Downregulation Of FoxM1 Expression Research Articles

Romidepsin and tamoxifen cooperatively induce senescence of pancreatic cancer cells through downregulation of FOXM1 expression and induction of reactive oxygen species/lipid peroxidation.

Although improvement has been made in therapeutic strategies against pancreatic carcinoma, overall survival has not significantly enhanced over the past decade. Thus, the establishment of better therapeutic regimens remains a high priority. Pancreatic cancer cell lines were incubated with romidepsin, an inhibitor of histone deacetylase, and tamoxifen, and their effects on cell growth, signaling and gene expression were analyzed. Xenografts of human pancreatic cancer CFPAC1 cells were medicated with romidepsin and tamoxifen to evaluate their effects on tumor growth. The inhibition of the growth of pancreatic cancer cells induced by romidepsin and tamoxifen was effectively reduced by N-acetyl cysteine and α-tocopherol, respectively. The combined treatment greatly induced reactive oxygen species production and mitochondrial lipid peroxidation, and these effects were prevented by N-acetyl cysteine and α-tocopherol. Tamoxifen enhanced romidepsin-induced cell senescence. FOXM1 expression was markedly downregulated in pancreatic cancer cells treated with romidepsin, and tamoxifen further reduced FOXM1 expression in cells treated with romidepsin. Siomycin A, an inhibitor of FOXM1, induced senescence in pancreatic cancer cells. Similar results were obtained in knockdown of FOXM1 expression by siRNA. Since FOXM1 is used as a prognostic marker and therapeutic target for pancreatic cancer, a combination of the clinically available drugs romidepsin and tamoxifen might be considered for the treatment of patients with pancreatic cancer.

Knockdown of UCHL3 inhibits esophageal squamous cell carcinoma progression by reducing CRY2 methylation.

UCHL3 (Ubiquitin carboxyl-terminal hydrolase L3), a member of deubiquitinating enzymes, has been implicated in various cancers. However, the role of UCHL3 in esophageal squamous cell carcinoma (ESCC) remains unknown. In the current study, we aimed to investigate the role of UCHL3 in ESCC growth and migration, and whether UCHL3 could modulate CRY2 methylation through FOXM1. The expression of UCHL3 and CRY2 in ESCC tissues was assessed using qRT-PCR, western blotting and immunohistochemistry (IHC). Cell viability was determined by CCK-8 and colony formation assays. Hoechst 33342 and flow cytometry were used to detect cell apoptosis. Transwell assay was performed to investigate cell migration and invasion. In vivo animal model was used to assess cell tumorigenesis. Methylation-Specific PCR (MSP) was applied to detect CRY2 methylation in the promoter region. The results showed that UCHL3 expression was elevated in ESCC tissues and cells, while CRY2 expression was decreased. UCHL3 silencing inhibited cell viability, invasion, migration and induced cell apoptosis in vitro, repressed tumor growth in vivo, and increased CRY2 expression and decreased FOXM1 expression. In addition, UCHL3 knockdown decreased CRY2 methylation through downregulating FOXM1, leading to an increase in the expression of CRY2. Moreover, CRY2 silencing abolished UCHL3 deficiency-mediated inhibition in cell growth and migration. In summary, this study reveals that knockdown of UCHL3 inhibits ESCC growth and migration by reducing CRY2 methylation through downregulation of FOXM1 expression.

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

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

Knockdown of MALAT1 expression inhibits HUVEC proliferation by upregulation of miR-320a and downregulation of FOXM1 expression.

Regulation of cancer angiogenesis could be a useful strategy in cancer therapy. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA (lncRNA), and can induce cancer cell proliferation, while lncRNAs, generally are able to act as microRNA (miRNA) sponges. The latter is a type of competitive endogenous RNA (ceRNA) that regulates expression of the targeting miRNAs and protein-coding genes. This study investigated the proliferative role of MALAT1 in human umbilical vein endothelial cells (HUVECs) and the underlying molecular events. The data showed that knockdown of MALAT1 expression using MALAT1 siRNA inhibited HUVEC proliferation and also significantly decreased levels of FOXM1 mRNA and protein in vitro, while knockdown of FOXM1 expression reduced HUVEC proliferation. Annotation of HUVEC microarray data revealed that seven miRNAs, including miR-320a, were upregulated after knockdown of MALAT1 expression in HUVECs. MALAT1 was shown to reciprocally interact with miR-320a, i.e., expression of one negatively regulated levels of the other, whereas knockdown of MALAT1 expression promoted miR-320a levels. Furthermore, miR-320a could directly target and inhibit FOXM1 expression in HUVECs. Knockdown of MALAT1 expression enhanced miR-320a expression but reduced FOXM1 expression resulting in downregulation of HUVEC proliferation. However, such an effect was inhibited by miR-320a depletion. In conclusion, this study demonstrates that miR-320a plays an important role in mediating the effects of MALAT1 on HUVEC proliferation by suppression of FOXM1 expression. Thus, targeting of this gene pathway could be a novel strategy in cancer therapy.

