The Biological Functions And The Molecular Mechanisms Of FLI-1 In Breast Cancer

Breast cancer is the most common cancer in women,approximately accounting for 20% of all cancer cases.Breast cancer represents a highly heterogeneous disease.Invasion,recurrence and metastasis remain the main causes of deaths,and breast cancer is the second leading cause of cancer-related deaths in women.Although mounting evidence has emerged,the pathogenesis and molecular mechanisms remain unclear.In the previous work of our team,we found that FLI-1 played its role as an oncogene in the malignant hematological disease.The constant outpouring studies in recent decades have showed that FLI-1 also participates in the tumorigenesis of some solid tumors.However,only few has been known about the biological functions of FLI-1 and the molecular mechanisms in breast cancer.Thus,the main aim of the present study is to explore the correlation between FLI-1 and tumorigenesis,progression,invasion and metastasis,to discover the underlying molecular mechanisms,and furthermore,to provide the foundation for being capable to find more effective molecular target drugs and improve the prognosis of breast cancer.Research background: FLI-1(Fli-1 Proto-Oncogene)is a member of Ets(E26 transformation-specific)transcription factor family,which located in chromosome 11q24,was firstly identified in F-Mu LV-induced mouse erythroleukemia cells by Ben-David Y.FLI-1 is preferentially expressed in hematopoietic cells,vascular endothelial cells,embryonic tissues and certain kinds of cancer tissues.As a transcription factor,FLI-1 could play its biological functions by recognizing and directly binding to sequence-specific DNA of many promoters and enhancers at the transcriptional level,such as hematopoiesis and neoangiogenesis.What's more,it also can play its biological functions through protein-protein interaction,such as GATA1?BRCA1?SMAD3,etc.In our previous work,we found that FLI-1 could promote cell growth and inhibit cell apoptosis in erythroleukemia cell line,and play a role as a protooncogene.In the aspect of solid tumors,the studies about firstly Ewing sarcoma and later successively reported endometrial cancer,epithelial ovarian cancer,melanoma,nasopharyngeal carcinoma and glioblastoma,etc,discovered that ectopic expression of FLI-1 is closely associated with proliferation,invasion and metastasis phenotypes of tumors,in addition,poor overall survival rate.High expression of FLI-1 often indicates poor prognosis,which can be a potential target for therapy.The above research demonstrated that FLI-1 is closely related to progression and metastasis of tumors.However,the biological functions and its mechanisms of FLI-1 in breast cancer remain unclear and urgently need to further be researched.Research objectives: 1.To analyze the correlation between expression of FLI-1 and clinicopathological characteristics.2.To evaluate the biological functions of FLI-1 in breast cancer cells: proliferation,apoptosis,invasion,EMT and stemness traits.3.To explore the mechanisms,through which FLI-1 can play its biological functions.Research methods:(1)To analyze the expression of FLI-1 in breast cancer tissues with tissue microarray by using immunohistochemical staining method and to statistically analyze the correlation between FLI-1 and clincopathological characteristics in breast cancer;(2)To transiently intervene the expression level of FLI-1 with small interfering RNA(si RNA)and apply MTT method,cell number count,flow cytometry,transwell assay and plate colony-forming assay to evaluate the influence of FLI-1 on cell proliferation,cell cycle,cell apoptosis,migration and colony-forming abilities.(3)To construct overexpression plamid and virus packaging stable interference plasmid of FLI-1 to test the related markers of epithelial mesenchymal transition(EMT)by western blot,and further to evaluate the stem cell traits by sphere-forming assay and western blot;(4)To construct the stable interfering FLI-1 cell line of MDA-MB231,and to inject the mammary fat pad in situ of nude mice.On the level of animal,to observe the influence of FLI-1 on tumorigenesis.(5)To apply the molecular biological methods,such as GST pulldown,Co-IP(CoImmunoprecipitation),luciferase reporter assay,western blot and RTPCR methods,etc for exploring the underlying mechanisms.Research results: We conducted the research about the functions of FLI-1 from the histological,cellular,animal and molecular levels.1.FLI-1 was positively related with the AJCC stage,metastasis of lymphnode;obviously overexpressed in the HER-2 positive and TNBC subtypes and negatively related with the oversurvival(OS)and disease-free survival(DFS).2.FLI-1 expressed higher in metastatic breast cancer cell line,compared to the non-metastatic cell line;Interfering FLI-1 expression could inhibit the cell proliferation,make the cell cycle arrest at G0/G1,make the number of colony forming decreased and induce cell apoptosis.Knockdown of FLI-1 could attenuate the abilities of migration,invasion and metastasis.FLI-1 could induce the process of EMT and sustain the stemness traits of breast cancer cells.3.On the animal level,FLI-1 could significantly promote the ability of tumorigeneses in vivo.Proliferation index(Ki-67 index)can further support the above conclusion that FLI-1 can promote cell proliferation and tumorigenesis.4.On the one hand,FLI-1,as a transcription factor,could bind to the promoter of mi R-17-92 by its ETS domain,promote the proliferation,inhibit the apoptosis by activating mi R-17-92,and then influence the downstream target genes,P21 and Bim.On the other hand,FLI-1 could influence Rho GDI,and then activate Rho A,Rac1 and CDC42 of Rho GTPase signal pathway.FLI-1 could also influence AKT pathway.Research conclusions: 1.FLI-1 could serve as an independent prognostic factor of breast cancer.2.FLI-1 could promote cell proliferation,colony forming ability,invasion ability,stemness and induce EMT,and meanwhile inhibit apoptosis of breast cancer cells.3.FLI-1 could play its biological functions via more than one pathways.On the one hand,FLI-1 can upregulate the transcription activity of mi R-17-92 cluster to promote the proliferation and inhibit apoptosis,and on the other hand,participate the regulation of Rho A/Rac1 and influence the level of phosphate-AKT.In conclusion,this research discovered that FLI-1 participated in the tumorigenesis and progression of breast cancer and explored the underlying mechanisms,which provided detailed and reliable data for searching for new target drugs to reduce the metastasis and drug-resistance and to achieve the precise treatments.