Role Of WT1in The Progression Of Breast Cancer And Its Mechanism

Breast cancer is the most common malignant tumor in women, with the highestmortality. There are1.3million people newly diagnosed as breast cancer and450,000diedof it each year worldwide. Invasion and metastasis is a major biological characteristic ofmalignant tumors, and the leading cause of death, but its process and mechanism is notclear so far. Therefore, it is necessary to further study breast cancer invasion and metastasismechanism and to explore effective regimen of disease prevention and control, which ishelpful to improve the survival and quality of life for breast cancer patients.The occurrence, invasion and metastasis of malignant tumors are multi-level,multi-factor mediated, and the imbalance of oncogenes and tumor suppressor gene plays akey role in the process. So searching for new genes or further revealing the functions ofexisting oncogene or tumor suppressor gene is important to demonstrate the developmentmechanism of malignancy and investigate the potential therapeutic targets.Wilms' tumor gene1, WT1, first identified as a tumor suppressor gene innephroblastoma, located at11p13. WT1encoding product is a zinc finger transcriptionfactor with double functions of transcription activation and inhibition. During the physicaldevelopment of human body, WT1regulates multiple target genes and signaling pathways,so as to be involved in the formation of the heart, kidneys, spleen and other organs. Theprevious study shows that the abnormal expression of WT1in adult tissues is closelyrelated to many kinds of malignant tumors, but it may act as oncogene or tumor suppressorgene depending on the different cellular characteristics. On one hand, in the childhoodmalignancies such as Wilms' tumor, WT1can inhibit cell growth by interfering cellproliferation signals, which can be regarded as tumor suppressor genes; on the other hand, WT1can enhance tumor cell resistance to apoptosis, and promote cell proliferation throughregulation of proto-oncogene K-ras, suggesting that WT1can also play an oncogenic role.It is previously reported that WT1is highly expressed in breast cancer tissue, but notexpressed in adjacent tissues, indicating that WT1acts as an oncogene in breast cancer, butits mechanism remains unclear, especially on the issues whether it is involved in breastcancer invasion and metastasis, as well as the specific role and molecular mechanisms. Upto now, there is no definite conclusion yet on the relation of WT1expression with theclinical and pathological features and prognosis of breast cancer patients, especially on thedifference of WT1expression in different molecular subtypes of breast cancer. In addition,up-stream mechanism of WT1expression regulation is largely unknown.This study performed bioinformatics analysis of the microarray data, which have beenpublished, to clarify the relationship between WT1mRNA and breast cancer molecularsubtypes and patients' prognosis; detected WT1protein expression in breast cancer tissueby immunohistochemistry (IHC) method and analyzed its relationship with clinicalpathological features. Then we applied RNA interfering (RNAi) technology todown-regulate WT1expression, RNA activating (RNAa) technology to up-regulate WT1expression, and accordingly analyzed the role of WT1in proliferation, apoptosis, cell cycle,invasion and migration of breast cancer cells. We also explored the molecular mechanismsof WT1's biological function, with the key molecule of the cell proliferation and metastasis–inhibitor of differentiation1(Id1) as target gene. In addition, we established aepithelial-mesenchymal transition (EMT) cell model to study the regulation of WT1bytransforming growth factor β1(TGF-β1), and a meta-analysis on the relationship betweenTGF-β1single nucleotide polymorphisms (SNPs) and onset risk of breast cancer wasconducted. By a series of experiments, this study explored the role of WT1in thedevelopment of breast cancer and its mechanism, providing experimental references forfurther study on biological behavior of WT1in malignant tumors, and new perspective onthe research and development of molecular targets in malignant tumors.Methods&Results1. Relationship between WT1and clinical and pathological features and prognosis of breast cancerMETHODS: The GSE21653microarray data involved in266breast cancer patientswere processed using bioinformatics method to analyze the relationship of WT1mRNAexpression with molecular subtypes and prognosis of breast cancer. Immunohistochemicalstaining was used to detect WT1protein expression in46cases of breast cancer tissues, andits relationship with clinical pathological features was analyzed.Results: In GSE21653, WT1mRNA expression was significantly increased in breastcancer patients with higher histological grading (G2vs. G1, P=0.0260; G3, vs. G1, P=8.04e-5), ER-negative (ER-negative vs. ER-positive, P=0.0500), Basal-like type andERBB2/HER-2overexpression type (Basal-like type vs. Luminal type A, P=0.0049;Basal-like vs. luminal-type B, P=0.0350; ERBB/HER-2overexpression type vs.luminal-type, P=0.0350). Univariate analysis showed