Knutson Todd P, Truong Thu H, Ma Shihong, Brady Nicholas J, Sullivan Megan E, Raj Ganesh, Schwertfeger Kathryn L, Lange Carol A
Departments of Medicine (Division of Hematology, Oncology, and Transplantation) and Pharmacology, Masonic Cancer Center, University of Minnesota, Delivery Code 2812, Cancer and Cardiovascular Research Building, 2231 6th St SE, Minneapolis, MN, 55455, USA.
Department of Urology, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, J8.130C, Dallas, TX, 75390-9110, USA.
J Hematol Oncol. 2017 Apr 17;10(1):89. doi: 10.1186/s13045-017-0462-7.
Estrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities.
To better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology.
Phospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate.
We conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence.
雌激素和孕激素是强效的乳腺有丝分裂原。除了甾体激素外，多种信号通路通过翻译后事件输入到雌激素受体（ER）和孕激素受体（PR）的作用中。在乳腺癌中通常被激活的蛋白激酶使甾体激素受体（SRs）磷酸化，并深刻影响其活性。
为了更好地理解修饰后的PRs在乳腺癌中的作用，我们在组织芯片上的2754个个体组织斑点所代表的209个人类乳腺肿瘤中测量了总PRs和磷酸化Ser294 PRs，并在体外培养的人类肿瘤外植体中检测了该位点的调控情况。为了补充这一分析，我们在T47D管腔型乳腺癌模型中，在受体受赖氨酸388 SUMO化调控（K388完整）或SUMO缺陷（通过K388R突变模拟持续的Ser294磷酸化）的条件下，用孕激素（普罗孕酮；R5020）和抗孕激素（米非司酮、奥那司酮或阿格列司酮）处理后，检测PR靶基因的调控情况。通过qRT-PCR以及在乳腺癌细胞系中敲低RUNX2后，对选定的磷酸化PR驱动的靶基因进行了验证。进行了原代和二代乳腺球试验，以阐明磷酸化Ser294 PRs、表皮生长因子信号传导和RUNX2在乳腺癌干细胞生物学中的作用。
磷酸化Ser294 PR种类在大多数（54%）管腔型乳腺肿瘤中含量丰富，并且PR启动子选择性对翻译后修饰极为敏感。磷酸化PR的表达和靶基因程序与浸润性小叶癌（ILC）显著相关。与我们发现激活的磷酸化PRs经历快速的配体依赖性周转一致，独特的磷酸化PR基因特征在临床上被指定为PR低至PR无（管腔B型）的乳腺肿瘤中最为普遍，并且包括与癌症干细胞生物学相关的基因集（HER2、PAX2、AHR、AR、RUNX）。验证研究表明，RUNX2是调控选定的磷酸化PR靶基因（SLC37A2）所必需的。体外乳腺球形成试验支持磷酸化Ser294-PRs通过生长因子（EGF）信号传导以及RUNX2作为乳腺癌干细胞命运的强效驱动因子发挥作用。
我们得出结论，PR Ser294磷酸化是乳腺癌进展中的常见事件，是维持乳腺癌干细胞命运所必需的，部分是通过与生长因子启动的信号通路以及包括SLC37A2和RUNX2在内的关键磷酸化PR靶基因合作实现的。磷酸化PRs的临床检测应被视为乳腺肿瘤干细胞潜能的有用标志物。或者，独特的磷酸化PR靶基因集可能提供有用的工具，用于识别可能对阻断PR Ser294磷酸化的选择性PR调节剂有反应的患者，作为合理联合（即与抗雌激素）内分泌疗法的一部分，旨在持久阻断乳腺癌复发。
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