Oncogenic Transcription Factor FOXM1 Research Articles

Abstract 3031: Overcoming drug resistance in cancer: A paradigm shift with FoxM1 targeting

Abstract In cancer treatment, overcoming drug resistance remains a formidable challenge. The oncogenic transcription factor FoxM1 is widely recognized for its crucial role in promoting drug resistance to various chemotherapeutic and target therapies. Its involvement is attributed to pivotal pathways, encompassing cell cycle progression, angiogenesis, and metastasis. Despite its prominent role in tumor drug resistance, achieving modulation of FoxM1 using small molecule therapeutics has proven elusive. 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent, is known to induce a significant upregulation of FoxM1. In this study, we demonstrate that our novel compound A3 leads to a robust dose-dependent reduction of FoxM1 driven by 5-FU in HT-29 colorectal cancer cells. Notably, exposure of 5-FU-treated HT-29 cells to A3 was shown to promote apoptosis via pro-apoptotic factors, including PUMA. The mechanism of action was further elucidated through a comprehensive analysis of cell signaling molecules. This study presents an innovative approach using small molecule modulators to control FoxM1 levels, providing profound insights for potential therapeutic interventions in cancer treatment. Citation Format: Martin Pettersson, Su Jung Bae, Kyu-kwang Cho, Hye-yeoung Yun, Hee-Sung Park. Overcoming drug resistance in cancer: A paradigm shift with FoxM1 targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3031.

Gastric coinfection with thiopeptide-positive Cutibacterium acnes decreases FOXM1 and pro-inflammatory biomarker expression in a murine model of Helicobacter pylori-induced gastric cancer.

H. pylori infects half of the world population and is the leading cause of gastric cancer. We previously demonstrated that gastric cancer risk is associated with gastric microbiota. Specifically, gastric urease-positive Staphylococcus epidermidis and Streptococcus salivarius had contrasting effects on H. pylori-associated gastric pathology and immune responses in germ-free INS-GAS mice. As gastritis progresses to gastric cancer, the oncogenic transcription factor Foxm1 becomes increasingly expressed. In this study, we evaluated the gastric commensal C. acnes, certain strains of which produce thiopeptides that directly inhibit FOXM1. Thiopeptide-positive C. acnes was isolated from Nicaraguan patient gastric biopsies and inoculated into germ-free INS-GAS mice with H. pylori. We, therefore, asked whether coinfection with C. acnes expressing thiopeptide and H. pylori would decrease gastric Foxm1 expression and pro-inflammatory cytokine mRNA and protein levels. Our study supports the growing literature that specific non-H. pylori gastric bacteria affect inflammatory and cancer biomarkers in H. pylori pathogenesis.

FDI-6 inhibits the expression and function of FOXM1 to sensitize BRCA-proficient triple-negative breast cancer cells to Olaparib by regulating cell cycle progression and DNA damage repair

Inducing homologous-recombination (HR) deficiency is an effective strategy to broaden the indications of PARP inhibitors in the treatment of triple-negative breast cancer (TNBC). Herein, we find that repression of the oncogenic transcription factor FOXM1 using FOXM1 shRNA or FOXM1 inhibitor FDI-6 can sensitize BRCA-proficient TNBC to PARP inhibitor Olaparib in vitro and in vivo. Mechanistic studies show that Olaparib causes adaptive resistance by arresting the cell cycle at S and G2/M phases for HR repair, increasing the expression of CDK6, CCND1, CDK1, CCNA1, CCNB1, and CDC25B to promote cell cycle progression, and inducing the overexpression of FOXM1, PARP1/2, BRCA1/2, and Rad51 to activate precise repair of damaged DNA. FDI-6 inhibits the expression of FOXM1, PARP1/2, and genes involved in cell cycle control and DNA damage repair to sensitize TNBC cells to Olaparib by blocking cell cycle progression and DNA damage repair. Simultaneously targeting FOXM1 and PARP1/2 is an innovative therapy for more patients with TNBC.

