FoxM1 Transcriptional Activity Research Articles

Epigenomic analyses identify FOXM1 as a key regulator of anti-tumor immune response in esophageal adenocarcinoma

Unlike most cancer types, the incidence of esophageal adenocarcinoma (EAC) has rapidly escalated in the western world over recent decades. Using whole genome bisulfite sequencing (WGBS), we identify the transcription factor (TF) FOXM1 as an important epigenetic regulator of EAC. FOXM1 plays a critical role in cellular proliferation and tumor growth in EAC patient-derived organoids and cell line models. We identify ERBB2 as an upstream regulator of the expression and transcriptional activity of FOXM1. Unexpectedly, gene set enrichment analysis (GSEA) unbiased screen reveals a prominent anti-correlation between FOXM1 and immune response pathways. Indeed, syngeneic mouse models show that FOXM1 inhibits the infiltration of CD8+ T cells into the tumor microenvironment. Consistently, FOXM1 suppresses CD8+ T cell chemotaxis in vitro and antigen-dependent CD8+ T cell killing. This study characterizes FOXM1 as a significant EAC-promoting TF and elucidates its novel function in regulating anti-tumor immune response.

Artemisinin Mediates Its Tumor-Suppressive Activity in Hepatocellular Carcinoma Through Targeted Inhibition of FoxM1.

The aberrant up-regulation of the oncogenic transcription factor Forkhead box M1 (FoxM1) is associated with tumor development, progression and metastasis in a myriad of carcinomas, thus establishing it as an attractive target for anticancer drug development. FoxM1 overexpression in hepatocellular carcinoma is reflective of tumor aggressiveness and recurrence, poor prognosis and low survival in patients. In our study, we have identified the antimalarial natural product, Artemisinin, to efficiently curb FoxM1 expression and activity in hepatic cancer cells, thereby exhibiting potential anticancer efficacy. Here, we demonstrated that Artemisinin considerably mitigates FoxM1 transcriptional activity by disrupting its interaction with the promoter region of its downstream targets, thereby suppressing the expression of numerous oncogenic drivers. Augmented level of FoxM1 is implicated in drug resistance of cancer cells, including hepatic tumor cells. Notably, FoxM1 overexpression rendered HCC cells poorly responsive to Artemisinin-mediated cytotoxicity while FoxM1 depletion in resistant liver cancer cells sensitized them to Artemisinin treatment, manifested in lower proliferative and growth index, drop in invasive potential and repressed expression of EMT markers with a concomitantly increased apoptosis. Moreover, Artemisinin, when used in combination with Thiostrepton, an established FoxM1 inhibitor, markedly reduced anchorage-independent growth and displayed more pronounced death in liver cancer cells. We found this effect to be evident even in the resistant HCC cells, thereby putting forth a novel combination therapy for resistant cancer patients. Altogether, our findings provide insight into the pivotal involvement of FoxM1 in the tumor suppressive activities of Artemisinin and shed light on the potential application of Artemisinin for improved therapeutic response, especially in resistant hepatic malignancies. Considering that Artemisinin compounds are in current clinical use with favorable safety profiles, the results from our study will potentiate its utility in juxtaposition with established FoxM1 inhibitors, promoting maximal therapeutic efficacy with minimal adverse effects in liver cancer patients.

Maslinic Acid Enhances Docetaxel Response in Human Docetaxel-Resistant Triple Negative Breast Carcinoma MDA-MB-231 Cells via Regulating MELK-FoxM1-ABCB1 Signaling Cascade.

Docetaxel (DOC) is the most important chemotherapeutic drug for the treatment of triple negative breast cancer (TNBC); however, acquired drug resistance upon the long-term treatment limits its therapeutic effect. Maslinic acid (MA), a natural triterpene from Olea europaea L., attracts increasing interest in recent years because of its promising anti-cancer activity, but the reversal effect of MA on drug resistance in cancer therapy is rarely explored. In this study, the combined effect of DOC and MA on human docetaxel-resistant triple negative breast carcinoma MDA-MB-231 (MDA-MB-231/DOC) cells was investigated. The enhanced effect of MA on DOC cytotoxicity and DOC accumulation was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and HPLC (high performance liquid chromatography) analysis in MDA-MB-231/DOC cells. Western blot, co-immunoprecipitation assay, luciferase reporter assay, and chromatin immunoprecipitation (ChIP) assay were performed for exploring the underlying mechanisms. Our data indicated that the co-treatment of MA could dose-dependently enhance DOC sensitivity and cellular DOC accumulation in MDA-MB-231/DOC cells. Moreover, MELK-FoxM1-ABCB1 signaling cascade was confirmed to contribute to DOC resistance in MDA-MB-231/DOC cells. In such process, MA directly suppressed expressions and interaction of MELK and FoxM1 as well as the transcriptional activity of FoxM1, and thus reducing the expression of ABCB1. Overall, our study suggests that the combined use of DOC and MA may be helpful for overcoming DOC resistance in human TNBC therapy.