that the disease-free survival wassignificantly lower in patients with WT1mRNA high expression than in patients with WT1mRNA low expression (hazard ratio [HR]=3.294,95%confidence interval [CI]=1.198-9.055, P=0.014). The multivariate analysis showed that WT1is an independent poorprognostic factors for breast cancer patients (HR=3.4539,95%CI=1.2029-9.918, P=0.0213; the patients with WT1mRNA high expression vs. the patients with WT1mRNAlow expression). In46cases of breast cancer, the WT1protein expression level wasdetected by immunohistochemistry. The positive expression rate of WT1protein in breastcancer was84.8%(39/46). There are no significant differences among WT1proteinexpression and other clinicopathological parameters of breast cancer (P＞0.05).2. Role of WT1in the proliferation, apoptosis, invasion and migration of breastcancer cells and its possible mechanismMethods: qRT-PCR, western blotting (WB) and immunocytochemical methods wereused to detect the expression of WT1in human breast cancer MCF-7, Bcap-37, SKBR-3and MDA-MB-321cells. The cells with WT1high expression and low expression werescreened. Three sequences established by foreign scholars were adopted to construct WT1small interfering RNA (siRNA); one sequence domenstrated to be able to up-regulate WT1expression was adopted to construct WT1double strands RNA (dsRNA). Cells were liposome-mediated transfected; qRT-PCR and western blotting were performed to selectsiRNA and dsRNA with obvious evidence of impacts on WT1expression. Using CCK8,Annexin V/PI and transwell methods, the proliferation, apoptosis, cycle, invasion andmigration of breast cancer cells after interfering WT1expression were observed. The effectof interfering WT1on Id1expression in cells was determined by WB.Results: The WT1expression in MDA-MB-321cells was significantly higher thanthat in MCF-7, Bcap-37and SKBR-3cells, so MDA-MB-231cell was taken as RNAimodel and MCF-7was taken as RNAa model. Three WT1siRNAs were successfullyconstructed and transfected into MDA-MB-231cells, which can effectively inhibit theexpression of WT1mRNA and protein in48h after transfection. Interfering WT1expression can decrease MDA-MB-231cell proliferation, so the proportion of apoptoticcells,cells in G1phase increased and cells in S and G2phase decreased, and the invasionand migration ability of the cells reduced, which may be attributed to the down-regulatedexpression of Id1. WT1dsRNA could increase the expression of WT1in MCF-7cell andpromoted the proliferation, invasion and migration of MCF-7cell.3. Functions of TGF-β1up-regulating WT1in breast cancer, and meta-analysison the relationship between TGF-β1single nucleotide polymorphisms and onset risk ofbreast cancerMethods: TGF-β1was used to induce WT1expression in MCF-7cell with10ng/mlfor48h, which was detected by qRT-PCR and WB. Using transwell methods to assessinvasion and migration of MCF-7cell after treatment of TGF-β1. The literature inMEDLINE, PubMed, Web of Science, EMBASE, CNKI and Articles, VIP and biomedicalservice system (SinoMed) database up to March2010was retrieved on the relationshipbetween TGF-β1T869C as well as TGF-β1C-509T and breast cancer risk. According tothe inclusion criteria, the effective data were extracted and then meta-analysis wasconducted using STATA software.Results: TGF-β1could result in the morphologic change of MCF-7cell, chaning fromepithelial-like to mesenchymanl-like. In addition, TGF-β1could up-regulate the expressionof WT1in MCF-7cell after treatment, and enhance the ability of invasion and migration. TGF-β1T869C is independent of the overall risk of breast cancer in general population (Cvs. T: OR=1.033,95%CI=0.996-1.072), but subgroup analysis showed that TGF-β1T869C is related to the risk of breast cancer in Caucasians (C vs. T: OR=1.051,95%CI=1.018-1.085; CC vs. TT+TC: OR=1.083,95%CI=1.019-1.151); TGF-β1C-509T hasnothing to do with the risk of breast cancer in general population (T vs. C: OR=0.986,95%CI=0.936-1.039).Conclusions1. WT1play an oncogenic-like role in the progression of breast cancer, promoting theproliferation, invasion and migration and increasing the degree of malignancy of breastcancer cells, which are based on the following evidences:①The WT1mRNA expression issignificantly increased in the breast cancer with higher histological grade, ER-negative andBasal-like type, and HER-2overexpression and WT1is an independent factor for poorprognosis of breast cancer patients.②Interfering WT1expression can decrease theproliferation, invasion and migration of MDA-MB-231cell, while up-regulating WT1expression can increase the proliferation, invasion and migration of MCF-7cell.2. TGF-β1might be the up-stream molecular for WT1because it could up-regulatethe expression of WT1MCF-7cell and promote its invasion and migration. Id1might thedown-stream molecular of WT1because WT1RNA interfering could down-regulate theexpression of WT1MDA-MB-231cell and inhibit its invasion and migration.3. TGF-β1T869C SNP is related to the high risk of breast cancer in Caucasians,which indicated that detection of TGF-β1SNP might contribute to the screening andprevention of breast cancer.