Role Of Forkhead Box M1 Research Articles

Role of FOXM1 and AURKB in regulating keratinocyte function in psoriasis.

This study investigated the effect of forkhead box M1 (FOXM1) and Aurora kinase B (AURKB) on the epidermal function of keratinocytes. Bioinformatics analysis was used to analyze the co-expression network of FOXM1 and its correlation with AURKB. The expression of FOXM1 and AURKB in tissues and cells was detected by immunofluorescence and real-time quantitative polymerase chain reaction, respectively. HaCaT cells were transfected with si-FOXM1 to knock down FOXM1. Cell proliferation was detected by cell counting kit-8 assay. Cell migration was detected by scratch assay. Cell invasion was detected by the Transwell invasion assay. Cell apoptosis and cell cycle were detected by flow cytometry. FOXM1 and AURKB were positively correlated and highly expressed in psoriatic lesions. After transfection of si-FOXM1, the expression levels of FOXM1 and AURKB genes significantly decreased. The proliferation of HaCaT cells decreased, the apoptosis rate increased significantly, and the proportion of cells in the G1 phase increased significantly, while the proportion of cells in the S phase decreased significantly. The scratch closure of HaCaT cells was reduced, and the number of cell invasions decreased significantly. FOXM1 and AURKB may affect the progression of psoriasis by regulating the proliferation, cell cycle, migration, and invasion of keratinocytes.

FOXM1: a new therapeutic target of extramammary Paget disease.

Extramammary Paget disease (EMPD) is a rare skin cancer that primarily affects older individuals predominantly in areas with apocrine sweat glands. Although most early EMPD lesions are indolent, patients with metastatic EMPD have a poor prognosis due to the lack of effective systemic treatment. In this study, we investigated the role of forkhead box M1 (FOXM1), a potent transcription factor, in EMPD and assessed the potential of FOXM1 as a therapeutic target. Immunohistochemistry of 112 primary and 17 metastatic EMPD samples revealed that FOXM1 expression increased with tumor progression. Patients in whom FOXM1 was expressed in more than 10% of tumor cells had significantly shorter disease-specific survival than the other patients (p = 0.0397). In in vitro studies using our newly established EMPD cell line, KS-EMPD-1, we found high expression of FOXM1. Knockdown of FOXM1 impaired tumor cell viability, migration, and invasion. Inhibition of FOXM1 using thiostrepton also reduced tumor cell viability in a dose-dependent manner. These findings suggest that FOXM1 is a promising therapeutic target for patients with EMPD.

Abstract 203: The role of FOXM1 in senescence suppression and radiation resistance in oral cavity squamous cell carcinoma

