Mechanism Of The Epigenetic Silencing Of CCL2-mediated Macrophage Infiltration In Small Cell Lung Cancer

Background:Characterized by being sensitive to chemotherapy,highly aggressive,and greatly prone to recurrence,small cell lung cancer(SCLC)is a special subtype of lung cancer with a very poor prognosis.The five-year survival rate of SCLC patients is limited to around 6%.However,in the face of the severe consequences caused by SCLC,its therapeutic strategies have remained almost still in the past three decades,prompting us to further investigate SCLC from multiple perspectives.The close relationship between the immune system and SCLC is being widely studied.And currently much of the research in SCLC is focusing on the acquired immunity,especially T cells,while innate immunity has received scant attention.Among innate immune cell populations,macrophages are capable of recognizing and presenting antigens,and killing/inhibition(M1 type)or repairing injuries(M2 type).Meanwhile,high level of macrophage infiltration has shown to confer different clinical outcomes in various types of tumors due to their heterogeneity and plasticity: high levels of macrophage infiltration are indicative of good prognosis in colorectal cancer,melanoma,but poor prognosis in breast,prostate and bladder cancer,and other tumors.Whereas in SCLC,the description of the extent of macrophage infiltration in the tumor microenvironment is controversial owing to the poor accessibility of postoperative specimens,which requires more detailed research.C-C motif chemokine ligand 2(CCL2)is a potent chemokine for monocyte and macrophages,and studies have found that CCL2 also plays an important role in the tumor development and progression.CCL2 is not only regulated by classical signaling pathways such as NF-?B,but also by epigenetic mechanisms,among which DNA methylation and histone lysine methylation are two important types.DNA methylation is mainly regulated by DNA methyltransferases(DNMTs),in which DNMT1 maintains DNA methylation level at the promoter region of tumor suppressor genes,silencing their expression and thus promoting tumor development.Meanwhile,numerous studies have shown that the histone methyltransferase EZH2(enhancer of zeste homolog 2)can facilitate tumor initiation and progression by affecting the methylation of histones,especially H3K27me3(histone H3 lysine 27 tri methylation).It has been well documented that EZH2 and DNMT1 act in combination to regulate the expression of CXCL9 and CXCL10,thereby affecting T lymphocyte infiltration.Therefore,whether the chemokine CCL2 is regulated by EZH2 induced H3K27me3 and DNMT1 mediated DNA methylation in SCLC also becomes a scientific question that is worth exploring.Objectives:1.To observe the degree of macrophage infiltration in SCLC tissues;2.To verify whether migration of monocytes in SCLC is affected by the chemokine CCL2;3.To explore whether CCL2 in SCLC is regulated by EZH2 induced H3K27me3 and DNMT1 mediated DNA methylation;4.To examine the phenotypic and functional changes of macrophages after co-culturing with SCLC cells;5.To investigate whether the expression of CCL2 can be restored through the treatment of epigenetic inhibitors,to regulate the infiltration of macrophages,thus leading to shrinkage of tumor in SCLC.Methods:1.To determine the degree of macrophage infiltration by CD68 staining in SCLC tissue microarrays using immunohistochemical staining(IHC);2.The expression of CCL2 was determined in SCLC tissues and cell lines by IHC,enzyme-linked immunosorbent assay(ELISA)and real-time quantitative PCR(RT-qPCR)techniques,respectively.Transwell assay was used to verify the chemotactic effect of CCL2 on monocytes;3.The expression of EZH2 or DNMT1 was measured in SCLC tissue samples using IHC;in cell lines,the expression of the corresponding CCL2 and the chemotactic effect on monocytes were measured after blocking the expression of these epigenetic factors using small molecule inhibitors or si RNAs;4.To further explore the epigenetic regulation on CCL2 in SCLC,whether the expression of CCL2 was affected by EZH2 mediated H3K27me3 was verified by chromatin immunoprecipitation(ChIP),and Mass Array methylation analysis was utilized to verify the role of DNMT1 mediated DNA methylation in this process;5.To probe the phenotype of macrophages in the SCLC microenvironment and thus infer their possible