Journal of Cancer Research and Immuno-Oncology
Open Access

In Silico Analysis of the FOXP3 Transcription Factor Associated with T-Cell Oncogenesis

Shouhartha Choudhury^{1,2,3* *}Correspondence: Shouhartha Choudhury, Department of Biotechnology, Assam University, Silchar, Assam, India, Email:

Author info »

Introduction

The genetic lesions of several autosomal tumour-suppressor genes intimates in the molecular pathogenesis of cancer. The cancer pathogenesis involves both inactivations of tumoursuppressor genes and activation of oncogenes. One of the most compelling aspects of cancer biology is the influence between caner-suppressor genes and oncogenes. The encoded protein of tumour-suppressor genes can inactivate oncogenes. Conversely, oncogenes may defeat the tumour-suppressor proteins. The epidemiology study suggested a role of X-linked genes controls the susceptibility of the cancers. The breakthrough of X chromosome encoded gene FOXP3 function in human leading autoimmune disorders. The FOXP3 gene function assigns critical novel insight into the biology of Treg and cellular mechanism of immune homeostasis. The immunosuppressive activity and regulatory T-cells (Treg) or CD4+ cells contribute to the progression of cancer and prevent the induction of specific immune response [1-3]. The regulatory T-cell has distinct lymphocyte lineage ability with an inhibitory property that affects the activation of the immune system. The FOXP3 gene characterizes by the specific forkhead box domain involved in the development of mammals [4,5]. The FOXP3 gene is a forkhead box DNA binding domain is conserved in evolution and consists of precise amino acid residues. The FOX (Forkheadbox) family contains 43 genes in the human genome [6]. A recent study suggested the FOXP3 gene expressed in normal breast, prostate, ovarian epithelium and also down-regulated expression in corresponding to the tumours [7-9]. The overexpression of FOXP3 gene reported in pancreatic adenocarcinoma [10], melanoma [11-13], hepatocellular carcinoma [14], leukaemia [15], bladder cancer [16], thyroid carcinoma [17] and cervical cancer [18] demonstrated critical question in the human cancer genome sequencing [19]. In contrast, the FOXP3 gene function control tumorigenesis by enabling tumour cells to lose tumour immunity. Thus previous reports supported the FOXP3 expression suppresses Treg proliferation in pancreatic carcinoma and melanoma cells [10, 13]. The FOXP3 gene expression in tumours suggested worse overall survival of the breast, bladder, and colorectal cancers [16, 20,21]. This study suggested either a pro-tumorigenic or antitumorigenic activity depend on clinical criteria. Numerous study in the animal model suggested that the inhibiting of Treg dramatically improves tumours and survival [22,23]. Treg plays a unique role in the prevention and development of effective autoimmunity. Therefore, the notable objective of this study is to evaluate the encoded FOXP3 gene that is immunogenic in nature and potential used as biomarker and target for immunotherapy. In this study, I reviewed the FOX family develops a novel therapeutics and preventives strategy in mammals. In contrast, FOXP3 gene functions play important roles in the biology of Treg and cellular mechanism of the immune homeostasis. One of the exciting developments in the cancer research findings prompted exploration of the fundamental genetic, immunogenic and molecular mechanism of the differentiation and function of Treg in oncogenesis.

Materials and Methods

Sequence and database

Primary sequence retrieves from the different specific database (UniProt, KEGG, GenBank, EMBL, DDBJ and NCBI), and performed web base application SMART for identification of the particular domain in the query sequence. Pfam searched for retrieving protein family information. SWISS-MODEL performs for the structure prediction. The SWISS-MODEL is structural bioinformatics web-server for comparative modelling of 3D structure. The most accurate method for generating rational 3D structure and routinely used in many practical applications. This method makes the experimental protein structure to a build model of evolutionarily related proteins. The SWISS-MODEL is a continuously updated database of homology or comparative model of the organism proteome for biomedical research. Genome: The genome sequences download from genomic data in different specialized databases (NCBI and Ensemble).

Standalone tools and GO annotation

HMMER executes using multiple sequence alignments of the specific domain as a profile search. HMMER is a statistical algorithm, making multiple sequence alignment (MSA) of the particular domain as a profile search and implemented methods using probabilistic models called the profile hidden Markov model. The standalone BLAST performs for homologs gene in selected organisms. The BLAST2GO performs for the gene ontology annotation, is a bioinformatics and computational tool for high-throughput gene ontology annotation of the particular sequence. The practical information of the genes retrieves via Gene Ontology (GO) annotation, a controlled vocabulary of the functional attribute.