Abstract AP12: FOXM1 INDUCES DNA REPLICATION STRESS AND PROMOTES GENOMIC INSTABILITY DOWNSTREAM OF CYCLIN E1 IN HIGH-GRADE SEROUS OVARIAN CANCER

Abstract PURPOSE: Genomic instability (GI) is a hallmark of high-grade serous ovarian cancer (HGSC), and induction of the FOXM1 transcription factor regulatory network is the second most frequent genetic alteration in HGSC. FOXM1 expression is associated with cancer GI, but the mechanistic link is unknown. DNA replication stress (DRS), which involves altered replication fork dynamics, is a key contributor to cancer GI. FOXM1 and cyclin E1 (CCNE1) are both expressed in STIC lesions in the fallopian tube epithelium (FTE), and CCNE1-CDK2 enhances FOXM1 transcriptional activity. Based on these observations, we hypothesized that: 1) FOXM1 is linked to cancer GI through the induction of DRS, and 2) FOXM1 promotes GI downstream of CCNE1, by promoting unscheduled M phase entry in cells that have not completed DNA synthesis. METHODS: We determined associations between FOXM1 and markers of DRS and GI in TCGA HGSC data. We overexpressed FOXM1c and a DNA binding domain mutant (DBD) in immortalized FTE cells and characterized the impact on DRS, DNA replication, and DNA damage. We determined the impact of CCNE1 on FOXM1 expression in immortalized FTE cells. We determined the combined effect of CCNE1 and FOXM1 on GI in FTE cells. RESULTS: FOXM1 was significantly associated with markers of DRS (CHK1-Ser345) and GI (fractional copy number altered genome (CNA)) in TCGA HGSC data. FOXM1 expression, but not its DBD mutant, induced biomarkers of DRS and DNA damage in hTERT/mutant Tp53 immortalized FTE cells. Unexpectedly, FOXM1c increased DNA replication fork rate in FTE cells. Increased fork rate is a novel and recently recognized cause of DRS. CCNE1 expression in FTE cells induced and activated FOXM1, promoting mitotic entry. FOXM1 and CCNE1 co-expression in FTE cells induced an expression signature of GI, and TCGA HGSC tumors with FOXM1 and CCNE1 co-expression showed significantly increased GI. CONCLUSIONS: We demonstrate that FOXM1 induces DRS and DNA damage in FTE cells, providing an explanation for the known link between FOXM1 and GI. Notably, FOXM1 induces a novel form of DRS involving increased replication fork rate. We show that CCNE1 activates FOXM1, which in turn promotes mitotic entry and GI. These data reveal a potentially major form of oncogenic collaboration in HGSC, and has implications for HGSC treatment approaches that promote DRS or inhibit the cellular DRS response. . Citation Format: Carter J Barger, Kajal Biswas, Linda Chee, Ronny Drapkin, Shyam Sharan, Adam R. Karpf. FOXM1 INDUCES DNA REPLICATION STRESS AND PROMOTES GENOMIC INSTABILITY DOWNSTREAM OF CYCLIN E1 IN HIGH-GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP12.

HMGA1 promotes breast cancer angiogenesis supporting the stability, nuclear localization and transcriptional activity of FOXM1

BackgroundBreast cancer is the most common malignancy in women worldwide. Among the breast cancer subtypes, triple-negative breast cancer (TNBC) is the most aggressive and the most difficult to treat. One of the master regulators in TNBC progression is the architectural transcription factor HMGA1. This study aimed to further explore the HMGA1 molecular network to identify molecular mechanisms involved in TNBC progression.MethodsRNA from the MDA-MB-231 cell line, silenced for HMGA1 expression, was sequenced and, with a bioinformatic analysis, molecular partners HMGA1 could cooperate with in regulating common downstream gene networks were identified. Among the putative partners, the FOXM1 transcription factor was selected. The relationship occurring between HMGA1 and FOXM1 was explored by qRT-PCR, co-immunoprecipitation and protein stability assays. Subsequently, the transcriptional activity of HMGA1 and FOXM1 was analysed by luciferase assay on the VEGFA promoter. The impact on angiogenesis was assessed in vitro, evaluating the tube formation ability of endothelial cells exposed to the conditioned medium of MDA-MB-231 cells silenced for HMGA1 and FOXM1 and in vivo injecting MDA-MB-231 cells, silenced for the two factors, in zebrafish larvae.ResultsHere, we discover FOXM1 as a novel molecular partner of HMGA1 in regulating a gene network implicated in several breast cancer hallmarks. HMGA1 forms a complex with FOXM1 and stabilizes it in the nucleus, increasing its transcriptional activity on common target genes, among them, VEGFA, the main inducer of angiogenesis. Furthermore, we demonstrate that HMGA1 and FOXM1 synergistically drive breast cancer cells to promote tumor angiogenesis both in vitro in endothelial cells and in vivo in a zebrafish xenograft model. Moreover, using a dataset of breast cancer patients we show that the co-expression of HMGA1, FOXM1 and VEGFA is a negative prognostic factor of distant metastasis-free survival and relapse-free survival.ConclusionsThis study reveals FOXM1 as a crucial interactor of HMGA1 and proves that their cooperative action supports breast cancer aggressiveness, by promoting tumor angiogenesis. Therefore, the possibility to target HMGA1/FOXM1 in combination should represent an attractive therapeutic option to counteract breast cancer angiogenesis.

Timp3 deficiency affects the progression of DEN-related hepatocellular carcinoma during diet-induced obesity in mice.

Obesity and low-grade inflammation are associated with an increased risk of hepatocellular carcinoma (HCC), a leading cause of cancer-related death worldwide. The tissue inhibitor of metalloproteinase (TIMP) 3, an endogenous inhibitor of protease activity that represents a key mediator of inflammation, is reduced in inflammatory metabolic disorders and cancer. In contrast, Timp3-deficient mice (Timp3-/-) are highly resistant to developing HCC in response to a diethylnitrosamine (DEN); therefore, we aimed to elucidate the biological role of genetic loss of Timp3 in obesity-related hepatocarcinogenesis. Fourteen-day-old male wild-type (wt) and Timp3-/- mice were injected with 25mg/kg DEN or an equal volume of saline. After 4weeks, mice were randomized into two dietary groups and fed either normal or high-fat diet and allowed to grow until 32weeks of age. Liver histological features were analyzed, and differentially expressed genes in the liver were quantified. In Timp3-/- mice fed with the obesogenic diet, despite the increase in liver steatosis and inflammation, both the number of tumors and the total tumor size are significantly reduced 30weeks post-DEN injection, compared to control mice. Moreover, Timp3 deletion in hepatocarcinogenesis during obesity is associated with a reduction in FoxM1 transcriptional activity through H19/miR-675/p53 pathway. This study suggests that Timp3 ablation leads to cell cycle perturbation, at least in part by repressing FoxM1 transcriptional activity through H19/miR-675/p53 pathway.

