The Role And Mechanism Of Tumor-expressed CD38 On NSCLC Tumor Cells Progression

Background:Immune checkpoint inhibitors,such as PD-1/PD-L1 antibodies,have made a significant breakthrough in tumor treatment via re-activating the function of immune cells to eliminate malignant tumor cells.However,the effecti ve response of PD-1/PD-L1 therapy only happened in minority of patients.Moreover,almost all those who initially benefited from the PD-1/PDL1 therapy will eventually develop acquired drug resistance after a certain period of time.To improve the efficacy of immune checkpoints inhibitors and explore the mechanism of acquired drug resistance of immune checkpoints are urgent to be resolved.Our groups have always been committed to explorate the mechanism of immune checkpoints inhibitors resistance.Through RNA-sequencing and bioinformatics analysis,we found that the expression of CD38 on tumor cells was significantly up-regulated in accompany with PD-L1 antibody resistance.The expression of CD38 increased the production of adenosine,a common immunosuppressive molecule in tumor microenvironment,to promote tumor immune escape.Our preliminary in vivo experiments also showed that PD-L1 antibody combined with CD38 antibody could overcome PD-L1 resistance.However,the regulatory mechanism and enzymatic activity of tumor-expressed CD38 on tumor cells has not been reported.Herein,this study is intended to explore the mechanism of tumor cells derived CD38 in regulating tumor progression.And we also want to explore the molecular mechanism of CD38 enzymatic metabolites and substrates in regulating the survival of tumor cells.Furthermore,we also identify that targeting CD38 enzymatic products and substrates might serve as a potential therapeutic intervention for lung cancer.Through this study,we hope to provide a basic foundation for the usage of CD38 antibody or combination with PD-L1 antibody in the treatment of solid tumors.Methods:In vitro experiments,mouse Lewis lung carcinoma cell line(LLC)and human non-small cell lung adenocarcinoma A549 cell line were used to construct CD38 knockout cell lines(CD38 KO)by CRISPR-Cas9 technology.On the basis of CD38 KO tumor cells,we further constructed CD38 point mutant cells and CD38 overexpression cells(CD38 MU and CD38 OE)with lentivirus transfection.Then,the expression and localization of CD38 in our model cell lines were verfied by flow cytometry and confocal laser scanning microscope.In addition,in order to monitor tumor metastasis,we also re-transfected a red fluorescent protein into LLC cells which co-expressed with diversity of CD38(LLC-T2).siRNA technology was used to down-regulate the expression of TRPM2 protein in mouse and human tumor cells.The changes of CD38 enzymatic activity,substrates and metabolites were detected by liquid chromatography and cADPR detection kit.The ability of cell proliferation was detected by CCK8 kit,cell stemness was confirmed by clone formation and the ability of cell migration was detected by Transwell kit.And the ratio of cell apoptosis was detected by annexin V plus PI kit.The changes of gene expression and cell signal pathways between CD38 wild type(WT)and KO of A549 cells were screened,analyzed and enriched by RNA sequencing and bioinformatics analysis.Western blotting analysis was used to verify the alteration of related pathways of CD38 enzymatic activity.The calcium probe kit was used to test the changes of intracellular calcium level caused by the manipulation of CD38.The changes of intracellular ROS caused by the manipulation of CD38 expression was detected by ROS detection kit.In vivo experiments,we carried out subcutaneous transplantation of CD38 differentially expressed cells in C57BL/6 wild-type mice,CD38 deficient mice and BABL/c nude mice and monitor the tumor growth in above animal models.The lung metastases in C57BL/6 mice was observed and quantified by fluorescence microscope.C57BL/6 mice were also used to verify the potential efficacy of CD38 enzymatic specific inhibitor 78C and its enzymatic product small molecule inhibitor 8-Br-cADPR.The numbers of tumor infiltrating T cells,myelogenous suppressor cells(MDSCs)and macrophages in the tumor microenvironment were measured by flow cytometry.We also collected 158 paired of lung adenocarcinoma and related adjacent tissues(including clinical information),and then detected the expression of CD38 in all of tissue microarrays by immunohistochemistry.Then we analyzed the relationship between the expression of CD38 with clinical stage,pathological stage and prognosis.In addition,we also collected pleural effusion from lung cancer and non-cancer patients.And we further measured the relative level of cADPR in cancerous and non-cancerous pleural effusion by cADPR kit.The correlation between the expression of CD38 and transient receptor potential cation channel subfamily M member 2(TRPM2)in lung cancer metastasis and prognosis was further analyzed by the public databases such as Kaplan Meier,TNM and GEPIA.Results:(1)Within in vivo and in vitro experiments,we found that both in A549 and LLC tumor cells,compared with WT cells,the proliferation,migration and clonogenic formation ability of CD38 KO and MU cells were significantly inhibited,but the ratio of apoptotic cells were obviously up-regulated.On the contrary,the proliferation,migration and clonogenic ability of CD38 OE cells were more strengthen than those of WT tumor cells.In vivo experiments,we demonstrated that the subcutaneous transplanted tumor growth of CD38 KO and MU cells in C57BL/6 and CD38 deficient mice were significantly slower than those of WT and OE cells.Consistently,the number of lung metastases originated from the CD38 KO and MU tumor were also significantly fewer than those of WT tumor,but the infiltration of CD3+T cells was increased.And there were no significant changes of infiltrated MDSC and macrophages in all groups.Similarly,compared with WT cells transplanted into BALB/c nude mice,we also found that CD38 KO and MU cells showed impaired tumor growth,while CD38 OE cells showed faster tumor growth.