Regulation Of FOXM1 Research Articles

FOXM1 Mediates Drug-Resistance and Represents a Therapeutic Target in Pre-B Acute Lymphoblastic Leukemia

Background & Significance: Pre-B acute lymphoblastic leukemia (ALL) emerges in virtually all cases from B cell precursors that are arrested at the pre-B cell receptor checkpoint. In a gene expression survey of early B cell development, we found specific upregulation of FOXM1 at this developmental stage. FOXM1 belongs to the forkhead box transcription factor family and is a key regulator of cell growth by promoting cell cycle progression and has been implicated in progression of solid tumors. Therefore, we characterized the function and regulation of FOXM1 in normal B cell development as well as in pre-B ALL.Results: First, we verified the upregulation of FOXM1 during B cell development by qRT-PCR on sorted human and murine B cell progenitor populations. Then, we crossed Mb1-Cre tg mice to a Foxm1 conditional knockout mouse model (Foxm1fl/fl) and analyzed the early B cell populations according to the Hardy fractions. Despite the observed high expression of Foxm1 mRNA in fraction C’ and D, Foxm1 deletion did not alter B cell development. In order to investigate a potential role of FOXM1 in transformed B cells, we compared FOXM1 protein levels in patient-derived pre-B ALL samples with healthy B cells and B cell precursors and found 10-60-fold higher expression in the transformed B cell progenitors. To evaluate a potential predictive value of FOXM1 levels in patient-derived ALL samples, we measured FOXM1 mRNA levels at the time of diagnosis which strikingly correlate with risk stratification of ALL (intermediate-risk ALL n=31 vs. high risk ALL n=21; P=7.3e-5; BFM-REZ 2002). To further study the function and regulation of FOXM1, we cultured murine B cell precursors in the presence of IL7 and induced transformation with a retroviral BCR-ABL1 expression vector. BCR-ABL1 expression increased levels of FOXM1 compared to the normal IL7-dependent pre-B cells. Short-term inhibition of BCR-ABL1 did not affected protein levels of FOXM1. However, after 4 days of tyrosine kinase inhibition (TKI) treatment, FOXM1 protein levels were significantly downregulated in a dose-dependent manner. BCL2 overexpression prevented apoptosis induction by TKI but FOXM1 downregulation was retained. In addition, we found evidence that inactivation of FOXO factors by the PI3K/AKT pathway contributes to high FOXM1 expression in Ph+ ALL. Overexpression of a constitutively active form of Akt to prevent activation of FOXO factors in the presence of TKI abrogated FOXM1 downregulation. Similarly, BCR-ABL1+ ALL derived from FOXO3a knockout mice prevented TKI-mediated FOXM1 reduction. Overexpression of a constitutively active form of FOXO3a but not FOXO1 significantly reduces levels of FOXM1 expression. In line with this, we found a significant inverse correlation of FOXM1 with FOXO3A mRNA levels in Ph+ ALL patients from the ECOG E2993 trial. However, the requirement of long-term treatment indicates, that, in addition to FOXO3a activation, epigenetic regulation of the FOXM1 promoter downstream of BCR-ABL1 is required. Consistent with this finding, the FOXM1 promoter region was found to be de-methylated in a large fraction of ALL. In order to further study FOXM1 function, we transduced pre-B cells derived from Foxm1fl/fl mice with BCR-ABL1 and with an inducible ERT2-Cre vector. Deletion of Foxm1 in BCR-ABL1-driven leukemia decreases cell viability, colony formation, and proliferative capacity in vitro as well as leukemia formation in vivo. FOXM1-deleted ALL cells revealed a strikingly higher sensitivity towards TKI-treatment compared to the control cells in Imatinib dose-response curves (IC50 EV:420 nM vs IC50 Cre-ERT2: 160 nM) as well as annexin V staining. We identified the ROS scavenger Catalase as a critical target of FOXM1 in mediating this drug resistance. As potential therapeutic agents to target FOXM1, we evaluated the effects of a previously described ARF peptide and the natural occurring antibiotic Thiostrepton. Both bind FOXM1 and inhibit its function as shown by reduced mRNA expression of FOXM1 target genes (Cyclin B1, PLK1, AURKB) and induced apoptosis in ALL and prolonged survival of patient-derived ALL transplant recipient mice.Conclusion: We have identified a critical function of the transcription factor FOXM1 in mediating proliferation and drug-resistance in B cell lineage ALL, but not in normal B cell progenitors and validated FOXM1 as a therapeutic target in a large fraction of drug-resistant B cell lineage ALL. DisclosuresNo relevant conflicts of interest to declare.