functions,monocytes isolated from peripheral blood were induced into macrophages by in vitro culture,and then we used RT-qPCR and fluorescence-activated cell sorter(FACS)to detect macrophage phenotypic markers to determine the phenotypic changes of macrophages before and after co-culturing with SCLC cell lines and to search for evidence of phagocytic and pro-apoptotic effects of macrophages on tumor cells;6.To establish xenograft models of SCLC in nude mice,exploring the effect of EPZ011989(EPZ),an inhibitor of EZH2,combined with Decitabine(DAC),an inhibitor of DNMT1,on tumor growth by recording tumor volume change,tumor weighing,IHC,immunofluorescence(IF),ELISA and RT-qPCR.Results:1.Compared with normal lung tissues,the degree of CD68 positive macrophage infiltration in SCLC tissues was significantly reduced in a stage-dependent manner,suggesting that the reduction of macrophage infiltration might be correlated with poor prognosis;2.Compared with normal tissue samples or cell lines,the expression of CCL2 was dramatically decreased in SCLC ones,presenting similar changes with the degree of CD68 positive macrophages infiltration.Correlation analysis revealed that the proportion of CD68 positive cells and the expression of CCL2 were positively associated.Transwell assay confirmed that CCL2 was the major chemokine for the recruitment of macrophages;3.Among the 101 SCLC patients we tested,a moderate to strong positive expression of EZH2 or DNMT1 was observed in 88.1% and 75.2% of SCLC patients,respectively,and both EZH2 and DNMT1 expression were negatively correlated with CCL2 expression or the proportion of CD68 positive cells;CCL2 expression level was significantly increased after using the corresponding inhibitors EPZ / DAC or si RNAs to interfere with EZH2 and(or)DNMT1 expression,especially in combination treatment.Meanwhile,the recruitment of monocytes was significantly enhanced after the combination treatment;4.ChIP-qPCR assay confirmed that EZH2 mediated tri-methylation of H3K27 was increased at the enhancer region of CCL2,and this enrichment could be relieved by EPZ;Mass Array methylation analysis confirmed that DNMT1 mediated methylation of Cp G sites was significantly increased at the promoter region of CCL2,and this methylation could be reversed by DAC;5.When co-culturing differentiated macrophages with SCLC cells in vitro,cell-cell contact promoted a significant increase in the expression levels of the M1-type markers,iNOS,IL-12,IL-23 and IL-8,and soluble factors secreted by tumor cells promoted CCL2 expression by macrophages while simultaneously inhibiting CCR2 expression;meanwhile,indirect contact resulted in the decreased expression of two important M2-type markers,Arg-1 and TGF-?,whereas cell-cell contact substantially promoted the expression of EGF and VEGFA;we also observed evidence of phagocytic and pro-apoptotic effects of macrophages on SCLC cell lines;6.In the combination treatment group of EPZ and DAC,the tumor volume of nude mice bearing SCLC was significantly reduced,the tumor weight was significantly reduced;ELISA results indicated an elevated expression of CCL2 in SCLC tumor tissues;IF results showed that intralesional macrophage infiltration was increased,and a trend towards M1 polarization was exhibited;IHC results indicated that the proliferation of tumor tissues was attenuated,while the apoptosis was enhanced.Conclusions:1.Significant reduction in macrophage infiltration due to low expression of chemokine CCL2 is evident in SCLC lesions,with higher stages associated with lower degrees of infiltration;2.The expression of CCL2 is regulated by epigenetic factors,i.e.,EZH2 mediated H3K27me3 on CCL2 enhancer regions and DNMT1 mediated DNA methylation of Cp G sites on the CCL2 promoter regions;3.Upon co-culturing between macrophages with SCLC cells,soluble factors and cell-cell contact promote macrophages to skew to M1 phenotype,leading to phagocytosis and apoptosis of tumor cells;4.The combination therapy of EPZ and DAC can restore the expression of CCL2 in SCLC lesions,thus increasing the infiltration of macrophages,and may play a potential anti-tumor function by promoting macrophage polarization to M1 type.Therefore,these findings indicate novel perspectives into the role of macrophages in the development of SCLC,and provide a sound basis for the construction of new therapeutic approaches for SCLC.