Progesterone receptor isoforms A and B exert different biological effects in breast cancer cells. Alteration of PRA/PRB ratio is often observed during breast cancer progression. High PRA/PRB ratios in breast cancer patients are associated with resistance to chemotherapy and poor prognosis. While it is well accepted that PRA and PRB regulate different sets of genes, how the expression of PRA and PRB alters breast cancer proteomes has not been fully investigated. To directly investigate the effects of PR isoform expression on the breast cancer proteome, both in the presence and absence of progestin, PRA and PRB were independently stably expressed in T47DC42 PR-null breast cancer cells using a doxycycline (Dox)-regulated promoter. Dox induction dose-dependently increased PRA and PRB expression. Dox-induced PRA and PRB showed normal receptor localization and were transcriptionally active. Differential quantitative proteomic analysis by stable isotope dimethyl labeling was performed to quantitatively examine how PR isoforms altered global breast cancer proteomes. Cells expressing PRA in the absence of progestin were enriched in proteins involved in the TCA cycle and enriched in proteins involved in glycolysis in the presence of progestin, whilst cells expressing PRB in the absence and presence progestin were significantly enriched in proteins involved in the cell cycle and cell apoptosis pathways. This proteomic data revealed a link between PR isoform expression and alteration in cell metabolism, cell proliferation, and apoptosis. The enrichment of proteins involved in the glycolytic pathway in breast cancer cells expressing PRA is consistent with stem cell-like properties, previously reported in PRA-rich breast cancer cells. Moreover, compared to liganded PRB, liganded PRA differentially upregulated proteins involved in chromatin remodeling, such as linker histone H1.2. Silencing H1.2 gene expression suppressed PRA-mediated cell proliferation and promoted G2/M and S phase entry of the cell cycle. Additionally, liganded PRA upregulated the expression of cathepsin D (CTSD) protease, whose expression is associated with poor prognosis in breast cancer patients. Together, our data demonstrated that the expression of PRA or PRB dramatically and differentially altered breast cancer cell proteomes. These isoform-specific changes in the breast cancer proteome will help to explain the distinct phenotypic properties of breast cancer cells expressing different levels of PRA and PRB.

中文翻译：

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差异定量蛋白质组学揭示了与表达孕激素受体A的乳腺癌细胞表型变化有关的关键蛋白。

孕激素受体同工型A和B在乳腺癌细胞中发挥不同的生物学作用。PRA / PRB比值的变化通常在乳腺癌进展过程中观察到。乳腺癌患者的高PRA / PRB比值与化疗耐药性和预后不良有关。虽然PRA和PRB调节不同的基因集已广为接受，但PRA和PRB的表达如何改变乳腺癌蛋白质组尚未得到充分研究。为了直接研究PR异构体表达对乳腺癌蛋白质组的影响，无论是否存在孕激素，均使用强力霉素（Dox）调控的启动子在T47DC42 PR无效的乳腺癌细胞中稳定稳定地表达PRA和PRB。Dox诱导剂量依赖性增加PRA和PRB表达。Dox诱导的PRA和PRB显示正常的受体定位，并具有转录活性。通过稳定同位素二甲基标记进行差异定量蛋白质组学分析，以定量检查PR亚型如何改变全球乳腺癌蛋白质组。在没有孕激素的情况下表达PRA的细胞富含TCA周期中涉及的蛋白质，并在存在孕激素的情况下富含参与糖酵解的蛋白，而在不存在或存在孕激素的情况下表达PRB的细胞显着富含细胞周期中涉及的蛋白。和细胞凋亡途径。蛋白质组学数据揭示了PR亚型表达与细胞代谢，细胞增殖和凋亡改变之间的联系。表达PRA的乳腺癌细胞中参与糖酵解途径的蛋白质的富集与干细胞样特性一致，以前在富含PRA的乳腺癌细胞中已有报道。而且，与配体PRB相比，配体PRA差异上调了参与染色质重塑的蛋白质，例如接头组蛋白H1.2。沉默H1.2基因表达抑制PRA介导的细胞增殖，并促进细胞周期的G2 / M和S期进入。此外，配体PRA上调组织蛋白酶D（CTSD）蛋白酶的表达，其表达与乳腺癌患者的不良预后相关。在一起，我们的数据表明PRA或PRB的表达大大和差异地改变了乳腺癌细胞蛋白质组。