The Roles Of β-arrestin1 In The Progression And Treatment Of Non-small Cell Lung Cancer

Lung cancer is the most lethal malignant cancer to human life and physical fitness worldwide, and it represents the leading cause of cancer mortality in China. Lung cancer comprises non-small lung cancer (NSCLC) and small cell lung cancer(SCLC), in which NSCLC accounts for approximately 80% and it is defined as the most dangerous and common malignant cancer. In recent years, the incidence of lung cancer continually rises.Cancer is a kind of diseases with many genes involved, and is multi-stage complex process. Many factors affect the prognosis of lung cancer, and the interactive relationship between these factors make cancer more complex. People aimed to study cancer prognostic factors in order to better understand the cancer process, to develop appropriate and effective treatments, and to predict the curative effect of patients with lung cancer. And it is of great significance for individualized treatment. Beta-arrestins came to light as mediators of G protein coupled receptor (GPCR) desensitization and internalization. GPCR is a kind of seven transmembrane receptors. G protein coupled receptor kinase (GRKs) specificity phosphorylate GPCRs, and recruitp-arrestins to activated receptors, and mediate receptor desensitization, internalization and signal transduction. GRK/β-arrestins system is the key factor of GPCR regulation. In addition to regulating the GPCR, it was found that β-arrestins were involved in a wide variety of tumor related biological processes, such as cell proliferation and apoptosis pathway.β-arrestins expression and dysfunction may play an important role in the process of cancer development. At present about the β-arrestins expression in tumor tissue and its relationship with prognosis in patients with study were less focused, and the role and mechanisms of different types of β-arrestins in tumor, has not yet came to a conclusion.In addition to study the prognostic value of β-arrestins in patients with lung cancer, this study will also further focus on the role and mechanism of β-arrestins in lung cancer development and treatment. At present, for non-small cell lung cancer, first and second-line chemotherapy regimens still is based on platinum. Platinum is cell cycle non-specific drugs, and it can cause DNA damage to cancer cells. Radiation therapy is to make a cell to absorb any form of radiation, and radiation can damage the structure of the cells, directly or indirectly. Thus, DNA is a common target of radiation and chemotherapy. How to increase the efficacy of radiation and chemotherapy in lung cancer, is a hot spot in the study of radiation and chemotherapy sensitivity. DNA damage occurs due to the external environment factors or within the normal physiological process. DNA damage response (DN A damage response, DDR) is one of the most important physiological mechanisms to protect the biological stability of the genome. Given the genome instability potentially cause huge harm, cells have to evolve to develop a set of complete system to ensure the integrity of the genome, and prevent the occurrence of tumor. With more and more studies suggesting that damage repair gene mutations occurred in germ cell DNA are more likely to cause cancer, and DNA damage response as cells of antitumor function and mechanism have been already recognized.Beta arrestinl is involved in a variety of cell biological process, and play an important role in the process of cancer development. The currentβ-arrestinl expression and prognosis in NSCLC study is less focused. We aim to study the role and mechanism of β-arrestin1 in NSCLC with radiotherapy and chemotherapy, and to explore the roles and mechanism of β-arrestinl in the development of NSCLC and in DNA damage response pathway. Section I the expression and prognostic value of β-arrestin1 in non-small cell lung cancerObjectives1. To investigate the β-arrestinl expression and prognosis value in NSCLC.2. To investigate the β-arrestinl and NF-κB co-expression in NSCLC and to study the prognosis value with NSCLC.3. To explore the impact of β-arrestin1 on NF-κB pathways.4.Methods1. A total of 152 patients with NSCLC underwent curative surgery (tumor-node-metastasis (TNM) stage:Ⅰa-Ⅲa) at our hospital, from January 2006 to December 2008. None had received preoperative adjuvant therapy. Our research was approved by Ethical Committee of our hospital, and the informed written consent was obtained from every investigated patient. All patients were pathologically staged according to the TNM classification system of the American Joint Committee for Cancer. Immunohistochemistry was used to test the expression of β-arrestinl in NSCLC.2. Tissue samples from 115 patients with primary lung adenocarcinoma who had undergone complete surgical resection were