Su1994 Loss of CFTR Results in Intestinal Stem Cell Hyperproliferation

Abstract

Standard methods such as western blot and flow-cytometry (protein expression); quantitative real time polymerase chain reaction (qPCR) (mRNA levels) and TCF/LEF reporter assay (βcatenin transcription) were used. Chromatin immunoprecipitation assay was performed with anti-CtBP2 antibody on spheroids. Results: Spheroid showed significant up regulation of CSCs factors (CD133, CXCR4, LGR5 and C-MYC), TCF/LEF activity as well as increased NADH/NAD ratio and enhanced binding of CtBP on p21 (a known CtBP target) promoter compared to monolayer controls. Depletion of CtBP2 inhibited, while its overexpression enhanced CSC growth (1° spheroid) and self-renewal (2°/3° spheroids). Similarly, MTOB caused a robust inhibition of CSC growth and self-renewal in a dose dependent manner (IC50: 250-700μM) in 4/5 cells tested. The corresponding IC50 for monolayer growth was 6-fold higher, suggesting selective targeting of CSCs. Moreover, MTOB treatment (1mM) caused robust apoptosis induction only in spheroids. Additionally, MTOB inhibited expression of CSCmarkers (CD133, CD44 and LGR5) and self-renewal factor (C-MYC) in spheroids. Moreover, MTOB inhibited basal as well as induced (GSK-3β inhibitor) TCF/LEF activity while inhibiting mRNA and protein levels of several β-catenin target genes (CD44, Snail, C-MYC and LGR5) and significantly reduced CtBP binding to LGR5 (a TCF/LEF target) and p21 promoters. Lastly, CtBP physically interacted with β-catenin. Conclusion: We have discovered a novel role of CtBP in promotion of CSC growth and self-renewal through direct regulation of TCF/LEF transcription. Moreover, small molecular inhibition of its function can selectively target CSCs, presenting a paradigm-shifting treatment approach for colorectal cancer.