The Role Of RBP2 And THOP1 In Progression Of Non-small Cell Lung Cancer And The Relative Mechanism

Background and significanceNon-small cell lung cancer (NSCLC) is among the most common malignancies leading to cancer-related death worldwide. Despite numerous improvements in surgical techniques and adjuvant chemoradiotherapy for NSCLC over the last decades, the prognosis remains relatively poor. A variety of new molecular markers and possible new targets have been found to treat the disease. However, tumor progression is a multistep process and the molecular mechanism underlying lung carcinogenesis is largely unclear. Biological therapy and gene therapy of tumor have become a hot research topic in tumor therapy.With the continuous progressions in research, it’s well known that the growth, invasion and metastasis of tumor cells need a large number of newly born blood vessels, tumor angiogenesis is the process of microvascular growth induced by tumor cells and establishment of blood circulation in tumor microenvironment, plays very important roles in the process of growth, invasion and metastasis of malignant tumors, and then puts forward the new approach to suppress tumor growth-antiangiogenic therapy, has become an important strategy in the modern therapeutic fields of tumors. Tumor angiogenesis is an extremely complex and coordinating process, regulated by a tight balance of various factors at different levels. Antiangiogenic therapy, with new blood vessels for acting targets, by cutting off the nutrients supply and metastatic path to prevent growth, invasion and metastasis of tumor cells, is a kind of important method of targeted therapy. It has been proposed that vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) play critical roles in tumor angiogenesis.Retinoblastoma binding protein 2 (RBP2), a member of the JARID family of proteins, is a nuclear phosphoprotein with demethylase activity for lysine 4 of histone H3 (H3-K4). It appeared that RBP2 exerts its function partly by repressing the transcription of target genes involved in differentiation and that binding to retinoblastoma protein (pRB) converts RBP2 from a transcriptional repressor to a transcriptional activator. Recent research in lung cancer has established that RBP2 is correlated to tumor migration and invasion by directly binding to integrin β1 (ITGB1) promoters. Another study demonstrated that RBP2 up-regulates the expression of N-cadherin and snail via the activation of Akt signaling. Moreover, ITGB1 and Akt signaling are significantly correlated with tumor angiogenesis. Taken together, these results suggest an oncogenic role for RBP2 in tumor angiogenesis and progression.Invasion and metastasis are the primary causes of death in patients with malignant tumors, and also are the problems have not yet been solved in the therapeutic field of malignant tumor, therefore, investigate the molecular mechanisms of invasion and metastasis, and corresponding therapeutic strategies is of very important significance. The exact mechanisms of malignant tumor invasion and metastasis are still incompletely clear, but is well known involving the bioadhesion reduction between tumor cells, close attachment of tumor cells to the basement membrane, degradation of the extracellular matrix, tumor cells penetrate into blood vessels or lymphatics, arrest at distant sites by adhesion to endothelial cells, invasion outward and induction of angiogenesis, evasion of host antitumor responses and continue to grow at the metastatic sites. Theoretically, blocking any link in the above multistage and continuous process may block the invasion and metastasis of malignant tumor cells. MMP-9 protein plays important regulating roles in promoting migration and invasive abilities of tumors.Thimet oligopeptidase (THOP1; EC3.4.24.15 or EP 24.15) is a characteristic metallopeptidases with a HEXXH zinc binding motif and associated with metabolism of several neuropeptides containing from 5 to 17 amino acids, such as bradykinin, gonadotropin releasing hormone, opioids and neurotensin. Accumulated evidence suggested that THOP1 was predominately expressed in the cytosol, endoplasmic reticulum, mitochondria and nucleus of different normal human tissues and tumor cells. Recent study demonstrated that high THOP1 mRNA expression level in the normal background liver of hepatocellular carcinoma (HCC) was significantly correlated with better survival. Furthermore, THOP1 could inhibit tumor angiogenesis and tumor proliferation promoted by BK in melanoma cells. These findings indicate THOP1 may be a potential anti-tumor agent for tumor therapy.Part ⅠThe relationship of RBP2 protein to tumor angiogenesis and prognosis of non-small cell lung cancer cellsObjective:To detect the expression levels of RBP2, HIF-la, VEGF and CD34 