Systematic functional interrogation of human pseudogenes using CRISPRi

BackgroundThe human genome encodes over 14,000 pseudogenes that are evolutionary relics of protein-coding genes and commonly considered as nonfunctional. Emerging evidence suggests that some pseudogenes may exert important functions. However, to what extent human pseudogenes are functionally relevant remains unclear. There has been no large-scale characterization of pseudogene function because of technical challenges, including high sequence similarity between pseudogene and parent genes, and poor annotation of transcription start sites.ResultsTo overcome these technical obstacles, we develop an integrated computational pipeline to design the first genome-wide library of CRISPR interference (CRISPRi) single-guide RNAs (sgRNAs) that target human pseudogene promoter-proximal regions. We perform the first pseudogene-focused CRISPRi screen in luminal A breast cancer cells and reveal approximately 70 pseudogenes that affect breast cancer cell fitness. Among the top hits, we identify a cancer-testis unitary pseudogene, MGAT4EP, that is predominantly localized in the nucleus and interacts with FOXA1, a key regulator in luminal A breast cancer. By enhancing the promoter binding of FOXA1, MGAT4EP upregulates the expression of oncogenic transcription factor FOXM1. Integrative analyses of multi-omic data from the Cancer Genome Atlas (TCGA) reveal many unitary pseudogenes whose expressions are significantly dysregulated and/or associated with overall/relapse-free survival of patients in diverse cancer types.ConclusionsOur study represents the first large-scale study characterizing pseudogene function. Our findings suggest the importance of nuclear function of unitary pseudogenes and underscore their underappreciated roles in human diseases. The functional genomic resources developed here will greatly facilitate the study of human pseudogene function.

FOXM1-Dependent Transcriptional Regulation of EZH2 Induces Proliferation and Progression in Prostate Cancer.

Prostate cancer is one of the most commonly diagnosed cancers and one of the most common causes of cancer-related deaths among men worldwide. Patients who are diagnosed with localized prostate cancer and treated with radical prostatectomy often respond well to therapy. The current standard therapy for prostate cancer involves maximal surgical resection, followed by radiotherapy and chemotherapy. Clarifying the molecular mechanism of tumor proliferation and recurrence becomes more and more important for clinical therapies of prostate cancer. Quantitative Real-Time PCR and Western-blot were used in the detection of mRNA and protein expression. Lentivirus infection was used to overexpress or knockdown the target gene. Flow cytometry analysis was performed to test protein expression and apoptosis level. Immunohistochemistry was used to identify protein expression in tissue. Statistical differences between the two groups are evaluated by two-tailed t-tests. The comparison among multiple groups is performed by one-way Analysis of Variance (ANOVA) followed by Dunnett's posttest. The statistical significance of the Kaplan-Meier survival plot is determined by log-rank analysis. In this study, we identified that FOXM1 expression was significantly enriched in prostate cancer compared with normal tissue. Additionally, FOXM1 was functionally required for tumor proliferation and its expression was associated with poor prognosis in prostate cancer patients. Mechanically, FOXM1-dependent regulation of EZH2 is essential for proliferation and progression in prostate cancer. Taken together, our data suggest that oncogenic transcription factor FoxM1 is up-regulated in prostate cancer, suggesting that the growth of cancer cells may depend on FOXM1 activity. FOXM1 may serve as a clinical prognostic factor and a therapeutic target for prostate cancer.

The FOXM1 Inhibitor RCM-1 Decreases Carcinogenesis and Nuclear β-Catenin.

