| Backgroud and objectiveColorectal cancer(CRC)is a leading cause of morbidity and mortality in developed countries.The cancer stem cells(CSC)which exhibit stem-like features,sustain tumor formation,metastasis,and resistance to therapy has been proposed to explain the functional heterogeneity and carcinogenesis of cancer.Achaete scute-like 2(Ascl2/Mash2/Hash2)plays a critical role in intestinal Lgr5+ cryptic stem cells and CRC progenitor cells.Ascl2 is overexpressed in colorectal cancer and has the potential to shift the hierarchy of stem and progenitor cells within liver metastases,resulting in self-renewal rather than differentiation and potentially affecting the clinical behavior of these tumors.Ascl2 is a downstream target of Wnt signaling pathway,but recently,Ascl2 was reported can be affected by Hippo signalling pathway.The Hippo signaling pathway is a critical pathway to determine cell growth rate and organ size.YAP1/TAZ,an effector of the Hippo signaling pathway,is activated in many human tumors,where YAP1/TAZ has been shown to be essential for cancer initiation,progression,or metastasis.These two pathways are known to be important for epithelial development and homeostasis and there is accumulating evidence that the Hippo cascade engages in crosstalk with Wnt signaling in epithelial tissues.Understanding the molecular mechanisms connecting these two signaling will require further investigation.We provided an evidence in this study to confirm the molecular mechanism how Hippo signaling pathway regulated Ascl2 expression in CRC progenitor cells.Mucin-type Core 3 O-glycan is biosynthesized by the enzyme β1,3-N-acetylglucosaminyltransferase-6(β3gnT6,Core 3 synthase),which is primarily expressed in the human stomach,colon and small intestine.Core 3 synthase is dramatically decreased in cancer cells,including gastric and colorectal cancer.Ectopic expression of Core 3 synthase in human pancreatic cancer inhibits metastasis via deregulation of α2β1 integrin.Forced expression of Core 3 synthase in human CRC cells has been shown to suppress their metastatic ability.But the mechanism how Core 3 O-glycan moderates tumor metastasis is still undefined.In this study,we found Core 3 synthase reexpression in CRC cells induces their mesenchymal-epithelial transition(MET).Methods1.Ascl2 regulation by Hippo signaling pathway in colon cancer1.1 Fluorescence-activated cell sorting and flow cytometry was used to separate HT-29 and Caco-2 cells into CD133+CD44+ and CD133-CD44-CRC cell population.The proliferation,colony formation and tumoursphere formation were investigated and Ascl2,KLF5,Hippo signaling pathway and ‘stemness’ associated genes(CD133,CD44,Bmi1,C-myc and Oct4)were tested by real-time PCR and WB in each groups.1.2 HT-29 and Caco-2 CD133+CD44+ were transfected with YAP1 si RNA and the efficiency was tested by WB.After transfection,proliferation and colony formation were tested.CD133+CD44+ percentages were detected by Flow cytometry.Ascl2,KLF5,Hippo signaling pathway and ‘stemness’ associated genes were tested by WB in each groups.1.3 HT-29 and Caco-2 were transfected with lentivirus particles using a LV5(EF-1a F/GFP&Puro)with YAP1 insert and the efficiency was tested by PCR and WB.After transfection,proliferation,colony formation and tumoursphere formation were tested.CD133+CD44+ percentages were detected by Flow cytometry.Ascl2,KLF5,Hippo signaling pathway and ‘stemness’ associated genes were tested by real-time PCR and WB in each group.1.4 Co-immunoprecipitation(Co-IP)was used to test whether YAP1 bound with KLF5 and vice versa in HT-29 and Caco-2 colon cancer cells.YAP1 and KLF5 bound to the proximal promoter of Ascl2 and transcriptionally