FoxM1 Inhibitor Research Articles

Abstract P1-07-07: Targeting reversal of epithelial-mesenchymal transition through FOXM1 inhibition in breast cancer cells

Abstract Purpose: Triple-negative breast cancer (TNBC), representing up to 20% of all breast cancer cases, is a highly diverse group of cancer that is associated with an aggressive phenotype, with affected patients having a poorer prognosis. Its treatment has been challenging due to its heterogeneity and the absence of well-defined molecular targets. Epithelial-to-mesenchymal transition (EMT) is one of the hallmarks of aggressive breast cancers and is associated with increased metastatic potential. TNBCs have been shown to exhibit elevated EMT. Thus, reversal of EMT has been identified as a viable target in the treatment of aggressive breast cancers. FOXM1 is an oncogenic transcription factor of the Forkhead family and it has a well-defined role in cell proliferation and cell-cycle progression. FOXM1 has been reported to be over-expressed in breast cancer. In prostate cancer, FOXM1 overexpression has been linked to increase EMT. We have previously shown that targeting NF-kB with panepoxydone (PP), an NF-kB inhibitor isolated from Lentinus crinitus (an edible mushroom), has significant antitumor activity against breast cancer cells with a resultant decrease in cell migration and invasion and induction of apoptosis. We have further identified that the FOXM1 promoter has a binding site for NF-kB. Thus, in this study, we investigated whether PP-induced inhibition of breast cancer cell growth could be attributed to FoxM1 activity and EMT reversal. Experimental Design: The estrogen receptor positive (MCF-7) and three different triple negative (MDA-MB-231, MDA-MB-453, and MDA-MB-468) breast cancer cell lines were selected to observe the effect of PP on FOXM1and EMT markers. Results: In the current study, we investigated whether panepoxydone- induced inhibition of breast cancer cell growth could be attributed to FoxM1activity and EMT reversal. Breast cancer cells were treated with increasing concentrations of PP and proteins collected after 24 hrs. Panepoxydone treatment significantly reduced FOXM1 expression in MCF-7 and MDA-MB-231 (1.7 and 2.4-fold, respectively, p < 0.05) but not in MDAMB-453 and MDAMB-468 cells when analyzed through western blot analysis. Furthermore, we showed that treatment with PP resulted in alteration of EMT-associated proteins with a significant increase in E-cadherin in all the cell lines (ranging from 1.8 to 4.2-fold, p < 0.05) and a decrease in mesenchymal markers vimentin and zeb1 in the MDA-MB-231 cells (2.5 and 2-fold respectively, p < 0.05). To further elucidate the relationship between FOXM1 on EMT reversal, we then silenced FOXM1 in MDA-MB-231 cells and evaluated its effect on the EMT-associated markers. Again we noted upregulation of E cadherin (3.5-fold, p< 0.01) and down-regulation of vimentin (4.5-fold, p <0.01) in FOXM1, which was very similar to what was observed in the PP-treated MDA-MB-231 cells. Conclusion: These studies show, for the first time, the role of FOXM1 in EMT reversal in breast cancer cells. Furthermore, it shows that use of an NF-kB inhibitor, such as panepoxydone, may be helpful in elucidating the relationship between these two transcriptional factors, in the reversal of the EMT phenotype in aggressive breast cancer cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-07.

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.

Abstract C76: FoxM1 inhibition sensitized BRCA-proficient triple-negative breast cancer to PARP inhibition.

