The Role And Mechanism Of CCL2 In Cisplatin-resistance Of Non-small Cell Lung Cancer

Background and objectives Lung cancer is the most common malignancies in China andhas become the main cause of tumor death.Non-small cell lung cancer(NSCLC)accounts for about 85% in lung cancer.Due to lacking early screening technologies,most patients are diagnosed at advanced stages,probably missing the best opportunity in treatment.Chemotherapy is the main approach in advanced patients' treatment.Disappointingly,the efficiency of chemotherapy for lung cancer is only 30% to 40%.Because of local tumor recurrence and distant metastasis,the 5-year overall survival rate is only 16.1%.Resistance to anticancer drugs is the main obstacle preventing the effective treatment of tumors.The causes of cisplatin-resistance include the reduced tumoral concentration of drugs,the intracellular degradation of drugs,enhanced DNA repair as well as the alterations in apoptotic signal transduction pathways.In recent years,much more attentions have been paid to tumor microenvironment(TME).As the major immune constituent of the TME,cytokines or chemokines play key roles in tumor progression and are intimately related with chemo-resistance.Largely relying on monocytes/macrophages recruit,monocyte chemotactic factor(MCP-1;also known as CCL2)is widely existed in TME and promotes tumor invasion and metastasis.However,it's not clear whether CCL2 is responsible for treatment failure of cytotoxic agents.This study aims to explore how CCL2 is involved in cisplatin resistance in NSCLC and the underlying mechanisms,and to provide new means to reverse chemo-resistance.Methods Protocols were approved by ethics committee of the first affiliated hospital of Zhengzhou university.Serums were collected from cisplatin-sensitive or resistant NSCLC patients.Then Multi-Analyte Flow assay and ELISA were perform,showing CCL2 was the chemokine changing most sharply between patients with different clinical responses.Tumor tissues from 81 cases of NSCLC patients which were treatmented in The First Affiliat Hospital of Zhengzhou University from 2013 to 2015 were collected and the CCL2 m RNA expression level in tumor tissues were dected by Real-time PCR assay.The CCL2 m RNA expression level in A549 cells and A549/Cis cells were also dected by Real-time PCR assay.In addition,Real-time PCR was also adopted to evaluate the stemness of A549 after CCL2 knockdown.The proliferation of CCL2-deficient A549 cells was monitored by CCK8.Using MACS CD14+ monocytes were purified from peripheral blood mononuclear cells from health donors.Then the sorted monocytes were cultured with the supernatant of A549 cells or A549/Cis cells in vitro.The percentage of CD163+ M2 macrophages were analyzed by flow cytometry.The effect of M2 macrophages on metastasis,angiogenesis and the resistance of cisplatin of A549 cells were analyzed by transwell,tubule formation and CCK-8 assay.PCR array was used to detect the transcription factors regulating CCL2 expression.Furthermore,the transcriptional regulations of CCL2 were confirmed using small interfering RNA.Results 1.Compared with cisplatin-sensitive patients,elevated CCL2 levels were found in serums from cisplatin-resistant subjects.2.The m RNA expression level of CCL2 was higher in A549/Cis cells than A549 cells.And there was positive relationships between the level of CCL2 m RNA in tumors with the clinical stages and prognosis.After silencing CCL2,the expression levels of Bim-1,c-Myc,Sox2,Oct-4 in A549/Cis cells were reduced.3.CCL2 enhances the stemness of NSCLC to facilitate the resistance to cisplatin.4.Compared with cultured with A549 cell supernatant,the proportions of CD163+ M2 were greater in CD14+ cells cultured with the supernatant of A549/Cis cells.5.M2 macrophages polarized in A549/Cis supernatant demonstrated enhanced migration and angiogenesis-promoting function,and augmented the chemo-resistant effects of A549 cells.6.Compared with A549 cells,the expression of FOS,EGR1 and STAT5 A were increased and the expression of MYF5,HAND2,HNF4 A were decreased in A549/Cis cells.7.After silencing the expression of c-Fos,CCL2 m RNA expression level was decreased in A549/Cis cells,enhanced the cytotoxicity of cisplatin to A549/Cis cells.Conclusion More than dictating CD14+ monocytes into tumor followed by M2 macrophage polarization,CCL2 facilitates the resistance to cisplatin in NSCLC by promoting the stemness of tumor cells.c-Fos regulates the transcription of CCL2,representing a potential therapeutic target to reverse the cisplatin resistance in NSCLC.