Study On The Role And Mechanism Of Apatinib Against Colon Cancer

Colorectal cancer(CRC)is the third most common cancer and a significant cause of death worldwide.At present,its incidence and mortality are still on the rise in China.For patients with metastatic Colorectal Cancer(m CRC),the current emphasis is fluoropyrimidine-based chemotherapy combined with targeted therapy.Immunotherapy is effective for colon cancer patients with d MMR/MSI-H,but this part of patients accounts for a minimal proportion of m CRC.Thus,novel and safe treatment strategies for CRC are urgently needed.Apatinib,a small molecule anti-angiogenesis drug targeting VEGFR2 independently developed by China,has been reported to have anti-cancer activity in various malignancies.In a previous study,we conducted a clinical trial assessing the efficacy of apatinib monotherapy treatment in m CRC patients who have failed three or more chemotherapy lines,which showed that the median PFS of apatinib was 3.9 months and the median OS was 7.9 months.Based on the effectiveness of apatinib in the clinical trial,this paper explores the effects of apatinib on colon cancer and the corresponding mechanism through a combination of cell experiments and animal experiments,and other methods and techniques.In the first chapter,we first evaluated the inhibitory effect of apatinib on colon cancer cells'malignant phenotype in vitro by CCK8,clone formation,cell cycle,Trans-well cell invasion test,and cell scratch test.Apatinib showed anti-proliferative and proapoptotic effects,induced G0/G1 arrest,and blocked cell migration and invasion in colon cancer cells.Simultaneously,a CT26 mouse xenograft model was established to study the effect of apatinib on colon cancer in vivo.RNA-seq(transcriptome)analysis was conducted on apatinib-treated HCT116 cells to elucidate the associated mechanism further.The differential genes were discovered using DESeq2.The potential signal mechanism by which apatinib exerts its influence was investigated using GO and KEGG enrichment research.In vitro and in vivo tests,including RT-PCR,Co-Immunoprecipitation,Immunohistochemistry,Immunofluorescence,were performed to confirm the potential mechanism.RNA-Seq analysis results showed that apatinib inhibits the activity of the Wnt-b-catenin pathway.Further analysis of the mechanism related to the activity of apatinib showed that apatinib reduced the level of p-GSK3a(ser9),thereby further increasing the ubiquitination of b-catenin and inhibiting the nuclear translocation of b-catenin.Besides,apatinib significantly inhibited tumor growth in the CT26 mouse xenograft model by inhibiting b-catenin signaling and angiogenesis.These results reveal a mechanism of apatinib activity in CRC and provide a new candidate for the treatment of m CRC in clinical practice.Immunotherapy has only shown its efficacy in the minority of CRC patients with d MMR/MSI-H.Studies have shown that the combination of anti-angiogenic drugs and immunotherapy has a particular synergistic effect.In analyzing the RNA-seq data,we also found a significant increase in the JAK-STAT pathway and PD-L1expression after apatinib treatment,indicating that apatinib may also regulate tumor immunity.Thus,in the second chapter,we investigated the effects of apatinib on the expression of PD-L1and anti-PD-1 immunotherapy in colon cancer.Firstly,we performed RT-PCR,flow cytometry,immunofluorescence,and western blot studies to estimate the effect of apatinib on the m RNA and protein expression levels of PD-L1 in colon cancer cells.The results indicated that apatinib increased PD-L1 expression at both the cellular and animal levels.Tumor cells treated with apatinib can significantly inhibit the level of IFN-?secreted by T cells in the spleen of mice in the co-culture system.This inhibition can be reversed by anti-PD-1 antibodies,which indicated that the elevation of PD-L1mediates the inhibition of apatinib to the immune system.Further,we explored the anti-tumor effects of apatinib combined with anti-PD-1 in a CT26 mouse xenograft model.The results showed that the apatinib combined with anti-PD-1 significantly improved anti-tumor immunity,as shown by a significant increase in the invasion of CD4⁺and CD8⁺T cells in tumor tissue,ultimately leading to a substantial increase in the effectiveness of combined use.Mechanism studies show that the significant increase in the activity level of STAT1 after apatinib treatment(increased phosphate level and increased nucleation)is the reason for the increase of PD-L1 expression.This chapter provides a clinical basis for a combination of apatinib and anti-PD-1 to treat colon cancer.We have confirmed that apatinib affects both the?-catenin and the STAT1signaling pathways in tumor cells in the previous two chapters.Through literature search analysis,we find that the two signaling pathways have a common junction-phosphatase SHP2.SHP2 can affect its binding to?-catenin by negatively regulating parafibromin's activity,activating the Wnt signal transducing pathway,promoting tumor development.Besides,SHP2 inhibits the activity of STAT1 by directly removing its phosphorylation.At the same time,when the laboratory used its self-built compound library to screen small molecules that interact with SHP2,it was found that apatinib can bind to SHP2.These prompted us to study further the role of apatinib on phosphatase SHP2 and related mechanisms.To this end,in Chapter Three,we explore whether apatinib affects the relevant signal pathway and mechanism by affecting the function of SHP2.Apatinib treatment does not affect the m RNA levels of SHP2 but significantly down-regulated its protein levels,which can be inhibited by MG132 treatment,suggesting that apatinib treatment can significantly induce SHP2degradation.The results of Cellular Thermal Shift Assay(CETSA)have shown that apatinib can bind directly to SHP2.Then,the purified SHP2 protein was separated to conduct SPR and MST experiments,which determined the binding constant of apatinib and SHP2 was K_D=16.5×10^-6?M/10.2×10^-6?M.The results of molecular docking showed that the binding sites of apatinib and SHP2 were ARG(arginine 111),GLU(glutamate 249),and PHE(phenylalanine 113).The enzymatic activity experiment results suggested that compared to PHPS1(an enzyme-active inhibitor of SHP2),apatinib does not affect the phosphatase activity of SHP2.Further CO-IP experiments verified that apatinib induced an increase in ubiquitination levels of SHP2,and the results of protein spectrometry showed that the E3 ubiquitin ligases that might bind to SHP2 were TRIM21/TRIM25.After overexpression of SHP2,the inhibitory effect of apatinib on colon cancer cells decreased.It was initially determined that the effect of apatinib on colon cancer might be achieved by inducing SHP2 degradation.In summary,based on the results of clinical trials,this paper discussed the role and mechanism of apatinib in treating CRC in basic research.In addition to binding to VEGFR2 inhibiting angiogenesis,we found that apatinib can directly bind to the phosphatase SHP2 and induce its ubiquitination degradation through TRIM21/25.Degradation of SHP2 brings two effects:First,it inhibits the b-catenin pathway and thus inhibits colon cancer;second,it up-regulates the expression of PD-L1 in colon cancer,the combination of anti-PD-1 enhances the anti-colon cancer effect.Our study thoroughly explored the inhibitory effect of apatinib on colon cancer and its mechanism,which provided a new candidate drug and strategy to treat colon cancer.Moreover,a new regulation mechanism of SHP2 degradation was proposed for the development of SHP2 inhibitors.