Abstract Breast cancer cells with mutations in breast cancer-associated 1 (BRCA1) or BRCA2 have defects in the homologous recombination (HR) DNA repair pathway and this confers sensitivity to inhibitors of poly (ADP) ribose polymerase (PARP). However, BRCA-deficient tumors represent only a small fraction of adult cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. The transcription factor Forkhead box M1 (FoxM1) is a key regulator of cell proliferation and is overexpressed in many forms of primary cancers, leading to uncontrolled cell division and genomic instability. FoxM1 is implicated in genotoxic drug resistance but its role and mechanism of action remain unclear. Our results revealed that triple-negative breast cancer (TNBC) cell lines depleted in FoxM1 levels by small interfering RNA transfection display increase DNA damage, as evidence by activation of checkpoint kinase 2 (Chk2) and phosphorylation of histone H2AX. These results shows a role for FoxM1 in the transcriptional response during DNA damage/checkpoint signaling and we hypothesized that FoxM1 inhibition would result in HR impairment and subsequent sensitization to PARP inhibitor. We found that knockdown of FoxM1 led to sensitization to AZD2281, PARP inhibitor, in MDA-MB-231 (BRCA-wild-type) and MDA-MB-436 (BRCA-mutated) TNBC cells. Combined PARP and FoxM1 inhibition had a synergistic anti-proliferative effect on BRCA1-defective and BRCA-proficient TNBC cell lines in vitro. Furthermore, the combination treatment of AZD2281 and thiostrepton (FoxM1 inhibitor) increased DNA repair-related gene expression such as H2AX, chk1, chk2 and p53 and downregulated BRCA2 expression in TNBC cell lines. Here, we show that depletion or inhibition of FoxM1 compromises the ability of cells to repair DNA by HR and sensitization to PARP inhibition in TNBCs without BRCA mutations, providing a rationale to combine FoxM1 and PARP inhibitors. Therefore, this combination may be effective for treating both BRCA1-defective and BRCA-proficient TNBC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C76. Citation Format: Jun Woo Lee, Kyung-Min Lee, Wonshik Han. FoxM1 inhibition sensitized BRCA-proficient triple-negative breast cancer to PARP inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C76.

Forkhead box transcription factor 1 expression in gastric cancer: FOXM1 is a poor prognostic factor and mediates resistance to docetaxel

BackgroundForkhead box transcription factor 1 (FOXM1) has been reported to overexpress and correlate with pathogenesis in a variety of human malignancies. However, little research has been done to investigate its clinical significance in gastric cancer.MethodsWe examined the expression of FOXM1 in 103 postoperational gastric cancer tissues and 5 gastric cell lines by immunohistochemistry and western blot analysis respectively. Data on clinic-pathological features and relevant prognostic factors in these patients were then analyzed. Moreover, the association of FOXM1 expression and chemosensitivity to docetaxel in gastric cancer cells was further explored.ResultsOur study demonstrated that the level of FOXM1 expression was significantly higher in gastric cancer than in para-cancer tissues (P < 0.001) and normal gastric cell lines (P = 0.026). No significant association was found between FOXM1 expression and any clinical pathological features (P > 0.1). FOXM1 amplification was identified as an independent prognostic factor in gastric cancer (P = 0.001), and its affection is more significant in patients with tumor size larger than 5 cm (P = 0.004), pT3-4 (P = 0.003) or pIII-IV (P = 0.001). Additionally, shown to mediate docetaxel resistance in gastric cancers by our research, FOXM1 was revealed to alter microtubule dynamics in response to the treatment of docetaxel, and the drug resistance could be reversed with FOXM1 inhibitor thiostrepton treatment.ConclusionsFOXM1 can be a useful marker for predicting patients’ prognosis and monitoring docetaxel response, and might be a new therapeutic target in docetaxel resistant gastric cancer.

Inhibition of FOXM1 by thiostrepton sensitizes medulloblastoma to the effects of chemotherapy

Medulloblastoma (MB) is the most common malignant brain tumor in children and is highly invasive and metastatic. Despite recent advances, most MB patients suffer significant therapy-related morbidity, and the survival rate for patients with metastatic MB remains unsatisfactory. Altered expression of FOXM1 has been detected in many types of cancers, and the inhibition of FOXM1 has been studied as a cancer therapy. In the present study, we evaluated the impact of the inhibition of FOXM1 by thiostrepton in Daoy MB cells. Cells were treated with different concentrations of thiostrepton alone or in combination with cisplatin. Cell viability was measured with CCK-8 assays, and cell cycle distribution and apoptosis were assessed by flow cytometric analysis. Changes in protein expression were examined by western blotting. RNAi experiments were performed using siRNA oligonucleotides. The invasion and migration studies were performed using 8-µm Transwell plates. Inhibition of FOXM1 by thiostrepton significantly decreased MB cell proliferation. Cell arrest at the G2/M phase and apoptosis were significantly increased in MB cell lines that were treated with thiostrepton or transfected with siRNA. Thiostrepton decreased the IC50 value of cisplatin for MB treatment by enhancing cisplatin-induced apoptosis. Thiostrepton also decreased cell invasion and migration, which are crucial steps for tumor progression. Our data suggest that targeting FOXM1 with small-molecule inhibitors results in potent antitumor activity and chemosensitizing effects in human medulloblastoma cells.

