ABSTRACT
FOXM1, a proto-oncogenic transcription factor, plays a critical role in cancer development and treatment resistance in cancers, particularly in breast cancer. Thus, this study aimed to identify potential FOXM1 inhibitors through computational screening of drug databases, followed by in vitro validation of their inhibitory activity against breast cancer cells. In silico studies involved pharmacophore modeling using the FOXM1 inhibitor, FDI-6, followed by virtual screening of DrugBank and Selleckchem databases. The selected drugs were prepared for molecular docking, and the crystal structure of FOXM1 was pre-processed for docking simulations. In vitro studies included MTT assays to assess cytotoxicity, and Western blot analysis to evaluate protein expression levels. Our study identified Pantoprazole and Rabeprazole as potential FOXM1 inhibitors through in silico screening and molecular docking. Molecular dynamics simulations confirmed stable interactions of these drugs with FOXM1. In vitro experiments showed both Pantoprazole and Rabeprazole exhibited strong FOXM1 inhibition at effective concentrations and that showed inhibition of cell proliferation. Rabeprazole showed the inhibitor activity at 10 µM in BT-20 and MCF-7 cell lines. Pantoprazole exhibited FOXM1 inhibition at 30 µM and in BT-20 cells and at 70 µM in MCF-7 cells, respectively. Our current study provides the first evidence that Rabeprazole and Pantoprazole can bind to FOXM1 and inhibit its activity and downstream signaling, including eEF2K and pEF2, in breast cancer cells. These findings indicate that rabeprazole and pantoprazole inhibit FOXM1 and breast cancer cell proliferation, and they can be used for FOXM1-targeted therapy in breast or other cancers driven by FOXM1.
Subject(s)
Breast Neoplasms , Cell Proliferation , Drug Repositioning , Forkhead Box Protein M1 , Molecular Docking Simulation , Rabeprazole , Humans , Forkhead Box Protein M1/antagonists & inhibitors , Forkhead Box Protein M1/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Female , Rabeprazole/pharmacology , MCF-7 Cells , Cell Proliferation/drug effects , Molecular Dynamics Simulation , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Pantoprazole/pharmacology , Cell Line, Tumor , Pyridines , ThiophenesABSTRACT
BACKGROUND: Genetic studies implicate the oncogenic transcription factor Forkhead Box M1 (FOXM1) as a potential therapeutic target in high-grade serous ovarian cancer (HGSOC). We evaluated the activity of different FOXM1 inhibitors in HGSOC cell models. RESULTS: We treated HGSOC and fallopian tube epithelial (FTE) cells with a panel of previously reported FOXM1 inhibitors. Based on drug potency, efficacy, and selectivity, determined through cell viability assays, we focused on two compounds, NB-73 and NB-115 (NB compounds), for further investigation. NB compounds potently and selectively inhibited FOXM1 with lesser effects on other FOX family members. NB compounds decreased FOXM1 expression via targeting the FOXM1 protein by promoting its proteasome-mediated degradation, and effectively suppressed FOXM1 gene targets at both the protein and mRNA level. At the cellular level, NB compounds promoted apoptotic cell death. Importantly, while inhibition of apoptosis using a pan-caspase inhibitor rescued HGSOC cells from NB compound-induced cell death, it did not rescue FOXM1 protein degradation, supporting that FOXM1 protein loss from NB compound treatment is specific and not a general consequence of cytotoxicity. Drug washout studies indicated that FOXM1 reduction was retained for at least 72 h post-treatment, suggesting that NB compounds exhibit long-lasting effects in HGSOC cells. NB compounds effectively suppressed both two-dimensional and three-dimensional HGSOC cell colony formation at sub-micromolar concentrations. Finally, NB compounds exhibited synergistic activity with carboplatin in HGSOC cells. CONCLUSIONS: NB compounds are potent, selective, and efficacious inhibitors of FOXM1 in HGSOC cells and are worthy of further investigation as HGSOC therapeutics.
Subject(s)
Antineoplastic Agents , Apoptosis , Forkhead Box Protein M1 , Ovarian Neoplasms , Forkhead Box Protein M1/metabolism , Forkhead Box Protein M1/antagonists & inhibitors , Humans , Female , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Ovarian Neoplasms/metabolism , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cystadenocarcinoma, Serous/drug therapy , Cystadenocarcinoma, Serous/pathology , Cystadenocarcinoma, Serous/metabolism , Cell Survival/drug effects , Neoplasm GradingABSTRACT
Inducing homologous recombination (HR) deficiency is a promising strategy to broaden the indication of PARP1/2 inhibitors in pancreatic cancer treatment. In addition to inhibition kinases, repression of the transcriptional function of FOXM1 has been reported to inhibit HR-mediated DNA repair. We found that FOXM1 inhibitor FDI-6 and PARP1/2 inhibitor Olaparib synergistically inhibited the malignant growth of pancreatic cancer cells in vitro and in vivo. The results of bioinformatic analysis and mechanistic study showed that FOXM1 directly interacted with PARP1. Olaparib induced the feedback overexpression of PARP1/2, FOXM1, CDC25A, CCND1, CDK1, CCNA2, CCNB1, CDC25B, BRCA1/2 and Rad51 to promote the acceleration of cell mitosis and recovery of DNA repair, which caused the generation of adaptive resistance. FDI-6 reversed Olaparib-induced adaptive resistance and inhibited cell cycle progression and DNA damage repair by repressing the expression of FOXM1, PARP1/2, BUB1, CDC25A, BRCA1 and other genes-involved in cell cycle control and DNA damage repair. We believe that targeting FOXM1 and PARP1/2 is a promising combination therapy for pancreatic cancer without HR deficiency.
