Breast cancer is a condition wherein the cells in the mammary gland grow in a dysregulated manner, resulting in the formation of a mass of cells called a tumor. The World Health Organization statistics revealed that, in 2020, the worldwide scale of breast cancer cases increased to 2.3 million, and, globally, 6,85,000 individuals lost their lives (1). Although breast cancer is generally considered benign, a delayed diagnosis can cause the cancer to spread to the surrounding lymph nodes, resulting in distant metastasis and impacting vital organs such as the brain, liver and lungs (2). Therefore, researchers are striving hard to develop effective therapeutic approaches; in this regard, the role of forkhead box proteins cannot be overlooked due to their involvement in cancer development and progression.

Forkhead box proteins, commonly known as forkhead box (FOX) proteins, belong to a family of transcription factors that are evolutionarily conserved, distinguished by their DNA binding domain, which is referred to as either ‘forkhead’ or ‘winged helix’ (3). These transcription factors play pivotal roles as essential regulators of gene expression in various pathological and physiological processes, encompassing cell proliferation, differentiation and survival (4). Interestingly, numerous studies have attempted to establish the links between FOX proteins and cellular events, including the cancer initiation and progression, alongside the emergence of resistance against anticancer drugs (5). These discoveries highlight the diverse functions of FOX proteins in the field of cancer biology.

The Forkhead box, or Fox, gene family is a group of transcriptional regulators known for their ancient evolutionary origins. They were named after the Drosophila melanogaster fork head gene (fkh). It has been reported that mutations in fkh can lead to abnormalities in head fold involution during embryonic development, causing adult flies to exhibit a characteristic spiked head phenotype (6). Fox genes are found across a vast range of organisms, from yeast to humans. They are further classified into subfamilies such as FOXA and FOXP. A defining feature of Fox proteins is their DNA-binding domain, the forkhead (FKH) region, which is remarkably similar across species and consists of roughly 100 amino acids. The high degree of conservation within the FKH domain extends across the entire Fox family. This feature served as the basis for the initial classification system, where Fox proteins were assigned to 15 classes, designated FOXA to FOXO, based on sequence similarity within the FKH domain. More recent evolutionary analyses have refined the Fox protein classification system, increasing the number of classes to 19, ranging from FOXP to FOXS. This categorization is based on sequence similarities within the FKH domain, which typically consists of three alpha helices, three beta sheets, and two unique ‘wing’ regions flanking the third beta sheet. The ‘forkhead’ or ‘winged helix’ terminology originates from the distinctive butterfly-like structure formed by these ‘wing’ regions in Fox proteins (7).

FOX transcription factors are crucial regulators in embryonic development and maintaining cellular balance, driven by evolutionary forces that shape their diverse functions (8). They act as master regulators of fundamental cellular functions, including cell cycle progression, proliferation, differentiation, DNA repair mechanisms, metabolism, blood vessel formation (angiogenesis), and ultimately, a cell's fate. Disruptions in Fox protein activity have been implicated in the onset, spread, advancement and resistance to therapy of cancer. They also influence pathways related to cancer, aiding cell survival in adverse conditions. The involvement of FOX proteins in cancer spans its entire spectrum, from initiation to metastasis, orchestrated through intricate networks. While the importance of Fox proteins is well-established, a comprehensive understanding of how mutations within FOX-binding sites in the regulatory regions of FOX-target genes remains incomplete. Further research is needed to unravel these mechanisms in detail.

FOX proteins can also be regulated by microRNAs (miRNAs), which are special non-coding RNAs whose role as a tumor suppressor and oncogene is gradually coming into stronger light (9,10). The present review has tried to provide a brief overview about miRNAs and FOX proteins as other multiple roles and association of FOX proteins have to be covered in more detail. However, a thorough analysis of the crosslinks between miRNAs and FOX proteins would help unlock the therapeutic hurdles around breast cancer treatment.

Currently, the understanding of the FOX family is at its nascent stage; therefore, a comprehensive study that unveils the complexity revolving around the FOX transcription factor and the detailed understanding of its association with breast cancer would aid in the discovery of cancer biomarkers and improve the existing treatment strategies. Keeping this in view, the article has tried to document the majority of important associations between multiple FOX proteins and breast cancer.