Domain, motif, and phylogeny

Multiple sequence alignment (MSA) method for calculate the best match of the homologs sequences and line them up so identities, similarities, and differences can observe. MSA of highest hits sequence analysis carried out by web-based tool MultAlin for identification of conserved domain. The identification of the molecular evolutionary relationship of the specific gene in the Homo sapiens and Musmusculus, MEGA7 performed for constructing a phylogenetic tree using Neighbor- Joining Methods. The MEME suite retrieves for the sequence motifs is a computational web-based tool for discovery and analysis of specific motifs.

Gene expression and chromosome location

The expression analysis carried out using the GENEVESTIGATOR tool is a high-performance search engine of gene expression in different biological contexts. GENEVESTIGATOR use to identify and validate novel targets. Chromosome location retrieves using gene card is a database of the human genes provide genomic information of all known and predicted human genes. This database is currently available for biomedical research such as gene, encoded protein and associated disease.

Results

Structural Analysis

The primary sequence demonstrated the specific composition of nucleotides and peptides. The sequence composed of 1296 nucleotides and 431 peptides with 83 peptides binding to the DNA sequence is well-known as a forkhead domain (Table 1a and 1b). The peptide structure illustrated the forkhead domain involved in DNA binding. The forkhead domain also is known as “ winged helix ” domain. The 3D structure demonstrated alpha and beta proteins binds with B-DNA as a monomer interacts with the DNA backbone. The alpha helices assume a compact structure that presents in the third helix to the major grove. The protein comprises a twisted antiparallel beta structure and random coil that interact with the minor groove (Figure 1).

Table 1 (a): Primary Structure –Nucleotide.

Table 1 (b): Primary Structure -Peptide.

Figure 1: Tertiary structure (FOXP3).

Genome-wide Analysis

The genome-wide analysis by HMMER algorithm results shown that the multiple hits of 114 and 88 of the particular forkhead domains in Homo sapiens and Musmusculus respectively (Table 2). The standalone BLAST results represent 116 and 88 of homologs in Homo sapiens and Musmusculus, respectively (Table 2). Multiple hits selected from both organisms for gene ontology (GO) annotation (Table 3). The gene ontology annotation confirms that a total of 6 and 4 of the FOXP3 gene in Homo sapiens and Musmusculus respectively (Table 2).

Table 2: Summary of the (a) Forkhead domain and (b) FOX family

Table 3: Summary of the Gene Ontology annotation (a) Homo sapiens and (b) Musmusculus.

Analysis of the domain, motifs, and phylogeny

The highest hits of the FOXP3 gene listed from both organisms for sequence alignment, a multiple sequence alignment (MSA) determine the conserved domain (Figure 2). The high consensus (90%) sequence indicates the extended forkhead domain and their specific motifs (Figure 3a, 3b, and 3c). The phylogenetic tree demonstrated the molecular evolutionary relationship of the FOXP3 gene in between Homo sapiens and Musmusculus. Particular clades represent the multifunctional forkhead domain involved genes in both organisms (Figure 4).

Figure 2: Multiple sequence alignment (Forkhead domain).

Figure 3: Sequence motifs (a, b, and c).

Figure 4: Phylogenetic tree (FOX family).

Analysis of the gene expression and chromosome location

The gene expression analysis of four anatomical cancer categories shown that the low level of FOXP3 expression and express in neoplasm of secondary/ill-defined, malignant neoplasm, malignant melanoma (unstable behavior) is shown in Figure 5. The chromosome location study demonstrated that the FOXP3 located band Xp11.23, start 49,250,436 bp and end 49,270,477 bp is shown in Figure 6.

Figure 5: Gene expression(FOXP3).

Figure 6: Chromosome location(FOXP3).

Discussion

The immune system is a subject of the self and no selfdiscrimination both is directly fighting against the pathogens and maintains tolerance to self-antigens in mammals. Immune tolerance of the T-cells is achieving through central and peripheral strategies. The identification and characterization of regulatory T-cells (Treg or CD4+ cells) play unique roles in immune tolerance to self and transplant antigens [24-28].

