The Impact Of Hypertension And Rennin-angiotensin System Blockers On Outcomes Of Lung Cancer Patients

Tumor is the most important public health problemworldwide.Despite ofbasic and clinical research of the tumor progress in the decades,its incidence and mortality remain high and the leading cause of death both in China and the world.At present there are about fourmillion new cases and nearly three million deaths in China each year.Lung cancer is one of the most common malignancies at present,although the incidence is the second most common in both men and women,but it has the highest mortality rate both among men and women.Although the diagnosis and treatment of lung cancer has greatly improved,the treatment of lung cancer has not achieved breakthrough in the past decade,and its overall five-year survival rate is still very low(15%).Therefore,the discovery of new therapeutic drugs or targets for lung cancer has profound implications for the development of new lung cancer therapies.Although the tumor has shown a certain trend of youth in recent years,most of the tumor patients,especially lung cancer;the aged(>55 years old)is still the highest peak population.Therefore,patients with lung cancer often coexist other comorbidities.In recent years,comorbidity has been regarded as an important factor affecting the treatment decision,treatment process and prognosis of lung cancer patients,but there are many controversies.Many comorbidities can affect lung cancer,such as hypertension and diabetes,among which hypertension is the most common comorbidity,and its most controversial influence on lung cancer patients is also the biggest.In the case of lung cancer,studies have shown that hypertension may increase the risk of lung cancer,but some studies have found no significant link between the hypertension and lung cancer.Due to hypertension patients by taking antihypertensive drugs to maintain blood pressure,studies have found that the affection of different antihypertensive drugs on the prognosis of patients with lung cancer is different,the effects of hypertension on the prognosis of lung cancer may also be related to the use of antihypertensive drugs.Among the antihypertensive drugs,renin angiotensin system blockers(RASBs)is one of the most common clinically used because of its well tolerated,but whether it will affect the prognosis of patients of China with lung cancer has not been determined.In this study we further evaluated the influence of hypertension and related antihypertensive drugs on prognosis of lung cancer through a population-based retrospective cohort analysis,and provided new ideas for the occurrence and treatment of lung cancer.Epithelial-mesenchymal transition(EMT)was closely related with the migration,invasion and metastasis of tumor,in the process the expression of epithelial type protein molecular marker E-cadherin decreased,while the mesenchymal marker N-cadherin molecules,FN1 and vimentin upregulated.Studies have shown that EMTis an important way to the development of NSCLC,had a great influence on the prognosis of lung cancer.We then further studied the effect of RASBs on EMT of Lung cancer cells,as so far there was no related research on this.This study provides a new theoretical basis for RASBs on the clinical treatment of lung cancer.Based on the clinical and experimental studies,the biological data mining and information analysis were carried out in the open database to understand the mechanism of the influence of hypertension and related therapeutic drugs on lung cancer.In recent years,with the rapid development of biological information technology,many large database of genetic and clinical were widely used,it is convenient for researchers to analyzedata of gene expression difference in certain types of cancers and the prognosis significance.In order to better understand the reason if RASBs improve the prognosis of lung Cancer from gene level,we used the c-BioPortal(an integrated the data of 126 tumor genome research)to analyze the genetic variation of the gene expression related to the renin angiotensin system(RAS).We alsoused tumor genome atlas(The Cancer Genome Atlas,TCGA,the world's largest cancer gene information database collected almost all human tumors and subtypes of genome usingthe genome sequencing technologyto analyze the RAS genes expressionin lung Cancer and normal tissues to analysis the prognosis of the genein RAS in order to understand the mechanism of RASBs on lung cancer from gene expression level.CHAPTER I:The prognosis of patients with lung cancer combined with hypertensionBackground:As lung cancer patients are more common in elderly patients,the median age is about 60 years old,about 51%of patients coexist less than or equal to 1 comordity,18%of patients had greater than or equal to 4 comordities,among the patients in the stage ?,comordity is the independent