Mechanism Of Cetuximab Regulating The Proliferation Of KRAS G13D Mutant Colorectal Cancer Cells By Mono-ADP-ribosylation Of H3R117

Backgroud:Colorectal cancer(CRC)is one of the most common gastrointestinal cancers,it is also one of the most deadly malignancies,and it is still the fourth leading cause of death worldwide,poseing a huge threat to human health.In recent decades,with the in-depth study of the pathogenesis of CRC,more and more therapeutic targets have been found,but the efficacy is still limited.Therefore,it is necessary to explore new mechanisms affecting the pathogenesis and carcinogenic factors of colorectal cancer facilitating to implement effective preventive measures and improve treatment.Over 40% of CRC are known to with KRAS mutation.KRAS gene is an indispensable biomarker for anti-epidermal growth factor receptor(EGFR)therapy,and is considered to be a predictive gene of EGFR biotherapy resistance.CRC is one of the first cancer types to be introduced with targeted therapy(anti-EGFR antibody),but anti-EGFR monoclonal antibody is only effective for patients with wild-type KRAS(wt KRAS),while resistant to patients harbouring mutant KRAS(mtKRAS).Cetuximab is a monoclonal antibody that is beneficial in individuals CRC patients with wt KRAS,but less than half of metastatic colorectal cancer(mCRC)patients with wt KRAS respond to anti-EGFR therapy,while the rest develop resistance to it.These results indicate that KRAS mutation is not the whole factor of anti-EGFR drug resistance.CRC patients with dual KRAS and PIK3 CA mutations showed stronger aggressiveness,poorer overall survival and increased drug resistance compared with KRAS mutations alone.PIK3 CA mutation is a biomarker for drug resistance in mCRC patients treated with anti-EGFR monoclonal antibody,especially KRAS wild-type patients.It is suggested that the effect of cetuximab on CRC may be related to whether KRAS mutation is accompanied by PI3KCA mutation.In addition,the prevailing view is that anti-EGFR therapy is ineffective in metastatic colorectal cancer(mCRC)patients with KRAS mutations.However,some studies have found that compared with KRAS G12 V mutation,tumors carrying KRAS G13D mutation have less impact on tumor cell growth and tumor progression,and are more sensitive to anti-EGFR antibody therapy.Cell experiments showed that the sensitivity of KRAS G13D mutation LoVo cells to cetuximab was between KRAS G12 V mutation SW480 and KRAS wild-type LIM1215.These results suggested that some patients or cell lines with subtype of KRAS mutation show some sensitivity to EGFR monoclonal antibody therapy.If we can find the difference of molecular mechanism and intervene,it will undoubtedly expand the application scope of anti-EGFR monoclonal antibody.To a certain extent,it can improve the therapeutic effect on some patients harbouring mutant KRAS.Adenosine diphosphate(ADP)-ribosylation is a reversible and site-specific posttranslational modification that transfers single or multiple ADP ribose to specific amino acid sites of the receptor protein.It is catalyzed by ADP-ribosyltransferases(ARTDs)and reversible by ADP-ribosylhydrolases.In human health and disease,ADP ribosylation is associated with key biological processes such as gene transcription and chromatin remodeling.At present,the research on ADP ribosylation is mainly based on the enzymes of ADP ribosylation.Our previous studies have shown that inhibition of poly-ADP-ribosylation can inhibit the malignant biological behavior of colorectal cancer cells,but upregulation of poly-ADP-ribosylation couldn’t obtaine the recovery effect.Studies have shown that poly(ADP-ribose)polymerase-1(PARP-1)was restricted by arginine-specific mono-ADP-ribosyltransferase 1(ART1)in colorectal cancer.Therefore,we further studied the role of mono-ADP-ribosylation in colorectal cancer.The results showed that ART1 could participate in PI3K/AKT/NF-κB,Erk/AKT/Tubb3,PARP-1-LKB1-AMPK-MTOR,etc signal pathways and promote the progression of colorectal cancer.As is known to all,the mainly downstream signal pathway of EGFR is RAS/RAF/MEK/ERK/MAPK and