Forkhead box M1 transcription factor: a novel target for pulmonary arterial hypertension therapy.

Forkhead box M1 (FoxM1), a member of forkhead family, plays a key role in carcinogenesis, progression, invasion, metastasis and drug resistance. Based on the similarities between cancer and pulmonary arterial hypertension, studies on the roles and mechanisms of FoxM1 in pulmonary arterial hypertension have been increasing. This article aims to review recent advances in the mechanisms of signal transduction associated with FoxM1 in pulmonary arterial hypertension. Articles were retrieved from PubMed and MEDLINE published after 1990, including-but not limited to-FoxM1 and pulmonary arterial hypertension. FoxM1 is overexpressed in pulmonary artery smooth muscle cells in both pulmonary arterial hypertension patients and animal models, and promotes pulmonary artery smooth muscle cell proliferation and inhibits cell apoptosis via regulating cell cycle progression. Multiple signaling molecules and pathways, including hypoxia-inducible factors, transforming growth factor-β/Smad, SET domain-containing 3/vascular endothelial growth factor, survivin, cell cycle regulatory genes and DNA damage response network, are reported to cross talk with FoxM1 in pulmonary arterial hypertension. Proteasome inhibitors are effective in the prevention and treatment of pulmonary arterial hypertension by inhibiting the expression and transcriptional activity of FoxM1. FoxM1 has a crucial role in the pathogenesis of pulmonary arterial hypertension and may represent a novel therapeutic target. But more details of interaction between FoxM1 and other signaling pathways need to be clarified in the future.

FOXM1 and the NPM-ALK/STAT3 Axis Form a Novel Positive Feedback Loop in Promoting the Oncogenesis of ALK-Positive Anaplastic Large Cell Lymphoma

Peng Wang1, Moinul Haque2, Jing Li2,3, Yung-Hsing Huang2, Meaad Almowaled2, Carter Bargar4, Adam Karpf4, Will Chen2, Suzanne Turner5 and Raymond Lai2,61Division of Hematology, Dept of Medicine, University of Alberta, Edmonton, 2 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton Alberta, Canada; 3Electron Microscopy Center, Basic Medical Science College, Harbin Medical University, Harbin, Heilongjiang, China; 4Eppley Institute and Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, USA; 5Division of Cellular and Molecular Pathology, Department of Pathology, University of Cambridge, Cambridge, UK;6Department of Oncology, University of Alberta, Edmonton, Alberta, Canada1. Backgrounds and AimsForkhead Box M1 (FOXM1) is a transcription factor implicated in the pathogenesis of solid tumors, and it has been shown to promote cell-cycle progression, stem cell renewal and chemotherapeutic resistance in cancer cells. Nonetheless, the biological significance of FOXM1 in hematologic malignancies has not been extensively studied. Here, we studied the expression and role of FOXM1 in ALK-positive anaplastic large cell lymphoma (ALK+ALCL).2 Methods and ResultsIn contrast with normal lymphocytes, FOXM1 was highly expressed in all ALK+ALCL cell lines (5/5), tumors from patients (6/6) and tumors arising from NPM-ALK transgenic mice. Experiments using nuclear/cytoplasmic fractionation, immunocytochemistry and reporter assays had provided evidence that FOXM1 is transcriptionally active in ALK+ALCL. Down-regulation of FOXM1 expression using shRNA and a pharmacologic agent (thiostrepton) resulted in a significant reduction in cell growth, colony formation in soft agar and cell-cycle arrest in ALK+ALCL cells. Further studies revealed that the oncogenic potential of FOXM1 is linked to substantial increases in the phosphorylation/activation status of NPM-ALK and STAT3, and the upregulations of a host of cytokines that have been previously shown to activate the NPM-ALK/STAT3 axis, including IGF-1, IL9 and IL21. Using co-immunoprecipitation, we found that NPM-ALK binds to FOXM1 in the nucleus of ALK+ALCL cells. Importantly, the binding of NPM-ALK to FOXM1 promotes the DNA binding ability and transcriptional activity of FOXM1, and functional inhibition of NPM-ALK using crizotinib or depletion of NPM-ALK using siRNA in ALK+ALCL cells significantly decreased the transcriptional activity of FOXM1.Conclusions:In conclusion, we have identified a novel oncogenic feedback loop involving FOXM1 and the NPM-ALK/STAT3 axis in ALK+ALCL. This study has revealed the first clear example in which NPM-ALK exerts important oncogenic functions in the nuclei of ALK+ALCL cells, by means of its binding to an oncogenic transcription factor so as to promote its DNA binding and transcription activity. DisclosuresNo relevant conflicts of interest to declare.