factors influencing the prognosis and treatment options.The most common comordity in men is hypertension,heart disease,diabetes,and the most common among women is lung disease,hypertension and heart disease.The burden of comordities of lung cancer is significant.Hypertension is the most common of these comordities.More and more evidence suggests that the proliferation of tumor cells is enhanced in patients with cardiovascular diseases,such as hypertension and atherosclerosis.lt has been suggested that hypertension is a prognostic factor for breast cancer,prostate cancer,moreore cancer combined with hypertension can lead to stroke and high heart disease incidenceand mortality.Some prospective epidemiological studies have madehypertension as an important cancer risk factors,but there are also some research suggests the opposite point of view,a study reported hypertension does not independently increase the risk of dying from lung cancer,but for lung cancer patientswith smoking is poor prognostic factors.The influence of hypertension in patients with tumor is still controversial,especially in patients with lung cancer,there is no systematic study,this study aims to identify the influence of hypertensionon the prognosis of lung cancer patients,which is of great significance to guide the individual therapy.METHODS:Between January 2006 and July 2012,1534 lung cancer patients undergoing complete surgical resection were consecutively selected from the Shandong Provincial Hospital,excluding 71 patients for clinical information incomplete,postoperative death in a month or for other reasons not to enroll,so a total of 1463 patients in our clinical research.All follow-up information involved in the study was obtained through telephone contact with patients or family members through a unified training staff.The first two years were followed up every 3 months,followed by every half years after 2 years.The definition of hypertension involved in this study was the first admission to hospotial to check for bloodpressure more than 130/85mmhg or previous history of antihypertensive therapy.In order to the analysis,we divided variables into several categories:gender(male or female),age(?60 and>60 years old),smoking history(with or without),histologic type(non-small cell lung cancer NSCLC and small cell lung cancer SCLC),and tumor size(?3 cm,>3cm),lymph node metastasis(withor without),pathologic differentiation degree(high,moderate,low,unknown),pathological staging(?,? or ?a),hypertension(with or without)and the use of RASBs or not.Divided the patients into two groups according to hypertension condition,to assess the effect of hypertension on the prognosisof lung patients,the study endpoints were overall survival(OS)and progression-free survival(PFS).OSwas calculated from surgery to the date of death from all causes or the data of the last follow-up.PFS was defined as the period from surgery till progression or death or the date of the last follow-up.The comparison of clinicopathological features were evaluated using Chi-square test and/or analysis ofvariance(ANOVA)test.The OS and PFS curveswere constructed using the kaplan-meier methods,Cox proportional risk models were was used to assessthe effect of clinicopathological features on survival.All data were analyzed using SPSS22.0 statistical software,A two-tailed p<0.05was considered statistically significant.RESULTS:In 1463 cases of lung cancer patients,386(26.38%)had hypertension,the hypertension patients were more likely to be older than thenon-hypertension patients(p<0.001),andcompared with the non-hypertensive group,the patients in the hypertension group were more likely to have smaller tumor size(p=0.002),to have no lymph node metastasis(p=0.001),and to have earlier stage disease(p=0.001).Other clinical pathological factors were not significantly different between the two groups,such as gender(p=0.439),pathological differentiation(p=0.419),and smoking(p=0.298).The kaplan-meier survival curve shows that the effect of hypertension on patients OS and PFS is not statistically significant.Considering the age and gender factors in the hypertension and non-hypertension group maybe the bias for the analysis of the survival rate,Propensity matching analysis were performed according the two factors,a total of 772 cases weresuccessfully matched,then divided the matched group to two subgroups according to the smoking status,the result suggested that the hypertensionin non-smoking group improve the survival(p=0.014),the univariable Cox regression analysis showed the HR:0.608,(95%CI,0.406 to 0.901,p=0.016),in smoking group there was no significant difference between the two subgroups(p>0.05).CONCLUSION:For patients with lung cancer,hypertension is not an independent prognostic factor,in contrast,the lung cancer patients with hypertension were 'more likely to be older,the tumor