Molecular Epidemiological Study On The Genetic Variation Of FoxA1 Binding Site To The Risk And Prognosis Of Breast Cancer

Breast cancer(BC)remains a global public health issue and the most common cancer in females worldwide.Genetic factors play an important role in the development of breast cancer.Remarkably,BC risk-associated SNPs identified by GWAS were enriched in the binding sites for the transcription factors FOXA1 and estrogen receptor 1,which suggested FOXA1 contributes to BC development by regulatory mechanisms related to estrogen receptor(ER)signaling.In this paper,we used bioinformatics analysis,hospital-based two-stage case-control study and molecular biology functional experiments,to do a deep exploration on the relationship between genetic variation of FoxAl binding site and the risk or prognosis.In the first part,we explore the effect of the SNP(which disrupts the recruitment of FOXA1 and associated with elevated BC risk in Europeans)on the development and progression of breast cancer in Chinese population.The results are negative,suggesting a distinct association related to genetic heterogeneity across ethnic populations;So in the next,we screening the FoxAl binding region of the whole genome,to find the potential functional genetic variation,which is associated with BC risk in Chinese population,and conduct biology experiments to confirm it’s molecular mechanism;In the second part we studied the impact of SNPs which modulating FOXA1-binding affinity on neoadjuvant chemotherapy prognosis,which gives a new thought to our individual breast cancer prevention.Part I Molecular epidemiological study on the genetic variation of FoxAl binding site to the risk of breast cancerObjective:To excavate and identify the functional variants which modulating FOXA1-binding affinity,and to explore the genetic predisposition of Chinese BC etiology in post-GWAS era.Methods：1.A hospital-based case-control study of 1227 breast cancer patients and 1285 control subjects was performed to expore the relationship between rs4442975 which modulating FOXA1-binding affinity and BC risk or progression in Chinese population.All subjects in this study are female and unrelated ethnic Han Chinese.The genetic variant rs4442975 was genotyped by TaqMan SNP Genotyping Assay.The odds ratios(ORs)and their 95%confidence intervals(CIs)were calculated by unconditional multivariate logistic regression analysis to estimate the association between genotypes and BC risk.Stratified analyses were performed based on estrogen or progesterone receptor expression,smoking or drinking habits,and menopausal status.Associations between rs4442975 and traditional progression factors such as tumor size,nodal status,distant metastasis,and TNM staging were observed.2.By step-wise bioinformatics analyses,we screened out the potentially functional variants in the FoxAl binding region of the whole genom.Then we performed a two-stage case-control study to investigate the associations between candidate SNPs and BC susceptibility in chinese population,with a total of 2164 BC cases and 2382 controls.Furthermore,we verified the biological functionality of the positive variants with EMSA,dual luciferase reporter gene assays,siRNA knockdown,tissue samples of e-QTL experiments and TCGA data analysis.Results:1.No significant associations between rs4442975 and BC risk were observed under any genetic models,with an odds ratio of 0.96(95%confidence interval =0.81-1.15,P=0.678)under the additive model.When stratified based on estrogen or progesterone receptor expression,smoking or drinking habits,or menopausal status,similar negative associations were observed for all subgroups.No significant associations were observed between rs4442975 and traditional progression factors under the additive model:tumor size(OR = 1.06,P = 0.823),lymph node involvement(OR =1.01,P = 0.933).distant metastasis(OR = 0.68,P = 0.154),and advanced TNM stage(OR = 0.95,P = 0.765).A similar negative result was observed in the dominant model.2.Four candidate genetic variants with potential function were chosen after the bioinformatics analyses.A risk SNP rs6506689 was found in the first and confirmed in the second stage.It was found that rs6506689-T could increase the risk of breast cancer in Chinese population,with additive mode OR =1.18(95%CI = 1.08-1.30,P=0.001).Stratified analyses also found that compared with individuals carrying wild homozygous GG genotype,the risk of breast cancer is more likely to increase in ER-positive TT genotype patients(OR = 1.43,95%CI = 1.13-1.79,P = 0.002)than in ER-negative TT genotype patients(OR = 1.33,95%CI = 0.99-1.78,P = 0.057).3.EMSA results showed that the DNA containing rs6506689-T allele might be more capable of binding to a transcription factor;Dual luciferase reporter assay indicated that compared with rs6506689-G,the minor allele rs6506689-T could significantly increase the transcriptional activity in two BC cell lines Mcf-7 and T47D;And the difference between the transcriptional activities of different alleles disappeared after siRNA knockdown on FoxAl;The cis-e-QTL analysis of TCGA data,circular plot anlysis by Hi-C,and the experiment on 60 peritumor tissues of breast cancer in China confirmed that the expression of RAB31 gene was associated with the polymorphism of rs6506689(P = 0.010),and the expression of RAB31 gene was higher in individuals with the risk genotype TT;Compared with peritumor tissues in TCGA,the expression of RAB31 and Fox A1 was significantly higher in breast cancer tissues(P<0.001).Conclusions:1.Our results reveal that European risk variant rs4442975 may not confer a risk of BC occurrence or progression in Chinese Han population,which indicates a distinct association related to genetic heterogeneity across ethnic populations.2.By the integration of bioinformatics analyses,two-stage case-control association study and biological function experiments,the functional variant rs6506689 which located in the FoxAl binding site loci,was found to be associated with BC risk in Chinese population.It was suggested that rs6506689 regulated the expression of the target tumor gene RAB31 by altering the binding of transcription factor FoxAl,and consequently influence the susceptibility of BC.Part Ⅱ The relationship between SNPs which modulating FOXA1-binding and the prognosis of neoadjuvant chemotherapy for luminal A subtype breast cancerObjective:Aim to expore the correlation between genetic variation which modulating FOXA1-binding affinity and neoadjuvant chemotherapy prognosis of luminal A subtype breast cancer in Chinese population,hoping to find some molecular markers of individualized treatment.Methods:The cases of luminal A subtype breast cancer was treated with anthracycline(epirubicin)and taxane(docetaxel).The genotype of candidate genetic variants was completed by TaqMan technique.Unconditional logistic regression analysis was used to estimate the association between genotypes and neoadjuvant chemotherapy prognosis.Results:1.175 patients with luminal A subtype breast cancer who underwent neoadjuvant chemotherapy were included in the study,unconditional logistic regression analysis showed no association between SNPs and neoadjuvant chemotherapy efficacy,with rs4442975 additive model OR was 0.73(95%CI=0.27-1.94,P= 0.527),and rs6506689 was 0.80(95%Cl = 0.43-1.47,P = 0.464).2.The association analysis showed that rs4442975 was associated with the toxicity of myelosuppression.The additive model suggested that the risk of myelosuppression was reduced by 62%for each additional mutation allele(OR = 0.38,95%CI = 0.18-0.69,P = 0.003).There was no correlation between rs6506689 and myelosuppression or gastrointestinal toxicity(OR = 0.89,95%CI = 0.56-1.41,P ＝0.611 and OR = 0.92,95%CI= 0.58-1.44.P = 0.707).Conclusions:The functional variant rs4442975 which modulating FOXA1-binding affinity may be related to the myelosuppressive effect of neoadjuvant chemotherapy of luminal A subtype breast cancer in Chinese population,but its specific biological function mechanism needs to be further explored and verified.