Abstract B36: FOXM1 induces DNA replication stress, and its bidirectional gene partner RHNO1 participates in the DNA replication stress response, in high-grade serous ovarian cancer

Abstract Purpose: FOXM1 is located at 12p13.33, which shows copy number gains in the majority of high-grade serous ovarian cancer (HGSC) cases. The 12p13.33 amplicon contains 33 genes, some of which may cooperate with FOXM1 to exert oncogenic function. Although FOXM1 overexpression is associated with genomic instability in cancer, whether FOXM1 contributes directly to this phenotype is unclear. We hypothesized that FOXM1 induces DNA replication stress (DRS), resulting in ATR-CHK1 activation. Intriguingly, we noted that RHNO1, a component of the 9-1-1 complex required for ATR-CHK1 activation, is contained within the 12p13.33 amplicon and is arranged in a head-to-head orientation with FOXM1. Here, we characterized the FOXM1/RHNO1 bidirectional promoter, FOXM1/RHNO1 expression, and determined the role of FOXM1/RHNO1 in DRS, the DRS response, and clonogenic growth of HGSC cells. Methods: We used reporter assays to characterize the FOXM1/RHNO1 bidirectional promoter and FOXM1 transcriptional activity. We analyzed copy number, mRNA, and protein expression datasets in TCGA, CCLE, and GTEX. We measured FOXM1/RHNO1 expression in HGSC cell lines and tumors. We overexpressed FOXM1 in human immortalized fallopian tube epithelial (FTE) cells to determine its contribution to DRS and DNA damage. We knocked down RHNO1 in HGSC cells to determine its contribution to the DRS response and clonogenic growth. We performed Western blot analyses to measure markers of DRS and DNA damage. We characterized FOXM1-induced DNA damage at single-cell resolution using Comet assays and flow cytometry measurement of γ-H2AX. Results: FOXM1/RHNO1 showed copy number gains or amplifications in 58% of TCGA HGSC tumors, and FOXM1/RHNO1 mRNA expression significantly correlated in TCGA HGSC tumors, CCLE HGSC lines, and GTEX normal tissues. The FOXM1/RHNO1 bidirectional promoter showed balanced activity in each direction in FTE and HGSC cells, and bidirectional promoter activity correlated with endogenous mRNA expression. TCGA HGSC data indicated that FOXM1 expression associates with markers of DNA replication, global copy number alterations, and CHK1-Ser345-P, a canonical marker of DRS. In agreement, FOXM1 overexpression in FTE cells induced phosphorylation of ATR-Thr1989, CHK1-Ser317/345, H2AX-Ser139, and the proportion of DNA in Comet tails. In contrast, a DNA-binding domain mutant of FOXM1 did not induce DRS or DNA damage. RHNO1 knockdown attenuated CHK1-Ser345-P in response to DRS and RHNO1 shRNA knockdown or CRISPR-Cas9 knockout severely reduced HGSC cell clonogenic growth. Importantly, the effects of RHNO1 disruption were rescued by WT RHNO1 expression, but not by a RHNO1 mutant defective for interaction with the 9-1-1 complex. Conclusion: Our data reveal that FOXM1/RHNO1 share a bidirectional promoter and that this may account for their frequent coexpression in HGSC. FOXM1 induces DRS, and this is dependent on its ability to bind DNA. Conversely, RHNO1 contributes to ATR-CHK1 signaling and promotes the clonogenic growth of HGSC cells. We hypothesize that balanced FOXM1/RHNO1 expression promotes HGSC development and progression. Ongoing studies are characterizing the impact of FOXM1/RHNO1 on DRS and genomic instability in model systems. Finally, our data suggest that the ATR-CHK1 pathway is a synthetic lethal vulnerability in HGSC tumors that overexpress FOXM1. Citation Format: Carter J. Barger, Linda Chee, Connor Branick, Kunle Odunsi, Ronny Drapkin, Adam R. Karpf. FOXM1 induces DNA replication stress, and its bidirectional gene partner RHNO1 participates in the DNA replication stress response, in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B36.

Abstract 28: The novel role of FOXM1 in AML

Abstract Forkhead box M1 (FOXM1) is a transcription factor of the Forkhead family that induces the expression of genes involved in the execution of the mitotic program of normal cells, while it overexpressed in the majority of human cancer cells. In addition, FOXM1 regulatory network was a major predictor of adverse survival outcomes. We have previously shown that FOXM1 interacts with nucleophosmin (NPM) in cancer cells and NPM determines cellular localization of FOXM1. Mutations in NPM1 result in cytoplasmic relocalization of NPM (NPM1mut) and favorable outcome for the patients. We have also shown by immunofluorescence in the AML cell line OCI-AML3 and in AML primary samples with NPM1mut, FOXM1 and mutant NPM co-localize in the cytoplasm. Here we show the evidence that improved outcomes in the subset of NPM1mut AML may be partially explained by the cytoplasmic relocalization and consequent functional inactivation of FOXM1. First, we confirmed the colocalization of FOXM1 and NPMmut in cytoplasm of AML primary patient samples using Vectra imaging of AML diagnostic bone marrow biopsies. Strong cytoplasmic expression of FOXM1 was seen only in NPM1mut AML cells. We proceeded to test the role of FOXM1 in mediating chemoresistance in leukemia cell lines with nuclear FOXM1. Stable knockdown of FOXM1 in AML cell lines KG-1 with nuclear FOXM1/wild-type NPM resulted in increased sensitivity to the chemotherapeutic agent cytarabine. We overexpressed FOXM1 in the OCI-AML3 cell line with cytoplasmic localization of FOXM1 and confirmed overexpression of exogenous FOXM1 in the nuclear compartment by fractionation. Furthermore, we showed that nuclear overexpression of FOXM1 switches the sensitivity of these leukemia cell line to resistant phenotype. These data imply that suppressing of FOXM1 in AML could increase sensitivity to standard chemotherapy, while overexpression of FOXM1 increases chemoresistance of AML cells. Our data suggest that FOXM1 inhibitors may be useful for AML patients with FLT3 wild-type/NPM1 wild type with nuclear FOXM1. We used two compounds to target FOXM1 in AML: honokiol and ixazomib. Honokiol inhibits FOXM1-mediated transcription and FOXM1 protein expression and honokiol's inhibitory effect on FOXM1 is a result of direct binding of honokiol to FOXM1. We found that that honokiol suppresses FOXM1 in AML cell lines and sensitizes AML cells with nuclear FOXM1 to cytorabine. We also tested novel proteasome inhibitor ixazomib (Takeda) as a potential FOXM1 inhibitor in AML. We found that similarly to several other proteasome inhibitors ixazomib inhibits transcriptional activity of FOXM1, FOXM1 mRNA, and FOXM1 protein expression in primary AML cells and in AML cell line. In addition, it shows potential synergy with AML's most common drug cytarabine. Combination of cytarabine and ixazomib in AML KG1 cells led to strong apoptosis correlated with FOXM1 suppression. Since ixazomib is already approved for patients with multiple myeloma and it works as FOXM1 inhibitor in AML, we propose to test it as FOXM1 inhibitor in vivo. These results substantiate FOXM1 targeting to improve chemosensitivity for AML treatment. Our work is based on an intriguing premise that nuclear export of FOXM1 by mutant NPM1 in a subset of favorable risk AML is responsible for outcome. Furthermore, the idea that FOXM1 inactivation leads to favorable outcome gives rise to several strategies to develop FOXM1 inhibitors to increase the efficacy of chemotherapy in AML patients. Citation Format: Irum Khan, Marianna Halasi, Andrei L. Gartel. The novel role of FOXM1 in AML [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 28.

