Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20814, USA.
Sci Rep. 2017 Oct 20;7(1):13690. doi: 10.1038/s41598-017-13989-7.
Transgenic mice expressing the Notch-4 intracellular domain (designated Int3) in the mammary gland have two phenotypes exhibited with 100% penetrance: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch-4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. Interestingly, WAP-Int3/Rbpj knockout mice have normal mammary gland development but still developed mammary tumors with a slightly longer latency than the WAP-Int3 mice. Thus, Notch-induced mammary tumor development is Rbpj-independent. Here, we show that Int3 activates NF-κB in HC11 cells in absence of Rbpj through an association with the IKK signalosome. Int3 induced the canonical NF-κB activity and P50 phosphorylation in HC11 cells without altering the NF-κB2 pathway. The minimal domain within the Int3 protein required to activate NF-κB consists of the CDC10/Ankyrin (ANK) repeats domain. Treatment of WAP-Int3 tumor bearing mice with an IKK inhibitor resulted in tumor regression. In a soft agar assay, treatment of HC11-Int3 cells with P50-siRNA caused a significant decrease in colony formation. In addition, Wap-Int3/P50 knockout mice did not develop mammary tumors. This data indicates that the activation of NF-κB canonical signaling by Notch-4/Int3 is ANK repeats dependent, Rbpj-independent, and is mediated by IKK activation and P50 phosphorylation causing mammary tumorigenesis.
在乳腺中表达 Notch-4 细胞内结构域（命名为 Int3）的转基因小鼠表现出两种表型，具有 100%的外显率：乳腺腺泡/小叶发育停滞和乳腺肿瘤发生。Notch-4 信号主要通过 Int3 与转录抑制因子/激活子 Rbpj 的相互作用来介导。有趣的是，WAP-Int3/Rbpj 敲除小鼠具有正常的乳腺发育，但仍发展出乳腺肿瘤，潜伏期比 WAP-Int3 小鼠略长。因此，Notch 诱导的乳腺肿瘤发展与 Rbpj 无关。在这里，我们表明 Int3 在没有 Rbpj 的情况下通过与 IKK 信号体的关联在 HC11 细胞中激活 NF-κB。Int3 在不改变 NF-κB2 途径的情况下诱导 HC11 细胞中的典型 NF-κB 活性和 P50 磷酸化。激活 NF-κB 所需的 Int3 蛋白的最小结构域包含 CDC10/Ankyrin（ANK）重复结构域。用 IKK 抑制剂治疗 WAP-Int3 荷瘤小鼠导致肿瘤消退。在软琼脂测定中，用 P50-siRNA 处理 HC11-Int3 细胞导致集落形成显著减少。此外，Wap-Int3/P50 敲除小鼠不会发生乳腺肿瘤。这些数据表明，Notch-4/Int3 通过 IKK 激活和 P50 磷酸化激活 NF-κB 经典信号通路，这是依赖于 ANK 重复、不依赖于 Rbpj 的，导致乳腺肿瘤发生。