Foxm1 Expression in Prostate Epithelial Cells Is Essential for Prostate Carcinogenesis

The treatment of advanced prostate cancer (PCa) remains a challenge. Identification of new molecular mechanisms that regulate PCa initiation and progression would provide targets for the development of new cancer treatments. The Foxm1 transcription factor is highly up-regulated in tumor cells, inflammatory cells, and cells of tumor microenvironment. However, its functions in different cell populations of PCa lesions are unknown. To determine the role of Foxm1 in tumor cells during PCa development, we generated two novel transgenic mouse models, one exhibiting Foxm1 gain-of-function and one exhibiting Foxm1 loss-of-function under control of the prostate epithelial-specific Probasin promoter. In the transgenic adenocarcinoma mouse prostate (TRAMP) model of PCa that uses SV40 large T antigen to induce PCa, loss of Foxm1 decreased tumor growth and metastasis. Decreased prostate tumorigenesis was associated with a decrease in tumor cell proliferation and the down-regulation of genes critical for cell proliferation and tumor metastasis, including Cdc25b, Cyclin B1, Plk-1, Lox, and Versican. In addition, tumor-associated angiogenesis was decreased, coinciding with reduced Vegf-A expression. The mRNA and protein levels of 11β-Hsd2, an enzyme playing an important role in tumor cell proliferation, were down-regulated in Foxm1-deficient PCa tumors in vivo and in Foxm1-depleted TRAMP C2 cells in vitro. Foxm1 bound to, and increased transcriptional activity of, the mouse 11β-Hsd2 promoter through the -892/-879 region, indicating that 11β-Hsd2 was a direct transcriptional target of Foxm1. Without TRAMP, overexpression of Foxm1 either alone or in combination with inhibition of a p19(ARF) tumor suppressor caused a robust epithelial hyperplasia, but was insufficient to induce progression from hyperplasia to PCa. Foxm1 expression in prostate epithelial cells is critical for prostate carcinogenesis, suggesting that inhibition of Foxm1 is a promising therapeutic approach for prostate cancer chemotherapy.

Activation of AMPK inhibits cervical cancer cell growth through AKT/FOXO3a/FOXM1 signaling cascade

BackgroundAlthough advanced-stage cervical cancer can benefit from current treatments, approximately 30% patients may fail after definitive treatment eventually. Therefore, exploring alternative molecular therapeutic approaches is imperatively needed for this disease. We have recently shown that activation of AMP-activated protein kinase (AMPK), a metabolic sensor, hampers cervical cancer cell growth through blocking the Wnt/β-catenin signaling activity. Here, we report that activated AMPK (p-AMPK) also inhibits cervical cancer cell growth by counteracting FOXM1 function.MethodsEffect of the activation of AMPK on FOXM1 expression was examined by hypoxia and glucose deprivation, as well as pharmacological AMPK activators such as A23187, AICAR and metformin. RT Q-PCR and Western blot analysis were employed to investigate the activities of AMPK, FOXM1 and AKT/FOXO3a signaling.ResultsConsistent with our previous findings, the activation of AMPK by either AMPK activators such as AICAR, A23187, metformin, glucose deprivation or hypoxia significantly inhibited the cervical cancer cell growth. Importantly, we found that activated AMPK activity was concomitantly associated with the reduction of both the mRNA and protein levels of FOXM1. Mechanistically, we showed that activated AMPK was able to reduce AKT mediated phosphorylation of p-FOXO3a (Ser253). Interestingly, activated AMPK could not cause any significant changes in FOXM1 in cervical cancer cells in which endogenous FOXO3a levels were knocked down using siRNAs, suggesting that FOXO3a is involved in the suppression of FOXM1.ConclusionTaken together, our results suggest the activated AMPK impedes cervical cancer cell growth through reducing the expression of FOXM1.

