Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, VIC, 8006, Australia.
Breast Cancer Res Treat. 2011 Nov;130(1):319-29. doi: 10.1007/s10549-011-1657-1. Epub 2011 Jul 14.
CBP/p300-interacting transactivator with ED-rich carboxy-terminal domain 4 (CITED4) inhibits HIF-1α transactivation by binding to CBP/p300. We hypothesised that either somatic mutation or hypermethylation of the CITED4 gene underlies CITED4 down-regulation and thus enhanced HIF-1α expression in some breast tumours. DNA sequencing was used to screen for somatic mutations. Methylation-sensitive high resolution melting was performed to identify CITED4 methylation. RT-qPCR was carried out to measure the expression of CITED4 and selected HIF downstream targets. HIF-1α and downstream gene expression was assessed with immunohistochemistry. No somatic mutations of CITED4 were identified in 10 tumour cell lines and 100 breast carcinomas. However, CITED4 promoter methylation was identified in 5/168 breast carcinomas (four infiltrating ductal carcinomas and one infiltrating lobular carcinoma) and in 3/10 breast cancer cell lines (MDA-MB-453, MDA-MB-231 and Hs578T). CITED4 mRNA expression in cell lines was inversely correlated with DNA methylation. CITED4 mRNA expression was significantly increased in all three cell lines after 5-aza-2-deoxycytidine (DAC) treatment. Treatment of the MDA-MB-231 cell line with DAC followed by hypoxia (0.1% O²) resulted in down-regulation of expression of the HIF-1α downstream genes VEGFA and SLC2A1 (P = 0.0029). HIF-1α downstream SLC2A1 was decreased (P = 0.021) after CITED4 was re-expressed under hypoxia. Loss of expression of CITED4 in breast cancer may be due to DNA methylation but is unlikely to be due to mutation. Demethylation and histone modification can potentially reactivate CITED4 gene expression in some breast cancers and lead to changes in tumour behaviour. Strategies such as HDAC inhibitors may overcome this effect.
CBP/p300 相互作用的转录激活因子与富含 ED 的羧基末端域 4（CITED4）通过与 CBP/p300 结合抑制 HIF-1α 的转录激活。我们假设，在一些乳腺癌中，CITED4 基因的体细胞突变或高甲基化导致 CITED4 下调，从而增强 HIF-1α 的表达。我们使用 DNA 测序筛选体细胞突变，用甲基化敏感高分辨率熔解曲线分析鉴定 CITED4 甲基化，通过 RT-qPCR 测量 CITED4 和选定的 HIF 下游靶基因的表达。用免疫组织化学评估 HIF-1α 和下游基因的表达。在 10 个肿瘤细胞系和 100 例乳腺癌中未发现 CITED4 的体细胞突变。然而，在 4 例浸润性导管癌和 1 例浸润性小叶癌以及 3 例乳腺癌细胞系（MDA-MB-453、MDA-MB-231 和 Hs578T）中发现了 CITED4 启动子甲基化。细胞系中 CITED4 mRNA 的表达与 DNA 甲基化呈负相关。在所有 3 个细胞系中，用 5-氮杂-2-脱氧胞苷（DAC）处理后，CITED4 mRNA 的表达显著增加。用 DAC 处理 MDA-MB-231 细胞系，然后在低氧（0.1% O²）条件下培养，导致 HIF-1α 下游基因 VEGFA 和 SLC2A1 的表达下调（P = 0.0029）。在低氧条件下 CITED4 重新表达后，HIF-1α 下游 SLC2A1 也降低（P = 0.021）。乳腺癌中 CITED4 的表达缺失可能是由于 DNA 甲基化，但不太可能是由于突变所致。去甲基化和组蛋白修饰可能在某些乳腺癌中重新激活 CITED4 基因表达，导致肿瘤行为发生变化。HDAC 抑制剂等策略可能会克服这种影响。
