Mechanisms Of The Crosstalk Between Tumor-associated Macrophages And Tumor Cells On Promoting Epithelial-mesenchymal Transition In Lung Adenocarcinoma

Ⅰ.Background Non–small cell lung cancer(NSCLC),which accounts for 80% to 85% of all lung cancers,is the most fatal cancer worldwide,with a 5-year survival as low as 13%.Therefore,further investigation on the mechanisms of the progression is crucial for improving the efficacy and prognosis of NSCLC.Recent evidence suggests the tumor microenvironment(TME)is critical for the initiation,progression and metastasis of tumor.Macrophages infiltrated in the tumor stroma are define as tumor-associated macrophages(TAMs),and they are the most abundant inflammatory cells in the TME.TAMs exhibit the macrophages M2 promote tumor angiogenesis,growth,metastasis,and immunosuppression by secreting a series of cytokines,chemokines,and proteases.Several studies have revealed that an increased density of TAMs was correlated with poor prognosis in various cancers including NSCLC.This association is linked to the typical presence of the M2-polarized TAMs,however,the exact mechanisms underlying the process is still not clear.Epithelial-mesenchymal transition(EMT)is a process,in which epithelial tumor cells lose epithelial feather and gain mesenchymal phenotypes.It is considered as the critical step by which tumor cells gain the higher capabilities of invasion and metastasis.Previous studies have demonstrated that EMT was correlated with carcinogenesis,metastasis,and poor prognosis in various types of cancers including NSCLC.Recent study suggests that TAMs play a protumor role and promote EMT in oral squamous cell carcinoma,pancreatic cancer,et al.However,the relationship between TAMs and EMT inlung adenocarcinomais still unclear.Abnormal glycosylation is a characteristic feature of tumors and is associated with the abnormal expression of glycosyltransferase enzymes.Le Y is a type of difucosylated oligosaccharide,and FUT4 is a key enzyme in the synthesis of the tumor-associated carbohydrate antigen Le Y.FUT4/Le Y was highly expressed in a variety of solid tumors including lung cancer.However,few studies have evaluated the relationship between TAMs and the fucosylation mediated by FUT4/Le Y.USP22 is a newly discovered member of the ubiquitin hydrolasefamily.USP22 deubiquitinatesthe H2 A and H2 B histone proteins and acetylates the H4 histone protein.UPS22 is significantly elevated in a variety of solid tumors,andis closely related tometastatic potential,therapy resistance and prognosis.However,the relationship between TAMs and the deubiquitination mediated by USP22 is still unclear.Our study aimed to explore the role of the fucosylation mediated by FUT4/Le Y in TAMs induced EMT in lung adenocarcinoma,and the role of the deubiquitination mediated by USP22 in the infiltration of TAMs.Ⅱ.Methods 1.Immunohistochemistry SP method was used to detect the expression of CD68,E-cadherin,Le Y and USP22 in 60 cases of lung adenocarcinoma paraffin histopathological tissues.The correlation among CD68,E-cadherin,Le Y,and the correlationbetween CD68 and USP22 was analyzed.SPSS20.0 statistical software was used for statistical analysis,and p-value less than 0.05 were considered significant.2.The vector was transfected into cells.Stable transfectants were selected with 800 μg/ml G418,and individual clones were isolated.The WT and T567D-mutant Ezrin overexpression plasmids were constructed.The H1299 and H358 cell lines with USP22 stably interfered or overexpressed were constructed by the same method.3.The expression of TGF-β1 in the culture supernatant of macrophage,and the expression of IL-1,IL-6,IL-8,CCL-2 and CXCL1 in the culture supernatant of tumor cells were detected by ELISA assay.4.The expression of FUT4,Le Y,mesenchymal protein markers,Ezrin,USP22,p65,H2B-ub1 and IκBwere detected by Western blot.5.The expression of E-cadherin,β-catenin and F-actinwas analyzed by indirect immunofluorescence staining.6.Le Y binding to Ezrin and the fucosylation level analyzed by UEA was detected by co-immunoprecipitation.7.The binding between H2B-Ubi and p65 target gene,RNA polymerase II and p65 target gene,p65 and its target gene was analyzed by chromatin immunoprecipitation.8.In vivo assays confirmed the relationship between FUT4/Le Y and TAMs mediated EMT,and the role of USP22 in the infiltration of TAMs.