Inhibition of CIP2A attenuates tumor progression by inducing cell cycle arrest and promoting cellular senescence in hepatocellular carcinoma

CIP2A is a recent identified oncogene that inhibits protein phosphatase 2A (PP2A) and stabilizes c-Myc in cancer cells. To investigate the potential oncogenic role and prognostic value of CIP2A, we comprehensively analyzed the CIP2A expression levels in pan-cancer and observed high expression level of CIP2A in majority cancer types, including hepatocellular carcinoma (HCC). Based on a validation cohort including 60 HCC and 20 non-tumorous tissue samples, we further confirmed the high mRNA and protein expression levels of CIP2A in HCC, and found high CIP2A mRNA expression level was associated with unfavorable overall and recurrence-free survival in patients with HCC. Mechanistic investigations revealed that inhibition of CIP2A significantly attenuated cellular proliferation in vitro and tumourigenicity in vivo. Bioinformatic analysis suggested that CIP2A might be involved in regulating cell cycle. Our experimental data further confirmed CIP2A knockdown induced cell cycle arrest at G1 phase. We found accumulated cellular senescence in HCC cells with CIP2A knockdown, companying expression changes of senescence associated proteins (p21, CDK2, CDK4, cyclin D1, MCM7 and FoxM1). Mechanistically, CIP2A knockdown repressed FoxM1 expression and induced FoxM1 dephosphorylation. Moreover, inhibition of PP2A by phosphatase inhibitor rescued the repression of FoxM1. Taken together, our results showed that CIP2A was highly expressed in HCC. Inhibition of CIP2A induced cell cycle arrest and promoted cellular senescence via repressing FoxM1 transcriptional activity, suggesting a potential anti-cancer target for patients with HCC.

Roles of FoxM1 in cell regulation and breast cancer targeting therapy.

Forkhead box M1 (FoxM1) is an oncogenic transcription factor involved in a wide variety of cellular processes, such as cell cycle progression, proliferation, differentiation, migration, metabolism and DNA damage response. It is overexpressed in many human cancers, especially in breast cancers. Posttranslational modifications are known to play an important role in regulating the expression and transcriptional activity of FoxM1. In this review, we characterize the posttranslational modifications of FoxM1, summarize modifications of FoxM1 by different kinases, explore the relationship between the different sites of modifications and comprehensively describe how posttranslational modifications to regulate the function of FoxM1 by changing protein stability, nucleus localization and transcriptional activity. Additionally, we systematically summarize the roles of FoxM1 in breast cancer occurrence, therapy and drug resistance. The purpose of this paper tries to give a better understanding of the regulatory mechanisms of FoxM1 in cell regulation and highlights potential of a new method for breast cancer therapy by targeting FoxM1.

Inhibition of FOXM1 By Ixazomib Confers Chemosensitivity in NPM1-Wild Type Acute Myeloid Leukemia