Oncogenic Human Papillomavirus 16E7 modulates SUMOylation of FoxM1b

The oncogenic transcription factor Forkhead box M1b (FoxM1b), a key regulator of cell cycle, is often overexpressed in many human cancers. Interestingly, posttranslational modifications are known to play important role in regulating the levels and activity of FoxM1b. The purpose of the present study was to characterize the SUMOylation of FoxM1b and identify the functional consequences including viral pathogenesis. Here, we report that FoxM1b interacts with SUMOylating enzymes Ubc9 and PIAS1 and acts as a substrate for SUMOylation. We also show that SUMOylation facilitates FoxM1b protein destabilization and nucleocytoplasmic shuttling. More importantly, we provide the first evidence for a role of E7 oncoprotein in high risk human papillomavirus (HPV) mediated upregulation of FoxM1b. The elevated expression of FoxM1 was determined to be posttranscriptional and was attributed to decreased SUMOylation of FoxM1b in the E7-expressing cells. Moreover, we demonstrate the involvement of SUMOylation in regulation of FoxM1 and present biochemical evidence that HPV16 E7 oncoprotein can modulate SUMOylation of FoxM1b by impairing its interaction with Ubc9. Together, these results provide a novel connection between SUMOylation of FoxM1b and HPV carcinogenesis. The findings may have important implications in the discovery of future anti-cancer therapeutics.

Abstract 1598: Grainyhead-like 2 (GRHL2) regulates the expression of forkhead box transcription factor M1 (FoxM1) in human oral cancer cells and determines tumorigenicity

Abstract GRHL2 is a proto-oncogene that regulates epithelial proliferation and differentiation through transcriptional regulation of large number of target genes. The current study was performed to determine its role in oral carcinogenesis through regulation of forkhead box 1 (FoxM1), which is a known oncogene for many solid tumors. GRHL2 expression was highly elevated in oral squamous cell carcinoma (OSCC) tissues compared with normal oral epithelium (NHOM), and its protein expression is localized in the proliferative cells at the outer border of invasive islands of OSCC. In cultured OSCCs, GRHL2 and FoxM1 levels were both highly upregulated compared with normal human oral keratinocytes (NHOK), supporting their roles in oral carcinogenesis. When GRHL2 was knocked down in SCC4 cells by RNA interference, the cells have lost their proliferative capacity and colony formation when plated at low density. GRHL2 knockdown also resulted in marked loss of tumor spheroid formation, which is hallmark of cancer self-renewal. GRHL2 knockdown also led to loss of tumorigenic ability of cells upon xenograft transplantation in immunocompromised mice. GRHL2 appeared to determine the epithelial phenotype in OSCC cells. GRHL2 knockdown led to loss of E-cadherin and miR-200 family genes, which are epithelial markers. These data indicate that GRHL2 is required to maintain the transformed phenotype in OSCC. We also noted that GRHL2 knockdown led to drastic loss of FoxM1 expression, suggesting that GRHL2 may regulate FoxM1. To test this possibility, we performed in vivo binding assay by chromatin immunoprecipitation (ChIP), which showed GRHL2 binding at the proximal promoter of FoxM1. Luciferase reporter plasmid under FoxM1 promoter showed that GRHL2 directly regulates the gene promoter activity. FoxM1 knockdown resulted in loss of OSCC proliferation and tumor spheroid formation, while its overexpression conferred transformed phenotype in non-tumorigenic human oral keratinocytes (HOKs). Therefore, our data indicate that GRHL2 is required for the maintenance of transformed phenotype and that it plays a role in oral carcinogenesis through regulation of FoxM1. This study was supported in part by the grants from NIDCR/NIH (DE18295 and DE18959) and Jack Weichman Endowed Fund. Note: This abstract was not presented at the meeting. Citation Format: Wei Chen, Jin-Kyu Yi, Ki-Hyuk Shin, Reuben Kim, Shebli Mehrazarin, No-Hee Park, Mo K. Kang. Grainyhead-like 2 (GRHL2) regulates the expression of forkhead box transcription factor M1 (FoxM1) in human oral cancer cells and determines tumorigenicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1598. doi:10.1158/1538-7445.AM2014-1598

FoxM1 is Overexpressed in Helicobacter pylori–Induced Gastric Carcinogenesis and Is Negatively Regulated by miR-370

Helicobacter pylori (H. pylori) infections are strongly implicated in human gastric mucosa-associated diseases. Forkhead box M1 (FoxM1), a key positive regulator of cell proliferation, is overexpressed in gastric cancer. MicroRNAs are important post-transcriptional regulators of gene expression. In this study, the effects of H. pylori infection on FoxM1 expression and possible mechanisms of carcinogenesis were explored. The expression of FoxM1 was gradually increased in human gastric specimens from inflammation to cancer. FoxM1 upregulation was time- and concentration-dependent in gastric epithelial-derived cell lines infected with H. pylori. CagA, a key virulence factor of H. pylori, was associated with increased FoxM1 expression. H. pylori and CagA inhibited the expression of p27(Kip1) (CDKN1B) and promoted cell proliferation by upregulating FoxM1. The expression of miR-370 was decreased in human gastritis and gastric cancer. FoxM1 was directly downregulated by miR-370 in gastric cell lines. H. pylori and CagA inhibited miR-370 expression, which led to overexpression of FoxM1 and cell proliferation. Furthermore, the overexpression of FoxM1 and reduced expression of miR-370 was confirmed in H. pylori-infected C57BL/6J mice. H. pylori infection and CagA upregulated FoxM1 expression, dependent on miR-370, altered the expression of p27(Kip1), and promoted proliferation in gastric cells. These findings delineate the mechanisms governing FoxM1 regulation and the role of H. pylori in the process of gastric carcinogenesis.