Abstract INTRODUCTION: Advanced oral cavity squamous cell carcinoma (OCSCC) relies on surgery and radiation for cure; with ~30% experiencing locoregional failure. Earlier studies suggest OCSCCs resist radiation response by suppressing senescence. CDKN2A, coding for p16INK4a (p16), a CDK4/6 inhibitory protein, is among the most commonly lost tumor suppressors in OCSCC. Forkhead Box M1 (FOXM1) is an understudied key regulatory protein in OCSCC whose activation, similar to Rb, is controlled by CDK4/6. FOXM1 activity not only impacts cell proliferation, but also promotes DNA damage repair and suppresses senescence. The current study examines how FOXM1 signaling plays a critical role in resistance to radiation in OCSCC. We demonstrate that CDK4/6 inhibition improves radiosensitivity in OCSCC via senescence as a result of FOXM1 suppression. METHODS: Microarray data were analyzed to investigate FOXM1 levels in OSCC tumors. Basal FOXM1 levels in immortalized OCSCC cell lines (HN5 and Cal27) were evaluated using western blot. Cells were treated with palbociclib, radiation (RT), or combination (1µM palbociclib and 4 Gy (P+RT)) to examine the effects on senescence and FOXM1 signaling. β galactosidase colorimetric (β-gal) staining was used to evaluate senescence. Western blot was used to measure protein levels and QPCR to measure FOXM1 expression. To demonstrate senescence as a function of FOXM1 inhibition, knockdown assays (KD) were performed with siRNA targeting FOXM1. To further eliminate off target drug effects knockdown was also performed with siRNA targeting CDK4/6. RESULTS: FOXM1 was upregulated ~2.2-fold in OCSCC tumors vs. matched normal mucosa samples (N=43, p=2.85008E-06). FOXM1 protein was upregulated 7-fold (HN5) and 5-fold (Cal27) compared to normal oral keratinocytes (HOK16B). β-gal staining revealed a dose dependent senescence induction in OCSCC, where P+RT vs. RT showed a robust 5-fold (HN5) and 3-folds (Cal27) increase. Interestingly, FOXM1 protein and transcript expression doubled in samples treated with RT. P+RT vs. RT demonstrated a decrease in FOXM1 protein levels in HN5 (10-fold) and Cal27 (3-fold), with corresponding decreases in mRNA levels. Knockdown of FOXM1 induced senescence, which further increased when combined with radiation: siFOXM1+4 Gy resulted in 60% (HN5) and 50% (CAL27) β-gal positive cells. Compared to untreated controls, CDK4/6 KD demonstrated a 2.5 and 10-fold decrease in FOXM1 protein levels in HN5 and CAL27, respectively. Furthermore, a concurrent inhibition of CDK4/6 with RT not only resulted in a decrease in FOXM1 expression but also resulted in a near-complete senescence in treated cells (95% β-Gal staining). CONCLUSION: Suppressing FOXM1 directly or via CDK4/6 inhibition appears to make OCSCC more susceptible to RT via potentiating senescence. We propose inhibition of FOXM1 as a rational and potentially effective strategy to sensitize OCSCC to radiation. Citation Format: Nitisha Shrivastava, Yisrael Wallach, Daniel Li, Carlos Thomas, Michael B. Prystowsky, Indranil Basu, Chandan Guha, Thomas J. Ow. The role of FOXM1 in senescence suppression and radiation resistance in oral cavity squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 203.

HDAC class I inhibitor domatinostat sensitizes pancreatic cancer to chemotherapy by targeting cancer stem cell compartment via FOXM1 modulation

BackgroundPancreatic ductal adenocarcinoma (PDAC) represents an unmet clinical need due to the very poor prognosis and the lack of effective therapy. Here we investigated the potential of domatinostat (4SC-202), a new class I histone deacetylase (HDAC) inhibitor, currently in clinical development, to sensitize PDAC to first line standard gemcitabine (G)/taxol (T) doublet chemotherapy treatment.MethodsSynergistic anti-tumor effect of the combined treatment was assessed in PANC1, ASPC1 and PANC28 PDAC cell lines in vitro as well as on tumor spheroids and microtissues, by evaluating combination index (CI), apoptosis, clonogenic capability. The data were confirmed in vivo xenograft models of PANC28 and PANC1 cells in athymic mice. Cancer stem cells (CSC) targeting was studied by mRNA and protein expression of CSC markers, by limiting dilution assay, and by flow cytometric and immunofluorescent evaluation of CSC mitochondrial and cellular oxidative stress. Mechanistic role of forkhead box M1 (FOXM1) and downstream targets was evaluated in FOXM1-overexpressing PDAC cells.ResultsWe showed that domatinostat sensitized in vitro and in vivo models of PDAC to chemotherapeutics commonly used in PDAC patients management and particularly to GT doublet, by targeting CSC compartment through the induction of mitochondrial and cellular oxidative stress. Mechanistically, we showed that domatinostat hampers the expression and function of FOXM1, a transcription factor playing a crucial role in stemness, oxidative stress modulation and DNA repair. Domatinostat reduced FOXM1 protein levels by downregulating mRNA expression and inducing proteasome-mediated protein degradation thus preventing nuclear translocation correlated with a reduction of FOXM1 target genes. Furthermore, by overexpressing FOXM1 in PDAC cells we significantly reduced domatinostat-inducing oxidative mitochondrial and cellular stress and abolished GT sensitization, both in adherent and spheroid cells, confirming FOXM1 crucial role in the mechanisms described. Finally, we found a correlation of FOXM1 expression with poor progression free survival in PDAC chemotherapy-treated patients.ConclusionsOverall, we suggest a novel therapeutic strategy based on domatinostat to improve efficacy and to overcome resistance of commonly used chemotherapeutics in PDAC that warrant further clinical evaluation.

