Nuclear Galectin-1-FOXP3 Interaction Dampens The Tumor-suppressive Properties Of FOXP3 In Breast Cancer

BackgroundThe transcription factor FOXP3 is a member of the FOX protein family,which contains a characteristic DNA-binding forkhead(FKH)domain.As a specific marker of immunoregulatory T cells,FOXP3 plays an important role in the differentiation and development of Treg cells.However,in recent years,a series of studies have found that FOXP3 is also expressed in normal breast epithelial cells,and plays an important suppressive role in the progression of breast cancer.As a transcription factor,FOXP3 exerts its anti-cancer function and inhibits the growth and metastasis of breast cancer by regulating the expression of some oncogenes or tumor suppressor genes which are related to the development of breast cancer.It is reported that FOXP3 is usually mutant,absent,or cytoplasmic distribution in breast cancer cells,which increases the risk of breast cancer.However,in our study the full-length FOXP3 transcript can be detected in breast cancer cells and nuclear FOXP3 is expressed in some breast cancer samples.The results suggest that the tumor-suppressive function of wild-type FOXP3 is negated in some breast cancers.Therefore,an important question is how the tumor-suppressive function of FOXP3 is negated during the development of breast cancer.AimsThe purpose of this study is to systematically elucidate the causes and molecular mechanisms of the loss of anti-cancer function of wild-type FOXP3 in breast cancer through clinical sample analysis,in vitro cytological experiments and bioinformatic analysis,so as to provide a theoretical basis for gene therapy of breast cancer targeting on FOXP3.Methods1.Clinical breast cancer specimens were collected,and the expression of FOXP3 in them was detected by immunohistochemistry.2.Co-Immunoprecipitation(Co-IP)experiment,fluorescence resonance energy transfer experiment(FRET)and some other experiments were conducted to verify the interaction between FOXP3 and Gal-1 in breast cancer.3.Bioinformatics analysis was performed to mimic the interaction between FOXP3 and Gal-1,and to predict the interaction sites between FOXP3 and Gal-1.Truncated or mutant FOXP3 or Gal-1 plasmids were transfected into cells to determine the interaction sites between FOXP3 and Gal-1.4.FOXP3-MDA-MB-231 cells were transfected with Gal-1 overexpressing plasmid or control vector,and a genome-wide ChIP-Seq analysis was performed to determine whether the nuclear expression of Gal-1 affects the binding of FOXP3 to DNA throughout the genome.Real-time quantitative PCR and ELISA were performed to verify the ChIP-Seq results in transcription and translation levels.5.Breast cancer cells overexpressing Gal-1 or Gal-1 mutant were subjected to analyze the effect of the Gal-1-FOXP3 interaction on the tumor suppressive function of FOXP3 through xCELLigence RTCA and Transwell assay.6.FOXP3-positive breast cancer samples and paired adjacent normal tissues were collected to observe the expression and subcellular localization of Gal-1 in breast cancer cells by immunohistochemistry.7.3D culture of breast cancer cells was performed by adding lactose or/and acidic culture medium to mimic different extracellular conditions.Confocal assay was conducted to observe the expression and localization of Gal-1.8.Sialic acid concentration in breast cancer cell acidic culture medium was detected to explore the cause of high expression of Gal-1 in the nuclear.Results1.About 32.1%(53/165)of the breast cancer specimens exhibited nuclear expression of FOXP3.2.FOXP3 could interact with Gal-1 in breast cancer cells,and their interaction was mainly located in the nucleus.3.Deletion of the FKH region of FOXP3 or mutation of the N-terminal domain of Gal-1 greatly abolished the interaction between FOXP3 and Gal-1.4.In cells transfected with Gal-1,the overall recruitment of FOXP3 to promoter regions was lower than that in the cells transfected with empty vector.The enriched signal pathways related to the target genes of FOXP3 are mainly involved in cancer-related pathways,and these were almost abolished by co-transfection with Gal-1.5.The interaction between Gal-1 and FOXP3 attenuated the ability of FOXP3 to inhibit breast cancer cell proliferation and invasion.6.In breast cancer tissues,Gal-1 was mainly expressed in the nuclear of breast cancer cells,while in normal tissues it was mainly expressed in the cytoplasm orextracellular matrix.7.By immunofluorescence experiments,it was found that lactose can reduce the nuclear localization of Gal-1 in breast cancer cells,while breast cancer cell acidic culture medium prevented lactose from redistributing Gal-1 to the extranuclear space.8.Sialic acid was significantly increased in breast cancer cell acidic culture medium.Conclusion1.In this study,we systematically verify that FOXP3 can interact with Gal-1 in breast cancer;and it is the FKH domain of FOXP3 and the N-terminal of Gal-1 that mediate their interaction.2.FOXP3 exerts its tumor suppressive role mainly through its FKH domain,and the interaction of Gal-1 and FOXP3 “masks” this region,which results in the loss of FOXP3 function of regulating cancer related genes.3.The interaction between Gal-1 and FOXP3 affects the inhibitory function of FOXP3 on the growth and metastasis of breast cancer cells.4.The acidic microenvironment in breast cancer tissue may promote the localization of Gal-1 in the nucleus,and excessive Gal-1 in the nucleus can interact with FOXP3,which may affect the anti-tumor function of FOXP3.