Small-molecule inhibitors targeting FOXM1: Current challenges and future perspectives in cancer treatments.

Forkhead box (FOX) protein M1 (FOXM1) is a critical proliferation-associated transcription factor (TF) that is aberrantly overexpressed in the majority of human cancers and has also been implicated in poor prognosis. A comprehensive understanding of various aspects of this molecule has revealed its role in, cell proliferation, cell migration, invasion, angiogenesis and metastasis. The FOXM1 as a TF directly or indirectly regulates the expression of several target genes whose dysregulation is associated with almost all hallmarks of cancer. Moreover, FOXM1 expression is associated with chemoresistance to different anti-cancer drugs. Several studies have confirmed that suppression of FOXM1 enhanced the drug sensitivity of various types of cancer cells. Current data suggest that small molecule inhibitors targeting FOXM1 in combination with anticancer drugs may represent a novel therapeutic strategy for chemo-resistant cancers. In this review, we discuss the clinical utility of FOXM1, further, we summarize and discuss small-molecule inhibitors targeting FOXM1 and categorize them according to their mechanisms of targeting FOXM1. Despite great progress, small-molecule inhibitors targeting FOXM1 face many challenges, and we present here all small-molecule FOXM1 inhibitors in different stages of development. We discuss the current challenges and provide insights on the future application of FOXM1 inhibition to the clinic.

FOXM1 increases hTERT protein stability and indicates poor prognosis in gastric cancer.

Gastric cancer is one of most lethal diseases across the world. However, the underlying mechanism of gastric cancer carcinogenesis and development is still not fully known. Forkhead box M1 (FOXM1) belongs to the FOX family and has crucial roles in transactivation of multiple oncogenes in several cancer types, including gastric cancer. Recent studies have also shown the non-transcriptional function of FOXM1 via protein-protein interactions. Human telomerase reverse transcriptase (hTERT) is the core subunit of telomerase that facilitates cancer initiation and progression by maintaining cell immortalization, promoting cell proliferation and inhibiting cell apoptosis. However, the relationship between FOXM1 and hTERT in gastric cancer is still unclear. In our study, we found that FOXM1 and hTERT were convergent to the cell cycle-related pathways and they were positively related with advanced gastric cancer stages and poor outcomes. Simultaneous high levels of FOXM1 and hTERT predicted the worst prognosis. FOXM1 could increase hTERT protein rather than mRNA levels in a non-transcriptional manner. Mechanistically, FOXM1 interrupted the interaction between the E3 ligase MKRN1 and hTERT and decreased hTERT protein degradation. Further studies revealed that FOXM1 interacted with hTERT through its DNA-binding domain (DBD) region. Finally, we found that hTERT played important roles in FOXM1-mediated activation of the Wnt/ß-catenin pathway to promote gastric cancer cell proliferation. Taken together, we found a novel non-classical function of FOXM1 to increase hTERT protein stability. Targeting the FOXM1-hTERT pathway may be a potential therapeutic strategy in treating gastric cancer.

Activation of Autophagy Induces Monocrotaline-Induced Pulmonary Arterial Hypertension by FOXM1-Mediated FAK Phosphorylation.

PURPOSE: It has been shown that activation of autophagy promotes the development of pulmonary arterial hypertension (PAH). Meanwhile, forkhead box M1 (FOXM1) has been found to induce autophagy in several types of cancer. However, it is still unclear whether FOXM1 mediates autophagy activation in PAH, and detailed mechanisms responsible for these processes are indefinite. METHOD: PAH was induced by a single intraperitoneal injection of monocrotaline (MCT) to rats. The right ventricle systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-smooth muscle actin (α-SMA) staining, and Ki67 staining were performed to evaluate the development of PAH. The protein levels of FOXM1, phospho-focal adhesion kinase (p-FAK), FAK, and LC3B were determined by immunoblotting or immunohistochemistry. RESULTS: FOXM1 protein level and FAK activity were significantly increased in MCT-induced PAH rats, this was accompanied with the activation of autophagy. Pharmacological inhibition of FOXM1 or FAK suppressed MCT-induced autophagy activation, decreased RVSP, RVHI and %MT in MCT-induced PAH rats, and inhibited the proliferation of pulmonary arterial smooth muscle cells and pulmonary vessel muscularization in MCT-induced PAH rats. CONCLUSION: FOXM1 promotes the development of PAH by inducing FAK phosphorylation and subsequent activation of autophagy in MCT-treated rats.

