Mnk2 As Prognostic Factors Of Non-small Cell Lung Cancer And Its Molecular Basis

Background and Purpose:Lung cancer has been to the leading cause of the mortality and morbidity of cancer in China and the worldwide in recent years, especially in male patients with the proportion increasing year by year . The treatment of lung cancer is unsatisfied because the majority of patients with lung cancer were in advanced stage, even they have the chance to do the radical resection with early or middle stage, but they also have to face the problem of recurrence and metastasis. The emergence of chemotherapy drugs supposed to find the hope of survival, but more and more date analysis of large sample from worldwide had shown that the chemotherapy drug can only increase the 5-year survival rate of patients with less than 10% in the no screen patients. Targeted therapy is the very popular treatment mode in the recent years since the Gefitinib (TKI) come to market in 2003, more and more targeted therapy drugs emerge to the clinic application include the Bevacizumab, Crizotinib and so on. Individualized treatment becomes the new mode for lung cancer. From the statistic analysis we can find that the PFS (progression-free survival) has been improved with the targeted therapy apparently, but the OS (overall survival) data still need more large clinical reports to verify and supplement.Targeted therapy make the scientists pay more attention to the signaling pathway associated with lung cancer research, the discovery of new signal pathway also provide basis for the development of targeted drugs. Mitogen activated protein kinases (MAPK) signal transduction pathway is the most active area in the research of signal transduction for the past few years. Studies have shown that the MAPK signal transduction pathway through the gene transcription and regulation, play an important role in the cell proliferation, differentiation, apoptosis, angiogenesis and tumor metastasis, in the progress of these studies will provide new targets for the treatment of tumor . In a sense, the occurrence of tumor reflects the imbalance between tumor cell proliferation and apoptosis, the tumor cells are often characterized by resistance to apoptosis and promote proliferation. Growth factor receptor mediated the MAPK signal transduction pathway, is closely related to cell proliferation and differentiation in many signaling pathway . Mitogen activated protein kinases (MAPK) is the important passer make the cell signal outside transmit from the cell surface to the nucleus. In mammals,14 MAPKs have been characterized into eight groups. Conventional MAPKs comprise the p38 isoforms, extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun amino (N)-terminal kinases1/2/3 (JNK1/2/3) and ERK5. Atypical MAPKs have nonconforming particularities and comprise ERK3/4, ERK7 and Nemo-like kinase (NLK). These subgroup comprise many signaling pathways, the p38 isoforms, extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun amino (N)-terminal kinases1/2/3 (JNK1/2/3) are the most important of them.Mnk(MAPK signal-integrating kinase) is the downstream tyrosine kinase of p38MAPK and ERK/MAPK signal pathway, as the subgroub of the ERK at the beginning, name the Mnkel and Mnk2. Mnkl/2 phosphorylate the eIF4E (p-eIF4E) lead to tumor cells with resistance to apoptosis, promote the synthesis of tumor associated protein. Mnk2 is the homologous structures of Mnk1. The study found that Mnk2 forming complexes with eIF4G like Mnk1, make it easier for eIF4E combining with 5-cap structure of mRNA through the 209th serine phosphorylation of eIF4E , in turn, increase the mRNA translation. But the Mnk2 phosphorylate the eIF4E through the signal pathway of ERK and p38 MAPKa/p, not the signal pathway of p38MAPK and JNK . No matter in vitro or in vivo experiments, Mnk2 showed the higher biological activity than Mnkl, it also present to be relatively insensitive to the inhibition of the upstream p38MAPK or ERK signaling pathway.Eukaryotic translation initiation factor 4E (eIF4E), is the core element of the translation initiation and regulation of eukaryotic cells, through the combined with the 5-cap structure