Forkhead Box Protein M1 Expression Research Articles

A role for YAP/FOXM1/Nrf2 axis in oxidative stress and apoptosis of cataract induced by UVB irradiation.

This study aims to investigate the hypothesis that Yes-associated protein (YAP) significantly regulates antioxidant potential and anti-apoptosis in UVB-induced cataract by exploring the underlying molecular mechanisms. To investigate the association between YAP and cataract, various experimental techniques were employed, including cell viability assessment, Annexin V FITC/PI assay, measurement of ROS production, RT-PCR, Western blot assay, and Immunoprecipitation. UVB exposure on human lens epithelium cells (HLECs) reduced total and nuclear YAP protein expression, increased cleaved/pro-caspase 3 ratios, decreased cell viability, and elevated ROS levels compared to controls. Similar Western blot results were observed in invivo experiments involving UVB-treated mice. YAP knockdown invitro demonstrated a decrease in the protein expression of FOXM1, Nrf2, and HO-1, which correlated with the mRNA expression, accompanied by an increase in cell apoptosis, caspase 3 activation, and the release of ROS. Conversely, YAP overexpression mitigated these effects induced by UVB irradiation. Immunoprecipitation revealed a FOXM1-YAP interaction. Notably, inhibiting FOXM1 decreased Nrf2 and HO-1, activating caspase 3. Additionally, administering the ROS inhibitor N-acetyl-L-cysteine (NAC) effectively mitigated the apoptotic effects induced by oxidative stress from UVB irradiation, rescuing the protein expression levels of YAP, FOXM1, Nrf2, and HO-1. The initial findings of our study demonstrate the existence of a feedback loop involving YAP, FOXM1, Nrf2, and ROS that significantly influences the cell apoptosis in HLECs under UVB-induced oxidative stress.

Expression of Forkhead Box M1 and Anticancer Effects of FOXM1 Inhibition in Epithelioid Sarcoma

Epithelioid sarcoma (ES) is a rare aggressive sarcoma that, unlike most soft-tissue sarcomas, shows a tendency toward local recurrence and lymph node metastasis. Novel antitumor agents are needed for ES patients. Forkhead box transcription factor 1 (FOXM1) is a member of the Forkhead transcription factor family and is associated with multiple oncogenic functions; FOXM1 is known to be overexpressed and correlated with pathogenesis in various malignancies. In this study, we immunohistochemically analyzed FOXM1 expression levels and their clinical, clinicopathologic, and prognostic significance in 38 ES specimens. In addition, to investigate potential correlations between FOXM1 downregulation and oncologic characteristics, we treated ES cell lines with thiostrepton, a naturally occurring antibiotic that inhibits both small interfering RNA (siRNA) and FOXM1. In the analyses using ES samples, all 38 specimens were diagnosed as positive for FOXM1 by immunohistochemistry. We separated specimens into high (n = 19) and low (n = 19) FOXM1–protein expression groups by staining index score, and into large (n = 12), small (n = 25), and unknown (n = 1) tumor-size groups using a cutoff of 5 cm maximum diameter. Although there were significantly more samples with high FOXM1 expression in the large tumor group (P = .013), there were no significant differences with respect to age (P = 1.00), sex (P = .51), primary site of origin (P = .74), histologic subtypes (P = 1.00), depth (P = .74), or survival rate (P = .288) between the high and low FOXM1–protein expression groups. In the in vitro experiments using ES cell lines, FOXM1 siRNA and thiostrepton successfully downregulated FOXM1 mRNA and protein expression. Furthermore, downregulation of FOXM1 inhibited cell proliferation, drug resistance against chemotherapeutic agents, migration, and invasion and caused cell cycle arrest in the ES cell lines. Finally, cDNA microarray analysis data showed that FOXM1 regulated cIAP2, which is one of the apoptosis inhibitors activated by the TNFα-mediated NF-κB pathway. In conclusion, the FOXM1 gene may be a promising therapeutic target for ES.