Background: FOXM1 represents an attractive therapeutic target owing to selective up-regulation in dividing cells. Proteasome inhibitors target FOXM1 by inhibiting FOXM1 through the stabilization of HSP70, which is the negative regulator of FOXM1 {Halasi. J Biol Chem. 2016}. Ixazomib has previously been shown to induce cell death in NPM1-mutant AML cells by oxidative stress {Garcia, Clin Cancer Research 2016} and is currently being tested in NPM1-mutant AML patients as a single agent. We have shown that FOXM1 is inactive in NPM1- mutant AML (Bhat UG. J Bio; Chem 2012) supporting an alternative mechanism of action such as reactive oxygen species induction.In this abstract we propose a FOXM1 dependent mechanism of action in NPM1-wild type AML. We demonstrate that the novel proteasome inhibitor ixazomib is an inhibitor of FOXM1 and can enhance sensitivity to standard chemotherapy drug cytarabine in AML. With its favorable toxicity profile and oral route of administration, this is an attractive option to study as a chemosensitizing drug in leukemia.Materials and Methods: Ixazomib was purchased from Selleck Chemicals. The doxycycline-inducible U2OS-derived C3-luc cell line expressing FOXM1-dependent firefly luciferase was developed in Dr. Gartel's lab (Radhakrishnan. Cancer Res. 2006). KG-1, MV4-11 and HL60 cell lines were purchased from ATCC. Patient samples were collected at the University of Illinois Cancer Center after informed consent using an IRB approved protocol.Results: Using the C3-luc cell line, we studied the effect of ixazomib on FOXM1 transcriptional activity. Cells were treated with 1μg/ml doxycycline and ixazomib doses from 300-1000 nM overnight. The luciferase activity was determined by the Luciferase Assay System (Promega). We demonstrate significant dose-dependent inhibition of FOXM1 transcriptional activity at doses as low as 300nM.Inhibition of FOXM1 protein expression was then confirmed in NPM1-wild type AML cells. We analyzed KG-1, MV4-11 and HL60 cell lines by immunoblotting for FOXM1 protein after 24 hour incubation with ixazomib at doses ranging from 10-1000 nM.Given the well-studied role of FOXM1 in mediating chemoresistance, we studied the effect of ixazomib on increasing sensitivity of AML cells to the chemotherapeutic agent cytarabine. The MTS assay was used to calculate the IC50 of ixazomib in KG-1 and MV4-11 cells. The IC50 for KG1 cells was 38 nM (95% CI: 18-78nM) and for MV4-11 cells it was 27nM (95% CI: 16-45nM). We then used a range of doses of ixazomib (0-100nM) in combination with cytarabine at a fixed ratio in AML cell lines to assess cell proliferation using the MTS assay. A combination index (CI) was calculated using CompuSyn and the combination was synergistic in inhibiting proliferation in KG-1 and MV4-11 cells at all doses tested. Inhibition of FOXM1 expression with combination therapy was confirmed by immunoblotting and was accompanied by increased caspase-3 cleavage after 24 hour exposure to the drug.Finally, to establish that this anti-leukemic effect is dependent on proteasome inhibition, we show a rescue effect with N-Acetyl L-Cysteine, which antagonizes the activity of proteasome inhibitors (Halasi. Biochemical J. 2013).In primary NPM1-wild type AML mononuclear cells, treatment with ixazomib ex vivo for 24 hours in liquid culture resulted in dose dependent cell death measured by flow cytometry and caspase-3 cleavage. We are currently investigating FOXM1 expression and transcriptional activity in primary AML cells treated with ixazomib.Conclusion:Ixazomib demonstrates anti-neoplastic activity against NPM1-wild type AML and potentiates the effect of standard chemotherapy cytarabine. We present a novel mechanism of action whereby ixazomib, through its effect as a proteasome inhibitor, suppresses FOXM1 an oncogenic transcription factor that confers chemoresistance in AML. We are performing animal experiments to confirm the in vitro data and link the anti-leukemic activity of ixazomib with suppression of FOXM1. Our findings lay the groundwork for a target driven trial in AML. Using a novel, well-tolerated oral proteasome inhibitor to target FOXM1 we would expect increased efficacy with lower doses of the chemotherapy drug cytarabine in the treatment of NPM1-wild type AML. DisclosuresNo relevant conflicts of interest to declare.

Siomycin A Induces Apoptosis in Human Lung Adenocarcinoma A549 Cells by Suppressing the Expression of FoxM1

Forkhead box M1 (FoxM1), a transcription factor of the Forkhead family, is demonstrated to be critical for proliferation, apoptosis, migration and invasion of lung cancer. In this study, we extensively investigated the anticancer effect of siomycin A, which was identified as an inhibitor of FoxM1 transcriptional activity, on human lung adenocarcinoma A549 cells. Our study indicated that treatment with siomycin A resulted in the suppression of FoxM1 expression, which consequently contributed to its effect of cell growth inhibition and cell apoptosis induction in A549 cells. Then the molecular mechanism of siomycin A's apoptotic action on A549 cells was further investigated. The results revealed that siomycin A induced apoptosis by influencing the downstream events of FoxM1, including inhibiting the expression of Bcl-2 and Mcl-1, as well as leading to caspase-3 cleavage. Taken together, our findings may be useful for understanding the mechanism of action of siomycin A on lung cancer cells and provide new insights into the possible application of such a compound in lung cancer therapy in the future.

Targeting FoxM1 transcription factor in T-cell acute lymphoblastic leukemia cell line

The Forkhead box protein M1 (FoxM1) is an important transcription factor having significant roles in various cellular events. FoxM1 overexpression has been reported to be related with many types of cancer. However, it is not known whether it contributes to oncogenesis of acute lymphoblastic leukemia. Siomycin A, a thiazol antibiotic, is known to inhibit FoxM1 transcriptional activity. In this study, we aimed to determine gene expression levels of FoxM1 in Jurkat cells (T-cell acute lymphoblastic leukemia cell line) and therapeutic potential of targeting FoxM1 by siomycin A alone and in combination with dexamethasone which improves the survival of children with T-cell acute lymphoblastic leukemia (ALL). We also examined the molecular mechanisms of siomycin A and dexamethasone-induced cell death in Jurkat cells. We demonstrated that FoxM1 mRNA is highly expressed in Jurkat cells. Dexamethasone and siomycin A caused a significant reduction in gene expression levels of FoxM1 in Jurkat cells. Targeting FoxM1 by siomycin A and dexamethasone caused a significant decrease in T-ALL cell line proliferation through induction of G1 cell cycle arrest. All these findings suggest a possible role of FoxM1 in T-cell ALL pathogenesis and represent FoxM1 as an attractive target for T-cell ALL therapy.

