Abstract 5028: The lymphovascular embolus of inflammatory breast cancer exhibits a selective notch3 addiction
Authors: Xiao Y , Ye Yin , Yearsley Kurtis , Barsky Sanford H. 
Published: February 2011

Abstract
Inflammatory breast carcinoma (IBC) is characterized by exaggerated lymphovascular invasion (LVI), recapitulated in our human xenograft, MARY-X. This model exhibited lymphovascular emboli in vivo and corresponding spheroids in vitro. Because of the morphological and gene profile resemblance of these spheroids to embryonal blastocysts, we wondered whether they might exhibit embryonic stem cell signaling. Specifically we investigated notch signaling and observed selective notch3 activation by expression profiling, RT-and real time PCR, Western blot and immunofluorescence in vitro and immunohistochemistry (IHC) in vivo. Notch3 was selectively overexpressed, cleavage of its intracellular domain (N3icd) nuclear translocated and target genes: HES-5, HEY-2, c-Myc, Deltex-1, NRARP and PBX1 were significantly upregulated. In IBC, notch3, in contrast to notch1, occupied a very central role in maintaining its stem cell signature as well as its embolic phenotype. For example CD133, a stem cell marker, was expressed in virtually 100% of MARY-X cells. Blockade of notch3 but not notch1 signaling dramatically decreased CD133 in MARY-X spheroids. Introduction of the notch3 intracellular domain (N3icd) but not the notch1 intracellular domain (N1icd) in human mammary epithelial cells (HMEC) partially reproduced the unique phenotype of the MARY-X spheroids, such as CD133 expression and anchorage-independent growth in vitro. Notch3 contained a specialized TAD domain in its C-terminal region, which would be expected to preferentially activate promoters with zinc-finger binding sites near a CSL-binding site, such as the HES-5 promoter. Interestingly, MARY-X exhibited the highest level of HES-5 (&gt;148 fold greater than all other cell lines tested). Initial notch3 processing differed from conventional notch1 processing. Notch1 is thought to be cleaved by furin which allows it be activated by EDTA. Notch3 was not cleaved by furin as demonstrated by furin inhibitor studies nor activated by EDTA. A significant percentage of MARY-X cells expressed aldehyde dehydrogenase (ALDH), a stem cell marker. Sorted ALDH+ cells expressed increased notch3 signaling of HES-5 and HEY-2. Inhibiting notch3 activation in vitro with γ-secretase inhibitors (GSI) or siRNA resulted in caspase 3-mediated apoptosis, independent of E-cadherin; on the other hand, inhibiting E-cadherin adhesion caused spheroid disadherence, notch3 signaling inhibition and apoptosis. GSI in vivo resulted in similar but diffusion-limited effects on notch3 signaling inhibition, caspase 3 activation and apoptosis resulting in xenograft growth reduction. The lymphovascular emboli of human IBC independent of molecular subtype exhibited dual N3icd and ALDH1 immunoreactivities. This unique notch3 addiction of lymphovascular emboli might be exploited in future GSI or gene knockdown therapeutic strategies aimed at IBC.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5028.