FOXO1 in cancer: context-dependent roles, microRNA regulation, and therapeutic opportunities

Abstract

Forkhead box O1 (FOXO1) is a key transcription factor involved in regulating apoptosis, cell cycle arrest, oxidative stress responses, and metabolic homeostasis. Although FOXO1 traditionally functions as a tumour suppressor, emerging evidence reveals its context-dependent oncogenic role, particularly in cancer stem cells (CSCs) and therapy-resistant cancers. This review explores the complex regulatory network by which microRNAs (miRNAs) modulate FOXO1 expression and activity. Oncogenic miRNAs downregulate FOXO1 or promote its cytoplasmic sequestration via the PI3K/AKT pathway, enhancing tumour proliferation, epithelial-mesenchymal transition, and metastasis. In contrast, tumour-suppressive miRNAs upregulate or activate FOXO1, reinstating cell cycle arrest and apoptosis. Beyond its tumour-suppressive roles, FOXO1 also supports CSC maintenance and therapeutic resistance, highlighting the duality of its function. Notably, FOXO1 overexpression has been shown to improve the metabolic fitness and persistence of chimeric antigen receptor (CAR) T cells in solid tumours, suggesting potential for immunotherapy enhancement. Therapeutic approaches targeting the miRNA-FOXO1 axis, including miRNA mimics, inhibitors, phosphatase modulators, and kinase inhibitors, are promising but require precision to avoid undesirable effects in non-malignant tissues. Comprehensive understanding of FOXO1’s context-specific roles is essential for advancing targeted cancer therapies.

Background

FOXO1 (Forkhead Box O1) transcription factor regulates many processes, including metabolism, cell cycle control, apoptosis, oxidative stress resistance, and longevity [1]. It is widely expressed in mammals. FOXO1 (Fig. 1) is structurally related to FOXO3, FOXO4 and FOXO isoforms as they have conserved, defining Forkhead domains or “winged helix structures”, to facilitate DNA target binding [2, 3].

Fig. 1.

Solution structure of the free FOXO1 DNA binding domain (PDB ID 6QVW) [4]

FOXO is triggered following cascades initiated by growth factors or cellular stress. FOXO proteins are typically localised in the nucleus of quiescent or growth factor-deprived cells, where they suppress gene transcription. Phosphorylation of FOXO proteins via the PI3K pathway causes their migration into the cytoplasm, then degradation through ubiquitination. In the absence of growth factor signalling, nuclear FOXO proteins increase, leading to gene transcription, facilitating cell cycle arrest, enhanced stress resistance, and apoptosis [5].

FOXO1 is important for mediating insulin and growth factor effects on cellular function. FOXO regulatory pathways are mainly triggered via phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) (Fig. 2). AKT directly phosphorylates FOXO1 at three conserved serine/threonine residues, creating docking sites for 14-3-3 proteins, which bind to FOXO1 and chaperone it from the nucleus to the cytoplasm [6], where it is unable to initiate transcriptional activity [7]. Thus, AKT prevents expression of pro-apoptotic genes by phosphorylating FOXO1. Conversely, depletion of growth factors or PI3K/AKT allows phosphatases to dephosphorylate FOXO1, allowing it to re-translocate to the nucleus. FOXO1 resumes its role in the nucleus as a transcriptional activator, upregulating genes involved in cell cycle arrest, apoptosis and oxidative stress resistance [8].

Fig. 2.

An Overview of PI3K/AKT/FOXO signalling pathway

Roles of FOXO1 in cancer

FOXO1 induces expression of endogenous cyclin-dependent kinase inhibitors (CDKis) such as p27/Kip1 (CDKN1B) and p21/Cip1 (CDKN1A), which are essential for cell cycle progression. CDKN1A and CDKN1B specifically inhibit cyclin E•CDK2 and cyclin D•CDK4/6 complexes, preventing G1 phase to S phase transition, G1 phase cell cycle arrest, cessation of DNA replication and cell division [9, 10] (Fig. 3).

Fig. 3.

An Overview of the roles of FOXO1 in cancer

FOXO1 also affects activation of pro-apoptotic genes during apoptosis by directly inducing BIM (BCL2L11) expression: BIM is a pro-apoptotic member of the B-cell lymphoma (BCL)−2 family. BIM promotes apoptosis by binding to, and neutralising, anti-apoptotic proteins like BCL-2, activating pro-apoptotic effectors like Bcl-2 Associated X-protein (BAX) and Bcl-2 homologues antagonist/killer (BAK). This leads to mitochondrial outer membrane permeabilisation (MOMP) and cytochrome c release, which activates caspases in the intrinsic apoptotic pathway [11, 12].

FOXO1 also upregulates expression of Fas Ligand (FasL), a component of the extrinsic apoptotic pathway. FasL binds to its receptor Fas (CD95) on the cell surface, triggering formation of the death-inducing signalling complex (DISC), caspase-8 activation, leading to apoptosis [13]. Figure 4 summarises how FOXO1 demonstrates its tumour suppressive function through two pathways. Recent studies also discovered the synergistic interaction between FOXO1 and the tumour suppressor p53 to enhance the expression of pro-apoptotic genes, amplifying the apoptotic response to cellular stress. This interaction is significant in DNA damage, where p53 and FOXO1 synergistically eliminate potentially malignant cells [14]. Figure 4 shows how the intrinsic and extrinsic pathways converge to induce apoptosis through caspases.