The critical role of dysregulated FOXM1–PLAUR signaling in human colon cancer progression and metastasis.

405 Background: The mammalian Forkhead Box (Fox) transcription factor FOXM1 is implicated in tumorigenesis including mouse intestinal cancer. However, the clinical significance of FOXM1 signaling in human colorectal cancer (CRC) pathogenesis remains unknown. Methods: We investigated FOXM1 expression in 203 cases of primary colon cancer and matched normal colon tissue specimens and explored the underlying mechanisms of altered FOXM1 expression and the impact of this altered expression on colon cancer growth and metastasis using in vitro and animal models of colon cancer. Results: We found weak expression of FOXM1 protein in the colon mucosa, whereas we observed strong FOXM1 expression in tumor-cell nuclei of colon cancer and lymph node metastases. A Cox proportional hazards model revealed that FOXM1 expression was an independent prognostic factor in multivariate analysis. Experimentally, overexpression of FOXM1 by gene transfer significantly promoted the growth and metastasis of colon cancer cells in orthotopic mouse models, whereas knockdown of FOXM1 expression by small interfering RNA did the opposite. Promotion of colon tumorigenesis by FOXM1 directly and significantly correlated with activation of urokinase plasminogen activator receptor (PLAUR) expression and elevation of invasion and metastasis. Conclusions: Given the importance of FOXM1 in regulation of the expression of genes key to cancer biology, dysregulated expression and activation of FOXM1 may play important roles in colon cancer progression and metastasis.

Thiazole Antibiotics Siomycin a and Thiostrepton Inhibit the Transcriptional Activity of FOXM1

I read with great interest the review article by Alvarez-Fernandez and Medema (2013). I was especially pleased to see that the authors comprehensively acknowledged our work by citing six of our recent papers in their review. However, when they discussed our findings on the identification of the thiazole antibiotics siomycin A and thiostrepton as inhibitors of FOXM1 transcriptional activity they made a significant mistake. They wrongly stated, “surprisingly, these inhibitors do not affect the transcriptional activity of FOXM1 per se.” In numerous publications we showed that indeed siomycin A and thiostrepton inhibit the transcriptional activity and the expression of FOXM1, and they also act as proteasome inhibitors (Radhakrishnan et al., 2006; Bhat et al., 2009a,b). Moreover, we demonstrated that even in the presence of high levels of exogenous FOXM1 siomycin A, thiostrepton, and known proteasome inhibitors were able to inhibit FOXM1 transcriptional activity, suggesting that this inhibition is a key initiating event for FOXM1 protein suppression (Radhakrishnan et al., 2006; Bhat et al., 2009a,b). In addition, the authors concluded that both the mRNA and protein expression of FOXM1 are downregulated by siomycin A and thiostrepton “through an unknown mechanism” (Alvarez-Fernandez and Medema, 2013). However, recently I have proposed a general model for FOXM1 inhibition by proteasome inhibitors, which links the transcriptional activity and expression of FOXM1 (Gartel, 2010, 2012). According to this model, siomycin A, thiostrepton, and other proteasome inhibitors hinder the proteasomal degradation of a negative regulator of FOXM1 (NRFM), which in return directly or indirectly inhibits the activity of FOXM1 as a transcription factor (Gartel, 2011). Because FOXM1 is involved in a positive feedback loop and activates its own transcription (Halasi and Gartel, 2009), inhibition of FOXM1 transcriptional activity leads to a decrease in its expression (Gartel, 2010). This model also anticipates that all proteasome inhibitors regardless of their structure will inhibit FOXM1 transcriptional activity through stabilizing the NRFM and subsequently (via the auto-regulatory loop) inhibit FOXM1 expression (Gartel, 2011, 2012). Overall, inhibition of FOXM1 transcriptional activity by siomycin A, thiostrepton, and other proteasome inhibitors is an essential part of their regulation of FOXM1. FOXM1 is without a doubt emerging as a critical regulator of cancer development (Halasi and Gartel, 2013) that may affect all hallmarks of cancer (Hanahan and Weinberg, 2012). Originally, it was identified as a key regulator of cell proliferation and cell cycle progression; however, in the past few years FOXM1 became one of the central contributors to tumorigenesis. Growing body of evidence suggests that targeting this single transcription factor may have great promise for inhibiting tumor development. Great efforts are currently being undertaken to find more specific inhibitors of FOXM1 and to elucidate their mechanism of action. I believe it is essential for the scientific community to have correct information about the regulation of FOXM1 by FOXM1 inhibitors in order to progress and accelerate research in the FOXM1 field.

