FoxM1, a critical regulator of oxidative stress during oncogenesis.

The transcription factor FoxM1 is over-expressed in most human malignancies. Although it is evident that FoxM1 has critical functions in tumour development and progression, the mechanisms by which FoxM1 participates in those processes are not understood. Here, we describe an essential role of FoxM1 in the regulation of oxidative stress that contributes to malignant transformation and tumour cell survival. We identify a negative feedback loop involving FoxM1 that regulates reactive oxygen species (ROS) in proliferating cells. We show that induction of FoxM1 by oncogenic Ras requires ROS. Elevated FoxM1, in turn, downregulates ROS levels by stimulating expression of ROS scavenger genes, such as MnSOD, catalase and PRDX3. FoxM1 depletion sensitizes cells to oxidative stress and increases oncogene-induced premature senescence. Moreover, tumour cells expressing activated AKT1 are 'addicted' to FoxM1, as they require continuous presence of FoxM1 for survival. Together, our results identify FoxM1 as a key regulator of ROS in dividing cells, and provide insights into the mechanism how tumour cells use FoxM1 to control oxidative stress to escape premature senescence and apoptosis.

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.

A cell-penetrating ARF peptide inhibitor of FoxM1 in mouse hepatocellular carcinoma treatment.

The forkhead box m1 (Foxm1) transcription factor is essential for initiation of carcinogen-induced liver tumors; however, whether FoxM1 constitutes a therapeutic target for liver cancer treatment remains unknown. In this study, we used diethylnitrosamine/phenobarbital treatment to induce hepatocellular carcinomas (HCCs) in either WT mice or Arf(-/-)Rosa26-FoxM1b Tg mice, in which forkhead box M1b (FoxM1b) is overexpressed and alternative reading frame (ARF) inhibition of FoxM1 transcriptional activity is eliminated. To pharmacologically reduce FoxM1 activity in HCCs, we subjected these HCC-bearing mice to daily injections of a cell-penetrating ARF(26-44) peptide inhibitor of FoxM1 function. After 4 weeks of this treatment, HCC regions displayed reduced tumor cell proliferation and angiogenesis and a significant increase in apoptosis within the HCC region but not in the adjacent normal liver tissue. ARF peptide treatment also induced apoptosis of several distinct human hepatoma cell lines, which correlated with reduced protein levels of the mitotic regulatory genes encoding polo-like kinase 1, aurora B kinase, and survivin, all of which are transcriptional targets of FoxM1 that are highly expressed in cancer cells and function to prevent apoptosis. These studies indicate that ARF peptide treatment is an effective therapeutic approach to limit proliferation and induce apoptosis of liver cancer cells in vivo.

Chk2 mediates stabilization of the FoxM1 transcription factor to stimulate expression of DNA repair genes.

The forkhead box M1 (FoxM1) transcription factor regulates expression of cell cycle genes essential for DNA replication and mitosis during organ repair and cancer progression. Here, we demonstrate that FoxM1-deficient (-/-) mouse embryonic fibroblasts and osteosarcoma U2OS cells depleted in FoxM1 levels by small interfering RNA transfection display increased DNA breaks, as evidenced by immunofluorescence focus staining for phosphospecific histone H2AX. FoxM1-deficient cells also exhibit stimulation of p53 transcriptional activity, as evidenced by increased expression of the p21(cip1) gene. FoxM1-deficient cells display reduced expression of the base excision repair factor X-ray cross-complementing group 1 (XRCC1) and breast cancer-associated gene 2 (BRCA2), the latter of which is involved in homologous recombination repair of DNA double-strand breaks. Furthermore, FoxM1 protein is phosphorylated by checkpoint kinase 2 (Chk2) in response to DNA damage. This phosphorylation of FoxM1 on serine residue 361 caused increased stability of the FoxM1 protein with corresponding increased transcription of XRCC1 and BRCA2 genes, both of which are required for repair of DNA damage. These results identify a novel role for FoxM1 in the transcriptional response during DNA damage/checkpoint signaling and show a novel mechanism by which Chk2 protein regulates expression of DNA repair enzymes.

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.

New and unexpected: forkhead meets ARF.

