Regulation Of FOXM1 Research Articles

Sublethal UV irradiation induces squamous differentiation via a p53-independent, DNA damage-mitosis checkpoint

The epidermis is a self-renewal epithelium continuously exposed to the genotoxic effects of ultraviolet (UV) light, the main cause of skin cancer. Therefore, it needs robust self-protective mechanisms facing genomic damage. p53 has been shown to mediate apoptosis in sunburn cells of the epidermis. However, epidermal cells daily receive sublethal mutagenic doses of UV and massive apoptosis would be deleterious. We have recently unravelled an anti-oncogenic keratinocyte DNA damage-differentiation response to cell cycle stress. We now have studied this response to high or moderate single doses of UV irradiation. Whereas, as expected, high levels of UV induced p53-dependent apoptosis, moderate levels triggered squamous differentiation. UV-induced differentiation was not mediated by endogenous p53. Overexpression of the mitosis global regulator FOXM1 alleviated the proliferative loss caused by UV. Conversely, knocking-down the mitotic checkpoint protein Wee1 drove UV-induced differentiation into apoptosis. Therefore, the results indicate that mitosis checkpoints determine the response to UV irradiation. The differentiation response was also found in cells of head and neck epithelia thus uncovering a common regulation in squamous tissues upon chronic exposure to mutagens, with implications into homeostasis and disease.

Downreguation of FoxM1 by miR-214 inhibits proliferation and migration in hepatocellular carcinoma.

The FoxM1 transcription factor plays an important role in the progression of HCC. Therefore, it is necessary to study cell regulation of FoxM1. In this study, we determined the expression of miR-214 and it was inversely associated with FoxM1 protein level in HCC; and suppression of FoxM1 translation by miR-214 mimics. We found that miR-214 targeted the 3'untranslated region of FoxM1 mRNA. In addition, the study found that DLX1 was the direct target of FoxM1 in HCC. Downregulation of FoxM1 inhibits the proliferation, migration, and invasion of HCC cells by miR-214. These results indicate that miR-214 may be used as a completely new molecular target by influencing FoxM1 expression in HCC.

Abstract 5425: Investigating the effects of p53 activation and HDAC inhibition on genes associated with poor prognosis prostate cancer

Abstract Background Prostate cancer (PCa) is now the most commonly diagnosed malignancy in men worldwide. SOC combines radiotherapy with ADT, however recurrence rate remains unacceptably high and many men present with late stage aggressive and metastatic disease, with poor prognosis and limited treatment options. Despite tumor suppressor p53 being mutated in approximately 50% of human tumors, it has been reported that p53 alteration occurs in only 12% of primary prostate tumors. Emerging data links epigenetic-mediated gene silencing as a major differentiator between aggressive and indolent disease that is associated with up-regulation of key cell cycle regulator FOXM1, which is known to be suppressed by p53. HDAC inhibitors (HDACi) are an emerging class of epigenetic modifying anti-cancer agents, which we hypothesises will enhance p53 activity, cell death and alter patterns of gene expression to targeting p53 dependent and independent vulnerabilities in PCa. Methods We used basally p53 deficient/proficient PCa models (PC3/LNCaP) and novel C4-2B and LNCap p53 CRISPR-Cas9 knockout models to investigate the combination effects of Nutlin 3A or radiation (IR) with class 1 HDACi Entinostat by Annexin V/PI Flow Cytometry and Western Blot. Transcriptional effects were evaluated by RT-PCR and RNA-seq analysis. TCGA mutational and RNA-seq data were used to link p53 mutation with altered gene expression and outcome. Results We have demonstrated that there is a synergistic induction of cell death when we combine N3A/IR with Entinostat that is p53-dependent. In addition, we see that p53 acts as a barrier to genes associated with poor prognosis including FOXM1 which is phenocopied by single-agent Entinostat independent of WT-p53. This suppression may potentially be downstream of p21 via the DREAM complex. Importantly p53 mutation in TCGA primary PCa patients is associated with poor outcome and elevated FOXM1 expression. Conclusion and Future Work Results to date suggest that Entinostat enhances p53 dependent cell death but also has p53 dependent and independent mechanisms to suppress genes associated with poor prognosis PCa. The mechanism looks to be dependent of p21 and may involve DREAM complex. Citation Format: Gemma M. Gregg, Fiammetta Falcone, Andrea Lees, Gerard Quinn, Peter Gallagher, Richard Kennedy, Ian I. Mills, Simon S. McDade. Investigating the effects of p53 activation and HDAC inhibition on genes associated with poor prognosis prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5425.

