Over-expression Of BAG-1 Is Associated With Cisplatinresistance In Head And Neck Squamous Cell Carcinomas

BACKGROUND In order to improve the therapy for head and neck squamous cell carcinoma(HNSCC),biomarkers associated with local and/or distant tumor relapses and cancer drug resistance are urgently needed.Conventional experimental methods are limited by the ability to study only the expressions of a single or a group of genes and cannot explain the biological behaviors of carcinoma cells very well.Gene expression Micro Array technology can provide almost all of the transcriptional activity of cells or tissues,and currently it is widely applied in cancer diagnosis,disease analysis,drug development and toxicity,and other fields.Genes do not act alone but regulate biological functions with multiple genes in a number of intricate reaction networks.Although the data gained from gene expression Micro Array is huge,the data itself does not directly reflect the differences in biological functions.In order to fully explore the significance of the data,we introduce a software termed IPA(Ingenuity Pathway Analysis)based on cloud computing to analyze the correlations between gene expression and cancer biological behaviors.In our preliminary experiments,hepatocellular growth factor(HGF)was used to simulate the tumor microenvironment of HNSCC.It was found that the cell movement and invasion abilities of the cancer cells were increased after HGF stimulation,along with the increase of BAG-1 protein expression.Thus,we suggest that BAG-1 may be associated with cisplatin resistance in HNSCC.In this study,the expression of BAG-1 in HNSCC cells was confirmed by gene expression microarray and conventional experiments,to identify BAG-1 is associated with cisplatin resistance in HNSCC.METHODS(1)Screening of cells: MTT assay was used to detect metabolic inhibition effect of cisplatin on primary HNSCC cells(Group A)and recurrence/metastatic cells(Group B)from 7 patients,all these cell lines were isolated from the tumor by University of Michigan;Clonogenic Survial Assay was performed to determine the reaction of cells to cisplatin;the expressions of cleaved Caspase 3 and 9 in these cells were detected by Western Blot.(2)HGF stimulation and HNSCC: MMP detection was applied to determine the effectors of HGF or HGF combined with tumor signal pathway inhibitors(MEK 1/2 inhibitor or PI3 K inhibitor)on cancer cell mobility;detecting BAG-1 expression changes after HGF or HGF combined with the inhibitor treatments by Western Blot;Transwell invasion experiment was applied to detect the invasive ability after HGF stimulation.(3)Gene microarray analysis: Comparison of all recurrence/metastatic cells(Group B)and primary cells(Group A);based on the previous data,14 B and 17 B cells were identified as cisplatin-insensitive cells,then compared(recurrence)and 17B(metastatic)between 14 A and 17 A cells;Import the differential expressed genes into IPA for further analysis.(4)BAG-1 and BCL-x L expression: Detecting BAG-1,BCL-x L and phosphorylated AKT expression in 14 A and B,17 A and B cells by Western Blot;immunohistochemistry was used to detect these proteins in tissue microarray.(5)Cisplatin resistance assay: The differences between 14 A and 14 B cells due to serum starvation or cisplatin were detected by MTT assay and DNA ladder assay;Western Blot was used to detect the expression of BAG-1,BCL-x L and other apoptosis-related proteins in 14 A and 14 B cells after 24 hours incubation with cisplatin.(6)BAG-1 and tumor signaling pathways: The phosphorylation proteins related to tumor signal pathways in 14 A and 14 B cells were detected by Western Blot after 24 hours incubation with different concentrations of cisplatin;the expression level of BAG-1 was measured after incubating 14 B cells by tumor signaling pathway inhibitors;MTT assay was used to detect the effect of cisplatin combined with inhibitors on the activity of 14 B cells;after incubated 14 B cells with BAG-1 si RNA,detecting the expression of BAG-1 and phosphorylated proteins;using MTT assay to detect the influence of BAG-1 si RNA on 14 B cells.RESULTS(1)The inhibitory effect of cisplatin on cell viability and colony formation was dose dependent,and the effect on 14 B and 17 B cells was significantly lower than other cells.After 24 hours incubation of cisplatin,cleaved Caspase 3 and 9 expression levels in 14 B and 14 B were lower than those of the primary cells 14 A and 17 A corresponding to the same concentration of cisplatin.(2)The expression of BAG-1M protein was increased with HGF incubation,but this phenomenon can be antagonized by adding MEK1/2 or PI3 K inhibitor.(3)The cell morphologies in HNSCC are similar,but the gene expressions are different.The pathogenesis of HNSCC may be associated to gender-related factors.A series of upstream regulatory factors associated with cancer recurrence/metastasis,including E2F3,MTOR,DEK were obtained.The expression of BAG-1 and BCL2L1(BCL-x L protein coding gene)were higher in 14 B and 17 B cells than 14 A and 17 A cells,and a series of upstream regulatory factors including IFG-?,prolactin and hepatocyte growth factors may be associated with increased cisplatin resistance.(4)The expression levels of BAG-1,BCL-x L and phosphorylated AKT in 14 B and 17 B cells are higher than those in 14 A and 17 B cells,and the three isoforms of BAG-1 protein were different in 14 and 17 groups.BCL-x L and phosphorylated AKT were not detected in 14 A cells.Only 2 cases of BAG-1 expression were positive in 60 cases of HNSCC samples,and BCL-x L and phosphorylated AKT were also positive in these two cases.(5)14B cells have higher tolerance compared with 14 A while culture with lower nutrition or treated by cisplatin.The expression of BAG-1 in 14 B cell line was higher than that in 14 A cell line after 24 hours incubation with cisplatin.BCL-2 and BCl-x L expressions demonstrated the similar trend,but BCL-2 was not detect in 14A(all concentrations)and 14 B incubated at the highest concentration;DNA damage marker protein,phosphorylated ?H2AX,was highly reduced in 14 B cell line compared with 14 A cells.(6)The expression levels of phosphorylated ERK,AKT,STAT remained higher in 14 B than those in 14 A after 24 hours incubation of cisplatin;PI3K and STAT3 inhibitors could decrease the expression of BAG-1 protein,but different BAG-1 isoforms have different changes,MEK1/2 inhibitor did not change the expression of BAG-1;Cisplatin combined with PI3 K or MEK 1/2 inhibitor in 14 B cells decreased the cell viability compared with cisplatin alone,cisplatin combined with STAT3 inhibitor has a certain inhibitory effect but lower than cisplatin combined with other PI3 K or MEK 1/2 inhibitors;BAG-1 si RNA treated cell line 14 B has lower expression of phosphorylated ERK,AKT and STAT expression,and the treated cell line is more sensitive to cisplatin.CONCLUSION BAG-1 is up-regulated in the cisplatin-resistant cells,and the mechanism is closely related to BCL-x L and multiple tumor signaling pathways(PI3K/AKT,MAPK/ERK,JAK/STAT).