The Critical Role of Dysregulated FOXM1–PLAUR Signaling in Human Colon Cancer Progression and Metastasis

The mammalian Forkhead Box (Fox) transcription factor FOXM1 is implicated in tumorigenesis including mouse intestinal cancer. However, the clinical significance of FOXM1 signaling in human colorectal cancer pathogenesis remains unknown. We investigated FOXM1 expression in 203 cases of primary colon cancer and matched normal colon tissue specimens and explored the underlying mechanisms of altered FOXM1 expression and the impact of this altered expression on colon cancer growth and metastasis using in vitro and animal models of colon cancer. We found weak expression of FOXM1 protein in the colon mucosa, whereas we observed strong FOXM1 expression in tumor-cell nuclei of colon cancer and lymph node metastases. A Cox proportional hazards model revealed that FOXM1 expression was an independent prognostic factor in multivariate analysis. Experimentally, overexpression of FOXM1 by gene transfer significantly promoted the growth and metastasis of colon cancer cells in orthotopic mouse models, whereas knockdown of FOXM1 expression by siRNA did the opposite. Promotion of colon tumorigenesis by FOXM1 directly and significantly correlated with activation of urokinase-type plasminogen activator receptor (PLAUR) expression and elevation of invasion and metastasis. Given the importance of FOXM1 in regulation of the expression of genes key to cancer biology, dysregulated expression and activation of FOXM1 may play important roles in colon cancer progression and metastasis.

Abstract LB-212: Ionizing radiation selectively promotes invasion of ErbB2-expressing breast cancer cells via regulation of FoxM1

Abstract ErbB2 is frequently amplified in premalignant breast cancers including ductal carcinoma in situ (DCIS); however, little is known about the signals or pathways it modulates in the progression into the invasive/malignant state. Radiotherapy is often used to treat early premalignant lesions regardless of ErbB2 status. Here we show that clinically relevant doses of ionizing radiation (IR) induce cellular invasion of ErbB2-expressing breast cancer cells, such as SKBR3 and BT-474, as well as MCF10A cells transduced to express ErbB2. This effect is specific to ErbB2 expression, as ErbB2-negative breast cancer cells, such as MCF7 and T47D, do not invade following treatment with IR, nor do parental MCF10A cells or MCF10A cells overexpressing EGF receptor alone. Exposure of cells to X-rays leads to ErbB2 phosphorylation at Y877 in a dose- and time-dependent manner, and activates signaling pathways, including Src and PI3K/Akt. Inhibition of these pathways, as well as inactivation of IR-induced reactive oxygen species with the antioxidant N-acetylcysteine, prevented invasion. Activation of ErbB2-dependent signaling resulted in the upregulation of the transcription factor, FoxM1, a member of the forkhead family which has previously been shown to have a role in invasion, in part through transcriptional regulation of MMP2. Indeed, we have seen upregulation of MMP2 in irradiated ErbB2-expressing breast cancer cells, which is abolished when Src or ErbB2 is inhibited. Inhibition of FoxM1 by shRNA abrogated the increase in MMP2 level and prevented induction of invasion by IR, and overexpression of FoxM1 alone in MCF10A cells was sufficient to promote IR-induced invasion. Our data show that low level IR stimulates invasion of ErbB2-expressing breast cancer cells in a FoxM1-dependent manner. 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 LB-212. doi:1538-7445.AM2012-LB-212

Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cell Proliferation Is Controlled by Forkhead Box M1

Pulmonary arterial hypertension (PAH) is a devastating disease, and no effective treatments are available. Hypoxia-induced pulmonary artery remodeling, including smooth muscle cell proliferation, contributes to PAH, but the exact mechanisms underlying this abnormal process are largely undefined. The forkhead box M1 (FoxM1) transcription factor regulates cancer cell growth by modulating gene expression critical for cell cycle progression. Here, we report for the first time, to the best of our knowledge, a novel function of FoxM1 in the hypoxia-stimulated proliferation of human pulmonary artery smooth muscle cells (HPASMCs). Exposure to hypoxia caused a marked up-regulation of FoxM1 gene expression, mainly at the transcription level, and this induction correlated with HPASMC cell proliferation. The knockdown of FoxM1 inhibited the hypoxia-stimulated proliferation of HPASMCs. We found that the knockdown of HIF-2α, but not HIF-1α, diminished FoxM1 induction in response to hypoxia. However, the knockdown of FoxM1 did not alter expression levels of HIF-2α or HIF-1α, suggesting that HIF-2α is an upstream regulator of FoxM1. Furthermore, the knockdown of FoxM1 prevented the hypoxia-induced expression of aurora A kinase and cyclin D1. Collectively, our results suggest that hypoxia induces FoxM1 gene expression in an HIF-2α-dependent pathway, thereby promoting HPASMC proliferation.