size was smaller and have no lymph node metastasis and have earlier stage,and the non-smoking lung cancer patients with hypertensionhad improved survival.CHAPTER ?.The prognostic effects of renin angiotensin system blockers in lung cancer patientsBackground:Due to tumor properties of continuous proliferation,escaping growth inhibitory factor,antiapoptosis,unlimited replication,inducing angiogenesis and activate the invasion and metastasis,utill now the treatment of tumor has no breakthrough.In previous research,we found that the tuomor size and lymph node metastasis rate and stage of lung cancer patients with hypertension were lower,moreover in the non-smokers hypertension is a good prognosis factor,this is very different from the traditional view,which was different from previous studies,we presume that the reasons for this difference may be the reason of the use of antihypertensive drugs in lung cancer patients.At present there are many kinds of antihypertensive drugs,the most commonly used were beta receptor blockers(BBS),RASBs(including Angiotensin converting enzyme inhibitors,ACEI and Angiotensin ? type 1 receptor blockers,ARB),thiazide diuretics(TD),calcium channel blockers(CCB)etc.Studies have shown that BBS has no significant effect on the survival rate of tumor patients compared with those who do not use antihypertensive drugs.The use of RASBs or CCB increased the mortality risk of breast cancer in some extent,and lung cancer patients with hypertension using CCBs improved the survival than the use of BBS.McMenamin et al reported that ACEI and ARB may have relations to the improved survival of cancer patients,such as in prostate cancer the use of RASBs reduces the risk of death,these studies shows the effects of antihypertensive drugsin the tumor tissues has specificity.RASBs are the most used drugs approved for the treatment of hypertension,heart failure,diabetic nephropathy,and well tolerated in patients,Previous studies have shown that RASBs is a favourable factor for the prognosis of lung cancer,but there are still enormous controversy,and research mainly concentrated in the combined radiation and chemotherapy or targeted therapy,or among patients with stage ? lung cancer patients.Whether or not it is an independent prognostic factorin early stage lung cancer patients,there is no related research.Based on our previous research,we believe that thereason for the good prognosis of non-smokers lung cancer patients with hypertension is due to the use of antihypertensive drugs.In this study,we still utilize preceding clinical data to further analyze the prognostic effects of anti-hypertensive drug RASBs on lung cancer patients.Epithelial mesenchymal transition(EMT)is an important process of tumorigenesis,invasion and metastasis.In this process,epithelial cells lose their connections and apical-basalpolarity,cytoskeletal reorganization,signal procedures change,epithelial phenotype loss and obtain the matrix properties.During the course of EMT,the expression of epithelial protein E-cadherin was downregulated,while the expression of mesenchymal markers N-cadherin,FN1 and vimentin was upregulated.So we further investage the influence of of RASBs on EMT of NSCIC cell from the cellular level,in order to verify the capability of cell proliferation and migration,provides a new theoretical basis for the clinical treatment of lung cancer.METHODS:The patients were same with the former hypertension part.In this study,the patients were divided into three groups:non-hypertension group,hypertension without RASBs group,and hypertension with RASBs group.In subgroup analysis,we divided lung cancer patients into two groups,non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC)according to the histological type.For each subgroup,evaluate the effects of hypertension and RASBs on prognosis of lung cancer.The study endpoints were OS and PFS,OS was defined as from surgery to the date of death from all causes or the date of last follow-up.PFS was calculated from surgery till progression or death or the date of last follow-up.The comparison of clinicopathological features was based on chi-square test or variance analysis.The OS and PFS curveswere constructed using the kaplan-meier methods,univariable and multivariable Cox proportional risk models were was used to assessthe effect of clinicopathological features and RASBs on survival.All data were analyzed using SPSS22.0 statistical software,A two-tailed p<0.05was considered statistically significant.Western blot was used to measure the expression of ACE1 and AT1R in the A549,H157,H1299,2B and H520 cellsrespectively.After inhibitionof ACE1 or AT1R expression using Captopril orvalsartan respectively,the change of EMT related protein E-cadherin,Vimentin,FN1 were observed.Using the Transwell and scratch test,the migration ability of A549,2B,H157,H1299 and H520 cells