of mRNA, play an important role in the process of the protein translation initiation. Research proves, the eIF4E excessive expression was found in a variety of human malignant tumors no matter in the carcinoma tissue or the tissue adjacent, the eIF4E was positively correlated with the ability of tumor invasion and metastasis. The non-small cell lung cancer patient include the bronchioloalveolar carcinoma (BAC), the expression of p-eIF4E in cancer cells is much higher than the normal tissue cells, the over expression in NSCLC patients has a lower 5-year survival rate, the p-eIF4E seems to be as a independent prognostic factor. Rosenwald found that the increased expression of eIF4E is easy to promote the growth and infiltration of bronchioloalveolar carcinoma cell. Seki found that after the immunohistochemical staining of eIF4E on 143 cases of peripheral lung adenocarcinoma and atypical adenomatous hyperplasia, the expression of eIF4E in adenocarcinoma tissue is significantly higher than normal tissue, it is associated with the tumor histological and infiltration degree.Because of the downstream kinase of MAPK, Mnk1/2 makes tumor cells with resistance to apoptosis and promotes the cell proliferation through the phosphorylation of eIF4E. As a result, the expression level of Mnk1/2 can reflect the ability of anti-apoptosis, invasion and proliferation of tumor cell. In breast cancer research, Mnk1/2 expression level was positively correlated with the over expression of HER2, are potential targets for cancer treatment. But, there is no detailed reports of Mnkl/2 in lung cancer on a global scale, whether like the research in breast cancer, high expression of Mnk1/2 related to the cell proliferation, invasion and anti-apoptosis; How about the relationship between the Mnk2 protein expression levels and clinical feature? Can it be an independent prognostic factor of lung cancer? All of these need to be further research.Mnk2 (mainly Mnk2A) has higher biological activity than MnklA . In our experiment, we found that the expression level of Mnk2 is higher than Mnkl no matter in the lung cancer sample or lung cancer cell lines. It became a research hotspot in recent years and also to be the focus of our research. We suppose to detect the Mnk2 level through the paraffin section, non-small cell lung cancer cell lines and transplanted tumor animal models, for the assessment of Mnk2 in significance to the tip of the prognosis in non-small cell lung cancer and its molecular basis.Chapter 1 The relationship between Mnk2 protein expression level and the postoperative survival rate, clinical pathological featuresMethods:We collected the tissue specimens of 367 cases with non-small cell lung cancer and 117 cases of normal lung tissue adjacent to carcinoma at the First Affiliated Hospital of Guangzhou Medical University from October 2006 to June 2009. All the specimens staining by HE, selecting the suitable area for making the paraffin block of tissue microarray. Divided into Mnk low expression group and Mnk high expression group according to the cell staining intensity under the microscope.Results:1. The Mnk2 protein located in the tumor cells and the cytoplasm of bronchial epithelial cells. The Mnk2 protein expression level of lung cancer tissues increased significantly relative to the tissue adjacent to carcinoma, the difference is statistically significant(log-rank test, t=-11.397, P<0.001);2. Mnk2 high expression in 220 patients and the higher expression rate was 59.5% (220/367) in 367 cases of non-small cell lung cancer tissues. The OS ratios was 45.74%, the PFS ratios was 43.83%;3. The OS ratios of Mnk2 low expression group (n=147) and high expression group (n=220) was 60.39% and 36.01%, the two groups have significant difference (log-rank test, X2=16.382, P<0.001); The PFS ratios was 57.26% and 35.04%, the two groups still have significant difference (log-rank test, X2= 16.982, P<0.001);4. In the early and mid stage (stage I and II) cases, the Mnk2 expression level was significantly correlated with the prognosis of patients. The OS ratios of the Mnk2 low expression group (n=102) and high expression group (n=131) was 67.43% and 51.54%, the