FOXM1 and CHD4 expression is associated with chemoresistance in hepatoblastoma

Hepatoblastoma (HB) is the most common malignant liver tumor in childhood. Although pre-operative cisplatin (CDDP)-based chemotherapy is often used in cases of HB, about 20％ of HB patients exhibit resistance to CDDP. Forkhead box protein M1 (FOXM1) and chromo-domain-helicase-DNA-binding protein 4 (CHD4) have been associated with CDDP resistance in various tumors. We here analyzed the immunohistochemical expression of FOXM1 and CHD4 in HB specimens of 33 patients (mean age: 20 months) post-chemotherapy. The differentiation of specimens was assessed using the digital pathology software QuPath®, and then the relation between the FOXM1 or CHD4 expression and the differentiation and various other clinicopathological parameters was investigated. The histological type was epithelial in 19 cases (57.6%) and mixed epithelial and mesenchymal in 14 cases (42.4%). Nine cases had only a fetal component, 1 case had only an embryonal component, 22 cases had both fetal and embryonal components, and 1 case had no viable tumor. Both the FOXM1 and CHD4 immunoexpressions were found significantly more frequently in the embryonal than fetal components (p<0.0001 and p<0.0001, respectively). Regarding chemotherapy efficacy, the alpha-fetoprotein (AFP) level after chemotherapy was correlated with both the imaging shrinkage rate (R=-0.52) and histological residual rate (the percentage of the viable tumors of HB after chemotherapy)(R=0.62). High FOXM1 score was correlated with a high-postoperative AFP value (p<0.01) and a low AFP attenuation rate (p<0.05), but the FOXM1 score was not correlated with the imaging shrinkage rate (p=0.4418) or histological residual rate (p=0.4418). High CHD4 score showed a nonsignificant trend toward correlation with high postoperative AFP value (p=0.0849) and was not significantly correlated with the other parameters. Collectively, our results showed that FOXM1 expression may be useful in evaluating the response to CDDP-based chemotherapeutic regimens. Accurate measurement of FOXM1 expression by our scoring system using QuPath® is important in cases with mixed HB components of various differentiation levels.

Circadian Rhythms Metabolically Regulate Bone Marrow Retained Primitive Hematopoietic Stem Cell Size and Function

Most primitive hematopoietic stem cells (pHSC; LSK SLAM CD34 - EPCR +) are bone marrow (BM) retained in a quiescent non motile state, and are the only chemotherapy resistant HSC which requires their rapid exit from homeostatic quiescence on demand to prevent lethal hematology failure. Circadian rhythms induce daily release of progenitor cells and low levels of rarely cycling pHSCs to replenish the blood and lymphatics with new maturing cells. Metabolically, pHSCs are characterized by their dependence on glycolysis, with low mitochondrial activity and membrane potential (MMP), associated with higher competitive repopulation potential. However, if their metabolism is influenced by circadian cues is currently unknown. We report here that pHSCs at night are more glycolytic with higher surface expression of the glucose transporter Glut1, and higher glucose uptake. Repeated administration of the glucose analog, 2-deoxyglucose (2-DG), during daylight mimicked the night phenotype and enhanced HSC engraftment potential. However, nocturnal administration of 2-DG did not further improve glucose uptake nor HSC engraftment potential, suggesting glycolytic saturation. To further understand these metabolic oscillations of glucose uptake, we investigated the circadian, transcriptional signatures by single pHSC RNA-seq analysis. We found higher expression of mitophagy-related genes at night. Genes regulating mitochondrial function, were also significantly down regulated at night leading to low MMP in pHSC. While mitochondrial ROS levels were higher, cytoplasmic ROS levels were significantly reduced in pHSC at night. Importantly, we further found at night, low-functioning pHSC mitochondria to also undergo higher levels of fission as assessed by increased expression of the active mitochondrial fission marker, phospho-Drp1 (S616). To better understand the molecular pathways mediating daily circadian metabolic reprogramming between glycolysis and mitochondrial activity, we monitored our gene set enrichment analysis. We found the Wnt/b-catennin and HIF-1a signaling pathways to be significantly increased at night in pHSC. Active Wnt signaling, leads to enhanced b-catenin localization to the nucleus which is enhanced by downstream effector of Wnt activation, FOXM1. We observed higher total protein expression of FOXM1 at night. While its nuclear and cytoplasmic localization were reduced, we observed significantly higher FOXM1 levels in the mitochondria. FOXM1 localization to the energy powerhouse has been shown to inhibit mitochondrial activity. Blocking mitochondrial recruitment of FOXM1 with a specific canonical Wnt inhibitor at night led to upregulation of MMP, reversing mitochondrial metabolic responses in pHSC. Finally, daily genetic changes between night Wnt, HIF-1a and glycolysis, versus daylight GSK3b, m-TOR signaling and enhanced mitochondrial activity in BM retained pHSC, also led to cell size changes. We documented reduced quiescent pHSC size at night associated with their increased BM maintenance and higher repopulation potential. While larger sized pHSCs in the morning during the time of blood replenishment, increased their migration and development potential, for their rapid exit from quiescence on demand. Our study reveals daily circadian cues dynamically determine Wnt-mediated FOXM1 bidirectional shuttling between the nucleus and the mitochondria, leading to metabolic switching of pHSCs to maintain their cellular fitness. Our data define key signaling pathways controlling pHSC quiescence and diverse circadian metabolic activities for accumulated ROS and old mitochondria clearance and turnover, keeping them ready for rapid activation in alarm situations.

