Fibroblast Growth Factor-inducible14is Involved In Cell Growth And Multi-drug Resistance Of Small Cell Lung Cancer

Lung cancer is one of the leading malignant tumors all around the world and small cell lung cancer (SCLC) occupies approximately15%of all lung cancers. Small cell lung cancer (SCLC) is characterized by a short cell doubling time, rapid progression, early occurrence of blood-borne and lymph metastasis and the malignancy is the highest of all lung cancer types. Although most SCLC patients have a relatively good initial response to chemotherapy (cisplatin, etoposide and doxorubicine) as well as radiotherapy, relapse or disease progression usually occur quickly because of multi-drug resistance (MDR). The mechanisms of MDR in SCLC are intricate and not fully understood.The fibroblast growth factor-inducible-14(Fnl4) gene, which was first described as one of the growth factor-inducible genes in murine fibroblasts, is the smallest member of the tumor necrosis factor (TNF) superfamily of receptors. Fn14encode a129-aa type Ⅰ transmembrane protein containing one cysteine-rich domain in the extracellular region and a TNFR-associated factor (TRAF) binding domain but lacking a cytoplasmic death domain. The expression level of Fn14is relatively low in a variety of normal tissues including kidney, lung, and pancreas and so on. In cancerous tissues, Fn14expression is dramatically elevated and closely associated with disease progression and poor patient outcome in glioma, breast cancer, esophageal cancer, prostate cancer, gastric cancer, bladder cancer and non-small cell lung cancer (NSCLC). Overexpression of Fn14enhanced cancer cells migration and invasion in vitro and increased experimental metastasis in vivo. These findings suggested Fn14can function as an oncogene in some cancers. In our previous study, we identified Fn14upexpression based on genome-wide gene expression analyses in SCLC multidrug resistance cells (H69AR) as compared to parental H69cells (unpublished data). Therefore, we propose that Fn14may confer apoptosis and resistance to chemotherapeutic drugs in SCLC.NF-κB has been implicated to play an important role in regulating cell proliferation, angiogenesis, adhesion, invasion and metastasis. There is also evidence that Fn14overexpression in tumor cells can activate NF-κB signaling pathways and stimulate cellular processes. In glioblastoma, Fn14signaling modulated cell survival through regulation of NF-κB activation resulting in increased expression of survival factors such as Bcl-xl/w. Similarly, Fn14-mediated NF-κB activity was associated with the expression of Bcl-xl in gastric cancer.To better understand the potential biological function of Fn14in SCLC, we determined the expression of the Fn14in human SCLC, and tested its potential role on tumor growth and chemoresistance by loss-and gain-of-function experiments. Additionally, we aimed to understand how Fn14affected chemoresistance of SCLC. We showed that Fn14was highly expressed in SCLC and correlated with the poor prognosis of SCLC. Enforced expression of Fn14in cancer cells promoted cell growth and enhanced multidrug resistance. Fn14-mediated NF-κB activation underlied the molecular basis for cell growth and drug resistance in SCLC, in part, by up-regulating Bcl-x1expression. Together, these data suggest that Fn14signaling contributes to cell growth and drug resistance in SCLC and that Fn14may be a new potential target for SCLC treatment.ObjectivesTo analyze the differentially expressed genes in small cell lung cancer cell line H69and its multi-drug resitant cell line H69AR and select the differentiated expressed Fn14gene for further study. To investigate its effect on growth and chemoresistance of small cell lung cancer by loss-and gain-of-function experiments, and analyze its regulatory mechanism, Which further enriches the molecular mechanism of SCLC MDR and provides new theoretical basis for clinical treatment for SCLC.Materials and methods1. Fn14expression in small cell lung cancer cell line H69and H446and its multi-drug resistant cell line H69AR and H446AR。We discover the differentially expressed gene Fn14between small cell lung cancer cell line H69and its multi-drug resistant cell line H69AR by using cDNA microarray. Deferential expressions of Fn14mRNA between the two drug-sensitive cells and its drug-resitant cells were verified by using quantitative real-time PCR, and protein by Western Blot.2. The effect of Fn14on growth of SCLCUsing Fn14ectopic overexpression and shRNA-mediated knockdown of Fn14expression levels in small cell lung cancer cell line H69and H446and its multi-drug resistant cell line H69AR and H446AR, Then Fn14mRNA and protein changes were assessed by RT-PCR and Western blot analysis, respectively. The effect of Fn14on growth of SCLC was analyzed through CCK-8assay and plate clone forming experiment, respectively, and was further tested in Tumor xenograft experiments.3. Effect of Fn14on multi-drug resistance of small cell lung cancerFn14expression plasmid or shRNA was transfected into cells to increased or inhibit Fn14expression in SCLC cell line H69and H446and its multi-drug resistant cells H69AR and H446AR, and the transfectant efficiency was measured by RT-PCR and Western blot, then the chemosensitivity of all the cells to adriamycin (ADM), cisplatin (DDP) and etoposide (VP-16) were analyzed by using cell counting kit-8(CCK8). Apoptosis and cell-cycle induced by the three drugs were analyzed by using flow cytometric analysis.4. Effect of NF-κB signaling pathway on the process of Fn14regulated growth and multi-drug resistance of small cell lung cancer(1) RT-PCR and Western blot were used to examine the changed levels of Bcl-xl mRNA and protein in Fn14overexpressed cells and Fn14knockdown cells, for clarifying the regulated role of Fn14on Bcl-xl expression.