The discovery of significant findings often sparks interest, and a similar scenario unfolded with FOX proteins, now recognized for their role in positive and negative regulation of breast carcinogenesis. Initially, the focus of the present review centered on the FOXA protein family, which is extensively engaged in hormonal signaling (10).

MiRNAs are small, non-coding RNAs that exert regulatory control over various biological processes. They have a length of 22 nucleotides and function by negatively regulating the mRNA transcription. miRNAs regulate mRNAs by interacting with the 3′ untranslated region (UTR) of the targeted mRNA (76). This interaction suppresses mRNA expression. Apart from the 3′ UTR, miRNA also interacts with other regions such as 5′ UTR, gene promoters and coding sequences (77).

The biogenesis of miRNA involves a two-step process, wherein DNA sequences are transcribed into primary miRNA (pri-miRNA), subsequently processed into precursor and mature miRNAs. The initial step entails transcription of the DNA sequence into pri-miRNA within the nucleus (78). A microprocessor complex, comprising DiGeorge syndrome Critical Region 8 (DGCR8) functioning as an RNA binding domain and Drosha, a ribonuclease III enzyme, is responsible for converting pri-miRNA into pre-miRNA. DGCR8 protein specifically binds to the N6-methylated GGAC sequence in pri-miRNA. This complex then recruits Drosha nuclease, which cleaves the pri-miRNA duplex, resulting in the generation of a pre-miRNA with a characteristic hairpin structure (79,80). Following formation, the pre-miRNA is transported to the cytoplasm via the Exportin 5 (XPO5)/RanGTP complex (81). In the cytoplasm, Dicer, an RNase III enzyme, matures the pre-miRNA by cleaving off the terminal loop, resulting in the formation of mature miRNA (82). The mature miRNA is loaded into the Argonaute protein, which subsequently regulates the targeted mRNA.

miRNAs play regulatory roles in various processes, including cell proliferation and apoptosis. The involvement of miRNAs in breast cancer, either as tumor suppressor miRNAs (tsmiRs) or oncogenic miRNAs (oncomiRs), has been underscored in numerous studies. OncomiRs are miRNAs capable of downregulating the expression of tumor suppressors and are therefore elevated in breast cancer (83). tsmiRs are miRNAs capable of downregulating the expression of oncogenes and are thereby downregulated in breast cancer (84). miRNAs are known for their regulatory roles in cellular mechanisms, including cell proliferation, metastasis, invasion and apoptosis; therefore, the altered miRNAs levels can induce cancer-like conditions. For instance, miR-16 and miR-497 are known to inhibit cell proliferation, thereby acting as tsmiRs (85). Contrarily, miR-200c/141 works as an oncogene, thereby promoting the metastasis of breast cancer cells (86).

FOX proteins are a group of transcription factors that play a regulatory role in controlling gene expression. FOX proteins function as oncogenes and tumor suppressors; therefore, any dysregulation in FOX protein levels results in cancer-like conditions.

On the other hand, miRNA is a small non-coding RNA that works by regulating target mRNA expression. In breast cancer, miRNAs are known to function as either oncomiRs or tsmiRs, thereby promoting the pathological condition. miRNA overexpression or downregulation results in increased cell proliferation and metastasis, resulting in tumorigenesis.

Research is being conducted to understand the interlinkage between miRNA and various classes of FOX proteins as miRNAs regulate the expression of FOX proteins, which leads to breast cancer initiation and progression. Therefore, detailed insights into the crosslinks between miRNA and FOX proteins and the role of miRNA in modulating the activity of FOX proteins would help in advancing the effective treatment for breast cancer.