FoxM1 promotes epithelial-mesenchymal transition, invasion, and migration of tongue squamous cell carcinoma cells through a c-Met/AKT-dependent positive feedback loop.

Forkhead box protein M1 (FoxM1) has been associated with cancer progression and metastasis. However, the function of FoxM1 in tongue squamous cell carcinoma (TSCC) remains largely unknown. The purpose of this study was to determine the role of FoxM1 in regulation of epithelial-mesenchymal transition (EMT) and migration of TSCC cells. We found that FoxM1 induced EMT and increased invasion/migration capacity in SCC9 and SCC25 cells. FoxM1 stimulation increased c-Met, pAKT, and vimentin levels but decreased E-cadherin level. Chromatin immunoprecipitation assay established that FoxM1 is bound to the promoter of c-Met to activate its transcription. In turn, c-Met promoted the expression of FoxM1 and pAKT. Blocking AKT signaling attenuated the invasion and migration of SCC9 and SCC25 cells stimulated by FoxM1 or c-Met. These results indicate that a positive feedback loop controls the EMT and migration of TSCC cells induced by FoxM1 and c-Met through AKT. Furthermore, the expression levels of FoxM1, pAKT, and c-Met were found to significantly increase in TSCC tissues compared with normal tissues, and these three biomarkers were concomitantly expressed in TSCC tissues. Clinical association analyses indicated that the expression of FoxM1, c-Met, and pAKT was associated with clinicopathological characteristics of patients with TSCC including tumor stage, tumor size, and lymph node metastasis. Taken together, our findings suggest that FoxM1 promotes the EMT, invasion and migration of TSCC cells, and cross-talks with c-Met/AKT signaling to form a positive feedback loop to promote TSCC development.

Cancer cell line specific co-factors modulate the FOXM1 cistrome.

ChIP-seq has been commonly applied to identify genomic occupation of transcription factors (TFs) in a context-specific manner. It is generally assumed that a TF should have similar binding patterns in cells from the same or closely related tissues. Surprisingly, this assumption has not been carefully examined. To this end, we systematically compared the genomic binding of the cell cycle regulator FOXM1 in eight cell lines from seven different human tissues at binding signal, peaks and target genes levels. We found that FOXM1 binding in ER-positive breast cancer cell line MCF-7 are distinct comparing to those in not only other non-breast cell lines, but also MDA-MB-231, ER-negative breast cancer cell line. However, binding sites in MDA-MB-231 and non-breast cell lines were highly consistent. The recruitment of estrogen receptor alpha (ERα) caused the unique FOXM1 binding patterns in MCF-7. Moreover, the activity of FOXM1 in MCF-7 reflects the regulatory functions of ERα, while in MDA-MB-231 and non-breast cell lines, FOXM1 activities regulate cell proliferation. Our results suggest that tissue similarity, in some specific contexts, does not hold precedence over TF-cofactors interactions in determining transcriptional states and that the genomic binding of a TF can be dramatically affected by a particular co-factor under certain conditions.

