| BackgroundLung cancer is the leading cause of cancer death around the world.NSCLC comprises approximately 80-85%of all lung cancers,with adenocarcinoma and squamous cell carcinoma being the predominant histological subtypes of NSCLC.Clinically,over 60%of lung cancer patients present with locally advanced or metastatic disease upon diagnosis.For NSCLC patients with advanced-stages,the multiple therapeutic choices including molecular targeted therapy and immunotherapy have emerged and reached the clinic,leading to improved clinical outcomes in recent years.However,the approved molecular targeted therapy or immunotherapy is limited to a small portion of NSCLC patients with advanced-stages who possess the genetic alteration.Therefore,the platinum-based chemotherapeutic agents continue to be the most widely prescribed chemotherapy for the vast majority of NSCLC patients,including those who failed in the genetic alteration-guided targeted therapies and patients of stage I to IIIa who have undergone the complete surgical resection.Thus,the use of platinum agents,including cisplatin(DDP),remains unshakable despite chemoresistance associated with treatment failures.Over the past three decades,great efforts have been made to explore the mechanisms for the cisplatin-resistant phenotype of tumor cells.Though the known mechanisms explain the cisplatin resistance at the molecular level to a certain extent,cisplatin resistance often exhibits a multifactorial nature.One of the hard facts is that the micromolecules targeting the currently known mechanisms of chemoresistance have not achieved the expected effect on the tumor suppression in the related clinical researches,exerting little or no impact on PFS or OS of NSCLC patients.Moreover,clinically,once the tumor cells present the cisplatin-resistant phenotype,the disease progresses malignantly and develops multidrug resistance to different chemotherapeutic agents with different pharmacological mechanisms,accompanied by distant metastasis,and relapse,eventually resulting in the treatment failure.Thus,there is an urgent need to continue exploring the mechanisms of chemoresistance in NSCLC.To excavating the mechanism of acquired drug resistance in NSCLC,based on our prior establishment of DDP resistant cells A549/DDP of human NSCLC,we carried out the studies as follows:1)Identification of multidrug resistant cells of NSCLC;2)Screening and identification of acquired multidrug resistant genes of NSCLC;3)Association of the candidate multidrug resistant gene with malignant progression of NSCLC;4)Molecular mechanisms of the candidate multidrug resistant gene in promoting drug resistance and malignant progression of NSCLC;5)Role of ICA and ATRA in enhancing the killing effect of cisplatin on tumor resistant cells.Methods1.To identify the chemoresistance of NSCLC,JC-1 staining was used to detect the MMP,ATP detection kit was used to assess the ATP production and DCFH-DA probe was utilized to detect ROS.Flow cytometry was utilized to detect apoptosis.CCK8 kit was used to detect the IC50 value of five chemotherapeutic drugs on cells.2.The differentially expressed genes were analyzed using microarray and integrated with the differentially methylated genes to screen candidate multidrug resistant genes.Expression of the candidate gene was analyzed in five chemoresistant cells and their corresponding progenitor cells of lung cancer.Promoter methylation status of the candidate gene was checked by BSP method.3.The recombinant lentivirus were used to overexpress or silence the candidate gene.IC50 values of five chemotherapeutic drugs on each cell lines were determined by CCK8reagent.Flow cytometry was used to detect the apoptosis induced by different concentration of DDP.Transwell assay was used to assess the invasion of cells.Wounding healing assay was used to evaluate the migration of cells.Anoikis assay was used to evaluate the colony forming and anti-apoptosis of cells.Ed U staining was utilized to analyze the proliferation of cells.Flow cytometry was used to measure the cell cycle of cells.The cytoskeleton was observed by confocal microscopy.Immunodeficient mouse xenograft model was established to verify the findings in vitro.4.The candidate gene was analyzed using Bioinformatics analysis to reveal the correlation of candidate