Subject(s)
Forkhead Box Protein M1/antagonists & inhibitors , Pancreatic Neoplasms/drug therapy , Phthalazines/therapeutic use , Piperazines/therapeutic use , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Pyridines/therapeutic use , Thiophenes/therapeutic use , Animals , Apoptosis/drug effects , BRCA1 Protein/genetics , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Comet Assay , Female , Forkhead Box Protein M1/genetics , Forkhead Box Protein M1/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice, Inbred BALB C , Mice, Nude , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Phthalazines/pharmacology , Piperazines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Pyridines/pharmacology , Signal Transduction/drug effects , Thiophenes/pharmacology , cdc25 Phosphatases/geneticsABSTRACT
Aim: Given the importance of FOXM1 in the treatment of ovarian cancer, we aimed to identify an excellent specific inhibitor and examined its underlying therapeutic effect. Materials & methods: The binding statistics for FDI-6 with FOXM1 were calculated through computer-aided drug design. We selected XST-119 through virtual screening, performed surface plasmon resonance and in vitro cell antiproliferative activity analysis and evaluated its antitumor efficacy in a mouse model. Results: XST-119 had significantly higher affinity for FOXM1 and antiproliferative activity than FDI-6. XST-119 had a definite inhibitory activity in a xenograft mouse model. Conclusion: We identified XST-119, a FOXM1 inhibitor, with better efficacy for treatment of ovarian cancer. FOXM1 binding sites for small molecules are also highlighted, which may provide the foundation for further drug discovery.
Subject(s)
Forkhead Box Protein M1/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Forkhead Box Protein M1/metabolism , Humans , Ligands , Mice , Molecular Docking Simulation , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Structure-Activity Relationship , ThermodynamicsABSTRACT
BACKGROUND: Increased expression of the transcription factor Forkhead box M1 (FOXM1) has been reported to play an important role in the progression and development of multiple tumors, but the molecular mechanisms that regulate FOXM1 expression remain unknown, and the role of FOXM1 in aerobic glycolysis is still not clear. METHODS: The expression of FOXM1 and NADPH oxidase 4 (NOX4) in normal brain tissues and glioma was detected in data from the TCGA database and in our specimens. The effect of NOX4 on the expression of FOXM1 was determined by Western blot, qPCR, reactive oxygen species (ROS) production assays, and luciferase assays. The functions of NOX4 and FOXM1 in aerobic glycolysis in glioblastoma cells were determined by a series of experiments, such as Western blot, extracellular acidification rate (ECAR), lactate production, and intracellular ATP level assays. A xenograft mouse model was established to test our findings in vivo. RESULTS: The expression of FOXM1 and NOX4 was increased in glioma specimens compared with normal brain tissues and correlated with poor clinical outcomes. Aberrant mitochondrial reactive oxygen species (ROS) generation of NOX4 induced FOXM1 expression. Mechanistic studies demonstrated that NOX4-derived MitoROS exert their regulatory role on FOXM1 by mediating hypoxia-inducible factor 1α (HIF-1α) stabilization. Further research showed that NOX4-derived MitoROS-induced HIF-1α directly activates the transcription of FOXM1 and results in increased FOXM1 expression. Overexpression of NOX4 or FOXM1 promoted aerobic glycolysis, whereas knockdown of NOX4 or FOXM1 significantly suppressed aerobic glycolysis, in glioblastoma cells. NOX4-induced aerobic glycolysis was dependent on elevated FOXM1 expression, as FOXM1 knockdown abolished NOX4-induced aerobic glycolysis in glioblastoma cells both in vitro and in vivo. CONCLUSION: Increased expression of FOXM1 induced by NOX4-derived MitoROS plays a pivotal role in aerobic glycolysis, and our findings suggest that inhibition of NOX4-FOXM1 signaling may present a potential therapeutic target for glioblastoma treatment.