(2)By detecting the enzymatic activity of CD38,we found that,compared with CD38 WT and OE tumor cells,CD38 KO and MU cells showed a significantly decreased level of adenosine(ADO)while increased the concentration of NAD+.However,exogenous administration of NAD+or ADO did not affect the proliferation and migration of tumor cells with different level of CD38.Furthermore,our results also showed that the level of cADPR in CD38 KO and MU cells was also significantly decreased.Exogenous adding of cADPR could restore the ability of proliferation and migration in CD38 KO and MU tumor cells,while a small molecule inhibitor,8-Br-cADPR showed an inhibition of proliferation and migration in CD38 WT and OE tumor cells.Within subcutaneous transplantation model,we further confirmed that both 78C and 8-Br-cADPR could significantly inhibit the growth of LLCCD38 WT cells and promote the intratumoral infiltration of CD3+T immnue cells,but did not affect the growth of LLC-CD38 KO tumor cells.(3)Subsequently,we explored the mechanism of CD38 enzymatic activity in regulating the survive of tumor cells.The results showed that both CD38 KO and MU exhibited a significantly decreased intracellular calcium,while CD38 OE showed an increased concentration of intracellular calcium.Exogenous administration of cADPR could promote calcium influx into cells,while 8-Br-cADPR was on the contrary.Subsequently,we identified that CD38-cADPR promoted the proliferation and migration of tumor cells by inducing the open of TRPM2 ion channel and the influx of extracellular calcium into cytoplasm.Compared with the matched control group,down-regulation or knockout of TRPM2 could result in a significant decreased intracellular calcium in CD38 WT and OE cells,and led to a significantly inhibition of cell proliferation and migration as well.But there were no significant changes happened in CD38 MU and KO cells by manipulating the expression of TRPM2.In BALB/c nude exprements,our results also found that all of TRPM2-deficient A549 tumor cells exhibited a significant reduction of tumor burden compared with that of control independent on the expression of CD38.(4)In order to explore the regulatory mechanism of CD38-cADPR-TRPM2 induced intracellular calcium on cell fate,we analyzed the differences of signal pathways between A549-CD38 WT and CD38 KO tumor cells by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).We identified that the pathways of cell cycle,cell proliferation,stress-activated MAPK signaling,cell metabolic were down-regulated.And further verification of differentially expressed genes by real-time PCR,we confirmed that CD38 promoted the mRNA expression of NRF2-target anti-oxidative genes GCLC,AKP1B10,GSTM3 and NQO1.Subsequently,we found that CD38 and its metabolic cADPR could promote the expression of NRF2 but inhibit the expression of KEAP1,which facilitated the progression of lung adenocarcinoma.On this basis,we further verified the regulation of NRF2/KEAP1 on cell redox homeostasis mediated by CD38-cADPR signal.Our results showed that the level of ROS was significantly up-regulated in CD38 KO and MU tumor cells,while a significantly decreased ROS was observed in the CD38 WT and OE cells.Consistently,cADPR could significantly inhibit the level of ROS,while 8-Br-cADPR resulted in an opposite consequence.(5)We also identified that the expression of CD38 was related to a potentiality of metastasis but a shortened survival in lung adenocarcinoma.Among the 158 paired adjacent and cancer tissue microarray,we found that 62%of cancer cases exhibited positive staining for CD38,while only 28.3%were positive for adjacent tissue.We found that the expression of CD38 in advanced tumors was significantly higher than that in early-stage tumors,but the CD38 expression was not associated with pathological characteristics.Our clinical data also demonstrated that the expression of CD38 was negatively correlated with the prognosis.Then we collected a small scale of cancer-pleural effusion and non-cancerous-pleural effusion from volunteer patients,and found that the concentration of cADPR in cancerous pleural effusion was significantly higher than that of non-cancerous patients.We also confirmed that the expression of CD38 was mainly responsible for the production of cADPR in malignant pleural effusion,since no obvious CD 157 positive cells was detected in pleural effusion.Finally,by analyzing public data from Kaplan Meier,TNM and GEPIA databases,we obtained that the expression of CD38 in cancer was also significantly higher than that of adjacent(p<0.05),and promoted cancer metastasis(p<0.0001),while no significant difference in predicting the prognosis of patients was found(p=0.26).Conclusion:In conclusion,we demonstrated a significant mechanism that the enzymatic of tumorexpressed CD38 could induce the open of TRPM2 by generating cADPR,then resulted in an elevated concentration of cytoplasm calcium and facilitated primary tumor progression and metastasis.We also found targeting enzymatic activity of CD38 and its enzymatic product cADPR might be a promising strategy for the lung cancer therapy.