gene with clinical characteristics of NSCLC.IHC was used to detect the expression of candidate gene in tissue samples of NSCLC patients and tissue chips of lung adenocarcinoma analyze its correlation with clinical characteristics of NSCLC patients.5.The expression profiles were compared between cells with upregulated candidate gene and cells with downregulated candidate gene.The differentially expressed genes were analyzed using GO,KEGG,and GSEA.WB was used to verify the expression of core genes in these pathways.Inhibitor and agonist of the related pathway were used to verify the findings.6.Morphological changes of co-cultured cells(parental and drug resistant cells with different fluorescent colors)were observed by high-content analysis system with treatment of ICA,ATRA and DDP,separately or combined,for 7 days.SA-?-GAL was used to stain the senescent cell induced by ICA,ATRA and DDP,separately or combined.Results1.ZNF300 mediates chemoresistance of NSCLCIC50 of cisplatin,gemcitabine,paclitaxel,docetaxel,and pemetrexed on A549/DDP cells was significantly higher than that of its progenitor A549 cells.Because mitochondrial apoptosis induction accounts for one of the primary mechanisms of cisplatin antitumor activity,the function of mitochondria between A549/DDP and A549 cells after treated with cisplatin were compared,displaying that mitochondrial functions of A549/DDP cells were less affected compared to A549 cells after DDP treatment.Similarly,H1650/DDP,H520/DDP,H1915/DDP,and H446/DDP cells all presented the chemoresistant phenotype.Because A549 cells were widely used in the scientific studies of lung cancer,A549 and A549/DDP cells were utilized to screen the potential chemoresistant genes by microarrays.Integration of the expression profile with the published Me DIP-Ch IP data revealed that77 genes with promoter hypomethylation were upregulated,and 63 genes with promoter hypermethylation were downregulated in A549/DDP cells compared to A549 cells.RT-PCR results confirmed that,out of the 140 genes,15 genes with hypomethylated promoters were upregulated,while 25 genes with hypermethylated promoters downregulated in A549/DDP cells related to A549 cells.Among the 15 upregulated genes,ZNF300 was selected as the candidate chemoresistant gene to be investigated comprehensively in the study because ZNF300 expression was negligible in A549 cells.However,higher expression of ZNF300was detected in A549/DDP cells.WB validated that ZNF300 expression was significantly higher in A549/DDP,H1650/DDP,and H520/DDP cells compared to the corresponding progenitor cells.ZNF300 was endogenously overexpressed in H1915 and H446 cells.No significant difference in ZNF300 expression was observed between H1915/DDP and H1915cells,or between H446/DDP and H446 cells.The findings suggested that ZNF300 might mediate the chemoresistance of NSCLC rather than SCLC.BSP results and changes of ZNF300 expression in A549,H1650,H520 cells after incubation of 5-Azacitidine or Belinostat or combined usage of both demonstrated that ZNF300 expression was regulated by promoter methylation.In the following experiments,the recombinant lentivirus vectors were utilized to overexpress ZNF300 in A549 cells(A549-ZNF300)or silence ZNF300 in A549/DDP cells(A549/DDP-sh ZNF300)to explore the role of ZNF300 in the chemoresistance of NSCLC.CCK8 analysis revealed that IC50 of cisplatin,gemcitabine,paclitaxel,docetaxel,and pemetrexed on A549-ZNF300 and A549/DDP-sh ZNF300-NC cells were significantly higher than that of A549-ZNF300-NC and A549/DDP-sh ZNF300 cells.The apoptosis of A549-ZNF300 and A549/DDP-sh ZNF300-NC cells induced by cisplatin were significantly lower than that of A549-ZNF300-NC and A549/DDP-sh ZNF300 cells,demonstrating that ZNF300 mediated the chemoresistance of NSCLC,which was confirmed in the subcutaneous tumor xenograft model.Additionally,the cancerous tissues originating from the tumor xenograft models injected subcutaneously with A549-ZNF300-NC and A549/DDP-sh ZNF300 cells presented a remarkable shrinkage in the tumor weight and volume when compared to that injected with A549-ZNF300 and A549/DDP-sh ZNF300-NC cells after cisplatin was applied.2.ZNF300 promotes aggressive growth of tumor cells correlating to poor prognosis of patients with NSCLCClinically,once tumor cells acquire