The oncogenic transcription factor FOXM1 has been previously shown to play a critical role in carcinogenesis by inducing cellular proliferation in multiple cancer types. A small-molecule compound, Robert Costa Memorial drug-1 (RCM-1), has been recently identified from high-throughput screen as an inhibitor of FOXM1 in vitro and in mouse model of allergen-mediated lung inflammation. In the present study, we examined antitumor activities of RCM-1 using tumor models. Treatment with RCM-1 inhibited tumor cell proliferation as evidenced by increased cell-cycle duration. Confocal imaging of RCM-1-treated tumor cells indicated that delay in cellular proliferation was concordant with inhibition of FOXM1 nuclear localization in these cells. RCM-1 reduced the formation and growth of tumor cell colonies in the colony formation assay. In animal models, RCM-1 treatment inhibited growth of mouse rhabdomyosarcoma Rd76-9, melanoma B16-F10, and human H2122 lung adenocarcinoma. RCM-1 decreased FOXM1 protein in the tumors, reduced tumor cell proliferation, and increased tumor cell apoptosis. RCM-1 decreased protein levels and nuclear localization of β-catenin, and inhibited protein-protein interaction between β-catenin and FOXM1 in cultured tumor cells and in vivo Altogether, our study provides important evidence of antitumor potential of the small-molecule compound RCM-1, suggesting that RCM-1 can be a promising candidate for anticancer therapy.

Honokiol is a FOXM1 antagonist

Honokiol is a natural product and an emerging drug for a wide variety of malignancies, including hematopoietic malignancies, sarcomas, and common epithelial tumors. The broad range of activity of honokiol against numerous malignancies with diverse genetic backgrounds suggests that honokiol is inhibiting an activity that is common to multiple malignancies. Oncogenic transcription factor FOXM1 is one of the most overexpressed oncoproteins in human cancer. Here we found that honokiol inhibits FOXM1-mediated transcription and FOXM1 protein expression. More importantly, we found that honokiol’s inhibitory effect on FOXM1 is a result of binding of honokiol to FOXM1. This binding is specific to honokiol, a dimerized allylphenol, and was not observed in compounds that either were monomeric allylphenols or un-substituted dihydroxy phenols. This indicates that both substitution and dimerization of allylphenols are required for physical interaction with FOXM1. We thus demonstrate a novel and specific mechanism for FOXM1 inhibition by honokiol, which partially may explain its anticancer activity in cancer cells.

Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cells by targeting the transcription factors FOXO3a and FOXM1

Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties that promote cancer cell invasive growth and metastasis, yet the mechanisms mediating these functions still largely remain elusive. We show here that overexpression of the GOF mutant p53 G245D and other GOF p53 mutants enhances the invasive cell growth of p53-deficient head and neck squamous cell carcinoma (HNSCC) UM-SCC-1 cells both in in vitro three-dimensional culture and in an in vivo orthotopic nude mouse model of HNSCC through a novel transcription-independent mechanism. We demonstrate that the expression of the oncogenic forkhead transcription factor FOXM1 is upregulated by GOF mutant p53s. Moreover, we show that overexpression of GOF mutant p53 G245D decreases the AMP-activated protein kinase (AMPK)-mediated phosphorylation of FOXO3a, a tumor suppressive forkhead transcription factor, leading to its cytoplasmic accumulation. This downregulation of FOXO3a’s activity, in turn, leads to de-repression of FOXM1 expression. Importantly, we show that either overexpression of FOXO3a or downregulation of FOXM1 impairs both GOF mutant p53-mediated cell invasion in vitro and pulmonary metastases of UM-SCC-1 cells in vivo. Finally, not only do oral cancer patients with p53 mutations exhibit higher levels of FOXM1 expression than patients with wild-type p53, but also HNSCC patients with TP53 mutations and high levels of FOXM1 expression have the poorest survival outcomes. Given our prior demonstration that GOF mutant p53s inhibit AMPK, our current study, establishes and demonstrates a novel transcription-independent GOF mutant p53-AMPK-FOXO3a-FOXM1 signaling cascade that plays an important role in mediating mutant p53s’ gain-of-function activities in HNSCCs.