PI3K/Akt(PKB)/mTOR.Thus,mono ADP ribosylation can be involved in the regulation of EGFR signal pathway.Therefore,we speculated that mono ADP ribosylation can affect the therapeutic effect of anti-EGFR monoclonal antibody.Histone is one of the targets of ADP ribosylation,and the main form of histone ADP ribosylation is mono-ADP-ribosylation.In our previous studies,it has been identified that the 117 th arginine of histone H3(H3R117)is a mono-ADP-ribosylated site in KRAS G13D LoVo cells.Mutating the mono-ADP-ribosylated 117 th arginine residue of histone H3(H3R117)to construction H3R117A mutation LoVo cells could inhibite the proliferation,invasion and metastasis of CRC cells.Meanwhile,the mutation also changed the gene expression profiles of LoVo cells by transcriptome sequencing(RNA-seq).There were more than 2,000 differential genes(DEGs)between LoVo WT and LoVo H3R117A cells.Further analysis of RNA-seq results showed that spt2 was one of the downregulation genes after mutation.According to the analysis of functional enrichment gene Ontolog(Go),only spt2 and parp10 were enriched in the biological process of "regulation of chromatin assembly".Parp10 is an intracellular mono-ADP-ribosyltransferase and can catalyze the formation of mono-ADP ribosylation of target proteins.Spt2 is a histone chaperone protein with a high-mobility group(HMG)-like domain.It can interact with H3/H4,maintenance of histone H3/H4 tetramer integrity through the RNA polymerase II,and maintain the function of H3/H4 tetramer during transcription.It can also lengthen RNA polymerase II and alter chromatin structure by reducing nucleosome density,thereby regulating histone-DNA interactions and playing an important regulatory role in DNA transcription and repair.Therefore,we speculated that spt2 interacts with mono ADP ribosylation in the maintenance of chromatin function.But further research is needed to confirm this。Parp10 could promote the proliferation of colorectal carcinoma by regulating the nuclear transfer of β-catenin.In addition,RNA-seq results showed that several significance DEGs between LoVo WT and H3R117A mutant LoVo cells were enriched in the glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate pathway.And the expression of β-catenin was decreased,the ability of migration and invasion were weakened after mutation.But the exact mechanism is unclear.Studies have shown that the glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate pathway interact with Wnt,resulting in increased expression of β-catenin,thus affecting tumor metastasis.β-catenin is a key nuclear effector of the classical Wnt signaling pathway in the nucleus,also known as Wnt/β-catenin signaling pathway.A large number of studies have shown that β-catenin is a key factor of cetuximab resistance in KRAS G13D colon cancer.TCF7L2 is a major transcription coactivator of the Wnt/β-catenin signaling pathway,which plays an important role in the progression of cetuximab resistance in mCRC.TCF7L2 interact with β-catenin in the nucleus to form TCF7L2/β-catenin complex,which activates the Wnt/β-catenin signaling pathway to regulate the proliferation of cancer cells.Correlative signaling pathways can positively regulate aerobic glycolysis in tumors and promote tumor progression.Therefore,we speculated that the effects of cetuximab on the proliferation of KRAS mutant colorectal cancer cells regulating by mono ADP ribosylation through the Wnt/β-catenin signaling pathway.Whether histone H3R117 mono-ADP-ribosylation regulate the proliferation of colorectal cancer cells by spt2 is the first question to be solved in this study.Secondly,we want to further explore the mechanism of cetuximab inhibition the proliferation of LoVo cells by histone H3R117mono-ADP-ribosylation in this process.To provide new ideas and preliminary experimental basis for improving the clinical treatment effect of colorectal cancer.Methods: This research is divided into three parts.1.Effects of cetuximab on the proliferation of KRAS mutant colorectal cancer cells regulating by mono ADP ribosylation.1.1 CCK8 was used to detect the effect of cetuximab