P.059 Identifying the role of trace amine-associated receptor 9 in behaviour, brain neurochemistry and blood biochemistry

Accelerated smooth muscle cell (SMC) proliferation is the primary cause of intimal hyperplasia (IH) following vascular interventions. Forkhead Box M1 (FOXM1) is considered a proliferation-associated transcription factor. However, the presence and role of FOXM1 in IH following vascular injury have not been determined.We examined the expression of FOXM1 in balloon-injured rat carotid arteries and investigated the effect of FOXM1 inhibition in SMCs and on the development of IH.FOXM1 was detected by immunofluorescent staining in balloon-injured rat carotid arteries where we observed an upregulation at day 7, 14, and 28 compared to uninjured controls. Immunofluorescence staining revealed FOXM1 coincided with proliferating cell nuclear antigen (PCNA). FOXM1 was also detectable in human carotid plaque samples. Western blot showed an upregulation of FOXM1 protein in serum-stimulated SMCs. Inhibition of FOXM1 using siRNA or chemical inhibition led to the induction of apoptosis as measured by flow cytometry and western blot for cleaved caspase 3. Perturbations in survival signaling were measured by western blot following FOXM1 inhibition, which showed a decrease in phosphorylated AKT and β-catenin. The chemical inhibitor thiostrepton was delivered by intraperitoneal injection in rats that underwent balloon injury and led to reduced intimal thickening compared to DMSO controls.FOXM1 is an important molecular mediator of IH that contributes to the proliferation and survival of SMCs following vascular injury.

Role of FOXM1 in vascular smooth muscle cell survival and neointima formation following vascular injury

BackgroundAccelerated smooth muscle cell (SMC) proliferation is the primary cause of intimal hyperplasia (IH) following vascular interventions. Forkhead Box M1 (FOXM1) is considered a proliferation-associated transcription factor. However, the presence and role of FOXM1 in IH following vascular injury have not been determined. ObjectiveWe examined the expression of FOXM1 in balloon-injured rat carotid arteries and investigated the effect of FOXM1 inhibition in SMCs and on the development of IH. Methods and resultsFOXM1 was detected by immunofluorescent staining in balloon-injured rat carotid arteries where we observed an upregulation at day 7, 14, and 28 compared to uninjured controls. Immunofluorescence staining revealed FOXM1 coincided with proliferating cell nuclear antigen (PCNA). FOXM1 was also detectable in human carotid plaque samples. Western blot showed an upregulation of FOXM1 protein in serum-stimulated SMCs. Inhibition of FOXM1 using siRNA or chemical inhibition led to the induction of apoptosis as measured by flow cytometry and western blot for cleaved caspase 3. Perturbations in survival signaling were measured by western blot following FOXM1 inhibition, which showed a decrease in phosphorylated AKT and β-catenin. The chemical inhibitor thiostrepton was delivered by intraperitoneal injection in rats that underwent balloon injury and led to reduced intimal thickening compared to DMSO controls. ConclusionsFOXM1 is an important molecular mediator of IH that contributes to the proliferation and survival of SMCs following vascular injury.

Knockdown of FOXM1 inhibits activation of keloid fibroblasts and extracellular matrix production via inhibition of TGF-β1/Smad pathway

Keloid is characterized by overactive fibroblasts. Forkhead box M1 (FOXM1) is transcription factor that plays important roles in the progression of fibrosis. However, the role of FOXM1 in keloid has not been elucidated. In the present study, we examined the expression levels of FOXM1 in clinical keloid tissue specimens and primary keloid fibroblasts (KFs). The results showed that FOXM1 levels were significantly increased in both keloid tissues and KFs. To further investigate the biological functions of FOXM1, FOXM1 was knocked down in KFs by transfection with small interfering RNA targeting FOXM1 (si-FOXM1). Knockdown of FOXM1 inhibited transforming growth factor-β1 (TGF-β1)-induced cell proliferation and migration of KFs. Besides, the increased expressions of collagen (coll I), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) in TGF-β1-induced KFs were suppressed by si-FOXM1 transfection. Furthermore, TGF-β1-induced increase in p-Smad2 and p-Smad3 expressions was attenuated by FOXM1 knockdown. These data indicated that knockdown of FOXM1 inhibited TGF-β1-induced KFs activation and extracellular matrix (ECM) accumulation, which was attributed to the inhibition of TGF-β1/Smad pathway.