USP5 promotes tumorigenesis and progression of pancreatic cancer by stabilizing FoxM1 protein

Increased ubiquitin-specific protease 5 (USP5) has been associated with tumorigenesis of malignancy including glioblastoma, melanoma and hepatocellular carcinoma. However, the role of USP5 in tumorigenesis of pancreatic ductal adenocarcinoma (PDAC) has not been studied yet. In this study, we demonstrated that USP5 was significantly upregulated in a panel of PDAC cell lines and correlated with FoxM1 protein expression. USP5 knockdown inhibited proliferation of PANC-1 and SW1990, two PDAC cell lines. In the mouse xenografted pancreatic tumor model, suppression of USP5 significantly decreased tumor growth, correlated with down regulation of FoxM1. Additionally, we found that overexpression of USP5 stabilized the FoxM1 protein in PDAC cells. Overexpression of USP5 extended the half-life of FoxM1. Knockdown of USP5 in PANC-1 cells decreased FoxM1 protein level while the proteasome inhibitor MG-132 treatment restored FoxM1 expression. We also found that endogenous USP5 was coimmunoprecipitated with an endogenous FoxM1 from PANC-1 cells while FoxM1 was also coimmunoprecipitated with USP5. Furthermore, we also confirmed that USP5 regulated proliferation of PDAC via FoxM1 by rescuing the inhibitory effect of USP5 knockdown with ectopic expression of FoxM1 in USP5-depleted cells. Taken together, our study demonstrates that USP5 plays a critical role in tumorigenesis and progression of pancreatic cancer by stabilizing FoxM1 protein, and provides a rationale for USP5 being a potential therapeutic approach against PDAC.

Akt/FoxM1 signaling pathway-mediated upregulation of MYBL2 promotes progression of human glioma

BackgroundMYB-related protein B (B-MYB/MYBL2), a member of the myeloblastosis family of transcription factors, has been reported for its role in the genesis and progression of tumors. Forkhead box M1 (FoxM1), another transcriptional factor, is considered to be an independent predictor of poor survival in many solid cancers. The aim of the present study was to investigate the clinical significance of the correlation between MYBL2 and FoxM1 in glioma and the possible mechanism of FoxM1and MYBL2 expression.MethodsMYBL2 and FoxM1expression in cancerous tissues and cell lines were determined by reverse transcription-PCR (RT-PCR), Western blotting and immunostaining. The co-expression of MYBL2 and FoxM1 was analyzed in low-grade glioma (LGG) and glioblastoma (HGG) cohorts of TCGA using cBioportal and UCSC Xena. And, the role of MYBL2 and FoxM1 in glioma cell progression and the underlying mechanisms were studied by using small interfering RNA (si-RNA) and pcDNA3.1 + HAvectors. Furthermore, the effects of MYBL2 and FoxM1 in cell proliferation, cell cycle progression, apoptosis, migration, invasion, and adhesion were determined by cell proliferation assays, flow cytometry analysis, transwell migration and cell adhesion assay.ResultsMYBL2 and FoxM1 expression are significantly associated with clinical stages and overall survival of glioma patients. In cohorts of TCGA, patients with high MYBL2 but without radio-chemotherapy had the highest hazard ratio (adjusted HR = 5.29, 95% CI = 1.475–18.969, P < 0.05). Meanwhile, MYBL2 closely related to the FoxM1 expression in 79 glioma tissues (r = 0.742, p < 0.05) and LGG (r = 0.83) and HGG (r = 0.74) cohorts of TCGA. Down regulation of FoxM1 and MYBL2 by siRNAs induced the cell cycle arrest, apoptosis and EMT of glioma cells. Furthermore, inactivations of Akt/FoxM1 signaling by Akt inhibitor and siRNA-FoxM1 reduce the expression of MYBL2 in glioma cells.ConclusionsMYBL2 is a key downstream factor of Akt/FoxM1 signaling to promote progression of human glioma, and could be a new candidate gene for molecular targeting therapy and biomarker for radiotherapy of glioma.Trial registrationCTXY-1300041-3-2. ChiCTR-COC-15006186. Registered date: 13 September 2013.

Oncogenic KRAS-associated gene signature defines co-targeting of CDK4/6 and MEK as a viable therapeutic strategy in colorectal cancer.