Since the T-cell deficiency is a subject of lack of Treg in individual, it remains unknown the Treg function in the adult develop the immune system is not critical in the newborn. According to the genome, sequencing study suggested the elimination of Treg or CD4+ cells in the adult’s restively mild immune-mediated lesions and compression associated with congenital T-cell deficiency. In this study, my findings suggested the fundamental roles of FOXP3 gene dependent for differentiation and Treg function in the immune system and raise a question, how numerous inherent mechanisms prevent differentiation of Treg in the thymus and restrain their activity in the periphery to ensure self-tolerance.

FOXP3 gene considers as a master regulatory function in typically derived naturally occurring Treg (CD4+ cells). The FOXP3 express in Treg cells found in the major histocompatibility complex (MHC) class 2 restricted CD4+ cells and express high levels of CD25 (IL-2). An addition of FOXP3 also express CD4+ cells and CD25+ cells appears in minor histocompatibility complex (MHC) class 1 restricted CD8+ cells expression in Treg [29,30].

Therefore, the expression of CD8+ cells can kill tumour cells by the release of perforin and granzymes. The Treg also included Type 1 regulatory cells (Th1 or CD4+ cells), T helper 3 cells (Th3), CD8, CD28, and HLA-E restricted T-cells. The Treg function is associated with CD28 and CTLA-4 receptor molecules both potentially bind with two natural ligands such as CD80 and CD86 are capable of Treg-suppressive capacity [31].

The CD86 (B7-2) strongly enhances suppression by CD4+ CD25+ and Treg cells, the blocking of CD80 (B7-1) enhance proliferative responses by reducing Treg suppression. Comparative expression of CD80 and CD86 on dendritic cells regulates during the progression of abnormal to a normal state with the ability of Treg-suppressive responses. The CD80 and CD86 have rival functions through CD28 and CD152 (CTLA-4) on Treg that has remarkable effect for control of immune responses. The initiation of Treg cells and expression of immune checkpoint molecules includes CTLA-4, PD-1 (CD279), TIM-3, LAG-3, and TIGIT [32,33].

The prominent rule of PD-1 receptor is mainly express on T cells and interacts with their two ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) play roles in the initiation and maintenance of peripheral tolerance mechanism [34]. The PDL1 and PD-L2 express on antigen-presenting cells (APCs) and tumours release a negative signal to T cells, which is called T-cell exhaustion. Antibodies binding CTLA-4, PD-1 and PD-L1 have striking ability to enhance anti-cancer immunotherapy [35,36].

Therefore, Treg activity can evoke anti-tumour immunity and acknowledge inhibition of the T-cells function. The peptide inhibiter protein probably binds to the forkhead/winged-helix transcription factor 3 (FOXP3) which necessary for the development and function of Treg (CD4+ cells). The CD4+ cells produce transforming growth factor-beta and IL10 for suppressive function in the immune system and allow cell survival. The peptide P60 enter into the cells and inhibits FOXP3 nuclear translocation and reduces its ability to suppress the transcription factors NF-kB and NFAT both regulate transcription of DNA, cytokine production and cell survival. The functional evidence of the FOXP3 gene represses the expression of CD340, SKP2, SATB1 and MYC oncogenes and induces expression of tumour suppressor genes P21 and LATS2. The FOXP3 gene also pioneers of the anti-tumourenzymes such as CD39 and CD8 [37].

The functional inhibition of Treg by the FOXP3-inhibitory peptide initiates a strategy to enhance antitumor immunotherapies [3]. This study describes the significant advances of the FOXP3 gene as an X-linked tumour suppressor gene in between Homo sapiens and Musmusculus. This work represents a compelling exception and widely accepted theory of the inactivation of tumour suppressor genes. The oncogenes are a heterogeneous group that is generally expressed in the cells and trophoblast and also aberrantly activated in various cancers [38].

A subset of oncogenes encodes antigens that are immunogenic and elicit humoral and cellular immune responses in cancers [39]. Limited evidence supported the FOXP3 gene is a tumour suppressor gene. In this report, my finding suggested the expression of FOXP3 gene in cancer cells provide evidence that this could be important in tumour escape mechanisms. In this study, I discuss the multiple in-silico analysis and comprehensive genome-wide survey of the FOX family is involved in the development process in mammals. In contrast, the restricted expression of FOXP3 gene in the genome is an immuneprivileged and therapeutic strategy in cancers. The conserved domain, motifs, phylogeny, gene expression, and chromosome location analysis suggested the mechanism of the FOXP3 gene is conserved in evolution. The phylogeny analysis has apparent the large gene family consist of specific genes is closely related to each other.