miR-132, a tumor suppressor, is downregulated in breast cancer. It regulates cell growth, proliferation and apoptosis, and is known to downregulate the expression of FOXA1 (87). The overexpression of miR-132 downregulates FOXA1, thereby affecting the expression of LIPG (subsequent target of FOXA1). miR-132 downregulation results in increased cell proliferation along with elevated levels of FOXA1 and LIPG, thereby resulting in tumorigenesis (88). In a study conducted by Wang et al (88), it was observed that introducing miR-132 significantly hindered colony formation in MDA-MB-468 cells compared with the control group whereas suppressing miR-132 enhanced colony formation efficiency in these cells. Reducing FOXA1 expression led to a notable decrease in colony formation efficiency in breast cancer cells compared with the anti-miR-132 group. The aforementioned study revealed an inverse correlation between miR-132 and FOXA1 expression in breast cancer tissues. Furthermore, miR-132 was shown to suppress FOXA1 expression in breast cancer cells. These findings suggested that miR-132 downregulation might contribute to FOXA1 upregulation in breast cancer. Therefore, targeting miR-132 and FOXA1 together could hold promise as therapeutic strategy for breast cancer treatment. miR-802 is a tumor suppressor miRNA, responsible for regulating cell proliferation (89). FOXM1, an oncogene, is negatively regulated by miR-802 because it contains miR-802 binding sites. miR-802 interacts with the 3′ UTR of FOXM1, downregulating its expression. The upregulation of miR-802 results in the reduced expression of FOXM1, thereby controlling cell proliferation, whereas the downregulated expression of miR-802 elevates FOXM1 expression, contributing to cancer-like conditions. Yuan and Wang (90) delved into the expression pattern of miR-802 in cancerous vs. adjacent non-cancerous tissues, revealing a significant decrease in miR-802 levels in cancer tissues. It was demonstrated that increasing miR-802 levels significantly reduced the ability of MCF-7 cells to proliferate after transfection, compared with control cells. To confirm that miR-802 directly targets FoxM1, they created a luciferase reporter vector containing potential miR-802 binding sites within the 3′ UTR of the FoxM1 gene. Their outcomes showed that increasing miR-802 levels significantly reduced luciferase activity in MCF-7 cells when the reporter contained the FoxM1 3′ UTR. These findings provide initial evidence that boosting miR-802 expression hinders breast cancer cell proliferation both in cell culture (in vitro) and in animal models (in vivo), likely by regulating FoxM1 expression. This highlights the significance of miR-802 in breast cancer development and suggests its potential as a target for breast cancer therapy. miR-199a, identified as an oncogenic miRNA, exhibits elevated levels in breast cancer cells. The tumor suppressor FOXP2 is negatively regulated by miR-199a (91). Overexpression of miR-199a causes a decrease in FOXP2 protein expression in breast cancer cells, consequently promoting increased proliferation, survival and metastasis of breast cancer cells. Cuiffo et al (92) demonstrated that manipulating specific miRNAs or silencing FOXP2 resulted in increased BCSC properties, leading to enhanced tumor initiation and metastasis. They validated their initial findings on FOXP2 expression using independent reverse transcription-quantitative PCR (ΔΔCq) analysis, revealing a significant decrease in FOXP2 protein levels in BCC199a, BCC199a/214 and BCCMSC cell lines. Since the decrease in FOXP2 was associated with maintaining progenitor cell identity and inhibiting differentiation in these cellular contexts, it was suggested that downregulation of FOXP2 might contribute to the emergence of CSC-like phenotypes in BCC199a, BCC199a/214 and BCCMSC. These findings contribute to a holistic comprehension of miRNA-mediated regulation in breast cancer, as outlined in Table II.

Despite the condition's heterogeneity, various therapeutics are employed to treat breast cancer. Although beneficial, the major difficulty faced by researchers is drug resistance. After completing a few drug cycles, patients start to develop resistance against the administered drug, thereby reducing its efficiency. The development of resistance involves various signaling pathways; recently, FOX proteins are also observed in this light. Alterations in the expression levels of various FOX proteins contribute to the development of resistance against anticancer drugs. Endocrine therapeutics are administered to target and regulate the estrogen response in breast cancer cells. Although favorable, the major setback of endocrine therapies revolves around the emergence of resistance against anticancer drugs. Tumors that develop resistance can regulate the elevated ER expression by employing various processes, including drug efflux, modulation of ER cofactor expression, and activation of other pathways, including growth factor signaling (97).

FOXA1 exhibits a positive association with all ER+ luminal breast cancer cells and works by interacting with DNA to open up chromatin, which then elevates the ER interaction to make its response element more accessible. The overall increased interaction is responsible for mediating resistance to endocrine therapeutics, including tamoxifen (98).