Reversal of endocrine resistance in breast cancer: interrelationships among 14-3-3ζ, FOXM1, and a gene signature associated with mitosis

IntroductionDespite the benefits of estrogen receptor (ER)-targeted endocrine therapies in breast cancer, many tumors develop resistance. 14-3-3 ζ/YWHAZ, a member of the 14-3-3 family of conserved proteins, is over-expressed in several types of cancer, and our previous work showed that high expression of 14-3-3ζ in ER-positive breast cancers was associated with a poor clinical outcome for women on tamoxifen. Therefore, we now probe the role of 14-3-3ζ in endocrine resistance, and we examine the functional dimensions and molecular basis that underlie 14-3-3ζ activities.MethodsFrom analyses of four independent breast cancer microarray datasets from nearly 400 women, we characterized a gene signature that correlated strongly with high expression of 14-3-3ζ in breast tumors and examined its association with breast cancer molecular subtypes and clinical-pathological features. We investigated the effects of altering 14-3-3ζ levels in ER-positive, endocrine sensitive and resistant breast cancer cells on the regulation of 14-3-3ζ signature genes, and on cellular signaling pathways and cell phenotypic properties.ResultsThe gene signature associated with high 14-3-3ζ levels in breast tumors encompassed many with functions in mitosis and cytokinesis, including aurora kinase-B, polo-like kinase-1, CDC25B, and BIRC5/survivin. The gene signature correlated with early recurrence and risk of metastasis, and was found predominantly in luminal B breast cancers, the more aggressive ER-positive molecular subtype. The expression of the signature genes was significantly decreased or increased upon reduction or overexpression of 14-3-3ζ in ER-positive breast cancer cells, indicating their coregulation. 14-3-3ζ also played a critical role in the regulation of FOXM1, with 14-3-3ζ acting upstream of FOXM1 to regulate cell division-signature genes. Depletion of 14-3-3ζ markedly increased apoptosis, reduced proliferation and receptor tyrosine kinase (HER2 and EGFR) signaling, and, importantly, reversed endocrine resistance.ConclusionsThis study reveals that 14-3-3ζ is a key predictive marker for risk of failure on endocrine therapy and serves a pivotal role impacting growth factor signaling, and promoting cell survival and resistance to endocrine therapies. Targeting 14-3-3ζ and its coregulated proteins, such as FOXM1, should prove valuable in restoring endocrine sensitivity and reducing risk of breast cancer recurrence.

Abstract 1267: Metabolic sensor O-GlcNAc Transferase is a novel regulator of prostate cancer invasion and angiogenesis via regulation of FoxM1

Abstract Cancer cells universally increase glucose and glutamine consumption, leading to the altered metabolic state known as the Warburg effect. One metabolic pathway highly dependent on glucose and glutamine is the Hexosamine Biosynthetic Pathway (HBP). Increased flux through the HBP leads to increases in the post-translational addition of O-linked-β-N-acetylglucosamine (O-GlcNAc) on a diverse population of nuclear and cytosolic proteins. A number of these target proteins are implicated in cancer, yet the role of O-GlcNAc modifications in cancer remains poorly defined. Here, we show that OGT is overexpressed in prostate cancer tissue compared to normal prostate epithelium and, that OGT protein and O-GlcNAc levels are elevated in prostate carcinoma cell lines compared to non-transformed prostate cells. Decreasing O-GlcNAc levels using OGT-targeted RNAi in the metastatic prostate cancer cell line PC3-ML inhibited growth in three-dimensional (3D) culture, as well as decreased colony formation in soft agar assays. Furthermore, decreasing O-GlcNAc levels was also associated with cell cycle arrest at G1 and increased expression of the CDK inhibitors p27 and p21. However, reducing O-GlcNAc levels in the non-tumorigenic prostate epithelial cell line RWPE-1 had minimal effects on growth in 3D culture. In addition to growth inhibition, reducing O-GlcNAcation in PC3-ML cells was associated with reduced expression of matrix metalloproteinase-2 (MMP-2), MMP-9 and vascular endothelial growth factor (VEGF), resulting in the inhibition of invasion and angiogenesis. OGT regulation of invasion and angiogenesis was dependent upon regulation of the oncogenic transcription factor FoxM1, as reducing OGT expression led to increased FoxM1 proteasomal degradation. Moreover, overexpression of a FoxM1 degradation-resistant mutant abrogated OGT RNAi inhibition of invasion, MMP levels, as well as angiogenesis and VEGF expression. Finally, the use of novel second-generation small molecule inhibitor of OGT led to the similar reduction in growth, invasion and inhibition of MMPs and VEGF levels. Current experiments are underway to test whether targeting OGT blocks prostate cancer metastasis. Altogether, these data suggest that as prostate cancer cells alter glucose and glutamine levels, O-GlcNAcation serves as nutrient sensitive modification contributing to oncogenesis and suggest that OGT is positioned as a novel target for therapeutic intervention for the treatment of human prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1267. doi:10.1158/1538-7445.AM2011-1267

Increased expression of FoxM1 transcription factor in respiratory epithelium inhibits lung sacculation and causes Clara cell hyperplasia

