Novel Role of ErbB-2 in Inhibition of Jagged-1-Mediated Trans-Activation of Notch in Breast Cancer

The ErbB-2 gene is amplified and the resulting protein product overexpressed in 15-30% of breast tumors, and associated with aggressive behavior and poor overall survival. Currently, there are two FDA approved therapies targeting ErbB-2 for the treatment of ErbB-2 positive breast cancer: trastuzumab, a humanized monoclonal antibody is directed against the extracellular domain of ErbB-2 and lapatinib, a dual EGFR/ErbB-2 tyrosine kinase inhibitor. Unfortunately, anti-ErbB-2 therapy resistance remains a major problem in metastatic breast cancer. Our data suggested that gene amplification or overexpression of ErbB-2 inhibits Notch-1 transcriptional activity and trastuzumab or lapatinib increased Notch-1 transcriptional activity. Furthermore, Notch-1 is a breast oncogene and a novel target for the treatment of trastuzumab resistant ErbB-2 positive breast cancer in vitro. The Notch-1 receptor is overexpressed with its ligand Jagged-1 in breast cancers with the poorest overall survival. We showed that ErbB-2 inhibition activates Notch-1 which results in a compensatory increase in Notch-1-mediated proliferation. However, we do not yet know the mechanism by which ErbB-2 overexpression suppresses Notch-1 activity and whether inhibition of Notch-1 would reverse resistance to trastuzumab in vivo. Our results demonstrated that trastuzumab or lapatinib treatment of SKBr3 cells increased the cell surface protein expression of Jagged-1 by flow cytometry and cell surface biotinylation. Moreover, confocal studies indicated that Jagged-1 and Notch-1 co-localized possibly in early endosomal antigen-1 (EEA-1) positive vesicles. However, upon treatment with trastuzumab, Jagged-1 and Notch-1 don\u27t co-localize. Jagged-1 is present at the plasma membrane and Notch-1 is distributed throughout the cell. In SKBr3 and MCF-7/HER-2 breast cancer cells, ErbB-2 stabilizes the protein levels of Jagged-1. We demonstrated for the first time that Jagged-1 inhibits Notch in cis. More interestingly, ErbB-2 prevents Jagged-1-mediated trans-activation of Notch signaling by limiting the association of Jagged-1 and Mib-1 and subsequent ubiquitylation of Jagged-1. Moreover, Mib-1 is the E3 ubiquitin ligase required for lapatinib-mediated ubiquitylation of Jagged-1 and induction of Notch activity. Additionally, ErbB-2 promotes an association between Jagged-1 and PKCá. Further, PKCá inhibits Notch transcriptional activity. Importantly combined inhibition of Jagged-1 by siRNA and ErbB-2 by trastuzumab significantly growth arrested SKBr3, BT474 HS, and BT474 HR cells in G1 phase of the cell cycle and induced cell death in vitro. Combined inhibition of Notch and ErbB-2 signaling pathways could decrease recurrence rates for ErbB-2 positive breast tumors and may be beneficial in the treatment of recurrent trastuzumab resistant disease. Our studies will elucidate the mechanism by which ErbB-2 and Notch pathways crosstalk in ErbB-2 positive breast cancer cells. Mechanisms underlying transactivation and cis-inhibition of Notch by its ligand even though not well characterized yet are critical processes regulating Notch activity. These findings will provide a mechanism and functional relevance of Jagged-1-Notch interactions in ErbB-2 positive breast cancer cells. Furthermore, these studies will identify Jagged-1 as a novel and better therapeutic target for the treatment of ErbB-2 positive breast cancer. Finally, these studies will provide a preclinical proof of concept for future clinical trials using combination of trastuzumab or lapatinib and a Notch pathway inhibitor (GSI or Jagged-1 targeted therapy) for the treatment of ErbB-2 positive breast cancer