Fig. 4.

Two key pathways through which FOXO1 achieves its tumour suppressive outcomes

Oxidative stress arises from imbalance between production of reactive oxygen species (ROS) and cellular capacity to detoxify them or repair damage. Effects of oxidative stress are most pronounced during the first step in the electron transfer, which is involved in ATP generation in mitochondria.

In response to oxidative stress, FOXO1 enhances resistance by upregulating target genes encoding antioxidant enzymes and proteins involved in ROS detoxification, DNA repair, and cell survival. Antioxidant enzymes include superoxide dismutase (MnSOD), a mitochondrial enzyme that converts superoxide anions (O₂⁻) into hydrogen peroxide (H₂O₂), which is less harmful [15, 16]. Catalase is another critical enzyme; it converts hydrogen peroxide (H₂O₂) into water and oxygen. FOXO1 upregulates catalase expression [16]. Moreover, FOXO1 activates DNA repair genes, upregulating growth arrest and DNA-damage-inducible 45 alpha (Gadd45a). Gadd45a helps repair oxidative DNA damage, preventing mutations which could lead to cell death or oncogenesis [17]. FOXO1 may also influence the expression of genes like damage-specific DNA-binding protein 1 (DDB1), which is involved in nucleotide excision repair, a pathway critical for repairing oxidative DNA lesions [18]. Together, these genes and factors regulated by FOXO1 help to protect cells from ROS-induced damage and mitochondrial dysfunction, thereby contributing to cell survival, longevity, and prevention of diseases associated with oxidative stress.

Established research focuses on FOXO1 in insulin/IGF responses and glucose homeostasis via genes involved in glucose production and storage. These efforts are directed towards treatments for type II diabetes, but there may be positive and negative spin-off considerations in cancer. For instance, cancerous cells typically use aerobic glycolysis and lactic acid fermentation to amplify energy generation (Warburg effect), and FOXO1 is involved in this process via upregulation of glucose-6-phosphatase (G6P) and phosphoenolpyruvate carboxykinase 1 (PCK1) gluconeogenesis [19–21] under fasting or low insulin conditions. Conversely, phosphorylation/inhibition of FOXO1 suppresses G6P and PCK1, decreasing hepatic glucose output [22, 23].

We conclude the multifunctionality of FOXO1 has many consequences in cell biology. In one respect this is desirable as it fuels expanding interest in strategies to modulate FOXO1 activity or treatment of metabolic diseases, ageing, and cancer. Conversely the multifaceted activity of FOXO1 is a “double-edged sword” because it will be difficult to hone impact of FOXO1 interventions on the desired therapeutic target, and avoid side effects.

Mechanisms by which FOXO1 becomes dysregulated

In healthy cells the suppressor role of FOXO1 helps regulate apoptosis, cell cycle arrest, and oxidative stress, thus maintaining normal cellular homeostasis and preventing malignant transformation. In cancer however, dysregulated phosphorylation of FOXO1 disrupts desirable negative regulatory transcription functions, thereby releasing the brake on cell growth control and consequently promoting uncontrolled proliferation and metastasis [24–26].

FOXO1 can becomes an oncogene via chromosomal translocations creating fusion proteins (e.g. FOXO1•PAX3/7 or MLL•FOXO1) which morph from a suppressor to a pro-oncogenic driver in cancer progression. Prevalent examples of this are in rhabdomyosarcoma (cancers in soft tissues, particularly skeletal muscles, originating from immature muscle cells) and leukemia. It can also undergo somatic mutations leading to constitutively active forms (gain of function) driving cancer cell survival and proliferation. For example, B-cell lymphomas particularly feature aberrant forms like this where altered gene expression prevents normal inactivation via PI3K/AKT, or by directly controlling oncogenic networks.

Aberrant fusions of FOXO1 with PAX-3 or -7 through chromosomal translocation promote oncogenic signalling in Alveolar rhabdomyosarcoma (ARMS), an aggressive paediatric cancer [27]. This maintains an aggressive cancer phenotype and contributes to therapy resistance via signal pathways in cell survival and proliferation. For instance, overexpression of the fusion proteins causes upregulation of receptor tyrosine kinases (RTKs) FGFR4 and IGF1R which enhance improve tumour cell survival despite therapy [28]. Further, secreted exosomes, key mediators of cellular signalling, contain increases miR-486, which supports cancer-associated fibroblast activities [29].

Disease consequences of dyregulated FOXO1

FOXO1-PAX3 is observed in immunohistochemical analyses of primary human ARMS tumours but not in normal tissues. In vivo invasion assays confirm phosphorylated FOXO1-PAX3 in tumour cells. Conversely, inhibiting phosphorylation of PAX3-FOXO1 at Ser201 and Ser205 by GSK3β inhibitors, or site-directed mutagenesis at these positions attenuates key ARMS phenotypes: reduced cell migration, invasion, proliferation and anchorage-independent growth as seen in Fig. 5 [30].

Fig. 5.

