FOXM1 (Forkhead box M1) in tumorigenesis: overexpression in human cancer, implication in tumorigenesis, oncogenic functions, tumor-suppressive properties, and target of anticancer therapy.

FOXM1 (Forkhead box M1) is a typical proliferation-associated transcription factor and is also intimately involved in tumorigenesis. FOXM1 stimulates cell proliferation and cell cycle progression by promoting the entry into S-phase and M-phase. Additionally, FOXM1 is required for proper execution of mitosis. In accordance with its role in stimulation of cell proliferation, FOXM1 exhibits a proliferation-specific expression pattern and its expression is regulated by proliferation and anti-proliferation signals as well as by proto-oncoproteins and tumor suppressors. Since these factors are often mutated, overexpressed, or lost in human cancer, the normal control of the foxm1 expression by them provides the basis for deregulated FOXM1 expression in tumors. Accordingly, FOXM1 is overexpressed in many types of human cancer. FOXM1 is intimately involved in tumorigenesis, because it contributes to oncogenic transformation and participates in tumor initiation, growth, and progression, including positive effects on angiogenesis, migration, invasion, epithelial-mesenchymal transition, metastasis, recruitment of tumor-associated macrophages, tumor-associated lung inflammation, self-renewal capacity of cancer cells, prevention of premature cellular senescence, and chemotherapeutic drug resistance. However, in the context of urethane-induced lung tumorigenesis, FOXM1 has an unexpected tumor suppressor role in endothelial cells because it limits pulmonary inflammation and canonical Wnt signaling in epithelial lung cells, thereby restricting carcinogenesis. Accordingly, FOXM1 plays a role in homologous recombination repair of DNA double-strand breaks and maintenance of genomic stability, that is, prevention of polyploidy and aneuploidy. The implication of FOXM1 in tumorigenesis makes it an attractive target for anticancer therapy, and several antitumor drugs have been reported to decrease FOXM1 expression.

The transcription factor FOXM1 (Forkhead box M1): proliferation-specific expression, transcription factor function, target genes, mouse models, and normal biological roles.

FOXM1 (Forkhead box M1) is a typical proliferation-associated transcription factor, which stimulates cell proliferation and exhibits a proliferation-specific expression pattern. Accordingly, both the expression and the transcriptional activity of FOXM1 are increased by proliferation signals, but decreased by antiproliferation signals, including the positive and negative regulation by protooncoproteins or tumor suppressors, respectively. FOXM1 stimulates cell cycle progression by promoting the entry into S-phase and M-phase. Moreover, FOXM1 is required for proper execution of mitosis. Accordingly, FOXM1 regulates the expression of genes, whose products control G1/S-transition, S-phase progression, G2/M-transition, and M-phase progression. Additionally, FOXM1 target genes encode proteins with functions in the execution of DNA replication and mitosis. FOXM1 is a transcriptional activator with a forkhead domain as DNA binding domain and with a very strong acidic transactivation domain. However, wild-type FOXM1 is (almost) inactive because the transactivation domain is repressed by three inhibitory domains. Inactive FOXM1 can be converted into a very potent transactivator by activating signals, which release the transactivation domain from its inhibition by the inhibitory domains. FOXM1 is essential for embryonic development and the foxm1 knockout is embryonically lethal. In adults, FOXM1 is important for tissue repair after injury. FOXM1 prevents premature senescence and interferes with contact inhibition. FOXM1 plays a role for maintenance of stem cell pluripotency and for self-renewal capacity of stem cells. The functions of FOXM1 in prevention of polyploidy and aneuploidy and in homologous recombination repair of DNA-double-strand breaks suggest an importance of FOXM1 for the maintenance of genomic stability and chromosomal integrity.

Cyclin D1/Cdk4 increases the transcriptional activity of FOXM1c without phosphorylating FOXM1c.

Anders et al. (2011) [11] reported that cyclinD1/Cdk4 and cyclinD3/Cdk6 enhance the transcriptional activity of FOXM1c by phosphorylating its TAD. They defined 12 Cdk consensus sites as essential for the activation of FOXM1c by cyclinD1/Cdk4 and cyclinD3/Cdk6 and stated that the 12 Cdk-sites are positioned within the TAD of FOXM1c. In contrast, this study demonstrates that all potential cyclin/Cdk phosphorylation sites S/T-P of FOXM1c are located outside its TAD so that the TAD of FOXM1c contains no potential cyclin/Cdk site, which excludes a phosphorylation of the FOXM1c-TAD by cyclinD1/Cdk4 and cyclinD3/Cdk6. This study shows that the activation of FOXM1c by cyclinD1/Cdk4 is lost without removal of any cyclin/Cdk site and gained without addition of any cyclin/Cdk site because it depends on a FOXM1c domain with no potential cyclin/Cdk site, namely on the interaction domain for the tumor suppressor RB, which binds to and represses FOXM1c. CyclinD1/Cdk4 activates FOXM1c because cyclinD1/Cdk4 releases FOXM1c from its repression by RB through removal of RB from FOXM1c. For this purpose, cyclinD1/Cdk4 phosphorylates only RB, but not FOXM1c, so that cyclinD1/Cdk4 increases the transcriptional activity of FOXM1c without phosphorylating FOXM1c and activates FOXM1c independently of cyclin/Cdk phosphorylation sites in FOXM1c. In summary, this study changes the model of Anders et al. (2011) [11] completely because it disproves their central conclusion that cyclinD1/Cdk4 and cyclinD3/Cdk6 enhance the transcriptional activity of FOXM1c by phosphorylating its TAD at the 12 Cdk-sites.

