Abstract 5506: NOTCH1 activation in head and neck squamous cell carcinoma leads to growth inhibition, changes in gene expression associated with early differentiation, and acquisition of stem cell-like properties
Authors: Huang Chenfei , Moorthy Shhyam , Li Qiuli , Saade Rami , Wang Jiping , Rao Xiayu , Tanaka Noriaki , Zhang Jiexin , Tang Lin , Pickering Curtis R. , Zweidler-McKay Patrick A. , Osman Abdullah A. , Xie Tong-Xin , Shinbrot Eve , Xi Liu , Wheeler David , El-Naggar Adel K. , Wang Jing , Myers Jeffrey N. , Frederick Mitchell J. 
Published: August 2018

Abstract
Background: The NOTCH1 gene functions as either an oncogene or tumor suppressor in cancer depending upon the tumor type. Our group previously characterized the genomic alterations in head and neck squamous cell carcinoma (HNSCC), discovering that NOTCH1 is frequently altered with a pattern of inactivating mutations suggesting it is a tumor suppressor in this cancer type. However, recent work by others suggests NOTCH1 signaling plays a more complex role, possibly promoting a more aggressive phenotype or cancer stem cell-like properties in HNSCCs with wild type NOTCH1. Our present study aimed to systematically compare the phenotypic consequences of NOTCH1 signaling in HNSCC to better understand its function in cancer, and identify targets downstream of NOTCH1 signaling. Methods: Established HNSCC cell lines wild type for NOTCH1 (PJ34, FADU) or harboring an inactivating mutation (UMSCC22A) were engineered to express activated cleaved NOTCH1 (cl-NOTCH1) from a doxycycline-inducible promoter. In vitro cell growth was measured with clonogenic assays. Stem cell-like properties were measured by orosphere formation and anoikis resistance. Stem cell markers for HNSCC including Aldehyde dehydrogenase activity (ALDH), CD133, and CD44 expression were measured by flow cytometry. NOTCH1-regulated downstream gene expression changes were examined by RNA-seq and qRT-PCR. Results: Activation of NOTCH1 inhibited clonogenic growth of all three cell lines, regardless of original NOTCH1 gene status. Growth inhibition was frequently accompanied by spontaneous formation of spheroid-like structures, characteristic of stem cells. NOTCH1 activation in UMSCC22A and FADU cells promoted orosphere formation and anoikis resistance, conveying some stem cell-like properties. However, classical stem cell markers including ALDH activity, CD133, and CD44 expression were not affected by NOTCH1 activation. Furthermore, RNA-seq demonstrated that critical cancer-associated pathways, including proliferation, differentiation, and migration, were regulated by NOTCH1. NOTCH1 activation downregulated gene expression of ITGA3, ITGA4, ITGB1, ITGB6, and LAMC2, which are key adhesion molecules that human basal keratinocytes use for attachment to the basement membrane and maintenance of the stem cell compartment. Concomitantly, NOTCH1 activation increased the basal/superbasal marker SOX2, but also the early differentiation markers KRT4 and KRT13. SiRNA-mediated SOX2 silencing blocked NOTCH1-promoted anoikis resistance. Conclusion: NOTCH1 activation inhibits in vitro growth regardless of mutational status. We hypothesize that stem cell-like properties associated with NOTCH1 activation in HNSCC may be a consequence of pathways that recapitulate early differentiation, rather than true stem cell maintenance.
Citation Format: Chenfei Huang, Shhyam Moorthy, Qiuli Li, Rami Saade, Jiping Wang, Xiayu Rao, Noriaki Tanaka, Jiexin Zhang, Lin Tang, Curtis R. Pickering, Patrick A. Zweidler-McKay, Abdullah A. Osman, Tong-Xin Xie, Eve Shinbrot, Liu Xi, David Wheeler, Adel K. El-Naggar, Jing Wang, Jeffrey N. Myers, Mitchell J. Frederick. NOTCH1 activation in head and neck squamous cell carcinoma leads to growth inhibition, changes in gene expression associated with early differentiation, and acquisition of stem cell-like properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5506.