MiR-361-5p suppresses lung cancer cell lines progression by targeting FOXM1.

Lung cancer is the most common type of cancer and the leading cause of death in worldwide. MicroRNAs are known to be key players in a variety of biological processes, including tumorigenesis. In present study, we investigated the effect of miR-361-5p on lung cancer progression. We found that miR-361-5p was down-regulated in lung cancer. Overexpression of miR-361-5p suppressed lung cancer proliferation and invasion. Mechanistically, FOXM1 was identified as a direct target of miR-361-5p. Furthermore, miR-361-5p inhibits EMT-like phenotype through down-regulation of FOXM1 expression in lung cancer cells. In conclusion, our results indicated that miR-361-5p acts as a tumor suppressor in lung cancer.

A novel peptide, 9R-P201, strongly inhibits the viability, proliferation and migration of liver cancer HepG2 cells and induces apoptosis by down-regulation of FoxM1 expression

Overexpression of FoxM1 was closely related to the proliferation, metastasis, chemo-resistance and poor prognosis of various cancers. FoxM1 was regarded as the Achilles’ heel of cancer and a potential target for anti-cancer drug discovery. We previously obtained several high affinity peptides from the phage random library against the DNA binding domain of FoxM1c (FoxM1c-DBD) protein. Here in this paper, we found that 9R-P201, one of the novel peptides, showed stronger inhibition to HepG2 cancer cells than those of DU145, HUVEC and L-02 cells with an IC50 of 43.6µg/ml (13.1µM). The peptide was highly effective to liver cancer cells with an IC50 for L-02 cells of 2855.9µg/ml. We confirmed that 9R-P201 aggregated in the cell nucleus and the expression of FoxM1 was significantly down-regulated at both transcriptional and translational levels in HepG2 cells, leading to the suppression of cell proliferation, migration, angiogenesis, and induction of apoptosis. Whole genomic RNA sequencing analysis revealed that 357 genes were significantly and differentially expressed, most of them were enriched in cancer-associated biological processes. Finally, treatment of HepG2 xenografts with 9R-P201 resulted in growth inhibition and down-regulation of foxM1 expression in tumors. Collectively, our findings suggested that 9R-P201 could strongly inhibit the viability, proliferation and migration of liver cancer HepG2 cells and induce apoptosis by down-regulation of FoxM1 and regulation of related gene expression in signal transduction passways. Thus, 9R-P201 holds great potential as a lead anti-cancer drug targeting FoxM1.

Expression and significance of FoxM1 in esophageal squamous cell carcinoma cells in vitro and in vivo

To investigate the expression and significance of FoxM1 in esophageal squamous cell carcinoma (ESCC) cell lines and tissues. Western blot assay was used to detect the expression of FoxM1 in human esophageal epithelial cells and esophageal squamous cell cancer cell lines TE1, TE10, TE11 and Eca109 cells. To determine whether down-regulation of FoxM1 expression could inhibit the aggressive phenotype of ESCC cells, we knocked down the expression of FoxM1 by using FoxM1-shRNA in TE1 cells. Then we detected the cell proliferation, migration and invasion of TE1 cells by MTT assay, scratch assay and transwell assay. Furthermore, the effect of FoxM1 knockdown on tumorigenicity in nude mice was evaluated. Finally, immunohistochemical staining was used to detect the expression of FoxM1 in 99 cases of ESCC tissues and adjacent normal esophageal tissues. χ(2) test was used to analyze the correlations between the expression of FoxM1 and clinicopathologic characteristics and prognosis of ESCC patients. Western blot data showed that FoxM1 expression was lower in normal esophageal epithelial cells and highly expressed in four esophageal cancer cell lines, especially in TE1 cells. Knockdown of FoxM1 inhibited the growth, invasion and migration of TE1 cells and reduced their tumorigenicity in nude mice.The positive expression rate of FoxM1 in ESCC was 61.6% (61/99), significantly higher than that in the paired adjacent normal tissues (24.2%, 24/99) (P<0.05). The positive expression rate of FoxM1 in ESCC tissues was 61.6% (61/99), significantly higher than that in the paired adjacent normal tissues (24.2%, 24/99) (P<0.05). FoxM1 expression was significantly and positively correlated with lymph node metastasis, clinical stage and invasive depth (P<0.05). The median survival time was 42.3 months in 38 cases of patients with negative FoxM1 expression, and 33.0 months in 61 cases of positive FoxM1 expression, and the difference was statistically significant (P=0.036). FoxM1 is highly expressed in ESCC, and significantly correlated with the initiation, development and prognosis of esophageal cancer. FOXM1 might be an indicator to predict the prognosis and serve as a potential target for therapy in esophageal cancer.

