FOXP3 regulatory T cells on prognosis of breast cancer

1. Introduction

Cancer is a disease caused by uncontrolled cell growth and proliferation. Breast cancer is a heterogeneous disease. This cancer is one of the most common female malignant tumors and the leading cause of death for women worldwide. Invasive breast cancer affects 1 in 8 women in the United States (12.4%) during their lifetime. In the United States, approximately 266,120 women will develop invasive breast carcinoma in 2018, and 63,960 will develop breast cancer in situ. About 40,920 US women died in 2018 from breast cancer. Based on data from the American Cancer Society (2019), around 41,760 women are estimated to die from breast cancer in 2019. Based on data from the World Health Organization (WHO) in 2020 also shows that the most common cancer cases in Indonesia are breast cancer, which is 58,256 out of a total of 384,809 cases of cancer [1,2].

The incidence of breast cancer increases with age, from 1.5 cases per 100,000 in women aged 20–24 years to a peak of 421.3 cases per 100,000 in women aged 75–79 years; 95% of new cases occur in women aged 40 years or older. The mean age of women at the time of breast cancer diagnosis is 61 years [2].

The immune response to tumors is very complex; immune cells can inhibit tumor growth (Immuno Immuno editing) and promote tumor cell growth and angiogenesis. Immuno-editing consists of 3 phases: the elimination phase, the equilibrium phase, and finally, the escape phase. The presence of Tumor infiltrating Lymphocytes (TIL) in tumors is known to give a better prognosis. Lymphocyte infiltration includes T cells, Tregulatory (Treg), and Natural Killer (NK) cells. Tregs play a role in preventing autoimmune diseases but can also inhibit anti-tumor immunity and promote tumor growth. Tregs also play a role in inhibiting cytotoxic T lymphocyte cells by inhibiting the release of granules from CD8⁺, where CD8⁺ is important in killing tumor cells. FOXP3 is a Treg-specific biomarker and plays an important role in the development and function of Tregs [3–5].

T regulatory cells suppress a variety of immune responses to self-antigens and play a role in peripheral tolerance maintenance by limiting autoimmune disorders, and other pathological immune responses such as limiting immune reactivity to oncoprotein encoded antigens. Forkhead box P3 (FOXP3) expression is required for Treg stability and affects functional activity [6].

Mutations in the master regulator FOXP3 and related components have been linked to human autoimmune diseases, such as IPEX, and a scurfy-like phenotype in mice. Several lines of evidence indicate that Treg uses a variety of immunosuppressive mechanisms to limit an immune response by targeting effector cells, including secretion of immunoregulatory cytokines, granzyme/perforin-mediated cell cytolysis, metabolic perturbation, directing the maturation and function of antigen-presenting cells (APC) and secretion of extracellular vesicles for the development of immunological tolerance [7,8].

This review discusses the FOXP3 Treg and breast cancer prognosis which is a health problem with low survival in women in the world, especially in developing countries.

2. Materials and methods

The literature study method was used, both from primary and secondary libraries. The library search was conducted using online-based search instruments such as NCBI-PubMed, Google Scholar, and Elsevier.

This scoping review was conducted in five stages, including developing research questions, searching and extracting related articles, selecting relevant studies, tabulating and summarizing data, and reporting results.

3. Research question development

The main research questions addressed in this review study are as follows:

“How FOXP3 Regulatory T Cells in Breast Cancer?”

4. Extraction of articles that met the inclusion criteria

The following are the inclusion criteria for this structured review: clinical trials and quasi-experimental studies with control and intervention groups published from 2017 to 2022, studies with specified sample sizes, articles focusing on the Effect of FOXP3 Regulatory T Cells on Breast Cancer (no pharmacological interventions were included in this study), and studies describing intervention outcomes. It should be noted that articles that did not meet the inclusion criteria, full-text studies in languages other than English and Indonesian, and summaries of articles presented at conferences that did not have full-text and case-control or cross-sectional studies were excluded.

5. Related article search and extraction

The researchers used the following keywords or their English equivalents in their search strategy for this study: Breast cancer, The immune response to tumors, T regulatory cells, Forkhead box P3 (FOXP3), FOXP3 is a Treg-specific biomarker and plays an important role in the development and function of Tregs.

The search was performed on international (Google Scholar, Science Direct, Web of Science, Cochrane Library, Springer, PubMed, Scopus, and Elsevier) and national databases (Scientific Information Database). Moreover, the references of the retrieved articles were manually searched in journals to extract studies on related topics.

After the search was completed, the researcher prepared a list of articles and then studied and reviewed the articles at each stage of screening the title, abstract and full text of the related studies.

6. Results

During the initial screening and search phase of articles, 86 related articles were obtained. After that, the full texts of 77 articles were examined, and 30 studies were deleted because they did not meet the inclusion criteria. Finally, Sixteen articles were included in this review.