FoxM1 as a potential therapeutic target for triple-negative breast cancer.

e22063 Background: Elevated expression or activity of the transcription factor forkhead box M1 (FoxM1) is associated with development and progression of breast cancer. In this study, we show that whether inhibition of FoxM1 in triple negative breast cancer (TNBC) cell lines causes the changes of cancer progress and response to the treatment. Methods: We inhibited FoxM1 using thiostrepton (FoxM1 inhibitor) and identified the activity of VEGF, cyclin B, cyclin D1, p27, CD44 and cleaved capase-3 in MDA-MB-231 cells. Invasion assay was evaluated in BT-20 cells and combination with cisplatin and inhibition of FoxM1 were treated in triple negative breast cancer (TNBC) cell lines including MDA-MB-231, MDA-MB-157, and BT-20. Results: When FoxM1 was inhibited using thiostrepton in MDA-MB-231 cells, expressions of VEFG, cyclin B, cyclin D1, p27, and CD44 were decreased. On the other hand, cleaved caspase-3 expression was increased. Intersetingly, response to inhibition of FoxM1 depended on the expression of p53 in each cell lines. Invasiveness of BT-20 cells is markedly reduced by inhibitioh of FoxM1. Co-treatment with thiotrepton and cisplatin synergistically induced apoptosis of breast cancer cells in MDA-MB-231, MDA-MB-157, and BT-20. Conclusions: Inhibition of FoxM1 increases apoptosis and decreases invasiveness and VEGF exprsssion of TNBC cells. And when inhibition of FoxM1 combines with cisplatin treament, synergistic effect was shown. Therefore, FoxM1 is valuable as a potential target for not only anticancer activity but also overcoming cisplatin resistance in TNBC.

Targeting FoxM1 Effectively Retards p53-Null Lymphoma and Sarcoma

The forkhead box transcription factor FOXM1 is considered to be a promising target for cancer therapy. However, the significance of FOXM1 in tumors harboring mutation in p53, which is very common, is unclear. In this study, we investigated the efficacy of FoxM1 targeting in spontaneous p53-null tumors using genetic ablation as well as using a peptide inhibitor of FOXM1. We show that conditional deletion of FoxM1 inhibits growth of the p53-null thymic lymphoma and sarcoma cells. In addition, deletion of FoxM1 induces apoptotic cell death of the p53-null tumors, accompanied by reduced expression of the FOXM1 target genes survivin and Bmi1. An ARF-derived peptide that inhibits the activity of FOXM1, by targeting it to the nucleolus, also induces apoptosis in the p53-null sarcoma and lymphoma, leading to a strong inhibition of their metastatic colonization. Together, our observations suggest that FOXM1 is critical for survival and growth of the p53-null lymphoma and sarcoma and provide proof-of-principle that FOXM1 is an effective therapeutic target for sarcoma and lymphoma carrying loss of function mutation in p53.

Abstract 1074: Genome-wide analysis of FOXM1 binding and involvement of FOXM1 in cancer stem cell and metastasis properties of endocrine-sensitive and resistant breast cancer cells.

Abstract FOXM1 is a transcription factor that coordinates expression of late cell cycle-related genes. Elevated expression of FOXM1 in breast cancer correlates with a more aggressive tumor phenotype and, as we have previously shown, is associated with resistance to endocrine treatment via 14-3-3ζ protein signaling (Bergamaschi A. et al. Breast Cancer Research 2011, 13:R7). To better understand the role that FOXM1 plays in endocrine resistance and aggressiveness we analyzed its genome-wide DNA binding and effects on gene expression. We mapped genome-wide FOXM1 binding events, by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), in hormone-sensitive and resistant breast cancer cell lines after estrogen or tamoxifen treatments. We also investigated the relationship between FOXM1, estrogen receptor-α and MAPK chromatin binding events by clustering analysis of Chip-seq data and identified specific clusters of colocalized binding events as identifiers for different functional gene classes. Clusters enriched in colocalized factor binding sites were associated with genes involved in maintenance of stem cell properties and genes involved in invasion and metastasis (Rho-GTPases). Binding profiles were also integrated with expression data from FOXM1 knockdown experiments, to reveal specific FOXM1 transcriptional networks. Cell studies showed the relevance of FOXM1 in breast cancer endocrine resistance and development of metastasis. We modulated the levels of FOXM1 and some newly discovered FOXM1 regulated genes in MCF7 cells and assessed their roles in hormone resistance and breast cancer aggressiveness by several functional studies. Increased expression levels of FOXM1 were associated with an increase in the cancer stem cell population. Moreover, FOXM1 overexpressing cell lines exhibited invadopodia which correlated with an increased invasion potential. The use of a specific and selective FOXM1 inhibitor proved to be especially effective in restoring endocrine sensitivity and in decreasing breast cancer aggressiveness. Collectively, our findings define FOXM1 as a master regulator of Rho-GTPase and stem cell marker expression. Dysregulation of the FOXM1 pathway promotes breast cancer progression and therefore makes FOXM1 a relevant therapeutic target to be considered for reducing invasiveness and enhancing response to endocrine and other therapies. Supported by Department of Defense grant W81XWH-09-1-0398 and a grant from The Breast Cancer Research Foundation). Citation Format: Anna Bergamaschi, Zeynep Madak-Erdogan, Hailing Lu, Benita S. Katzenellenbogen. Genome-wide analysis of FOXM1 binding and involvement of FOXM1 in cancer stem cell and metastasis properties of endocrine-sensitive and resistant breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1074. doi:10.1158/1538-7445.AM2013-1074