Recent genetic studies demonstrate that mice deficient in the forkhead box m1b (Foxm1b) transcription factor are highly resistant to developing hepatocellular carcinoma, which is among the most lethal cancers worldwide. In addition, the Foxm1b transcription factor was identified as a novel inhibitory target of the p19ARF tumor suppressor during early stages of liver tumorigenesis, but p19ARF expression is extinguished in hepatic tumors that develop at later stages. Structure-function studies demonstrate that amino acids 26-46 of the p19ARF protein are sufficient to bind Foxm1b and reduce Foxm1b transcriptional activity by targeting it to the nucleolus. A peptide containing amino acids 24-46 of p19ARF, which was modified to enhance cellular uptake, is an effective inhibitor of Foxm1b transcriptional activity and prevents Foxm1b stimulation of anchorage-independent growth of cells on soft agar. Thus, the p19ARF peptide is an effective inhibitor of Foxm1b and represents a potential therapy for hepatocellular carcinoma.

Forkhead box M1 regulates the transcriptional network of genes essential for mitotic progression and genes encoding the SCF (Skp2-Cks1) ubiquitin ligase.

The Forkhead box m1 (Foxm1) gene is critical for G(1)/S transition and essential for mitotic progression. However, the transcriptional mechanisms downstream of FoxM1 that control these cell cycle events remain to be determined. Here, we show that both early-passage Foxm1(-)(/)(-) mouse embryonic fibroblasts (MEFs) and human osteosarcoma U2OS cells depleted of FoxM1 protein by small interfering RNA fail to grow in culture due to a mitotic block and accumulate nuclear levels of cyclin-dependent kinase inhibitor (CDKI) proteins p21(Cip1) and p27(Kip1). Using quantitative chromatin immunoprecipitation and expression assays, we show that FoxM1 is essential for transcription of the mitotic regulatory genes Cdc25B, Aurora B kinase, survivin, centromere protein A (CENPA), and CENPB. We also identify the mechanism by which FoxM1 deficiency causes elevated nuclear levels of the CDKI proteins p21(Cip1) and p27(Kip1). We provide evidence that FoxM1 is essential for transcription of Skp2 and Cks1, which are specificity subunits of the Skp1-Cullin 1-F-box (SCF) ubiquitin ligase complex that targets these CDKI proteins for degradation during the G(1)/S transition. Moreover, early-passage Foxm1(-)(/)(-) MEFs display premature senescence as evidenced by high expression of the senescence-associated beta-galactosidase, p19(ARF), and p16(INK4A) proteins. Taken together, these results demonstrate that FoxM1 regulates transcription of cell cycle genes critical for progression into S-phase and mitosis.

ARF directly binds DP1: interaction with DP1 coincides with the G1 arrest function of ARF.

The tumor suppressor ARF inhibits cell growth in response to oncogenic stress in a p53-dependent manner. Also, there is an increasing appreciation of ARF's ability to inhibit cell growth via multiple p53-independent mechanisms, including its ability to regulate the E2F pathway. We have investigated the interaction between the tumor suppressor ARF and DP1, the DNA binding partner of the E2F family of factors (E2Fs). We show that ARF directly binds to DP1. Interestingly, binding of ARF to DP1 results in an inhibition of the interaction between DP1 and E2F1. Moreover, ARF regulates the association of DP1 with its target gene, as evidenced by a chromatin immunoprecipitation assay with the dhfr promoter. By analyzing a series of ARF mutants, we demonstrate a strong correlation between ARF's ability to regulate DP1 and its ability to cause cell cycle arrest. S-phase inhibition by ARF is preceded by an inhibition of the E2F-activated genes. Moreover, we provide evidence that ARF inhibits the E2F-activated genes independently of p53 and Mdm2. Also, the interaction between ARF and DP1 is enhanced during oncogenic stress and "culture shock." Taken together, our results show that DP1 is a critical direct target of ARF.

The Forkhead Box m1 transcription factor stimulates the proliferation of tumor cells during development of lung cancer.