Therapeutic strategies against KRAS mutant colorectal cancers are developed using cell line models, which do not accurately represent the transcriptome driven by oncogenic KRAS in tumors. We sought to identify a KRAS-associated gene signature from colorectal tumors to develop a precise treatment strategy. Integrative analysis of quantitative KRAS mutation detection and matched gene expression profiling in 55 CRC bulk tumors was carried out to define a gene signature enriched in CRC tumors with high KRAS mutation. The KRAS-associated gene signature identified exhibits functional enrichment in cell cycle and mitosis processes, and includes mitotic transcription factor, FOXM1. Combination treatment of CDK4/6 inhibitor Palbociclib and MEK inhibitor PD0325901 was tested in KRAS-mutant, BRAF-mutant CRC, normal colon epithelial lines and xenografts models to determine their efficacy and toxicity and to monitor the changes in the gene signature. Inhibiting CDK4/6, an upstream regulator of FOXM1, and MEK synergistically depleted FOXM1 and KRAS-associated gene signature, suggesting that CDK4/6 and MEK regulate the KRAS gene signature. The combined inhibition of CDK4/6 and MEK elicited a robust therapeutic response in KRAS-dependent and BRAF-mutant CRC, both in vitro and in vivo and this correlated with downregulation of the KRAS-associated gene signature. Our preclinical study demonstrated the efficacy of Palbociclib and PD0325901 combinatorial treatment selectively in KRAS-dependent and BRAF-mutant CRC but not in normal colon epithelial cells. The KRAS-associated gene signature could facilitate the identification of responsive metastatic CRC to this therapeutic strategy in clinical settings.

Inhibition of FOXM1 By Ixazomib Confers Chemosensitivity in NPM1-Wild Type Acute Myeloid Leukemia

Background: FOXM1 represents an attractive therapeutic target owing to selective up-regulation in dividing cells. Proteasome inhibitors target FOXM1 by inhibiting FOXM1 through the stabilization of HSP70, which is the negative regulator of FOXM1 {Halasi. J Biol Chem. 2016}. Ixazomib has previously been shown to induce cell death in NPM1-mutant AML cells by oxidative stress {Garcia, Clin Cancer Research 2016} and is currently being tested in NPM1-mutant AML patients as a single agent. We have shown that FOXM1 is inactive in NPM1- mutant AML (Bhat UG. J Bio; Chem 2012) supporting an alternative mechanism of action such as reactive oxygen species induction.In this abstract we propose a FOXM1 dependent mechanism of action in NPM1-wild type AML. We demonstrate that the novel proteasome inhibitor ixazomib is an inhibitor of FOXM1 and can enhance sensitivity to standard chemotherapy drug cytarabine in AML. With its favorable toxicity profile and oral route of administration, this is an attractive option to study as a chemosensitizing drug in leukemia.Materials and Methods: Ixazomib was purchased from Selleck Chemicals. The doxycycline-inducible U2OS-derived C3-luc cell line expressing FOXM1-dependent firefly luciferase was developed in Dr. Gartel's lab (Radhakrishnan. Cancer Res. 2006). KG-1, MV4-11 and HL60 cell lines were purchased from ATCC. Patient samples were collected at the University of Illinois Cancer Center after informed consent using an IRB approved protocol.Results: Using the C3-luc cell line, we studied the effect of ixazomib on FOXM1 transcriptional activity. Cells were treated with 1μg/ml doxycycline and ixazomib doses from 300-1000 nM overnight. The luciferase activity was determined by the Luciferase Assay System (Promega). We demonstrate significant dose-dependent inhibition of FOXM1 transcriptional activity at doses as low as 300nM.Inhibition of FOXM1 protein expression was then confirmed in NPM1-wild type AML cells. We analyzed KG-1, MV4-11 and HL60 cell lines by immunoblotting for FOXM1 protein after 24 hour incubation with ixazomib at doses ranging from 10-1000 nM.Given the well-studied role of FOXM1 in mediating chemoresistance, we studied the effect of ixazomib on increasing sensitivity of AML cells to the chemotherapeutic agent cytarabine. The MTS assay was used to calculate the IC50 of ixazomib in KG-1 and MV4-11 cells. The IC50 for KG1 cells was 38 nM (95% CI: 18-78nM) and for MV4-11 cells it was 27nM (95% CI: 16-45nM). We then used a range of doses of ixazomib (0-100nM) in combination with cytarabine at a fixed ratio in AML cell lines to assess cell proliferation using the MTS assay. A combination index (CI) was calculated using CompuSyn and the combination was synergistic in inhibiting proliferation in KG-1 and MV4-11 cells at all doses tested. Inhibition of FOXM1 expression with combination therapy was confirmed by immunoblotting and was accompanied by increased caspase-3 cleavage after 24 hour exposure to the drug.Finally, to establish that this anti-leukemic effect is dependent on proteasome inhibition, we show a rescue effect with N-Acetyl L-Cysteine, which antagonizes the activity of proteasome inhibitors (Halasi. Biochemical J. 2013).In primary NPM1-wild type AML mononuclear cells, treatment with ixazomib ex vivo for 24 hours in liquid culture resulted in dose dependent cell death measured by flow cytometry and caspase-3 cleavage. We are currently investigating FOXM1 expression and transcriptional activity in primary AML cells treated with ixazomib.Conclusion:Ixazomib demonstrates anti-neoplastic activity against NPM1-wild type AML and potentiates the effect of standard chemotherapy cytarabine. We present a novel mechanism of action whereby ixazomib, through its effect as a proteasome inhibitor, suppresses FOXM1 an oncogenic transcription factor that confers chemoresistance in AML. We are performing animal experiments to confirm the in vitro data and link the anti-leukemic activity of ixazomib with suppression of FOXM1. Our findings lay the groundwork for a target driven trial in AML. Using a novel, well-tolerated oral proteasome inhibitor to target FOXM1 we would expect increased efficacy with lower doses of the chemotherapy drug cytarabine in the treatment of NPM1-wild type AML. DisclosuresNo relevant conflicts of interest to declare.