Knockdown of FOXM1 attenuates inflammatory response in human osteoarthritis chondrocytes

Osteoarthritis (OA) is the most common inflammatory joint disease that is mainly characterized by articular cartilage destruction. Forkhead box M1 (FOXM1) is a transcription factor that acts as a critical mediator of inflammatory response. However, the role of FOXM1 in OA has not been investigated. Interleukin (IL)-1β is a major proinflammatory cytokine, which is associated with cartilage destruction in the pathophysiology of OA. In the present study, we used IL-1β to stimulate chondrocytes for the establishment of OA in vitro model. We found that FOXM1 was up-regulated in IL-1β-induced chondrocytes. Knockdown of FOXM1 attenuated IL-1β-caused decrease in cell viability. Knockdown of FOXM1 suppressed the IL-1β-induced production of inflammatory cytokines including tumor necrosis factor (TNF)-α, and IL-6. Besides, several inflammatory mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthases (iNOS), and cyclooxygenase-2 (COX-2) were also repressed by knockdown of FOXM1. FOXM1 silencing also inhibited the production of matrix metalloproteinases (MMPs) including MMP-3 and MMP-13. Furthermore, we found that knockdown of FOXM1 blocked the IL-1β-induced NF-κB activation in chondrocytes. These findings indicated that FOXM1 might play an important role in the pathogenesis of OA, suggesting that FOXM1 might be a potential therapeutic target for the treatment of OA.

Abstract 505: Targeting FOXM1 in Vascular Smooth Muscle Cells Induces Apoptotic Cell Death

Objectives: Vascular smooth muscle cell (VSMC) proliferation and dedifferentiation are key contributors to the initiation and progression of vascular diseases including restenosis. Forkhead Box M1 (FOXM1) is a proliferation-associated transcription factor shown to play a role in a variety of biological processes including cell cycle progression, cell survival, and apoptosis in many cell types. However, the role of FOXM1 in VSMC phenotypic transformation as a result of vascular injury following balloon angioplasty has not been studied. We hypothesize that FOXM1 modulates VSMC response by enhancing pro-proliferative and pro-survival signaling following vascular injury. Methods & Results: The rat carotid artery balloon injury was used to model vascular injury. Immunofluorescence staining was carried out on sections from injured carotid arteries or uninjured controls collected at 3, 7, or 14 days post injury (dpi). We observed that FOXM1 expression was upregulated in injured arteries at 7 and 14 dpi. Expression of FOXM1 in injured arteries co-localized with PCNA, a marker of proliferation. The upregulation of FOXM1 protein was replicated in cultured rat VSMCs by serum stimulation. Using thymidine synchronization, we observed that FOXM1 expression followed a cell cycle specific pattern in vitro . Chemical inhibition of FOXM1 using thiostrepton or FDI-6, or via siRNA resulted in decreased VSMC viability as measured by CCK-8, induction of apoptosis as measured by flow cytometry, and cleaved caspase 3 induction. More detailed immunocytochemistry analysis revealed that FDI-6 treated VSMCs exhibit disorganized microtubule networks. Furthermore, FDI-6 treatment decreased expression of Eg5, a motor protein essential for microtubule orientation and critical for mitosis execution. Additionally, FOXM1 inhibition decreased levels of β-catenin, a known activator of cell cycle-related genes and proliferation. Conclusions: In conclusion, our data suggest FOXM1 is critical to VSMC viability and may modulate VSMC proliferation and survival via regulation of important cell cycle and pro-proliferative proteins, and therefore may serve as a novel therapeutic target to prevent VSMC pathophysiology in the context of restenosis.

FoxM1 is associated with metastasis in colorectal cancer through induction of the epithelial-mesenchymal transition.