The proliferation-specific Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) regulates expression of cell cycle genes essential for progression into DNA replication and mitosis. Expression of Foxm1 is found in a variety of distinct human cancers including hepatocellular carcinomas, intrahepatic cholangiocarcinomas, basal cell carcinomas, ductal breast carcinomas, and anaplastic astrocytomas and glioblastomas. In this study, we show that human Foxm1 protein is abundantly expressed in highly proliferative human non-small cell lung cancers (NSCLC) as well as in mouse lung tumors induced by urethane. To determine the role of Foxm1 during the development of mouse lung tumors, we used IFN-inducible Mx-Cre recombinase transgene to delete mouse Foxm1 fl/fl-targeted allele before inducing lung tumors with urethane. We show that Mx-Cre Foxm1-/- mice exhibit diminished proliferation of lung tumor cells causing a significant reduction in number and size of lung adenomas. Transient transfection experiments with A549 lung adenocarcinoma cells show that depletion of Foxm1 levels by short interfering RNA caused diminished DNA replication and mitosis and reduced anchorage-independent growth of cell colonies on soft agar. Foxm1-depleted A549 cells exhibit reduced expression of cell cycle-promoting cyclin A2 and cyclin B1 genes. These data show that Foxm1 stimulates the proliferation of tumor cells during progression of NSCLC.

Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice.

The proliferation-specific Forkhead Box M1 (FoxM1 or FoxM1b) transcription factor is overexpressed in a number of aggressive human carcinomas. Mouse hepatocytes deficient in FoxM1 fail to proliferate and are highly resistant to developing carcinogen-induced liver tumors. We previously developed a transgenic (TG) mouse line in which the ubiquitous Rosa26 promoter was used to drive expression of the human FoxM1b cDNA transgene in all mouse cell types. To investigate the role of FoxM1b in prostate cancer progression, we bred Rosa26-FoxM1b mice with both TRAMP and LADY TG mouse models of prostate cancer. We show that increased expression of FoxM1b accelerated development, proliferation, and growth of prostatic tumors in both TRAMP and LADY double TG mice. Furthermore, development of prostate carcinomas in TRAMP/Rosa26-FoxM1b double TG mice required high levels of FoxM1 protein to overcome sustained expression of the alternative reading frame tumor suppressor, a potent inhibitor of FoxM1 transcriptional activity. Depletion of FoxM1 levels in prostate cancer cell lines PC-3, LNCaP, or DU-145 by small interfering RNA transfection caused significant reduction in proliferation and anchorage-independent growth on soft agar. This phenotype was associated with increased nuclear levels of the cyclin-dependent kinase inhibitor protein p27(Kip1) and diminished expression of S-phase promoting cyclin A2 and M-phase promoting cyclin B1 proteins. Finally, we show that elevated levels of FoxM1 protein correlate with high proliferation rates in human prostate adenocarcinomas. Our results suggest that the FoxM1 transcription factor regulates development and proliferation of prostate tumors, and that FoxM1 is a novel target for prostate cancer treatment.

Forkhead box M1B transcriptional activity requires binding of Cdk-cyclin complexes for phosphorylation-dependent recruitment of p300/CBP coactivators.

Previous liver regeneration studies demonstrated that the mouse forkhead box M1B (FoxM1B) transcription factor regulates hepatocyte proliferation through expression of cell cycle genes that stimulate cyclin-dependent kinase 2 (Cdk2) and Cdk1 activity. In this study, we demonstrated that disruption of the FoxM1B Cdk1/2 phosphorylation site at Thr residue 596 significantly reduced both FoxM1B transcriptional activity and Cdk phosphorylation of the FoxM1B T596A mutant protein in vivo. Retention of this FoxM1B 596 Cdk phosphorylation site was found to be essential for recruiting the histone acetyltransferase CREB binding protein (CBP) to the FoxM1B transcriptional activation domain. Consistent with these findings, dominant negative Cdk1 protein significantly reduced FoxM1B transcriptional activity and inhibited FoxM1B recruitment of the CBP coactivator protein. Likewise, Cdc25B-mediated stimulation of Cdk activity together with elevated levels of the CBP coactivator protein provided a 6.2-fold synergistic increase in FoxM1B transcriptional activity. Furthermore, mutation of the FoxM1B Leu 641 residue within an LXL motif (residues 639 to 641) inhibited recruitment of Cdk-cyclin complexes and caused significant reduction in both FoxM1B transcriptional activity and in vivo Cdk phosphorylation of the FoxM1B Thr 596 residue. We demonstrated that FoxM1B transcriptional activity requires binding of either S-phase or M-phase Cdk-cyclin complexes to mediate efficient Cdk phosphorylation of the FoxM1B Thr 596 residue, which is essential for recruitment of p300/CBP coactivator proteins.