Abstract 73: Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cell by targeting forkhead transcription factors FOXO3a and FOXM1

Abstract Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties in promoting cancer cell invasive growth and metastasis, but the mechanisms mediating these functions largely remain elusive. We show here that overexpression of the GOF mutant p53 G245D and other GOF p53 mutants enhances invasive cell growth of p53-deficient head and neck squamous cell carcinoma (HNSCC) UM-SCC-1 cells both in vitro 3D culture and in vivo orthotopic tumor mouse model. We demonstrate that the expression of the oncogenic forkhead transcription factor FOXM1 is upregulated by GOF mutant p53s in UM-SCC-1 cells and tumors. Moreover, we show that overexpression of GOF mutant p53 G245D decreases the inhibitory phosphorylation of FOXO3a, a tumor suppressive forkhead transcription factor, leading to its cytoplasmic accumulation. Consistent with the observation that FOXO3a negatively regulates FOXM1 expression, we demonstrate that overexpression FOXO3a decreases GOF mutant p53-mediated FOXM1 expression, whereas downregulation of FOXO3a partially restores decreased FOXM1 expression due to loss of GOF mutant p53. In support of the roles of FOXO3a-FOXM1 signaling axis in mutant p53's GOF signaling, we further show that overexpression of FOXO3a or downregulation of FOXM1 impairs GOF mutant p53-mediated cell invasion, whereas downregulation of FOXO3a, in part, rescues impaired cell invasion due to downregulated GOF mutant p53. Finally, given that AMP-activated protein kinase (AMPK) directly phosphorylates and promotes FOXO3a function, and that our prior demonstration that GOF mutant p53s inhibit AMPK, our current study establishes that GOF mutant p53-AMPK-FOX3a-FOXM1 signaling is one of important mechanisms through which mutant p53s gain oncogenic functions in HNSCCs. Citation Format: Noriaki Tanaka, Mei Zhao, Jiexin Zhang, Jing Wang, Ge Zhou, Jeffrey N. Myers. Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cell by targeting forkhead transcription factors FOXO3a and FOXM1 [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 73.

MiR-149 suppresses human non-small cell lung cancer growth and metastasis by inhibiting the FOXM1/cyclinD1/MMP2 axis.

Valid evidence has demonstrated that microRNAs have critical functions in cancer genesis and tumor progression. In the present study, aberrant expression of microRNA-149 (miR-149) was confirmed in non-small cell lung cancer (NSCLC) tissues. Low expression of miR-149 was associated with malignant clinical features and poor survival. Gain-and loss-of-function experiments demonstrated that miR-149 inhibited NSCLC tumor growth and metastasis invitro and invivo. Furthermore, oncogenic transcription factor FOXM1 was confirmed as a direct target of miR-149 in NSCLC. CyclinD1 and MMP2 served as downstream targets of FOXM1 and were also inhibited by miR-149. In summary, the present study indicated that downregulation of miR-149 in NSCLC predicted poor clinical outcomes. miR-149 suppresses tumor growth and metastasis in NSCLC by inhibiting the FOXM1/cyclinD1/MMP2 signaling pathway.

VprBP/DCAF1 Regulates the Degradation and Nonproteolytic Activation of the Cell Cycle Transcription Factor FoxM1.

The oncogenic transcription factor FoxM1 plays a vital role in cell cycle progression, is activated in numerous human malignancies, and is linked to chromosome instability. We characterize here a cullin 4-based E3 ubiquitin ligase and its substrate receptor, VprBP/DCAF1 (CRL4VprBP), which we show regulate FoxM1 ubiquitylation and degradation. Paradoxically, we also found that the substrate receptor VprBP is a potent FoxM1 activator. VprBP depletion reduces expression of FoxM1 target genes and impairs mitotic entry, whereas ectopic VprBP expression strongly activates a FoxM1 transcriptional reporter. VprBP binding to CRL4 is reduced during mitosis, and our data suggest that VprBP activation of FoxM1 is ligase independent. This implies a nonproteolytic activation mechanism that is reminiscent of, yet distinct from, the ubiquitin-dependent transactivation of the oncoprotein Myc by other E3s. Significantly, VprBP protein levels were upregulated in high-grade serous ovarian patient tumors, where the FoxM1 signature is amplified. These data suggest that FoxM1 abundance and activity are controlled by VprBP and highlight the functional repurposing of E3 ligase substrate receptors independent of the ubiquitin system.