Foxm1 is a member of the Forkhead Box (Fox) family of transcription factors. Foxm1 (previously called Foxm1b, HFH-11B, Trident, Win, or MPP2) is expressed in multiple cell types and plays important roles in cellular proliferation, differentiation and tumorigenesis. Genetic deletion of Foxm1 from mouse respiratory epithelium during initial stages of lung development inhibits lung maturation and causes respiratory failure after birth. However, the role of Foxm1 during postnatal lung morphogenesis remains unknown. In the present study, Foxm1 expression was detected in epithelial cells of conducting and peripheral airways and changing dynamically with lung maturation. To discern the biological role of Foxm1 in the prenatal and postnatal lung, a novel transgenic mouse line that expresses a constitutively active form of FoxM1 (FoxM1 N-terminal deletion mutant or FoxM1-ΔN) under the control of lung epithelial-specific SPC promoter was produced. Expression of the FoxM1-ΔN transgene during embryogenesis caused epithelial hyperplasia, inhibited lung sacculation and expression of the type II epithelial marker, pro-SPC. Expression of FoxM1-ΔN mutant during the postnatal period did not influence alveologenesis but caused focal airway hyperplasia and increased proliferation of Clara cells. Likewise, expression of FoxM1-ΔN mutant in conducting airways with Scgb1a1 promoter was sufficient to induce Clara cell hyperplasia. Furthermore, FoxM1-ΔN cooperated with activated K-Ras to induce lung tumor growth in vivo. Increased activity of Foxm1 altered lung sacculation, induced proliferation in the respiratory epithelium and accelerated lung tumor growth, indicating that precise regulation of Foxm1 is critical for normal lung morphogenesis and development of lung cancer.

FOXM1 is a transcriptional target of ERα and has a critical role in breast cancer endocrine sensitivity and resistance

Previous data have shown that FOXM1 expression is elevated in breast cancer tissues and is strongly correlated with the expression pattern of ERα in breast cancer cells. The expression of ERα is a good prognostic factor in breast cancer, as about two-thirds of these ERα-positive patients respond to treatment with antiestrogens. However, approximately one-half of the patients that initially respond to hormonal therapy develop resistance. Since FOXM1 is critical for the progression of the cell cycle, we investigated the regulation of FOXM1 by ERα and its role in endocrine sensitivity and resistance in breast cancer cells. We firstly observed by quantitative RT-PCR a strong and significant positive correlation between ERα and FOXM1 mRNA expression in breast cancer patient samples. We showed that FOXM1 protein and mRNA expression was regulated by ER ligands. We also demonstrated that ectopic conditional expression of ERα, in the presence of estrogens, leads to induction of FOXM1 expression in ER-negative U2OS cells. Using reporter gene assays, we demonstrated that ERα activates FOXM1 transcription through an estrogen-response element site. The direct binding of ERα to the FOXM1 promoter was confirmed in vitro by mobility shift and DNA pull-down assays and in vivo by chromatin immunoprecipitation analysis. Importantly, silencing of FOXM1 by RNA interference abolishes the estrogen-mediated MCF-7 cell proliferation. Conversely, ectopic expression of a constitutively active FOXM1 can abrogate the cell cycle arrest mediated by the antiestrogen tamoxifen. Taken together, the results clearly demonstrate FOXM1 as a key mediator of the mitogenic functions of ERα and estrogen in breast cancer cells. Our findings that antiestrogens repress FOXM1 expression in endocrine-sensitive but not endocrine-resistant breast carcinoma cell lines and that ectopic expression of an active FOXM1 can abrogate the anti-proliferative effects of tamoxifen also suggest that deregulation of FOXM1 may also contribute to antiestrogen insensitivity.

Abstract LB-28: Regulation of FOXM1 by NADPH oxidases in malignant mesothelioma cells

Abstract Malignant mesothelioma (MM) is linked to the occupational exposure to asbestos with an estimated median survival of less than two years; effective therapy is lacking. Due to long latency periods, incidence is expected to rise in regions where asbestos use has been banned, as well as where worker protection is lacking. MM cells exhibit changes in oxidant and energy metabolism, with elevated expression of the NADPH oxidase NOX4. We hypothesize that NOX4 produces reactive oxygen species (ROS) that promote uncontrolled growth through redox-dependent signaling to core cell cycle factors such as FOXM1 and FOXO3a, transcription factors involved in responses in oxidative stress and cell cycle progression. Targeted microarrays were used to assess the expression levels of 96 genes involving redox metabolism in four human MM cell lines and one control mesothelial line (LP9) immortalized with hTERT. Gene expression profiles from MM cell lines indicate tumorigenesis commonly results in elevated expression of NOX activity and adaptation to constitutive production of ROS, including up-regulation of antioxidant enzymes and cytoprotective factors. mRNA for FOXM1, a transcription factor that regulates the G2/M transition in the cell cycle, was up-regulated in all four MM cell lines, and FOXM1 continued to be expressed in MM cells, but not LP9 cells, in the absence of growth factors. Inhibitors of NOX activity markedly reduced expression of FOXM1, which correlated with dose-dependent effects on MM cell proliferation and viability. In the HM MM cell line, thiostrepton (TS), a thiazole antibiotic that inhibits the expression of FOXM1 via an unknown mechanism, increased the rate of oxidation of DCF, hyper-activated ERK1/2, and showed dose-dependent effects on MM cell viability in vitro and MM tumor growth in Fox Chase SCID mice. N-acetyl-L-cysteine blocked activation of ERK1/2 and cell death by TS, indicating the cytotoxic effects of TS in MM cells may be dependent on induction of intolerable levels of oxidative stress. In support of this possibility, inhibitors of p38 MAPK accentuated the cytotoxic effects of TS on cell viability. Our studies indicate that altered oxidant metabolism that supports expression of cell cycle factors such as FOXM1 is a common property of MM cells, and suggest that either inhibiting or accentuating the production of ROS in MM cells may be useful therapeutic approaches in the treatment of MM. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-28.