obtained in this study. Pathological stage was determined according to TNM staging system of AJCC 2010. Specimens were collected from patients in the Department at our hospital from June 2008 to December 2009. None of these patients received preoperative adjuvant therapy. Tissue specimens from primary tumor site of the lung were stored in archived paraffin-embedded blocks. All eligible patients received written informed consent prior to the collection of specimens. Follow-up data were available for all patients. Immunohistochemistry was used to test the expression of β-arrestinl and NF-κB in NSCLC.3. Western Blot technique was used to detect pathways activation and related protein expression level.4. We conducted plasmid and siRNA of β-arrestinl transfection to study the impact of P-arrestin1 status on NF-KB pathway.Results1. Squamous cell lung cancer (SCC) patients with loss of β-arrestinl expression showed significantly poorer OS (log rank, P= 0.026) than the patients who did not. For 64 adenocarcinoma (ADC) patients enrolled in our analysis, only 4 patients showed loss expression of β-arrestinl, which also predicted poor survival (log rank, P= 0.006). P-arrestin1 expression status was independent risk factors of prognosis in SCC patients.2. ADC patients with high β-arrestin1 expression had shorter overall survival (OS) and disease-free survival (DFS) than those with low P-arrestinl expression. The OS rate of the patients with p65-overexpression tumors was significantly lower than that of the patients with p65 underexpression tumors. The DFS rate of the patients with p65 overexpression tumors was also significantly lower than that of the patients with p65 underexpression tumors. The co-expression group had a significantly poor OS and DFS.3. Overexpression of P-arrestin1 enhanced chemotherapy and radiotherapy induced NF-kB phosphorylation, and knockdown of β-arrestinl subrogate chemotherapy and radiotherapy induced NF-κB phosphorylation.Conclusion1. β-arrestinl expression in NSCLC is an independent prognostic factor, especially in SCC.2. Co-expression of β-arrestinl and NF-κB were better prognostic factor of NSCLC.3. The state of β-arrestin 1 regulates the activation of NF-κB. Section II The roles of β-arrestinl in the treatment of Non-small cell lung cancerObjectives1. To explore the impact of β-arrstin1 status on chemotherapy and radiotherapy for NSCLC.2. To test the DNA damage and apoptosis induced by chemotherapy and radiotherapy through β-arrstinl regulation.3. To explore the mechanism of regulation of β-arrstinl on DDR.4. To explore the specific binding domain of β-arrstin1 with DDR related proteins.Methods1. We conducted plasmid and siRNA of β-arrestinl transfection and used MTT and colony formation assay to study the impact of β-arrestinl status on cell apoptosis and proliferation with treatment of chemotherapy or radiotherapy.2. We conducted Comet assay to study the impact of β-arrestinl status on DNA damage with treatment of chemotherapy or radiotherapy.3. We conducted Flow Cytometry and Western Blot assay to test the impact of β-arrestin1 status on the apoptosis and cell cycle with treatment of chemotherapy or radiotherapy.4. We conducted Western Blot assay to test the impact of β-arrestinl status on the DDR pathway with treatment of chemotherapy or radiotherapy.5. We searched the public database to explore the potential interact proteins of P-arrestin 1.6. We used co-immunoprecipitation assay to test the interaction between β-arrestinl and potential proteins.7. Genetic mutation technique would be used to identify the specific binding domain of β-arrestin1, and co-immunoprecipitation assay or GST PULL-DOWN assay would be conducted to verify.Results1. The apoptosis was enhanced with β-arrestinl overexpressed and cell proliferation was inhibited with β-arrestinl overexpressed with treatment of chemotherapy and radiotherapy.2. Knock down of β-arrestinl reduced the DNA damage and apoptosis with chemotherapy, whereas it inhibited cell proliferation with radiation.3. Overexpression of β-arrestinl enhanced DDR activation.4. Knock down of β-arrestin1 reduced the phosphorylation of ERK and NF-κB.5. We searched data base including https://www.predictprotein.org; http://swissmodel.expasy.org; http://consurf.tau.ac.il; http://www.russelllab.org; http://www.ebi.ac.uk, and we also searched National Center of Biotechnology Information to explore the potential proteins interacting with β-arrestinl, and we found ATR, H2AX,etc.6. β-arrestin1 interacted with ATR and H2AX.7. Knock down of β-arrestin1 reduced the phosphorylation of H2AX.Conclusion1. β-arrestinl mediates the DNA damage and apoptosis induced by chemotherapy or radiotherapy.2. β-arrestin1 regulates DDR pathway initiated by chemotherapy of radiotherapy.3. β-arrestin1 regulates cell proliferation via ERK and NF-κB pathway.