The transcription factor FOXM1c is activated by protein kinase CK2, protein kinase A (PKA), c-Src and Raf-1.

The transcription factor FOXM1c possesses a very strong C-terminal TAD (transactivation domain), but full-length FOXM1c is only a weak transactivator because the TAD is completely inhibited by the auto-inhibitory N-terminus. The N-terminus blocks the TAD by directly binding to the TAD. Accordingly, FOXM1c deletion mutants without N-terminus are strong transactivators. Therefore, the question arises whether signals exist, which activate full-length FOXM1c by releasing the FOXM1c-TAD from its inhibition by the N-terminus. Indeed, full-length FOXM1c is strongly activated by protein kinase CK2 and PKA (protein kinase A). Both CK2 and PKA do not activate a FOXM1c deletion mutant without N-terminus demonstrating that the activation of FOXM1c by CK2 and PKA depends on the presence of the N-terminus. Consequently, CK2 and PKA activate FOXM1c by alleviating the inhibition of FOXM1c by its N-terminus. The presence of two potential CK2 phosphorylation sites and two potential PKA phosphorylation sites in the N-terminus of FOXM1c suggests that CK2 and PKA may activate FOXM1c through phosphorylation of the FOXM1c N-terminus. Thus, CK2 and PKA strongly activate full-length FOXM1c because they alleviate the repression of FOXM1c by its own auto-inhibitory N-terminus. Also c-Src activates full-length FOXM1c by relieving the inhibition of FOXM1c by its N-terminus. In contrast, Raf-1 activates FOXM1c independently of the FOXM1c N-terminus. In summary, this study shows for the first time that FOXM1c is activated by the four kinases CK2, PKA, c-Src and Raf-1.

The transcription factor FOXM1c binds to and transactivates the promoter of the tumor suppressor gene E-cadherin.

This study demonstrates for the first time that FOXM1c transactivates the murine E-cadherin promoter. It shows also that the purified DNA-binding domain of FOXM1c binds to the murine and human E-cadherin promoters in vitro, namely to a perfectly conserved FOXM1 site. Thus, this study identifies E-cadherin as a new direct FOXM1c target gene. This finding is surprising because E-cadherin is a tumor suppressor gene whereas FOXM1 is a proliferation-associated and tumorigenesis-promoting transcription factor. The transmembrane glycoprotein E-cadherin mediates cell-cell adhesion in adherens junctions. Its expression is frequently lost or reduced in human tumors, which correlates with poor prognosis. Downregulation of E-cadherin represents a central event in epithelial-to-mesenchymal transition. In contrast, FOXM1 contributes to oncogenic transformation and participates in tumor initiation and progression. It is overexpressed in many human cancers and a high FOXM1 level correlates with poor prognosis. FOXM1 stimulates cell proliferation and promotes cell cycle progression at the G1/S- and G2/M-transitions. The surprising finding that FOXM1c transactivates the promoter of the tumor suppressor gene E-cadherin points to a tumor-suppressive property of FOXM1. This view is supported by FOXM1's new tumor suppressor role as others reported that urethane-induced lung tumorigenesis is increased in mice with an endothelial cell-specific foxm1 deletion.

Cyclin E/Cdk2, P/CAF, and E1A regulate the transactivation of the c-myc promoter by FOXM1.

FOXM1c transactivates the c-myc promoter by binding directly to its TATA-boxes. The present study demonstrates that the transactivation of the c-myc promoter by FOXM1c is enhanced by the key proliferation signal cyclin E/Cdk2, but repressed by P/CAF and the adenoviral oncoprotein E1A. Furthermore, FOXM1c interacts with the coactivator and histone acetyltransferase P/CAF. This study shows that, on the c-myc-P1 TATA-box, FOXM1c does not function simply as normal transcription factor just binding to an unusual site. Moreover, the inhibitory N-terminus of FOXM1c does not inhibit its transrepression domain or its EDA. Others reported that a cyclin/Cdk-binding LXL-motif of the splice variant FoxM1b is required for its interaction with Cdk2, Cdk1, and p27, its phosphorylation by Cdk1 and its activation by Cdc25B. In contrast, we now demonstrate that this LXL-motif is not required for the activation of FOXM1c by cyclin D1/Cdk4, cyclin E/Cdk and cyclin A/Cdk2 or for the repression of FOXM1c by p27.