Subject(s)
Brain Neoplasms/metabolism , Forkhead Box Protein M1/metabolism , Glioblastoma/metabolism , NADPH Oxidase 4/metabolism , Reactive Oxygen Species/metabolism , Warburg Effect, Oncologic , Adenosine Triphosphate/metabolism , Animals , Blotting, Western , Brain/metabolism , Brain Neoplasms/therapy , Cell Line, Tumor , Forkhead Box Protein M1/antagonists & inhibitors , Glioblastoma/therapy , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lactic Acid/biosynthesis , Mice , Mice, Inbred BALB C , Mice, Nude , Mitochondria/metabolism , NADPH Oxidase 4/antagonists & inhibitors , Neoplasm Proteins/metabolism , Neoplasm TransplantationABSTRACT
AIMS: 5-Fluorouracil (5-FU), a thymidylate synthase (TS) inhibitor, has been used as the first-line chemotherapeutic drug for cholangiocarcinoma (CCA). The side effects and drug resistance have developed the limits of the clinical application of 5-FU in CCA treatment. Upregulation of Forkhead box M1 (FOXM1) and TS were shown to play a significant role in 5-FU resistance. In this study, the effect of Siomycin A (SioA), a FOXM1 inhibitor, on enhancing 5-FU cytotoxicity and reversing 5-FU resistance in CCA cell lines were demonstrated. MAIN METHODS: Human CCA cell lines, KKU-100 and KKU-213A were used. Cell viability was determined using MTT assay. Expression of FOXM1 and TS proteins were determined using Western blotting. FOXM1 mRNA expression was quantitated using real-time PCR. The combination and dose reduction (DRI) were analyzed according to the Chou and Talalay method. KEY FINDING: Single drug treatment of 5-FU and SioA effectively inhibited CCA cell growth in dose and time dependent fashions. The two CCA cell lines had different responses to 5-FU but exhibited similar sensitivity to SioA. FOXM1 and TS expression were increased in the 5-FU treated cells but were suppressed in the SioA treated cells. A direct binding of SioA, to TS and 5,10-methylene-tetrahydrofolate as an inactive ternary complex was simulated. The combined treatment of 5-FU with SioA showed a synergistic effect with a high DRI and restored 5-FU sensitivity in the 5-FU resistant cells. SIGNIFICANCE: Targeting FOXM1 using SioA in combination with 5-FU might be a strategy to overcome the 5-FU resistance in CCA.
Subject(s)
Cholangiocarcinoma/drug therapy , Peptides/pharmacology , Thymidylate Synthase/metabolism , Apoptosis/drug effects , Bile Duct Neoplasms/pathology , Bile Ducts, Intrahepatic/drug effects , Bile Ducts, Intrahepatic/metabolism , Bile Ducts, Intrahepatic/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cholangiocarcinoma/metabolism , Drug Resistance, Neoplasm/drug effects , Fluorouracil/pharmacology , Forkhead Box Protein M1/antagonists & inhibitors , Forkhead Box Protein M1/metabolism , Gene Expression/genetics , Gene Expression Regulation, Neoplastic/genetics , Humans , Peptides/metabolism , Thymidylate Synthase/physiologyABSTRACT
Electrophoretic mobility shift assay (EMSA) technology has been widely employed for the analysis of transcription factors such as Forkhead box protein M1 (FOXM1). However, the application of high-throughput screening (HTS) in performing, such analyses are limited as it uses time consuming electrophoresis procedure and radioisotopes. In this study, we developed a FOXM1-DNA binding domain (DBD) binding assay based on time-resolved fluorescence energy transfer (TR-FRET) that enables HTS for the inhibitors of FOXM1-DNA interaction. This assay was robust, highly reproducible and could be easily miniaturized into 384-well plate format. The signal-to-background (S/B) ratio and Z' factor were calculated as 7.46 and 0.74, respectively, via a series of optimization of the assay conditions. A pilot library screening of 1019 natural compounds was performed using the FOXM1-DBD binding assay. Five hit compounds, namely, AC1LXM, BRN5, gangaleoidin, leoidin, and roemerine were identified as the inhibitors of FOXM1. In a cell viability assay, it was demonstrated that cell proliferation of FOXM1 overexpressed cell lines was suppressed in cell lines such as MDA-MB-231 and MCF-7 by five hit compounds. These results indicate that developed FOXM1-DBD binding assay can be applied to highly efficiency HTS of compound libraries.
Subject(s)
Forkhead Box Protein M1/metabolism , High-Throughput Screening Assays/methods , DNA/metabolism , Drug Discovery/methods , Fluorescence Resonance Energy Transfer , Forkhead Box Protein M1/antagonists & inhibitors , Humans , MCF-7 Cells , Protein Binding/drug effects , Protein Interaction Domains and MotifsABSTRACT
The FOXM1 protein controls the expression of essential genes related to cancer cell cycle progression, metastasis, and chemoresistance. We hypothesize that FOXM1 inhibitors could represent a novel approach to develop 18 F-based radiotracers for Positron Emission Tomography (PET). Therefore, in this report we describe the first attempt to use 18 F-labeled FOXM1 inhibitors to detect triple-negative breast cancer (TNBC). Briefly, we replaced the original amide group in the parent drug FDI-6 for a ketone group in the novel AF-FDI molecule, to carry out an aromatic nucleophilic (18 F)-fluorination. AF-FDI dissociated the FOXM1-DNA complex, decreased FOXM1 levels, and inhibited cell proliferation in a TNBC cell line (MDA-MB-231). [18 F]AF-FDI was internalized in MDA-MB-231 cells. Cell uptake inhibition experiments showed that AF-FDI and FDI-6 significantly decreased the maximum uptake of [18 F]AF-FDI, suggesting specificity towards FOXM1. [18 F]AF-FDI reached a tumor uptake of SUV=0.31 in MDA-MB-231 tumor-bearing mice and was metabolically stable 60â min post-injection. These results provide preliminary evidence supporting the potential role of FOXM1 to develop PET radiotracers.