Forkhead Box M1 Regulates the Proliferation,Invasion,and Drug Resistance of Gastric Cancer Cells via circ_NOTCH1

Objective To investigate the impacts of forkhead box M1(FOXM1)on the proliferation,invasion,and drug resistance of gastric cancer cells by regulating the circular RNA circ_NOTCH1.Methods Western blotting and real-time quantitative PCR were performed to determine the expression of FOXM1 protein and circ_NOTCH1,respectively,in the gastric cancer tissue,para-carcinoma tissue,human normal gastric mucosa epithelial cell line GES-1 and gastric cancer cell lines MGC-803,HGC-27,and BGC-823.BGC-823 cells were classified into the following groups:control,short hairpin RNA FOXM1(sh-FOXM1)and negative control(sh-NC),small interfering RNA circ_NOTCH1(si-circ_NOTCH1)and negative control(si-NC),and sh-FOXM1+circ_NOTCH1 overexpression plasmid(sh-FOXM1+pcDNA-circ_NOTCH1)and sh-FOXM1+negative control(sh-FOXM1+pcDNA).CCK-8 assay and clone formation assay were employed to measure the cell proliferation,and Transwell assay to measure cell invasion.After treatment with 1.0 mg/L adriamycin for 48 h,the cell resistance in each group was analyzed.Western blotting was employed to determine the expression levels of FOXM1,proliferating cell nuclear antigen(PCNA),Bax,multi-drug resistance-associated protein 1(MRP1),and multi-drug resistance gene 1(MDR1).RNA pull-down and RNA immunoprecipitation were employed to examine the binding of circ_NOTCH1 to FOXM1 protein.Results Compared with those in the para-carcinoma tissue,the expression levels of FOXM1 protein and circ_NOTCH1 in the gastric cancer tissue were up-regulated(all P<0.001).Compared with GES-1 cells,MGC-803,HGC-27,and BGC-823 cells showed up-regulated expression levels of FOXM1 protein and circ_NOTCH1(all P<0.001).Compared with the control group and sh-NC group,the sh-FOXM1 group with down-regulated expression of FOXM1 protein and circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with the control group and the si-NC group,the si-circ_NOTCH1 group with down-regulated expression of circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with sh-FOXM1 group and sh-FOXM1+pcDNA group,the sh-FOXM1+pcDNA-circ_NOTCH1 group with up-regulated expression of circ_NOTCH1 showed increased optical density value,clone formation rate,cell invasion number,and cell viability,up-regulated expression of PCNA,MRP1,and MDR1,and down-regulated expression of Bax protein(all P<0.001).FOXM1 protein was able to interact with circ_NOTCH1.Conclusion Interference with FOXM1 may inhibit the proliferation,invasion,and drug resistance of gastric cancer cells by silencing circ_NOTCH1 expression.

Domatinostat Targets the FOXM1-Survivin Axis to Reduce the Viability of Ovarian Cancer Cells Alone and in Combination with Chemotherapeutic Agents.

The deregulation of the FOXM1 transcription factor is a key molecular alteration in ovarian cancer, contributing to the development and progression of ovarian cancer via activation of the target genes. As such, FOXM1 is a highly attractive therapeutic target in the treatment of ovarian cancer, but there has been no clinically tested FOXM1 inhibitor to date. We investigated in this study the effects of domatinostat, a class I-selective HDAC inhibitor currently in the clinical stage of development as a cancer therapeutic, on the expression of FOXM1 and viability of ovarian cancer cells. Cell viability, as well as protein and mRNA expression of FOXM1 and its transcriptional target survivin, was examined after domatinostat treatment of TOV21G and SKOV3 ovarian cancer cell lines in the absence or presence of cisplatin and paclitaxel. The effect of FOXM1 knockdown on survivin expression and those of genetic and pharmacological inhibition of survivin alone or in combination with the chemotherapeutic agents on cell viability were also examined. Domatinostat reduced the protein and mRNA expression of FOXM1 and survivin and also the viability of ovarian cancer cells alone and in combination with cisplatin or paclitaxel at clinically relevant concentrations. Knockdown experiments showed survivin expression was dependent on FOXM1 in ovarian cancer cells. Survivin inhibition was sufficient to reduce the viability of ovarian cancer cells alone and in combination with the chemotherapeutic agents. Our findings suggest that domatinostat, which effectively targets the FOXM1-survivin axis required for the viability of ovarian cancer cells, is a promising option for the treatment of ovarian cancer.

