Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor-suppressor activities

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Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor-suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor-suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated β-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16INK4A-Rb pathway. Loss of p16INK4A or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as transforming growth factor-β-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor-suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.

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We thank Dr Hiroshi Shirasawa (Chiba University, Chiba, Japan) for the gift of EN60 cells. We are grateful to the Molecular Pathology & Imaging, Molecular Biology/Gene Expression and Cell Culture Core Facilities of the NIH/NIDDK Center for Molecular Studies in Digestive and Liver Diseases (P30-DK050306) and of the NIH P01CA098101. This study was supported in part by NIH Grants P01CA098101 (to SK, MN, KAW, DB, HK, SN, SO, PAG, AJK-S, AB, K-KW, JAD, HN and AKR), U01CA143056 (to AKR), R01DK077005 (to HN), K26 RR032714 (to HN), Pennsylvania CURE Program Grant (to HN), F32-CA174176 (to KAW), F32-DE024685 to (NF), K08DE022842, Trio/ACS Career Award and VA CPPF Grant (to DB), K07CA137140 (to AME), University of Pennsylvania University Research Foundation Award (to HN), University of Pennsylvania, Abramson Cancer Center Pilot Project Grant (to HN), and the American Cancer Society RP-10-033-01-CCE (to AKR).

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Correspondence to H Nakagawa.

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Kagawa, S., Natsuizaka, M., Whelan, K. et al. Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor-suppressor activities. Oncogene 34, 2347–2359 (2015) doi:10.1038/onc.2014.169

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