Study On The Mechanism Of Epinephrine Inducing M2-Type Polarization Of Macrophages Via TRIM2 To Promote The Progression Of Colorectal Cancer

Part I TRIM2 promotes colorectal cancer progression by inducing M2-type polarization in macrophagesBackground Previous studies from our group have shown that long-term chronic stress stimulation increase the level of epinephrine(EPI)in experimental mice and promote the development of colorectal cancer through TRIM2 pathway.Objective To investigate the effect of TRIM2 on the progression of colorectal cancer and macrophage M2 polarization in tumor microenvironment.Methods The expression of TRIM2 in 65 pairs of colorectal cancer tissues and normal tissues was detected by immunohistochemistry.GEPIA2,UALCAN and TIMER databases were retrieved and compared with the experimental results of IHC.The possible relationship between TRIM2 and immune cell infiltration was explored in the TIMER database.Different markers of macrophage polarization in human colorectal cancer tissues were detected by immunohistochemistry.TRIM2 expression was detected in several colorectal cancer cell lines(Lo Vo,SW480,DLD1,SW62 and HCT1160).Lo Vo cells were transfected with si-TRIM2 and SW480 cells were transfected with the overexpressed plasmid of TRIM2.The role of TRIM2 in colorectal cancer was studied by wound healing assays,Transwell assays and colony formation assays.Subcutaneous xenograft implantation model and pulmonary metastasis model were used to explore the effect of TRIM2 on tumor proliferation in vivo.Immunohistochemical staining for Ki67,TUNEL assay and flow cytometry were used to evaluate proliferation and apoptosis of xenograft tumors.The markers of TAM polarization in xenograft tumors were detected by immunohistochemical staining.After activation with PMA,THP-1 cells were cocultured with treated colorectal cancer cells.Then the M2 polarization of TAMs was analyzed by flow cytometry and IF.Results First,immunohistochemical detection of TRIM2 showed that TRIM2 expression was significantly increased in colorectal cancer tissues.Data from the GEPIA2,UALCAN,and TIMEER databases also support this result.TRIM2 expression was closely related to tumor stage and lymph node metastasis.Data from the TIMEER database showed that TRIM2 expression was associated with the infiltration of a variety of immune cells,including macrophages,in colorectal cancer.A similar number of CD68+ cells were found in tumor tissues with high and low TRIM2 expression by IHC assay.More CD206+/CD163+ cells were found in tumor tissues with high TRIM2 expression,while more i NOS+/CD86+ cells were found in tumor tissues with low TRIM2 expression.The expression of TRIM2 in several colorectal cancer cell lines(SW480,DLD1,Lo Vo,HCT116 and SW620)and the expression of TRIM2 in colorectal cancer cells indicate that TRIM2 acts as a tumor-promoting factor in colorectal cancer.In addition,TRIM2 may influence macrophage differentiation in human colorectal cancer tissues.Lo Vo cells were transfected with si-TRIM2,and SW480 cells were transfected with the TRIM2 overexpression plasmid.Then,the function of TRIM2 in colorectal cancer was investigated through wound healing assays,Transwell assays,colony formation assays,and CCK-8 assays,all of which indicated that TRIM2 can enhance the migration and proliferation abilities of colorectal cancer cells.A subcutaneous xenograft implantation model showed that TRIM2 promoted the growth of xenograft tumors.Immunohistochemical staining for Ki67,TUNEL assay and flow cytometry showed that TRIM2 promoted tumor proliferation and inhibited tumor apoptosis in vivo.A pulmonary metastasis model suggest that TRIM2 promotes tumor metastasis in vivo.Immunohistochemical detection of TAM polarization markers in xenograft tumors showed that similar numbers of CD68+ cells were found in sh NC group and sh-TRIM2 groups. When TRIM2 expression was inhibited,there were fewer CD206+/CD163+ cells and more i NOS+/CD86+ cells.After activation with PMA,THP-1 cells were cocultured with treated colorectal cancer cells.Then,flowcytometric and IF analyses were performed,and the results indicated that TRIM2 promoted M2 polarization of TAMs.Part II Study on the mechanism of epinephrine inducing M2 Type polarization of macrophages via TRIM2 to promote the progression of colorectal cancerObjective To further elucidate the molecular mechanism of epinephrine regulating TRIM2 protein expression and promoting M2-type polarization of macrophages leading to proliferation and metastasis of colorectal cancer.Methods The proteins interacting with TRIM2 were detected by immunocoprecipitation and analyzed by liquid chromatography and high-throughput mass spectrometry analysis.Ubibrowser database was used to predict TRIM2 substrates and to explore the same genes between the two groups.The expression level of TRIM2 and IκBα in colorectal cancer and their relationship were detected by Western blot,and the effect of TRIM2 on P65 was evaluated.After silencing or overexpressing TRIM2,color ectal cancer cells were further stimulated with cycloheximide to investigate the effect of TRIM2 on IκBα protein.The effect of TRIM2 on IκBα was studied after MG132 treatment.Experiments were performed to