Key functional changes associated with PAX3/7-FOXO1 fusion

FOXO1-PAX3 overexpression in cervical neoplasia samples correlates with unfavourable outcomes [31]. Moreover, this phenotype shows increased resistance to chemotherapy and radiotherapy. Conversely, silencing FOXO1 expression in vitro reduces cell viability, migration and metastasis, which suggests FOXO1 is a causative, not just associative.

Aberrant FOXO1 may also promote malignant progression in low-grade gliomas (LGGs) [32]. This assertion is based on observed upregulation of FOXO1 (observed in RT-qPCR), which has proto-oncogenic effects due to dysregulation of STAT3 in LGGs compared to normal astrocytes. STAT3 activation upregulates FOXO1 expression, binding to the corresponding promoter region (Chromatin immunoprecipitation assay), promoting malignant progression of LGG.

FOXO1 is significant role in acute myeloid leukaemia (AML) t(8;21) subtype. This is characterised by chromosomal translocation of the AML1 (RUNX1) gene on chromosome 21 with the ETO (RUNX1T1) gene on chromosome 8 [33], resulting in the AML1-ETO (AE) fusion protein [34]. This oncoprotein disrupts normal AML1 function, blocking myeloid differentiation and promoting the survival of immature hematopoietic cells [33, 35]. AE has been shown to directly bind to the promoter region of the FOXO1 gene, leading to its upregulation and further suppression of myeloid differentiation, thereby expanding the pool of pre-leukemic progenitor cells (Fig. 6) [36]. Lin et al. [37] demonstrated FOXO1 expression increases in the presence of AE and decreases when AE is removed, indicating direct transcriptional regulation. Chromatin immunoprecipitation confirmed AE physically associates with the FOXO1 promoter, providing strong evidence AE can drive FOXO1 overexpression in AML.

Fig. 6.

A schematic overview of AML1-ETO formation and impact

AML1-ETO helps tumours survive through two other mechanisms. First, AE is associated with downregulation of miR-193a, which represses PTEN, a negative regulator of the PI3K axis [38]. PTEN loss results in a sustained PI3K/AKT activation [39]. Second, PI3K/AKT signalling is also promoted through increase in IGF1R, which is a PI3K activator [39]. Increased IGF1R is facilitated by downregulation of miR-223 due to AE. Both pathways converge to activate PI3K/AKT signalling, which promotes cell survival and anti-apoptosis signalling [39].

In AML1-ETO, (AE)–associated leukemia, AE upregulates FOXO1 expression in early hematopoietic progenitor cells (CD34+), promoting self-renewal and blocking differentiation. This leads to accumulation of immature cells, a defining feature of leukemia [37]. Thus FOXO1 is co-opted to support leukemogenesis rather than suppress it.

Overall, function of FOXO1 in disease is context-dependent. Further investigation of abberant FOXO1 roles, guide development of more precise therapeutic strategies.

FOXO1 in maintaining stemness of cancer

Cancer stem cells (CSCs) are a distinct subpopulation of tumour cells characterized by their ability to self-renew, differentiate and drive tumour initiation, progression and recurrence. Stem cell-like properties contribute to chemoresistance and metastasis in cancer. Many transcription factors implicated in the regulation of CSC characteristics, but FOXO1 particularly contributes to CSC properties by promoting transcription of key pluripotency genes such as OCT4 and SOX2. Chromatin immunoprecipitation (ChIP) studies have confirmed FOXO1 binding to the regulatory regions of these genes, reflecting a mechanism similar to its role in embryonic stem cells [40]. Uniquely, in cancer cells, FOXO1 retains its nuclear localization and transcriptional activity even in the presence of inhibitory signals from pathways like PI3K/AKT, which would normally induce its phosphorylation and cytoplasmic sequestration. This aberrant regulatory behavior allows FOXO1 to sustain expression of stemness-associated genes, thereby enabling CSCs to evade differentiation and maintain their self-renewal capacity [40].

Hyperactivated PI3K/AKT signalling promotes phosphorylation of FOXO1, sequestering it in the cytoplasm and inhibiting its function [41]. However, in CSCs, FOXO1 remains active in the nucleus despite this hyperactivation, suggesting alternative regulatory mechanisms that allow it to bypass AKT-mediated inhibition. Such persistent nuclear localisation is essential for the continued expression of pluripotency factors in CSCs. By preventing differentiation, FOXO1 enhances the aggressive nature of tumours and cancer recurrence [41, 42].

Another notable aspect of FOXO1’s role in CSCs is maintaining balance between self-renewal and differentiation [43]. CSCs must carefully regulate this balance to drive tumour growth. FOXO1 ensures this by promoting a stem-like state while inhibiting differentiation signals [43].

In summary, although FOXO1 functions as a tumour suppressor in normal cells by promoting apoptosis and cell cycle arrest in response to stress or DNA damage, its role in CSCs is more complex and can contribute to tumour progression. In CSCs, FOXO1-mediated regulation of the cell cycle may facilitate entry into a quiescent state, rendering these cells resistant to therapies that target actively dividing populations. This quiescence allows CSCs to persist during treatment and later drive tumour recurrence. The ability of FOXO1 to maintain this dormant state is a key factor in CSC survival and represents a potential target for strategies aimed at their eradication [5, 44, 45]. These observations suggest that the impact of FOXO1 is not solely determined by genetic aberrations but is also heavily influenced by the cellular context in which it functions.