The aim of the present study was to investigate the role of forkhead box M1 (FoxM1) in epithelial-mesenchymal transition (EMT) and metastasis in colorectal cancer (CRC). Immunohistochemical assays were performed to detect FoxM1 and epithelial (E-) cadherin protein expression in 92 CRC, 61 colonic adenoma and 32 wild-type colonic tissue samples. Quantitative polymerase chain reaction (qPCR) assays were performed to determine the expression levels of FoxM1 and E-cadherin mRNAs in 30 CRC and adjacent normal mucosal tissues. RNA interference was used to knock down endogenous FoxM1 expression in CRC cell lines, and the migratory and invasive capacity of the CRC cells was analyzed. The expression of FoxM1, E-cadherin and neuronal (N-) cadherin in the CRC cell lines was evaluated using qPCR and Western blot analysis. The relative expression levels of FoxM1 mRNA and protein were significantly increased in the CRC tissues compared with those in the colonic adenoma and wild-type mucosal tissue samples (P<0.01). In contrast, the relative expression levels of E-cadherin mRNA and protein were significantly decreased in the CRC tissues compared with in the colonic adenoma and normal mucosal tissues (P<0.01). FoxM1 overexpression and decreased E-cadherin expression were significantly associated with poor colonic tissue differentiation, lymph node metastasis and an advanced tumor-node-metastasis stage. Additionally, the increased expression of FoxM1 was associated with a decrease in E-cadherin expression (P<0.01). Furthermore, RNA interference-mediated FoxM1 knockdown significantly inhibited the proliferation, migration and invasion of CRC cells. Downregulation of FoxM1 expression significantly increased E-cadherin expression and decreased N-cadherin expression. The results of the present study suggest that FoxM1 overexpression in tumor tissues is significantly associated with metastasis in CRC through the induction of EMT.

Abstract 4848: The functional synergism and pro-metastatic role of FOXM1 and CENPF in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide and metastasis is regarded as the major cause of HCC-associated lethality. In this study, we have analysed the TCGA whole-transcriptome sequencing data of paired human HCC samples (n=50), and identified Forkhead Box M1 (FOXM1) and Centromere Protein F (CENPF) to be the top-listing upregulated genes. Interestingly, both of them are the essential components in cell-cycle progression. FOXM1 encodes for the cell-cycle-dependent transcription factor that regulates genes for DNA replication and mitosis, while CENPF encodes for the centromere protein that is required for kinetochore function and chromosome segregation in mitosis. We hypothesized that the upregulation of FOXM1-CENPF signaling axis may drive hepatocarcinogenesis. In our human HCC cohort (n=34), FOXM1 and CENPF were shown to be upregulated compared with the non-tumorous liver tissues, and their mRNA expressions were positively correlated (p&amp;lt;0.0001). Co-upregulation of FOXM1 and CENPF in patient samples was demonstrated to be positively correlated with the absence of tumor encapsulation (p=0.035) in our clinico-pathological correlation analysis, whereas the samples without their co-upregulation was less likely to be correlated with venous invasion (p =0.045). This indicated that FOXM1 and CENPF are likely to be associated with HCC metastasis synergistically. Stable single and co-knockdown clones of FOXM1 and CENPF in four HCC cell lines (BEL7402, SMMC-7721, MHCC-97L, Huh7) were established using short-hairpin (sh) RNA approach for subsequent functional characterization. Both single and co-knockdown of FOXM1 and CENPF cells have shown reduced proliferation rate (p&amp;lt;0.05) in cell proliferation assay. Moreover, all single and co-knockdown of FOXM1 and CENPF cell lines were found to have a at least 2-fold reduction in migration and invasion rate (p&amp;lt;0.0001) in transwell cell migration and invasion assay. These suggested that FOXM1 and CENPF are critical for HCC cell proliferation, migration and invasion. To assess the translational significance of targeting FOXM1 and CENPF, seven HCC cell lines (BEL7402, SMMC-7721, MHCC-97L, Huh7, Hep3B, HepG2, PLC/PRF/5) were challenged with thiostrepton and zoledronic acid, which are inhibitors against FOXM1 and CENPF, respectively. Intriguingly, both individual and combined treatments of these inhibitors effectively inhibited HCC cell growth. It was also demonstrated that such inhibition acts via suppressing the respective endogenous transcript and protein expression of FOXM1 and CENPF. Taken together, our study has demonstrated that FOXM1 and CENPF play a critical oncogenic role in HCC and they may function as an attractive molecular therapeutic target. This study is supported in part by Health and Medical Research Fund (03142836). Citation Format: Wai Ling Macrina Lam, Lo Kong Chan, Daniel Wai-Hung Ho, Charles Shing Kam, Irene Oi-Lin Ng. The functional synergism and pro-metastatic role of FOXM1 and CENPF in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4848. doi:10.1158/1538-7445.AM2017-4848