O-GlcNAcylation regulates breast cancer metastasis via SIRT1 modulation of FOXM1 pathway.

Tumors utilize aerobic glycolysis to support growth and invasion. However, the molecular mechanisms that link metabolism with invasion are not well understood. The nutrient sensor O-linked-β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) modifies intracellular proteins with N-acetylglucosamine. Cancers display elevated O-GlcNAcylation and suppression of O-GlcNAcylation inhibits cancer invasion and metastasis. Here, we show that the regulation of cancer invasion by OGT is dependent on the NAD+-dependent deacetylase SIRT1. Reducing O-GlcNAcylation elevates SIRT1 levels and activity in an AMPK-dependent manner. Reduced O-GlcNAcylation in cancer cells leads to SIRT1-mediated proteasomal degradation of oncogenic transcription factor FOXM1 in a MEK/ERK-dependent manner. SIRT1 is critical for OGT-mediated regulation of FOXM1 ubiquitination and reducing SIRT1 activity reverses OGT-mediated regulation of FOXM1. Moreover, we show that SIRT1 levels are required for OGT-mediated regulation of invasion and metastasis in breast cancer cells. Thus, O-GlcNAcylation is a central component linking metabolism to invasion and metastasis via a SIRT1/ /FOXM1 axis.

A Novel Function of Molecular Chaperone HSP70: SUPPRESSION OF ONCOGENIC FOXM1 AFTER PROTEOTOXIC STRESS

The oncogenic transcription factor FOXM1 is overexpressed in the majority of human cancers, and it is a potential target for anticancer therapy. We identified proteasome inhibitors as the first type of drugs that target FOXM1 in cancer cells. Here we found that HSP90 inhibitor PF-4942847 and heat shock also suppress FOXM1. The common effector, which was induced after treatment with proteasome and HSP90 inhibitors or heat shock, was the molecular chaperone HSP70. We show that HSP70 binds to FOXM1 following proteotoxic stress and that HSP70 inhibits FOXM1 DNA-binding ability. Inhibition of FOXM1 transcriptional autoregulation by HSP70 leads to the suppression of FOXM1 protein expression. In addition, HSP70 suppression elevates FOXM1 expression, and simultaneous inhibition of FOXM1 and HSP70 increases the sensitivity of human cancer cells to anticancer drug-induced apoptosis. Overall, we determined the unique and novel mechanism of FOXM1 suppression by proteasome inhibitors.

EZH2 Protects Glioma Stem Cells from Radiation-Induced Cell Death in a MELK/FOXM1-Dependent Manner

SummaryGlioblastoma (GBM)-derived tumorigenic stem-like cells (GSCs) may play a key role in therapy resistance. Previously, we reported that the mitotic kinase MELK binds and phosphorylates the oncogenic transcription factor FOXM1 in GSCs. Here, we demonstrate that the catalytic subunit of Polycomb repressive complex 2, EZH2, is targeted by the MELK-FOXM1 complex, which in turn promotes resistance to radiation in GSCs. Clinically, EZH2 and MELK are coexpressed in GBM and significantly induced in postirradiation recurrent tumors whose expression is inversely correlated with patient prognosis. Through a gain-and loss-of-function study, we show that MELK or FOXM1 contributes to GSC radioresistance by regulation of EZH2. We further demonstrate that the MELK-EZH2 axis is evolutionarily conserved in Caenorhabditis elegans. Collectively, these data suggest that the MELK-FOXM1-EZH2 signaling axis is essential for GSC radioresistance and therefore raise the possibility that MELK-FOXM1-driven EZH2 signaling can serve as a therapeutic target in irradiation-resistant GBM tumors.

PTPROt-mediated regulation of p53/Foxm1 suppresses leukemic phenotype in a CLL mouse model.