Casticin induces ovarian cancer cell apoptosis by repressing FoxM1 through the activation of FOXO3a

Casticin, a polymethoxyflavone, is reported to have anticancer activities. The aim of the present study was to examine the molecular mechanisms by which casticin induces apoptosis in ovarian cancer cells. The human ovarian cancer cell lines SKOV3 and A2780 were cultured in vitro. Various molecular techniques, including histone/DNA enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), western blot analysis and gene transfection, were used to assess the expression of FOXO3a and forkhead box protein M1 (FoxM1) in casticin-treated ovarian cancer cell lines. Casticin-induced apoptotic cell death was accompanied by the activation of transcription factor FOXO3a, with a concomitant decrease in the expression levels of FoxM1 and its downstream target factors, namely survivin and polo-like kinase 1 (PLK1), and an increase in p27KIP1. A small inhibitory RNA (siRNA) knockout of FoxM1 potentiated casticin-induced apoptosis in ovarian cancer cells. Silencing FOXO3a expression using siRNA increased FoxM1 expression levels and clearly attenuated the induction of apoptosis by casticin treatment. These results show that casticin-induced apoptosis in ovarian cancer may be caused by the activation of FOXO3a, leading to FoxM1 inhibition.

Abstract C85: FOXM1-dependent gene expression program controls cancer stem cell and metastasis properties of breast cancer cells

Abstract The transcription factor FOXM1 coordinates expression of late cell cycle-related genes. Elevated expression of FOXM1 in breast cancer correlates with a more aggressive tumor phenotype and, as we have previously shown, is associated with resistance to endocrine treatment via 14-3-3ζ protein signaling (Bergamaschi A. et al. Breast Cancer Research 2011, 13:R7). To better understand the role that FOXM1 plays in endocrine resistance and aggressiveness, we analyzed its genome-wide DNA binding and effects on gene expression. We mapped genome-wide FOXM1 binding events, by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), in breast cancer cell lines after estrogen or tamoxifen treatments. We also investigated the relationship between FOXM1, estrogen receptor-α and MAPK chromatin binding events by clustering analysis of Chip-seq data and identified specific clusters of colocalized binding events as identifiers for different functional gene classes. Clusters enriched in colocalized factor binding sites were associated with genes involved in maintenance of stem cell properties and genes involved in invasion and metastasis (Rho-GTPases). Binding profiles were also integrated with expression data from FOXM1 knockdown experiments, to reveal specific FOXM1 transcriptional networks. Cell studies showed the relevance of FOXM1 in breast cancer endocrine resistance and development of metastasis. We modulated the levels of FOXM1 and some newly discovered FOXM1 regulated genes in MCF7 cells and assessed their roles in hormone resistance and breast cancer aggressiveness by several functional studies. Increased expression levels of FOXM1 were associated with an increase in the cancer stem cell population. Moreover, FOXM1 overexpressing cell lines exhibited invadopodia which correlated with an increased invasion potential. The use of a specific and selective FOXM1 inhibitor proved to be especially effective in restoring endocrine sensitivity and in decreasing breast cancer aggressiveness. Collectively, our findings define FOXM1 as a master regulator of Rho-GTPase and stem cell marker expression. Dysregulation of the FOXM1 pathway promotes breast cancer progression and therefore makes FOXM1 a relevant therapeutic target to be considered for reducing invasiveness and enhancing response to endocrine and other therapies. Citation Format: Anna Bergamaschi, Zeynep Madak-Erdogan, Hailing Lu, Benita S. Katzenellenbogen. FOXM1-dependent gene expression program controls cancer stem cell and metastasis properties of breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C85.