Therapeutic potential of FOXO1

The dual role of FOXO1 as a tumour suppressor in normal cells and a promoter of stemness in CSCs poses significant challenges for therapeutic targeting. Inhibiting FOXO1 may impair survival and self-renewal of CSCs, but such interventions also risk disrupting critical functions in normal tissue homeostasis. Therefore, the development of strategies that selectively modulate FOXO1 activity in CSCs, while preserving its tumour-suppressive role in healthy cells, is essential for achieving effective and safe cancer therapies [40].

In addition to its roles in the solid tumours, FOXO1 also has implications for improving therapies targeting haematological malignancies, such as chimeric antigen receptor CAR T-cell therapy [46]. CAR T-cell therapy can treat blood cancers but has struggled to achieve similar efficacy in solid tumours, primarily due to the immunosuppressive tumour microenvironment. However, promoting a stem-like phenotype through FOXO1 overexpression significantly enhances CAR T-cells’ fitness and therapeutic efficacy. T-cells modified to overexpress FOXO1 demonstrate improved mitochondrial fitness, persistence, and an enhanced ability to combat solid tumours. Thus, FOXO1 contributes to CAR T-cell longevity and effectiveness in the tumour microenvironment by modulating the metabolic phenotype of CAR T-cells. This is achieved through oxidative phosphorylation and mitochondrial biogenesis. FOXO1 tends to upregulate mitochondrial function and cellular metabolism to ensure CAR T-cells survive in low-nutrient environments such as tumours [46]. These findings highlight FOXO1’s potential as a target for genetic engineering strategies to enhance CAR T-cell responses in solid tumours. Despite this potentially favourable outcome, caution is warranted to ensure that such enhancement of FOXO1 does not promote CSCs. This highlights the complexity of exploiting FOXO1 due to its dual nature.

Besides PI3K/AKT, FOXO1 modulates Wnt/β-Catenin signalling in CSCs [47]. Non-coding RNA (lncRNA) microarray analyses have identified differentially expressed lncRNAs in FOXO1-overexpressing PANC1 cells compared to human pancreatic ductal cells (HPNE). The multifunctional long non‐coding RNA LINC01197 was assessed through quantitative PCR, northern blotting, and fluorescence in situ hybridisation (FISH). LINC01197 was significantly decreased in malignant pancreatic ductal adenocarcinoma (PDAC) tissues, for which low expression correlates with poor patient prognosis. LINC01197 was shown to interact with β-catenin, inhibiting Wnt/β-catenin signalling by preventing β-catenin from binding to TCF4 (transcription factor 4) in PDAC cells. This suggests it as a potential target for clinical therapy and prognostic prediction in PDAC patients [47]. To explore if targeting this pathway can prove beneficial, validation in pre-clinical models is imperative. In the clinical setting, the focus should be on looking at the expression of LINC01197 in the patient cohorts to observe if there is any correlation. These steps can lay the groundwork for therapeutic approaches. However, modulating gene expression requires being mindful of potential problems likerisk of off-target effects before moving to human trials. In addition, the complexity of the tumour microenvironment (TME) cannot be ignored. Heterogeneity of cells in the TME means that targeting a gene can have a favourable outcome in one kind of cell, while promoting an aggressive phenotype in a different kind of cell requires careful weighing of overall benefit and harm.

By targeting FOXO1 through the inhibition of polo-like kinase 1 (PLK1), a key regulator of FOXO1 activity, appears to be promising in inducing apoptosis in PCa, as demonstrated by Gheghiani et al. [48]. By blocking PLK1-dependent inhibition of FOXO1 using BI2536 - PLK1-BRD4 kinase inhibitor alongside treatment with nocodazole, a mitotic inhibitor, it worked synergistically to induce apoptosis in the tumour, while sparing normal prostate epithelial cells. This demonstrates that there is therapeutic promise of strategically targeting the PLK1-FOXO1 axis in anticancer drug development. It is, however, also worth mentioning that currently, there are no approved drugs to enhance FOXO1 activity directly.

microRNAs and FOXO1

MicroRNAs in general

Noncoding RNAs are implicated in many human diseases, particularly cancer [49]. MicroRNAs (miRNA) are short noncoding RNA derived from these. They are generated by transcription by RNA polymerases II and III, then are cleaved to generate mature miRNAs averaging ~ 22 nucleotides [50, 51]. They are highly conserved across species.

miRNAs control expression of specific genes, primarily via interactions with the 3’ untranslated region (3’ UTR) of messenger RNA (mRNA), repressing translational activities [52]. This mechanism is crucial for cellular functions such as growth [53] and proliferation [54]. Nevertheless, studies have reported interaction with other areas, including 5’ UTR, coding sequence and gene promoters [50]. miRNAs can also cause targeted mRNA degradation [55]. More than 2588 discovered mature human miRNAs modulate ~ 60% protein-coding genes [56, 57]. These comprise ~ 5,300 human genes, many of which are associated with proliferation, metastasis and apoptosis of cancer cells [58, 59]. miRNAs can act on oncogenes or tumour suppressors [60], where they can promote tumorigenesis of diverse cancers, but they also have potential therapeutic applications (Fig. 7) [61–63].

