677 Inactivation of the Retinoblastoma Protein-Dependent Senescence Checkpoint Functions Permits Notch1 to Gain Oncogenic Tumor Promoting Activity

Abstract

Introduction:Notch1-mediated signaling influences cell fate decisions, cell cycle and carcinogenesis via a transcription activation complex consisting of ICN1 (the activated form of Notch1), the co-activatorMAML and the transcription factor CSL. However, the consequences of Notch activation are complex and context dependent. The role of Notch signaling in esophageal epithelial biology andmalignant transformation remain elusive. Methods:Human esophageal keratinocytes immortalized by telomerase (EPC1-hTERT and EPC2-hTERT) as well as non-transformed and transformed derivatives including EPC2T overexpressing EGFR, cyclin D1 and p53R175H; transformed human esophageal keratinocytes carrying a human papilloma virus (HPV)-E6E7 fusion oncogene (EN60) and esophageal squamous cell carcinoma (ESCC) cell lines were genetically engineered to express tetracycline-inducible ICN1; dominant-negative MAML1 (DNMAML1), a genetic pan-Notch inhibitor; or short hairpin RNA (shRNA) directed against CSL, HPV-E7 or cyclin-dependent kinase inhibitor p16INK4A. Notch activity was determined by transfection assays of an 8xCSL-luciferase reporter construct. Notch target genes were determined by quantitative RT-PCR. Western blotting determined retinoblastoma (RB) protein phosphorylation status. Cell proliferation was determined by colorimetric assays. Anchorage-independent colony formation was evaluated in soft agar. Tumor formation was assessed by xenograft transplantation in immunodeficient mice. Senescence associated β-galactosidase (SABG) activity was determined. Results: ICN1 activated CSL-dependent transcription in all cell lines tested and this activation was antagonized by either DNMAML1 expression or CSL knockdown. In response to ICN1 induction, nontransformed cells underwent growth arrest displaying flat and enlarged cell morphology with SABG positive staining (40-80%), suggesting senescence. ICN1-mediated senescence was found to be CSL-dependent and was associated with decreased RB protein phosphorylation and p16INK4A upregulation. ICN1-mediated senescence was inhibited by p16INK4A knockdown, but not the presence of p53R175H. ICN1 failed to induce senescence in EN60 cells expressing EPV-E7 protein or ESCC cell lines lacking the INK4A locus. ICN1 stimulated colony formation and tumor growth in these transformed cell lines. HPV E7 knockdown in EN60 cells resulted in senescence with activation of endogenous Notch1. Conclusions: These novel data indicate that loss of the RB protein-dependent senescence checkpoint function may be permissive for Notch1 to facilitate malignant transformation in human esophageal epithelial cells.