&lt;b&gt;Anticancer activity of eugenol is not related to regulation of the oncogenic transcription factor &lt;i&gt;Forkhead Box M1

Genome-wide gene expression profiling of cancers has consistently identified the FOXM1 as one of the most commonly upregulated genes in cancer cells that plays an essential role in the regulation of a wide spectrum of biological processes, including inhibition of apoptosis. Since the anticancer activity of EUG reported in the literature is related to induction of apoptosis in cancer cells, we hypothesized that there is a correlation between the EUG-induced apoptosis effect and downregulation of FOXM1. A series of experiments were conducted to evaluate the effect of EUG on cellular viability of cancer cells (MTT) and its potential regulatory effect on FOXM1 protein levels (western blots). Our findings confirm the anticancer effect of EUG on different human cancer cell lines as previously reported in the literature (SKBR3 LC50: 318.6; HT29 LC50: 525.5; and HepG2 LC50: 2090.0 µM). However, we demonstrated that EUG does not regulate the FOXM1. The results evidenced the anticancer effect of EUG on three cancer cell lines and showed that the EUG- apoptosis induced effect is not related to regulation of FOXM1 at the protein level. Further studies must be done to provide information on the mechanism of action of this agent.

FBXO31 protects against genomic instability by capping FOXM1 levels at the G2/M transition

F-box proteins in conjunction with Skp1, Cul1 and Rbx1 generate SCF complexes that are responsible for the ubiquitination of proteins, leading to their activation or degradation. Here we show that the F-box protein FBXO31 is required for normal mitotic progression and genome stability due to its role in regulating FOXM1 levels during the G2/M transition. FBXO31-depleted cells undergo a transient delay in mitosis due to an activated spindle checkpoint concomitant with an increase in lagging chromosomes and anaphase bridges. FBXO31 regulates mitosis in part by controlling the levels of FOXM1, a transcription factor and master regulator of mitosis. FBXO31 specifically interacts with FOXM1 during the G2/M transition, resulting in FOXM1 ubiquitination and degradation. FBXO31 depletion results in increased expression of FOXM1 transcriptional targets and mimics the FOXM1 overexpression. In contrast, co-depletion of FBXO31 and FOXM1 restores the genomic instability phenotype but not the delay in mitosis, indicating that FBXO31 probably has additional mitotic substrates. Thus, FBXO31 is the first described negative regulator of FOXM1 during the G2/M transition.