two groups have significant difference (log-rank test, X2=5.123, P=0.024). The PFS ratios was 66.30% and 50.42%, the two groups have significant difference (log-rank test, X2=5.058, P=0.025, P=0.025);5. In the late stage (stage I and II) cases, the OS ratios of the Mnk2 low expression group (n=45) and high expression group (n=89) was 44.44% and 13.18%, the two groups have significant difference (log-rank test, X2=9.192, P=0.002). The PFS ratios was 36.02% and 12.56%, the two groups have significant difference (log-rank test, X2=11.083, P=0.001);6. In the lung adenocarcinoma, the OS ratios of Mnk2 low expression group (n=88) and high expression group (n=154) were 61.22% and 34.00%, the two groups have significant difference (log-rank test, X2=12.255, P<0.001); PFS ratios were 57.79% and 33.09%, the two groups have significant difference (log-rank test, X2=12.041, P=0.001). While the Mnk2 expression level in squamous cell carcinoma and large cell carcinoma has nothing to do with the OS and PFS (P>0.05);7. The results shown that high Mnk2 expression level (P<0.001,log-rank test) is one of the poor prognosis indicator of non-small cell lung cancer through the COX single factor regression analysis in 367 cases. In addition, the age (P=0.017, log-rank test), T stage (P=0.003, log-rank test), N stage (P<0.001, log-rank test) and clinical stage (P<0.001, log-rank test) are also the poor prognosis indicator;8. The COX multiple regression analysis showed that high Mnk2 expression level (Hazard ratio=1.832,95% confidence interval:1.358-2.472, P=0.003) and N stage (Hazard ratio=3.275,95% confidence interval:2.437-4.401, P<0.001) are one of the independent indicator of non-small cell lung cancer with poor prognosis respectively;9. In 367 cases, the application of Cross-table analysis showed that the expression of Mnk2 levels has significant correlation with N stage (P=0.008), but not with age, sex, clinical stage, tissue types, smoking and T stage (P>0.05).10. In the 174 cases of the gene microarray of NSCLC, the results show that the Mnk2 and eIF4E (r=0.216, P=0.006),P-eIF4E (r=0.241, P=0.002) protein expression has a positive correlation correlation through the Pearson correlation statistical analysis.Conclusion:1. Mnk2 is high expression in non-small cell lung cancer cell tissue, Mnk2 protein locate inside the tumor cells and cell plasma of bronchial epithelial cells.2. The OS and PFS of the patient with Mnk2 high expression was significantly lower than that with low expression in the 367 cases;3. The expression level of Mnk2 is significant correlation with the prognosis in the Clinical stage Ⅰ-Ⅱ (early) and Ⅲ-Ⅳ(late).4. The Mnk2 expression level was significantly associated with the prognosis in lung adenocarcinoma.5. Mnk2 high expression and N stage is one of the independent poor prognosis indicator of non-small cell lung cancer respectively.6. Mnk2 expression level has significant correlation with N stage but not the age, sex, clinical stage, tissue types, smoking and T stage.7. The Mnk2 and eIF4E, P-eIF4E protein expression has a positive correlation. Prompt that Mnk2 maybe play a role in the MAPK pathway through the eIF4E and phosphorylation eIF4E.Chapter 2 The Mnk2 expression in non-small cell lung cancer cell lines and silence of Mnk2 in cell function in vitro experimentMethods:1. Choose the logarithmic phase non-small cell lung cancer cell lines (A549, NCI-H460, NCI H1975, NCI H520, NCI-H1650, SPC-A-1) and normal bronchial epithelial cell line 16HBE, using the Real-time PCR to detect the Mnk2 expression level respectively;2. Mnk2 gene silence using the siRNA sequences with chemical synthesis, the sequence is:sense 5’UCGUCAAGAUCAUUGAGAATT--3’and anti-sense5’-UUCUCAAUGAUCUUGACGGTT-3’,it was transfected the A549 and NCI-h460 respectively;3. Using the Mnk2-siRNA and Negative control for the in vitro experiments, including:CCK cell proliferation experiment, tablet clone formation experiment and Transwell cell migration experiment.Results:1. In non-small cell lung cancer cell lines (A549, NCI-H460, NCI-H1975, NCI-H520, SPC-A-1, NCI-H1650) and normal bronchial epithelial cell line (16HBE), NCI-H1975 has the highest Mnk2 expression