The gene encoding PTPROt is methylated and suppressed in Chronic Lymphocytc Leukemia. PTPROt exhibits in vitro tumor suppressor characteristics through the regulation of B-cell receptor signaling. Here, we generated transgenic (Tg) mice with B-cell specific expression of PTPROt. While lymphocyte development is normal in these mice, crossing them with TCL1 Tg mouse model of CLL results in a survival advantage compared to the TCL1 Tg mice. Gene expression profiling of splenic B-lymphocytes before detectable signs of CLL followed by Ingenuity Pathway Analysis revealed that the most prominently regulated functions in TCL1 Tg vs non-transgenic (NTg) and TCL1 Tg vs PTPROt/TCL1 double Tg are the same and also biologically relevant to this study. Further, enhanced expression of the chemokine Ccl3, the oncogenic transcription factor Foxm1 and its targets in TCL1 Tg mice were significantly suppressed in the double Tg mice suggesting a protective function of PTPROt against leukemogenesis. This study also showed that PTPROt mediated regulation of Foxm1 involves activation of p53, a transcriptional repressor of Foxm1, which is facilitated through suppression of B-cell receptor signaling. These results establish the in vivo tumor suppressive function of PTPROt, and identify p53/Foxm1 axis as a key downstream effect of PTPROt-mediated suppression of BCR signaling.

Abstract 470: Artemisinin derivatives synergize with paclitaxel by targeting FOXM1 through Raf/MEK/MAPK signaling pathway in ovarian cancer

Abstract Objective: Artemisinin, the active principle of the Chinese medicinal herb Artemisia annua and its derivatives have the most efficacies against Plasmodium falciparum malaria. Artemisinin contains an endoperoxide bridge that reacts with ferrous iron to generate free radicals which leads to macromolecular damage and cell death. In this study, we aim to determine if artemisinin derivatives dihydroartemisinin (DHA) can improve the efficacy of paclitaxel and cisplatinum in human ovarian cancer cells in vitro and its possible underlying molecular mechanism and novel molecular targets. Methods: Ovarian cancer cell lines SKOV3 and OVCAR3 were treated with paclitaxel/cisplatinum/DHA or a combination for 72 hrs and cytotoxicity was assessed by sulfarodamine B assay. Drug combination effects were determined by calculating the combination index (CI) values using the Chou and Talalay method (CI <0.9 synergy, CI 0.9-1.10 additive and CI >1.10 antagonism). Quantitative RT-PCR and western blot were performed to determine changes in FOXM1 expression and its downstream molecules. In MAPK signaling studies, cells were starved for 24 hrs, stimulated with 20% FBS for 30min with pre-incubation of DHA for 30 min, and then probed with anti-phospho-MAPK for western blot. Results: Application of DHA to ovarian cancer cells induced cytotoxicity with IC50 between 1µM to 2µM. Drug combination studies demonstrated that DHA synergized with paclitaxel (CI value 0.6-0.73) and additive to cisplatinum (CI value 0.98-1.11) in SKOV3. In addition, low dose DHA (0.5µM) significantly synergized to the combination of paclitaxel and cisplatinum in SKOV3 cells (CI 0.40-0.83). To reveal the underlying molecular mechanism, we analyzed the cell cycle upon DHA treatment which proved that DHA alone arrested cells in G2/M phase (30.8%) compare to control (17.3%) after 24 hrs treatment. Furthermore, DHA significantly downregulated oncogenic transcription factor FOXM1 expression and the transcription of downstream molecules such as CCNB1, BIRC5 and STMN1. Therefore, targeting FOXM1 by DHA delay G2/M transition, induce apoptosis and stabilize microtubule dynamics to sensitize the tumor cells to paclitaxel-induced apoptosis, contributing to the synergistic effects. Finally, FOXM1 is phosphorylated for nuclear translocation before entry into G2/M, and one of the major kinase is MAPK. The western blot demonstrated that DHA inhibit serum-induced phosphorylation of MAPK, indicating the inhibitory effects of DHA toward Raf/MEK/MAPK signaling pathway. Conclusion: These results demonstrate that inhibition of Raf/MEK/MAPK signaling by DHA led to suppression of FOXM1 target gene expression and contribute to cellular cytotoxicity and drug synergism in ovarian cancer. In conclusion, we provide the new information in the knowledge of artemisinin derivatives involved signaling pathways and potential molecular targets. Citation Format: Yi Chen, Brian W. Chin, Marcia M. Bieber, Xianjie Tan, Nelson N.h Teng. Artemisinin derivatives synergize with paclitaxel by targeting FOXM1 through Raf/MEK/MAPK signaling pathway in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 470. doi:10.1158/1538-7445.AM2014-470

