Abstract 746: Notch-1 expression in human cancer cell lines and solid tumors by validated immunohistochemistry
Authors: Nguyen Dat , Witter Sara , Tomaszewski Joseph E. , Ivy Percy , Doroshow James H. , Yang Sherry X. 
Published: August 2012

Abstract
Notch-1 is a transmembrane receptor whose activation upon ligand binding or calcium depletion leads to translocation of the Notch-1 intracellular domain (NICD) to the nucleus where it activates transcription of Notch target genes. Targeting Notch signaling, such as with inhibitors of γ-secretase that is required for Notch-1 activation, is currently in early clinical development. It is critical to characterize the expression of Notch-1, an important member of Notch family of receptors in human cancers with a validated assay suitable to monitor pharmacodynamic responses to Notch inhibitors and to carry out clinical validation investigation of Notch-1 as predictive/prognostic biomarker. This could improve our understanding of pathobiology of Notch signaling and in turn increase the odds of success of targeting Notch signaling. The aims of this study were to develop an immunohistochemistry assay for Notch-1/NICD (activated Notch-1), and to evaluate expression levels and patterns of Notch-1/NICD in paraffin-embedded tumor samples. Human breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468, and colorectal cancer HCT-116 cells were treated with and without a calcium chelator EDTA for 10 min and embedded in paraffin after formalin fixation. Expression levels and patterns of Notch-1 were examined by Western blot, and immunocytochemistry as well as immunohistochemistry and were quantitated using an Automated Cellular Imaging System. There was an increase in Notch-1/NICD expression in cells treated with EDTA versus without EDTA in MCF-7 (staining index: 28 vs. 10) and MDA-MB-231 (81 vs. 62) cells by immunocytochemistry and confirmed by Western blot. Interestingly, membranous/cytoplasmic Notch-1 was in part translocated into the nucleus in MCF-7 and MDA-MB-468 cells following EDTA treatment, suggesting Notch-1 activation and specific detection of Notch-1 in distinct cellular compartments. In addition, a predominant cytoplasmic/membranous expression of Notch-1 was observed in MDA-MB-231 and HCT-116 cells with and without EDTA treatment. Low to intermediate levels of Notch-1 with a predominantly cytoplasmic localization, relative to moderate to high levels in cell lines, were detected in 13% (6/45), 4% (2/48), 16% (7/45) of tumor cells in carcinomas of the breast, lung, and ovary. Nuclear/cytoplasmic staining of Notch-1 was observed in one case of lung cancer. In addition, Notch-1 expression was found in a significant fraction of endothelial cells of human solid tumors. We have established a fit-for-use immunohistochemistry assay with an antibody that specifically detects Notch-1 in formalin-fixed and paraffin-embedded human tumors. Its application could facilitate the evaluation of pharmacodynamic responses to various Notch targeting agents undergoing clinical development and subsequent clinical validation studies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 746. doi:1538-7445.AM2012-746