FOXM1 regulates expression of eukaryotic elongation factor 2 kinase and promotes proliferation, invasion and tumorgenesis of human triple negative breast cancer cells.

Eukaryotic elongation factor 2 kinase (eEF2K), an emerging molecular target for cancer therapy, contributes to cancer proliferation, cell survival, tumorigenesis, and invasion, disease progression and drug resistance. Although eEF2K is highly up-regulated in various cancers, the mechanism of gene regulation has not been elucidated. In this study, we examined the role of Forkhead Box M1 (FOXM1) proto-oncogenic transcription factor in triple negative breast cancer (TNBC) cells and the regulation of eEF2K. We found that FOXM1 is highly upregulated in TNBC and its knockdown by RNA interference (siRNA) significantly inhibited eEF2K expression and suppressed cell proliferation, colony formation, migration, invasion and induced apoptotic cell death, recapitulating the effects of eEF2K inhibition. Knockdown of FOXM1 inhibited regulators of cell cycle, migration/invasion and survival, including cyclin D1, Src and MAPK-ERK signaling pathways, respectively. We also demonstrated that FOXM1 (1B and 1C isoforms) directly binds to and transcriptionally regulates eEF2K gene expression by chromatin immunoprecipitation (ChIP) and luciferase gene reporter assays. Furthermore, in vivo inhibition of FOXM1 by liposomal siRNA-nanoparticles suppressed growth of MDA-MB-231 TNBC tumor xenografts in orthotopic models. In conclusion, our study provides the first evidence about the transcriptional regulation of eEF2K in TNBC and the role of FOXM1 in mediating breast cancer cell proliferation, survival, migration/invasion, progression and tumorgenesis and highlighting the potential of FOXM1/eEF2K axis as a molecular target in breast and other cancers.

Abstract P6-19-03: Overexpression of FOXM1 is a potential prognostic marker in male breast cancer

Abstract Background The Forkhead box M1 (FOXM1) transcription factor is known to play an important role in the development and progression of many cancer types including breast cancer. The important role of FOXM1 in cancer affirms its significance for clinical use and therapeutic intervention. Elevated expression of FOXM1 in female breast cancer correlates with undifferentiated tumor phenotype and negative clinical outcome. However, whether FOXM1 has any indication for prognosis in male breast cancer (MBC) patients is still unknown. The purpose of this study was to examine the expression levels of FOXM1 in MBC and to identify the relationship between FOXM1 expression and patient survival. Methodsl Immunohistochemical analysis for FOXM1 was performed in a total of 80 male breast cancer specimens, all with linked clinical outcome data. Kaplan-Meier method and Cox proportional hazards analysis were used to relate FOXM1 expression to clinicopathological variables and overall survival (OS). Results We observed high expression of the FOXM1 protein in 39 % of MBC samples (31/80). FOXM1 overexpression was significantly associated with higher histological grade (p=0.05), lymph node metastasis (p=0.04), tumor size (p=0.05), and estrogen receptor expression (p=0.04). Patients with FOXM1 expression had a significantly poorer overall survival than those without FOXM1 expression (p=0.02). Multivariate analyses indicated that FOXM1 positivity was an independent prognostic factor for OS (p=0.03). Conclusion These results show that FOXM1 may represent a novel MBC marker with prognostic significance that could be included into the limited marker panels for MBC. Citation Format: Rahal K, Abdeljaoued S, Bettaieb I, Bouzaiene H, Adouni O, Miladi S, Goucha A, Bouaziz H, Makhlouf R, Hechiche M, Benna F, Boussen H, El May A, Gamoudi A. Overexpression of FOXM1 is a potential prognostic marker in male breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-19-03.