level.Applicate the two independent samples t test, we found that the Mnk2-mRNA was in high expression in non-small cell lung cancer cell lines NCI-H460, NCI-H1975, NCI-H520, A549 and SPC-A-1 compared with the 16HBE(P*<0.001, F*值 =43.494);2. The detection of Mnk2 protein expression level through the Immunofluorescence in non-small cell lung cancer cell lines (A549, NCI-H1975, NCI-H460, SPC-A-1, NCI-H1650) and normal bronchial epithelial cell line (16HBE) show that the expression of Mnk2 in the A549, NCI-H1975 and NCI-H460 is elevate but lower than that in SPC-A-1, NCI-H1650,16HBE;3. The detection of Mnk2 protein expression level through the Western blot in non-small cell lung cancer cell lines (A549, NCI-H1975, NCI-H460, SPC-A-1, NCI-H1650) and normal bronchial epithelial cell line (16HBE) show that the expression of Mnk2 in the A549, NCI-H1975, NCI-H460 and NCI-H1299 is elevate but reduce in SPC-A-1, NCI-H1650,16HBE;4. The detection of Mnk2 expression level in Mnk2-siRNA group and Negative control group of A549 using the Real-time PCR show that the Mnk2 expression level of Mnk2-siRNA group was obviously lower than Negative control group, the difference is statistically significant (P t=-13.742, P<0.001, Independent-Samples t test);5. The detection of Mnk2 expression level in Mnk2-siRNA group and Negative control group of NCI-H460 using the Real-time PCR show that the Mnk2 expression level of Mnk2-siRNA group was obviously lower than Negative control group, the difference is statistically significant (t=-15.179, P0.001);6. The detection of Mnk2 expression level in Mnk2-siRNA group and Negative control group of NCI-H1975 using the Real-time PCR show that the Mnk2 expression level of Mnk2-siRNA group was obviously lower than Negative control group, the difference is statistically significant (t=-44.946, P0.001);7. Adopt CCK8 colorimetry to detect the proliferation ability of A549 after the transfection of Mnk2-siRNA and Negative control. The cell growth difference in Mnk2-siRNA group and Negative control group in 1,2,3,4 days has statistical significance (P<0.05, Independent-Samples t test);8. Adopt CCK8 colorimetry to detect the proliferation ability of NCI-H460 after the transfection of Mnk2-siRNA and Negative control. The cell growth difference in Mnk2-siRNA group and Negative control group in 2,3,4,5 days has statistical significance (P<0.05, Independent-Samples t test);9. Adopt CCK8 colorimetry to detect the proliferation ability of NCI-H1975 after the transfection of Mnk2-siRNA and Negative control. The cell growth difference in Mnk2-siRNA group and Negative control group inl,2,3,4,5 days has statistical significance (P<0.05, Independent-Samples t test);10. Using the clone formation experiment for testing the cell proliferation of A549/Mnk2-SiRNA group and A549/Negative control group in vitro, the number of clone forming of A549/Mnk2-SiRNA is 24±4.041 and the number of clone forming of A549/Negative control is 164±5.132, the differences between two groups of cells have significant difference (t=-36.946, P<0.001, Independent-Samples t test);11. Using the clone formation experiment for testing the cell proliferation of NCI-H460/Mnk2-SiRNA group and NCI-H460/Negative control group in vitro, the number of clone forming of NCI-H460/Mnk2-SiRNA is 55±7.00 and the number of clone forming of NCI-H4609/Negative control is 147±6.557, the differences between two groups of cells have significant difference (t=-16.613, P<0.001, Independent-Samples t test);12. Using the clone formation experiment for testing the cell proliferation of NCI-H1975/Mnk2-SiRNA group and NCI-H19750/Negative control group in vitro, the number of clone forming of NCI-H1975/Mnk2-SiRNA is 31±4.583 and the number of clone forming of NCI-H1975/Negative control is 118±6.000, the differences between two groups of cells have significant difference (t=-19.959. P<0.001, Independent-Samples t test);13. Using the transwell experiment for testing the migration ability of Mnk2-siRNA group or negative control group of A549 in vitro. The numbers of cellpermeating septum in Mnk2-siRNA group is less than the negative control group, the difference is statistically significant (t=-6.360, P=0.003, Independent-Samples t test);14. Using the transwell experiment for testing the migration ability of Mnk2-siRNA group or negative control group of NCI-H460 in vitro. The numbers of cellpermeating septum in Mnk2-siRNA group is less than the negative control group, the difference is statistically significant (t=-12.932, P<0.001, Independent-Samples t test);15. Using the transwell experiment for testing the migration ability of Mnk2-siRNA group or negative control group of NCI-H1975 in vitro. The numbers of cellpermeating septum in Mnk2-siRNA group is less than the negative control group, the difference is statistically significant (t=-5.480, P=0.005, Independent-Samples t test);Conclusion:1. In lung cancer cell lines, expression level of Mnk2 was obviously higher than that of normal cells, it means that the high expression level of Mnk2 in lung cancer cells is a common phenomenon.2. After the transfection of Mnk2-siRNA in lung cancer cell lines, it can be detected that the cell proliferation, cell cloning and in vitro migration ability was restrained, prompt that Mnk2 is one of the important factor for the occurrence, development, invasion and metastasis in malignant tumor.Chapter 3 The animal tumorigenicity experiments of Mnk2-shRNAMethods:1. Through the way of lentivirus transfection build the Mnk2-shRNA-A549 and Mnk2-shRNA-H460;2. Choose the A549 and NCI-H460 cells in logarithmic phase and then transfect the lentivirus Mnk2-siRNA or negative control in it. Dilute the concentration of A549 and NCI-H460 to 2 x 106/ml and 3 x 106/ml in DMEM medium;3. The cells were injected into the flank side of nude mice, the tumor size was observed four days later and made a record every 3 days. The mice were killed 6 times later, winkled the tumor and weight it, then used paraffin embedding it, stain with HE and immunohistochemical, observed by microscope;4. The cells was injected into the nude mice via tail vein, the status of nude mice was observed after 3 days. The mice were killed at 30 days and the number of metastasis tumor was calculated in liver tissue.Results:1. After the injection of A549 cell line in all seven nude mice, we can find that in the NC group all nude mice have the tumor, but the tumor appear in only 6 (85.7%) nude mice in the Mnk2-shRNA group, the tumor appear in dya 10. The speed of tumor appear in two group is significant differences (P<0.05). The tumor weight of Mnk2-shRNA group is 0.05±0.049g, less than the NC group (0.149+0.722 g) (t=-2.976, P=0.012, two-independent t test);2. After the injection of NCI-H460 cell line in all seven nude mice, we can find that in the NC group all nude mice have the tumor, but the tumor appear in only 6 (85.7%) nude mice in the Mnk2-shRNA group, the tumor appear in dya 7. The speed of tumor appear was slower in Mnk2-shRNA group, the speed of tumor appear is significant differences only in the day 7(P=0.031, Independent-Samples t test). The tumor weight of Mnk2-shRNA group is 0.217±0.152g, less than the NC group (0.343±0.078g) (t=-1.947, P=0.075, Independent-Samples t test);3. After the injection of A549 cell line in all five nude mice, the number of metastatic tumors in the liver in Mnk2-shRNA group(3±1) is less than the NC group(10±1) obviously, the speed of tumor appear has statistically significant differences (t=-5.259, P=0.001, Independent-Samples t test);4.1. After the injection of NCI-H460 cell line in all six nude mice, the number of metastatic tumors in the liver in Mnk2-shRNA group(4±1) is less than the NC group(19±1) obviously, the speed of tumor appear has statistically significant differences (t=-3.415, P=0.018, Independent-Samples t test).Conclusion:1. The subcutaneous injection of Mnk2-shRNA transfected into A549 cell line can inhibit the growth of nude mice tumor, prompt that the Mnk2 gene silencing can inhibit the tumor growth. But we can find that the result is unsatisfactory in NCI-H460 cells, it need more experimental data to make clear;2. The tail intravenous transfection of Mnk2 shRNA transfected into A549 and NCI-H460 cell line can inhibit the tumor metastasis ability, prompt that silencing the Mnk2 gene can inhibit tumor metastasis.