Mechanistic And Functional Studies Of Notch Signaling In T-ALL And PDA

Objective Notch exerts important functions in cell proliferation, survival anddifferentiation,which plays a critical role in tumor development when aberrantlyactivated. Howerver, the mechanism whereby Notch activates tumor-relevant targetgenes and induces tumorigenesis is largely unknown. Using liquid tumors acuteT-lymphoblastic leukemia (T-ALL) and solid tumors pancreatic ductal carcinoma,combining approaches of cell biology and molecular biology, we studied themolecular mechanism by which Notch directly activates target genes in T-ALL andexamined anti-tumor activity of Notch inhibitors to block cell proliferation in PDA.The studies will help understand the molecular pathogenesis of Notch-mediatedtumorigenesis and may also direct toward the identification of small molecules orother bioactive agents to modulate NOTCH1with a view toward therapeuticapplications.Methods Notch in T-ALL: Murine T-ALL line G4A2and T6E and were treatedwithγ-secretase inhibitor compound E (GSI) for NOTCH1inhibition, then Rag1,Rag2mRNA and Rag1protein were detected by real-time PCR and Western Blot. Tomeasure the mRNA levels of Rag1and Rag2in the “Notch-off” condition, T6E cellswere transduced with DNMAML1or empty vector MigR1surrogated with a GFPmarker, followed by real-time PCR to analyze Rag1and Rag2abundance.MurineT-ALL line were treated with GSI,then we determined whether Notch1directly bindsand stimulates Rag1and Rag2genes by chromatin immunoprecipitation.Notch in PDA: The survival rate of human PDA cells was determined by MTTupon GSI treatments. Similarly, effects of GSI and gemcitabine double treatmentswere monitored with MTT. mRNA of tentative Notch targets c-Myc, Hes1, Jagged1and Jagged2was detected by real-time PCR in the presence or absence of GSI.Results Notch in T-ALL:Transcripts of Rag1and Rag2in T6E and G4A2cellswere dramatically decreased upon GSI-mediated Notch inhibition, and the Rag1protein level went down as well. Expression of a dominant negative MAML1(DNMAML1) to repress the activity of Notch1transcriptional complex to turn off gene expression consistently resulted in sharp decrease of Rag1and Rag2mRNAabundance. We analyzed the genome-wide Chip-seq data which revealed Notch1andCSL binding regions in the murine T-ALL cell line T6E, and used conventional ChIPassay to confirm the Notch1binding to5’ upstream of Rag2gene.Notch in PDA: Although GSI treatment alone resulted in a marginal decrease incell numbers in various PDA cell culture, combination of GSI nd gemcitabinemanifested an appreciable decrease in terms of survival rate. RT-PCR shows that theexpression level of the Notch1downstream genes c-Myc, Hes1, Jagged1and Jagged2mRNA declined. Hes1appeared downregulated in all PDA cells tested, while c-Mycseemed more specifically affected in PDA cells in later stage.Conclusion Notch signaling pathway directly regulated Rag1and Rag2in acuteT-lymphoblastic leukemia, suggesting their roles in mediating Notch function indeveloping and/or transforming T cells. In pancreatic ductal adenocarcinoma, theeffect of GSI inhibiting pancreatic ductal adenocarcinoma cancer cell proliferation isnot obvious, combination therapy with gemcitabine enhance its role in inhibition ofcell proliferation. Hes1were dramatically decreased in different PDA，and it may playan important role in carcinogenesis. c-Myc specifically regulated in later stage PDAcells,which suggested c-Myc medicates important functions of Notch downstream.