Ⅲ.Results Section1 Tumor-associated macrophages promote Ezrin phosphorylation mediated epithelial-mesenchymal transition in lung adenocarcinoma through FUT4/Le Y up-regulation 1.The density of TAMs correlates with E-cadherin level and Le Y level in human lung adenocarcinoma tissues.The staining intensity of E-cadherin in the TAMs negative group(CD68 negative)was higher than that in the TAMs positive group(CD68 positive)(P<0.01),while Le Y expression was lower than that in the TAMs positive group(P<0.01).The positive expression of CD68 correlated with a loss of E-cadherin expression(r=-0.505,P<0.01)and positive expression of Le Y(r=0.55,P<0.01).There is a negative correlation between the expression of E-cadherin and Le Y(r=-0.798,P<0.01).2.M2 macrophages promoted FUT4/Le Y expression through the TGF-β1/Smad2/3 signaling pathway in in vitro assays ELISA assay showed that the TGF-β1 expression was elevated in TCM.TCM significantly promoted FUT4/Le Y expression and Smad2/3 phosphorylation in lung adenocarcinoma cell lines in a dose-dependent manner.When A549 and H1299 cells were incubated with TCM and treated with LY364947 and SB431542,FUT4/Le Y expression was partially suppressed.TGF-β1 had significant dose-dependent effects on the FUT4/Le Y expression.3.FUT4/Le Y was indispensable in M2 macrophages-mediated cytoskeletal remodeling and EMT.After TCM induction,the morphology of A549 and H1299 cells changed from a pebble-like shape to an elongated shape mesenchymal phenotype,and themesenchymal markers N-cadherin,Vimentin and Fibronectin were elevated.Upon FUT4 down-regulation,the morphology of A549 and H1299 cells no longer change significantly after TCM treatment,and the expression of mesenchymal markers significantly decreased.After down-regulation of Le Y through either FUT4 interference or the use of exogenous Le Y antibodies to directly block the Le Y antigens on the surface of A549 and H1299 cells,TCM could not effectively promote the EMT process.Under TCM induction,the epithelial marker E-cadherin of A549 cells was significantly down-regulated,and β-catenin was accumulated and translocated from the cell membrane and cytoplasm into the nucleus.After FUT4 down-regulation by RNAi,E-cadherin expression levels gradually increased and β-catenin relocalized to the cell membrane and cytoplasm.After TCM induction,F-actin cytoskeleton was altered with increased polymerization and extended cellular protrusions.After FUT4 down-regulation by RNAi,TCM no longer elicited the above changes.4.FUT4/Le Y-mediated fucosylation of Ezrin was closely associated with the phosphorylation of Ezrin During the EMT induced by TCM,the Ezrin phosphorylation(T567)increased significantly in a dose-dependent manner in A549 and H1299 cells.After down-regulation of Le Y through FUT4 interference,the Ezrin phosphorylation(T567)decreased significantly,which was accompanied by the decreased expression of mesenchymal markers.Ezrin phosphorylation(T567)induced by TCM was inhibited after tunicamycin pretreatment,which was also accompanied by the decreased expression of mesenchymal markers.Bioinformatics analysis(http://www.cbs.dtu.dk/services/Net NGlyc/)showed that Ezrin protein was composed of 585 amino acids,and the sites of 23-26 and 247-250 were the potential N-glycosylation sites of Ezrin.Co-immunoprecipitation result showed that Ezrin protein indeed had a certain amount of Le Y glycoproteins.Lectin blotting with UEA lectin recognizes fucosylation on N-glycans.The fucosylation of Ezrin was decreased in A549-sh FUT4/H1299-sh FUT4 cells,compared with that in A549/H1299 cells.The down-regulation of Ezrin phosphorylation and mesenchymal markers after knockdown of FUT4 can be counteracted by the overexpression of WT Ezrin but not T567D-mutant Ezrin.5.In vivo assays confirmed that M2 macrophages promoted EMT through the up-regulation of Le Y and p-Ezrin M2 or M0 macrophages were pre-mixed with A549 cells at a 1:4 