CD147-K148me2 Drives The Formation Of Immunosuppressive Microenvironment In NSCLC

Tumor microenvironment(TME)is a critical infrastructure for tumor initiation,proliferation and metastasis,which includes cellular and non-cellular components,such as tumor cells,immune cells,fibroblasts,and extracellular matrix.The differences in the composition and function of TME exerts different impacts on tumor progression.Lung cancer is one of the most common malignant tumors around the world,with high morbidity and mortality.Previous studies have shown that immunosuppressive TME with abundant immunosuppressive cells and inflammatory factors is an important hallmark for non-small cell lung cancer(NSCLC),which plays an important role in regulating tumor immune tolerance and immunotherapy resistance.Tumor-associated macrophages(TAMs)are an important component of TME,which spin off pro-inflammatory TAMs(M1-TAMs)and anti-inflammatory TAMs(M2-TAMs)under the conditions full of different mediators.M2-TAMs promote the formation of immunosuppressive microenvironment by releasing a variety of important cellular mediators such as cytokines and chemokines,thus leading to tumor progression.Therefore,exploring the remodeling of immune microenvironment mediated by the interaction between tumor cells and M2-TAMs is helpful to further reveal the immune escape mechanism of NSCLC,and provides new ideas and strategies for optimizing immunotherapy programs and improving anti-tumor immunotherapy effects.Tumor-associated antigen CD147 is an important carcinogenic factor,which is widely expressed in various tumors,including NSCLC,and is involved in tumor invasion and metastasis,drug therapy resistance,metabolic reprogramming and other malignant biological behaviors.With the rapid development of liquid chromatography-tandem mass spectrometry(LC-MS/MS)and proteomics,more and more studies have focused on the roles of protein post-translational modifications(PTMs)in tumor progression.Previous studies have shown that CD147 has several PTMs such as glycosylation,phosphorylation,and di-methylation,which are closely linked to tumor progression.However,the following scientific questions remain to be resolved:(1)Does CD147-PTM participate in the regulation of M2-TAM-mediated tumor immune microenvironment remodeling?(2)How does CD147-PTM regulate M2-TAM-mediated tumor immune microenvironment remodeling?In our study,we screened and identified the potential PTM of CD147 in NSCLC tissues using LC-MS/MS,and analyzed the relationship between its expression level and clinical characteristics or prognosis of individuals with NSCLC.Meanwhile,we systematically explored the biological function and molecular mechanism of CD147-PTM in regulating M2-TAM-mediated tumor immune microenvironment remodeling.Our findings provide a novel theoretical basis for tumor therapy targeting protein PTMs.Part Ⅰ: Screening and identification and clinical significance analysis of CD147di-methylation in NSCLC.CD147 is a transmembrane protein that plays a crucial role in tumor progression.The implementation of its biological functions mainly depends on its extracellular domain(ECD),which is stimulated with corresponding ligands or extracellular signals,initiating a series of intracellular biological chemical reactions and signal transduction.The results of LC-MS/MS showed that CD147-ECD had multiple di-methylated lysine sites,and the di-methylation of CD147-K148(CD147-K148me2)with high frequency occurrence was significantly overexpressed in NSCLC tissues compared with their para-cancer tissues.Hence,we chose CD147-K148me2 for the further investigation.Subsequently,the monoclonal antibody 12C8 targeting this modification was successfully generated by peptide immunization.Immunohistochemical(IHC)staining showed that the level of CD147-K148me2 markedly increased in NSCLC tissues compared with their adjacent tissues,and was significantly correlated with the gender,tumor size,and pathological staging of lung adenocarcinoma(LUAD)suffers.Survival analysis results showed that the high CD147-K148me2 level was markedly associated with poor prognosis in patients with NSCLC,especially those with advanced NSCLC.Our findings indicate that CD147-K148me2 significantly increases in NSCLC tissues and suggests poor prognosis in NSCLC patients.Part Ⅱ: Screening and identifying methyltransferase for CD147-K148me2.Protein methylation modification is usually generated by specific methyltransferase catalyzation.Methyltransferase screening experiment showed that the silencing of methyltransferase NSD2 reduced the level of CD147-K148me2 in NSCLC cells.The molecular docking model also demonstrated that NSD2 might be a potential methyltransferase for the generation of CD147-K148me2.Immunofluorescence assay showed a co-localization of NSD2 and CD147 in NSCLC tissues.Meanwhile,co-immunoprecipitation(co-IP)experiments demonstrated an interaction between NSD2 and CD147 in NSCLC cells,which could be weakened by CD147-K148 R mutation.NSD2 overexpression enhanced CD147-K148me2 levels in wild-type cells,but no change was observed in cells with CD147-K148 R mutation.In vitro methyltransferase assay showed that CD147 protein and peptide K148 could be di-methylated by co-incubation with NSD2 protein in methyltransferase buffer,but no di-methylated signal was detected in CD147-K148 R group.These