EREG/EGFR Signaling Pathway Promotes Pulmonary Metastasis Of Bladder Cancer

Background:Bladder cancer(BC)is one of the most common malignant tumors of the urinary system.The latest statistics published in the 2017 Clinical Oncology Journal show that the incidence of bladder cancer has ranked the fourth most common malignant tumor in men in the United States.According to statistics from the China Cancer Registry in 2016,bladder cancer ranks fifth among the most common malignant tumors in men,and its incidence and mortality have increased significantly in recent years.There are two main types of bladder cancer,non-muscle invasive bladder cancer(NMIBC)and muscle invasive bladder cancer(MIBC).Non-muscle-invasive bladder cancer(NMIBC)is characterized by repeated relapses and eventually progresses to a more malignant type of muscle invasive bladder cancer(MIBC).In muscle invasive bladder cancer(MIBC)patients,cancer cells invade the muscle.Although the standard treatment plan to remove the bladder combined with systemic chemotherapy,adjacent organs and distant metastases can still occur,which seriously affect the patient's health;Common metastatic sites of bladder cancer are lung,bone,liver,etc.The five-year survival rate of bladder cancer patients once transferred is less than 50%.At present,there is no good treatment and target for metastatic bladder cancer except systemic chemotherapy based on platinum drugs.Therefore,to develop effective targeted drugs,control bladder cancer metastasis,and improve the efficacy of treatment of advanced patients,we intend to test and analyze the in vivo,in vitro and clinical tissue specimens to find the molecular mechanism of distant metastasis of bladder cancer.Methods:We first established a high-metastasis model for bladder cancer and demonstrated that high-metastatic cell lines have stronger stemness and metastasis abilities through related experiments;we confirmed the secretion of tumor cells by Array Analysis,ELISA,and related experiments.EREG is a key molecule that causes the metastasis of bladder cancer cells;then,,we elaborated the role of EREG/EGFR signaling pathway in the metastasis of bladder cancer through a series of experiments;later we found that miR-20b is lower expressed in high metastatic bladder cancer cell lines,which can simultaneously regulate EREG and EGFR;then,we will explore the application of EGFR inhibitor gefitinib in vivo to observe its anti-tumor efficacy,and analysis of EREG in bladder cancer patients in the prediction of bladder cancer and The role of guiding the prognosis.Finally,we examined serum EREG expression in patients with bladder cancer,and analyzed the relationship between EREG and EGFR expression and survival of bladder cancer patients,as well as clinicopathological parameters.(1)Establishment and validation of highly metastatic bladder cancer cell lines1 We injected 5x104 MB49 bladder cancer cell into the tail vein of mice.Two weeks later,we killed the mice and removed the lungs under aseptic conditions.After the lung metastases,the two colonies were cut and digested with trypsin for 5 minutes.The supernatant was centrifuged and resuspended in a tissue block.Primary cultures were performed using DMEM/F12 medium containing 10%FBS;respectively,tail vein injections were performed again,and after 3 transfers,highly metastatic cell lines were obtained and named MB49-LM1 and MB49-LM2.All cells are used within ten generations.2 After obtaining high-metastatic bladder cancer cell line(MB49-LM),we performed relevant validation.We used MB49 and MB49-LM cells to simultaneously inject the same number of cells into the tail vein,12 mice in each group;two weeks later,6 mice were randomly selected from each group and the lung metastasis was observed after sacrifice.The number of metastases in highly metastatic strains was significantly increased by the naked eye and stained with HE slices.The survival curve of the remaining mice showed that the survival time of mice with high metastatic strains significantly decreased.Through the above experiments,we verified the metastatic ability of highly metastatic bladder cancer cell lines.