Abstract A09: Targeting of FOXM1 recapitulates NPM1 mutations in chemo-sensitization of AML

Abstract NPM1 mutations are among the most common mutations in CN-AML (Cytogenetically Normal-AML) and are known to enhance chemosensitivity in AML patients. Across multiple co-operative group studies, in the absence of deleterious mutations, patients with NPM1 mutations show superior overall and relapse-free survival. The FOXM1 regulatory network is a major predictor of poor prognosis in human cancers of different origin, including AML. Early AML cell line and patient sample data from our lab demonstrated that nuclear NPM colocalizes with nuclear FOXM1 while mutant NPM sequesters FOXM1 in the cytoplasm. Applying RNAseq differential expression results from published FOXM1 knockout experiments on KG-1 cells and MLL-AF9-transformed mice to Beat AML RNAseq data, we developed a ~200-gene FOXM1 signature that was significantly associated with inferior CR/CRi rate (OR=0.44 [95% CI: 0.21–0.95], p=0.037) in wild type NPM1, wild type FLT3 de novo AML patients. Notably, this FOXM1 signature was not significantly associated with CR/CRi in mutated NPM1, wild type FLT3 patients (OR=1.78 [95% CI: 0.15–21.51], p=0.651). These data suggest that inactivation of FOXM1 may underlie the chemosensitivity conferred by the highly prevalent NPM1 mutation in favorable risk AML. Using our novel network-centric approach, we found a first-in-class small molecule (STL427944) that affects a pathway that limits FOXM1 expression and significantly disrupts the associated regulatory network. To directly address the role of FOXM1 in chemosensitivity of AML cells, we tested two FOXM1-specific inhibitory compounds: STL427944 and its derivative, STL001 (their structures will be disclosed). From a medicinal chemistry standpoint, STL427944 has two main metabolic liabilities, a hydrolytically labile furane-carboxylic acid phenolic ester and a hydrazone. We applied several paths of structural activity relationship (SAR) optimization to overcome these issues from removal of liabilities to bio-isosteric replacement to cyclizations. The ring replacement is likely to have significantly improved the overall stability observed in STL001, resulting in at least !0-fold more active compound with better ‘drug-like’ properties and thus enhanced potency. We showed that 500nM of STL001 partially suppressed expression of FOXM1 protein in AML, while 5mM of STL001 (versus 50mM of STL427944) fully suppressed FOXM1 and sensitized AML cells to venetoclax and cytarabine treatment. Ex vivo treatment of AML patient samples with STL427944 and STL001 resulted in inhibition of nuclear FOXM1 expression according to IHC and downregulation of canonical FOXM1 transcriptional targets as indicated by real-time PCR. This treatment potentiated the efficacy of venetoclax in inhibiting colony forming activity in AML patient samples. Our data suggest that targeting FOXM1 in AML patients with wild type NPM1 may recapitulate NPM1 mutations and will have a robust therapeutic effect leading to more favorable outcome for AML patients. Citation Format: Irum Khan, Sandjeev Raghuwanshi, Andy Kaempf, Jeff Tyner, Alex Domling, Carlos Camacho, Andrei Gartel. Targeting of FOXM1 recapitulates NPM1 mutations in chemo-sensitization of AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A09.

Increased FOXM1 Expression was Associated with the Prognosis and the Recruitment of Neutrophils in Endometrial Cancer.

Although the biological functions of Forkhead box protein M1 (FOXM1) were explored in a variety of cancer, to date, however, little attention has been paid to the situation of FOXM1 in EC endometrial cancer (EC). Bioinformatics analysis, including GEPIA, TIMER, cBioPortal, LinkedOmics, and STRING were used to analyze the FOXM1 gene expression, genetic alteration, and immune cell infiltration in EC. IHC staining, qPCR, cell viability, and migration assay were applied to identify the functions of FOXM1 in EC. FOXM1 was highly expressed in EC tissues and closely correlated with the prognosis of EC patients. FOXM1 knockdown inhibited EC cell proliferation and invasion as well as migration. FOXM1 genetic alteration was verified in EC patients. Coexpression network of FOXM1 indicated that it had roles in the EC cell cycle and the infiltration of immune cells in EC. Furthermore, bioinformatic and immunohistochemical analysis indicated that FOXM1 induced the increased CD276 expression and also enhanced the neutrophil recruitment in EC. Our present study discovered a novel role of FOXM1 in EC, suggesting FOXM1 could be treated as a potential prognostic biomarker and immunotherapeutic target in EC diagnosis and treatment.

FOXM1 promotes TGF-β2-induced injury of human lens epithelial cells by up regulating VEGFA expression.