Subject(s)
Antineoplastic Agents/pharmacology , Forkhead Box Protein M1/antagonists & inhibitors , Pyridines/pharmacology , Thiophenes/pharmacology , Triple Negative Breast Neoplasms/drug therapy , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Forkhead Box Protein M1/metabolism , Humans , Mammary Neoplasms, Experimental/diagnosis , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/metabolism , Mice , Molecular Structure , Positron-Emission Tomography , Pyridines/chemical synthesis , Pyridines/chemistry , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/chemistry , Triple Negative Breast Neoplasms/diagnosis , Triple Negative Breast Neoplasms/metabolismABSTRACT
The development of radioresistance during radiotherapy is a major cause of tumor recurrence and metastasis. To provide new insights of the mechanisms underlying radioresistance, we established radioresistant cell lines derived from two different subtypes of breast cancer cells, HER2-positive SK-BR-3 and ER-positive MCF-7 breast cancer cells, by exposing cells to 48 ~ 70 Gy of radiation delivered at 4-5 Gy twice weekly over 9 ~ 10 months. The established radioresistant SK-BR-3 (SR) and MCF-7 (MR) cells were resistant not only to a single dose of radiation (2 Gy or 4 Gy) but also to fractionated radiation delivered at 2 Gy/day for 5 days. Furthermore, these cells exhibited tumor-initiating potential in vivo and high CD24-/CD44 + ratio. To identify novel therapeutic molecular targets, we analyzed differentially expressed genes in both radioresistant cell lines and found that the expression of ACSL4 was significantly elevated in both cell lines. Targeting ACSL4 improved response to irradiation and inhibited migration activities. Furthermore, inhibition of ACLS4 using ASCL4 siRNA or triacsin C suppressed FOXM1 expression, whereas inhibition of FOXM1 using thiostrepton did not affect ACSL4 expression. Targeting the ACSL4-FOXM1 signaling axis by inhibiting ASCL4 or FOXM1 overcame the radioresistance by suppressing DNA damage responses and inducing apoptosis. This is the first study to report that ACSL4 plays a crucial role in mediating the radioresistance of breast cancer by regulating FOXM1. We propose the ACSL4-FOXM1 signaling axis be considered a novel therapeutic target in radioresistant breast cancer and suggest treatment strategies targeting this signaling axis might overcome breast cancer radioresistance.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/radiotherapy , Coenzyme A Ligases/metabolism , Forkhead Box Protein M1/metabolism , Radiation Tolerance/physiology , Animals , Coenzyme A Ligases/antagonists & inhibitors , Female , Forkhead Box Protein M1/antagonists & inhibitors , Humans , MCF-7 Cells , Mice , Mice, Inbred BALB C , Mice, NudeABSTRACT
FOXM1 transcription factor is an oncogene and a master regulator of chemoresistance in multiple cancers. Pharmacological inhibition of FOXM1 is a promising approach but has proven to be challenging. We performed a network-centric transcriptomic analysis to identify a novel compound STL427944 that selectively suppresses FOXM1 by inducing the relocalization of nuclear FOXM1 protein to the cytoplasm and promoting its subsequent degradation by autophagosomes. Human cancer cells treated with STL427944 exhibit increased sensitivity to cytotoxic effects of conventional chemotherapeutic treatments (platinum-based agents, 5-fluorouracil, and taxanes). RNA-seq analysis of STL427944-induced gene expression changes revealed prominent suppression of gene signatures characteristic for FOXM1 and its downstream targets but no significant changes in other important regulatory pathways, thereby suggesting high selectivity of STL427944 toward the FOXM1 pathway. Collectively, the novel autophagy-dependent mode of FOXM1 suppression by STL427944 validates a unique pathway to overcome tumor chemoresistance and improve the efficacy of treatment with conventional cancer drugs.
Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Drug Resistance, Neoplasm/drug effects , Forkhead Box Protein M1/antagonists & inhibitors , Gene Expression Profiling , Neoplasms/drug therapy , Cell Line, Tumor , Forkhead Box Protein M1/genetics , Forkhead Box Protein M1/metabolism , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Protein Stability , Protein Transport , Proteolysis , RNA-Seq , TranscriptomeABSTRACT
Triple-negative breast cancer (TNBC) presents an important clinical challenge, as it does not respond to endocrine therapies or other available targeting agents. FOXM1, an oncogenic transcriptional factor, has reported to be upregulated and associated with poor clinical outcomes in TNBC patients. In this study, we investigated the anti-cancer effects of FDI-6, a FOXM1 inhibitor, as well as its molecular mechanisms, in TNBC cells. Two TNBC cell lines, MDA-MB-231 and HS578T, were used in this study. The anti-cancer activities of FDI-6 were evaluated using various 2D cell culture assays, including Sulforhodamine B (SRB), wound healing, and transwell invasion assays together with 3D spheroid assays, mimicking real tumour structural properties. After treatment with FDI-6, the TNBC cells displayed a significant inhibition in cell proliferation, migration, and invasion. Increased apoptosis was also observed in the treated cells. In addition, we found that FDI-6 lead to the downregulation of FOXM1 and its key oncogenic targets, including CyclinB1, Snail, and Slug. Interestingly, we also found that the FDI-6/Doxorubicin combination significantly enhanced the cytotoxicity and apoptotic properties, suggesting that FDI-6 might improve chemotherapy treatment efficacy and reduce unwanted side effects. Altogether, FDI-6 exhibited promising anti-tumour activities and could be developed as a newly effective treatment for TNBC.
Subject(s)
Antineoplastic Agents/pharmacology , Forkhead Box Protein M1/antagonists & inhibitors , Pyridines/pharmacology , Thiophenes/pharmacology , Antineoplastic Agents/chemistry , Caspase 3/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyridines/chemistry , Thiophenes/chemistry , Triple Negative Breast Neoplasms/metabolismABSTRACT
The activation of Wnt/ß-catenin pathway plays a pivotal role in promoting renal fibrosis. The activation of Wnt/ß-catenin pathway relies on the binding of Wnts to Frizzled receptors on cell membrane. However, the factor regulating Wnts production remains unclear. Here, we demonstrated that transcriptional factor FoxM1 was significantly increased in obstructed kidneys and patients' kidneys with fibrosis. The up-regulation of FoxM1 mainly distributed in tubular epithelial cells. Pharmacological inhibition of FoxM1 down-regulated multi-Wnts elevation in UUO mice and attenuated renal fibrosis. In cultured renal tubular epithelial cells, overexpression of FoxM1 promoted 8 Wnts expression, while knock-down on FoxM1-suppressed multi-Wnts including Wnt1, Wnt2b and Wnt3 expression induced by Ang II. Chromatin immunoprecipitation PCR confirmed that FoxM1 bound to Wnt1, Wnt2b, Wnt3 promoters and luciferase assay further identified that the transcriptions of Wnt1, Wnt2b and Wnt3 were regulated by FoxM1. Thus, our findings show that multi-Wnt family members were regulated by transcriptional factor FoxM1. FoxM1 might be a key switch for activating ß-catenin pathway and renal fibrosis. Therefore, FoxM1 might be a potential therapeutic target in manipulating renal fibrosis.
Subject(s)
Forkhead Box Protein M1/metabolism , Gene Expression Regulation , Kidney Diseases/genetics , Kidney Diseases/metabolism , Wnt Proteins/genetics , Wnt Proteins/metabolism , Wnt Signaling Pathway , Angiotensin II/metabolism , Angiotensin II/pharmacology , Animals , Biomarkers , Cells, Cultured , Disease Models, Animal , Disease Susceptibility , Epithelial Cells/metabolism , Fibrosis , Forkhead Box Protein M1/antagonists & inhibitors , Forkhead Box Protein M1/genetics , Gene Expression Regulation/drug effects , Humans , Immunohistochemistry , Kidney Diseases/pathology , Kidney Tubules/metabolism , Male , MiceABSTRACT
BACKGROUND: Pancreatic Ductal Adenocarcinoma (PDAC) is the most common and deadly cancer. Surgical resection is the only possible cure for pancreatic cancer but often has a poor prognosis, and the role of adjuvant therapy is urgently explored. METHODS: MicroRNAs (miRNAs) play a very important role in tumorigenesis by regulating the target genes. In this study, we identified miR-320b lower-expressed in human pancreatic cancer tissues but relatively higher-expressed in the adjacent non-tumor tissues. RESULTS: Consistently, the expression of miR-320b in different pancreatic cancer cell lines was significantly lower than the normal pancreatic cells. In order to identify the effects of miR-320b on cell growth, we overexpressed miR-320b in PANC-1 and FG pancreatic cancer cell lines, CCK8 and BrdU incorporation assay results showed that miR-320b inhibited cell proliferation. DISCUSSION: We next predicted miR-320b targeted FOXM1 (Forkhead box protein M1) and identified the negative relationship between miR-320b and FOXM1. We also demonstrated that elevated miR- 320b expression inhibited tumor growth in vivo. CONCLUSION: All of these results showed that miR-320b suppressed pancreatic cancer cell proliferation by targeting FOXM1, which might provide a new diagnostic marker for pancreatic cancer.