7. Discussion

Breast cancer is perhaps one of the most studied cancer types in the tumor microenvironment (TME) context. Although survival rates for breast cancer patients are steadily increasing, it is still the leading cause of cancer-related deaths in women worldwide (Bray et al., 2018, DeSantis et al., 2019). The vast majority of breast cancer-related mortality is due to the incurable metastatic stage of the disease. Understanding, preventing, and treating metastatic breast cancer are unmet needs. As such, mechanistic insights into the complex interactions of key players in the TME could pave the way for novel, innovative treatments and improved patient stratification [9,10].

Breast carcinoma, like other types of cancer, is a clonal proliferation of epithelial cells due to multiple genetic changes due to hormonal exposure and the presence of inherited susceptibility genes. T regulatory cells (Tregs) are immunosuppressive subsets of CD4⁺ T cells, characterized by the expression of the master regulatory transcription factor, Forkhead box protein P3 (FOXP3), which plays an indispensable role in regulating Treg differentiation and development. Tregs orchestrate cellular and molecular networks to induce an immunosuppressive environment favoring tumorigenesis. Tregs within the tumor microenvironment (TME) are highly activated and immunosuppressive, characterized by upregulated levels of FOXP3 and Helios. Tregs exert suppressive activities on effector cells, such as T effector cells (Teffs), natural killer (NK) cells, monocytes/macrophages, and antigen-presenting cells (APCs), via various mechanisms leading to the induction of apoptosis and inhibition of effector cell activation/proliferation. These mechanisms include increased consumption of IL-2 and Teff deprivation, the release of high levels of interleukin (IL)-10, IL-35, transforming growth factor-beta (TGF-β), granzyme B, and perforin. Upregulated levels of inhibitory immune checkpoints (ICs), such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), programmed cell death-1 (PD-1), lymphocyte activation gene-3 (LAG-3), T-cell immunoreceptor with Ig and ITIM domains (TIGIT) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3). Tregs constitute 5–10% of circulating CD4⁺ T cells in healthy individuals. At the same time, they represent significant levels of circulating CD4⁺ T cells in the peripheral blood of cancer patients, including those with lung, ovarian and gastric cancers and melanoma. In cancer patients, the frequency of circulating Tregs increases by approximately 2-fold, compared to healthy individuals.

Additionally, increased tumor-infiltrating Tregs have been positively correlated with poor prognosis and low survival rates in various cancer patients, including those with lung, ovarian, gastric, breast, and pancreatic cancers, head and neck squamous cell carcinoma (HNSCC), and melanoma. In solid tumors, the predictive value of tumor-infiltrating lymphocytes (TILs) is one of the most important aspects, reflecting the existing immune response’s ability to eradicate tumor cells. It predicts the clinical response to a particular cancer therapy. The association between patient survival and the presence of TILs has been demonstrated in many tumor types. Increased intratumoral CD8⁺ T cell infiltration has been associated with increased anti-tumor responses and better clinical outcomes following therapy. However, higher ratios of FOXP3⁺ Tregs: CD8⁺ T cell within the TME contributes to tumor progression and immunosuppression. Additionally, FOXP3⁺ Tregs play a central role in tumor progression and tumor progression and the development of tumor resistance to immunotherapies, such as immune checkpoint inhibitors (ICIs), in tumor progression and poor clinical outcomes. Therefore, targeting Tregs alone or in combination with ICIs could be beneficial in treating cancer, overcoming Treg-mediated tumor resistance, and maximizing the therapeutic efficacy and immune responses in cancer patients [4,11,12].

8. FOXP3 and treg cell development

Treg cells were not recognized as a distinct lineage of T cells until the identification of FOXP3, an X-chromosome encoded forkhead transcription factor family member, whose high expression level defines Treg cells in experimental animals and humans. Loss-of-function mutations in the FOXP3 gene in humans result in the IPEX syndrome, and similarly, in mice, the spontaneous FOXP3 mutant scurfy model is characterized by fatal, multi-system autoimmunity. In humans, FOXP3 is highly expressed in suppressive CD25⁺CD4⁺ T cells. However, activated conventional T cells also upregulate Foxp3, but only transiently and significantly lower [9,13].

Expression of FOXP3 serves as a dominant regulatory pathway in Treg development and function and is vital for Treg cell lineage identity. FOXP3 is a member of the forkhead/winged-helix family of transcriptional factors. The gene is located on the X-chromosome and is highly conserved among different species. The FOXP3 protein is a 431 amino acid structure encoded by the FOXP3 gene in humans. The protein has four domains: an amino-terminal proline-rich domain mediating transcriptional repression, a central zinc finger, and leucine zipper domains facilitating homo or hereto- dimerization and the C-terminal forkhead domain implicated in nuclear localization and DNA binding activity [7,14].

