Numb is a well known negative regulator of the Notch pathway. Numb is a cytoplasmic adaptor protein containing a phosphotyrosine-binding (PTB) domain, which acts as a negative regulator of Notch, through its interaction with the intracellular domain. Direct physical interactions involving the Numb PTB domain with the RAM23 and PEST domains of the Notch Intra Cellular Domain (NICD) have been reported in mammals. Numb‑Notch association promotes ubiquitination of Notch1. It has been demonstrated that Numb interacts with the cytosolic HECT domain of E3 ligase Itch, and that Numb and Itch act cooperatively to promote ubiquitination of Notch1. On the other hand, Numb may inhibit Notch signaling by altering the function of Tropomodulin, which is a positive regulator of the pathway. Tropomodulin encodes a four‑pass transmembrane protein, (with cytoplasmically located N‑ and C‑termini), which physically interact with the full length Notch receptor on the cell surface. Numb physically interacts with Tropomodulin and inhibits the membrane localization of Tropomodulin preventing its association with Notch.
To establish the role of Notch signaling in human breast cancer, the NICD protein levels and the expression of Numb were examined in many types and grades of human breast cancer samples. The expression of Numb was abolished in all breast cancer samples. In contrast, expression of NICD was upregulated in all breast cancer samples when compared to normal samples. More importantly, we have demonstrated that the attenuation of Notch signaling by Numb reverts the transformed phenotype of human breast cancer cell lines, including the high metastatic MDA‑MB231, and blocks tumor formation in vivo, indicating that inhibiting Notch signaling will prevent in vivo growth of human breast tumors.
|Attenuation of notch signaling pathway using numb inhibits tumor formation in immuno-deficient nude mice.|
Based on the above results, we genetically engineered a fusion protein, consisting of Antennapedia-Numb, and will be tested for its ability to target tumor cells in vivo.