Fig. 7.

miRNAs can promote or suppress tumours

Tumour-promoting effects of miRNAs on FOXO1

Some miRNAs selectively interact with the FOXO1 receptor in cancer cells, perturbing expression of several key molecular intermediates in cell signalling to promote tumorigenesis (Table 1).

Table 1.

Pro-tumour effects of different miRNAs in cancer cells

miRNA	Type of Cancer Cell Line	Targeted Pathway Mechanism of Action	miRNA-Targeted Binding sites on FOXO1 3’ UTR	Outcome	References
miR-204	Breast Cancer MDA-MB-231	PI3K/AKT and MAPK Downregulate FOXO1 and upregulate phosphorylated AKT, JNK and ERK proteins	miR-204: 3’-UCCGUAUCCUACU-GUUUCCCUU-5’ FOXO1: 5’-GUUUGUA-GAUCAGAAAAGGGAG-3’	Significant reduction in apoptosis (Apoptotic rate of 27.27% ± 1.15% in comparison with 36.17% ± 0.91% in control group) Significant reduction in inhibitory effects at 72 h (p < 0.001)	[64]
miR-27a miR-96 miR-182	Breast Cancer MCF-7	PI3K/AKT Downregulate FOXO1 and inactivation by AKT-mediated phosphorylation	miR-27a: 3’-CGCCUUGAAUCGGUGACACUU-5’ FOXO1: 5’-UACUCUUUUUGUAAUACUGUGAU-3’ miR-96: 3’-UCGUUUUUACACGAUCACGGUUU-5’ FOXO1: 5’-UUUCAUUACAAUGAAGUGCCAAA-3’ miR-182: 3’-UCACACUCAAGAUGGUAACGGUUU-5’ FOXO1: 5’-UUCAUUACAAUGAAGUGCCAAAC-3’	Increase in proliferation in all miRNA groups at 24, 48 and 72-hr time points	[65]
miR-223	Liver, Cervical and Colon Cancer HuH7 HeLa HCT116	PI3K/AKT Downregulate phosphorylated FOXO1 and cyclin D1 and upregulate p21, p27 gene transcription	miR-223: 3’-UUGACUG-5’ FOXO1: 5’-UUUGACAAACUGAC-3’	Significant reduction in cell proliferation in treated groups (all cell lines) in comparison with the control groups (p < 0.01)	[66]
miR-552	Gastric Cancer MKN-45 cells	EMT and PI3K/AKT Downregulate FOXO1, increase expression of N-cadherin, decrease expression of E-cadherin, increase expression of BAX, decrease expression of BCL-2, and increase expression of p-PI3K and p-AKT.	miR-552: 3’-AACAGAUUGGUCAGUGGACAA-5’ FOXO1: 5’-UAUUACUUUCCAAUUACCUGUAA-3’	Significant increase in cell proliferation (p < 0.05), migration (p < 0.01) and invasion (p < 0.01)	[67]
miR-196a	Cervical Cancer C33A CaSKi	PI3K/AKT Downregulate FOXO1, promote G1/S cell cycle transition, resulting in decreased expression of p21 and p27 and increased expression of cyclin D1	miR-196a: 3’-GGGUUGUUGUACUUUGAUGGAU-5’ FOXO1: 5’-UAAACUUUUGUUUGUACUACCUG-3’	Significant increase in proliferation (p < 0.05)	[68]
miR-135b	Cervical Cancer HT-3 SiHa	PI3K/AKT Negatively regulate FOXO1, promote G1/S cell cycle transition, resulting in decreased expression of p21 and p27 and increased expression of cyclin D1.	miR-135b: 3’-AUACCGAAAUAUGUUAAUAUCGU-5’ FOXO1: 5’-UAUGGCUUUUCAUUCCUAUGUGA-3’	Significant increase in proliferation (p < 0.01)	[69]
miR-196a	Liver Cancer Huh7 MHCC-97 H Hep3B HepG2 SMMC-7721	PI3K/AKT Downregulate FOXO1	miR-196a: 3’-GGGUUGUUGUACUUUGAUGGAU-5’ FOXO1: 5’-UAAACUUUUGUUUGUACUACCUG-3’	Upregulation of miR-196a promotes proliferation and inhibits apoptosis	[70]
miR-629	Lung Cancer A549 H1299	EMT/PI3K/AKT Downregulate FOXO1, promote the expression of N-cadherin, vimentin, p-AKT, and inhibit the expression of E-cadherin.	miR-629: 3’-UCAAGAGGGUUGCAUUUGGGU-5’ FOXO1: 5’-CAUAGCUGGUUUUAGAAACCCAA-3’	Promotion of proliferation (p < 0.05), migration (p < 0.01) and invasion (p < 0.01)	[71]
miR-370	Prostate Cancer PC3 DU145	AKT/PKB Downregulate FOXO1, resulting in cell cycle progression, leading to decreased p21Cip1 and p27Kip1, and increased expression of cyclin D1	miR-370: 3’-UGGUCCAAGGUGGGGUCGUCCG-5’ FOXO1: 5’-CUUCAGAUUGUCUGACAGCAGGA-3’	Significant increase in cell proliferation (P < 0.05) Significant increase in anchorage-independent growth (P < 0.05)	[72]

