Institute for Biochemistry II, Jena University Hospital, Friedrich Schiller University, 07743 Jena, Germany.
Department of Internal Medicine II, Jena University Hospital, Friedrich Schiller University, 07743 Jena, Germany.
Cells. 2020 Feb 12;9(2):427. doi: 10.3390/cells9020427.
Paracrine interactions between malignant estrogen receptor positive (ER) breast cancer cells and breast adipose fibroblasts (BAFs) stimulate estrogen biosynthesis by aromatase in BAFs. In breast cancer, mainly the cAMP-responsive promoter I.3/II-region mediates excessive aromatase expression. A rare single nucleotide variant (SNV) in this promoter region, which caused 70% reduction in promoter activity, was utilized for the identification of novel regulators of aromatase expression. To this end, normal and mutant promoter activities were measured in luciferase reporter gene assays. DNA-binding proteins were captured by DNA-affinity and identified by mass spectrometry. The DNA binding of proteins was analyzed using electrophoretic mobility shift assays, immunoprecipitation-based in vitro binding assays and by chromatin immunoprecipitation in BAFs in vivo. Protein expression and parylation were analyzed by western blotting. Aromatase activities and RNA-expression were measured in BAFs. Functional consequences of poly (ADP-ribose) polymerase-1 (PARP-1) knock-out, rescue or overexpression, respectively, were analyzed in murine embryonic fibroblasts (MEFs) and the 3T3-L1 cell model. In summary, PARP-1 and histone H1 (H1) were identified as critical regulators of aromatase expression. PARP-1-binding to the SNV-region was crucial for aromatase promoter activation. PARP-1 parylated H1 and competed with H1 for DNA-binding, thereby inhibiting its gene silencing action. In MEFs (PARP-1 knock-out and wild-type) and BAFs, PARP-1-mediated induction of the aromatase promoter showed bi-phasic dose responses in overexpression and inhibitor experiments, respectively. The HDAC-inhibitors butyrate, panobinostat and selisistat enhanced promoter I.3/II-mediated gene expression dependent on PARP-1-activity. Forskolin stimulation of BAFs increased promoter I.3/II-occupancy by PARP-1, whereas SIRT-1 competed with PARP-1 for DNA binding but independently activated the promoter I.3/II. Consistently, the inhibition of both PARP-1 and SIRT-1 increased the NAD/NADH-ratio in BAFs. This suggests that cellular NAD/NADH ratios control the complex interactions of PARP-1, H1 and SIRT-1 and regulate the interplay of parylation and acetylation/de-acetylation events with low NAD/NADH ratios (reverse Warburg effect), promoting PARP-1 activation and estrogen synthesis in BAFs. Therefore, PARP-1 inhibitors could be useful in the treatment of estrogen-dependent breast cancers.
旁分泌相互作用，恶性雌激素受体阳性（ER）乳腺癌细胞和乳腺脂肪成纤维细胞（BAFs）刺激芳香酶在 BAFs 中的雌激素生物合成。在乳腺癌中，主要是 cAMP 反应启动子 I.3/II 区域介导过度芳香酶表达。在这个启动子区域中，一种罕见的单核苷酸变异（SNV）导致启动子活性降低 70%，被用于鉴定芳香酶表达的新型调节剂。为此，在荧光素酶报告基因检测中测量了正常和突变启动子的活性。通过 DNA 亲和力捕获 DNA 结合蛋白，并通过质谱鉴定。使用电泳迁移率变动分析、基于免疫沉淀的体外结合测定以及体内 BAFs 的染色质免疫沉淀分析，分析蛋白质的 DNA 结合。通过蛋白质印迹分析蛋白质表达和聚（ADP-核糖）聚合酶-1（PARP-1）的翻译后修饰。测量 BAFs 中的芳香酶活性和 RNA 表达。在鼠胚胎成纤维细胞（MEFs）和 3T3-L1 细胞模型中，分别分析 PARP-1 敲除、拯救或过表达的功能后果。总之，PARP-1 和组蛋白 H1（H1）被鉴定为芳香酶表达的关键调节剂。PARP-1 结合到 SNV 区域对于芳香酶启动子的激活至关重要。PARP-1 翻译后修饰 H1，并与 H1 竞争 DNA 结合，从而抑制其基因沉默作用。在 MEFs（PARP-1 敲除和野生型）和 BAFs 中，PARP-1 介导的芳香酶启动子的诱导在过表达和抑制剂实验中分别表现出双相剂量反应。HDAC 抑制剂丁酸钠、帕比司他和塞利司他增强了依赖 PARP-1 活性的启动子 I.3/II 介导的基因表达。佛司可林刺激 BAFs 增加了 PARP-1 对启动子 I.3/II 的占据，而 SIRT-1 与 PARP-1 竞争 DNA 结合，但独立激活启动子 I.3/II。一致地，PARP-1 和 SIRT-1 的抑制均增加了 BAFs 中的 NAD/NADH 比值。这表明细胞 NAD/NADH 比值控制 PARP-1、H1 和 SIRT-1 的复杂相互作用，并调节翻译后修饰和乙酰化/去乙酰化事件与低 NAD/NADH 比值（反向沃伯格效应）的相互作用，促进 PARP-1 激活和雌激素在 BAFs 中的合成。因此，PARP-1 抑制剂可用于治疗雌激素依赖性乳腺癌。