activated Ascl2 gene were clarified by chromatin immunoprecipitation(ChIP)assay and dual luciferase experiment.1.5 Correlation between Ascl2,YAP1 and KLF5 expression levels in CRC samples.2.Core 3 O-glycan induces MET in CRC2.1 Glycosyl transferase expression in various colorectal cancer cell lines detected by real-time PCR.2.2 HT-29 and LS174 T were transfected with lentivirus particles using a LV5(EF-1aF/GFP&Puro)with Core 3 synthase insert and the transfection efficiency were detected by real-time PCR and WB.2.3 The MTT,migration and invasion assays,and in vivo tumor xenografts were used to investigate the effect of Core 3 synthase re-expression in HT-29 and LS174 T cells on their cellular biological behavior.2.4 EMT-related indicators(N-cadherin,E-cadherin,snail,slug,ZEB1 and ZEB2)were detected by real-time PCR and WB inβ3gn T6/HT-29 or β3gnT6/LS174 T cells and their tumor xenografts.Results1.Ascl2 regulation by Hippo signaling pathway in colon cancer1.1 CD133+CD44+ CRC cell population from HT-29 and Caco-2 cells had higher abilities of proliferation,colony formation and tumorsphere formation.Expression of Ascl2,KLF5,YAP1,and ‘stemness’ associated genes were higher in CD133+CD44+ CRC cell population,MST1 and p-YAP1 were lower in CD133+CD44+ CRC cell population1.2 YAP1 knockdown in CD133+CD44+ CRC cell population reduced their proliferation,colony formation,CD133+CD44+ percentages,Ascl2,KLF5 and ‘stemness’ associated genes expression and YAP1 nucleus translocation.1.3 YAP1 enforced expression in HT-29 and Caco-2 cells increased their proliferation,colony formation,percentages of CD133+CD44+ population,tumorsphere formation,Ascl2,KLF5 and ‘stemness’ associated genes expression and YAP1 nucleus translocation.1.4 YAP1 and KLF5 interacted and bound to the human Ascl2 promoter,their binding was decreased in YAP1 interfered CD133+CD44+ CRC cell population and increased in lv-YAP1/HT-29 and lv-YAP1/Caco-2 cells.Luciferase assay and Ch IP assay indicated that both YAP1 and KLF5 bound to the first two loci with GC-box residing at the proximal promoter of Ascl2 and induced its transcriptional activation.We found that the decreased Ascl2 transcription by YAP1 interference required an intact KLF5 binding site(GC-box,GGGCGG)in the Ascl2 promoter.Increased Ascl2 promoter activity in lv-YAP1/HT-29 and lv-YAP1/Caco-2 cells was evident from luciferase reporter assays than their control cells,GC-box mutation led to a significant reduction of Ascl2 promoter activity.1.5 Ascl2,YAP1 and KLF5 mRNA levels in cancerous tissues were significantly higher than normal mucosa.Ascl2 mRNA expression level was correlated with KLF5 and YAP1 expression in human CRC samples.2.Core 3 O-glycans induce MET in CRC2.1 Extremely low Core 3 synthase mRNA level was observed in HT-29 and LS174 T colon cancer cell lines.2.2 The screening of the most efficient clones(clone1-3)for stably transfection using lentivirus particles expressing Core 3 synthase were determined by Core 3 synthase mRNA level and its protein level.The clone 1 had the highest expression of Core 3 synthase.2.3 Core 3 synthase enforced expression in HT-29 and LS174 T cells attenuated their proliferation,migration and invasion in vitro and suppressed tumor formation in vivo.2.4 The induction of E-cadherin and the repression of N-cadherin,snail,and slug in β3gnT6/HT-29 or β3gnT6/LS174 T cells and their tumor xenografts indicate the reversal of epithelial-mesenchymal transition(EMT).Conclusions1.YAP1 interaction with KLF5 transcriptionally activates Ascl2 expression and induces self-renewal of colon cancer progenitor cells.2.Core 3 O-glycans induce MET in CRC. |
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