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

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

Gene Expression Database May Lead to More-Accurate Prognoses

Abstract A new database that combines gene expression measurements from 166 data sets enables researchers to identify prognostic variables across 39 types of cancer. For multiple cancer types, high expression of the cell-cycle regulator FOXM1 and the presence of neutrophils in the tumor suggest a poor prognosis. The immune cell marker KLRB1 and the presence of plasma cells in tumors are strong indicators that patients are likely to survive.

FOXM1 and its Oncogenic Signaling in Gastric Cancer.

The transcription factor FOXM1 is overexpressed in many human cancers, including liver, stomach, prostate, brain, breast, lung, colon, pancreas, cervix, ovary and nervous system. Recent data have demonstrated that FOXM1 is involved in cell initiation, progression, and metastasis and in responses to anticancer drugs. In this review, the effects of FOXM1 on gastric cancer, especially the function and regulation of FOXM1 in tumorigenesis and clinical chemotherapy, will be summarized and discussed together with recent relevant patents on the role of FOXM1 in tumor therapy.

Abstract 518: O-GlcNAcylation regulates breast cancer cell invasion via the NAD+-dependent deacetylase SIRT1

Abstract Cancer cells increase nutrient consumption leading to the altered metabolic state known as the Warburg effect. One pathway dependent on glucose, glutamine and acetyl-CoA is the Hexosamine Biosynthetic Pathway (HBP). Increased flux through the HBP leads to elevated post-translation addition of O-linked-β-N-acetylglucosamine (O-GlcNAc) on a diverse population of nuclear and cytosolic proteins, many of which are implicated in cancer. Recently, our lab provided the first evidence that breast and prostate cancers increases total O-GlcNAcylation by increasing O-GlcNAc Transferase (OGT) levels. Importantly, reducing OGT activity inhibits cancer cell invasion in vitro and metastasis in vivo. OGT inhibition reduces breast and prostate cancer cell invasion through, in part, inhibition of the oncogenic transcription factor, FOXM1 and its transcriptional target matrix metalloproteinase 2 (MMP2). Here, we show that OGT regulation of FOXM1 and cancer cell invasion requires regulation of the NAD+-dependent deacetylase, SIRT1. SIRT1 is a NAD+-dependent deacetylase regulated by changes in energy metabolism and its levels have been found to be reduced during breast cancer progression. We demonstrate an inverse relationship between O-GlcNAcylation and SIRT1 protein level and activity in multiple breast cancer cells and show that SIRT1 regulation by O-GlcNAcylation occurs in an AMPK-dependent manner. Consistent with this idea that reducing O-GlcNAcylation increases SIRT1 activity, we found that reducing OGT levels results in increased NAD+ metabolite levels in breast cancer cells. Importantly, we demonstrate that SIRT1 is critical for OGT-mediated regulation of FOXM1 protein ubiquitination and proteosomal degradation as reducing SIRT1 levels or activity reverses OGT-mediated regulation of FOXM1 and invasion in aggressive triple negative breast cancer cells. Furthermore, we show that SIRT1 regulates FOXM1 to modulate its transcriptional target MMP2 to promote breast cancer cell invasion. Collectively, these data indicate that flux through HBP, via O-GlcNAcylation, connects this nutrient sensing pathways with SIRT deactylases to regulate cellular invasion signaling via regulation of FOXM1. Citation Format: Christina M. Ferrer. O-GlcNAcylation regulates breast cancer cell invasion via the NAD+-dependent deacetylase SIRT1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 518. doi:10.1158/1538-7445.AM2015-518