on the survival fraction of three KRAS mutant colorectal cancer cells: SW480(KRAS G12 V without PI3KCA mutation),HCT116(KRAS G13D with PI3KCA mutation)and LoVo(KRAS G13D without PI3KCA mutation)cells.1.2 CCK8 was used to detect the effect of cetuximab on the survival fraction of three KRAS mutant colorectal cancer cells: SW480(KRAS G12 V without PI3KCA mutation),HCT116(KRAS G13D with PI3KCA mutation)and LoVo(KRAS G13D without PI3KCA mutation),after inhibiting mono ADP ribosylation by MIBG.1.3 EdU was used to detect the effect of cetuximab on the proliferation of three KRAS mutant colorectal cancer cells: SW480(KRAS G12 V without PI3KCA mutation),HCT116(KRAS G13D with PI3KCA mutation)and LoVo(KRAS G13D without PI3KCA mutation),after inhibiting mono-ADP ribosylation by MIBG.1.4 Western blotting was used to detect the level of mono-ADP ribosylation of histone H3 in three KRAS mutant colorectal cancer cells:SW480(KRAS G12 V without PI3KCA mutation),HCT116(KRAS G13D with PI3KCA mutation),and LoVo(KRAS G13D without PI3KCA mutation)cells.1.5 Colony-forming efficiency assay was used to evaluate the ability of a single of LoVo(KRAS G13D without PI3KCA mutation)or SW480(KRAS G12 V without PI3KCA mutation)cell to grow into a colony,after inhibiting mono-ADP ribosylation by MIBG.1.6 Western blotting was used to detect the level of mono-ADP ribosylation of histone H3 in LoVo WT,empty vector transfection group LoVo cells and H3R117A LoVo cells.1.7 CCK8,flow cytometry and colony-forming efficiency assay were used to detect the effect of cetuximab on the proliferation of LoVo cells and H3R117A LoVo cells.1.8 The tumor subcutaneous transplantation model of nude mice was used to evaluate the effect of cetuximab on the transplanted tumor of KRAS G13D LoVo cells or H3R117A LoVo cells.2.Effect of H3R117 mono ADP ribosylation on spt2 in colorectal cancer with KRAS G13D mutation2.1 Western blotting and q PCR were used to detect the expression of spt2 in LoVo(KRAS G13D)and H3R117A LoVo cells.2.2 Agarose magnetic bead immunoprecipitation(IP)was used to detect the interaction between spt2 and histone H3 in LoVo(KRAS G13D)and H3R117A LoVo cells.2.3 Cellular immunofluorescence was used to detect the intensity of fluorescence of spt2.2.4 Immunohistochemistry was used to detect the expression of spt2 in colorectal cancer.2.5 Western blotting was used to detect the efficiency of knockdown of spt2 in LoVo cells.2.6 CCK8 was used to detect the survival fraction of LoVo cells after spt2 knockdown.2.7 Flow cytometry was used to detect the cell cycle and apoptosis of LoVo after spt2 knockdown.3.Effects of spt2 changes on the Wnt/β-catenin signal pathway and its downstream glycolytic enzymes such as PDK1 and LDHA etc regulating by histone H3R117 MARylation.3.1 The online colorectal cancer database of cBio Portal and Gene Expression Profiling Interactive Analysis(GEPIA)was used to analyze spt2 in CRC.3.2 Western blotting was used to detect the protein level of TCF7L2 andβ-catenin,and q PCR was used to detect the m RNA level of TCF7L2 in LoVo(KRAS G13D)and H3R117A LoVo cells.3.3 Chromatin immunoprecipitation(CHIP)was used to determine the enriched H3K56 ac of the TCF7L2 promoter in LoVo(KRAS G13D)and H3R117A LoVo cells.3.4 Lactic acid and ATP assay kits were used to investigate the concentrations of lactic acid and ATP in LoVo(KRAS G13D)and H3R117A LoVo cells.3.5 Western blotting was used to determine the expression of enzymes associated with glycolysis and genes associated with cell proliferation in LoVo(KRAS G13D)and H3R117A LoVo cells.3.6 Western blotting was used to determine the expression of enzymes associated with glycolysis and genes associated with cell proliferation in the LoVo(KRAS G13D)and H3R117A LoVo cell allograft transplanted tumors.Results:1.Effects of cetuximab on proliferation of KRAS mutant colorectal cancer cells regulating by mono-ADP-ribosylation.1.1 CCK8 results showed that these three cell lines were primarily resistant to cetuximab,especially the HCT116 cells,harboring KRAS G13D and PIK3 CA mutations.1.2 CCK8 results showed that cetuximab combined with MIBG