chemoresistance,the disease will progress malignantly.Hence,the biological characteristics were compared between chemoresistant cells and chemosensitive cells.The transwell assay showed that the invasion of chemoresistant cells was significantly enhanced related to chemosensitive cells.The wounding healing assay presented that the migration of chemoresistant cells was significantly increased compared to chemosensitive cells.And the anoikis resistance assay demonstrated that the colony forming and the anti-anoikis apoptosis of chemoresistant cells was strengthened compared to chemosensitive cells.To evaluate the potential role of ZNF300 in lung cancer,we retrieved ZNF300 in normal human tissues,lung cancer cell Lines and lung cancer tissues.The retrieval revealed that no expression of ZNF300 was detected in the normal lung tissues.The median expression of ZNF300 in cell lines of NSCLC and SCLC was 121.68 and 266.32,respectively.Based on the threshold of p<0.05,FC>2,gene rank:top 10%,data type:m RNA,no ZNF300 data of lung cancer were available in the datasets of cancer tissues versus normal tissues through an Oncomine Research for ZNF300.Interestingly,two ZNF300 datasets of lung cancer appeared in the outlier analysis(Broet-Lung and Larsen-Lung).Because only age,sex,and stage were listed in Broet-Lung,we used the data from Larsen-Lung to analyze the association of ZNF300 with the clinical characteristics of lung cancer patients.The analysis revealed that ZNF300 was associated with poor OS of NSCLC patients when ZNF300 expression was stratified into three levels of<0.00,0-0.25,and>0.25.Additionally,the expression of ZNF300 in anaplastic oligodendroglioma,glioblastoma,skin basal cell carcinoma,invasive ductal breast carcinoma,and invasive lobular breast carcinoma was significantly higher than that of the corresponding normal tissues.The analysis of biological characteristics and bioinformatics mentioned above strongly suggested that the upregulated ZNF300 induced by cisplatin might be of special importance in the invasion,metastasis,and other malignant progression of NSCLC because SCLC,anaplastic oligodendroglioma,glioblastoma,skin basal cell carcinoma,invasive ductal breast carcinoma,and invasive lobular breast carcinoma are all highly aggressive and malignant tumors.ZNF300 staining on specimens from NSCLC patients and the tissue microarray by IHC validated that ZNF300 expression was positively correlated with stage,lymph node metastasis,and relapse of NSCLC patients.Additionally,ZNF300 was displayed to be associated with the poor OS of patients with lung adenocarcinoma,confirming that ZNF300mediated the malignancy of NSCLC.3.ZNF300 inhibits MAPK/ERK signaling pathway and activates CDK1 by inhibiting WEE1 and MYT1 and modulating MYC/AURKA/BORA/PLK1 axisZNF300 was reported to function as a transcriptional regulator;hence,we compared the expression profiles between A549-ZNF300-NC and A549-ZNF300 cells.Because the cells with upregulated ZNF300 manifested relatively slower proliferation and cell cycle arrest at G2 phase compared to the cells with downregulated ZNF300,we screened the genes that might regulate these biological characteristics based on the keywords of proliferation,growth,differentiation,and cell cycle in the columns of gene.title or pathway of the expression profiles.MAPK was also screened as a keyword in the column of pathway because of its important function in growth and differentiation of cells.The screening displayed that most of the genes linked with MAPK/ERK pathways were downregulated,while most of the genes associated with cell cycle and cancer stemness were upregulated in A549-ZNF300 cells compared to A549-ZNF300-NC cells.According to their functions in cell cycle,the cell cycle related genes were classified into kinase inhibitor,kinase,cell cycle checkpoint,cell division cycle,cell cyclin,and G2/M DNA damage checkpoint,and found that most of the genes related with cyclin-dependent kinase inhibitor were downregulated,while most of the genes related with cyclin-dependent kinase,cell cycle checkpoint,cell division cycle,cell cyclin,and G2/M DNA damage checkpoint were upregulated in A549-ZNF300 compared to A549-ZNF300-NC.The core genes were verified by the WB of the key genes.Moreover,the WB and IHC staining of the key genes on the tumor tissues