Role of forkhead box M1 in pathogenesis of pancreatic cancer

Role of forkhead box M1 in pathogenesis of pancreatic cancer

Role of MTDH, FOXM1 and microRNAs in Drug Resistance in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies due to underlying co-morbid cirrhosis and chemo-resistance. Vaccination and improved treatment for hepatitis are the most effective means to reduce the burden of liver cancer worldwide. Expression of biomarkers such as AFP (alpha-fetoprotein), DDK1 (Dickkopf WNT Signaling Pathway Inhibitor 1) and microRNAs in blood are being tested for early screening of liver cancer. Since 2008, sorafenib has been used as the standard molecular targeting agent for HCC. However, overall outcomes for sorafenib alone or in combination with other tyrosine kinase inhibitors are unsatisfactory. Whether simultaneously or sequentially, addiction switches and compensatory pathway activation in HCC, induced by sorafenib treatment, may induce acquired resistance. Forkhead box M1 (FOXM1) and metadherin (MTDH) have been shown to be master regulators of different aspects of tumorigenesis, including angiogenesis, invasion, metastasis and drug resistance. Elevated expression of both FOXM1 and MTDH is known to be a consequence of both activating mutations in oncogenes such as PI3K, Ras, myc and loss of function mutations in tumor suppressor genes such as p53 and PTEN in various types of cancers including HCC. The role of FOXM1 and MTDH as potential prognostic markers as well as therapeutic targets in HCC will be discussed. In addition, microRNAs (miRNAs), endogenous small non-coding RNAs involved in the regulation of gene expression, are involved in HCC and interact with both FOXM1 and MTDH in several ways. Thus, altered expression of miRNAs in HCCs will also be discussed as potential tools for diagnosis, prognosis and therapy in HCC.

Abstract A92: The role of FOXM1 and NBS1 in DNA damage-induced senescence and epirubicin resistance.

Abstract Background: The Forkhead box M1 (FOXM1) transcription factor is responsible for the regulation of a wide range of biological processes, including cell cycle, cell proliferation, apoptosis and tumorigenesis. Deregulation of FOXM1 has been suggested to be associated with drug resistance and poor prognosis in breast cancer patients and this could occur through an enhancement of DNA damage repair as well as a reduced propensity to undergo cell death or senescence. However, the exact mechanisms of how FOXM1 modulates drug resistance still remain unclear. The aim of this study is to investigate the new roles of FOXM1 and its downstream targets in DNA damage response and cellular senescence, which are vital for both the tumor progression and the development of chemotherapeutic drug resistance. Materials and Methods: MCF-7 human breast carcinoma cell lines and mouse embryonic fibroblasts (MEFs) were exposed to epirubicin and long-term cell viability was assessed by clonogenic assay. DNA double strand break repair was examined by γH2AX foci and DR-GFP repair assay. Analysis of cellular senescence was based on cellular β-galactosidase activity and changes in cell morphology. Expression levels of key DNA repair genes were assessed by using quantitative real-time PCR and western blot analysis. Results: We previously found that FOXM1 is required for DNA repair via homologous recombination repair. Consistently, reconstitution of FOXM1 in FOXM1-deficient MEFs decreased the accumulation of senescence-associated γH2AX foci after epirubicin treatment. Here, we shows that FOXM1-depletion can sensitize both human breast cancer MCF-7 cells and MEFs into entering epirubicin-induced senescence as indicated by the loss of long-term cell proliferation ability, the accumulation of persistent γH2AX foci, and the induction of senescence-associated β-galactosidase activity. Interestingly, we also demonstrate that FOXM1 regulates NBS1, a key component for the MRN complex-mediated ATM activation in response to DNA double stranded break (DSBs), at the transcriptional level. Using HeLa cell lines harbouring the DR-GFP reporter, overexpression of FOXM1 failed to augment HR activity in the background of NBS1 depletion, suggesting that NBS1 is indispensable for the HR function of FOXM1. Similar to FOXM1, NBS1 is strongly up-regulated in the epirubicin-resistant MCF-7 (MCF-7 EpiR) cells compared with parental MCF-7 cells and the depletion of FOXM1 or NBS1 also renders MCF-7 and MCF-7 EpiR cells more sensitive to epirubicin-induced cellular senescence. Conclusion: Taken together, these results indicate that NBS1 is a key FOXM1 downstream target gene involved in DNA damage response, DNA damage-induced senescence and epirubicin resistance. The FOXM1-NBS1 axis can be a reliable prognostic marker and a potential therapeutic target for overcoming genotoxic agent resistance in cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A92. Citation Format: Pasarat Khongkow, Upekha Karunarathna, Eric Lam. The role of FOXM1 and NBS1 in DNA damage-induced senescence and epirubicin resistance. [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 A92.