Abstract 3039: The MELK/FOXM1 axis is a master regulator of proneural to mesenchymal transition (PMT) in glioma stem cells by controlling EZH2 transcriptional activity

Abstract In the Central Nervous System, stem cells are present in the normal brain and primary brain tumor, glioblasotoma multiforme (GBM). Both normal and oncogenic stem cells play central roles in organogenesis and tumorigenesis, respectively. Previously we identified that the mitotic kinase MELK binds and phosphorylates the oncogenic transcription factor FOXM1 to regulate the growth of both brain-derived and GBM-derived stem cells (GSCs). Here we demonstrate that the Enhancer of Zeste Homolog 2 (EZH2), the lysine methyltransferase of Polycomb repressive complex 2, is the major transcriptional target of the MELK-FOXM1 complex in GSCs. PCR-based analysis of FOXM1 immunoprecipitated chromatin determined the binding of FOXM1 to the EZH2 promoter region to drive its transcriptional activity. This signaling is mediated through the protein complex formation of FOXM1 and MELK in a MELK kinase-dependent manner. The MELK-FOXM1 driven EZH2 signals are required to retain the immature stem cell trait and mesenchymal identity in GBM, as well as GSCs' radiation resistant phenotype in a MELK kinase-dependent manner. Clinically, GBM tumors co-express MELK and EZH2 with significant elevation in post-irradiation recurrent tumors. This post-irradiation elevation of MELK and EZH2 was observed specifically in GSCs. Irradiation induces transformation of proneural (PN) GSCs into a Mesenchymal (Mes)-like GSC phenotype (PMT) that is highly resistant to radiation treatment, and inhibition of EZH2 by shRNA reverses the radiation-resistant phenotype of Mes GSCs. Elimination of MELK or FOXM1 by shRNA reduced EZH2 expression resulting in attenuated stemness and radiation resistance in vitro and in vivo. These phenotypes were completely restored by exogenous expression of EZH2. To our knowledge, this study provides the first evidence of the role of MELK-FOXM1-EZH2 signaling axis for maintenance of stem cell trait and PMT regulation in GSCs. Identification of the role of transcriptional regulator EZH2 in PMT by the mitotic kinase MELK and transcription factor FOXM1 complex sheds lights on a novel therapeutic target for aggressive cancers including GBM. Citation Format: Kaushal Joshi, Sunghak Kim, Jeongwu Lee, Ronald Waclaw, Lisa Salcini, Ichiro Nakano. The MELK/FOXM1 axis is a master regulator of proneural to mesenchymal transition (PMT) in glioma stem cells by controlling EZH2 transcriptional activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3039. doi:10.1158/1538-7445.AM2014-3039

Suppression of the Oncogenic Transcription Factor FOXM1 by Proteasome Inhibitors.

