PTP1B Contributes To The Proliferation And Metastasis Of Non-small Cell Lung Cancer

Background and ObjectiveLung cancer incidence ranks second place in a variety of tumors in United States, and the mortality of lung cancer has become the highest which is one of the largest malignant threating to human health. And non-small cell lung cancer accounts for 85% of lung cancer. In recent years, although the development of treatment and drug, including the platinum-based chemotherapy, molecular targeted drugs, the five-year survival rate was still only about 15%. The reason was that 85% of NSCLC was advanced or local advanced at the time of diagnosis. The proliferation and metastasis of lung cancer cells was one of the important reasons. Therefore, people continue to explore the factors that affect the proliferation and metastasis of lung cancer to look forward to control the occurrence and development of lung cancer. The proliferation and metastasis of lung cancer were regulated by many signal pathways.Protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) maintain the balance of protein activity through dephosphorylation and phosphorylation of a protein in the signal pathway, and to maintain the balance of the signal pathway. When PTPs and PTKs functions were imbalance, it will cause imbalance in the signaling pathway, leading to the occurrence of the disease. Many previous studies have focused on PTKs, found over-expression or mutation of many PTKs will lead to the development of lung cancer. However, our study focused on the PTPs. PTP1 B is an important member of the family of PTPs. Studies has shown that PTPIB played an important role in the metabolic disease (diabetes and obesity). And the role of PTPIB in tumors had two sides which can promote tumor progression in breast cancer, colon cancer, and prostate cancer. And PTPIB was tumor suppressor in other tumors such as:esophageal cancer, lymphoma. The two side’s role of PTP1B suggested that PTP1B was tissue-specific. And no study researched the role of PTP1B in NSCLC. The aims of the study were as follows:1.PTPIB regulates of the proliferation and metastasis of NSCLC; 2. the mechanism of PTPIB regulation of proliferation and metastasis of in NSCLC.Materials and Methods1. We detected the expression of PTPIB in pathological paraffin of postoperative non-small cell lung cancer and benign lung disease by immunohistochemistry then compared the different expression between NSCLC tissues and benign lesions tissues. The relation between the expression of PTPIB and clinical pathological characteristics and prognosis of patients was analyzed.2. A further study to confirm the role and mechanism of PTPIB in regulating proliferation and migration of NSCLC in vitro was made. First of all, we transfected lung cancer cells with siRNA-PTPIB to inhibit PTPIB expression and studied the effect of it on the proliferation of NSCLC by MTT. Meanwhile, we researched the influence of decreased PTP1B expression on migration of NSCLC by scratch test and Transwell study. Moreover, the Annexin V/PI flow cytometer and PI flow cytometer were used to detect the impact of decreased PTP1B expression on the apoptosis and cell cycle. Second, we infected NSCLC cells with adeno virus vector to increase PTP1B expression and studied the effect of it on the proliferation of NSCLC by MTT. Also, we researched the influence of PTPIB overexpression on migration of non- small cell lung cancer by scratch test and Transwell study. Third, we study PTP1B mechanism for regulating NSCLC cell proliferation and migration. Western-blot was used to detect PTPIB protein expression and the landmark protein expression of epithelial- mesenchymal transition (epithelial mesenchymal transition, EMT) such as E-Cadherin, N-Cadherin, β-Catenin, Vimentin in NSCLC cells. In. this way, relationship between PTP1B and EMT in lung cancer cell were explored. Further, PTP1B and p-Akt (Ser473) expression were respectively detected by Western-blot to investigate the role of AKT in the way of PTP1B. Then, PTP1B and p-src (Tyr527) expression were respectively detected to explore the role of src in the way of PTP1B and the downstream protein of the src.3. On the basis of the clinical studies and experiments in vitro, the role of PTP1B in regulating the proliferation of NSCLC in vivo by animal experiments. Ten 7-week-old male nude mice were divided into PTP1B siRNA interference group and control group on average. Then, we made with PBS 5 ×107/mL lung cancer cell suspension and used subcutaneous injections of 0.1 mL to the right anterior axillary of nude mice. The maximum and minimum diameter of the tumor was measured periodically and two groups of tumor growth curve were draw. After 26 days, the mice were sacrificed to remove