reduced the survival fraction of HCT116(KRAS G13D with PI3KCA mutation),SW480(KRAS G12 Vwithout PI3KCA mutation)and LoVo(KRAS G13D without PI3KCA mutation)cells,and the survival fraction of LoVo cells decreased most obviously(P<0.05).1.3 EdU results showed that cetuximab combined with MIBG reduced the proliferation of HCT116(KRAS G13D with PI3KCA mutation),SW480(KRAS G12 Vwithout PI3KCA mutation)and LoVo(KRAS G13D without PI3KCA mutation)cells(P<0.05).1.4 Western blotting results showed that the level of mono-ADP ribosylation of histone H3 in LoVo cells was highest in the three cells(P<0.05).1.5 Colony-forming assay results showed that LoVo cells had a lower colony-forming efficiency than SW480 cells,after treatment with cetuximab combined with MIBG(P<0.05).1.6 Western blotting results showed that the level of mono-ADP ribosylation of histone H3 was decreased in H3R117A LoVo cells(P<0.05).There was no significant difference between LoVo WT and empty vector transfection group.1.7 CCK8 revealed that cetuximab was more sensitive to H3R117A LoVo cells(P<0.05).Flow cytometry indicated the G1 phase arrest of H3R117A mutant LoVo cells(P<0.05),it was more pronounced than LoVo WT cells with cetuximab treatment.Colony-forming efficiency of H3R117A mutant LoVo cells was significantly decreased after cetuximab treatment(P<0.05).1.8 Tumor weight was measured in each group 4 weeks after the LoVo cells were transplanted.The tumor weight in the H3R117A group was much lower than that in the LoVo WT groups,and cetuximab treatment was more effective in the H3R117A group(P<0.05).2.Effect of H3R117 mono-ADP-ribosylation on spt2 in colorectal cancer with KRAS G13D mutation2.1 Compared to the LoVo WT or empty vector-transfected LoVo cells,Western blotting and q PCR showed that the protein and m RNA levels of spt2 were decreased in the H3R117A LoVo cells(P<0.05).There was no significant difference between LoVo WT and empty vector transfection groups.2.2 Agarose magnetic bead immunoprecipitation(IP)assay was conducted to verify the interaction between endogenous spt2 and H3 in H3R117A,LoVo WT,and empty vector-transfected LoVo cells.Histone H3 was immunoprecipitated from LoVo cell lysates by spt2 antibody.Ig G was used as a negative control.Western blotting showed that the protein level of histone H3 was enriched in the IP fractions from three groups and indicated that spt2 immunoprecipitation was successfully obtained from cell extracts.Furthermore,compared to LoVo WT and empty vector-transfected LoVo cells,histone H3 enrichment was the least in the H3R117A LoVo cells.The results confirmed the interaction between spt2 and histone H3.Moreover,the mutation of MARylation on H3R117 reduced the interaction between spt2 and histone H3.2.3 Cell immunofluorescence showed that spt2 was located in the nucleus of LoVo cells.The fluorescence intensity of spt2 in cells treated with MIBG combined with cetuximab was lower than that in the control group(P<0.05).There is no statistical difference between the treated with cetuximab alone and the control group.The fluorescence intensity of spt2 in H3R117A mutant LoVo cells was significantly decreased after cetuximab treatment(P<0.05).2.4 Immunohistochemical staining identified spt2 in both colorectal adenocarcinoma and normal colorectal tissues,and expressed in both the nucleus and the cytoplasm.The nuclear expression was mainly distributed in highly differentiated colorectal adenocarcinoma and the adjacent normal tissues.Both the cytoplasmic and nuclear expression were mainly distributed in moderately and poorly differentiated colorectal adenocarcinoma.As per the detected acinus positivity degree,when compared with that in adjacent normal tissues,the level of spt2 in colorectal adenocarcinoma was significantly increased(P < 0.05).The clinicopathological characteristics indicated that the level of spt2 was not related to sex,age,and location,but rather to the TNM stage indexes.When compared with READ,strongly positive(+++)expression of spt2 was found to be more correlated with deeper invasion depth in COAD(P<0.05).2.5 Compared with the control