in patients with stage Ⅰ non-small cell lung cancer, and investigate the relationship of RBP2 protein to the clinicopathologic factors and prognosis. To further investigate the relative mechanism of RBP2 in tumor angiogenesis.Methods:A total of 102 patients (71 men and 31 women, mean age 62±3.56 years) with stage Ⅰ NSCLC who underwent complete tumor resection (lobectomy or pneumonectomy) with regional lymph node dissection between January 2006 and December 2008 at the department of Thoracic Surgery, Qilu Hospital, were included in the study. Immunohistochemical staining for RBP2, HIF-la, VEGF and CD34 were carried out using the streptavidin-peroxidase method. Western blot was used to detect the protein level of RBP2 in SK-MES-1, A549, SPCA-1 and H1975 cell lines. Two pieces of siRNAs and pcDNA3-HA-RBP2 targeting RBP2 were employed for a functional analysis in H1975 and SK-MES-1 cells. Endothelial Cell Tube Formation Assay was used to evaluate the functional significance of RBP2 in tumor angiogenesis The VEGF levels in the different conditioned media were determined using an ELISA kit according to the manufacturer’s instructions and analyzed using a Labsystems Multiscan reader. Real-time PCR was used to examine the expression of the transcription factors HIF-la and VEGF in RBP2-overexpressing and -depleted NSCLC cells.Proteins were extracted using RIPA lysis buffer. Equal amounts (20μug) of protein were subjected to SDS-PAGE analysis, transferred onto nitrocellulose membranes and probed with primary antibodies against RBP2, HIF-1α, VEGF, Akt and phospho-Akt.Results:1. RBP2 was detected as being overexpressed in 52 (51%) of 102 NSCLC specimens according to the abovementioned criteria. The chi-square test showed that RBP2 overexpression was associated with tumor size (P=0.030), high HIF-1αexpression (P=0.028) and high VEGF expression (P=0.048).2. The average number of microvessels of MVD in each tumor sample ranged broadly, from 6.4 to 102. As described previously, tumors with microvessels≥57 were classified as high MVD, and 46 cases (45.1%) showed high MVD. The chi-square test showed that high MVD was associated with high HIF-1αexpression (P=0.034) and high VEGF expression (P=0.001).3. High MVD was detected more frequently in tumors with RBP2 protein overexpression than in those without overexpression (P=0.033, Mann-Whitney U test).4. A Kaplan-Meier analysis of overall survival also demonstrated a poor 5-year overall survival rate in patients with RBP2 protein overexpression (53.8% versus 72.0%,P=0.037) and high MVD (52.2% versus 71.4%, P=0.040). The multivariate analysis indicated that only RBP2 had an independent influence on the survival of patients with stage Ⅰ NSCLC (P=0.044).5. The protein level of RBP2 was up-regulated in lung cancer cell lines SK-MES-1, A549, SPCA-1 and H1975 compared to the human bronchial epithelial cell line BEAS2B. The expression level of RBP2 in H1975 cells was higher than that in BEAS2B, SK-MES-1, SPCA-1 and A549 cells.6. To evaluate the functional significance of RBP2 in tumor angiogenesis, RBP2 was knocked down in H1975 cell lines. The results demonstrated that the number of complete tubes induced by the conditioned medium of RBP2-siRNA1 H1975 cells (12.33+3.06) and RBP2-siRNA2 H1975 cells (9.67+1.53) was significantly reduced compared to that of the control siRNA H1975 cells (38.67+2.52, P<0.01).7. The results demonstrated that the VEGF protein levels in the conditioned media of RBP2-siRNAl (0.99±0.11 ng/ml) and RBP2-siRNA2 (0.94±0.14 ng/ml) H1975 cells were significantly lower than in the control siRNA H1975 cells (1.87±0.10 ng/ml). In addition, our results suggested that the tube formation induced by the conditioned medium of the control siRNA H1975 cells was blocked with the addition of a VEGFR inhibitor (sunitinib malate,2.5 μM,10.33+2.22, P＜0.01); the reduced tube formation induced by the conditioned medium of RBP2-siRNA2 H1975 cells was rescued by the addition of VEGF-165 (2 ng/ml,41.03+3.25, P<0.01).8. The enforced expression of RBP2 up-regulated the expression of the HIF-la protein in a time-dependent manner under normoxic conditions, and the peak value of HIF-1α expression appeared at 36 hours after transfection was performed. The down-regulation of RBP2 in H1975 cells led to the decreased expression of HIF-la and VEGF, whereas ectopic RBP2 expression in SK-MES-1 cells by pcDNA3-HA-RBP2 led to the up-regulation of HIF-la and VEGF.9. RBP2 induction of VEGF is dependent on HIF-lα.10. RBP2 activates HIF-la via the PI3K/Akt signaling pathway. In the presence of recombinant human VEGF-165 stimulation, the activation of Akt was increased in RBP2-depleted H1975 cells and RBP2-overexpressing SK-MES-1 cells. Thus, our results suggested that VEGF indeed increases the activation of Akt, as regulated by