Abstract 1992: Evidence for modulation of FoxM1 by p21 in ovarian cancer

Abstract The oncogenic transcription factor forkhead box M1 (FoxM1) is overexpressed in many cancers, including 84% of ovarian cancer and plays a role in DNA repair, mitotic checkpoint, cell proliferation, and cancer drug resistance. Similar to Her2 in breast cancer, the constitutive expression of FoxM1 makes it a plausible gene target for novel anti-cancer therapies, but the regulation of FoxM1 has yet to be elucidated. Evidence suggests FoxM1 up-regulation is a result of TP53 mutations, however, no TP53 response element has been found within the FoxM1 promoter, thus there is likely another molecule mediating p53-induced FoxM1 overexpression. We hypothesize that cyclin-dependent kinase inhibitor 1a, p21, is involved in the regulation of FoxM1 by p53 in ovarian cancer. To test this hypothesis, we knocked down p21 expression with siRNA and shRNA in two wild-type p53 cancer cell lines, NCI-H23 and A2780. Knock down of p21 resulted in a mirrored increase in FoxM1 protein level in both cell lines as detected by Western blot. The addition of nutlin-3 (10 μM), a p53 stabilizer, to these p21 knocked down cell lines restored some p21 expression 24 hour post-treatment, which led to a subsequent decrease in FoxM1 levels compared to the corresponding DMSO-treated p21 knock down. These results suggest p21 is a negative regulator of FoxM1 in cancer cell lines. To further investigate this pathway, the same two cancer cell lines were treated with the FoxM1 inhibitor thiostrepton (10 μM) and protein levels were measured over a time course of 48 hours. FoxM1 protein levels were reduced following 8 hours, a time point where p21 protein levels peaked. This finding was further support by qRT-PCR analysis of A2780 ovarian cancer cell mRNA collected 0, 6, 8, 9, 10, 11, and 12 hours following thiostrepton (10 μM) treatment. In this experiment, p21 mRNA levels peaked 6 hours post-treatment, with FoxM1 mRNA levels reduced at 8 hours post-treatment, relative to control. Although thiostrepton has been shown to directly bind and inhibit the transcription of FoxM1, these results suggest that thiostrepton may also induce p21, potentiating FoxM1 inhibition. Thiostrepton has previously been shown to induce cancer cell death and reduce tumor burden, therefore further supporting FoxM1 as a potential drug target as well as encouraging the need to understand regulators involved in this pathway. Citation Format: Jill Madden, Jeremy Chien. Evidence for modulation of FoxM1 by p21 in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1992. doi:10.1158/1538-7445.AM2015-1992

Abstract 55: Over-expression of FoxM1 in breast cancer can be therapeutically targeted using thiostrepton

Abstract Breast cancer incidence is increasing worldwide and is one of the most common cancers among women in Saudi Arabia. In spite of conventional treatment regimes in breast cancer, the prognosis is still poor. However, over the last decade, outcome of breast cancer has improved due to increased awareness among the population leading to early detection. Forkhead box protein 1 (FoxM1), is an important member of Forkhead family protein, plays an important role in the pathogenesis of various cancer including basal cell carcinoma. In addition, FoxM1 has also been shown to be involved in the pathogenesis of breast cancer from other regions. However, the role of FoxM1 in Saudi Arabia breast cancer has not been fully elucidated. Therefore in this study, we first evaluated the expression of FoxM1 in a panel of patients with breast cancer samples in Saudi Arabia by immuno histo chemistry. Our data showed that FoxM1 is over expressed in 79% (770/975) of breast cancer and directly associated with clinical parameters such as younger age (&amp;lt;30yrs)(p = 0.00172),high Grade(p&amp;lt;0.0001), mucinous histology (p&amp;lt;0.0001), and triple negative phenotype (p&amp;lt;0.0001). In addition, FoxM1over-expession was also significantly associated with activated AKT (p = 0.0001), as well as high Ki67 expression(p&amp;lt;0.0001),matrix metalloproteinase-9 (MMP-9) (p &amp;lt;0.0001), and XIAP (p &amp;lt;0.0001). In vitro, using breast cancer cell lines, we found that treatment with thiostrepton resulted in inhibition of cell viability and induction of apoptosis in a dose dependent manner. In addition, in these cell lines, thiostrepton treatment caused down regulation of FoxM1, in activation of AKT and decreased the expression of XIAP, and MMP-9. Furthermore pharmacological inhibition of FoxM1 triggered Caspase dependent apoptosis in breast cancer cell lines. Altogether, our results suggest that FoxM1 and its associated signaling pathway may be a potential target for therapeutic intervention in the treatment of breast cancer. Citation Format: Maqbool Ahmed, Azhar Hussain, Rafiya Begum, Saravanan Thangavel, Dahish S. Ajarim, Shaham Beg, Shahab Uddin, Khawla S. Al-Kuraya. Over-expression of FoxM1 in breast cancer can be therapeutically targeted using thiostrepton. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 55. doi:10.1158/1538-7445.AM2015-55