Association between hepatocyte nuclear factor 6 (HNF-6) and FoxA2 DNA binding domains stimulates FoxA2 transcriptional activity but inhibits HNF-6 DNA binding.

In previous studies we used transgenic mice or recombinant adenovirus infection to increase hepatic expression of forkhead box A2 (FoxA2, previously called hepatocyte nuclear factor 3beta [HNF-3beta]), which caused diminished hepatocyte glycogen levels and reduced expression of glucose homeostasis genes. Because this diminished expression of FoxA2 target genes was associated with reduced levels of the Cut-Homeodomain HNF-6 transcription factor, we conducted the present study to determine whether there is a functional interaction between HNF-6 and FoxA2. Human hepatoma (HepG2) cotransfection assays demonstrated that HNF-6 synergistically stimulated FoxA2 but not FoxA1 or FoxA3 transcriptional activity, and protein-binding assays showed that this protein interaction required the HNF-6 Cut-Homeodomain and FoxA2 winged-helix DNA binding domains. Furthermore, we show that the HNF-6 Cut-Homeodomain sequences were sufficient to synergistically stimulate FoxA2 transcriptional activation by recruiting the p300/CBP coactivator proteins. This was supported by the fact that FoxA2 transcriptional synergy with HNF-6 was dependent on retention of the HNF-6 Cut domain LXXLL sequence, which mediated recruitment of the p300/CBP proteins. Moreover, cotransfection and DNA binding assays demonstrated that increased FoxA2 levels caused a decrease in HNF-6 transcriptional activation of the glucose transporter 2 (Glut-2) promoter by interfering with the binding of HNF-6 to its target DNA sequence. These data suggest that at a FoxA-specific site, HNF-6 serves as a coactivator protein to enhance FoxA2 transcription, whereas at an HNF-6-specific site, FoxA2 represses HNF-6 transcription by inhibiting HNF-6 DNA binding activity. This is the first reported example of a liver-enriched transcription factor (HNF-6) functioning as a coactivator protein to potentiate the transcriptional activity of another liver factor, FoxA2.

The FoxM1 transcription factor is required to maintain pancreatic beta-cell mass.

The FoxM1 transcription factor is highly expressed in proliferating cells and activates several cell cycle genes, although its requirement appears to be limited to certain tissue types. Embryonic hepatoblast-specific inactivation of Foxm1 results in a dramatic reduction in liver outgrowth and subsequent late gestation lethality, whereas inactivation of Foxm1 in adult liver impairs regeneration after partial hepatectomy. These results prompted us to examine whether FoxM1 functions similarly in embryonic outgrowth of the pancreas and beta-cell proliferation in the adult. We found that FoxM1 is highly expressed in embryonic and neonatal endocrine cells, when many of these cells are proliferating. Using a Cre-lox strategy, we generated mice in which Foxm1 was inactivated throughout the developing pancreatic endoderm by embryonic d 15.5 (Foxm1(Deltapanc)). Mice lacking Foxm1 in their entire pancreas were born with normal pancreatic and beta-cell mass; however, they displayed a gradual decline in beta-cell mass with age. Failure of postnatal beta-cell mass expansion resulted in impaired islet function by 6 wk of age and overt diabetes by 9 wk. The decline in beta-cell mass in Foxm1(Deltapanc) animals is due to a dramatic decrease in postnatal beta-cell replication and a corresponding increase in nuclear localization of the cyclin-dependent kinase inhibitor, p27(Kip1), a known target of FoxM1 inhibition. We conclude that Foxm1 is essential to maintain normal beta-cell mass and regulate postnatal beta-cell turnover. These results suggest that mechanisms regulating embryonic beta-cell proliferation differ from those used postnatally to maintain the differentiated cell population.

On the epigenetic regulation of the human reelin promoter.