from subcutaneous xenograft further proved that p-p38 MAPK,p-ERK1/2 and CD61 were inhibited while p15 and Nanog were upregulated in the tumor tissues of xenograft models injected with A549-ZNF300 and A549/DDP-sh ZNF300-NC cells compared to that injected with A549-ZNF300-NC and A549/DDP-sh ZNF300 cells.To clarify the relationship of ZNF300 with MAPK/ERK pathways,AZD6244,an inhibitor,and hesperetin,an agonist of MAPK/ERK pathways were used to treat the relevant cells,demonstrating that p-ERK1/2,p-p38 MAPK,and CD61 decreased while p15 and Nanog increased in A549-ZNF300-NC and A549/DDP-sh ZNF300 cells after treatment with AZD6244.Additionally,the migration,invasion,anti-apoptosis,and resistance to cisplatin of A549-ZNF300-NC and A549/DDP-sh ZNF300 cells were significantly enhanced,but proliferation was subdued after treatment with AZD6244.In contrast,hesperetin could reverse the above results.No effect of AZD6244 and hesperetin was observed on the ZNF300expression,strongly indicated that ZNF300 was the upstream regulator of MAPK/ERK pathways.ZNF300 was predicted to interact with E2F3 and SPI1.CO-IP showed that ZNF300might interact with E2F3 to function in the chemoresistance and aggressive behavior of tumor cells.No significant difference of E2F3 expression between the cells with upregulated ZNF300 and the cells with downregulated ZNF300 implied that E2F3 might not be regulated by ZNF300.4.ICA and ATRA improve the anti-tumor effect of cisplatin on chemoresistant cells by inducing differentiationATRA and Icariin ICA were reported to induce the cell differentiation.Thus,the two chemical compounds were used to test whether they could enhance the anti-tumor effect of cisplatin on the chemoresistant cells by inducing differentiation.Compared to the chemosensitive cells,CD235a and CD61 upregulated in the chemoresistant cells after treatment with ICA and ATRA,respectively.Observation of the co-cultured cells for seven days demonstrated that the chemoresistant cells were more sensitive to cisplatin combined with ATRA or ICA compared to the chemosensitive cells,suggesting that ATRA and ICA could enhance the anti-tumor effect of cisplatin on the chemoresistant cells by inducing the differentiation of tumor cells.Additionally,the synergistic cytotoxic effect of cisplatin with ICA on the chemoresistant cells was stronger than that of the combination of cisplatin and ATRA.Intriguingly,the treatment with cisplatin,ATRA,and ICA,separately or combined,led to a remarkable mutual phagocytosis response in both the chemoresistant cells and chemosensitive cells.It is noteworthy here that the ratio of the chemoresistant cells engulfed by the chemosensitive cells was relatively higher than that of the chemosensitive cells engulfed by the chemoresistant cells.The senescence of tumor cells induced by chemotherapy was reported to enhance their survival via engulfing both neighboring senescent or non-senescent tumor cells.Thus,SA-?-GAL staining was utilized to determine whether the mutual engulfment of the chemosensitive cells and the chemoresistant cells was related with the senescence induced by chemotherapeutic agents.The staining manifested that the positively stained chemoresistant cells of SA-?-GAL were more abundant than that of the chemosensitive cells after treated by cisplatin combined with ATRA and ICA,especially ICA.The expression detection of genes related with senescence and SASP showed that the expression of CDKN2A,CDKN1A,IL6,IL8,CXCL10,MMP2,and MMP9 significantly increased in chemoresistant cells after treatment with cisplatin compared to chemosensitive cells,confirming that cisplatin could induce more chemoresistant cells into senescent stage compared to chemosensitive cells.Conclusions1.ZNF300 is a chemoresistant gene of human NSCLC.2.ZNF300 promotes aggressive growth of tumor cells and correlates to poor prognosis of patients with NSCLC.3.ZNF300 inhibits MAPK/ERK signaling pathway to regulate the slow cycling phenotype and differentiation of chemoresistant cells.4.ZNF300 activates CDK1 by inhibiting WEE1 and MYT1 and modulating MYC/AURKA/BORA/PLK1 axis to regulate cell cycle of chemoresistant cells.5.ICA and ATRA improves anti-tumor effect of cisplatin on chemoresistant cells by inducing differentiation. |
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