Departments of Medicine (Division of Hematology, Oncology, and Transplantation) and Pharmacology, Masonic Cancer Center, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455 USA.
Breast Cancer Res. 2012 Jun 14;14(3):R95. doi: 10.1186/bcr3211.
Progesterone receptors (PR) are emerging as important breast cancer drivers. Phosphorylation events common to breast cancer cells impact PR transcriptional activity, in part by direct phosphorylation. PR-B but not PR-A isoforms are phosphorylated on Ser294 by mitogen activated protein kinase (MAPK) and cyclin dependent kinase 2 (CDK2). Phospho-Ser294 PRs are resistant to ligand-dependent Lys388 SUMOylation (that is, a repressive modification). Antagonism of PR small ubiquitin-like modifier (SUMO)ylation by mitogenic protein kinases suggests a mechanism for derepression (that is, transcriptional activation) of target genes. As a broad range of PR protein expression is observed clinically, a PR gene signature would provide a valuable marker of PR contribution to early breast cancer progression.
Global gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild-type (SUMOylation-capable) or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Gene sets were validated by RT-qPCR. Recruitment of coregulators and histone methylation levels were determined by chromatin immunoprecipitation. Changes in cell proliferation and survival were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and western blotting. Finally, human breast tumor cohort datasets were probed to identify PR-associated gene signatures; metagene analysis was employed to define survival rates in patients whose tumors express a PR gene signature.
'SUMO-sensitive' PR target genes primarily include genes required for proliferative and pro-survival signaling. DeSUMOylated K388R receptors are preferentially recruited to enhancer regions of derepressed genes (that is, MSX2, RGS2, MAP1A, and PDK4) with the steroid receptor coactivator, CREB-(cAMP-response element-binding protein)-binding protein (CBP), and mixed lineage leukemia 2 (MLL2), a histone methyltransferase mediator of nucleosome remodeling. PR SUMOylation blocks these events, suggesting that SUMO modification of PR prevents interactions with mediators of early chromatin remodeling at 'closed' enhancer regions. SUMO-deficient (phospho-Ser294) PR gene signatures are significantly associated with human epidermal growth factor 2 (ERBB2)-positive luminal breast tumors and predictive of early metastasis and shortened survival. Treatment with antiprogestin or MEK inhibitor abrogated expression of SUMO-sensitive PR target-genes and inhibited proliferation in BT-474 (estrogen receptor (ER)+/PR+/ERBB2+) breast cancer cells.
We conclude that reversible PR SUMOylation/deSUMOylation profoundly alters target gene selection in breast cancer cells. Phosphorylation-induced PR deSUMOylation favors a permissive chromatin environment via recruitment of CBP and MLL2. Patients whose ER+/PR+ tumors are driven by hyperactive (that is, derepressed) phospho-PRs may benefit from endocrine (antiestrogen) therapies that contain an antiprogestin.
孕激素受体（PR）正在成为重要的乳腺癌驱动因素。乳腺癌细胞中常见的磷酸化事件影响 PR 的转录活性，部分是通过直接磷酸化。PR-B 而不是 PR-A 同工型可被丝裂原活化蛋白激酶（MAPK）和细胞周期蛋白依赖性激酶 2（CDK2）磷酸化 Ser294。磷酸化 Ser294 PR 对配体依赖性 Lys388 SUMOylation（即抑制性修饰）具有抗性。有丝分裂原蛋白激酶对 PR 小泛素样修饰（SUMO）化的拮抗作用表明，靶基因去抑制（即转录激活）的机制。由于临床上观察到广泛的 PR 蛋白表达，因此 PR 基因特征将为 PR 对早期乳腺癌进展的贡献提供有价值的标志物。
在表达野生型（SUMOylation 能力）或 K388R（SUMOylation 缺陷）PR 的 T47D 和 MCF-7 乳腺癌细胞中测量全基因表达模式，并进行途径分析。通过 RT-qPCR 验证基因集。通过染色质免疫沉淀测定核心调节剂的募集和组蛋白甲基化水平。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐（MTT）测定和 Western blot 测定来确定细胞增殖和存活的变化。最后，探测人类乳腺肿瘤队列数据集以识别与 PR 相关的基因特征；采用荟萃基因分析来定义肿瘤表达 PR 基因特征的患者的生存率。
“SUMO 敏感”PR 靶基因主要包括增殖和生存信号所需的基因。去 SUMOylated K388R 受体优先募集到去抑制基因（即 MSX2、RGS2、MAP1A 和 PDK4）的增强子区域，与类固醇受体共激活剂 CREB（cAMP 反应元件结合蛋白）-结合蛋白（CBP）和混合谱系白血病 2（MLL2）结合，MLL2 是核小体重塑的组蛋白甲基转移酶介体。PR SUMOylation 阻止了这些事件，表明 PR 的 SUMO 修饰阻止了与“封闭”增强子区域中早期染色质重塑介体的相互作用。SUMO 缺陷（磷酸化 Ser294）PR 基因特征与人类表皮生长因子 2（ERBB2）阳性腔乳腺癌显著相关，并预测早期转移和缩短生存。用抗孕激素或 MEK 抑制剂治疗可消除 SUMO 敏感 PR 靶基因的表达，并抑制 BT-474（雌激素受体（ER）+/PR+/ERBB2+）乳腺癌细胞的增殖。
我们得出结论，PR 的可逆 SUMOylation/deSUMOylation 可极大地改变乳腺癌细胞中的靶基因选择。磷酸化诱导的 PR deSUMOylation 通过募集 CBP 和 MLL2 有利于允许的染色质环境。其 ER+/PR+ 肿瘤由过度活跃（即去抑制）的磷酸化 PR 驱动的患者可能受益于含有抗孕激素的内分泌（抗雌激素）治疗。
