza-2dC, whilst unmethylated lines showed no difference in level of expression before and after 5-aza-2dC treatment. RASSF2A was also frequently methylated in NSCLC tumours 44% (22/50). Methylation in breast tumours and NSCLC was tumour specific. We did not detect RASSF2A methylation in ovarian tumours (0/17). Furthermore no mutations were found in the coding region of RASSF2A in these ovarian tumours. RASSF2A suppressed breast tumour cell growth in vitro (through colony formation and soft agar assays) and in vivo. We identified a highly conserved putative bipartite nuclear localisation signal (NLS) between amino acids 151 and 167 in the RASSF2A sequence and demonstrated that endogenous RASSF2A localised to the nucleus. Mutation of the putative nuclear localisation signal abolished the nuclear localisation so RASSF2A became predominantly cytoplasmic. Our data indicates that RASSF2A is frequently methylated in colorectal, breast and NSCLC tumours, furthermore, the methylation is tumour specific. Hence we have identified RASSF2A as a novel methylation marker for multiple malignancies and it has the potential to be developed into a valuable marker for screening several cancers in parallel using promoter hypermethylation profiles. We also demonstrate that RASSF2 has a functional NLS signal. Furthermore this is the first report demonstrating that RASSF2 suppresses growth of cancer cells in vivo. Hence providing further evidence for its role as a tumour suppressor gene located at 20p13.
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