(2)The mechanism of high metastatic cells promoting bladder cancer metastasis through EREG/EGFR signaling pathway1 In order to determine whether cytokines have differential expression between high-metastatic and low-metastatic bladder cancer cells,we first sent two highly metastatic and parental cells for qPCR microarray analysis.Subsequently,different cell RNAs were extracted and the genes screened according to the chip analysis results were subjected to qPCR detection.Finally,the most relevant cytokine EREG was determined based on the results of both.Array data revealed that EREG was the most highly expressed in secretory factors;further detection by qPCR confirmed the high expression of EREG.2 During cell culture of MB49-P and MB49-LM cells,we will collect cell supernatants,ie conditional media(CM),and use PBS as a negative control.Further,ELISA was used to detect the auto-secretory increase of EREG in highly metastatic bladder cancer cell line;and through the WB experiment,EEGFR downstream signaling pathways such as EGFR,STAT3 and ERK1/2 were abnormally activated in high-metastatic cell lines.3 Next,we used exogenous EREG protein to stimulate MB49 cells and observed changes in stemness,invasiveness,and further WB detection of related protein markers.4 We used EREG neutralizing antibody to block EREG to observe the changes of stem metastasis and invasive ability of highly metastatic cell lines,further demonstrating that EREG can function by activating p-EGFR of bladder cancer cells.5 We have confirmed that EREG can increase the level of p-EGFR in bladder cancer cells.We use EGFR inhibitors to block its downstream effects and observe the migration and invasion of bladder cancer cells.6 In vivo application of EGFR inhibitors to observe their inhibition of lung metastasis in mice.(3)miR-20b regulates EREG and EGFR expressionThrough literature analysis and the TargetScan website,we found miR-20b,which regulates EREG and EGFR.First,the luciferase activity assay experiments confirmed that miR-20b could knock down the 3'UTR of EREG and EGFR and inhibit protein synthesis;then miR-20b was over-expressed in highly metastatic cell lines.miR-20b can suppress the 3'UTR of EREG and EGFR and inhibit protein synthesis,thereby inhibiting bladder cancer lung metastasis.(4)Clinical significance of EREG and EGFR expression1 We further validated the therapeutic effect of the EGFR inhibitor Gefitinib on metastatic bladder cancer in a tumor-bearing cancer cell line.Through the tail vein metastasis model,mice bearing MB49-LM were established and randomly divided into two groups with 12 animals in each group.The oral administration method(1 mg/kg)was used.Two weeks later,6 mice were randomly sacrificed to observe the lungs.Metastasis and statistical analysis;remaining mice were used for survival analysis.2 Given that our institutional sample bank has been collecting blood samples and relevant prognostic information before and after treatment for bladder cancer patients,we used this platform to preliminarily obtain blood samples from 405 patients with bladder cancer and analyze the relationship between serum EREG levels and the patient's clinical features in patients with bladder cancer.The association of pathological features was evaluated by EREG to predict the occurrence of bladder cancer and determine the applicability of prognosis.Results:We first established a high-metastasis model for bladder cancer and demonstrated that high-metastatic cell lines have stronger stemness and metastasis capacity through relevant experiments.Second,through literature learning,we identified and validated the activation of the tumor's own EGFR signaling pathway to promote bladder cancer stemness.The ability of metastasis was improved;we confirmed the EREG secreted by tumor cells through gene Array analysis,ELISA,and related experiments to promote the bladder cancer stemness and metastasis.Next we further elaborated the molecular mechanism of EREG/EGFR signaling pathway in the experiment.Later,we found that EREG and EGFR are miR-20b target genes and confirmed by in vivo and in vitro experiments.Finally,we will explore the anti-tumor efficacy of EGFR inhibitor Gefitinib in vivo.EREG in 405 bladder cancer patients was used to predict the occurrence of bladder cancer and to guide the prognosis.Conclusion:The results of the study indicate that high metastatic bladder cancer cell lines can be screened through the tail vein lung metastasis model.The EREG/EGFR signaling pathway plays a key role in enhancing the tumor's ability to metastasize and promote lung tumor colonization,and EREG?EGFR is regulated by miR-20b.The EREG/EGFR signaling pathway is a key target for the metastasis of bladder cancer,which can improve the colonization ability of the lung and accelerate the lung metastasis of bladder cancer.It is expected to be a therapeutic target for the treatment of metastatic bladder cancer.