Reln mRNA and protein levels are reduced by approximately 50% in various cortical structures of post-mortem brain from patients diagnosed with schizophrenia or bipolar illness with psychosis. To study mechanisms responsible for this down-regulation, we have analyzed the promoter of the human reelin gene. We show that the reelin promoter directs expression of a reporter construct in multiple human cell types: neuroblastoma cells (SHSY5Y), neuronal precursor cells (NT2), differentiated neurons (hNT) and hepatoma cells (HepG2). Deletion constructs confirmed the presence of multiple elements regulating Reln expression, although the promoter activity is promiscuous, i.e. activity did not correlate with expression of the endogenous gene as reflected in terms of reelin mRNA levels. Co-transfection of the -514 bp human reelin promoter with either Sp1 or Tbr1 demonstrated that these transcription factors activate reporter expression by 6- and 8.5-fold, respectively. Within 400 bp of the RNA start site there are 100 potential CpG targets for DNA methylation. Retinoic acid (RA)-induced differentiation of NT2 cells to hNT neurons was accompanied by increased reelin expression and by the appearance of three DNase I hypersensitive sites 5' to the RNA start site. RA-induced differentiation was also associated with demethylation of the reelin promoter. To test if methylation silenced reelin expression, we methylated the promoter in vitro prior to transfection. In addition, we treated NT2 cells with the methylation inhibitor aza-2'-deoxycytidine and observed a 60-fold increase in reelin mRNA levels. The histone deacetylase inhibitors trichostatin A (TSA) and valproic acid also induced expression of the endogenous reelin promoter, although TSA was considerably more potent. These findings indicate that one determinant responsible for regulating reelin expression is the methylation status of the promoter. Our data also raise the interesting possibility that the down-regulation of reelin expression documented in psychiatric patients might be the consequence of inappropriate promoter hypermethylation.

Sustained hepatic expression of FoxM1B in transgenic mice has minimal effects on hepatocellular carcinoma development but increases cell proliferation rates in preneoplastic and early neoplastic lesions.

Increased hepatic expression of the Forkhead transcription factor FoxM1B in adult mice accelerates hepatocyte proliferation after partial hepatectomy, while in hepatocytes in intact liver the transgenic (Tg) protein is inactive and has no effect on proliferation. To investigate the influence of FoxM1B on liver tumor formation, we examined the effect of sustained enrichment of FoxM1B in the hepatocytes of mice treated with a diethylnitrosamine (DEN)/phenobarbital tumor induction protocol. Tg enrichment of FoxM1B in hepatocytes did not increase the proliferation rate in normal liver tissue even when the protein was localized to the nucleus. However, it did cause an increase in the proliferation rate and size of preneoplastic and early neoplastic lesions, although having no effects on the total numbers of these lesions. As tumors progressed to hepatocellular carcinomas, the additional Tg FoxM1B protein had no effect on cell proliferation, and there was no increase in tumor burden compared to wild-type animals. This suggests that the artificial enrichment of FoxM1B in the liver, which has been suggested as a gene therapy protocol for liver dysfunction with aging, may not be tumorigenic in that organ.

The forkhead box F1 transcription factor is expressed in brain and head mesenchyme during mouse embryonic development.

The forkhead box f1 (Foxf1) transcription factor is expressed in septum transversum mesenchyme and splanchnic (visceral) mesoderm, which give rise to mesenchymal cells of gut-derived organs such as liver, gall bladder, lung, stomach, and intestine. Foxf1 -/- embryos die in utero and haploinsufficiency of the Foxf1 gene resulted in a variety of developmental abnormalities of the lung, gall bladder, esophagus, and trachea and caused defects in liver and lung repair. In this study, we used Foxf1 +/- mouse embryos containing the beta-Galactosidase (beta-Gal) gene knocked into the Foxf1 locus to examine whether Foxf1 is expressed in the developing brain and head region. In addition to previously reported Foxf1 expression patterns, beta-Gal staining was also found in the mesenchyme of developing pituitary gland, eye, pancreas, heart, notochord, genital tubercle, Meckel's cartilage, and cartilage primordia of nasal septum and vertebral bodies. In adult Foxf1 +/- mice, beta-Gal staining was detected in anterior and posterior pituitary gland, astrocytes of the cerebellum and cerebral cortex, lens and retina of the eye, tracheal cartilage, parathyroid, cortical layer of thymus, capsule of the spleen, coronary arteries, and pancreatic blood vessels.

Rapid hepatocyte nuclear translocation of the Forkhead Box M1B (FoxM1B) transcription factor caused a transient increase in size of regenerating transgenic hepatocytes.

