Abstract
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.
Highlights
Notch plays an oncogenic role in human breast cancer development
We investigated the effects of Notch4/Int[3] on NF-κB activation/signaling by analyzing the effects of Int[3] on activation of NF-κB promoter driven-reporter gene in HC11-Int[3] cells in presence of increasing doses of Int3-siRNA (Fig. 1C)
We have previously shown that this phenotype is dependent on the interaction of the Notch4-ICD/Int[3] with the transcription repressor/activator Rbpj[13]
Summary
Notch plays an oncogenic role in human breast cancer development. The increase of intracellular Notch and enhanced Notch signaling were observed in a variety of human breast carcinomas[1,2,3,4]. A total of 8 gene fusion rearrangements involving either Notch-1 or Notch-2 were discovered in a panel of 89 breast cancer cell lines and tumors. These gene fusions lead to the expression of the Notch intracellular domain[14]. The aberrant activation of Notch-4 signaling pathway has been proven to be associated with the development and progression of breast cancers. The data shows that Notch-4/Int[3] activation of NF-κB canonical signaling complex is Rbpj-independent. This activation is dependent on the CDC10/Ankyrin repeats in Int[3] which induces mammary tumors but not blockage of mammary gland development
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