Despite advances in early detection and treatment, invasion and metastasis of breast tumors remains a major hurdle. Cystatin A (CSTA, also called stefin A), an estrogen-regulated gene in breast cancer cells, is an inhibitor of cysteine cathepsins, and a purported tumor suppressor. Loss of CSTA expression in breast tumors evidently shifts the balance in favor of cysteine cathepsins, thereby promoting extracellular matrix remodeling, tumor invasion and metastasis. However, the underlying mechanism behind the loss of CSTA expression in breast tumors is not known. Here, we have analyzed CSTA expression, and methylation of upstream and intron-2 CpG sites within the CSTA locus in human breast cancer cell lines and breast tumors of the TCGA cohort. Results showed an inverse relationship between expression and methylation. Sequence analysis revealed a potential estrogen response element (ERE) in the intron-2. Analysis of ChIP-seq data (ERP000380) and our own ChIP experiments showed that 17β-estradiol (E2) enhanced ERα binding to this ERE in MCF-7 cells. This ERE was located amidst the differentially methylated intron-2 CpG sites, which provoked us to examine the possible conflict between estrogen-regulation of CSTA and DNA methylation in the intron-2. We analyzed the expression of CSTA and its regulation by E2 in MDA-MB-231 and T47D cells subjected to global demethylation by 5-azacytidine (5-aza). 5-aza significantly demethylated intron-2 CpGs, and enhanced estrogen-induced ERα occupancy at the intron-2 ERE, leading to restoration of estrogen-regulation. Taken together, our results indicate that DNA methylation-dependent silencing could play a significant role in the loss of CSTA expression in breast tumors. The potential of DNA methylation as an indicator of CSTA expression or as a marker of tumor progression can be explored in future investigations. Furthermore, our results indicate the convergence of ERα-mediated estrogen regulation and DNA methylation in the intron-2, thereby offering a novel context to understand the role of estrogen-ERα signaling axis in breast tumor invasion and metastasis.

中文翻译：

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内含子 2 中 ERα 结合和 DNA 甲基化的相互作用决定了乳腺癌细胞中胱抑素 A 的表达和雌激素调节。

尽管在早期检测和治疗方面取得了进展，但乳腺肿瘤的侵袭和转移仍然是一个主要障碍。胱抑素 A（CSTA，也称为 stefin A）是乳腺癌细胞中的一种雌激素调节基因，是半胱氨酸组织蛋白酶的抑制剂，据称是一种肿瘤抑制因子。乳腺肿瘤中 CSTA 表达的丧失显然使平衡转向半胱氨酸组织蛋白酶，从而促进细胞外基质重塑、肿瘤侵袭和转移。然而，乳腺肿瘤中 CSTA 表达缺失背后的潜在机制尚不清楚。在这里，我们分析了 CSTA 表达，以及 TCGA 队列的人类乳腺癌细胞系和乳腺肿瘤中 CSTA 基因座内上游和内含子 2 CpG 位点的甲基化。结果显示表达和甲基化之间呈负相关。序列分析揭示了内含子 2 中潜在的雌激素反应元件 (ERE)。对 ChIP-seq 数据 (ERP000380) 和我们自己的 ChIP 实验的分析表明，17β-雌二醇 (E2) 增强了 ERα 与 MCF-7 细胞中该 ERE 的结合。该 ERE 位于差异甲基化的内含子 2 CpG 位点之间，这促使我们检查 CSTA 的雌激素调节与内含子 2 中的 DNA 甲基化之间可能存在的冲突。我们分析了 CSTA 的表达及其在 MDA-MB-231 和 T47D 细胞中 E2 的调节，这些细胞经过 5-氮杂胞苷 (5-aza) 全局去甲基化。5-aza 显着去甲基化内含子 2 CpG，并增强雌激素诱导的内含子 2 ERE 的 ERα 占据，导致雌激素调节的恢复。综合起来，我们的结果表明 DNA 甲基化依赖性沉默可能在乳腺肿瘤中 CSTA 表达的丧失中发挥重要作用。DNA 甲基化作为 CSTA 表达的指标或作为肿瘤进展的标志物的潜力可以在未来的研究中进行探索。此外，我们的结果表明内含子 2 中 ERα 介导的雌激素调节和 DNA 甲基化的收敛，从而为了解雌激素-ERα 信号轴在乳腺肿瘤侵袭和转移中的作用提供了新的背景。