ratio and were subcutaneously implanted into nude mice.The M2 group already had tumor formation on day 6;in contrast,the M0 control grouphad tumor formation on day 11.The volume of the tumors in the M2 group were also higher than those in the M0 control group(P<0.01).Immunohistochemistry experiments showed that the E-cadherin expression at the cell membrane in the M2 group was significantly lower than that in the M0 control group,which was accompanied by up-regulation of Vimentin.Andthe expression of Le Y and p-Ezrinwas higher in the M2 group than in the M0 control group.Section 2 USP22 promotes tumor-associated macrophage infiltration in lung adenocarcinoma through NF-κB pathway 1.The level of USP22 correlates withthe density of TAMsin human lung adenocarcinoma tissues.The positive rate of TAMs in USP22 positive group is higher than USP22 nagative group(84.2% vs.18.2%,P<0.01).There is a positive correlation between USP22 and the density of TAMs(r=0.649,P<0.01).2.USP22 promoted NF-κB target gene transcription in lung adenocarcinoma We stably inhibit USP22 in H1299 cells and overexpressed USP22 in H358 cells.After 10ng/ml TNF-αtreatment,down-regulation of USP22 can inhibit the expression of IL-1,IL-6,IL-8,CCL-2and CXCL-1 in the culture supernatant of H1299 cells,up-regulation of USP22 can promote the expression of these the above factors.There was no change without TNF-α treatment.After 10ng/ml TNF-αtreatment,down-regulation of p65 can inhibit the up-regulation of these factor mediated by USP22.3.USP22 promoted macrophage infiltration through NF-κB pathway in lung adenocarcinoma After TNF-αtreatment,down-regulation of USP22 in H1299 cells promoted macrophage infiltration,and up-regulation of USP22 in H358 cells inhibited macrophage infiltration.There was no change without TNF-α treatment.Down-regulation of p65 by p65-si RNA in the H358 cells which stably overexpressed USP22 can inhibit the macrophage infiltration mediated by USP22 overexpression.4.USP22 deubiquitinated histone H2 B and enhanced the binding between p65 and its target gene Down-regulation or up-regulation of USP22 did not alter the nuclear localization of IκBand p65 with or without TNF-α treatment.After TNF-αtreatment,the down-regulation of USP22 can inhibit the binding between H2B-ub1 and p65target gene,RNA polymerase II and p65 target gene,p65 and its target gene.5.In vivo assays confirmed that USP22 can promote TAMs infiltrationin lung adenocarcinoma USP22-sh1 and sh-con H1299 cells were subcutaneously implanted into nude mice.The volume of the tumors in USP22-sh1 group were also lower than those in the USP22-sh-con group.Immunohistochemistry experiments showed that the H2B-ubi expression in USP22-sh1 group was significantly higher than that in the USP22-sh-con group.Andthe expression of IL-1,IL-6,CCL-2and CXCL-1 was significantly higher thanthat in the USP22-sh-con group,and there was no change on IL-8.Ⅳ.Conclusion Section 1 Tumor-associated macrophages promote Ezrin phosphorylation mediated epithelial-mesenchymal transition in lung adenocarcinoma through FUT4/Le Y up-regulation 1.The density of TAMs correlates with E-cadherin level and Le Y level in human lung adenocarcinoma tissues.2.M2 macrophages promoted FUT4/Le Y expression through the TGF-β1/Smad2/3 signaling pathway in in vitro assays 3.FUT4/Le Y was indispensable in M2 macrophages-mediated cytoskeletal remodeling and EMT.4.FUT4/Le Y-mediated fucosylation of Ezrin was closely associated with the phosphorylation of Ezrin 5.In vivo assays confirmed that M2 macrophages promoted EMT through the up-regulation of Le Y and p-Ezrin Section 2 USP22 promotes tumor-associated macrophage infiltration in lung adenocarcinoma through NF-κB pathway 1.The level of USP22 correlates withthe density of TAMsin human lung adenocarcinoma tissues.2.USP22 promoted NF-κB target gene transcription in lung adenocarcinoma 3.USP22 promoted macrophage infiltration through NF-κB pathway in lung adenocarcinoma 4.USP22 deubiquitinated histone H2 B and enhanced the binding between p65 and its target gene 5.In vivo assays confirmed that USP22 can promote TAMs infiltrationin lung adenocarcinoma...