Tamoxifen is an FDA-approved ER modulator used as an endocrine therapy for breast cancer treatment. Tamoxifen does not entirely inhibit the function of ER; instead, it binds to ER, and this complex is recruited to chromatin, subsequently leading to the suppression of ER target gene transcription. Similar to normal estrogen-bound ER, tamoxifen-bound ER also requires FOXM1 to access the chromatin and perform its function (99).

Dysregulated FOXA1 expression promotes resistance to tamoxifen, thereby limiting its effectiveness. The overexpression of FOXA1 leads to the upregulation of IL-8, a cytokine responsible for metastases and survival of cancer cells and contributes to resistance to ER inhibitors. Fu et al (98) investigated the contribution of FOXA1 to endocrine therapy resistance in ER-positive (ER+) breast cancer. They employed various endocrine-resistant (Endo-R) breast cancer cell lines derived from parental ER+ lines. Notably, they observed elevated FOXA1 expression in these Endo-R cell lines compared with their ER+ counterparts. Additionally, elevated levels of FOXA1 mRNA were predictive of unfavorable outcomes in patients with ER+ tumor receiving tamoxifen. Importantly, IL-8 emerged as a critical mediator of the FOXA1/ER transcriptional reprogramming process, promoting the growth and invasion of Endo-R cells. The study suggests that targeting the IL-8 signaling pathway might be a promising therapeutic approach for treating ER+ breast cancers with elevated levels of FOXA1.

The downregulation of FOXA1 results in upregulation of IL-6, thus promoting cancer-like properties in tamoxifen-resistant breast cancer cells (100). In a study by Yamaguchi et al (100), tamoxifen-resistant (TAM-R) breast cancer cells were developed by prolonged exposure of ER+ MCF7 cells to tamoxifen. Interestingly, TAM-R cells displayed lower levels of both ERα, a major ER form in breast cancer, and its co-regulator FOXA1. By contrast, these cells displayed increased activation of the transcription factor NF-κB and its target gene, IL6. Through experimental interventions, stable expression of FOXA1, but not ERα, led to reduced IL6 levels in both MDA-MB-231 cells (lacking FOXA1 and ERα) and TAM-R cells, without significantly affecting NF-κB activity. Conversely, depletion of FOXA1 increased IL6 expression in MCF7 cells.

FOXM1, a key regulator in therapeutic resistance among breast cancer cells, is associated with various cancer conditions due to its influence on cell cycle regulation, autophagy and senescence. Elevated FOXM1 levels contribute to drug resistance, notably against the anthracycline drug epirubicin (101). OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) acts as a regulator, with upregulated FOXM1 resulting in increased cell proliferation and resistance development against epirubicin. Lys48-linked polyubiquitin chains, pivotal in proteasomal regulation, interact with FOXM1 to modulate its levels. However, FOXM1-OTUB1 interaction prevails, enhancing FOXM1 expression and conferring resistance (102). Elevated FOXM1 levels also facilitate improved DNA damage repair induced by epirubicin. Additionally, FOXM1 regulates antiapoptotic genes XIAP and survivin, fostering resistance against doxorubicin, epirubicin, docetaxel and paclitaxel (103). Understanding the intricate role of FOXM1 provides insights for targeted therapeutic interventions.

The FOXO3-FOXM1 axis serves as an informative marker to comprehend the resistance of drug, as its dysregulation impacts the cellular fate. FOXO3 is recognized as a tumor suppressor that regulates cell growth, proliferation and apoptosis (30). The activity of FOXO3 is downregulated by Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)-Akt/Protein Kinase B (PKB) signaling pathways in response to various external stimuli and growth factors. Apart from all the targets, FOXO3 downregulates the FOXM1 expression levels and competes with FOXM1 to interact with the same binding sites at the gene promoters (102). FOXM1 is an oncogene responsible for cell growth and proliferation, thereby promoting tumorigenesis (104). The downregulation of FOXO3 induces an upregulation of FOXM1, promoting drug efflux and cell survival. This upregulation contributes to an increase in reactive oxygen species levels, ultimately leading to the development of resistance against the administered drug (105) (Fig. 4).