Liang et al. [64] transfected MDA-MB-231 cells with miR-204. Flow cytometry revealed a lower apoptotic rate (27%) in the treated cells compared to the untreated control (36%). This demonstrates that miR-204 has a tumorigenic role as it promotes cell survival for the tumour. The wound healing assay by the researchers showed that miR-204-treated MDA-MB-231 cells had lower migration of cells in contrast to the untreated cells at 72 h. A sequence analysis performed using miRWalk database revealed that miR-204‘s conserved binding site was complementary to the 3’-UTR of the FOXO1 mRNA. This implies there is a targeted binding post-transcriptional level. When the FOXO1 mRNA levels were compared between the groups, LV miR-204-treated cells had the lowest levels (p < 0.05), confirming the inverse relationship between FOXO1 and miR-204. Similarly, it has been reported that miR-27a, miR-96, and miR-182 target and suppress FOXO1 in MCF-7 cells, impairing its ability to regulate cell proliferation and apoptosis [65].

miRNA-223 targets FOXO1 receptors in HuH7, HeLa and HCT116 cells via the PI3K pathway [66]. Thus, pFOXO1 and cyclin D1 are downregulated, and p21 and p27 gene transcription is upregulated; both are associated with cell cycle suppression leading to apoptosis. Interestingly, miR-223 mainly affects cytoplasmic FOXO1, as the proportion of nuclear FOXO1 is higher in cells transfected with miR-223 in comparison with the control (immunofluorescence staining). This suggests miR-223 can reduce cytoplasmic FOXO1 or upregulate the nuclear form. However, upregulation of the nuclear form better explains this finding, because a significant reduction in cell proliferation in treated groups was observed relative to controls (p < 0.01) for all the cell lines listed above [66]. However, other mechanisms which we have not articulated may also be involved.

Zhao et al. [67] state miR-552 selectively targets FOXO1 in MKN-45 gastric cancer cells, promoting carcinogenesis by downregulating FOXO1. This is associated with a decrease in N-cadherin and BCL-2 expression, along with an increase in E-cadherin, Bax, p-PI3K, and p-AKT expression, in the epithelial-to-mesenchymal transition (EMT) and PI3K/AKT pathway. These changes increased in MKN-45 cell proliferation, migration and invasion. In a control experiment, transfection of miR-552 and FOXO1 vector reversed the carcinogenic effects from miR-552 alone, further implicating FOXO1 as the target [67].

Furthermore, FOXO1 was also reported to interact with miR-196a in C33A and CaSKi cells [68]. Initial transfection with miR-196a revealed a downregulation of FOXO1, which was eventually overcome via an additional transfection of FOXR. Using the PI3K/AKT pathway, cells transfected with miR-196a accelerated G1/S cell cycle transition, decreased expression of p21 and p27, and increased expression of cyclin D1, resulting in a significant increase in cell proliferation (p < 0.05). In a control experiment, co-transfection of miR-196a with FOXO1 exhibited anti-tumour effects, indicating that the tumour-promoting function of miR-196a can be offset by the anti-tumour role of FOXO1. In accord with this, Xu et al. [69] showed that miR-135b targeting HT-3 and SiHa cervical cancer cells, where FOXO1 is also downregulated, resulted in a significant increase in cell proliferation (p < 0.01).

In another study, FOXO1, when inhibited via miR-196a, reversed its effects, thus promoting cancer cell proliferation and invasion in liver cancer cells (Huh7, MHCC-97 H, Hep3B, HepG2 and SMMC-7721) [70]. This occurred via impacts on p21, p27 and cyclin D1 in the PI3K/AKT pathway. Thus, upregulation of miR-196a inhibits FOXO1, resulting in increased proliferation and inhibition of apoptosis [70]. In controls, knockdown of the FOXO1 gene in the cancer cells promoted cell proliferation, consistent with the anti-tumour role of FOXO1.

Zhu et al. [71] reported miRNA-629 inhibited FOXO1 in A549 and H1299 non-small cell lung carcinoma cells, where it promoted expression of N-cadherin, vimentin and p-AKT while inhibiting the expression of FOXO1 and E-cadherin. These effects resulted in promotion of proliferation (p < 0.05), migration (p < 0.01) and invasion (p < 0.01). Consistent with this, a study by Wang et al. [73] reported miRNA-629 may also serve as a novel biomarker for lung metastases of triple-negative breast cancer.

miR-370 was significantly upregulated in the prostate cancer cell lines PC3, DU145, 22Rv1, LNCaP, and Tsu-Pr1 when compared to normal prostate epithelial cells (PrEC) (p < 0.05) [72]. It bound directly to the 3’-UTR of FOXO1 mRNA, leading to decreased FOXO1 protein and mRNA levels. Downregulation of FOXO1 by miR-370 was connected to increased cell proliferation. In controls, reintroduction of FOXO1 lacking the 3’-UTR partially restored proliferation rates. This finding suggests that miR-370 has a role in controlling the expression of FOXO1, which ultimately has an impact on cell proliferation.

Tumour-suppressing effects of miRNAs in FOXO1

The previous section focuses on how miRNAs that directly target FOXO1 results in its downregulation and promotion of tumorigenesis. However, some miRNAs in several cancers may positively modulate the expression of FOXO1, resulting in enhanced anti-tumour effects instead (Table 2).

Table 2.