was observed.And MTT was used to detectwhethersuppressed the the proliferation ability of A549,2B,H157,H1299 and H520 cells.RESULTS:Of the 386 hypertensive patients,143(9.77%)used RASBs,108 used ACEIs,30 used ARBs,and 5 had both ACE1s and ARBs.Compared with the non-hypertension group and hypertension without RASBs group,RASBs group were with smaller tumor size(p=0.01),lower lymph node metastasis rate(p=0.001)andearlier pathological stage(p<0.001),otherclinical pathological parameters had no significant difference between the three groups,such as gender(p=0.467),pathological grade(p=0.577),smoking(p=0.306).In univariate Cox regression analysis with the"non-hypertension" group as referent,"hypertension without RASBs" group had no effect on OS(HR 1.09,95%CI 0.88 1.35,p=0.453)and PFS(HR 1.12,95%CI 0.89 1.42,p=0.344),and "hypertension with RASBs" group have significantly improved the OS(HR 0.63,95%CI 0.46-0.88,p=0.006)and PFS(HR0.64,95%CI 0.44-0.91,p=0.014).In multivariate Cox regression analysis,there is still a good OS(HR 0.74,95%CI 0.53-1.02,p= 0.07)and PFS(HR0.71,95%CI0.49-1.02,p=0.07),whereasno significant difference between the"non-hypertension" group and "hypertension without RASBs" group.According to the histology,the patients were divided inito NSCLC and SCLC groups,in NSCLC group,the "non-hypertension" group as referent,the univariable Cox regression analysis found "hypertension with RASBs" group have better OS(p= 0.004)and PFS(p=0.008),and "hypertension without RASBs" group has no effect on the OS and PFS.After adjusting for standardization of the clinical pathological parameters,in the multivariable Cox regression analysis,compared with "non-hypertension"group,"hypertension with RASBs" group still have better PFS(p=0.043),but the OS has no obvious difference between two groups.Further analysis,we subdivided RASBs into ACEIs and ARBs groups,in the uni variable Cox regression analysis,ACEIs had better OS(p=0.004)and PFS(p=0.019),but ARBs did not.In the multivariable Cox regression analysis,ACEIs and ARBs had no statistically significant impact on survival.In the SCLC group,the use of hypertension and RASBs in univariable andmultivariate Cox regression analysis also had no effect on OS and PFS.Western blot detected that ACE1 and ATIR were increasingly expressed in 2B,H157,H1299 and H520 cells,but their expression in A549 cells was low.In A549 cells,Captopril inhibited the expression of mesenchyme cell marker FN1 molecules,whereas the expression ofE-cadherin increased,but no effect on the expression of Vimentin;expression of Vimentin was inhibited by valsartan,but had no effect on FN1 and E-cadherin.In H520 cells,Captopril inhibited the effects of FN1 and Vimentin higher than valsartan.In 2B cells,both Captopril and Valsartan can inhibit the expression of FN1 and Vimentin and increase the expression of E-cadherin.In H157 cells,Captopril and Valsartan inhibit the expression of mesenchymal cell marker FN1 and Vimentin.After treatment of Captopril,the expression of E-cadherin increased,but after treatment of valsartan,the expression of E-cadherin decreased.In H1299 cells,the effect of Captopril was more pronounced than that of valsartan.Transwell and scratch experiment detected after inhibited ACE1 and AT1R by Captopril or valsartan respectively,that except the A549 cells,the effect of captopril inhibited the cell migration of 2B H157,H1299 and 2B in H520 cells more than valsartan.MTT detected that after inhibited ACE1 and AT1R by Captopril or valsartan respectively,Captopril inhibited the proliferation of A549,H520 more than valsartan,but in 2B,H157 and H1299 cells,the results did not make a trend.CONCLUSIONS:In lung cancer patients,RASBs is an independent prognostic factor and can significantly improve the survival of patients with lung cancer.Furthermore analysis found it was mainly that ACEIs improved the prognosis of patients with NSCLC,in SCLC group,no significant difference were observed.Although captopril and valsartan in different cell lines were not exactly the same,the general trend is that the captopril was more effective than valsartan in inhibitingthe ability of cell proliferation and migration of Lung cancer.CHAPTER ?.The expression difference and prognosis value of the genes in RAS of lung cancerBackground:Previous studies have confirmed the impact of hypertension and RASBs on the prognosis of lung cancer.Clinical data suggest that the hypertension with RASBs use hadsignificantly improved the survival of lung cancer patients?Further experiments had shown that RASBs has an effect on the proliferation and migration ability of NSCLC,but its research on gene expression is not clear.The human RAS is mainly composed of circulating RAS and local RAS,local RAS is an important component of the tumor microenvironment and in tumor metabolism,survival,angiogenesis and invasion process plays an important role,mutationof renin gene(deletion,amino acid substitution etc.)will hinder the function of renin,and lead to a variety of inflammation state and disease(such as hyperuricemia,anemia,chronic renal failure),In this RAS system,REN gene encodes REN,ANGPT1 gene encodes Angiotensin ?