Chemotherapy, a widely used treatment for breast cancer, targets and destroys cancer cells, with ongoing research aimed at enhancing its effectiveness against the disease. Distinct subclasses within the FOX family are identified as either oncogenes or tumor suppressors, exerting regulatory control over cellular fate through diverse mechanisms. This underscores their pivotal role in the initiation and progression of tumors (106). Targeting FOX proteins as a therapeutic intervention holds promise in the development of drugs responsible for regulation of FOX protein levels, thereby effectively inhibiting the growth of breast cancer cells which may potentially improve the efficacy of existing treatment strategies.

FOXM1 emerges as a crucial therapeutic target for breast cancer, particularly in HER2-positive cases. Lapatinib, an FDA-approved tyrosine kinase inhibitor, effectively downregulates elevated HER2 levels, concurrently decreasing FOXM1 expression. Francis et al (51) elucidated the connection between HER2 and FOXM1, establishing the ability of lapatinib to regulate FOXM1 at both mRNA and protein levels. Alternatively, honokiol, a plant-derived compound, demonstrates anti-inflammatory, antioxidative and anticancer properties, including FOXM1 inhibition. Honokiol disrupts the positive feedback loop regulating FOXM1 transcription, leading to reduced mRNA and protein levels. This dual approach, targeting FOXM1 with lapatinib and honokiol, offers a promising avenue for developing comprehensive breast cancer therapeutics, complementing existing strategies by Halasi et al (107).

Imipramine blue (IB), an anticancer derivative of the FDA-approved antidepressant imipramine, demonstrates efficacy in downregulating elevated FOXM1 expression in breast cancer, as demonstrated by Rajamanickam et al (108). The ability of IB to target FOXM1 and inhibit homologous recombination-mediated DNA repair positions it as a potential therapeutic agent for regulating tumor growth and metastasis in breast cancer. Integrating IB into treatment protocols could enable effective targeting of FOXM1 and improve the outcomes of breast cancer therapies.

Combination therapy has become pivotal in cancer treatment due to the limited efficacy of single-agent targeted drugs in clinical trials despite the expanding array of options available (109). Combining targeted therapies with hormone therapies (endocrine treatments) has been successful in improving the outlook for patients with breast cancer. However, some combinations of these drugs may have a higher chance of causing adverse effects. Therefore, identifying precise combinations and dosing regimens to optimize therapeutic efficacy while minimizing side effects is crucial in both preclinical and clinical investigations (110). A notable success in breast cancer combination therapy involves incorporating the anti-HER2 monoclonal antibody trastuzumab into first-line chemotherapy, significantly enhancing treatment benefits in patients with HER2-overexpressing metastatic breast cancer (111). Currently, the trastuzumab and paclitaxel combination is a potent regimen for treating low-risk HER2-positive tumors, yielding sustained positive outcomes (112). In a recent study, researchers investigated the synergistic potential of FOXM1 inhibitors alongside proteasome inhibitors such as Bortezomib, revealing significant inhibition of proliferation in both ER+ and TNBC (113). Similarly, the combined efficacy of FOXM1 inhibitors with CDK4/6 inhibitors including palbociclib, ribociclib and abemaciclib was demonstrated in ER+ breast cancer cells (113). These findings offer promise for advancing translational efforts and initiating clinical trials investigating agents targeting FOXM1, potentially improving outcomes for patients with breast cancer.