The c-myc promoter: still MysterY and challenge.

The transcription factor c-Myc is a key regulator of cell proliferation, cell growth, differentiation, and apoptosis. Deregulated c-myc expression possesses a high transformation potential and the proto-oncogene c-myc represents a promising target in anticancer therapy. This review on the c-myc promoter describes its organization, the different levels of its normal regulation (including initiation and elongation of transcription, the dual P1/P2 promoters, chromatin structure, c-Myc autosuppression) as well as its deregulation in Burkitt's lymphoma. Furthermore, it summarizes the many different transcription factors, signal transduction pathways, and feedback loops that activate or repress c-myc transcription. Finally, a concept for regulation of the c-myc promoter in different biological settings, for example, immediate-early induction, constant expression throughout the cell cycle in continuously cycling cells, repression during terminal differentiation and deregulation in cancer, is formulated.

FOXM1, a typical proliferation-associated transcription factor.

FOXM1 is a typical proliferation-associated transcription factor: it stimulates proliferation by promoting S-phase entry as well as M-phase entry and is involved in proper execution of mitosis. Accordingly, FOXM1 regulates genes that control G1/S-transition, S-phase progression, G2/M-transition and M-phase progression. Consistently, its expression and its activity are antagonistically regulated by many important proliferation and anti-proliferation signals. Furthermore, FOXM1 is implicated in tumorigenesis and contributes to both tumor initiation and progression. In addition to its function as a conventional transcription factor, FOXM1 transactivates the human c-myc P1 and P2 promoters directly via their TATA-boxes by a new transactivation mechanism, which it also employs for transactivation of the human c-fos, hsp70 and histone H2B/a promoters. This review summarizes the current knowledge on FOXM1, in particular its two different transactivation mechanisms, the regulation of its transcriptional activity by proliferation versus anti-proliferation signals and its function in normal cell cycle progression and tumorigenesis.

The central domain of transcription factor FOXM1c directly interacts with itself in vivo and switches from an essential to an inhibitory domain depending on the FOXM1c binding site.

We have previously shown that FOXM1c can transactivate its target genes by two different mechanisms, depending on the FOXM1c binding site. In the present study, by introducing a small 46-aa deletion, we confirm that the central domain of FOXM1c is essential for transactivation of the minimal c-myc P1 and P2 promoters via their TATA boxes, but functions as an inhibitory domain on conventional FOXM1c binding sites. Thus, distinct FOXM1c binding sites determine opposite functions of the central domain, suggesting allosteric control of its conformation by the DNA binding site. This is strongly supported by the identification of a direct in vivo interaction of the central domain with itself in the present study. In contrast, the DNA binding domain binds neither to itself nor to any other domain of FOXM1c. Transrepression by the central domain is unlikely to be achieved by recruitment of co-repressors, but instead seems to be mediated by direct interference with the basal transcription complex. Direct binding of the central domain to itself should be involved in transrepression. Finally, FOXM1c transactivates the chicken mim-1 promoter, whose TATA box represents a conventional FOXM1c binding site, so that transactivation follows neither of the above two mechanisms, but shows intermediate behavior.

FOXM1c and Sp1 transactivate the P1 and P2 promoters of human c-myc synergistically.

We have previously shown that FOXM1c transactivates the c-myc P1 and P2 promoters via their TATA-boxes by a new transactivation mechanism, namely by directly binding to the P1 and P2 TATA-boxes and to TBP, TFIIA, and TFIIB. We now confirm this surprising mechanism by demonstrating that FOXM1c transactivates the human c-myc P1 and P2 promoters synergistically with Sp1, a transcription factor known to bind and transactivate these two promoters. This synergism requires the P1 or P2 TATA-boxes as well as the respective Sp1-binding sites. Moreover FOXM1c binds directly to Sp1. Cooperative DNA binding, if it should occur, is not sufficient for synergism of Sp1 and FOXM1c at P1, but their contacts to multiple components of the basal transcription complex (TFIID, TFIIA, TFIIB) seem to be essential. However, FOXM1c does not synergize with Sp1 if it transactivates via its conventional binding site.

FOXM1c transactivates the human c-myc promoter directly via the two TATA boxes P1 and P2.

