Research On The Mechanisms Of Molecular Effects Of Zinc Finger Protein 70 In The Development Of Colitis-associated Colorectal Cancer And Anti-cancer Activity Of Convallatoxin In Colorectal Cancer

Background: IL-1? is a key pro-inflammatory mediator crucial for local and systemic inflammation.NLRP3 inflammasome and STAT3 activation could promote IL-1?secretion in macrophages.IL-1? could promote inflammation associated cancer.Aberrant activation of STAT3 is frequently encountered and promotes survival,cellular proliferation,migration,invasion and angiogenesis in tumor cell.ZNF70,a member of the zinc finger protein family,has a variety of pharmacological activities.Convallatoxin,triterpenoid ingredient,exhibits anticancer pharmacological properties.Aim of the study:(1)We identified a previously unknown role for Zinc finger protein70(ZNF70)in upregulating IL-1? secretion of macrophages to promote proliferation of HCT116,and investigated its underlying mechanism.(2)In this work,we investigated the anticancer potential of convallatoxin and explored whether convallatoxin mediates its effect through interference with the STAT3 activation in colorectal cancer cells.Materials and methods:(1)In vitro,the underlying mechanisms of ZNF70 at promoting the secretion of IL-1? and HCT116 proliferation were investigated by ELISA,RT-PCR,immunoblot,co-immunoprecipitation,proximity ligation assay,immunofluorescence,EdU labelling and colony formation assay.In vivo,the AAV-mediated genetic approaches combined with AOM/DSS model were used.(2)In vitro,the underlying mechanisms of convallatoxin at inhibiting STAT3 activation were investigated by homology modeling and molecular docking,luciferase reporter assay,MTT assay,RT-PCR,Western blotting and immunofluorescence assays.Changes in cellular proliferation,apoptosis,migration,invasion and angiogenesis were analyzed by EdU labeling assay,colony formation assay,flow cytometry assay,wound-healing assay,matrigel transwell invasion assay and tube formation assays.And in vivo,antitumor activity of convallatoxin was assessed in a murine xenograft model of HCT116 cells.Results:(1)In LPS plus ATP induced THP-1 cell,we found ZNF70 activated the NLRP3 inflammasome,resulting in robust IL-1? secretion.Interestingly,we discovered Zn F domain of ZNF70 could interact with NLRP3 and decrease K48-linked ubiquitination of NLRP3.Moreover,ZNF70 could activate STAT3,thereby promoting the synthesis of pro-IL-1?.Noteworthy,we found ZNF70 enhanced proliferation by upregulated STAT3 activation in HCT116 cells cultured in the conditioned medium from THP-1 macrophages treated with LPS plus ATP.Finally,In vitro observations were confirmed by showing AAV-mediated ZNF70 knockdown improved colitis-associated colorectal cancer in AOM/DSS model.(2)Convallatoxin decreased the viability of colorectal cancer lines.Moreover,convallatoxin reduced the P-STAT3(T705)via the JAK1,JAK2,and Src pathways and inhibited serine-727 phosphorylation of STAT3 via the PI3K-AKT-mTOR-STAT3 pathways in colorectal cancer cells.Interestingly,we discovered the crosstalk between mTOR and JAK2 in mTOR/STAT3 and JAK/STAT3 pathways,which collaboratively regulated STAT3 activation and convallatoxin play a role in it.Convallatoxin also downregulated the expression of target genes involved cell survival(e.g.,Survivin,Bcl-xl,Bcl-2),proliferation(e.g.,Cyclin D1),metastasis(e.g.,MMP-9),and angiogenesis(e.g.,VEGF).Indeed,we found that convallatoxin inhibited tube formation,migration,and invasion of endothelial cells,and inhibited the proliferation.Finally,in vivo observations were confirmed by showing antitumor activity of convallatoxin in a murine xenograft model.Conclusions:(1)Our research describes a mechanism that ZNF70 regulates IL-1?secretion of macrophages to promote HCT116 proliferation via activation of NLRP3 inflammasome and Src/STAT3 pathway and suggests that ZNF70 may be a promising therapeutic target for intervention in colitis-associated colorectal cancer.(2)The result of the current study shows that convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3(T705)and mTOR/STAT3(S727)signaling pathways in colorectal cancer cells and indicate that convallatoxin could be a valuable candidate for the development of colorectal cancer therapeutic.