OTUB1 inhibits the ubiquitination and degradation of FOXM1 in breast cancer and epirubicin resistance.

The forkhead transcription factor FOXM1 has a key role in DNA damage response, and its deregulated overexpression is associated with genotoxic drug resistance in breast cancer. However, little is known about the posttranslational mechanisms by which FOXM1 expression is regulated by genotoxic agents and how they are deregulated in resistant cells. Initial co-immunoprecipitation studies verified previous proteomic analysis finding that the OTUB1 is a novel FOXM1-interacting protein. Western blot analysis showed that both OTUB1 and FOXM1 expression reduced upon genotoxic agent treatment in MCF-7 cells, but remained relatively constant in resistant cells. FOXM1 expression reduced upon OTUB1 depletion by siRNA and increased with OTUB1 overexpression in MCF-7 cells, arguing that OTUB1 positively regulates FOXM1 expression. In agreement, co-immunoprecipitation experiments demonstrated that FOXM1 expression is associated with OTUB1 binding but inversely correlates with conjugation to the protein degradation-associated Lys-48-linked ubiquitin-chains. Overexpression of wild-type (WT) OTUB1, but not the OTUB1(C91S) mutant, disrupted the formation of Lys48-linked ubiquitin-conjugates on FOXM1. Importantly, knockdown of OTUB1 by siRNA resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide, whereas overexpression of WT OTUB1, but not the OTUB1(C91S) mutant, significantly enhances the half-life of FOXM1. In addition, proliferative and clonogenic assays also show that OTUB1 can enhance the proliferative rate and epirubicin resistance through targeting FOXM1, as OTUB1 has little effect on FOXM1-deficient cells. The physiological relevance of the regulation of FOXM1 by OTUB1 is further underscored by the significant correlations between FOXM1 and OTUB1 expression in breast cancer patient samples. Cox-regression survival analysis indicates that OTUB1 overexpression is linked to poorer outcome in particular in patients treated with chemotherapy. Collectively, these data suggest that OTUB1 limits the ubiquitination and degradation of FOXM1 in breast cancer and has a key role in genotoxic agent resistance.

Mutual Regulation of FOXM1, NPM and ARF Proteins.

ARF, NPM and FOXM1 proteins interact with each other in mammalian cells. We showed previously that proteasome inhibitors suppress not only FOXM1 expression, but also the expression of ARF and NPM proteins. Using RNA interference we found that the depletion of each of these proteins by RNAi in human cancer HeLa cells leads to down-regulation of the two other partners, suggesting that these proteins stabilize each other in human cancer cells. Since the suppression of FOXM1 is one of hallmarks of proteasome inhibition, suppression of ARF and NPM by proteasome inhibitors may be explained in part as a secondary effect of downregulation of FOXM1 that modulate stability of ARF and NPM1 proteins.

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

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