Abstract A28: Analyses of FOXM1 isoforms in ovarian cancer

Abstract Introduction &amp; Objectives: Epithelial ovarian cancer (OC) is the most lethal gynaecological malignancy and the fifth leading cause of cancer death in women in the Western world. Forkhead box M1 (FOXM1) transcription factor was already been described to be overexpressed in various malignancies, playing a key role in cell-cycle progression, chromosomal segregation and genomic stability, and oxidative stress. Moreover, it can also promote drug resistance to herceptin, paclitaxel and cisplatin in breast cancer cells. A recent report from our group has shown, for the first time, that FOXM1 is also overexpressed in OC, being associated with tumor grade. This result seem to indicate an interesting role of this molecule in the progression and resistance to therapy in OC. Our aim is to characterize FOXM1 isoforms in OC through the use of human tissue samples. Material &amp; Methods: Fresh ovarian primary tumors, ascites and peritoneal metastases from patients suffering advanced serous OC were collected from surgery room and processed for microarray analysis to detect FOXM1 expression levels. FOXM1 isoforms were analysed at mRNA expression level by using RTqPCR in a set of new frozen tissue samples. Results: We found that FOXM1 overexpression is increased in OC peritoneal metastases. Now, we are characterizing the isoforms of FOXM1 in primary, ascites and metastatic tumors. Nevertheless, more samples need to be analysed in order to have conclusive results. Conclusions: The present study highlights the role of FOXM1 isoforms in OC dissemination and pursues the idea to elucidate the biologic role of FOXM1 in OC tumorigenesis. Citation Format: Tatiana Altadill, Marina Rigau, Blanca Majem, Lucia Lanau, Josep Castellví, Ángel García, Assumpció Pérez-Benavente, José Luis Sánchez, Silvia Cabrera, Jordi Xercavins, Josep Maria del Campo, Antonio Gil-Moreno, Marta Llauradó, Jaume Reventós. Analyses of FOXM1 isoforms in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A28.

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.

Endothelial cell–restricted disruption of FoxM1 impairs endothelial repair following LPS-induced vascular injury

Recovery of endothelial integrity after vascular injury is vital for endothelial barrier function and vascular homeostasis. However, little is known about the molecular mechanisms of endothelial barrier repair following injury. To investigate the functional role of forkhead box M1 (FoxM1) in the mechanism of endothelial repair, we generated endothelial cell-restricted FoxM1-deficient mice (FoxM1 CKO mice). These mutant mice were viable and exhibited no overt phenotype. However, in response to the inflammatory mediator LPS, FoxM1 CKO mice displayed significantly protracted increase in lung vascular permeability and markedly increased mortality. Following LPS-induced vascular injury, FoxM1 CKO lungs demonstrated impaired cell proliferation in association with sustained expression of p27(Kip1) and decreased expression of cyclin B1 and Cdc25C. Endothelial cells isolated from FoxM1 CKO lungs failed to proliferate, and siRNA-mediated suppression of FoxM1 expression in human endothelial cells resulted in defective cell cycle progression. Deletion of FoxM1 in endothelial cells induced decreased expression of cyclins, Cdc2, and Cdc25C, increased p27(Kip1) expression, and decreased Cdk activities. Thus, FoxM1 plays a critical role in the mechanism of the restoration of endothelial barrier function following vascular injury. These data suggest that impairment in FoxM1 activation may be an important determinant of the persistent vascular barrier leakiness and edema formation associated with inflammatory diseases.