| Background:Colorectal cancer(CRC)is one of the most common gastrointestinal malignancies.According to the latest research report,the incidence of CRC in the Chinese population ranks second among all cancers,and the mortality rate ranks fourth.In metastatic colorectal cancer(m CRC),15-25% of CRC patients are accompanied by distant metastasis at first diagnosis.With the development of chemotherapy drugs and the application of targeted drugs,the survival of m CRC patients is significantly improved.Cetuximab targeting the epidermal growth factor receptor(EGFR)has shown survival benefits in patients with m CRC either monotherapy or in combination with chemotherapy.Cetuximab is a monoclonal antibody targeting EGFR extracellular region,which can competitively bind EGFR extracellular region and block intracellular signal transduction pathway,thus inhibiting the proliferation and inducing apoptosis of tumor cells and playing an anti-tumor role.However,cetuximab therapy is only effective in wild-type m CRC patients with RAS/BRAF/PIK3 CA,and even KRAS wild-type m CRC patients do not fully benefit from cetuximab therapy due to tumor heterogeneity and primary or secondary drug resistance problems.Therefore,it is an urgent problem to investigate the mechanism of cetuximab resistance and find effective intervention targets in m CRC treatment.Current screening for drug sensitivity is mainly based on traditional cancer cell lines that undergo genomic changes such as genetic drift after long-term in vitro culture,eventually losing the original molecular characteristics of the tumor in the parent.Although the human tissue xenotransplantation model can largely restore the biological behavior of tumors,its application is limited due to factors such as high cost.Organoid models have been widely used in cancer research in recent years.Organoids not only retain part of the functions of internal organs and maintain the heterogeneity of patient-derived tumors,but also have the advantages of extensive sources and large-scale expansion,making them a good model for drug screening and other studies.Cancer treatment resistance is a complex process,and epigenetics plays an important role in cancer treatment resistance.Among them,RNA m6 A methylation can change the stability of some key gene transcription products,activate or inhibit certain signaling pathways,and affect the occurrence of cancer treatment resistance in the complex process of oncogene activation,DNA damage repair,tumor stem cells,autophagy and other mechanisms.Therefore,in-depth exploration of the key role of m6 A modification in tumor resistance can provide new ideas for the development of drugs or combination therapies.Methods:First,we constructed 6 CRC organoids and screened 2 cetuximab sensitive CRC organoids to induce drug resistance.The paired sensitively-resistant organoids were subjected to RNA-seq,showing low expression of METTL14 in drug-resistant organoids.Subsequently,it was verified that low expression of METTL14 was associated with cetuximab resistance in CRC organoids,cell lines and clinical samples,and the effect of altered expression of METTL14 on cetuximab sensitivity was verified in vivo and in vitro functional experiments.Secondly,we screened the genes regulated by m6 A through the combined analysis of Me RIP-seq and protein spectrometry,and verified that METTL14 affected the m RNA stability of IFT22 through m6 A modification through q PCR,RNA-seq,Me RIP-seq,double luciferase reporter gene assay,and RNA stability assay.Then,we overexpressed/knockdown IFT22 in Caco-2,induced drug-resistant Caco-2-CR,and primary drug-resistant HCT116 cells,and verified the effect of IFT22 expression on cetuximab resistance by using CCK8,clonal formation,flow detection of apoptosis,and subcutaneous tumor bearing in nude mice.The association between IFT22 or cetuximab resistance and ciliary regeneration was verified by immunofluorescence.Finally,we overexpressed IFT22 in Caco-2-CR,HCT116 and Caco-2 cells to detect changes in the downstream signaling pathway of EGFR,and verified the proteins interacting with IFT22 by protein profiling,protein co-immunoprecipitation and immunofluorescence co-localization detection.The IFT22/CDK18/p-ERK activation pathway was verified by RESCUE experiment.Results:In the first part of the study,we