To explore whether Fork head box protein M1 (FOXM1) is involved in TGF-β2-induced injury of human lens epithelial cells and its related mechanism. Human lens epithelium samples from cataract patients and healthy controls were collected. A cellular epithelial injury model was established by treating HLE-B3 cells with TGF-β2. QPCR, immunoblot assays were performed to detect the levels of FOXM1 in human cataract samples and the lens epithelial injury cell model. FOXM1 siRNA and pcDNA3.1-FOXM1 plasmids were transfected into the cells to knockdown and overexpress FOXM1, respectively. MTT and wound closure and transwell assays were performed to analyze cell proliferation and migration in HLE-B3 cells. Immunoblot assays were also conducted to detect the effects of FOXM1 on EMT, VEGFA and MAPK/ERK signaling. We found high expression of FOXM1 in lens tissues of cataract patients. Silencing of FOXM1 in TGF-β2-induced HLE-B3 cells suppressed cell proliferation, migration, and the EMT process. Mechanistically, we found that downregulation of FOXM1 inhibited the VEGFA/MAPK signaling pathway in TGF-β2-induced HLE-B3 cells. FOXM1 promoted TGF-β2-induced injury of human lens epithelial cells (hLECs) by promoting VEGFA expression. FOXM1 could be a potential drug target for the treatment of ocular diseases.

High expression of cytoplasmic FOXO3 protein associated with poor prognosis of rectal cancer patients: A study from Swedish clinical trial of preoperative radiotherapy to big database analysis

IntroductionAccumulating evidence has implicated a pivotal role for FOXO3, FOXM1 and SIRT6 in cancer progression. The majority of researches focused on the functions of these proteins in drug resistance, but their relationships with radiotherapy (RT) response remain unclear. In this study, we examined protein expression of FOXO3, FOXM1 and SIRT6 and their clinical significance in a Swedish rectal cancer trial of preoperative RT. MethodsExpression of FOXO3, FOXM1 and SIRT6 protein was examined by immunohistochemistry in patient samples. Genetic analysis of FOXO3, FOXM1 and SIRT6 were performed by cBioportal and MEXPRESS database. Gene-gene network analysis was conducted using GeneMANIA. Functional enrichment analysis was performed based on LinkedOmics and Metascape online software. ResultsFOXO3 and FOXM1were mainly expressed in the cytoplasm in both normal and tumour tissues, and SIRT6 in both the cytoplasm and nucleus in normal and tumour tissues. FOXO3 and FOXM1 expression increased from normal mucosa to primary cancer (P < 0.001), while SIRT6 expression decreased from normal mucosa to primary cancer (P < 0.001). High FOXO3 expression correlated with late TNM stage (P = 0.040), distant metastasis (P = 0.032) and independently with disease free survival (DFS) in the RT patients (HR = 7.948; P = 0.049; 95% CI = 1.002–63.032) but not in non-RT patients (P > 0.05). Genetic analysis indicated that DNA methylation status contributed to FOXO3 overexpression. Functional enrichment analysis demonstrated that FOXO3 was closely related to metabolism-related signalling pathway which in turn associated with cancer radioresistance. Moreover, there were strong gene–gene interactions between FOXO3 and metabolism-related signalling. ConclusionsOur findings suggest that FOXO3 may be a prognostic factor in rectal cancer patients with RT.

FOXM1 Contributes to Chemotherapy Sensitivity in Cervical Cancer by Regulating TTK.

The emergence of chemotherapy resistance usually causes therapeutic failure in advanced cervical cancer. Forkhead box protein M1 (FOXM1) and threonine tyrosine kinase (TTK) are closely associated with cancer drug sensitivity, but the mechanism of FOXM1 on TTK involvement in chemo-treated cervical cancer remains unclear. Here, we aimed to observe the effects of FOXM1 on TTK and on chemotherapy sensitivity in cervical cancer. The expressions of FOXM1 and TTK in cervical cancer tissues and para-cancerous tissues were analyzed by immunohistochemistry. SiHa and Hela cells were transfected with human lentivirus-FOXM1, small interfering RNA (siRNA) or pcDNA3.1/FOXM1 to analyze the changes in TTK protein expression. Furthermore, the cells were treated with paclitaxel (8 μM) or cisplatin (10 μM) to analyze the effects of FOXM1 on chemotherapy sensitivity. SiHa cells were used to construct a xenograft model to study the effects of FOXM1 expression in response to paclitaxel treatment. The tumor size and weight were observed. The expressions of Ki-67, FOXM1, and TTK protein in tumor tissues were measured by immunohistochemistry. High expression of FOXM1 and TTK were found in the cervical cancer tissues (p < 0.05). The TTK protein expressions were decreased by FOMX1-siRNA transfection in SiHa and Hela cells (p < 0.01). The cell viability and cell cycle were also suppressed by FOMX1-siRNA transfection (p < 0.01) but enhanced by pcDNA3.1/FOXM1 transfection (p < 0.01). For paclitaxel or cisplatin treatment, the cell viability and cell DNA damage were improved due to the FOXM1 overexpression (p < 0.01). TTK inhibitor significantly suppressed the effects of FOXM1 overexpression (p < 0.01). FOXM1 regulated TTK and affected the therapeutic efficacy of cisplatin and paclitaxel in cervical cancer.