The immune system is a sophisticated defense network, evolved to withstand innumerable pathogenic challenges at any anatomical location. To do so, complex cellular interactions coordinate pathogen recognition, immune cell activation, and the execution of effector programs. In order to return to or maintain homeostasis, immunosuppressive signals are essential to dampen immune responses to prevent pathological immune responses such as chronic inflammation or autoimmunity. An essential cell type involved in this process is the T_reg. The importance of T_regs in immune tolerance has become evident through the characterization of so-called scurfy mice, which suffer from a severe lethal autoimmune syndrome characterized by inflamed skin, red eyes, enlarged lymphoid organs, and early death. Scurfy mice were first reported in 1949, but it was not until the early 2000s that a mutation in the FOXP3 gene, and consequential loss of T_regs, was identified as a direct cause for the severe immune pathology. Further research showed that FOXP3 is the master transcription factor (TF) for the previously identified specialized immunosuppressive CD4⁺ CD25⁺ T lymphocytes, now known as T_regs. Since then, it has become clear that reduced T_reg numbers or impaired T_reg functionality stand at the basis of autoimmune and inflammatory diseases such as diabetes, multiple sclerosis, and inflammatory bowel disease. In contrast, their activation and accumulation in tumors are considered detrimental, as we explore below in depth [15 ].

Many studies have reported increased frequencies of Tregs in the peripheral blood of breast cancer patients across subtypes, indicating that breast tumors can systemically engage Tregs. Still, their significance remained elusive for a long time until a recent in-depth analysis was performed on Tregs isolated from the blood and tumors of breast cancer patients. It was found that a subpopulation of Tregs (FOXP3^hi CD45RA^neg), comprising approximately 19% of the total Treg population in the peripheral blood of patients, strongly resembles intratumoral Tregs based on phenotype, TCR repertoire, and CCR8 expression. This may suggest that intratumoral Tregs derive from FOXP3hi CD45RAneg Tregs in peripheral blood or vice versa. These Tregs from blood had superior suppressive potential in vitro, compared to FOXP3^low CD45RA^pos/neg Tregs. FOXP3^hi CD45RA^neg Tregs were heterogeneous between patients in their signaling response to both immunosuppressive and inflammatory cytokines. High Treg responsiveness to immunosuppressive cytokines correlated with poor survival, whereas high responsiveness to inflammatory cytokines had the opposite effect. This exposes the potential clinical significance of Tregs in the peripheral blood of breast cancer patients but also highlights how Treg heterogeneity may potentially influence disease outcomes [2,16].

Breast cancer accumulates Foxp3⁺ Treg cells upon tumor progression, and we have demonstrated that transient ablation of Treg cells in established, highly immuno-suppressive breast tumors results in a significant increase in anti-tumor immunity in primary and metastatic tumors. In this context, while cytotoxic T and NK cell activity are dispensable for the anti-tumor effect, IFNγ-dependent reprogramming of the tumor microenvironment is required. In contrast, intraductal immune cell accumulation is rarely detected in early DCIS lesions, and Treg cell frequency in normal and neoplastic 8-weeks mammary glands is similar, suggesting the microenvironment is more similar to the normal gland. In this study, we found that transient Treg cell ablation at this pre-invasive breast tumor stage accelerates the rate of tumor progression to invasive cancer, increasing the number of mammary glands harboring tumors and promoting the development of early invasive carcinoma. In addition, Treg cell ablation heightened reactive mammary stroma, characterized by a higher desmoplasia and collagen deposition. In line with our observations, this stromal change has been associated with the activation of angiogenic programs, recruitment of inflammatory cells, invasive phenotype, and metastatic progression [9,17,18].

It is now well-established that Treg cells play critical roles in maintaining non-lymphoid tissue homeostasis. More recently, a relationship between Treg cells and tissue-specific stem cells has been identified. In the bone marrow, Treg cells create an immune-privileged site enabling allo-hematopoietic stem/progenitor cell persistence and quiescence. In addition, skin Treg cells play a major role in hair follicle biology by promoting the function of hair follicle stem cells. Cancer stem cells are required for the initiation, progression, metastatic dissemination, and response to therapy in breast cancers. Here, we describe a previously unrecognized effect of Treg cells on mammary cancer stem/progenitor cells during the early stages of tumorigenesis. Specifically, Treg cell ablation induced expansion of CD45⁻ CD24⁻/lo CD44⁺, CD45⁻ CD24⁺ CD49f⁺, and CD45⁻ CD24⁺ CD29hi stem cell like-populations, as well as an immature luminal progenitor-enriched population (CD45⁻ CD24⁺ CD29lo CD61⁺). The murine CD44⁺ CD24⁻ cancer stem cell population found in the primary tumors of MMTV-PyMT transgenic mice exhibit functional characteristics of human breast cancer stem cells, which highlights the clinical impact of our finding. Our data suggest that Treg cells negatively regulate the early cancer stem cell niche. Supporting this, we demonstrated that dissociated mammary glands from Treg cell ablated mice progressed into tumors faster and with increased penetrance after transplantation into naïve hosts. Additionally, Treg cell ablated mice mammospheres were significantly larger when cultured under non-adherent conditions [2,13,19].

9. Conclusion

In conclusion, the higher expression of FOXP3 represents the higher activity of Treg cells in patients with breast cancer, which may play a role in tumor development. These findings emphasize that targeting Treg cells with the markers above may be a suitable strategy for breast cancer immunotherapy.