Thus study suggested the FOXP3 gene acts as transcriptional activator and repressor and also targets makeup as a T-cell dependent gene. The fundamental contribution of the FOXP3 gene in tumour cells may represent a novel mechanism in the immune system. This study is consistent with the results of an in-silico analysis of FOXP3 gene target in T-cells. Therefore, my finding supported the genetic, immunologic and molecular mechanisms of the X chromosome encoded FOXP3 gene associated with T-cell oncogenesis.

Conclusion

My analysis data concluded that the FOX family of transcription factors developed therapeutics and preventives strategy in mammals. In contrast, the restricted expression of FOXP3 in the cell is an immune-privilege and therapeutic strategy. The ultimate transcriptional regulation of FOXP3 in tumour cells may represent a novel mechanism in the immune system.

Acknowledgement

The author is grateful to the editor and anonymous reviewers for valuable comments and suggestions. Also thanks to the Assam University, Silchar, Assam, India for providing the requisite lab facilities in carrying out this research work.

References

1. Zuo T, Liu R, Zhang H, Chang X, Liu Y, Wang L, et al. FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2. J Clin Invest. 2007;117(12):3765-3773.
2. Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity. 2005;22(3):329-341.
3. Casares N, Rudilla F, Arribillaga L, Llopiz D, Riezu-Boj JI, Lozano T, et al. A peptide inhibitor of FOXP3 impairs regulatory T cell activity and improves vaccine efficacy in mice. J Immunol.2010;185(9):5150-5159.
4. Wan YY, Flavell RA. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression. Nature. 2007;445(7129):766-770.
5. Williams LM, Rudensky AY. Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3. Nature Immunol. 2007;8(3):277-284.
6. Katoh M, Katoh M. Human FOX gene family. Int J Oncol. 2004;25(5):1495-1500.
7. Zuo T, Wang L, Morrison C, Chang X, Zhang H, Li W, et al. FOXP3 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene. Cell. 2007;129(7):1275-1286.
8. Wang L, Liu R, Li W, Chen C, Katoh H, Chen GY, et al. Somatic single hits inactivate the X-linked tumor suppressor FOXP3 in the prostate. Cancer cell. 2009;16(4):336-346.
9. Zhang HY, Sun H. Up-regulation of Foxp3 inhibits cell proliferation, migration and invasion in epithelial ovarian cancer. Cancer lett. 2010;287(1):91-97.
10. Hinz S, Pagerols-Raluy L, Oberg HH, Ammerpohl O, Grüssel S, Sipos B, et al. Foxp3 expression in pancreatic carcinoma cells as a novel mechanism of immune evasion in cancer. Cancer Res. 2007;67(17):8344-8350.
11. Ebert LM, Tan BS, Browning J, Svobodova S, Russell SE, Kirkpatrick N, et al. The Regulatory T Cell–Associated Transcription Factor FoxP3 Is Expressed by Tumor Cells. Cancer Res. 2008;68(8):3001-3009.
12. Quaglino P, Abate OS, Marenco F, Nardò T, Gado C, Novelli M, et al. FoxP3 expression on melanoma cells is related to early visceral spreading in melanoma patients treated by electrochemotherapy. Pigment Cell Melanoma Res. 2011;24(4):734-736.
13. Niu J, Jiang C, Li C, Liu L, Li K, Jian Z, et al. Foxp3 expression in melanoma cells as a possible mechanism of resistance to immune destruction. Cancer ImmunolImmunother. 2011;60(8):1109-1118.
14. Wang WH, Jiang CL, Yan W, Zhang YH, Yang JT, Zhang C, et al. FOXP3 expression and clinical characteristics of hepatocellular carcinoma. World J Gastroenterol. 2010;16(43):5502.
15. Kim MS, Chung NG, Yoo NJ, Lee SH. No mutation in the FOXP3 gene in acute leukemias. Leukemia research. 2011;35(1):e10.