The anti-tumour effects of different miRNAs in cancer cells

miRNA	Type of Cancer	Targeted Pathway Mechanism of Action	miRNA-Targeted Binding sites on FOXO1 3’ UTR	Outcome	References
miR-3188	Nasopharyngeal Cancer SUNE1 HONE-1	p-PI3K/p-AKT/p-mTOR Upregulation of FOXO1, p27 and p21 G1/S cell cycle arrest Downregulation of c-JUN, CCND1, mTOR, p-PI3K and p-AKT	Not specified	Significant reduction in proliferation (p < 0.05)	[74]
	Lung Cancer A549 H1299	mTOR-p-PI3K/AKT-c-JUN Upregulation of FOXO1, p27 and p21 G1/S cell cycle arrest Downregulation of c-JUN, CCND1, mTOR, p-PI3K and p-AKT	Not specified	Reduction of cells in the S phase Reduction in colony formation Reduction in tumour size in mice	[75]
miR-486-5p	Blood Cancer K562 Kasumi-1 THP-1	Akt/PI3K Upregulation of FOXO1 and caspase-3 activity	miR-486: 3’-GAGCCCCGUCGAGUCAUGUCCU-5’ FOXO1: 5’-AAAGACAUUUUUCCUGUACAGGA-3’	Significant reduction in cell density after 24 h (p < 0.05) Significant increase in apoptosis in K562 (p < 0.05), THP-1 and Kasumi-1 cells (p < 0.001)	[76]
miR-124	Gastric Cancer MGC-803 SGC7901	SPHK1/AKT/FOXO1 Targets and downregulates SPHK1 by binding to its 3′-UTR. Decreased AKT activity, leading to reduced phosphorylation and degradation of FOXO1. Increased FOXO1 activity and the expression of cell-cycle inhibitors.	Not specified	Reduced cell proliferation and tumourigenicity (p < 0.05)	[77]
miR-200-3p	Bone Cancer Saos-2 U2OS	Not specified Upregulation of FOXO1	Not specified	Inhibition of cell proliferation Significant increase in apoptosis (p < 0.05)	[78]

Zhao et al. [74] reported that FOXO1 is a direct target of miR-3188 in SUNE1 and HONE-1 nasopharyngeal cancer cells, but this interaction resulted in upregulated expression of FOXO1, p21 and p27, leading to G1/S cell cycle arrest. Additionally, this also downregulated jun-proto-oncogene (c-JUN), cyclin D1 (CCNDI), mammalian target of rapamycin (mTOR), p-PI3K and p-AKT (p < 0.05) [74]. Similar observations were made for miR-3188 in A549 and H1299 non-small cell lung carcinoma (NSCLC), where this miRNA was found to upregulate FOXO1 expression, resulting in fewer cells in the S phase, reduction in colony formation and decline of tumour size in mice [75]. Thus, this miRNA can exert anti-tumour effects in vitro and in vivo.

Consistent findings were also reported for K562, Kasumi-1 and THP-1 leukaemia cells, where miR-486-5p was found to upregulate FOXO1 expression, and, consequently, caspase-3 activity. This resulted in a significant reduction in the cell density 24 h post-treatment (p < 0.05) due to a significant increase in apoptosis (p < 0.001) [76].

In gastric cancer, SPHK1 expression is elevated and associated with poorer patient survival; however, the exact mechanism behind SPHK1 upregulation is not well understood. Xia et al. [77] found overexpression of miR-124 increased FOXO1 activity in gastric cancer by directly targeting the 3′-untranslated region (3′-UTR) of SPHK1 mRNA, leading to its downregulation. Inverse correlation between miR-124 and SPHK1 in gastric cancer supports this finding. They postulate reduction of SPHK1 by miR-124 leads to decreased AKT activity, reducing the phosphorylation and degradation of FOXO1. As a result, FOXO1 becomes more active and translocate into the nucleus, where it promotes the expression of cyclin-dependent kinase inhibitors such as p21 and p27 and suppresses cell proliferation and tumorigenicity both in vivo and in vitro. Anti-tumour effects were also exerted by miR-200-3p in osteosarcoma Saos-2 and U2OS cells, for which FOXO1 was also upregulated [78], giving increased apoptosis (p < 0.05).

Conclusion

miRNA-mediated regulation of FOXO1 represents a critical and context-dependent axis in cancer biology, governing processes ranging from apoptosis and cell-cycle arrest to cancer stem cell (CSC) maintenance, metabolic adaptation, and therapy resistance. In its wild-type, nuclear form, FOXO1 functions predominantly as a tumour suppressor, enforcing cell-cycle arrest and apoptosis. However, oncogenic signalling and tumour microenvironmental cues can reprogram FOXO1 activity, shifting it toward pro-tumourigenic functions. Aberrant phosphorylation, oncogenic gene fusions, and miRNA-dependent repression collectively act as molecular switches that determine whether FOXO1 restrains tumour progression or instead supports CSC stemness and therapeutic resistance (Fig. 8).

Fig. 8.