(Ang?),ANGPT2 gene encodes Angiotensin ?(Ang ?),AGTR1 gene encodes Angiotensin ? typel receptor(AT1),it has two highly homologous subtypes(AGTR1a and AGTR1b),AGTR2 gene encodes Angiotensin ? typel receptor,AT2),ACE gene encodes Angiotensin-converting enzyme,(ACE),ACE2 gene encodes Angiotensin converting enzyme2(ACE2).AGTR1 receptors are widely distributed in the human body,and the AGTR2 is often exists in period of embryo,there is delicaterestrict relationship between the AGTR1 and AGTR2,regulating the proliferation and apoptosis of tumor,ACE has polymorphism,including the insertion/deletion(I/D)genotype,these genotypes changed activity and physiology,ACE2 is a newly discovered component of RAS,42%of the amino acid was homologouswith ACE.Previous studies have reported the expression of ACE2 protein in NSCLC tissues decreased with anti-tumor effect.To systematically investigate the genes changes of RAS in lung cancer,we used the TCGA database.The TCGA database is sponsored by the U.S.government,and by the National Cancer Institute(NCI)and the National Human Genome Research Institute(NHGRI)technology applying analysis of genome,in particular the use of large-scale genome sequencing,almost drawn out all human cancer genome variation map to systematic analysis,in order to find all carcinogenic and tumor suppressor genes tiny variations,understanding the mechanism of the occurrence and development of cancer cells.We will analyze the genes differential expression of RAS in TCGA in lung adenocarcinoma and squamous cell carcinoma,and its effect on prognosis was analysed using kaplan-Meier plotter online website,to further explain the RAS in lung cancer and its possible mechanism.METHODS:We used c-BioPortal to evaluate the variation of REN?ANGPTI?ANGPT2?AGTR1?AGTR2?ACE?ACE2 in all seven genes in lung adenocarcinoma(LUSC)(TCGA,Nature 2014)and lung squamous cell carcinoma(LCSC)(TCGA,Nature 2012).The variation included amplification,deletion,missense mutation,the expression up-regulattion and down-regulation of mRNA).With the help of the Onco Query Language(OQL),the cancer genome data can be visualized.Data was obtained from Genomic Portal(GDC,https://cancergenome.nih.gov/newsevents/newsannouncements/genomic-data-commo ns-launch)to analyze expression data and related survival data.Differentiallyexpressedgenes(DEG)analysis was used to anylyze the data by using R software and DEseq packages.The DEG analysis uses the DEseq method to standardize the raw data.The use of heatmap software(Heatmap Illustrator,HemI version 1)to draw the heapmap of the genes.Using KM plotter(http://kmplot.com/analysis/index.php?P=service&cancer=lung)datasets to perform online survival analysis of gene.RESULTS:OncoPrints showed characteristic genomic variation,including somatic mutations,copy number variation(CNV),and changes in mRNA expression.In LUAD,the mostly variation was REN(8%),ANGPT1(7%)and ANGPT2(7%),mutual exclusion and co-occurrence analysis showed no significant(p>0.05).Overall survival analysis showed no significant difference between the mutation group and non-mutation group(p=0.765).The variation in LUSC is at most AGTR1(27%)and ANGPT1(10%),and mutual exclusion analysis showed that AGTR1 and REN are mutually exclusive expression(p=0.039),ANGPT1 and ACE2 was co-expression correlated(p=0.027).By downloading LUAD and LUSC mRNA expression data from the Genomic Data Commons Portal(GDC),DEG analysis is performed using R software and DEseq packages.We defined log2foldchange>1 or<-1and the corrected p value<0.05 as significant.According to DEG analysis,ANGPT1,AGTR1,AGTR2 and ACE were foundsignificantly down-regulated in both LUAD and LUSC.The use of KMplotter(online database www.kmplot.com,a prognosis dataset including gene expression data and clinical data)was used to analyze the mRNA expression of REN,ANGPT1,ANGPT2,AGTR1,AGTR2,ACE and ACE2 genes on the prognostic of NSCLC,the OS of mRNA expression of ANGPT1(HR0.77(0.67-0.87,p=7e-05),ANGPT2(HR1.37(1.15-1.62,p=3e-04(HR0.72),AGTR1(0.63-0.83,p=1.3e-06),ACE(HR0.73(0.62-0.87,p=0.00035)had significant difference on prognosis of NSCLC.CONCLUSIONS:Compared with normal tissues,the expression of ANGPT1,AGTR1,AGTR2 and ACE in LUAD and LUSC is markedly decreased in lung cancer.The high expression of ANGPT1,AGTR1 and ACE had a better prognosis,and the prognosis of low expression of ANGPT2 was better.The expression of ANGPT1,AGTR1 and ACE may be a favorable factor for lung cancer,and the expression of ANGPT2 and AGTR2 may be a risk factor.Drugs may affect the prognosis of patients by acting on different targets.