Promotion Of Colorectal Cancer Development By Fusobacterium Nucleatum Through Modulation Of CYP2J2-EpOMEs Axis

Background and objective:Fusobacterium nucleatum(Fn),a potential carcinogenic bacterium,is receiving increased attention for its tumor-promoting role in colorectal cancer(CRC).Specific pathogenic bacteria can exert carcinogenic effects either through their own toxic metabolites,or by inducing endogenous metabolic abnormalities and accumulation of specific metabolites in cancer cells.Recent studies have suggested that high concentrations of oxylipin metabolites such as epoxyoctadecamonoenoic acids(EpOMEs)in the tumor microenvironment can promote tumor angiogenesis and malignant progression.However,the regulatory relationship between Fn and oxylipin metabolites remains unclear.The expression of cytochrome P450(CYP)monooxygenase,a key enzyme in EpOMEs biosynthesis,has been reported to be increased in CRC tissues.Therefore,whether Fn regulates oxylipin metabolites(EpOMEs)by activating CYP in CRC progression also need to be further elucidated.Methods:Firstly,metagenomics and metabolomics were used to explore the features of gut microbiota and serum oxylipin metabolites in CRC patients,and to verify the enrichment and correlation of Fn and 12,13-EpOME.Secondly,the altered expression of CYP2J2 was identified through clinical data and tissue samples collection,in vitro cell models and CRC animal models,and its association with Fn abundance and clinical prognostic significance were analyzed in the validation cohort.Moreover,CYP2J2-overexpressed or-silenced CRC cell lines,nude mice CRC liver metastasis model,adeno-associated virus CYP2J2-overexpressed CRC mouse model and gene knockout CRC germ-free mouse model were constructed to verify the regulatory effect of Fn on the CYP2J2-EpOMEs axis.We also verified the effects of Fn-stimulated CYP2J2-EpOMEs axis on CRC cell proliferation,cell migration and invasion,epithelial-mesenchymal transition(EMT)signaling in vitro,and tumor progression and metastasis in vivo.Finally,differentially expressed genes in CRC cells infected with Fn were screened by transcriptome sequencing.And the TLR4/AKT and Keap1/Nrf2 mediated CYP2J2 upregulation by Fn stimulation was verified by gene silencing and overexpression analysis.Results:1.Metagenomics analysis revealed that fecal microbial diversity was significantly lower in CRC patients than that in healthy volunteers.In addition,Fn,Clostridium sp.CAG:567 and Prevotella sp.CAG:386 were significantly more abundant in CRC patients than controls,while Firmicutes bacterium CAG:41 was higher in healthy volunteers.Notably,Fn was identified as the key phylotype in CRC and its level was about 24.67 times higher than that of healthy volunteers.Then,metabolomic analysis showed significant differences in oxylipin profiles between the two groups.In particular,the serum levels of 12,13-EpOME and 9,10-EpOME in CRC patients were2.71 and 2.08 times higher than those in healthy volunteers,respectively.The correlation analysis of differential species and metabolites demonstrated that Fn level was positively correlated with 12,13-EpOME level in CRC patients.Thus,we will further focus on the biological and clinical significance of Fn and 12,13-EpOME in CRC.2.It is known that CYP monooxygenases were key enzymes in 12,13-EpOME biosynthesis,so we further explored whether CYP monooxygenase metabolism abnormality occurs in CRC.Firstly,we found CYP2J2 expression was significantly higher in human CRC tissues than that in normal colon tissue,and the expression of most CYP monooxygenases,especially CYP2J2,was significantly higher in CRC cell lines than that in normal colon epithelial cell lines and colon fibroblast cell lines.And CYP2J2 expression in high metastatic potential Lo Vo cells was 4.78 times higher than that in low metastatic potential HCT-116 cells.In AOM/DSS-induced CRC mouse model,Cyp2j5 expression(corresponding to human CYP2J2)in tumor tissue and12,13-EpOME level in both tumor tissue and serum were significantly higher than those in healthy control mice.Secondly,we analyzed the survival of 228 patients with stage III/IV CRC in the TCGA database and found that patients with higher CYP2J2 expression had significantly lower overall survival.In addition,correlation analysis revealed that Fn abundance were positively correlated with CYP2J2 expression in both fresh-frozen CRC tissue and paraffin-embedded CRC tissue,and elevated Fn and CYP2J2 levels were also associated with increased tumor invasion T stage,lymphatic metastasis,neural invasion and distant metastasis.Finally,co-culture of Fn with CRC cell lines showed that Fn infection increased CYP2J2 expression in a time-dependent manner.Fn infection also significantly increased 12,13-EpOME levels in CRC cell culture supernatant,but not in Fn culture supernatant,suggesting that EpOMEs are