ErbB2, FoxM1 and 14-3-3ζ prime breast cancer cells for invasion in response to ionizing radiation

ErbB2 is frequently highly expressed in premalignant breast cancers, including ductal carcinoma in situ (DCIS); however, little is known about the signals or pathways it contributes to progression into the invasive/malignant state. Radiotherapy is often used to treat early premalignant lesions regardless of ErbB2 status. Here, we show that clinically relevant doses of ionizing radiation (IR)-induce cellular invasion of ErbB2-expressing breast cancer cells, as well as MCF10A cells overexpressing ErbB2. ErbB2-negative breast cancer cells, such as MCF7 and T47D, do not invade following treatment with IR nor do MCF10A cells overexpressing epidermal growth factor receptor. ErbB2 becomes phosphorylated at tyrosine 877 in a dose- and time- dependent manner following exposure to X-rays, and activates downstream signaling cascades including PI3K/Akt. Inhibition of these pathways, as well as inhibition of reactive oxygen species (ROS) with antioxidants, prevents IR-induced invasion. Activation of ErbB2-dependent signaling results in upregulation of the forkhead family transcription factor, FoxM1, and its transcriptional targets, including matrix metalloproteinase 2 (MMP2). Inhibition of FoxM1 by RNA interference prevented induction of invasion by IR, and overexpression of FoxM1 in MCF10A cells was sufficient to promote IR-induced invasion. Moreover, we found that 14-3-3ζ is also upregulated by IR in cancer cells in a ROS-dependent manner, is required for IR-induced invasion in ErbB2-positive breast cancer cells and together with FoxM1 is sufficient for invasion in ErbB2-negative breast cancer cells. Thus, our data show that IR-mediated activation of ErbB2 and induction of 14-3-3ζ collaborate to regulate FoxM1 and promote invasion of breast cancer cells and furthermore, may serve as therapeutic targets to enhance radiosensitivity of breast cancers.

Foxm1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition

Alveolar epithelial cells (AECs) participate in the pathogenesis of pulmonary fibrosis, producing pro-inflammatory mediators and undergoing epithelial-to-mesenchymal transition (EMT). Herein, we demonstrated the critical role of Forkhead Box M1 (Foxm1) transcription factor in radiation-induced pulmonary fibrosis. Foxm1 was induced in AECs following lung irradiation. Transgenic expression of an activated Foxm1 transcript in AECs enhanced radiation-induced pneumonitis and pulmonary fibrosis, and increased the expression of IL-1β, Ccl2, Cxcl5, Snail1, Zeb1, Zeb2 and Foxf1. Conditional deletion of Foxm1 from respiratory epithelial cells decreased radiation-induced pulmonary fibrosis and prevented the increase in EMT-associated gene expression. siRNA-mediated inhibition of Foxm1 prevented TGF-β-induced EMT in vitro. Foxm1 bound to and increased promoter activity of the Snail1 gene, a critical transcriptional regulator of EMT. Expression of Snail1 restored TGF-β-induced loss of E-cadherin in Foxm1-deficient cells in vitro. Lineage-tracing studies demonstrated that Foxm1 increased EMT during radiation-induced pulmonary fibrosis in vivo. Foxm1 is required for radiation-induced pulmonary fibrosis by enhancing the expression of genes critical for lung inflammation and EMT.

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.

FOXM1 Promotes Allergen-Induced Goblet Cell Metaplasia and Pulmonary Inflammation

Chronic airway disorders, including chronic obstructive pulmonary disease (COPD), cystic fibrosis, and asthma, are associated with persistent pulmonary inflammation and goblet cell metaplasia and contribute to significant morbidity and mortality worldwide. While the molecular pathogenesis of these disorders is actively studied, little is known regarding the transcriptional control of goblet cell differentiation and mucus hyperproduction. Herein, we demonstrated that pulmonary allergen sensitization induces expression of FOXM1 transcription factor in airway epithelial and inflammatory cells. Conditional deletion of the Foxm1 gene from either airway epithelium or myeloid inflammatory cells decreased goblet cell metaplasia, reduced lung inflammation, and decreased airway resistance in response to house dust mite allergen (HDM). FOXM1 induced goblet cell metaplasia and Muc5AC expression through the transcriptional activation of Spdef. FOXM1 deletion reduced expression of CCL11, CCL24, and the chemokine receptors CCR2 and CX3CR1, resulting in decreased recruitment of eosinophils and macrophages to the lung. Deletion of FOXM1 from dendritic cells impaired the uptake of HDM antigens and decreased cell surface expression of major histocompatibility complex II (MHC II) and costimulatory molecule CD86, decreasing production of Th2 cytokines by activated T cells. Finally, pharmacological inhibition of FOXM1 by ARF peptide prevented HDM-mediated pulmonary responses. FOXM1 regulates genes critical for allergen-induced lung inflammation and goblet cell metaplasia.