RBP2.Conclusions:We provided evidence showing that high RBP2 expression and high MVD were common in stage I NSCLC tissues and closely associated with poor prognosis. In addition, high RBP2 expression was closely associated with tumor size, high HIF-la expression, high VEGF expression and increased tumor angiogenesis. Multivariate analysis indicated that RBP2 had an independent influence on the survival of patients with stage I NSCLC. The RBP2 protein may play a critical role in NSCLC tumor angiogenesis by enhancing HIF-1α and VEGF expression under normoxia via the PI3K/Akt signaling pathway. Moreover, VEGF could increase the activation of Akt regulated by RBP2. These findings indicate that RBP2 could serve as an attractive therapeutic target against angiogenesis for early-stage NSCLC patients.Part ⅡThe role of THOP1 protein in regulating migration and invasion ability of non-small cell lung cancer cellsObjective:The study was designed to detect the expression level of thimet oligopeptidase (THOP1) protein in non-small cell lung cancer (NSCLC) and investigate its correlation with clinicopathologic features and prognosis. To suppress THOP1 protein expression in non-small cell lung cancer cells by RNA interference in vitro, and investigate the roles of THOP 1 protein in regulating migration and invasion ability.Methods:Tumor samples (n=120, mean age 61±4.35 years) and distant normal lung tissues (n=53,5 cm from the margin of the tumor) were collected from patients with NSCLC who underwent complete tumor resection (lobectomy or pneumonectomy) with regional lymph node dissection between January 2006 and December 2007 at the department of Thoracic Surgery, Qilu Hospital. Chi-square test was performed to examine the association of THOP1 and various clinicopathologic factors. Follow-up time was censored if the patient was lost to follow-up. Survival curves were drawn using the Kaplan-Meier method and compared by the log-rank test. Multivariate Cox regression analysis was used to identify significant independent prognostic factors. Quantitative real-time PCR and western blotting were employed to measure the expression of THOP 1 in 16 pairs of primary NSCLC and corresponding normal tissues. Wound healing assay was performed to detect cell migration ability; Transwell invasion assay was performed to detect cell invasive ability; Zymography assay was performed to detect MMP-9 activity.Results:1. Analysis of immunohistochemical staining suggested low THOP1 expression was found in 71 (59.2%) of the 120 NSCLC specimens and significantly correlated with positive lymph node metastasis (P=0.048). However, low THOP1 expression was found in 22 (41.5%) of the 53 normal lung tissues. Chi-square test suggested that the expression of THOP1 was significantly higher in the normal lung tissues than that in the NSCLC specimens (P= 0.032). Real-Time PCR and western blotting showed that NSCLC specimens had decreased THOP1 mRNA and protein expression compared to corresponding normal tissues.2. Univariate analysis demonstrated that low THOP1 expression significantly predicted decreased 5-year disease-free survival (P= 0.038) and overall survival (P= 0.017). In addition, positive lymph node metastasis (P= 0.025) and advanced TNM stage (P= 0.009) significantly predicted decreased 5-year overall survival. However, multivariate Cox regression analysis showed that only low THOP1 expression retained its significance as an independent prognostic factor for unfavorable 5-year disease-free survival (P= 0.046) and overall survival (P= 0.021).3. Expression levels of THOP1 protein were detected in these 4 non-small cell lung cancer cell lines, of which HI299 cell line showed the highest expression level. Forty-eight hours after THOP1 siRNA transfection, the results of western blot showed that the expression of THOP1 protein (P<0.01) in H1299 cells was inhibited significantly.4. After THOP1 protein expression was decreased by siRNA, wound healing assay showed that the migration ability of HI 299 cells was significantly inhibited (P<0.01);5. After THOP1 protein expression was decreased by siRNA, Transwell invasion assay showed that the invasive ability of H322M cells was significantly inhibited (P<0.01).4. After THOP1 protein expression was decreased by siRNA, Zymography assay showed that the MMP-9 activity was significantly decreased (P<0.0\).Conclusions:THOP1 may have clinical potentials to be employed as a promising biomarker to identify individuals with better prognosis and a novel antitumor agent for therapy of patients with NSCLC. Inhibition of THOP1 protein expression could effectively up-regulate the migration and invasive abilities of H1299 cells via MMP-9 in vitro, suggesting that THOP1 protein plays important regulating roles in promoting migration and invasive abilities of NSCLC.