16. Winerdal ME, Marits P, Winerdal M, Hasan M, Rosenblatt R, Tolf A, et al. FOXP3 and survival in urinary bladder cancer. BJU international. 2011;108(10):1672-1678.
17. Cunha LL, Morari EC, Nonogaki S, Soares FA, Vassallo J, Ward LS. Foxp3 expression is associated with aggressiveness in differentiated thyroid carcinomas. Clinics. 2012;67(5):483-488.
18. Zeng C, Yao Y, Jie W, Zhang M, Hu X, Zhao Y, et al. Up-regulation of Foxp3 participates in progression of cervical cancer. Cancer ImmunolImmunother. 2013;62(3):481-487.
19. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov. 2012;2(5):401–404.
20. Merlo A, Casalini P, Carcangiu ML, Malventano C, Triulzi T, Menard S, et al. FOXP3 expression and overall survival in breast cancer. J ClinOncol. 2009;27(11):1746-52.
21. Kim M, Grimmig T, Grimm M, Lazariotou M, Meier E, Rosenwald A, et al. Expression of Foxp3 in colorectal cancer but not in Treg cells correlates with disease progression in patients with colorectal cancer. PloS one. 2013;8(1).
22. Beyer M, Schultze JL. Regulatory T cells in cancer. Blood. 2006;108(3):804-811.
23. Zou W. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol. 2006;6(4):295-307.
24. Von Boehmer H, Kisielow P. Self-nonself discrimination by T cells. Science. 1990;248(4961):1369-73.
25. Jiang S, Lechler RI. Regulatory T cells in the control of transplantation tolerance and autoimmunity. Am J Transplant. 2003;3(5):516-524.
26. Sakaguchi S. Naturally arising Foxp3-expressing CD25+ CD4+ regulatory T cells in immunological tolerance to self and non-self. Nature Immunol. 2005;6(4):345-352.
27. Cobbold SP, Castejon R, Adams E, Zelenika D, Graca L, Humm S, et al. Induction of foxP3+ regulatory T cells in the periphery of T cell receptor transgenic mice tolerized to transplants.JImmunol. 2004;172(10):6003-6010.
28. Chung KY, Shia J, Kemeny NE, Shah M, Schwartz GK, Tse A, et al. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J ClinOncol. 2005;23(9):1803-1810.
29. Doherty PC, Knowles BB, Wettstein PJ. Immunological Surveillance of Tumors in the Context of Major Histocompatibility Complex Restriction of T cell Function. Adv Cancer Res. 1984;42:1-65.
30. Dhodapkar MV, Steinman RM. Antigen-bearing immature dendritic cells induce peptide-specific CD8+ regulatory T cells in vivo in humans. Blood. 2002;100(1):174-177.
31. Zheng Y, Manzotti CN, Liu M, Burke F, Mead KI, Sansom DM. CD86 and CD80 Differentially Modulate the Suppressive Function of Human Regulatory T Cells. J Immunol. 2004;172(5):2778-2784.
32. Dyck L, Kingston HG Mills. Immune checkpoints and their inhibition in cancer and infectious diseases. Eur J Immunol. 2017;47(5):765-779.
33. Nicholas L Syn, Michele W L Teng, Tony S K Mok, Ross A Soo. De-novo and acquired resistance to immune checkpoint targeting. Lancet Oncol. 2017;18(12):e731-e741.
34. Ohue Y, Nishikawa H. Regulatory T (Treg) cells in cancer: Can Treg cells be a new therapeutic target? Cancer Sci. 2019;110(7):2080–2089.
35. Ishida Y, Agata Y, Shibahara K, Honjo T. Induced expression of PD‐1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 1992;11(11):3887-3895.
36. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhof K, Arce F,  et al. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti–CTLA-4 therapy against melanoma. J Exp Med. 2013;210(9):1695–1710.
37. Hori S, Nomura T, Sakaguchi S. Control of Regulatory T Cell Development by the Transcription. Science. 2003;299(5609):1057-1061.
38. Scanlan MJ, Simpson AJ, Old LJ. The cancer/testis genes: review, standardization, and commentary. Cancer Immun. 2004;4(1):1.
39. Simpson AJ, Caballero OL, Jungbluth A, Chen YT, Old LJ. Cancer/testis antigens, gametogenesis and cancer. Nature Reviews Cancer. 2005;5(8):615-625.