understand whether TRIM2 altered the polyubiquitination of endogenous IκBα.Plasmids expressing TRIM2,IκBα,Ub,Ubk48 or Ubk63 were transfected into 293 T cells to investigate the total ubiquitination,K48-linked ubiquitination and K63-linked ubiquitination of TRIM2 on IκBα.The direct interaction between TRIM2 and IκBα was investigated by Co-IP method.The interaction between TRIM2 and IκBα was investigated by Co-IP and GST pull-down analysis and immunofluorescence staining.We further constructed plasmids with the full-length TRIM2 sequence or sequences with deletion of different domains of TRIM2.Co-IP was applied to explore the interaction between each truncated mutant and IκBα,the full-length sequence of IκBα or the plasmids with different domain sequences of IκBα,and to know the domain of interaction between IκBα and TRIM2.Rescue experiments were conducted to investigate whether TRIM2 expressed in colorectal cancer cells promotes colorectal cancer progression and M2 polarization of TAMs by regulating IκBα expression.Results A total of 176 proteins were identified by immunocoprecipitation,liquid chromatography and high-throughput mass spectrometry.we predicted the top 300 substrates for TRIM2 through the Ubibrowser database.The same genes between the two groups were explore,and the results indicated that TRIM2 may bind to NFKBIA(IκBα),TRIM3 and ACTN4.Therefore,IκBα was selected to further investigation.We found that TRIM2 expression was up-regulated and IκBα expression was down-regulated in colorectal cancer tissues.In addition,the expression level of TRIM2 was negatively correlated with that of IκBα.Next,the results of WB indicated that TRIM2 inhibited the expression of IκBα in colorectal cancer cells.Further studies showed that TRIM2 promoted the nuclear translocation of P65.colorectal cancer cells were stimulated with cycloheximide(CHX)after silencing or overexpression of TRIM2.The results showed that TRIM2 may decrease the stability of IκBα protein.In addition,we found that TRIM2 induced the degradation of IκBα in a proteasome-dependent manner and promoted the polyubiquitination of endogenous IκBα.plasmids expressing TRIM2,IκBα,Ub,Ubk48 or Ubk63 were transfected into 293 T cells.We found that the total and K48-linked ubiquitination but not the K63-linked ubiquitination of IκBα was obviously decreased by TRIM2,suggesting that TRIM2 improve the K48-linked polyubiquitination of IκBα.The results of Co-IP,GST pull-down assays and immunofluorescence staining indicated that TRIM2 physically interacted with IκBα.Amino acid 1-111 of TRIM2 was found to be involved in the interaction,and amino acid 181-317 of IκBα might be involved in the interaction between IκBα and TRIM2.Finally,the plasmids expressing full-length TRIM2 or TRIM2 with deletion of amino acids 1–111 were transfected into CRC cells,and we found that the amino acids 1–111played an indispensable role in TRIM2-mediated inhibition of IκBα expression,promotion of P65 nuclear translocation,and promotion of IκBα degradation.It was found that silencing IκBα could reverse the inhibitory effect of TRIM2 on IκBα expression,promoting nuclear translocation of P65,tumor cell proliferation and metastasis,and promoting M2 polarization of TAMs.In this study,we also confirmed that epinephrine promoted the expression of TRIM2,inhibited the expression of IκBα,and promoted the nuclear translocation of P65 in colorectal cancer cells.The results of the wound healing,Transwell,CCK-8 and colony formation assays showed that epinephrine enhanced the migration and proliferation ability of colorectal cancer cells.we downregulated the expression of TRIM2 in cells treated with epinephrine for five days,which suppressed the regulatory effects of epinephrine on IκBα expression,migration and proliferation,indicating that epinephrine promotes the proliferation and metastasis of colorectal cancer cells and M2 polarization of THP1 cells by upregulating the expression of TRIM2 in colorectal cancer cells.The results of subcutaneous tumor formation in nude mice showed that epinephrine promoted the proliferation of colorectal cancer cells,the expression of TRIM2 in tumor tissues,and the M2 polarization of macrophages.Conclusion The increased secretion of epinephrine under chronic stress up-regulates the expression of TRIM2 in colorectal cancer cells,and the binding of TRIM2 amino acid sequence 1-111 to IκBα amino acid sequence 181-317 leads to the ubiquitination of IκBα and the decrease of its stability,which leads to nuclear translocation of P65 and M2 polarization of macrophages.Thus,it promotes tumor proliferation and metastasis.In addition to promoting tumor proliferation and metastasis through the above IκBα pathway,epinephrine can also directly enhance the migration and proliferation of colorectal cancer cells and directly cause M2 polarization of macrophages.Our study explored the role of epinephrine in the development of colorectal cancer,and revealed that epinephrine release promoted colorectal cancer proliferation and metastasis directly through the TRIM2/IκBαpathway or through the TRIM2-IκBα/ M2 polarization of macrophages.This study provides a new theoretical basis for further exploring the mechanism of colorectal cancer development.