FOXM1 Inhibition Enhances the Therapeutic Outcome of Lung Cancer Immunotherapy by Modulating PD-L1 Expression and Cell Proliferation.

Programmed death-ligand 1 (PD-L1) is a major target to cancer immunotherapy, and anti-PD-L1 and anti-PD-1 antibody-mediated immunotherapy are being increasingly used. However, immune checkpoint inhibitors (ICIs) are ineffective in treating large tumors and cause various immune-related adverse events in nontarget organs, including life-threatening cardiotoxicity. Therefore, the development of new therapeutic strategies to overcome these limitations is crucial. The focus of this study is the forkhead box protein M1 (FOXM1), which is identified as a potential therapeutic target for cancer immunotherapy and is associated with the modulation of PD-L1 expression. Selective small interfering RNA knockdown of FOXM1 or treatment with thiostrepton (TST) significantly reduces PD-L1 expression in non-small-cell lung cancer (NSCLC) cells and inhibits proliferation. Chromatin immunoprecipitation-PCR reveals that FOXM1 selectively upregulates PD-L1 expression by binding directly to the PD-L1 promoter. In vivo animal studies have shown that TST treatment significantly downregulates PD-L1 expression in human NSCLC tumors, while greatly reducing tumor size without side effects on normal tissues. Combined treatment with TST and anti-4-1BB antibody in the LLC-1 syngeneic tumor model induces synergistic therapeutic outcomes against immune resistant lung tumors as well as 2.72-folds higher CD3+ T cells in tumor tissues compared to that in the anti-4-1BB antibody treatment group.

A database of integrated molecular and phytochemical interactions of the foxm1 pathway for lung cancer.

The FoxM1 pathway is an oncogenic signaling pathway involved in essential mechanisms including control cell-cycle progression, apoptosis and cell growth which are the common hallmarks of various cancers. Although its biological functions in the tumor development and progression are known, the mechanism by which it participates in those processes is not understood. The present work reveals images of the oncogenic FoxM1 pathway controlling the cell cycle process with alternative treatment options via phytochemical substances in the lung cancer study. The downstream significant protein modules of the FoxM1 pathway were extracted by the Molecular Complex Detection (MCODE) and the maximal clique (Mclique) algorithms. Furthermore, the effects of post-transcriptional modification by microRNA, transcription factor binding and the phytochemical compounds are observed through their interactions with the lung cancer protein modules. We provided two case studies to demonstrate the usefulness of our database. Our results suggested that the combination of various phytochemicals is effective in the treatment of lung cancer. The ultimate goal of the present work is to partly support the discovery of plant-derived compounds in combination treatment of classical chemotherapeutic agents to increase the efficacy of lung cancer method probably with minor side effects. Furthermore, a web-based system displaying results of the present work is set up for investigators posing queries at http://sit.mfu.ac.th/lcgdb/index_FoxM1.php.Communicated by Ramaswamy H. Sarma.

FOXM1 regulates the proliferation, apoptosis and inflammatory response of keratinocytes through the NF-κB signaling pathway.