findings suggest that methyltransferase NSD2 catalyzes CD147-K148 to generate CD147-K148me2.Part Ⅲ: CD147-K148me2-mediated intercellular crosstalk promotes the migration of M2-TAMs.To explore the biological function of CD147-K148me2 in NSCLC cells,RNA sequencing(RNA-seq)analysis was performed using NSCLC cells with wild type CD147 and mutant CD147-K148 R.The data from RNA-seq showed that chemokine CCL5 was the most significantly differentially expressed gene(DEG)between the two groups.Western blot,PCR,and ELISA showed that the level of CCL5 in CD147-K148 R cells was markedly lower than that in wild type cells,suggesting that CD147-K148me2 could promote the expression of CCL5 in NSCLC cells.Further analysis showed that this PTM was closely related to macrophage chemotaxis,immune response,and cytokine-mediated signaling pathway.Previous studies have shown that CCL5-CCR5 axis contributes to M2-TAM chemotaxis and tumor progression.Therefore,we examined the relationship between CD147-K148me2 level and M2-TAM infiltration in NSCLC tissues.The multi-color immunofluorescence test showed a significant positive correlation between CD147-K148me2 expression and M2-TAM infiltration in NSCLC tissues.The cell chemotaxis assay revealed that M2-TAMs in wild type group exhibited stronger migratory ability compared to CD147-K148 R group,which could be inhibited by the addition of CCL5 antibody or CCR5 inhibitor in wild type group,while no significant change was observed in CD147-K148 R group.These findings indicate that CD147-K148me2 facilitates CCL5 secretion in NSCLC cells and enhances M2-TAM migration by CCL5-CCR5 axis-mediated intercellular crosstalk.Previous studies have shown that CyPA-CD147 signaling promotes the secretion of inflammatory mediators such as cytokines and chemokines by activating MAPK pathway.Therefore,we explored the effect of CD147-K148me2 on CyPA-CD147 interaction.Molecular docking model showed that this PTM promoted CyPA-CD147 interaction by increasing hydrogen bond formation and interacting interface area.Co-IP experiments showed that CyPA-CD147 interaction was attenuated in CD147-K148 R cells compared with wild type cells.In wild type cells,CyPA stimulation promoted CCL5 expression by activating p38 signaling pathway,which could be blocked by p38 inhibitor.RNA-seq data showed that the expression of transcription factor ZBTB32 was significantly correlated with CCL5 expression in NSCLC cells.Overexpression of ZBTB32 promoted CCL5 expression and vice versa.The dual-luciferase reporter assay showed that ZBTB32 promoted the transcription of CCL5 gene by binding to the-387--378 region of CCL5 gene promoter.In wild type cells,CyPA stimulation increased the level of CCL5 by promoting ZBTB32 expression and nuclear translocation,which could be blocked by p38 inhibitor.In CD147-K148 R cells,the decrease of ZBTB32 and CCL5 caused by CD147-K148 R mutation was reversed by p38 activator.These findings suggest that CyPA-CD147-K148me2 interaction enhances CCL5 secretion in NSCLC cells by activating p38-ZBTB32 signaling,leading to CCL5-CCR5 axis-dependent M2-TAM migration.Part Ⅳ: CD147-K148me2 promotes lung cancer progression by driving the formation of immunosuppressive tumor microenvironment.In order to explore the role of CD147-K148me2 in the progression of NSCLC,a mouse subcutaneous xenograft model was constructed using LLC cells with wild type CD147 or mutant CD147-K148 R.The results showed that compared with the wild type group,the tumor volume,tumor weight,and the proportion of Ki-67+ cells and M2-TAMs in tumor tissues were significantly reduced in CD147-K148 R group.Meanwhile,the expressions of ZBTB32 and CCL5 in tumor tissues were also detected and the results showed that the expressions of ZBTB32 and CCL5 both decreased in CD147-K148 R group compared with the wild type group.These results suggest that CD147-K148me2 promotes tumor progression by increasing M2-TAM infiltration in tumor tissues.We also evaluated the efficacy of 12C8 in inhibiting tumor progression.The results showed that12C8 treatment significantly reduced the tumor volume and tumor weight,as well as the proportion of Ki-67+ cells and M2-TAMs in tumor tissues compared with IgG treatment.These findings demonstrate that CD147-K148me2 promotes tumor progression by remodeling tumor immunosuppressive microenvironment,which provides a potential target for lung cancer treatment.Conclusion: In our study,we identify a highly expressed PTM of CD147,CD147-K148me2,in NSCLC tissues,which is catalyzed by methyltransferase NSD2 and values significantly in tumor progression and unsatisfying survival outcomes among NSCLC sufferers.Mechanically,CD147-K148me2 enhances the interaction between CyPA and CD147,and in turn promotes p38-ZBTB32 signaling-mediated CCL5 gene transcription,further leading to increased NSCLC cell-derived CCL5 secretion.Subsequently,CD147-K148me2-mediated CCL5 elevation facilitates M2-TAM infiltration in NSCLC tissues by CCL5-CCR5 axis-dependent intercellular crosstalk,and reformats immunosuppressive microenvironment,which can be inhibited by CD147-K148me2 blockade using targeted antibody 12C8.Collectively,our study reveals the role of CD147-K148me2-driven intercellular crosstalk in the formation of NSCLC immunosuppression,and provides a potential interventional strategy for PTM-targeted NSCLC therapy.