metabolites of CRC cells stimulated by Fn,rather than direct metabolites of Fn.3.To further clarify the roles of CYP2J2-EpOMEs axis in the progression and metastasis of CRC,assays in vitro and in vivo were performed.We firstly found that CYP2J2 overexpression could increase cell supernatant 12,13-EpOME levels and promote proliferative activity,migration and invasion abilities in vitro,whereas it is opposite for CYP2J2-silenced cells.In addition,CYP2J2 overexpression induced the EMT phenotype of CRC cells,with a spindle-shaped mesenchymal appearance,while CYP2J2-silenced CRC cells had no obvious EMT phenotype,with a cobblestone-like appearance.In nude mice CRC liver metastasis model,CYP2J2 overexpression promoted the metastatic capacity of CRC cells and increased the metastatic tumor burden,whereas it is opposite for CYP2J2-silenced cells.To explore whether the cancer-promoting properties of CYP2J2 overexpression are associated with 12,13-EpOME accumulation,we treated CRC cells with different concentration gradients of 12,13-EpOME(1n M,10 n M and 100 n M).The ATP cell viability assay showed that 10 n M 12,13-EpOME,which was similar to the average concentration in patients’ serum,exerted the best pro-proliferation effect on CRC cells.12,13-EpOME also promoted migration and invasion ability of CRC cells,as well as enhanced the EMT phenotype.In nude mice CRC liver metastasis model,12,13-EpOME promoted the metastatic capacity of CRC cells and increased the metastatic tumor burden.Furthermore,CYP2J2-overexpressed CRC mice on a high linoleic acid diet(Linoleic acid is the substrate of EpOMEs biosynthesis by CYP2J2)exhibited increased tissue and circulating levels of 12,13-EpOME,a higher incidence of anorectal prolapse,larger tumor size,and more tumor number compared to CYP2J2-overexpressed CRC mice on a normal diet and empty vehicle controls.To preferably examine whether CYP2J2-EpOMEs axis is involved in Fn-induced CRC progression,we next employed the CYP2J2-silenced CRC cells and Cyp2j5-knockout germ-free CRC mouse model.Compared to Fn-infected empty vehicle CRC cells,the migration and invasion abilities of Fn-infected CYP2J2-silenced CRC cells were decreased,and there was no obvious EMT phenotype.In nude mice CRC liver metastasis model,the metastatic ability of Fn-infected CYP2J2-silenced CRC cells was lower than that of the Fn-infected empty vehicle CRC cells.In the Cyp2j5-knockout germ-free CRC mouse model,Fn-infected Cyp2j5-knockout mice showed decreased serum 12,13-EpOME levels,a lower incidence of anorectal prolapse,smaller tumor size,and fewer tumor number compared to the Fn-infected wild-type mice,indicating that Cyp2j5 knockout alleviated Fn infection-induced CRC progression.4.To further understand the signal transduction pathways of Fn regulating CYP2J2,we firstly explored the changes in gene expression profiles of Fn-infected CRC cells,and found that both AKT signaling related genes and Keap1/Nrf2 signaling related genes were induced when Fn was treated.Then,based on KEGG pathway analysis and validation of in vitro experiments,we found that Fn significantly upregulated the TLR4/AKT and Keap1/Nrf2 pathways in CRC cells.Considering that Keap1 is a negative regulator of nuclear transcription factor Nrf2,which plays an important role in tumor progression and metastasis,we speculated that the altered expression of CYP2J2 and tumor progression induced by Fn may be related to the transcriptional regulation of this key nuclear transcription factor Nrf2.As expected,Keap1 overexpression significantly downregulated Nrf2 and CYP2J2 expression in CRC cells,and Nrf2 silencing also decreased the expression of CYP2J2,indicating that CYP2J2 expression depends on the activation of the Keap1/Nrf2 pathway.Bioinformatic analysis indicated that the upstream of CYP2J2 promoter contained a putative binding site of Nrf2.The luciferase reporter assay showed that CYP2J2 upregulation are Nrf2 dependence at the transcription level,and co-transfecting with overexpressed Lv-Nrf2 led to an increase in the CYP2J2 promoter reporter activity.Ch IP assay illustrated that the amount of the Nrf2 antibody binding to the promoter region of CYP2J2 was more than that of Ig G.In addition,the immunoprecipitated Nrf2 binding chromatin could be reduced by co-transfecting with Lv-sh RNA Nrf2.Conclusion:We are the first to identify the Fn-stimulated CYP2J2-EpOMEs axis mediating progression of CRC.Our findings demonstrated that Fn up-regulates CYP2J2 expression through TLR4/AKT and Keap1/Nrf2 signaling pathways,leading to the accumulation of 12,13-EpOME in CRC cells,initiating EMT and promoting tumor progression and metastasis.This study not only gives a novel insight into the cancerpromoting mechanism of Fn,but also highlights that the CYP2J2-EpOMEs axis may serve as a new clinical biomarker and therapeutic target for Fn-infected CRC patients.