constructed two paired organoids with initial sensitization and secondary resistance to cetuximab,conducted RNA-seq on them and found that the expression of m6 A methyltransferase METTL14 was down-regulated in drug-resistant organoids,and verified the above findings by q PCR and Western Blot.Subsequently,we constructed cell lines with METTL14 knockdown/overexpression,confirming that low METTL14 expression promoted cetuximab resistance,and further clarified the correlation between METTL14 expression and cetuximab resistance in clinical samples.In the second part of the study,we conducted Me RIP-seq and protein spectrum identification on 2 drug-resistant and sensitive CRC organoids,and found that m6 A modification level was low in drug-resistant organoids,and further screening found that IFT22 may be involved in cetuximab resistance.Subsequent studies showed that IFT22 m RNA was negatively regulated by the m6 A methyltransferases METTL3 and METTL14.Subsequently,we verified the presence of m6 A modification in IFT22 by Me RIP-q PCR,and the maximum binding site was located at site +857 in the 3 ’UTR region after the stop codon.Double luciferase reporter gene assay confirmed that METTL14 affected the expression of IFT22 through m6 A modification.Subsequently,it was found by q PCR and actinomycin D that METTL14 could affect the m RNA stability of IFT22 through m6 A modification.In the third part of the study,we first verified the high expression of IFT22 in the cetuximab resistant group in clinical samples,and it was negatively correlated with METTL14.Knockdown/overexpression IFT22 cell lines were further constructed,and it was found that knockdown IFT22 increased cetuximab sensitivity while overexpression had the opposite effect.At the same time,we found that overexpression of IFT22 can promote the proliferation of tumor cells under cetuximab and down-regulate the apoptosis of tumor cells.Further,we constructed a cetuximab secondary resistant cell line Caco-2-CR,in which IFT22 knockdown could reverse Cetuximab resistance and functional phenotype,but IFT22 knockdown could not reverse the resistance phenotype of the primary resistant cell HCT116.IFT22 is a cilia-related protein.We have found that Caco-2-CR has more cilia and longer cilia,and low/overexpression of IFT22 can reduce/increase the length and number of cilia.Overexpression of IFT22 in Caco-2/HCT116 cells also shows corresponding changes in cilia.In the fourth part of the study,we overexpressed IFT22 in Caco-2-CR and HCT116 cells,and found that the downstream signal of EGFR was up-regulated p-ERK.Subsequently,we compared Caco-2 and Caco-2-CR,and also found that p-ERK up-regulated expression.Treating Caco-2 with cetuximab inhibited p-ERK expression,but p-ERK showed continuous activation after overexpression of IFT22 in Caco-2.Protein kinases can regulate protein phosphorylation in cells.In order to explore the molecular mechanism,a total of 17 protein kinases were found to be differentially expressed after overexpression of IFT22 by protein spectrometry.The IFT22 interacting proteins were further screened by protein immunoprecipitation-mass spectrometry,and CDK18 was found to be involved in the above process.Subsequently,we verified that IFT22 could bind CDK18 by protein co-immunoprecipitation and immunofluorescence,and Western Blot confirmed that overexpression of IFT22 caused up-regulation of CDK18 expression.Finally,we used RESCUE experiments to verify that knocking down CDK18 in IFT22-overexpressed cells can reverse IFT22-induced p-ERK upregulation.In summary,we used multi-omics techniques such as RNA-seq,Me RIP-seq and protein profiling to conduct in-depth studies on CRC cetuximab resistance in organoid,cell line and clinical samples,and the results showed that low expression of METTL14 could enhance the stability of IFT22 m RNA through m6 A modification dependent approach.Recruitment of protein kinase CDK18 and up-regulation of its expression promoted the reactivation of EGFR downstream ERK signaling pathway,promoted the proliferation of CRC cells,inhibited apoptosis,promoted cilia regeneration,and promoted cetuximab resistance.These results provide new thinking for the study of CRC cetuximab resistance and the exploration of reversal strategies. |
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