the tumors which were weighed. PTP1B expression in the lung cancer tissue by immunohistochemistry and western-blotResults1. With the immunohistochemical study of clinical tissue samples, we found that the positive expression of PTP1B was brown staining in the cytoplasm of lung cancer cells. The expression of PTP1B in 32 patients was positive among 63 patients with non-small cell lung cancer, while that of 9 patients with pulmonary benign lesions were all negative. The expression of PTP1B in NSCLC tissues was significantly higher than that of lung benign disease (x2= 8.229, P= 0.004). That suggested that PTP1B may play a role in the occurrence and development of NSCLC. Studies of relationship between PTP1B and clinical and pathological features of lung cancer patients have shown that the expression of PTP1B in NSCLC patients was associated with pTNM stage (x2=6.426, P= 0.040), with the clinical stage gradually increased PTP1B positive rate also increased (I 35.0%, Ⅱ 42.1%, Ⅲ 70.8%). Kaplan-Meire survival analysis showed that the overall survival of patients with negative PTPIB were significantly better than those of patients with positive PTPIB (Log-rank test P= 0.047).And COX multivariate regression analysis showed the expression of PTPIB suggested a poor prognosis (HR= 2.050,95% CI:1.020-4.118). The risk of death of patients with PTP1B-positive was twice than the PTP1B-negative patients.2. In vitro, after the inhibition of PTP1B by siRNA, the PC9 cell proliferation was significantly decreased, and wound healing assay and Transwell indicated that the inhibition of PTPIB can significantly reduce the migration of lung cancer cell. Flow cytometer detected the inhibition of PTPIB could promote apoptosis of lung cancer cells. It suggested that the inhibition of lung cancer cell proliferation and migration maybe partly due to apoptosis. However, flow cytometer also found that the inhibition of PTPIB did not affect lung cancer cell cycle. The over-expression of PTPIB by the adenovirus vector infection could increase lung cancer cell proliferation and migration. Then we explored the mechanism of PTPIB regulation of non-small cell lung cancer cell proliferation and migration. First, Western-blot was used to detect the effect of PTPIB on the iconic proteins (E-Cadherin, N-Cadherin, β-Catenin, Vimentin) of EMT (Epithelial mesenchymal transition, EMT). The results showed that the inhibition or overexpression of PTP1B did not significantly affect the expression of E-cadherin, N-cadherin, beta-catenin and Vimentin, suggesting that the regulation lung cancer cell migration by PTPIB was no significantly associated with EMT. Then we detected the effect of PTPIB on p-Akt (Ser473). The results showed that the inhibition or overexpression of PTP1B did not significantly affect the expression of p-Akt (Ser473), which displayed the PTPIB could not affect the phosphorylation of AKT. Finally, we found that in NSCLC cells, the over-expression of PTPIB could reduce the expression of p-src (Tyr527), which activated src and ERK1/2. And that was not seen in the inhibition of PTPIB and HBE cells. That suggested the inhibition lung cancer cell proliferation and migration by the inhibition of PTP1B might be in other ways and the roles of PTPIB in the lung cancer cells and normal bronchial epithelial cells were different3. On the basis of the clinical studies and experiments in vitro, we studied the role of PTPIB in regulating the proliferation of NSCLC in vivo by animal experiments. The research showed that the tumor size and weight of PTPIB inhibition group were significantly less than the normal control group, suggesting that the inhibition of PTPIB in vivo could still inhibit the proliferation of lung cancer cells. Two cases of PTPIB in siRNA interference group were lower than the normal control group and the expression of PTPIB among siRNA interference group was different, suggesting that the expression of PTPIB was influenced by the in vivo environment.Conclusions1. This study was the first study to explore the role of PTPIB in the proliferation and metastasis of NSCLC. Our study confirmed that PTP1B could promote NSCLC proliferation and metastasis from clinical specimen’s studies, in vitro experiments and animal experiments. And high expression of PTPIB in lung cancer was associated with a poor prognosis.2. Our study preliminarily confirmed PTPIB increased cell apoptosis to promote the proliferation and metastasis NSCLC. And the dephosphorylation of src Tyr527 by PTP1B activated src and then activated the ERK1/2. There was no significant correlation with the cell cycle.3. The research of PTP1B molecule inhibitors was hot. And our study provided a theoretical basis for its applications in the treatment of NSCLC in the future.