MKL1 fuels ROS-induced proliferation of vascular smooth muscle cells by modulating FOXM1 transcription

Reactive oxygen species (ROS) promotes vascular injury and neointima formation in part by stimulating proliferation of vascular smooth muscle cells (VSMC). The underlying transcriptional mechanism, however, is not completely understood. Here we report that VSMC-specific deletion of MKL1 in mice suppressed neointima formation in a classic model of vascular injury. Likewise, pharmaceutical inhibition of MKL1 activity by CCG-1423 similarly mollified neointima formation in mice. Over-expression of a constitutively active MKL1 in vascular smooth muscle cells enhanced proliferation in a ROS-dependent manner. On the contrary, MKL1 depletion or inhibition attenuated VSMC proliferation. PCR array based screening identified forkhead box protein M1 (FOXM1) as a direct target for MKL1. MKL1 interacted with E2F1 to activate FOXM1 expression. Concordantly, FOXM1 depletion ameliorated MKL1-dependent VSMC proliferation. Of interest, ROS-induced MKL1 phosphorylation through MK2 was essential for its interaction with E2F1 and consequently FOXM1 trans-activation. Importantly, a positive correlation between FOXM1 expression and VSMC proliferation was identified in arterial specimens from patients with restenosis. Taken together, our data suggest that a redox-sensitive phosphorylation-switch of MKL1 activates FOXM1 transcription and mediates ROS fueled vascular smooth muscle proliferation. Targeting the MK-2/MKL1/FOXM1 axis may be considered as a reasonable approach for treatment of restenosis.

Sequential Treatment with Activin and Hepatocyte Growth Factor Induces FOXM1 to Promote Colorectal Cancer Liver Metastasis.

Cancer stem cells (CSCs) are involved in liver metastasis in colorectal cancer (CRC). Activin and hepatocyte growth factor (HGF) are important regulators of stem cell properties. This study was performed to explore the effect of activin and HGF on CRC invasion and metastasis. The key genes involved in the action of activin and HGF in CRC were identified. HCT116 CRC cells were sequentially treated with activin and HGF and examined for migration and invasion in vitro and liver metastasis in vivo. RNA sequencing was performed to identify differentially expressed genes in response to activin and HGF. Sequential treatment with activin and HGF-enhanced CRC cell migration, invasion, and metastasis. CXCR4 and AFP expressions were increased by activin and HGF treatment. Knockdown of FOXM1 blocked liver metastasis from HCT116 cells pretreated with activin and HGF and suppressed CXCR4 and AFP expression. Activin alone increased the mRNA and protein expression of FOXM1. In contrast, HGF alone enhanced the phosphorylation of FOXM1, without altering the total protein level of FOXM1. SMAD2 was required for activin-mediated FOXM1 induction. FOXM1 transactivated CXCR4 by directly binding to the promoter of CXCR4. Additionally, CXCR4 regulated AFP expression through the NF-κB pathway. Sequential treatment with activin and HGF accelerates CRC invasion and liver metastasis, which involves the upregulation and activation of FOXM1 and induction of CXCR4 and AFP.

Evaluation of Forkhead BOX M1 (FOXM1) gene expression in colorectal cancer.

Forkhead Box M1 (FOXM1)-a key cell cycle regulator is a member of the Forkhead transcription factor family. It plays a key role in embryogenesis and cell proliferation and has been strongly linked to various solid tumors. We sought to understand the regulation of FOXM1 in colorectal cancer (CRC), as well as if and to what extent other clinicopathological characteristics are associated with FOXM1. The investigation comprised 98 CRC samples and normal tissues (controls). All colon cancer patients had a colonoscopy and targeted biopsy. All rectal cancer patients had a CT and MRI. Real-time PCR, Immunohistochemistry, and Western blotting were used to evaluate FOXM1 expression, and the findings were analyzed using SPSS (v.26). FOXM1 mRNA and protein expression were substantially upregulated in tumor tissues, with the majority of these proteins localized in nucleo-cytoplasm. Elevated protein levels of FOXM1 were strongly correlated with lower education level, larger tumor size, lymph node status, lymphovascular invasion (LVI), perineural invasion (PNI), lymph node metastasis (LNM), tumor invasion depth (subserosal and serosal invasion), late stage (III and IV), localization (nucleo-cytoplasmic), intensity (strong) and recurrence. Based on survival analysis, FOXM1 overexpression and nucleo-cytoplasmic localization were associated with shorter disease-free survival while stage and PNI were linked to poorer overall and disease-free survival. According to the results of the Cox regression analysis, stage and PNI were significant predictors of prognosis in CRC patients. FOXM1 expression was elevated in CRC and was linked to reduced disease-free survival. These findings support prior reports and hence FOXM1 can be an important prognostic marker for CRC and a promising therapeutic target. Additionally, we found a link between poor disease-free survival and FOXM1's nucleo-cytoplasmic localization. However, since the sample size of this study was small, further research is needed to validate our findings.