Targeting Fox proteins, transcription factors linked to breast cancer, is a challenge due to selectivity issues (4). Ideally, therapies should target only the cancer-promoting functions of Fox proteins in tumors, leaving out the healthy tissues. However, this objective presents several key challenges. Inhibitor off-target effects can disrupt similar proteins in healthy cells, causing side effects. Lin et al (114) demonstrated that FOXC1 promotes EMT and metastasis in TNBC cell lines. Their study revealed that inhibiting FOXC1 with a specific inhibitor significantly suppressed TNBC cell invasion and metastasis in preclinical models. However, a critical challenge identified in the aforementioned study is the potential for off-target effects of the inhibitor. Further optimization is necessary to minimize adverse consequences on healthy cells (114). FOXA1 plays a crucial role in ER signaling and tumor progression in hormone receptor (HR)+ breast cancer. Robinson et al (17) employed whole-genome analysis of HR+ breast cancer and identified FOXA1 as a key regulator of ER-mediated transcription. While targeting FOXA1 holds promise for disrupting ER signaling and inhibiting tumor growth, a major challenge lies in developing selective inhibitors that specifically target FOXA1 within cancer cells without compromising its normal functions in ER+ healthy breast tissue (17). It is important to understand that the functional similarity between FOX proteins in healthy and cancerous cells possess a significant challenge. The development of inhibitors that precisely target the tumorigenic functions of FOX proteins is challenging. This underscores the challenge of specifically targeting the cancer-promoting functions of FOX proteins without affecting their essential physiological roles in healthy tissues; therefore, a deeper understanding of Fox protein activity unique to cancer cells is crucial to overcome this (4).

Although therapeutics targeting specific miRNAs have not yet been applied clinically in breast cancer, notable progress is being made. For example, a miR-122 antagonist is currently undergoing Phase II clinical trials for hepatitis C treatment, suggesting the potential for miRNAs to serve as viable therapeutic targets in breast cancer (115). Efforts are also being directed towards developing therapeutics targeting miR-21 and miR-221. These microRNAs have been linked to drug resistance in breast cancer, and scientists are investigating their potential as therapeutic targets for hepatocellular carcinoma and other cancers (116). Clinical trials testing miRNA inhibitors as a treatment for patients with breast cancer with multidrug resistance could be initiated in the near future. Addressing the epigenetic alterations associated with drug resistance in breast cancer presents significant challenges. However, histone deacetylation inhibitors are emerging as promising candidates for all breast cancer subtypes (117,118). It is crucial to proceed cautiously, as these inhibitors may inadvertently alter the expression of key receptors such as ERα, HER2 and MDR1, potentially exacerbating drug resistance. Ongoing clinical trials of various histone deacetylation inhibitors will provide valuable insights into their efficacy and safety profiles (118,119).

Understanding the factors involved in cancerous cell initiation, invasion and metastasis is essential for developing new treatment strategies and improving existing ones. Numerous factors have been discovered so far that have been targeted to develop various therapeutics. FOX proteins are an ideal biomarker and therapeutic target that would aid in understanding and improving the existing therapies for breast cancer. Clinical trials targeting FOX proteins, therapeutics and breast cancer, that are currently underway are summarized in Table III.

In recent years, significant advancements have been achieved in the realm of breast cancer research, owing to researchers' adeptness in selectively addressing multiple signaling pathways whose dysregulation contributes to the initiation and progression of breast cancer. Apart from the pathways, diverse transcription factors are involved in breast cancer tumorigenesis. FOX proteins function as transcription factors and have been linked to numerous processes, including cell growth and apoptosis. The altered levels of distinct subclasses of FOX proteins can act as either oncogenes or tumor suppressors, thus inducing abnormal proliferation in breast cancer cells. miRNAs regulate FOX proteins, thereby controlling their expression, and the dysregulation of FOX protein levels caused by altered miRNA levels contributes to tumorigenesis.

Research focusing on FOX factors has provided valuable insights into the underlying mechanisms driving cancer progression. FOX factors often play diverse roles in cancer progression, influencing various aspects of tumor development and metastasis. Understanding the specific functions of FOX factors in cancer modulation and identifying potential therapeutic targets capable of modulating their activities could expand treatment options for cancer patients. Inhibiting FOX factors or the pathways they regulate holds potential for reversing drug resistance, overcoming immune evasion and halting metastatic spread. Therapeutic strategies targeting FOX factors may involve inhibitors of the factors themselves, the kinases involved in FOX-mediated pathways, or agents utilizing RNA interference to silence FOX factor expression. These approaches represent promising avenues for treating various types of cancer and offer hope for improved patient outcomes. Additionally, FOX proteins can potentially relay drug resistance, making them an appropriate therapeutic target, with only few FOX proteins being investigated as potential therapeutic targets. Thus, a thorough understanding of the mechanisms behind the various classes of FOX proteins would help to develop drugs that target the FOX proteins, control their levels, and thereby assist in the development of efficient treatment approaches.