Subject(s)
Carcinoma, Pancreatic Ductal/pathology , Forkhead Box Protein M1/antagonists & inhibitors , MicroRNAs/biosynthesis , Pancreatic Neoplasms/pathology , Carcinogenesis/genetics , Carcinogenesis/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolismABSTRACT
This research article describes an approach to modify the thiazolidinedione scaffold to produce test drugs capable of binding to, and inhibit, the in vitro transcriptional activity of the oncogenic protein FOXM1. This approach allowed us to obtain FOXM1 inhibitors that bind directly to the FOXM1-DNA binding domain without targeting the expression levels of Sp1, an upstream transcription factor protein known to activate the expression of FOXM1. Briefly, we modified the chemical structure of the thiazolidinedione scaffold present in anti-diabetic medications such as pioglitazone, rosiglitazone and the former anti-diabetic drug troglitazone, because these drugs have been reported to exert inhibition of FOXM1 but hit other targets as well. After the chemical synthesis of 11 derivatives possessing a modified thiazolidinedione moiety, we screened all test compounds using in vitro protocols to measure their ability to (a) dissociate a FOXM1-DNA complex (EMSA assay); (b) decrease the expression of FOXM1 in triple negative-breast cancer cells (WB assay); (c) downregulate the expression of FOXM1 downstream targets (luciferase reporter assays and qPCR); and inhibit the formation of colonies of MDA-MB-231 cancer cells (colony formation assay). We also identified a potential binding mode associated with these compounds in which compound TFI-10, one of the most active molecules, exerts binding interactions with Arg289, Trp308, and His287. Unlike the parent drug, troglitazone, compound TFI-10 does not target the in vitro expression of Sp1, suggesting that it is possible to design FOXM1 inhibitors with a better selectivity profile.
Subject(s)
Antineoplastic Agents/chemical synthesis , Carcinogenesis/drug effects , Forkhead Box Protein M1/antagonists & inhibitors , Thiazolidinediones/chemical synthesis , Triple Negative Breast Neoplasms/drug therapy , Amino Acid Sequence , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Forkhead Box Protein M1/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Models, Molecular , Protein Binding , Sp1 Transcription Factor/metabolism , Thiazolidinediones/chemistry , Thiazolidinediones/pharmacology , Troglitazone/chemistryABSTRACT
The present study aimed to analyze the compensatory signaling pathways induced by forkhead domain inhibitor6 (FDI6), which is a forkhead box protein M1 (FOXM1) inhibitor, in ovarian cancer cells and evaluate the effectiveness of simultaneous inhibition of FOXM1 and the compensatory signaling pathway in decreasing the survival of ovarian cancer cells. The present study identified the proteins involved in the compensatory mechanism activated by FDI6 in HeyA8 ovarian cancer cells using western blot analysis and a reversephase protein array. In addition, a cell viability assay was performed to determine the effects of FDI6 and the compensatory signaling pathway on cancer cell viability. All experiments were performed in threedimensional cell cultures. The present study observed that FDI6 stimulated the upregulation of NRas, phosphoprotein kinase Cδ (pPKCδ) (S664) and HER3 in HeyA8 cells. Tipifarnib as an NRas inhibitor, rottlerin as a pPKCδ (S664) inhibitor and sapitinib as a HER3 inhibitor were selected. The combination of FDI6 with tipifarnib attenuated the upregulation of NRas induced by FDI6 and the combination of FDI6 with sapitinib also attenuated HER3 downstream signaling pathway in HeyA8 cells, as shown by on western blot analysis. Rottlerin downregulated pPKCδ (S664) by inhibiting the activity of a Srcrelated tyrosine kinase that transfers a phosphate group to PKCδ. Compared with FDI6 alone, the addition of tipifarnib or rottlerin to FDI6 was significantly more effective in reducing the growth of HeyA8 cells. However, the combination of FDI6 and sapitinib did not induce a significant decrease in survival of HeyA8 cells. In conclusion, the addition of tipifarnib or rottlerin to inhibit NRas or pPKCδ (S664), respectively, inhibited the compensatory signaling pathway response induced by FDI6 in HeyA8 cells. These inhibitors increased the efficacy of FDI6, which inhibits FOXM1, in reducing ovarian cancer cell viability.