Mutant p53 driven-LINC00857, a protein scaffold between FOXM1 and deubiquitinase OTUB1, promotes the metastasis of pancreatic cancer

Tumour metastasis is the major adverse factor for recurrence and death in pancreatic cancer (PC) patients. P53 mutations are considered to be the second most common type of mutation in PC and significantly promote PC metastasis. However, the molecular mechanisms underlying the effects of p53 mutations, especially the regulatory relationship of the protein with long noncoding RNAs (lncRNAs), remain unclear. In the present study, we demonstrated that the lncRNA LINC00857 exhibits a significantly elevated level in PC and that it is associated with poor prognosis; furthermore, TCGA data showed that LINC00857 expression was significantly upregulated in the mutant p53 group compared with the wild-type p53 group. Gain- and loss-of-function experiments showed that LINC00857 promotes the metastasis of PC cells. We further found that LINC00857 upregulates FOXM1 protein expression and thus accelerates metastasis in vitro and in vivo. Mechanistically, LINC00857 bound simultaneously to FOXM1 and to the deubiquitinase OTUB1, thereby serving as a protein scaffold and enhancing the interaction between FOXM1 and OTUB1, which inhibits FOXM1 degradation through the ubiquitin–proteasome pathway. Interestingly, we found that mutant p53 promotes LINC00857 transcription by binding to its promoter region. Finally, atorvastatin, a commonly prescribe lipid-lowering drug, appeared to inhibit PC metastasis by inhibiting the mutant p53-LINC00857 axis. Taken together, our results provide new insights into the biology driving PC metastasis and indicate that the mutant p53-LINC00857 axis might represent a novel therapeutic target for PC metastasis.

Exploring the prognostic significance of FOXM1 gene expression in human breast cancer by bioinformatics analysis

Breast cancer (BRCA) is one of the leading causes of cancer-related death worldwide. However, the preventive intervention of this disease is often obstructed by a lack of knowledge related to BRCA, later-stage diagnosis and lack of feasible treatment options. Here, the prognostic value of Forkhead Box M1 (FOXM1) was examined in BRCA to evaluate its potential as a prognostic marker. Initially, the gene was found to be noticeably upregulated (log2FC > 4) in BRCA tissues compared to adjacent normal breast tissues. Immunohistochemistry observation also revealed higher levels of FOXM1 protein expression in BRCA samples than in normal specimens. A number of somatic mutations with 0.2 % frequency, including 8 missense mutations, were observed inside the coding region of FOXM1. Moreover, the promoter of this gene was predicted to be hypomethylated, and its structural misorientation appeared to influence the patient's clinical outcome. Furthermore, the higher level of FOXM1 expression was also linked to a lower survival rate (HR > 1) of BRCA patients. Gene co-expression and further ontology and pathway analysis revealed that co-expressed genes of FOXM1 in BRCA tissue were involved in functions related to cell cycle maintenance. Altogether, the experimental findings of this study exert that FOXM1 could be a potential prognostic and therapeutic target for BRCA.

FOXM1-CD44 Signaling Is Critical for the Acquisition of Regorafenib Resistance in Human Liver Cancer Cells.

Regorafenib is a multikinase inhibitor that was approved by the US Food and Drug administration in 2017. Cancer stem cells (CSCs) are a small subset of cancer-initiating cells that are thought to contribute to therapeutic resistance. The forkhead box protein M1 (FOXM1) plays an important role in the regulation of the stemness of CSCs and mediates resistance to chemotherapy. However, the relationship between FOXM1 and regorafenib resistance in liver cancer cells remains unknown. We found that regorafenib-resistant HepG2 clones overexpressed FOXM1 and various markers of CSCs. Patients with hepatocellular carcinoma also exhibited an upregulation of FOXM1 and resistance to regorafenib, which were correlated with a poor survival rate. We identified a close relationship between FOXM1 expression and regorafenib resistance, which was correlated with the survival of patients with hepatocellular carcinoma. Thus, a strategy that antagonizes FOXM1–CD44 signaling would enhance the therapeutic efficacy of regorafenib in these patients.

CDI Exerts Anti-Tumor Effects by Blocking the FoxM1-DNA Interaction.