FOXM1c transactivates the c-myc promoter via the P1 and P2 TATA boxes using a new mechanism. Whereas the P1 TATA box TATAATGC requires its sequence context to be FOXM1c responsive, the P2 TATA box TATAAAAG alone is sufficient to confer FOXM1c responsiveness to any minimal promoter. FOXM1c transactivates by binding to the TATA box as well as directly to TATA-binding protein, transcription factor IIB and transcription factor IIA. This new transactivation mechanism is clearly distinguished from the function of FOXM1c as a conventional transcription factor. The central domain of FOXM1c functions as an essential domain for activation via the TATA box, but as an inhibitory domain (retinoblastoma protein-independent transrepression domain and retinoblastoma protein-recruiting negative regulatory domain) for transactivation via conventional FOXM1c-binding sites. Each promoter with the P2 TATA box TATAAAAG is postulated to be transactivated by FOXM1c. This was demonstrated for the promoters of c-fos, hsp70 and histone H2B/a. A database search revealed almost 300 probable FOXM1c target genes, many of which function in proliferation and tumorigenesis. Accordingly, dominant-negative FOXM1c proteins reduced cell growth approximately threefold, demonstrating a proliferation-stimulating function for wild-type FOXM1c.

FOXM1c is activated by cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1, but repressed by GSK-3alpha.

Two different inhibitory domains, N-terminus and central domain, keep FOXM1c almost inactive despite its strong transactivation domain. Here, we demonstrate that cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1 activate FOXM1c. Cyclin E/Cdk2 does not target its transactivation domain or its DNA-binding domain. Instead, its activating effect strictly depends on the presence of either the central domain or the N-terminus of FOXM1c and thus is completely lost if both inhibitory domains are deleted. Cyclin E/Cdk2 activates FOXM1c by releasing its transactivation domain from the repression by these two inhibitory domains. However, it does not directly increase the transactivation potential of the TAD. The DNA-binding is not affected by cyclin E/Cdk2, neither directly nor indirectly. These two activating effects of cyclin E/Cdk2 via central domain and N-terminus are additive. Cyclin A/Cdk2 and cyclin A/Cdk1 show similar characteristics. GSK-3alpha, another proliferation-associated kinase, represses FOXM1c.

Transcription factor FOXM1c is repressed by RB and activated by cyclin D1/Cdk4.

The proliferation-stimulating transactivator FOXM1c (MPP2) is repressed by RB and activated by cyclin D1/Cdk4 and therefore behaves like E2F. Despite its strong transactivation domain, FOXM1c is kept almost inactive by two different inhibitory domains, the N-terminus and the central domain. The tumor suppressor RB binds directly to the central domain of FOXM1c and thereby indirectly represses the transactivation domain, so that the central domain of FOXM1c functions as an RB-recruiting negative-regulatory domain. Cyclin D1/Cdk4 releases FOXM1c from this repression by RB and from the repression by its own inhibitory N-terminus, thereby strongly activating FOXM1c. However, cyclin D1/Cdk4 does not directly affect the transactivation domain or the DNA-binding domain. By phosphorylation of RB, but not FOXM1c, cyclin D1/Cdk4 interrupts their direct interaction and thus abrogates the repression of FOXM1c by RB. Cyclin D1/Cdk4 also eliminates the inhibition of the transactivation domain by the N-terminus of FOXM1c, probably by interruption of their direct interaction. Consequently, the G1-phase proliferation signal cyclin D1/Cdk4 converts FOXM1c from an almost inactive form into a strong transactivator in G1-phase, i.e., just at the time point at which the transcriptional activity of FOXM1 is required for stimulation of the G1/S-transition.

Despite its strong transactivation domain, transcription factor FOXM1c is kept almost inactive by two different inhibitory domains.

FOXM1c (MPP2) is an activating transcription factor with several nuclear localization signals, a forkhead domain for DNA binding, and a very strong acidic transactivation domain. Despite its very strong transactivation domain, FOXM1c is kept almost inactive by two different independent inhibitory domains, the N-terminus and the central domain. The N-terminus as a specific negative-regulatory domain directly binds to and thus inhibits the transactivation domain completely. However, it lacks any transrepression potential. In contrast, the central domain functions as a strong RB-independent transrepression domain and as an RB-recruiting negative-regulatory domain. The N-terminus alone is sufficient to eliminate transactivation, while the central domain alone represses the transactivation domain only partially. This hierarchy of the two inhibitory domains offers the possibility to activate the almost inactive wild type in two steps in vitro: deletion of the N-terminus results in a strong transactivator, while additional deletion of the central domain in a very strong transactivator. We suggest that the very high potential of the transactivation domain has to be tightly controlled by these two inhibitory domains because FOXM1 stimulates proliferation by promoting G1/S transition, as well as G2/M transition, and because deregulation of such potent activators of proliferation can result in tumorigenesis.