Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Br J Cancer. 2010 Jan 19;102(2):351-60. doi: 10.1038/sj.bjc.6605486. Epub 2009 Dec 15.
Epithelial-to-mesenchymal transition (EMT) is associated with decreased adhesion and acquisition of metastatic potential of breast cancer cells. Epithelial-to-mesenchymal transition is mediated, in part, by two transcription repressors, Snail and Slug, that are known to be targets of the Notch signaling pathway, and JAGGED1-induced Notch activation increases EMT. However, the events that lead to increased Notch activity during EMT of breast cancer cells are unknown.
The accumulation of hypoxia inducible factors (HIFs) under hypoxia was detected by western blot analysis, and their effects on Notch signaling were measured by an in vitro Notch reporter assay. The expression of Notch target genes under hypoxia was tested by real-time PCR. The knockdown of HIF-1alpha was mediated by retroviral delivery of shRNA. The expression of Slug and Snail under hypoxia was measured by real-time PCR. Breast cancer cell migration and invasion under hypoxia were tested with cell migration and invasion kits.
Hypoxia increased the expression of Notch target genes such as HES1 and HEY1 in breast cancer cells, as was expression of Notch receptors and ligands. The mechanism is likely to involve the accumulation of HIF-1alpha and HIF-2alpha in these cells by hypoxia, which synergised with the Notch co-activator MAML1 in potentiating Notch activity. Hypoxia inducible factor-1alpha was found to bind to HES1 promoter under hypoxia. Knockdown of HIF-1alpha with shRNA inhibited both HES1 and HEY1 expression under hypoxia. Hypoxia increased the expression of Slug and Snail, and decreased the expression of E-cadherin, hallmarks of EMT. Notch pathway inhibition abrogated the hypoxia-mediated increase in Slug and Snail expression, as well as decreased breast cancer cell migration and invasion.
Hypoxia-mediated Notch signaling may have an important role in the initiation of EMT and subsequent potential for breast cancer metastasis.
上皮-间充质转化（EMT）与乳腺癌细胞黏附性降低和获得转移潜能有关。 EMT 部分由两个转录抑制因子 Snail 和 Slug 介导，它们是 Notch 信号通路的已知靶点，Jagged1 诱导的 Notch 激活增加 EMT。然而，在乳腺癌细胞 EMT 过程中导致 Notch 活性增加的事件尚不清楚。
通过 Western blot 分析检测缺氧诱导因子（HIFs）在缺氧下的积累，并通过体外 Notch 报告基因测定测量它们对 Notch 信号的影响。通过实时 PCR 测试 Notch 靶基因在缺氧下的表达。通过逆转录病毒递送 shRNA 介导 HIF-1α 的敲低。通过实时 PCR 测量缺氧下 Slug 和 Snail 的表达。通过细胞迁移和侵袭试剂盒测试缺氧下乳腺癌细胞的迁移和侵袭。
缺氧增加了 Notch 靶基因如 HES1 和 HEY1 在乳腺癌细胞中的表达，以及 Notch 受体和配体的表达。其机制可能涉及缺氧时这些细胞中 HIF-1α 和 HIF-2α 的积累，这与 Notch 共激活子 MAML1 协同增强 Notch 活性。发现缺氧诱导因子-1α 在缺氧下结合 HES1 启动子。用 shRNA 敲低 HIF-1α 抑制缺氧下 HES1 和 HEY1 的表达。缺氧增加了 Slug 和 Snail 的表达，并降低了 E-钙粘蛋白的表达，这是 EMT 的标志。Notch 通路抑制消除了缺氧介导的 Slug 和 Snail 表达增加以及乳腺癌细胞迁移和侵袭的减少。
缺氧介导的 Notch 信号可能在 EMT 的起始和随后的乳腺癌转移潜能中起重要作用。
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