Abstract 70: Loss of cellular senescence checkpoint functions reveals the oncogene characteristics of Notch1 in squamous cell carcinomas

Abstract

Abstract Introduction: Notch signaling regulates cell fates that are dependent upon the CSL transcription factor. Both tumor suppressor and oncogenic roles of Notch have been implicated in the pathogenesis of squamous cell carcinomas (SCCs). We investigated the functional consequences of Notch activation in head and neck as well as esophageal SCCs (HNSCC and ESCC). Methods: Primary tumor tissues annotated with known clinical outcomes were analyzed. Human esophageal cells immortalized with telomerase or human papilloma virus E6/E7 genes, their derivatives expressing mutant p53 and ESCC cell lines were stably transduced with ICN1, an active form of Notch1 in a regulatable manner (Tet-On system). Notch was inhibited by dominant negative mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor or γ-secretase inhibitors (GSI). 8xCSL-luciferase reporter was used to assess Notch-mediated transcriptional activity. RNA interference was done targeting either CSL or p16INK4A. Senescence was determined by cell growth inhibition and senescence-associated β-galactosidase assays. Cell growth was tested in soft agar and immunodeficient mice. Gene expression was determined by quantitative RT-PCR, Western blotting and immunohistochemistry (IHC). Results: In primary tumors, the active form of Notch1 (ICN1Val1744) was detected as intense nuclear staining of tumor cells in the invasive fronts in 29% of HNSCC (n=17) and 56% of ESCC (n=171). Nuclear ICN1Val1744 was significantly associated with a poor 5-year survival in postsurgical ESCC patients (n=115). In culture, ICN1 induced cellular senescence through CSL-dependent p16INK4A induction, which was antagonized by DNMAML1 or knockdown of either CSL or p16INK4A. Moreover, ICN1 failed to induce senescence in immortalized cells expressing E6/E7 or ESCC cells with a deleted INK4 locus. However, p53 mutation did not prevent ICN1-induced senescence. In soft agar and xenograft transplantation, ICN1 stimulated colony formation and tumor growth of ESCC cells that negated senescence. Histology revealed an increased number of less-differentiated tumor cells upon ICN1 induction. Moreover, GSI treatment of mice with xenografted ESCC cells resulted in tumor necrosis. Conclusions: These data indicate that Notch activation contributes to disease progression in ESCC. While ICN1 induces senescence, loss of p16INK4A-mediated senescence checkpoint point functions may be required in ICN1-mediated malignant transformation, thus providing a novel mechanistic insight into how Notch signaling may contribute to the pathogenesis of SCCs. 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 70. doi:1538-7445.AM2012-70