Uncovering SUMOylation Dynamics during Cell-Cycle Progression Reveals FoxM1 as a Key Mitotic SUMO Target Protein

Loss of small ubiquitin-like modification (SUMOylation) in mice causes genomic instability due to the missegregation of chromosomes. Currently, little is known about the identity of relevant SUMO target proteins that are involved in this process and about global SUMOylation dynamics during cell-cycle progression. We performed a large-scale quantitative proteomics screen to address this and identified 593 proteins to be SUMO-2 modified, including the Forkhead box transcription factor M1 (FoxM1), a key regulator of cell-cycle progressionandchromosome segregation. SUMOylation of FoxM1 peaks during G2 and M phase, when FoxM1 transcriptional activity is required. We found that aSUMOylation-deficient FoxM1 mutant was less active compared to wild-type FoxM1, implying that SUMOylation of the protein enhances its transcriptional activity. Mechanistically, SUMOylation blocks the dimerization of FoxM1, thereby relieving FoxM1autorepression. Cells deficient for FoxM1 SUMOylation showed increased levels of polyploidy. Our findings contribute to understanding the role of SUMOylation during cell-cycle progression.

Targeting FOXM1 Transcription Factor In T-Cell Acute Lymphoblastic Leukemia

The Forkhead box protein M1(FoxM1) is an important transcriptional factor that takes play in regulation of cell cyle, proliferation, DNA repair, apoptosis, and angiogenesis. FoxM1 overexpression has been reported to be related with many types of cancer. Since many studies have reported that FOXM1 is an important target for cancer therapy, many researchers are studying on the identification of FOXM1 inhibitors. Siomycin A, a thiazol antibiotic, is known to inhibit FoxM1 transcriptional activity. Dexamethasone is a glucocorticoid that is very important in treatment of acute lymphoblastic leukemia (ALL) and is known to be more potent compared to other steroids in the treatment of T-cell ALL. In this study, our aims were to determine the gene expression levels of FoxM1 in Jurkat cells (T-ALL cell line), to find out the possible synergistic and apoptotic effects of siomycin A and dexamethasone on this cell line and to investigate the changes in expression profiles of some important genes that have vital roles in cellular processes by targeting FoxM1 with siomycin A and dexamethasone on Jurkat cells.The gene expression levels of FoxM1 were studied with reverse transcriptase polymerase chain reaction (RT-PCR). The cytotoxic effects of siomycin A and dexamethasone on Jurkat cells were assesed by MTT cell proliferation test. The possible synergistic, additive, neutral, and antagonistic effect of combination of dexamethasone and siomycin A was determined with isobologram analysis. The apoptotic effects of these two agents were evaluated by Caspase-3 activity, loss of mitochondrial membrane potential and localisation of phosphatidilserine on plasma membrane. For this purpose, Caspase-3 calorimetric assay kit, JC-1 mitochondrial membrane potential assay kit, and Annexin V-FITC apoptosis detection kit were used, respectively. For cell cycle analysis, Jurkat cells treated with siomycin A alone or in combination with dexamethasone were stained by propidium iodide and then analyzed by flow cytometry. Expression profiles of Jurkat cells treated with siomycin A alone or in combination with dexamethasone were determined by Cancer Pathway Finder PCR Array.We found that FoxM1 gene is overexpressed in T-ALL cell line and dexamethasone and siomycin A caused a reduction in gene expression levels of FoxM1 in Jurkat cells. 8% to 13% decrease in proliferation of Jurkat cells were observed when these cells were treated with 1 and 10 µM doses of dexamethasone for 72 hours, respectively. The same doses of dexamethasone combined with siomycin A caused 74% and 75% decrease in proliferation of Jurkat cells. Isobologram analysis revealed very strong synergy between dexamethasone and siomycin A. Apoptotic tests showed no apoptotic activity of dexamethasone and siomycin A on Jurkat cells. Cell cycle analysis demonstrated that, reduction of FOXM1 expression by combination of dexamethasone and siomycin A in Jurkat cells inhibited cell proliferation through induction of G1 phase arrest. PCR Array results showed that apoptotic CASPASE-2, CASPASE-7, and CASPASE-9 genes and XIAP and CYCLIN D3 genes were upregulated in response to the treatment. ETS2 gene, which is known as a protooncogene and shown to be involved in regulation of telomerase, was downregulated in response to siomycin A and dexamethosone alone and in combined treatment. TERF1 gene, which inhibits telomerase activity, was upregulated by the treatment. Combination of Siomycin A and dexamethasone downregulated the MCM-2 gene, which is a key component of the pre-replication complex and involved in the formation of DNA replication fork. Moreover, combined treatment resulted in the downregulation of MKI67 gene encoding a nuclear protein associated with cellular proliferation. WEE1 gene, which inhibits G2/M phase transition in cell cycle, was also upregulated.These data indicate that FoxM1 gene is strongly overexpressed in T-ALL cell line and targeting FoxM1 by siomycin A and dexamethasone causes a significant decrease in T-ALL cell proliferation through induction of G1 cell cycle arrest. Importantly, PCR array analyses also showed that siomycin A and dexamethasone treatment affects Jurkat cells via upregulating or downregulating the key genes of cell cycle, apoptosis, cell proliferation, telomere, and telomerase function. All these findings suggest a possible role for FoxM1 in T-ALL pathogenesis and represent FoxM1 as an attractive target for T-ALL therapy. Disclosures:No relevant conflicts of interest to declare.

