Abstract B11: Cyclin D1 overexpression antagonizes Notch inhibition in the esophageal squamous epithelium

Abstract

Abstract Cyclin D1 overexpression is common in esophageal squamous cell carcinoma (ESCC) and other squamous cell cancers. Notch signaling is implicated in cell proliferation and differentiation, and may act as a tumor suppressor. While squamous differentiation is maintained in ESCC and its precursor lesions, the role of Notch in esophageal carcinogenesis remains elusive. The role of genetic interactions between cyclin D1 and the Notch signaling were explored in this study. Immortalized human esophageal cells EPC2-hTERT (keratinocytes) were retrovirally transduced with tetracycline-regulatable (tet-cyclin D1. Notch was activated by the Notch1 intracellular domain and inhibited by dominant negative mastermind-like1 (DN-MAML1), a genetic pan-Notch inhibitor. Notch activity was assessed by a 8xCSL-luciferase reporter assay. Quantitative RT-PCR, Western blotting and immunohistochemistry were performed. The squamous epithelium was reconstituted in organotypic (3-D) culture, a form of tissue engineering. Notch activation led to inhibition of cell proliferation and terminal differentiation in EPC2-hTERT with a robust induction of involucrin (an early differentiation marker). By contrast, DN-MAML1 inhibited the ICN-induced CSL-dependent transcriptional activity and Notch target genes, including Hey1, Hes5 as well as involucrin. In organotypic culture, the parental EPC2-hTERT cells formed a well-organized stratified squamous epithelium. In organotypic culture, DN-MAML did not affect basal cell proliferation, but severely impaired squamous maturation and terminal differentiation with loss of filaggrin expression (a late differentiation marker) in the suprabasal layer. In 3-D culture, Cyclin D1 overexpression resulted in basal cell hyperplasia and antagonized the DN-MAML1 effect. Thus, cyclin D1 appears to stimulate cell proliferation while restraining the Notch-mediated squamous differentiation. Citation Information: Cancer Res 2009;69(23 Suppl):B11.