MiR-370 sensitizes chronic myeloid leukemia K562 cells to homoharringtonine by targeting Forkhead box M1

BackgroundHomoharringtonine (HHT) is a kind of cephalotaxus alkaloid used in traditional Chinese medicine. Although HHT has been successfully used as a therapeutic agent for leukemia, the drug resistance and toxicity are major concerns. MicroRNAs (miRNAs) have been identified to modulate cellular sensitivity to anticancer drugs. We examined the synergistic action between miR-370 and HHT in vitro and in vivo.MethodsThe synergistic action between miR-370 and HHT was examined by flow cytometry. The effect of HHT on miR-370 expression was determined by quantitative RT-PCR (qRT-PCR). The expression of miR-370 and Forkhead box M1 (FoxM1) in 23 patients with newly diagnosed chronic-phase chronic myeloid leukemia (CML-CP) and 10 patients with blast-crisis CML (CML-BP) as well as miR-370–targeted FoxM1 was determined by qRT-PCR and western blot analysis.ResultsEctopic expression of miR-370 sensitized the CML K562 cell line to HHT by targeting FoxM1, the major regulator in cell proliferation and apoptosis. miR-370 significantly promoted HHT-mediated cell apoptosis and miR-370 and HHT cooperated in affecting FoxM1 expression. As well, miR-370 was moderately upregulated after HHT treatment in K562 cells. In addition, the expression of miR-370 was significantly reduced in CML patients as compared with healthy controls. Furthermore, the expression of miR-370 was lower in CML-BP than CML-CP patients.ConclusionsMiR-370 sensitized K562 cells to HHT by inducing apoptosis in part by downregulation of FoxM1 expression. These findings may provide further information for CML treatment with HHT.

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.

Abstract 1429: Forkhead box M1 (FoxM1) is a direct transcriptional target of EWS/FLI1 and a regulator of Ewing's sarcoma cell morphology, adhesiveness and tumorigenicity

Abstract Ewing's sarcomas (EWS) are tumors typically formed by undifferentiated small round cells that predominantly affect children and young adults. The aggressiveness of EWS is illustrated by both the fact that about 25-30% patients present with metastases at diagnosis and the elevated rate of lethal recurrence among them. In spite of therapeutic combinations including chemotherapy, radiation and surgery, the 5-year disease-free survival rate for EWS patients has remained low for many years. Poor prognosis, lethal recurrence and long-term side effects point to the urgent requirement of novel EWS targeted therapies. EWS is cytogenetically characterized by the presence of chromosomal translocations resulting in the fusion of the EWS gene with several ETS genes, most frequently with FLI1, leading to the expression of chimeric proteins that contain the transcriptional activation domain of EWS and the FLI1 DNA binding domain. The EWS/FLI1 aberrant transcription factors are responsible for the malignant phenotype of EWS and regulate a large repertoire of target genes, many of which are critical determinants for EWS development, and thus have the potential of providing novel therapeutic options for EWS. In order to understand mechanisms of cell cycle regulation by EWS/FLI1, we performed comparative array analyses of EWS cells before and after knocking down EWS/FLI1 expression. Results showed that EWS/FLI1 knockdown consistently down-regulated FoxM1, and chromatin immunoprecipitation (ChIP) assays demonstrated that FoxM1 is a novel direct transcriptional target of EWS/FLI1. Western hybridization and immunohistochemical analyses demonstrated that FoxM1 is indeed abundantly expressed in EWS cell lines, patient tumors and EWS cell derived tumors in nude mouse xenografts. Down-regulation of FoxM1 expression in by shRNA-mediated gene knockdown caused morphological alterations, slowed the growth of EWS cells, and increased their adhesion to the substrate in culture-plates. Accordingly, FoxM1 down-regulation in EWS cells diminished their migration and invasiveness as well as their tumorigenicity in nude mice. Alterations in the pattern of integrin expression observed in FoxM1 knocked-down EWS cells may likely contribute to their altered morphology, increased adhesion and the overall downgrading of their malignant phenotype. These data identify FoxM1 as a direct EWS/FLI1 target and as an important regulator of the proliferation, morphology and tumorigenicity of EWS cells, and imply that targeting FoxM1 may allow the development of new molecular therapeutic strategies for EWS patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1429. doi:1538-7445.AM2012-1429