Negative regulation of transcription factor FoxM1 by p53 enhances oxaliplatin-induced senescence in hepatocellular carcinoma

Previous studies have demonstrated the involvement of transcriptional factor forkhead box M1 (FoxM1) in cellular senescence of hepatocellular carcinoma (HCC). In the present study, we revealed that oxaliplatin could induce senescence in HCC cells, since advanced HCC patients with lower expression of FoxM1 were more sensitive to oxaliplatin therapy. Our data indicated that due to the repression by p53, FoxM1 played a critical role in oxaliplatin-induced senescence via regulating cycle-related proteins p21, p27, cyclins B1 and D1. Furthermore, inhibition of FoxM1, combined with oxaliplatin treatment, could significantly promote the senescence of HCC cells. Taken together, our findings suggest that FoxM1 may represent a promising therapeutic target for the medication of the chemosensitivity to oxaliplatin in HCC patients.

FoxM1 expression is significantly associated with cisplatin-based chemotherapy resistance and poor prognosis in advanced non-small cell lung cancer patients

BackgroundThe transcription factor Forkhead box M1 (FoxM1) is known to play an important role in the development and progression of many malignancies including lung cancer. However, the relationship of FoxM1 expression and the clinical response to chemotherapy and prognosis in non-small cell lung cancer (NSCLC) remains unknown. MethodsTotal 162 NSCLC (stages IIIB and IV) patients who had tumor specimens available before treatment were assessed for FoxM1 expression using immunohistochemistry. Clinical significance was analyzed by Kaplan–Meier curves, log-rank test and multivariate Cox regression analysis. Sensitivities to cisplatin were detected by the MTT assay and drug-resistance related genes were analyzed by real-time PCR and western blot between DDP-sensitive A549 and the corresponding DDP-resistant cell subline (A549/DDP). Furthermore, small interfering RNA (siRNA) targeting FoxM1 was transfected into A549 and A549/DDP cell lines in vitro and migration and invasion were examined separately by Transwell chamber assay. ResultsPatients with FoxM1 expression had a significantly lower response rate (P=0.009) and poor progression-free survival (PFS, P=0.002) and overall survival (OS, P=0.007) than those without FoxM1 expression. Multivariate analyses indicated that FoxM1 positivity was an independent prognostic factor for PFS (P=0.006) and OS (P=0.021), respectively. Moreover, the expression of FoxM1 was significantly higher in A549/DDP cell subline than in A549 cells at both mRNA and protein levels. The FoxM1 inhibitor thiostrepton also showed efficacy in causing cell death and proliferative arrest in the cisplatin-resistant cells through the downregulation of FoxM1 expression. Knockdown of FoxM1 by siRNA suppressed cell migration and invasion in A549 and A549/DDP cells. Cisplatin resistance in A549/DDP cells could be partially reversed through siRNA-mediated FoxM1 inhibition. ConclusionsThe expression of FoxM1 might be an independent prognostic marker for advanced NSCLC patients and FoxM1 inhibition would be a potential strategy for chemosensitization of NSCLC cells.

Identification of FoxM1/Bub1b signaling pathway as a required component for growth and survival of rhabdomyosarcoma.

We identified Bub1b as an essential element for the growth and survival of rhabdomyosarcoma (RMS) cells using a bar-coded, tetracycline-inducible short hairpin RNA (shRNA) library screen. Knockdown of Bub1b resulted in suppression of tumor growth in vivo, including the regression of established tumors. The mechanism by which this occurs is via postmitotic endoreduplication checkpoint and mitotic catastrophe. Furthermore, using a chromatin immunoprecipitation assay, we found that Bub1b is a direct transcriptional target of Forkhead Box M1 (FoxM1). Suppression of FoxM1 either by shRNA or the inhibitor siomycin A resulted in reduction of Bub1b expression and inhibition of cell growth and survival. These results show the important role of the Bub1b/FoxM1 pathway in RMS and provide potential therapeutic targets.

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.