The Mechanism Of STAT3 And ERR-? Interaction Promoting Migration And Invasion Of Triple-Negative Breast Cancer

Breast cancer ranks first in female malignant tumors,and distal metastasis is the main cause of death.Breast cancer has features of muti-subtypes and rapid onsets.Especially,triple negative breast cancer(TNBC)is lack of estrogen receptor(ER),progestogen receptor(PR)and human epidermal growth factor receptor 2(HER2),which causes TNBC could not benefit from endocrine and targeted therapy.TNBC has a high invasiveness,the worst total survival rate and the shortest disease-tree survival.From now,the Food and Drug Administration of the United States(FDA)has not approved any specific targeted drugs for TNBC and traditional chemotherapy is the most important treatment for-TNBC patients.Once metastasis and drug resistance occur,the prognosis of patients will become very poor.Therefore,in view of the characteristics of the occurrence and development of TNBC,it worth great work and meaning to find key targets and corresponding regulatory signals for the clinical diagnosis and treatment of TNBC.Estrogen-mediated biological effects play an important role in the occurrence and development of breast cancer.Recent studies have shou n that estrogen not only functions through the genomic effects of classical estrogen receptor alpha/beta,but also directly recognizes estrogen response elements(ERE),binds to estrogen and activates downstream signals through non-classical estrogen signals,like estrogen-related receptor alpha/beta/gamma and G protein-coupled estrogen receptor(GPER).Estrogen-related receptors(ERRs)are the first nuclear orphan receptors discovered.It is over-expressed in high energy-consuming or fast metabolic tissues such as embryo and reproduction.It has a DNA binding domain highly similar to estrogen receptor and can regulate invasion and metastasis of triple negative breast cancer through FN signaling pathway.Non-classical estrogen signaling may play an important role in TNBC lacking ER alpha/beta expression.Signal transduction and activator of transcription(STATs)are family of transcription factors activated by cytokines,growth factors and other polypeptide ligands,which have extremely important biological functions.STAT3 is one of the most important members of STATS family,and its expression intensity is closely related to tumor proliferation,invasion and metastasis.STAT3 has been recognized as an oncogene and has become an important target for cancer treatment and discovery of anticancer drugs.Epithelial-mesenchymal transition(EMT)is a key step in the invasion and metastasis of breast cancer.This process is manifested in cancers where,under mutation or external stimulation,closely arranged epithelial phenotypic cells transform into dispersed mesenchymal phenotypic cells,which detach from the primary lesion,enter the vascular endothelium and travel to the secondary lesion.The changing of molecular markers of epithelial-mesenchymal transition were decreased E-cadherin and increased N-cadherin and Vimentin.Based on tissue microarray and immunohistochemical method,we found that the expression of STAT3 in TNBC was significantly higher than that in non-TNBC and normal tissue.At the same time,we also found that estrogen-related receptor alpha(ERR-?)increased accordingly.Furthermore,we used Western Blot and real-time quantitative PCR to find that there was a positive correlation between the expression of STAT3,p-STAT3 and ERR-alpha in TNBC cell lines.Based on the above results,we speculated that STAT3 might be associated with ERR-a.Around this hypothesis,we carried out the verification work.Through the establishment of STAT3 cell lines with high expression and interference,ERR-a was found to be positively regulated by STAT3.In the cell lines with high STAT3 expression,the expression of ERR-alpha increased,and vice versa.In order to reveal which pathway causes STAT3 to affect the expression of ERR-alpha,JAK2-STAT3 signaling pathway inhibitor AG490 and stimulator IL-6 were used to process the signaling pathway.Western Blot,real-time quantitative PCR and immunofluorescence staining were used to detect the expression of related proteins and mRNA after treatment.The positive results confirmed the effect of this pathway on the regulation of ERR-a by STAT3.After confirming the positive correlation and regulatory relationship,we constructed a luciferase reporter gene plasmid stably transfected in the promoter region of ERR-a.Through the luciferase reporter gene detection experiment,we verified that STAT3 can regulate its expression by activating the promoter region of ERR-?.Furthermore,we found that phosphorylated STAT3 could bind to the promoter region of ERR-? in the nucleus and activate the expression of ERR-a by immunoprecipitation of chromatin.In order to determine the relationship between the signal pathway and invasion and metastasis abilities in TNBC,we constructed overexpressed STAT3 cell lines with stable interfered STAT3 cell lines to carry out invasion and metastasis related experiments.Transwell assay and wound healing assay showed that STAT3-ERR-?signaling pathway could improve the invasive and metastatic ability of TNBC,and the inhibition of this pathway could also reduce the invasive and metastatic ability of TNBC.At the same time,in order to determine whether it affects the invasive and metastatic ability of TNBC through the process of EMT,we detected the expression of key genes and proteins of EMT by Western Blot,real-time quantitative PCR and immunofluorescence staining.The results showed that STAT3-ERR-? signaling pathway could promote EMT,and inhibition of this pathway could also reduce the ability of EMT in TNBC.In conclusion,we conclude that p-STAT3 can bind to ERR-? promoter,region and regulate the expression of ERR-? through JAK2-STAT3-ERR-a pathway inTNBC.This pathway can also promote EMT and enhance its invasion and metastasis ability.This study revealed that STAT3 regulates ERR-alpha and its mechanism of affecting EMT and invasion and metastasis of TNBC.STAT3-ERR-? signaling pathway was found in triple-negative breast cancer.These results provide experimental basis and data for STAT3-ERR-? signaling pathway as a clinical target of TNBC.At the same time,it provides potential background for the screening and application of ERR-a inhibitors.