Context-dependent dual functions of FOXO1 in cancer. Wild-type FOXO1 acts as a tumour suppressor by inducing cell-cycle arrest and apoptosis, whereas oncogenic signalling, miRNA-mediated repression, aberrant phosphorylation, or gene fusion events reprogram FOXO1 to support cancer stemness, metabolic adaptation, and therapy resistance

From a translational perspective, therapeutic strategies targeting miRNA-FOXO1 can be broadly categorized into two. The first involves inhibiting oncogenic miRNAs that suppress FOXO1 expression, thereby restoring its tumour-suppressive transcriptional programs. The second strategy leverages tumour-suppressive miRNAs or upstream pathway modulation to indirectly enhance FOXO1 activity. Recent studies demonstrating inhibition of specific oncogenic miRNAs can restore FOXO1 expression and suppress tumour growth highlight the promise of this approach [79]. In parallel, indirect modulation of FOXO1 activity through targeting upstream regulators in AKT–FOXO1 signalling enhances chemosensitivity and induces apoptosis in resistant tumours, implying pathway-level intervention is viable [80].

While microRNA-based strategies offer a tractable means of modulating FOXO1 expression, they should be viewed as complementary to, rather than replacements for, upstream signalling modulation and other therapeutic approaches targeting FOXO1 function. In our view, miRNA-based modulation and indirect restoration of FOXO1 function via upstream kinase or phosphatase targeting currently appear the most feasible for clinical translation, as they allow pathway-level correction without permanently altering FOXO1 expression in normal tissues. In contrast, direct FOXO1 activation remains challenging due to the risk of unintended effects on metabolic homeostasis and stemness programs.

Future research directions

A major obstacle to clinical implementation relates to therapeutic precision. The key is to target aberrant FOXO1 and not the wild type form. This requires detailed understanding of how the suppressor becomes dysregulated as a function of disease type; several examples are outlined in this review, but the field would progress faster with more intense investigations of this aspect of FOXO1 in tumour cell biology. Emerging delivery technologies, including nanoparticle-based miRNA mimics and inhibitors, offer promising avenues to reduce off-target toxicity. Indeed, miRNA expression profiling may serve as a valuable diagnostic and stratification tool, enabling identification of patient subgroups most likely to benefit from FOXO1-centred interventions.

In our view, future research perhaps should prioritise:

• (i)characterizing cancer- and stage-specific FOXO1 regulatory networks;
• (ii)elucidating FOXO1 interactions with cooperating transcription factors such as p53 and MYC;
• (iii)validating miRNA-FOXO1 predictive biomarkers in patient cohorts; and,
• (iv)targeted delivery platforms for selectively modulating FOXO1 activity within the tumour microenvironment. Notably, nanoparticle-based miRNA delivery systems, particularly lipid and polymeric nanoparticles, have shown preclinical promise for miRNA therapeutics, and FOXO1 modulation using self-assembled DNA nanostructures has been demonstrated [81]. However, biodistribution, immune activation, and reliable target engagement remain key translational challenges [82].

Systems-level understanding of FOXO1’s dynamic integration within broader transcriptional and signalling networks will be valuable for resolving its context-dependent functions and safely exploiting its therapeutic potential [56]. Expanding mechanistic understanding of FOXO1 may automatically lead to safe and effective precision-based cancer therapies.

Abbreviations

AKT

Protein kinase B

BAK

Bcl-2 homologues antagonist/killer

BAX

Bcl-2 Associated X-protein

BCL

B-cell lymphoma 2

CAR

Chimeric antigen receptor

CCNDI

Cyclin D1

CDK

Cyclin-dependent kinase

ChIP

Chromatin immunoprecipitation

c-JUN

Jun-Proto-Oncogene

CSC

Cancer stem cell

DiSC

Death-inducing signalling complex

EMT

Epithelial-to-mesenchymal transition

ERK

Extracellular signal-regulated kinase

ETO

Eight-Twenty-One

RUNX1T1

RUNX1 Partner Transcriptional Co-repressor 1

ESC

Embryonic stem cell

FasL

Fas ligand

FOXO

Forkhead box O protein

G6P

Glucose-6-phosphate

JNK

Jun N-terminal kinase

MAPK

Mitogen-activated protein kinase

miRNA

MicroRNA

MLL

Mixed lineage leukemia

MOMP

Mitochondrial outer membrane permeabilisation

mTOR

Mammalian target of rapamycin

NSCLC

Non-small cell lung carcinoma

PARP

Poly (ADP-ribose) polymerase

PCa

Human prostate cancer

PCK

Phosphoenolpyruvate carboxykinase

PDAC

Pancreatic ductal adenocarcinoma

PrEC

Normal human prostate epithelial cells

PLK

Polo-like kinase

PI3K

Phosphatidylinositol 3-kinase

Author contributions

WFW, KB and CYL contributed to the conceptualisation and framework of the manuscript. ICCN, TK, SYCC, SKN, AFA, and HM wrote the manuscript using the input from KB, CYL and WFW. All authors contributed to the manuscript revision, read, and approved the final submitted version.

Funding

The authors thank the Malaysian Ministry of Higher Education (MOHE) for funding through the Fundamental Research Grant Scheme (FRGS/1/2024/SKK15/TAYLOR/02/1). Irene Ngu is a postgraduate student whose work was supported by Taylor’s University through its TAYLOR’S RESEARCH SCHOLARSHIP Programme. Financial support for this project was provided by NIH R01EY029645, NIH R21NS130471, NIH 1R21NS13834-01A1, The Robert A. Welch Foundation AU-2182-20240404, and Texas A&M University T3-Grants Program (246292-00000).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.