雌激素相关受体 α、β 和 γ 的表达和功能与乳腺癌中的转录抑制因子 EZH2 有关。
Estrogen-related receptors alpha, beta and gamma expression and function is associated with transcriptional repressor EZH2 in breast carcinoma.
机构信息
Cancer Biology Laboratory, Department of Gene Function and Regulation, Institute of Life Sciences, Bhubaneswar. Utkal University, Bhubaneswar, Odisha, India.
Department of Pathology, AIIMS, Bhubaneswar, Odisha, India.
出版信息
BMC Cancer. 2018 Jun 26;18(1):690. doi: 10.1186/s12885-018-4586-0.
BACKGROUND
Orphan nuclear receptors ERRα, ERRβ and ERRγ that belong to NR3B or type IV nuclear receptor family are well studied for their role in breast cancer pathophysiology. Their homology with the canonical estrogen receptor dictates their possible contributing role in mammary gland development and disease. Although function and regulation of ERRα, ERRγ and less about ERRβ is reported, role of histone methylation in their altered expression in cancer cells is not studied. Transcriptional activity of nuclear receptors depends on co-regulatory proteins. The present study for the first time gives an insight into regulation of estrogen-related receptors by histone methylation specifically through methyltransferase EZH2 in breast cancer.
METHODS
Expression of ERRα, ERRβ, ERRγ and EZH2 was assessed by immunohistochemistry in four identical tissue array slides that were prepared as per the protocol. The array slides were stained with ERRα, ERRβ, ERRγ and EZH2 simultaneously. Array data was correlated with expression in MERAV expression dataset. Pearson correlation coeficient r was calculated from the partial matrix expression values available at MERAV database to study the strength of association between EZH2 and three orphan nuclear receptors under study. By western blot and real time PCR, their correlated expression was studied in breast cancer cell lines MCF-7, MDA-MB-231, T47D and MDA-MB-453 including normal breast epithelial MCF-10A cells at both protein and RNA level. Regulation of ERRα, ERRβ, ERRγ by EZH2 was further investigated upon overexpression and silencing of EZH2. The interaction between ERRs and EZH2 was validated in vivo by CHIP-qPCR.
RESULTS
We found a negative correlation between estrogen-related receptors and Enhancer of Zeste Homolog 2, a global repressor gene. Immunohistochemistry in primary breast tumors of different grades showed a correlated expression of estrogen-related receptors and EZH2. Their correlated expression was further validated using online MERAV expression dataset where a negative correlation of variable strengths was observed in breast cancer. Ectopic expression of EZH2 in low EZH2-expressing normal breast epithelial cells abrogated their expression and at the same time, its silencing enhanced the expression of estrogen-related receptors in cancerous cells. Global occupancy of EZH2 on ERRα and ERRβ was observed in-vivo.
CONCLUSION
Our findings identify EZH2 as a relevant coregulator for estrogen-related receptors in breast carcinoma.
背景
孤儿核受体 ERRα、ERRβ 和 ERRγ 属于 NR3B 或 IV 型核受体家族，它们在乳腺癌病理生理学中的作用得到了广泛研究。它们与经典雌激素受体的同源性决定了它们在乳腺发育和疾病中的可能作用。尽管已经报道了 ERRα、ERRγ 的功能和调节，以及 ERRβ 的部分调节，但它们在癌细胞中表达改变的组蛋白甲基化作用尚未得到研究。核受体的转录活性依赖于共调节蛋白。本研究首次深入了解了组蛋白甲基化对乳腺癌中 ERRα、ERRβ 和 ERRγ 表达的调节作用，特别是通过甲基转移酶 EZH2 进行调节。
方法
采用免疫组织化学法，在按照方案制备的 4 个相同组织阵列切片上评估 ERRα、ERRβ、ERRγ 和 EZH2 的表达。同时用 ERRα、ERRβ、ERRγ 和 EZH2 对阵列切片进行染色。将阵列数据与 MERAV 表达数据集进行相关。从 MERAV 数据库中可用的部分矩阵表达值计算 Pearson 相关系数 r，以研究 EZH2 与研究中的三种孤儿核受体之间的关联强度。通过 Western blot 和实时 PCR，在乳腺癌细胞系 MCF-7、MDA-MB-231、T47D 和 MDA-MB-453 中（包括正常乳腺上皮 MCF-10A 细胞），以及在蛋白质和 RNA 水平上，研究了它们在 EZH2 表达的相关性。通过过表达和沉默 EZH2，进一步研究了 ERRα、ERRβ、ERRγ 对 EZH2 的调节作用。通过 CHIP-qPCR 验证了 ERRs 和 EZH2 之间的体内相互作用。
结果
我们发现，雌激素相关受体与整体抑制基因 Enhancer of Zeste Homolog 2 之间存在负相关。不同分级的原发性乳腺癌肿瘤的免疫组织化学染色显示，雌激素相关受体和 EZH2 的表达呈相关性。利用在线 MERAV 表达数据集进一步验证了它们的相关性，在乳腺癌中观察到了不同强度的负相关。在低 EZH2 表达的正常乳腺上皮细胞中过表达 EZH2 会使其表达缺失，同时沉默 EZH2 可增强癌细胞中雌激素相关受体的表达。体内观察到 EZH2 对 ERRα 和 ERRβ 的整体占据。
结论
我们的研究结果表明，EZH2 是乳腺癌中雌激素相关受体的一个相关共调节因子。
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