The Forkhead box protein M1 (FoxM1) is an appealing target for anti-cancer therapeutics as this cell proliferation-associated transcription factor is overexpressed in most human cancers. FoxM1 is involved in tumor invasion, angiogenesis, and metastasis. To discover novel inhibitors that disrupt the FoxM1-DNA interaction, we identified CDI, a small molecule that inhibits the FoxM1–DNA interaction. CDI was identified through an assay based on the time-resolved fluorescence energy transfer response of a labeled consensus oligonucleotide that was bound to a recombinant FoxM1-dsDNA binding domain (FoxM1-DBD) protein and exhibited potent inhibitory activity against FoxM1-DNA interaction. CDI suppressed cell proliferation and induced apoptosis in MDA-MB-231 cells obtained from a breast cancer patient. Furthermore, it decreased not only the mRNA and protein expression of FoxM1 but also that of downstream targets such as CDC25b. Additionally, global transcript profiling of MDA-MB-231 cells by RNA-Seq showed that CDI decreases the expression of FoxM1-regulated genes. The docking and MD simulation results indicated that CDI likely binds to the DNA interaction site of FoxM1-DBD and inhibits the function of FoxM1-DBD. These results of CDI being a possible effective inhibitor of FoxM1-DNA interaction will encourage its usage in pharmaceutical applications.

Bioinformatics Analysis Reveals FOXM1/BUB1B Signaling Pathway as a Key Target of Neosetophomone B in Human Leukemic Cells: A Gene Network-Based Microarray Analysis.

Abnormal expression of Forkhead box protein M1 (FOXM1) and serine/threonine kinase Budding uninhibited by benzimidazoles 1 (BUB1B) contributes to the development and progression of several cancers, including chronic myelogenous leukemia (CML). However, the molecular mechanism of the FOXM1/BUB1B regulatory network and the role of Neosetophomone-B (NSP-B) in leukemia remains unclear. NSP-B, a meroterpenoid fungal secondary metabolite, possesses anticancer potential in human leukemic cells lines; however, the underlying mechanism has not been elucidated. The present study aimed to explore the role of NSP-B on FOXM1/BUB1B signaling and the underlying molecular mechanism of apoptosis induction in leukemic cells. We performed gene expression profiling of NSP-B-treated and untreated leukemic cells to search for differentially expressed genes (DEGs). Interestingly BUB1B was found to be significantly downregulated (logFC -2.60, adjusted p = 0.001) in the treated cell line with the highest connectivity score among cancer genes. Analysis of TCGA data revealed overexpression of BUB1B compared to normal in most cancers and overexpression was associated with poor prognosis. BUB1B also showed a highly significant positive correlation with FOXM1 in all the TCGA cancer types. We used human leukemic cell lines (K562 and U937) as an in vitro study model to validate our findings. We found that NSP-B treatment of leukemic cells suppressed the expression of FOXM1 and BUB1B in a dose-dependent manner. In addition, NSP-B also resulted in the downregulation of FOXM1-regulated genes such as Aurora kinase A, Aurora kinase B, CDK4, and CDK6. Suppression of FOXM1 either by siRNA or NSP-B reduced BUB1B expression and enhanced cell survival inhibition and induction of apoptosis. Interestingly combination treatment of thiostrepton and NSP-B suppressed of cell viability and inducted apoptosis in leukemic cells via enhancing the activation of caspase-3 and caspase-8 compared with single-agent treatment. These results demonstrate the important role of the FOXM1/BUB1B pathway in leukemia and thus a potential therapeutic target.

LINC00174 promotes cell proliferation and metastasis in renal clear cell carcinoma by regulating miR-612/FOXM1 axis

Background Kidney renal clear cell carcinoma (KIRC) is the most common pathological subtype of kidney tumor. Reportedly, LINC00174 is a key regulator in cancer progression. This study aims to clarify the role and molecular mechanism of LINC00174 in the progression of KIRC. Methods LINC00174 expression in KIRC and its prognostic value were analyzed by bioinformatics. LINC00174, miR-612 and FOXM1 mRNA expression levels in KIRC clinical samples and cell lines were detected by qRT-PCR. After LINC00174 was overexpressed or knocked down, CCK-8, BrdU and Transwell assays were adopted to evaluate the proliferation and metastatic potential of KIRC cells. Bioinformatics and dual luciferase reporter assays were employed to validate the targeting relationship between miR-612 and LINC00174 or FOXM1 mRNA, respectively. Western blot assay was performed to detect FOXM1 protein expression in KIRC cells. Results LINC00174 expression and FOXM1 expression were up-regulated in 42 cases of KIRC tissues (p < 0.001), while miR-612 expression was down-regulated (p < .001). LINC00174 overexpression or miR-612 inhibitor promoted the viability and proliferation of KIRC cells (p < .01). Migration and invasion of KIRC cells were promoted when the cells were transfected with LINC00174 overexpression or miR-612 inhibitor (p < .05). LINC00174 can competitively bind with miR-612 to repress the expression of miR-612, in turn up-regulate the expression of FOXM1 mRNA. Conclusion LINC00174 facilitates the proliferation and metastatic potential of KIRC cells via regulating the miR-612/FOXM1 axis.