Reduction of Raf Kinase Inhibitor Protein Expression by Bcr-Abl Contributes to Chronic Myelogenous Leukemia Proliferation

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The Ras/Raf-1/MEK/ERK pathway is constitutively activated in Bcr-Abl-transformed cells, and Ras activity enhances the oncogenic ability of Bcr-Abl. However, the mechanism by which Bcr-Abl activates the Ras pathway is not completely understood. Raf kinase inhibitor protein (RKIP) inhibits activation of MEK by Raf-1 and its downstream signal transduction, resulting in blocking the MAP kinase pathway. In the present study, we found that RKIP was depleted in CML cells. We investigated the interaction between RKIP and Bcr-Abl in CML cell lines and Bcr-Abl(+) progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of RKIP and reduced the pERK1/2 status, resulting in inhibited proliferation of CML cells. Moreover, RKIP up-regulated cell cycle regulator FoxM1 expression, resulting in G(1) arrest via p27(Kip1) and p21(Cip1) accumulation. In colony-forming unit granulocyte, erythroid, macrophage, megakaryocyte, colony-forming unit-granulocyte macrophage, and burst-forming unit erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced RKIP and reduced FoxM1 expressions, and inhibited colony formation of Bcr-Abl(+) progenitor cells, whereas depletion of RKIP weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl(+) progenitor cells. Thus, Bcr-Abl represses the expression of RKIP, continuously activates pERK1/2, and suppresses FoxM1 expression, resulting in proliferation of CML cells.

A new target for proteasome inhibitors: FoxM1

Importance of the field: The proteasome is responsible for ubiquitin- and ATP-dependent proteolysis of cellular proteins. The latest advances in proteasome studies led to the development of proteasome inhibitors as drugs against human cancer. It has been shown that proteasome inhibitors selectively kill cancer, but not normal cells. However, the exact mechanisms of the anticancer activity of proteasome inhibitors are not well understood. The oncogenic transcription factor Forkhead Box M1 (FoxM1) is overexpressed in a majority of human carcinomas, while its expression is usually low in normal cells. In addition, FoxM1 may also drive tumor invasion, angiogenesis and metastasis. For these reasons, FoxM1 is an attractive target for anticancer drugs.Areas covered in this review: My aim is to discuss recent publications that point out novel mechanism of action of proteasome inhibitors. In addition, I describe the identification of new types of proteasome inhibitors, called thiazole antibiotics. Using a cell-based screening system, the thiazole antibiotics siomycin A and thiostrepton were isolated as inhibitors of FoxM1 transcriptional activity and expression. Paradoxically, it has been shown that these drugs also stabilize the expression of other proteins and act as proteasome inhibitors in vitro. Moreover, it was found that well-known proteasome inhibitors, such as MG115, MG132 and bortezomib, inhibit FoxM1 transcriptional activity and FoxM1 expression.What the reader will gain: It has been shown that proteasome inhibitors suppress FoxM1 expression and simultaneously induce apoptosis in human tumor cell lines. This review describes the correlation between negative regulation of FoxM1 by proteasome inhibitors and apoptosis, and suggests that negative regulation of FoxM1 is a universal feature of these drugs and may contribute to their anticancer activity.Take home message: Oncogenic transcription factor FoxM1 is upregulated in a majority of human cancers, suggesting that growth of cancer cells may depend on FoxM1 activity. A short time ago, it has been shown that proteasome inhibitors simultaneously inhibit FoxM1 expression and induce apoptosis in human cancer cells. This effect may explain specificity of proteasome inhibitors to induce apoptosis in cancer, but not in normal cells. Now, it is critical to determine the role of suppression of FoxM1 in apoptosis induced by proteasome inhibitors and to establish how significant the inhibition of FoxM1 is for the anticancer activity of proteasome inhibitors.