group(LoVo WT),the highest efficiency target site was determined to be SPTY2D1-homo-432(P < 0.05),which was used in the subsequent experiment(SPT2-sh RNA)(the experimental group).No significant difference was noted between the positive control(sh GAPDH)and the negative control groups.2.6 Compared with the control group,CCK8 revealed that the survival fraction of LoVo cells was reduced after spt2 knockdown at 24,48,and 72 h.It was more pronounced at 48 h and 72 h,and the difference was statistically significant(P < 0.05),while no significant difference was noted at 24 h.2.7 Compare with the control group,flow cytometry indicated that the percent of spt2 knockdown LoVo cells in the G1 phase was increased and the proportion of the G2+S phase was decreased(P <0.05).Furthermore,the apoptotic rate of spt2 knockdown LoVo cells was significantly increased(P < 0.05)3.Effects of spt2 changes on the Wnt/β-catenin signaling pathway and its downstream glycolytic enzymes such as PDK1 and LDHA etc regulating by histone H3R117 MARylation.3.1 We first examined the genetic alteration variants of spt2 in CRC with reference to the cBio Portal online database.The genetic alteration profiling of spt2 revealed that deep deletion was one of the single factors of alteration in CRC.In addition,the mutation frequency was the highest,while the amplification frequency was the least in CRC.The copy number alteration(CNA)of spt2 was changed in the CRC.The possible pathways of spt2 in cell proliferation are associated with TCF7L2.Next,we assessed the expression and prognostic value of spt2 for CRC in GEPIA.Our result indicated that the expression of spt2 in COAD and READ was higher than that in adjacent normal tissues.3.2 Compared to the LoVo WT and empty vector-transfected LoVo cells,Western blotting showed that the mutation of MARylation on H3R117 reduced the histone protein levels of transcription factor 7-like 2(TCF7L2)and β-catenin(P<0.05);meanwhile,q PCR showed that the mutation of MARylation on H3R117 reduced the m RNA level of TCF7L2(P<0.05).There was no difference between untreated and empty vector-transfected LoVo cells.3.3 Compared to the LoVo WT cells,chromatin immunoprecipitation(CHIP)showed that the enriched H3K56 ac of the TCF7L2 promoter was decreased in the H3R117A LoVo cells(P<0.05).3.4 Compared to the LoVo WT and empty vector-transfected LoVo cells,the concentrations of lactic acid and ATP were significantly decreased in the H3R117A LoVo cells(P<0.05).3.5 Compared to the LoVo WT or empty vector-transfected LoVo cells,Western blotting showed that the expressions of PDK1,LDHA,c-Myc,and cyclin D1 were decreased in the H3R117A LoVo cells;there was no difference between LoVo WT and empty vector-transfected LoVo cells(P<0.05).However,there was no variation in PKM2 protein levels between different groups.3.6 In nude mice,compared to the LoVo WT cells allograft transplanted tumors,Western blotting revealed that the protein levels of c-Myc,PDK1,LDHA,and cyclin D1 were decreased slightly in the H3R117A LoVo cell allograft transplanted tumors.Compared to the control group,levels of these four proteins were significantly decreased in H3R117A transplanted tumors treated with cetuximab(P<0.05).Conclusion:1.The effect of cetuximab on the proliferation of KRAS G13D mutant colorectal cancer was regulated by H3R117 MARylation.2.The expression of spt2 in colorectal cancer carrying KRAS G13D mutation was regulated by H3R117 MARylation.3.The specific mono-ADP-ribosylated site of arginine-117 of histone 3 in KRAS G13D colon cancer cells may play an important role to help resistance to cetuximab treatment.H3R117 MARylation may enhance the aerobic glycolysis by up-regulating the Wnt/β-catenin pathway via increasing the transcription of TCF7L2 and the nuclear accumulation ofβ-catenin.Ultimately it promoted the growth of colon cancer cells.Therefore,inhibition of H3R117 MARylation may be a combined target for overcoming cetuximab resistance in KRAS G13D CRC.We expect that combination therapy with these specific markers will improve therapeutic efficacy in KRAS mutant CRC in the future.