Psoriasis is a common immune-mediated and genetic skin disease. Forkhead box M1 (FOXM1) is a member of FOX family that has been found to modulate skin disorders. However, its role in psoriasis remains unknown. Thus, we aimed to investigate the effect of FOXM1 on keratinocytes in response to tumor necrosis factor-α (TNF-α). The expression levels of FOXM1 in psoriasis tissues and normal skin tissues were examined using qRT-PCR and western blot. HaCaT cells were stimulated by TNF-α to mimic psoriasis in vitro. MTT assay was performed to assess cell proliferation. The caspase-3 activity and expression levels of bcl-2 and bax were determined to indicate cell apoptosis. The mRNA and secretion levels of IL-6, IL-23 and TGF-ß were determined by qRT-PCR and ELISA, respectively. The NF-κB activation was assessed using western blot analysis. Our results demonstrated that FOXM1 was highly upregulated in psoriatic skin tissues and TNF-α-stimulated HaCaT cells. Knockdown of FOXM1 repressed cell proliferation of TNF-α-stimulated HaCaT cells. Knockdown of FOXM1 caused significant increases in caspase-3 activity, bax expression and decrease in bcl-2 expression in TNF-α-stimulated HaCaT cells. Moreover, FOXM1 knockdown also suppressed the TNF-α-induced production of IL-6, IL-23, and TGF-ß in HaCaT cells. However, FOXM1 overexpression showed the opposite effect. Furthermore, the TNF-α-induced NF-κB activation was prevented by FOXM1 knockdown. Additionally, inhibition of NF-κB reversed the effects of FOXM1 on HaCaT cells. Taken together, these findings indicated that FOXM1 regulated cell proliferation, apoptosis and inflammation in TNF-α-induced HaCaT cells. The effects of FOXM1 were mediated by NF-κB pathway.

A YAP/FOXM1 axis mediates EMT-associated EGFR inhibitor resistance and increased expression of spindle assembly checkpoint components.

Acquired resistance to tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) remains a clinical challenge. Especially challenging are cases in which resistance emerges through EGFR-independent mechanisms, such as through pathways that promote epithelial-to-mesenchymal transition (EMT). Through an integrated transcriptomic, proteomic, and drug screening approach, we identified activation of the yes-associated protein (YAP) and forkhead box protein M1 (FOXM1) axis as a driver of EMT-associated EGFR TKI resistance. EGFR inhibitor resistance was associated with broad multidrug resistance that extended across multiple chemotherapeutic and targeted agents, consistent with the difficulty of effectively treating resistant disease. EGFR TKI-resistant cells displayed increased abundance of spindle assembly checkpoint (SAC) proteins, including polo-like kinase 1 (PLK1), Aurora kinases, survivin, and kinesin spindle protein (KSP). Moreover, EGFR TKI-resistant cells exhibited vulnerability to SAC inhibitors. Increased activation of the YAP/FOXM1 axis mediated an increase in the abundance of SAC components in resistant cells. The clinical relevance of these finding was indicated by evaluation of specimens from patients with EGFR mutant lung cancer, which showed that high FOXM1 expression correlated with expression of genes encoding SAC proteins and was associated with a worse clinical outcome. These data revealed the YAP/FOXM1 axis as a central regulator of EMT-associated EGFR TKI resistance and that this pathway, along with SAC components, are therapeutic vulnerabilities for targeting this multidrug-resistant phenotype.

Nanoparticle Delivery of Proangiogenic Transcription Factors into the Neonatal Circulation Inhibits Alveolar Simplification Caused by Hyperoxia.

Rationale: Advances in neonatal critical care have greatly improved the survival of preterm infants, but the long-term complications of prematurity, including bronchopulmonary dysplasia (BPD), cause mortality and morbidity later in life. Although VEGF (vascular endothelial growth factor) improves lung structure and function in rodent BPD models, severe side effects of VEGF therapy prevent its use in patients with BPD.Objectives: To test whether nanoparticle delivery of proangiogenic transcription factor FOXM1 (forkhead box M1) or FOXF1 (forkhead box F1), both downstream targets of VEGF, can improve lung structure and function after neonatal hyperoxic injury.Methods: Newborn mice were exposed to 75% O2 for the first 7 days of life before being returned to a room air environment. On Postnatal Day 2, polyethylenimine-(5) myristic acid/polyethylene glycol-oleic acid/cholesterol nanoparticles containing nonintegrating expression plasmids with Foxm1 or Foxf1 cDNAs were injected intravenously. The effects of the nanoparticles on lung structure and function were evaluated using confocal microscopy, flow cytometry, and the flexiVent small-animal ventilator.Measurements and Main Results: The nanoparticles efficiently targeted endothelial cells and myofibroblasts in the alveolar region. Nanoparticle delivery of either FOXM1 or FOXF1 did not protect endothelial cells from apoptosis caused by hyperoxia but increased endothelial proliferation and lung angiogenesis after the injury. FOXM1 and FOXF1 improved elastin fiber organization, decreased alveolar simplification, and preserved lung function in mice reaching adulthood.Conclusions: Nanoparticle delivery of FOXM1 or FOXF1 stimulates lung angiogenesis and alveolarization during recovery from neonatal hyperoxic injury. Delivery of proangiogenic transcription factors has promise as a therapy for BPD in preterm infants.