The oncogenic transcription factor FOXM1 is one of the key regulators of tumorigenesis. We found that FOXM1 upregulates its own transcription and its protein stability depends on its interaction with the chaperone nucleophosmin. We also determined that FOXM1 is negatively regulated by the tumor suppressor p53. We identified the thiazole antibiotics Siomycin A and thiostrepton as inhibitors of transcriptional activity and FOXM1 expression via proteasome inhibition. In addition, we found that all tested proteasome inhibitors target FOXM1. We showed synergy between thiostrepton and bortezomib in different human cancer cell lines and in vivo. We generated isogenic human cancer cell lines of different origin with wild-type p53 or p53 knockdown and we demonstrated that proteasome inhibitors induce p53-independent apoptosis in these cells. Using RNA-interference or proteasome inhibitors to inhibit FOXM1 we found that suppression of FOXM1 sensitized human cancer cells to apoptosis induced by DNA-damaging agents or oxidative stress. We encapsulated thiostrepton into micelle-nanoparticles and after injection we detected accumulation of nanoparticles in tumors and in the livers of treated mice. This treatment led to inhibition of human xenograft tumor growth in nude mice. Our data indicate that targeting FOXM1 increases apoptosis and inhibits tumor growth.

Phosphoglycerate dehydrogenase induces glioma cells proliferation and invasion by stabilizing forkhead box M1

Phosphoglycerate dehydrogenase (PHGDH) is the first enzyme branching from glycolysis in the three-step serine biosynthetic pathway. Recent evidence has shown that PHGDH is amplified in human breast cancer and melanoma and plays a key role in cancer metabolism. However, PHGDH expression in glioma and a potential non-metabolic role in tumorigenesis have not been reported. We analyzed PHGDH levels in specimens from glioma patients and found that PHGDH, although negative in normal brain tissues, was highly expressed in astrocytic tumors and increasingly expressed in more aggressive cancer types. Inhibition of PHGDH expression in glioma cells downregulated the expression of VEGF, MMP-2, CHK2 and cyclin D1 and reduced glioma cell proliferation, invasion and tumorigenicity in vitro and in vivo. Interestingly, we found that the oncogenic transcription factor FOXM1 was also downregulated in PHDGH-silenced glioma cells. Using LC/LC MS analysis, we identified PHGDH as a novel binding partner of FOXM1. PHGDH interacted with and stabilized FOXM1 at the protein level, promoting the proliferation, invasion and tumorigenicity of glioma cells. Our data identified PHGDH as a potential prognostic marker of glial brain tumors and identified a non-metabolic role for PHGDH in glioma tumorigenesis, providing a novel angle of targeting the PHGDH–FOXM1 axis in future brain tumor therapy.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-012-1018-x) contains supplementary material, which is available to authorized users.

FoxM1 Inhibition Sensitizes Resistant Glioblastoma Cells to Temozolomide by Downregulating the Expression of DNA-Repair Gene Rad51

Recurrent glioblastoma multiforme (GBM) is characterized by resistance to radiotherapy and chemotherapy and a poor clinical prognosis. In this study, we investigated the role of the oncogenic transcription factor FoxM1 in GBM cells' resistance to alkylator temozolomide (TMZ) and its potential molecular mechanism. FoxM1 expression levels were measured by immunohistochemical analysis in 38 pairs of primary and recurrent GBM tumor samples. Expression levels were also measured in primary recurrent GBM cell lines, and their responses to TMZ were characterized. In a mechanistic study, an siRNA array was used to identify downstream genes, and a chromatin immunoprecipitation assay was used to confirm transcriptional regulation. Recurrent tumors that were TMZ resistant expressed higher levels of FoxM1 than did primary tumors. Recurrent GBM cell lines expressed higher levels of FoxM1 and the DNA damage repair gene Rad51 and were resistant to TMZ. TMZ treatment led to increased FoxM1 and Rad51 expression. FoxM1 knockdown inhibited Rad51 expression and sensitized recurrent GBM cells to TMZ cytotoxicity. FoxM1 directly regulated Rad51 expression through 2 FoxM1-specific binding sites in its promoter. Rad51 reexpression partially rescued TMZ resistance in FoxM1-knockdown recurrent GBM cells. A direct correlation between FoxM1 expression and Rad51 expression was evident in recurrent GBM tumor samples. Targeting the FoxM1-Rad51 axis may be an effective method to reverse TMZ resistance in recurrent GBM.