The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA binding domain. Liver regeneration studies with the -3 kb transthyretin (TTR) promoter-driven FoxM1B transgenic (TG) mice demonstrated that premature hepatocyte nuclear localization of the FoxM1B transgene protein at 16 h following partial hepatectomy (PHx) caused an 8-h acceleration in the onset of hepatocyte DNA replication (S-phase) and mitosis by stimulating earlier expression of cell cycle genes. Whether the FoxM1B transgene protein participates in immediate early events during liver regeneration remains to be determined. Here, we found that the FoxM1B transgene protein translocated to hepatocyte nuclei immediately following PHx, that its nuclear staining persisted for the first 6 h after surgery, and that this translocation was associated with an increase in size of regenerating TG hepatocytes. However, regenerating TTR-FoxM1B liver did not exhibit altered expression of proteins that have been implicated in mediating increased cell size, including serum-and-gucocorticoid-inducible protein kinase (SGK), protein kinase-B/Akt, the tumor suppresser gene PTEN (negative regulator of the PI3K/Akt pathway), c-Myc, or peroxisome proliferation. Moreover, we demonstrated that hepatocyte nuclear translocation of the FoxM1B transgene protein was rapidly induced during the hepatic acute phase response, which occurs during the immediate early stages of liver regeneration. Analysis of cDNA expression arrays identified a number of genes such as immediate early transcription factors (ID-3, Stat3, Nur77), matrix metalloproteinase-9 (MMP-9), and several glutathione S-transferase (GST) isoforms and stress response genes, whose expression is elevated in regenerating TTR-FoxM1B TG livers compared with regenerating wild-type (WT) liver. These liver regeneration studies demonstrate that hepatocyte nuclear translocation of the FoxM1B transgene protein was sustained for the first 6 h after PHx, and was associated with transient hypertrophy of regenerating TG hepatocytes and increased expression of genes that may enhance hepatocyte proliferation.

FoxM1 in tumorigenicity of the neuroblastoma cells and renewal of the neural progenitors.

Malignant neuroblastomas contain stem-like cells. These tumors also overexpress the Forkhead box transcription factor FoxM1. In this study, we investigated the roles of FoxM1 in the tumorigenicity of neuroblastoma. We showed that depletion of FoxM1 inhibits anchorage-independent growth and tumorigenicity in mouse xenografts. Moreover, knockdown of FoxM1 induces differentiation in neuroblastoma cells, suggesting that FoxM1 plays a role in the maintenance of the undifferentiated progenitor population. We showed that inhibition of FoxM1 in malignant neuroblastoma cells leads to the downregulation of the pluripotency genes sex determining region Y box 2 (Sox2) and Bmi1. We provided evidence that FoxM1 directly activates expression of Sox2 in neuroblastoma cells. By using a conditional deletion system and neurosphere cultures, we showed that FoxM1 is important for expression of Sox2 and Bmi1 in the mouse neural stem/progenitor cells and is critical for its self-renewal. Together, our observations suggested that FoxM1 plays an important role in the tumorigenicity of the aggressive neuroblastoma cells through maintenance of the undifferentiated state.

Deregulation of FoxM1b leads to tumour metastasis.

The forkhead box M1b (FoxM1b) transcription factor is over-expressed in human cancers, and its expression often correlates with poor prognosis. Previously, using conditional knockout strains, we showed that FoxM1b is essential for hepatocellular carcinoma (HCC) development. However, over-expression of FoxM1b had only marginal effects on HCC progression. Here we investigated the effect of FoxM1b expression in the absence of its inhibitor Arf. We show that transgenic expression of FoxM1b in an Arf-null background drives hepatic fibrosis and metastasis of HCC. We identify novel mechanisms of FoxM1b that are involved in epithelial-mesenchymal transition, cell motility, invasion and a pre-metastatic niche formation. FoxM1b activates the Akt-Snail1 pathway and stimulates expression of Stathmin, lysyl oxidase, lysyl oxidase like-2 and several other genes involved in metastasis. Furthermore, we show that an Arf-derived peptide, which inhibits FoxM1b, impedes metastasis of the FoxM1b-expressing HCC cells. The observations indicate that FoxM1b is a potent activator of tumour metastasis and that the Arf-mediated inhibition of FoxM1b is a critical mechanism for suppression of tumour metastasis.