Fig. 1

ASPH activates Notch signaling pathway in breast cancer. (a) First Row Left: Histopathology of 2 representative tumors derived from breast cancer patients; First Row Right: Patient #1 and #2 had ASPH negative vs. positive tumor, respectively, by IHC. Second to Fourth Row: Consistent downregulation vs. upregulation of Activated Notch1, MMP-2, MMP-9, and ADAM17/TACE in ASPH negative vs. positive breast cancer patients. (b-c) ASPH activates Notch signaling pathways through physical interaction with Notch receptors, ligands and regulators in breast cancer. ASPH physically interacts with Notch1 extracellular domain (ECD), JAG1, ADAM10 and ADAM17 as demonstrated by co-IP. ASPH enhances physical interaction between Notch1 and JAG1. (d) Expression profiling of Notch signaling core components in response to SMI. (e) Luciferase reporter to detect the activation of Notch signaling in breast cancer, in the presence of full-length (FL) Notch1 and/or ASPH. Reporter alone served as a negative control whereas active Notch1 (Notch intracellular domain; NICD) alone as a positive control. ASPH activates Notch signaling in presence of FL Notch1, which was inhibited by SMI and DAPT. (f) ECM degradation/remodeling in response to SMI and DAPT, respectively. (g) 3-D invasion in response to SMI and DAPT, respectively. (h) Mammosphere formation in response to SMI and DAPT, respectively. (i) Scheme of in vitro metastasis assay. (j) Transendothelial migration (intravasation/extravasation); (k) Invasion through basement membrane and subsequent mammosphere formation in response to SMI and DAPT, respectively. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001