The CXCL12/CXCR4 axis confers temozolomide resistance to human glioblastoma cells via up-regulation of FOXM1.

Glioblastoma multiforme (GBM) is the most common primary malignancy in the adult central nervous, and is characterized by high aggressiveness and a high mortality rate. The high mortality rate is largely due to the development of drug resistance. Temozolomide (TMZ) resistance is considered to be one of the major reasons responsible for GBM therapy failure. CXCL12/CXCR4 has been demonstrated to be involved in cell proliferation, migration, invasion, angiogenesis, and radioresistance in GBM. However, its role in TMZ resistance in GBM is unknown. In this study, we aimed to evaluate the role of CXCL12/CXCR4 in mediating the TMZ resistance to GBM cells and explore the underlying mechanisms. We found that the CXCL12/CXCR4 axis enhanced TMZ resistance in GBM cells. Further study showed that CXCL12/CXCR4 conferred TMZ resistance and promoted the migration and invasion of GBM cells by up-regulating FOXM1. This resistance was partially reversed by suppressing CXCL12/CXCR4 and FOXM1 silencing. Our study revealed the vital role of CXCL12/CXCR4 in mediating the resistance of GBM cells to TMZ, and suggested that targeting CXCL12/CXCR4 axis may attenuate the resistance to TMZ in GBM.

Multiple therapeutic peptide vaccines for patients with advanced gastric cancer.

We performed a clinical trial using HLA-A24-binding peptide vaccines containing a combination of novel cancer-testis antigens and anti-angiogenic peptides for advanced gastric cancer (GC). Thirty-five GC patients who had shown resistance to the standard therapy were enrolled in this clinical trial using vaccinations with a mixture of multiple peptides derived from DEPDC1, URLC10, FoxM1, Kif20A and VEGFR1. The safety, the overall survival (OS), and the immunological responses based on an ELISPOT assay were determined to assess differences in patients who were HLA-A24-positive [24(+)] and HLA-A24-negative [24(-)]. No severe adverse effects were observed except for severe skin reactions in 4 patients. The differences in OS were not significant between patients who were 24(+) and 24(-). In the 24(+) group, patients who showed T cell responses specific to antigen peptides had a tendency towards better survival than those who showed no response, especially to the DEPDC1 peptide. The patients with local skin reactions had significantly better OS than the others. Peptide vaccine therapy was found to be safe and is expected to induce specific T cell responses in patients with advanced GC. The survival benefit of peptide vaccine monotherapy may not have been shown and further trials are needed to confirm these results.

Immunotherapy based on dendritic cells pulsed with CTPFoxM1 fusion protein protects against the development of hepatocellular carcinoma.

Application of dendritic cells (DCs) pulsed with tumor-associated antigens is considered attractive in immunotherapy for hepatocellular carcinoma (HCC). In order to efficiently prime tumor-associated antigens specific for cytotoxic T lymphocytes (CTLs), it is important that DCs present tumor-associated antigens on MHC class I. MHC class I generally present endogenous antigens expressed in the cytosol. In this study, we developed a new antigen delivery tool based on cross presentation of exogenous antigens in DCs by using cytoplasmic transduction peptide (CTP). CTP protein could transduce FoxM1 tumor antigen into the cytosol of DCs, and CTP-FoxM1 fusion protein could stimulate activation and maturation of DCs. DCs pulsed with CTP-FoxM1 could induce specific CTLs. More importantly, the immunity induced by DCs loaded with CTP-FoxM1 could significantly inhibit tumor growth and metastasis in HCC-bearing mice, which was more potent than that induced by DCs loaded with FoxM1 or CTP, alone. Our results indicate that DCs pulsed with CTP-FoxM1 might be a promising vaccine candidate for HCC therapy and provide new insight into the design of DC-based immunotherapy.