Tumor suppressor and T-regulatory functions of Foxp3 are mediated through separate signaling pathways

Abstract

Abstract. Foxp3 is a nuclear transcription factor that is both a tumor suppressor factor and regulator of T-regulatory cell (Treg) function, and is a potential therapeutic target in both autoimmunity and cancer. In order to distinguish molecular pathways responsible for these separate Foxp3 functions, deletion mutants of Foxp3 proteins were transduced and analyzed for cytotoxic activity in human cancer cell lines Skov3, MDA-MB-231, MCF-7 and Jurkat. Human Foxp3 cDNA mutants were amplified and ligated to produce plasmids for direct cell transfection. Constructs were produced and confirmed by DNA sequencing. Lipofectamine 2000 was used for plasmid transfection. Foxp3 cells were then examined. The results of our experiments reveal retention of tumor suppressor function in the absence of NFAT binding and transcriptional activation required for Treg function. Our results have significant implications for the design of autoimmune and cancer therapies that target Foxp3 and Treg cells. IntroductionFoxp3 is an X-linked nuclear transcription factor essential to the development and program of T-regulatory cells (Tregs) that protect against the development of autoimmunity (1,2). The tumor suppressor activity of Foxp3 was identified when it was serendipitously observed that female mice heterozygous for the mutation in the X-linked scurfin gene (homolog to human Foxp3) have high rates of mammary cancer as well as other types of cancers (3). Additional studies confirmed a relationship between mutations of Foxp3 and breast cancer in humans, and identified Foxp3-mediated repression of HER-2/ ErbB2 and SKP2 oncogenes as a potential mechanism for the tumor suppressor effect (3,4). Although Tregs suppress the development of autoimmunity, they are thought to be permissive for the development of cancer (5,6). Thus, Foxp3 has dual but opposing roles with respect to autoimmunity and cancer. Tregs and Foxp3 have become significant therapeutic targets in autoimmunity and cancer. Defining Foxp3 pathways responsible for its divergent functions should be helpful in the design of therapies that specifically target Foxp3. Development of the Treg function requires transcriptional activation and binding of Foxp3 to the transcription factor, NFAT (7). In this study, Foxp3 was found to exhibit tumor suppressor activity independent of transcriptional activation and binding NFAT. Materials and methodsCell lines. Human cancer cell lines Skov3 (ovarian), MDA-MB-231 (breast), MCF-7 (breast) and Jurkat (T cells) were obtained from the American Type Tissue Collection (Rockville, MD, USA).Antibodies. The antibodies used included mouse monoclonal IgG1 to human Foxp3 (sc-56680) and mouse anti-goat IgG-HRP (sc-2354) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).Plasmid constructs. Human Foxp3 cDNA was amplified by PCR from plasmid 13250 (Addgene, Cambridge, MA, USA) and ligated into pIRES2-ZsGreen1 (Clontech, Mountain View, CA, USA) to produce the plasmid pIRES2-ZsGreen1-Foxp3, pIRES2-ZsGreen1-Foxp3C (C-terminal deletion of residues 328-431) and pIRES2-Zs...

Clinical immunology Examination of correlation between vitamin D3 (25-OHD3) concentration and percentage of regulatory T lymphocytes (FoxP3) in children with allergy symptoms

“…Recent studies have shown that there is subpopulation of T cells called regulatory cells (Tregs) responsible for the strength and scope regulation of the immune system response. Numerous studies have demonstrated that an increased Tregs subpopulation percentage reduces the sensitivity of the immune system to external or changed in the disease process antigens, which allows the growth of tumors [21,22]. On the other hand decreased Tregs percentage increases the strength of immune response, which may lead to chronic inflammatory diseases and autoimmune diseases [23][24][25].…”

Searching for Peptide Inhibitors of T Regulatory Cell Activity by Targeting Specific Domains of FOXP3 Transcription Factor

“…FOXP3 has various distinguishable functional domains: (i) an N-terminal domain (from a.a. 1 to 193) responsible for transcriptional repression, (ii) a zinc finger (a.a. 200–223), a leucine-zipper (LZ)-like motif (a.a. 240–261), which facilitates the formation of FOXP3 homo-dimers or tetramers, and (iii) the highly conserved carboxy terminal forkhead domain (FKH; from a.a. 338 to 421) responsible for the DNA binding [ 30 ]. It has been described that the tumor suppressor activity of FOXP3 is located in the N-terminal region of the protein (Aa 1-196) [ 31 ]. In a recent work, Gong et al have shown that the ectopic expression of FOXP3 inhibited hepatocarcinoma cell line (HCC) proliferation, migration, and invasion, while FOXP3 downregulation promoted HCC growth [ 32 ].…”

Searching for the Achilles Heel of FOXP3

“…It has been described that this tumor suppressor activity is located in the N-terminal region of Foxp3, since a C-truncated versions of the protein do not retain this property (54). The authors described that this activity was independent of NFAT-FOXP3 interaction which is located in the FKH region of FOXP3 (55).…”

“…As mentioned above, FOXP3 expression is not restricted to the lymphocyte lineage but it is also present in cancer cells, especially in breast cancer cells, where it has been demonstrated to be a cancer suppressor gene and an important regulator of the HER2/ErbB2 and SKP2 oncogenes ( 27 , 30 ). It has been described that this tumor suppressor activity is located in the N-terminal region of Foxp3, since a C-truncated versions of the protein do not retain this property ( 54 ). The authors described that this activity was independent of NFAT-FOXP3 interaction which is located in the FKH region of FOXP3 ( 55 ).…”