Subject(s)
Forkhead Box Protein M1/antagonists & inhibitors , Ovarian Neoplasms/drug therapy , Pyridines/pharmacology , Thiophenes/pharmacology , Acetophenones/pharmacology , Benzopyrans/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Female , Forkhead Box Protein M1/analysis , GTP Phosphohydrolases/metabolism , Humans , Membrane Proteins/metabolism , Ovarian Neoplasms/pathology , Quinazolines/pharmacology , Quinolones/pharmacology , Signal Transduction/drug effectsABSTRACT
PURPOSE: Malignant rhabdoid tumor (MRT) is a rare, highly aggressive sarcoma with an uncertain cell of origin. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is very poor. Novel antitumor agents are needed for MRT patients. Forkhead box transcription factor 1 (FOXM1) is overexpressed and is correlated with the pathogenesis in several human malignancies. In this study, we identified the clinicopathological and prognostic values of the expression of FOXM1 and its roles in the progression of MRT. METHODS: We investigated the FOXM1 expression levels and their clinical significance in 23 MRT specimens using immunohistochemistry and performed clinicopathologic and prognostic analyses. We also demonstrated correlations between the downregulation of FOXM1 and oncological characteristics using small interfering RNA (siRNA) and FOXM1 inhibitor in MRT cell lines. RESULTS: Histopathological analyses revealed that primary renal MRTs showed significantly low FOXM1 protein expression levels (p = 0.032); however, there were no significant differences in other clinicopathological characteristics or the survival rate. FOXM1 siRNA and FOXM1 inhibitor (thiostrepton) successfully downregulated the mRNA and protein expression of FOXM1 in vitro and the downregulation of FOXM1 inhibited cell proliferation, drug resistance to chemotherapeutic agents, migration, invasion, and caused the cell cycle arrest and apoptosis of MRT cell lines. A cDNA microarray analysis showed that FOXM1 regulated FANCD2 and NBS1, which are key genes for DNA damage repair. CONCLUSION: This study demonstrates that FOXM1 may serve as a promising therapeutic target for MRT.
Subject(s)
Antineoplastic Agents/pharmacology , Forkhead Box Protein M1/antagonists & inhibitors , Forkhead Box Protein M1/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Rhabdoid Tumor/drug therapy , Rhabdoid Tumor/genetics , Apoptosis/drug effects , Apoptosis/genetics , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , DNA Damage/drug effects , DNA Damage/genetics , Down-Regulation/drug effects , Down-Regulation/genetics , Fanconi Anemia Complementation Group D2 Protein/genetics , Female , Humans , Immunohistochemistry/methods , Infant , Male , Nuclear Proteins/genetics , Prognosis , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Survival Rate , Thiostrepton/pharmacologyABSTRACT
Peritoneal spread is the primary mechanism of metastasis of ovarian cancer, and survival of ovarian cancer cells in the peritoneal cavity as nonadherent spheroids and their adherence to the mesothelium of distant organs lead to cancer progression, metastasis, and mortality. However, the mechanisms that govern this metastatic process in ovarian cancer cells remain poorly understood. In this study, we cultured ovarian cancer cell lines in adherent and nonadherent conditions in vitro and analyzed changes in mRNA and protein levels to identify mechanisms of tumor cell survival and proliferation in adherent and nonadherent cells. EGFR or ERBB2 upregulated ZEB1 in nonadherent cells, which caused resistance to cell death and increased tumor-initiating capacity. Conversely, Forkhead box M1 (FOXM1) was required for the induction of integrin ß1, integrin-α V, and integrin-α 5 for adhesion of cancer cells. FOXM1 also upregulated ZEB1, which could act as a feedback inhibitor of FOXM1, and caused the transition of adherent cells to nonadherent cells. Strikingly, the combinatorial treatment with lapatinib [dual kinase inhibitor of EGFR (ERBB1) and ERBB2] and thiostrepton (FOXM1 inhibitor) reduced growth and peritoneal spread of ovarian cancer cells more effectively than either single-agent treatment in vivo. In conclusion, these results demonstrate that FOXM1 and EGFR/ERBB2 pathways are key points of vulnerability for therapy to disrupt peritoneal spread and adhesion of ovarian cancer cells. SIGNIFICANCE: This study describes the mechanism exhibited by ovarian cancer cells required for adherent cell transition to nonadherent form during peritoneal spread and metastasis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5554/F1.large.jpg.
Subject(s)
ErbB Receptors/metabolism , Forkhead Box Protein M1/metabolism , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Peritoneal Neoplasms/metabolism , Peritoneal Neoplasms/secondary , Receptor, ErbB-2/metabolism , Signal Transduction/genetics , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/genetics , Disease Models, Animal , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Female , Forkhead Box Protein M1/antagonists & inhibitors , Forkhead Box Protein M1/genetics , Gene Knockdown Techniques , Humans , Lapatinib/pharmacology , Lapatinib/therapeutic use , Mice , Peritoneal Neoplasms/prevention & control , Receptor, ErbB-2/antagonists & inhibitors , Receptor, ErbB-2/genetics , Signal Transduction/drug effects , Thiostrepton/pharmacology , Thiostrepton/therapeutic use , TransfectionABSTRACT
Diabetic foot ulcers (DFUs) are a life-threatening disease that often result in lower limb amputations and a shortened lifespan. However, molecular mechanisms contributing to the pathogenesis of DFUs remain poorly understood. We use next-generation sequencing to generate a human dataset of pathogenic DFUs to compare to transcriptional profiles of human skin and oral acute wounds, oral as a model of "ideal" adult tissue repair due to accelerated closure without scarring. Here we identify major transcriptional networks deregulated in DFUs that result in decreased neutrophils and macrophages recruitment and overall poorly controlled inflammatory response. Transcription factors FOXM1 and STAT3, which function to activate and promote survival of immune cells, are inhibited in DFUs. Moreover, inhibition of FOXM1 in diabetic mouse models (STZ-induced and db/db) results in delayed wound healing and decreased neutrophil and macrophage recruitment in diabetic wounds in vivo. Our data underscore the role of a perturbed, ineffective inflammatory response as a major contributor to the pathogenesis of DFUs, which is facilitated by FOXM1-mediated deregulation of recruitment of neutrophils and macrophages, revealing a potential therapeutic strategy.