The C-Rel Subunit of Nuclear Factor-κB Regulates Murine Liver Inflammation, Wound-Healing, and Hepatocyte Proliferation

In this study, we determined the role of the nuclear factor-kappaB (NF-kappaB) subunit c-Rel in liver injury and regeneration. In response to toxic injury of the liver, c-Rel null (c-rel(-/-)) mice displayed a defect in the neutrophilic inflammatory response, associated with impaired induction of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted; also known as CCL5). The subsequent fibrogenic/wound-healing response to both chronic carbon tetrachloride and bile duct ligation induced injury was also impaired and this was associated with deficiencies in the expression of fibrogenic genes, collagen I and alpha-smooth muscle actin, by hepatic stellate cells. We additionally report that c-Rel is required for the normal proliferative regeneration of hepatocytes in response to toxic injury and partial hepatectomy. Absence of c-Rel was associated with blunted and delayed induction of forkhead box M1 (FoxM1) and its downstream targets cyclin B1 and Cdc25C. Furthermore, isolated c-rel(-/-) hepatocytes expressed reduced levels of FoxM1 and a reduced rate of basal and epidermal growth factor-induced DNA synthesis. Chromatin immunoprecipitation revealed that c-Rel binding to the FoxM1 promoter is induced in the regenerating liver. c-Rel has multiple functions in the control of liver homeostasis and regeneration and is a transcriptional regulator of FoxM1 and compensatory hepatocyte proliferation.

A Conserved Phosphorylation Site within the Forkhead Domain of FoxM1B Is Required for Its Activation by Cyclin-CDK1

The Forkhead box M1 (FoxM1) transcription factor is critical for expression of the genes essential for G(1)/S transition and mitotic progression. To explore the cell cycle regulation of FoxM1, we examined the phosphorylation profile of FoxM1. Here, we show that the phosphorylated status and the activity of FoxM1 increase as cells progress from S to G(2)/M phases. Moreover, dephosphorylation of FoxM1 coincides with exit from mitosis. Using mass spectrometry, we have identified a new conserved phosphorylation site (Ser-251) within the forkhead domain of FoxM1. Disruption of Ser-251 inhibits phosphorylation of FoxM1 and dramatically decreases its transcriptional activity. We demonstrate that the Ser-251 residue is required for CDK1-dependent phosphorylation of FoxM1 as well as its interaction with the coactivator CREB-binding protein (CBP). Interestingly, the transcriptional activity of the S251A mutant protein remains responsive to activation by overexpressed Polo-like kinase 1 (PLK1). Cells expressing the S251A mutant exhibit reduced expression of the G(2)/M phase genes and impaired mitotic progression. Our results demonstrate that the transcriptional activity of FoxM1 is controlled in a cell cycle-dependent fashion by temporally regulated phosphorylation and dephosphorylation events, and that the phosphorylation at Ser-251 is critical for the activation of FoxM1.

FoxM1 Is a General Target for Proteasome Inhibitors

Proteasome inhibitors are currently in the clinic or in clinical trials, but the mechanism of their anticancer activity is not completely understood. The oncogenic transcription factor FoxM1 is one of the most overexpressed genes in human tumors, while its expression is usually halted in normal non-proliferating cells. Previously, we established that thiazole antibiotics Siomycin A and thiostrepton inhibit FoxM1 and induce apoptosis in human cancer cells. Here, we report that Siomycin A and thiostrepton stabilize the expression of a variety of proteins, such as p21, Mcl-1, p53 and hdm-2 and also act as proteasome inhibitors in vitro. More importantly, we also found that well-known proteasome inhibitors such as MG115, MG132 and bortezomib inhibit FoxM1 transcriptional activity and FoxM1 expression. In addition, overexpression of FoxM1 specifically protects against bortezomib-, but not doxorubicin-induced apoptosis. These data suggest that negative regulation of FoxM1 by proteasome inhibitors is a general feature of these drugs and it may contribute to their anticancer properties.

Gefitinib (Iressa) represses FOXM1 expression via FOXO3a in breast cancer

Gefitinib (Iressa) is a specific and effective epidermal growth factor receptor inhibitor. An understanding of the downstream cellular targets of gefitinib will allow the discovery of biomarkers for predicting outcomes and monitoring anti-epidermal growth factor receptor therapies and provide information for overcoming gefitinib resistance. In this study, we investigated the role and regulation of FOXM1 in response to gefitinib treatment in breast cancer. Using the gefitinib-sensitive breast carcinoma cell lines BT474 and SKBR3 as well as the resistant lines MCF-7, MDA-MB-231, and MDA-MB-453, we showed that gefitinib represses the expression of the transcription factor FOXM1 in sensitive, but not resistant, cells. FOXM1 repression by gefitinib is associated with FOXO3a activation and is mediated at the transcriptional level and gene promoter level. These results were verified by immunohistochemical staining of biopsy samples from primary breast cancer patients obtained from a gefitinib neoadjuvant study. We also showed that ectopic expression of an active FOXO3a represses FOXM1 expression, whereas knockdown of FOXO3a expression using small interfering RNA can up-regulate FOXM1 and its downstream targets polo-like kinase, cyclin B1, and CDC25B and rescue sensitive BT474 cells from gefitinib-induced cell proliferative arrest. These results suggest that gefitinib represses FOXM1 expression via FOXO3a in breast cancer. We further showed that overexpression of a wild-type FOXM1 or a constitutively active FOXM1, DeltaN-FOXM1, abrogates the cell death induced by gefitinib, indicating that FOXM1 has a functional role in mediating the gefitinib-induced proliferative arrest and in determining sensitivity to gefitinib. In summary, our study defined FOXM1 as a cellular target and marker of gefitinib activity in breast cancer.