Ret Finger Protein-like 3 Promotes Tumor Growth In Non Small Cell Lung Cancer Via Upregulation Telomerase Reverse Transcriptase Activity

Lung cancer is the leading cause of cancer-related death in the world, and non-small cell lung cancer (NSCLC) accounts for approximately 80% of lung cancer diagnoses. Although the treatment of NSCLC has been improved greatly, the five-year overall survival rate for patients with NSCLC remains at a low level disappointmently. Thus, there is an urgent need for identifying a new therapeutic target for NSCLC patients.Telomerase is an intracellular ribonucleoprotein, which possesses reverse transcriptase activity and contributes to maintain the stability of the chromosome and genome integrity. Telomerase activity is regulated by hTERT gene transcription mainly, which serves as the catalytic subunit of telomerase. Moreover, hTERT is correlated with cell immortalization and malignant transformation. Telomerase activity mediated by hTERT activation play a crucial role in the tumorigenesis of lung cancer. Because the reactivation and up-regulation of hTERT were primarily regulated at the transcriptional level in tumor, so we tried to find a new protein that specifically binds to hTERT promoter, and further explore its functions in regulating hTERT activity and tumor growth in NSCLC.In this study, we used streptavidin-agarose pulldown assay combined with high-throughput proteomics to pull down the potential hTERT promoter-binding proteins in lung cancer cells and identified the one as ret finger protein-like 3 (RFPL3). RFPL3 gene locates at human 22ql2.3, and the proteins encoded by them are members of a large protein family with zinc finger motifs. However, the expression and potential role of other RFPL family members including RFPL3 in tumors are unknown.Part ⅠRet finger protein-like 3 activates hTERT expression in non-small cell lung cancer as promoter regulatory proteinsObjectives:To filter out the new hTERT promoter binding protein RFPL3 in non-small cell lung cancer cells, and to explore the role of RFPL3 expression in regulation of hTERT expression and transcriptional activity.Methods:Firstly, we isolated large protein complexes that bound to hTERT genes promoter from cancer and normal cells by the streptavidin-agarose pulldown. Candidate proteins were separated by SDS-PAGE, visualized by silver staining and identified by proteomics. Chromatin immunoprecipitation method also validated the specific binding between RFPL3 and hTERT promoter. Using cell transfection techniques, RFPL3 siRNA and RFPL3 plasmid were transfected respectively into normal lung cell line (HBE) and lung cancer cell lines (H1299, A549). Expressions of hTERT and RFPL3 protein were shown by western blot. Further, we studied how RFPL3 regulated hTERT promoter and telomerase activity as hTERT gene promoter binding protein by luciferase reporter assay method and TRAP telomerase activity analysis.Results:RFPL3 is tumor-specific hTERT promoter binding protein. Comparing with normal lung cell lines WI-38 and HBE, the expression of hTERT and RFPL3 of lung cancer cell lines (H1299, A549 and H322) were significantly upregulated. If RFPL3 was activated or inhibited, the expressions of hTERT protein would also increase or decrease. Similarly, when RFPL3 was over-expressed, hTERT promoter activity and telomerase activity were significantly increased, while RFPL3 was knockdown, hTERT promoter activity and telomerase activity was significantly reduced.Conclusions:Western blot experiments showed increased expression of RFPL3 in lung cancer cell lines.RFPL3 serving as a novel hTERT promoter regulatory proteins, can promote the transcription and expression of hTERT, and enhance the hTERT promoter activity and telomerase activity in non-small cell lung cancer.Part ⅡRFPL3 and CBP synergetically upregulate hTERT activity and promote tumor growth in non small cell lung cancersObjectives:To investigate the interaction between CBP and RFPL3 in non-small cell lung cancer, and further research the mechanisms by which they coordinately regulate the hTERT transcriptional activity, telomerase activity and tumor cell proliferation.Methods:Immunoprecipitation analysis was used to verify the interaction between RFPL3 and CBP, and explore the impact of CBP on RFPL3 acetylation