(2) The SCLC and its transfectants were co-transfected with pNF-κB Luc reporter plasmid and pRL-CMV Vector using Lipofectamine2000, then Luciferase activity was determined using the Dual-Luciferase Reporter Assay System to validate the relationship between Fn14and NF-κB signaling pathway.(3) Celastrol, the inhibitor of NF-κB pathway, was used to block NF-κB signaling pathway for observing the changes of Bcl-xl expression levels and growth and multi-drug resistance of SCLC.5. Relationship between Fn14expression and clinical features of small cell lung cancer.We collected51cases of paraffin-embedded small cell lung cancer tissue. They were immunohistochemical stained. Relationship between Fnl4expression and clinical features of patients with small cell lung cancer were analyzed.6. Statistical analysisAll the statistical analyses were performed using SPSS version16.0software. Results are presented using means±SD. Student’s t test was used to compare the means between two samples and Statistical comparisons of more than two groups were performed using one-way analysis of variance (ANOVA). Factorial experiment ANOVA was used to analyze results of CCK-8experiment. The association between Fn14expression and clinical features were analyzed by Fisher’s exact test. Survival curves were obtained by Kaplain-Meier method. Prognostic factors were examined by univariate and multivariate analyses (Cox proportional hazards model). P values<0.05were considered statistically significant.Results1. Fn14expression was significantly increased in SCLC multi-drug resistant cell lines H69AR and H446ARWe performed cDNA microarray to compare the gene expression between the parental cell H69and the multi-drug resistant cell H69AR. The results showed that the levels of Fn14expression in H69AR was5.4-fold of that in H69cell line. Then qRT-PCR and Western blot were used to confirm the expression of Fn14in drug resistant cells (H69AR and H446AR) and parental SCLC cells (H69and H446). The results by RT-PCR displayed that Fn14mRNA in SCLC multi-drug resistant cells H69AR and H446AR was respectively7.56-fold (P<0.05) and6.31-fold (P<0.05) of that in their parental cells H69and H446, which was verified by using Western blot, the protein levels of Fn14in H69AR and H446AR was3.50and2.48times of that in H69and H446(P<0.05).2. Overexpression of Fn14can induce the increased growth ability of cells(1)We successfully constructed Fn14expression plasmid pcDNA3.1-Fn14and its negative control plasmid pcDNA3.1.(2) Estabishement of stably transfected cell line H69-Fn14、H446-Fn14. The pcDNA3.1-Fn14or negative control pcDNA3.1was transfected into the drug-sensitive H69and H446cells respectively, After1mouth of selection in G418, four independent stable transfectants (H69-Fn14, H69-NC, H446-Fn14, H446-NC) were developed. The efficiency of transfection was confirmed by qRT-PCR and Western blot. As shown in results, the expression of Fn14mRNA in H69-Fn14and H446-Fnl4cells were4.52times and3.45times higher compared with that of corresponding control groups by qRT-PCR. Furthermore, H69-Fn14and H446-Fn14cells expressed4.05-fold and2.26-fold more Fn14protein than did the control cells respectively.(3) The results of CCK-8assay and plate clone forming experiment indicated that overexpression of Fn14can significantly increase the growth ability of cells as compared with control cells(P<0.05),which was also proved by Tumor xenograft experiment.3. Knockdown of Fn14expression greatly suppressed cell proliferation and growth ability.(1) The pGPU6/GFP/Neo-shFnl4or negative control GPU6/GFP/Neo-shNC was transfected into the drug-resistant H69AR and H446AR cells respectively, After1mouth of selection in G418, four independent stable transfectants (H69AR-shFn14, H69AR-shNC, H446AR-shFn14, H446AR-shNC) were developed. The expression of Fn14mRNA in H69AR-shFnl4and H446AR-shFnl4cells were decreased by (71.49±4.48)%and (64.39±5.20)%compared to their relevant control cells respectively. The protein levels of Fn14in H69AR-shFn14and H446AR-shFn14cells were significantly decreased by (65.23±5.35)%and (70.19±4.57)%compared with the H69AR-shNC and H446AR-shNC cells respectively, These results are consistent with those obtained from mRNA analysis.