Activated ERK/FOXM1 pathway by low-power laser irradiation inhibits UVB-induced senescence through down-regulating p21 expression

Cellular senescence is a growth-arrest program that limits cell proliferation. Low-power laser irradiation (LPLI) has been demonstrated to promote cell proliferation. However, whether LPLI can inhibit cellular senescence remains unknown. In the present study, to investigate the functional role of LPLI against skin aging, we used ultraviolet radiation b (UVB) to induce cell senescence. We first report that LPLI can delay UVB-induced cell senescence. The senescence-associated β-galactosidase (SA-β-Gal) activity and p21 expression, hallmarks of senescent cells, were decreased in the Forkhead box transcription factor FOXM1-dependent manner under treatment with LPLI. The effect of LPLI was further enhanced with an overexpression of FOXM1, and abolished when FOXM1 was knockdown with short hairpin RNA (shRNA). Furthermore, LPLI activated the extracellular regulated protein kinases (ERK) that was upstream of FOXM1. This led to FOXM1 phosphorylation and nuclear translocation. Nuclear translocation enhanced FOXM1 transcriptional activity and promoted its downstream target gene c-Myc expression that could inhibit p21 expression. These findings highlight the protective effects of ERK/FOXM1 pathway against UVB-induced cell senescence, suggesting a potential protecting strategy for treating skin aging by LPLI.

Thiazole Antibiotics Siomycin a and Thiostrepton Inhibit the Transcriptional Activity of FOXM1

I read with great interest the review article by Alvarez-Fernandez and Medema (2013). I was especially pleased to see that the authors comprehensively acknowledged our work by citing six of our recent papers in their review. However, when they discussed our findings on the identification of the thiazole antibiotics siomycin A and thiostrepton as inhibitors of FOXM1 transcriptional activity they made a significant mistake. They wrongly stated, “surprisingly, these inhibitors do not affect the transcriptional activity of FOXM1 per se.” In numerous publications we showed that indeed siomycin A and thiostrepton inhibit the transcriptional activity and the expression of FOXM1, and they also act as proteasome inhibitors (Radhakrishnan et al., 2006; Bhat et al., 2009a,b). Moreover, we demonstrated that even in the presence of high levels of exogenous FOXM1 siomycin A, thiostrepton, and known proteasome inhibitors were able to inhibit FOXM1 transcriptional activity, suggesting that this inhibition is a key initiating event for FOXM1 protein suppression (Radhakrishnan et al., 2006; Bhat et al., 2009a,b). In addition, the authors concluded that both the mRNA and protein expression of FOXM1 are downregulated by siomycin A and thiostrepton “through an unknown mechanism” (Alvarez-Fernandez and Medema, 2013). However, recently I have proposed a general model for FOXM1 inhibition by proteasome inhibitors, which links the transcriptional activity and expression of FOXM1 (Gartel, 2010, 2012). According to this model, siomycin A, thiostrepton, and other proteasome inhibitors hinder the proteasomal degradation of a negative regulator of FOXM1 (NRFM), which in return directly or indirectly inhibits the activity of FOXM1 as a transcription factor (Gartel, 2011). Because FOXM1 is involved in a positive feedback loop and activates its own transcription (Halasi and Gartel, 2009), inhibition of FOXM1 transcriptional activity leads to a decrease in its expression (Gartel, 2010). This model also anticipates that all proteasome inhibitors regardless of their structure will inhibit FOXM1 transcriptional activity through stabilizing the NRFM and subsequently (via the auto-regulatory loop) inhibit FOXM1 expression (Gartel, 2011, 2012). Overall, inhibition of FOXM1 transcriptional activity by siomycin A, thiostrepton, and other proteasome inhibitors is an essential part of their regulation of FOXM1. FOXM1 is without a doubt emerging as a critical regulator of cancer development (Halasi and Gartel, 2013) that may affect all hallmarks of cancer (Hanahan and Weinberg, 2012). Originally, it was identified as a key regulator of cell proliferation and cell cycle progression; however, in the past few years FOXM1 became one of the central contributors to tumorigenesis. Growing body of evidence suggests that targeting this single transcription factor may have great promise for inhibiting tumor development. Great efforts are currently being undertaken to find more specific inhibitors of FOXM1 and to elucidate their mechanism of action. I believe it is essential for the scientific community to have correct information about the regulation of FOXM1 by FOXM1 inhibitors in order to progress and accelerate research in the FOXM1 field.

Novel interactions between FOXM1 and CDC25A regulate the cell cycle.

FOXM1 is a critical regulator of the G1/S and G2/M cell cycle transitions, as well as of the mitotic spindle assembly. Previous studies have suggested that FOXM1 regulates CDC25A gene transcription, but the mechanism remains unknown. Here, we provide evidence that FOXM1 directly regulates CDC25A gene transcription via direct promoter binding and indirect activation of E2F-dependent pathways. Prior literature reported that CDC25B and CDC25C activate CDK1/cyclinB complexes in order to enable phosphorylation of FOXM1. It was unknown if CDC25A functions in a similar manner. We report that FOXM1 transcriptional activity is synergistically enhanced when co-expressed with CDC25A. The increase is dependent upon CDK1 phosphorylation of FOXM1 at T600, T611 and T620 residues. We also report a novel protein interaction between FOXM1 and CDC25A via the C-terminus of FOXM1. We demonstrate that the phosphorylation of Thr 600 and Thr 611 residues of FOXM1 enhanced this interaction, and that the interaction is dependent upon CDC25A phosphatase activity. Our work provides novel insight into the underlying mechanisms by which FOXM1 controls the cell cycle through its association with CDC25A.