Subject(s)
Diabetic Foot/genetics , Diabetic Foot/immunology , Forkhead Box Protein M1/immunology , Wound Healing/immunology , Adult , Aged , Animals , Cell Proliferation , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/immunology , Diabetic Foot/pathology , Disease Models, Animal , Female , Forkhead Box Protein M1/antagonists & inhibitors , Forkhead Box Protein M1/metabolism , Humans , Inflammation/genetics , Inflammation/immunology , Male , Mice, Inbred Strains , Middle Aged , Mouth Mucosa/physiology , Pyridines/pharmacology , Thiophenes/pharmacology , Transcriptome/physiology , Wound Healing/geneticsABSTRACT
Hepatocellular cancer (HCC) is one of the leading causes of mortality worldwide. Unfortunately, a limited choice of anti-cancer drugs is available for treatment, owing to their minimal efficacy and development of acquired resistance. Autophagy, a cellular survival pathway, often exhibits a pleiotropic role in HCC progression. Studies show increased autophagy in established HCC, promoting the survival of HCC cells in the tumour microenvironment. Therefore, novel anti-autophagy drugs hold promise for preventing HCC progression. Here, using a non-biased transcriptomics analysis in HepG2 cells we demonstrate the existence of an autophagy-FOXM1 nexus regulating growth in HepG2 cells. Additionally, we show that suppression of autophagy by an Unc-51 Like Autophagy Activating Kinase 1(ULK1) inhibitor not only attenuates the expression of FOXM1 and its transcriptional targets, but also has a synergistic effect on the inhibition of HepG2 growth when combined with FOXM1 inhibitors. Thus, the autophagic protein, ULK1, is a promising candidate for preventing HCC progression. Collectively, our results provide new insight into the role of autophagy in HCC growth and are a proof-of concept for combinatorial therapy using ULK1 and FOXM1 inhibitors.
Subject(s)
Autophagy-Related Protein-1 Homolog/metabolism , Autophagy , Carcinoma, Hepatocellular/metabolism , Forkhead Box Protein M1/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Liver Neoplasms/metabolism , Autophagy/genetics , Autophagy-Related Protein-1 Homolog/antagonists & inhibitors , Carcinoma, Hepatocellular/genetics , Cell Proliferation , Forkhead Box Protein M1/antagonists & inhibitors , Gene Silencing , Hep G2 Cells , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Liver Neoplasms/genetics , Signal Transduction/drug effectsABSTRACT
Pancreatic cancer has a high mortality rate due to poor rates of early diagnosis. One tumor suppressor gene in particular, p53, is frequently mutated in pancreatic cancer, and mutations in p53 can inactivate normal wild type p53 activity and increase expression of transcription factor forkhead box M1 (FoxM1). Overexpression of FoxM1 accelerates cellular proliferation and cancer progression. Therefore, inhibition of FoxM1 represents a therapeutic strategy for treating pancreatic cancer. Broussoflavonol B (BF-B), isolated from the stem bark of Broussonetia kazinoki Siebold has previously been shown to inhibit the growth of breast cancer cells. This study aimed to investigate whether BF-B exhibits anti-pancreatic cancer activity and if so, identify the underlying mechanism. BF-B reduced cell proliferation, induced cell cycle arrest, and inhibited cell migration and invasion of human pancreatic cancer PANC-1 cells (p53 mutated). Interestingly, BF-B down-regulated FoxM1 expression at both the mRNA and protein level. It also suppressed the expression of FoxM1 downstream target genes, such as cyclin D1, cyclin B1, and survivin. Cell cycle analysis showed that BF-B induced the arrest of G0/G1 phase. BF-B reduced the phosphorylation of extracellular signal-regulated kinase ½ (ERK½) and expression of ERK½ downstream effector c-Myc, which regulates cell proliferation. Furthermore, BF-B inhibited cell migration and invasion, which are downstream functional properties of FoxM1. These results suggested that BF-B could repress pancreatic cancer cell proliferation by inactivation of the ERK/c-Myc/FoxM1 signaling pathway. Broussoflavonol B from Broussonetia kazinoki Siebold may represent a novel chemo-therapeutic agent for pancreatic cancer.