The Role Of STAT5 In Breast Cancer Differentiation And Metastasis

Background:Breast cancer is a group of diseases in which cells in breast tissue change and divide uncontrolled.As the most common cancer in women around the world,approximately 1 in 8 women（13%）will be diagnosed with invasive breast cancer in their lifetime and 1 in 39 women（3%）will die from breast cancer.Besides family history,precancerous lesions（atypical hyperplasia and carcinoma in situ）and pathogenic genetic variations（e.g.BRCA1,BRCA2,PALB2,TP53,PTEN）,other breast cancer risk factors including lifetime exposure of breast tissues to hormones（early menarche,late menopause,obesity,and hormone uptake）,nulliparity,no lactation,radiation exposure of chest,hyperplasia are also factors that increase the risk for invasive breast cancer in women.What’s more,age,obesity,alcohol,tobacco and physical inactivity,which are risk factors for most cancers,also increase breast cancer risk.Although complicated,some crucial molecules in tumorigenesis are reported and can work as therapy targets.Currently,inhibitors targets HER2,CDK4/6,PARP and PIK3 are available for treatment of select patients with advanced disease.Signal transducers and activators of transcription 5（STAT5）belongs to the STAT family of transcriptional factors,which are related to cell proliferation,differentiation,apoptosis,and inflammation.STAT5 has two highly related isoforms,STAT5a and STAT5b,and it is the principal STAT expressed in the mammary glands at all stages of mammary development except during involution.Mouse lacks STAT5 is incapable for lactation.STAT5 is usually phosphorylated and activated by Janus kinase 2（JAK2）,which is activated following prolactin and other cytokines hormones binding to their cognate receptors.Phosphorylated STAT5 proteins then homodimerize（or sometimes heterodimerize with another family member）,translocate to the nucleus,and transactivate genes that regulate epithelial cells differentiation,lobuloalveolar formation and milk production.Aberrant STAT5 signaling is reported to be implicated in multiple cancer types,including breast cancer,prostate cancer,head and neck cancer and hepatocellular carcinoma.STAT5 can stimulate tumor formation in different cancers,including breast cancer.However,STAT5 shows a dual role in breast cancer.While STAT5 instigates breast cancer formation,it can promote the differentiation of tumors and higher p STAT5levels are correlated with better patient overall and disease-specific survivals.Objectives:In this study,we investigated the causal roles of STAT5a（the predominant isoform of STAT5）in alveolar and lactogenic differentiation during mammary tumorigenesis and progression in different mouse models of breast cancer and using cultured human breast cells in vitro and as xenograft.We also studied potential mechanisms and human patient implications.Our study aims to uncover a dichotomous function of STAT5 in breast tumorigenesis and metastasis.Methods:（1）Two pairs of viral vectors overexpressing oncogenes or STAT5a were amplified:retrovirus carrying constitutively activated Erb B2(RCAS-Erb B2^ca)and carrying constitutively activated Stat5a(RCAS-Stat5a^ca);lentivirus carrying PIK3CA mutant(FUCGW-PIK3CA^H1047R)and carrying constitutively activated Stat5a(FUW-Stat5a^ca).（2）By introductal injection,ductal epithelial cells of MMTV-tva（MA）mice were co-infected with and RCAS-Stat5^ca,and glands were collected 2 weeks post injection for precancerous lesions.（3）HER2+breast cancer mouse model was built by co-injecting MA mice introductally with RCAS-Erb B2^caand RCAS-Stat5^ca,and tumors were monitored by palpation twice a week.Tumors and lungs were collected when mice reached ethical endpoint.HE and IHC of tumor tissues were used for histological features and STAT5a^ca expression.HE of lungs were used to detect lung metastasis.（4）ER+breast cancer mouse model was built by injecting FVB mice introductally with FUCGW-PIK3CA^H1047R or co-injected with FUW-Stat5^ca,and tumors were monitored palpation twice a week.Tumors were collected when mice reached ethical endpoint.HE and IHC of tumor tissues were used for histological features and STAT5a expression.（5）Overexpress STAT5a in MCF-10A and MCF-7 human breast cancer cell lines:MCF-10A cell line was infected with FUCGW vector,FUW-Stat5^ca,FUCGW-PIK3CA^H1047R separately or co-infected with FUW-Stat5^ca and FUCGW-PIK3CA^H1047R.MCF-7 cells were infected with FUW-Stat5a or vector.All infected cells were selected by flow cytometry.（6）MCF10A-PIK3CA^H1047R cells with or without Stat5a^ca were transplanted and grow in NSG mice as xenograft by intraductal injection.Tumor size was monitored by IVIS-imaging.After mice reached ethical endpoint,luciferin signals of soft organs and bones were acquired by IVIS-imaging ex vivo after euthanasia.（7）IHC and IF staining were used to detect two differentiation markers,β-casein and PLIN-2 in precancerous and cancerous tissues.（8）Transwell assays were used to test the impact of STAT5a on the migration and invasion ability of MCF-10A and MCF-7 cells in vitro.（9）Reverse Phase Protein Array（RPPA）was used for protein profile to detect pathway signal changes induced by STAT5a overexpression in ER+breast cancer tissues.And the changes of EMT related markers were confirmed by western blotting and RT-q PCR in mouse ER+tumors and MCF-10A cell line.（10）Clinical data were downloaded from two public datasets,and survival R package was used to compare the prognosis between STAT5 m RNA high and low expression groups.Results:（1）The role of STAT5 in HER2+breast cancer tumorigenesisKiplan-Meier tumor-free survival curve showed that STAT5 activation stimulated HER2+tumor formation in MA mice.Besides,two anti-apoptosis molecules,BCL-x L and BCL-2 were higher in STAT5 overexpressed tumor tissues.So STAT5 stimulates tumorigenesis by suppressing apoptosis anticancer barrier.（2）STAT5 and differentiation of breast precancerous and cancerous tissuesSTAT5 overexpressed murine mammary glands with precancerous lesions,HER2+and ER+breast tumors were harvest.IHC and IF found that STAT5 activation increased the expression of two milk production markers,β-casein and PLIN-2,in precancerous lesions,HER2+and ER+tumors.STAT5 activation can induce the functional differentiation of breast precancerous and cancerous tissues.（3）The role of STAT5 in breast cancer metastasisUsing two breast cancer cell lines,MCF-7 and MCF10A-PIK3CA^H1047R,STAT5activation suppressed the transwell ability of both cell lines in vitro.In mouse models,lung metastasis was decreased in the STAT5 overexpressed group of HER2+breast cancer.Besides,IVIS imaging showed MCF10A-PIK3CA^H1047R can grow into tumors in the ducts of NSG mice,and STAT5 activation suppressed overall metastasis burden.Bioinformatic analysis showed that higher STAT5A m RNA expression was associated with better prognosis of breast cancer patients,especially among ER+breast cancer patients.RPPA data showed that slug and vimentin,two EMT key regulator/marker,were decreased by STAT5 activation in ER+breast cancer.Western blot and RT-q PCR confirmed that in both ER+tumor tissues and MCF10A-PIK3CA^H1047R cells,EMT was suppressed by STAT5a activation.So STAT5 restrains metastatic potential of breast cancer by suppressing EMT.Conclusions:（1）STAT5 stimulates tumorigenesis by suppressing apoptosis anticancer barrier.（2）STAT5 induces the functional differentiation of breast tumors and precancerous lesions.（3）STAT5 restrains metastatic potential of breast cancer by suppressing EMT.（4）STAT5 is a favorable biomarker in breast cancer prognosis.