levels. Dual immunofluorescence analysis was used to further analyze the colocalization of RFPL3 and CBP. Using the technology of transient transfection to build H1299 cells with stable CBP overexpression plasmid, and RFPL3 expression was knockdown by transfecting RFPL3siRNA in such cells. H1299 cells stably expressing RFPL3 were transfected by CBPsiRNA in order to knockdown the expression of CBP. The binding of RFPL3 to hTERT promoter mediated by CBP was investigated by streptavidin-agarose pulldown assay. The coordinated regulation of hTERT promoter activity by RFPL3 and CBP was studied through luciferase reporter gene assay. The coordinated control of telomerase activity by RFPL3 and CBP was further measured by telomeric repeat amplification protocol (TRAP). MTT assays were used to explore the coordinated regulation of cell proliferation by RFPL3 and CBP in lung cancer cells.Results:CBP was found in all the lung cancer cell lines in the complexes pulled down by anti-RFPL3 antibody, but not found in IgG treated samples, indicating that RFPL3 indeed interacted directly with CBP in the nucleus of lung cancer cell lines. Duel immunofluorescence analysis shows that RFPL3 and CBP are co-located in the nucleus of lung cancer cells. When one of them was upregulated or downregulated, whereas the other one remains unchanged, hTERT expression, telomerase activity and cell proliferation were activated or repressed accordingly. The expression level of HAT activity of CBP affected the binding of RFPL3 to hTERT promoter region accordingly as shown in pulldown assay. The overexpression of CBP elevated levels of RFPL3 acetylation, conversely, the inhibition of its acetyltransferase activity reversed this elevation.Conclusions:Tumor-specific acetylation of RFPL3 was mediated by CBP in non-small cell lung cancer. CBP and RFPL3 coordinately regulated hTERT expression, hTERT promoter activity, telomerase activity and tumor cell proliferation.Part ⅢThe expression and clinical significance of RFPL3 and CBP in tumor tissues of non-small cell lung cancerObjectives:To explore the expression correlations of RFPL3, CBP and hTERT in lung tissues of patients with NSCLC, and study the clinical significance and prognostic value of RFPL3 and CBP in patients with NSCLCMethods:Western blot experiments were used to explore the expressions of RFPL3、 CBP and hTERT proteins in tumor tissues of patients with NSCLC.The expressions of RFPL3 and hTERT proteins were further analyzed in 100 primary human non small cell lung cancer tissues by IHC using tissue micro arrays. Survival curves were constructed using the Kaplan-Meier method and were compared using the log-rank test. Multivariate Cox proportional hazards analyses used "backwards" modeling to generate models predictive of outcome.Results:It is obvious that RFPL3 and CBP proteins are upregulated in tumor tissues compared with normal lung tissues, and they are positively correlated with hTERT proteins. The Pearson’s correlation coefficient analysis showed that RFPL3 and CBP upregulation together was positively correlated with the percentage of hTERT-positive stained cells (P<0.01). CBP and RFPL3 upregulation had no significantly associations with patient’s gender (P= 0.554,χ2 tests), age (P= 0.861,χ2 tests), T classification (P= 0.179,χ2 tests) and lymph node metastasis (P= 0.075,χ2 tests). Survival analyses showed that high expression of CBP and RFPL3 significantly correlated with shorter overall survival time in patients with NSCLC (P< 0.001). Moreover, the lung adenocarcinoma patients with high CBP, RFPL3 and hTERT expression had a significantly shorter OS than those with low CBP, RFPL3 and hTERT expression (P< 0.001). By univariate analysis, CBP and RFPL3 upregulation (P< 0.001), T3 stage (P= 0.045) and presence of lymph node metastasis (P= 0.016) were significant inferior prognostic factors for OS in patients with NSCLC. Multivariate analysis further indicated that CBP and RFPL3 upregulation (P= 0.001) was independent prognostic predictors for OS in patients with lung adenocarcinoma enrolled in this study.Conclusion:RFPL3 and CBP expressions are positively correlated with hTERT in tumor tissues of patients with NSCLC. RFPL3 and CBP overexpression simultaneously is an independent predictor of short OS in patients with NSCLC.