(2) The results of CCK-8assay、plate clone forming experiment and Tumor xenograft experiment suggested that knockdown of Fn14notably suppressed SCLC growth compared with negative control and the blank (P<0.05) 4. Forced Fn14expression increased chemoresistance of H69and H446cells(1) After treatment of ADM, DDP and VP-16, survival and half maximal inhibitory concentration of a substance (IC50) value of H69-Fn14cell increased significantly compared with H69-NC and H69. So did the H446-Fn14cell compared with H446-NC and H446.(2) After treatment of ADM, DDP and VP-16, apoptosis of H69-Fn14and H446-Fn14cells decresed compared with their controls, while G2-M phase cells increased.5.Reduction of Fn14expression in H69AR and H446AR cells increased its chemosensitivity(1) the survival rates of H69AR-shFn14and H446AR-shFn14to ADM, DDP and VP-16was remarkably reduced in comparison with corresponding negative control (NC) or mock control, which also indicated by a decrease in the IC50values. These results indicated that Fn14can affect sensitivity of SCLC cells to chemotherapeutic drugs.(2) After treatment of ADM, DDP and VP-16, apoptosis of H69AR-shFn14and H446AR-shFn14cells incresed compared with their controls, and G1-S phase arrest increased.6. Fn14regulated the growth and multi-drug resistance of SCLC via NF-κB pathway(1) The cells with stable Fn14upregulation showed a significant elevation of Bcl-xl protein expression, and the inhibition of Fn14level led to the opposite changes in Bcl-xl protein expression, which suggested a positive correlation between FnI4and Bcl-xl.(2) Regulation of NF-κB pathway by Fn14The pNF-κB Luc reporter plasmid and pRL-CMV empty Vector were co-transfected into SCLC and its transfectants, then Dual luciferase reporter assay was used to detected the NF-κB luciferase activity. NF-κB reporter luciferase activity was enhanced in cells with Fn14upregulation (P<0.05), conversely, its activity was inhibited upon Fn14knockdown (P<0.05).In brief, NF-κB activity was increased in Fn14dependent manner. The results indicated a possible involvement of NF-κB signaling in Fn14induced up-regulation of Bcl-xl.(3) NF-κB pathway has an important role in Fn14modulated the growth and MDR of SCLCWe utilized Celastrol, the inhibitor of NF-κB, to block the NF-κB pathway. As shown in results, Bcl-x1mRNA and protein expressions were reduced after treated with Celastrol in H69-Fn14, H446-Fn14cells. Cancer cell survival was decreased after treated with Celastrol in H69-Fnl4, H446-Fnl4cells. The IC50values of H69-Fn14cells treated with Celastrol for ADM, DDP, VP-16were dramatically declined respectively as compared with the control cells, Similar results were verified in H446-Fnl4cells. In conclusion, all these results provided convincing evidence that Fn14up-regulated the expression of Bcl-x1via NF-κB pathway to promote cancer cell growth and enhance multidrug resistance.7. Relationship between Fn14expression and clinical pathological features in SCLC tissues.To research the Relationship between Fn14expression and clinical pathological features, we examined Fn14expression in51SCLC tissues by Immunohistochemistry staining.The results showed that Positive Fnl4expression in SCLC tissues was located in the cytoplasm. Its positive rate was51%(26/51).Positive rate of Fn14expression in male patients was54.5%(24/44), while in female ones28.6%(2/7). There was no significant difference between them (P=0.202). Positive rate of Fn14expression in patients younger than58-year-old was50.0%(15/30), while in over58-years old52.4%(11/21). There was no significant difference between them (P=0.867).Positive rate of Fn14expression in patients in limited stage was48.0%(12/25), while in extensive stage100.0%(14/14). There was significant difference between them (P<0.001).We used Kaplan-Meier method and Univariate analysis to estimate the relationship between Fn14and the survival rate of SCLC patients. The results indicated that There was significant difference between the survival rate of SCLC patients and Fn14(P<0.001).Multivariate analysis results suggested that there were no significant difference between the survival time of male patients and female patients (P=0.685); there were no significant difference between survival rate of patients younger58-years-old and patients older than58-years-old (P=0.520); Survival rate of patients in limited stage was significantly higher than that of patients in extensive stage (P<0.001); Survival rate of patients with positive Fn14expression was significantly lower than patients with negative Fn14expression (P=0.011<0.05)Conclusion1. The expression levels of Fn14in multi-drug resistant cell lines H69AR and H446AR were significantly higher than that of their parental cell lines, and elevation or inhibition of Fn14could induce the changes of cell growth and multi-drug resistance, which indicated Fn14was involved in regulating cell growth and multi-drug resistance of SCLC.2. NF-κB pathway has an important role in Fn14modulated the growth and MDR of SCLC via modulating the downstream gene Bcl-xl expression. 3. Fn14expression in SCLC tissues correlated with stage and survival of patients. Fn14may be a prognostic factor of SCLC patients.