Transcriptional induction of cell-cycle regulatory proteins ensures proper timing of subsequent cell-cycle events. Here we show that the Forkhead transcription factor FoxM1 regulates expression of many G2-specific genes and is essential for chromosome stability. Loss of FoxM1 leads to pleiotropic cell-cycle defects, including a delay in G2, chromosome mis-segregation and frequent failure of cytokinesis. We show that transcriptional activation of cyclin B by FoxM1 is essential for timely mitotic entry, whereas CENP-F, another direct target of FoxM1 identified here, is essential for precise functioning of the mitotic spindle checkpoint. Thus, our data uncover a transcriptional cluster regulated by FoxM1 that is essential for proper mitotic progression.

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The Forkhead transcription factor FoxM1 is an important regulator of gene expression during the G2 phase. Here, we show that FoxM1 transcriptional activity is kept low during G1/S through the action of its N-terminal autoinhibitory domain. We found that cyclin A/cdk complexes are required to phosphorylate and activate FoxM1 during G2 phase. Deletion of the N-terminal autoinhibitory region of FoxM1 generates a mutant of FoxM1 (ΔN-FoxM1) that is active throughout the cell cycle and no longer depends on cyclin A for its activation. Mutation of two cyclin A/cdk sites in the C-terminal transactivation domain leads to inactivation of full-length FoxM1 but does not affect the transcriptional activity of the ΔN-FoxM1 mutant. We show that the intramolecular interaction of the N- and C-terminal domains depends on two RXL/LXL motifs in the C terminus of FoxM1. Mutation of these domains leads to a similar gain of function as deletion of the N-terminal repressor domain. Based on these observations we propose a model in which FoxM1 is kept inactive during the G1/S transition through the action of the N-terminal autorepressor domain, while phosphorylation by cyclin A/cdk complexes during G2 results in relief of inhibition by the N terminus, allowing activation of FoxM1-mediated gene transcription.

Metastasis confronts clinicians with two major challenges: estimating the patient's risk of metastasis and identifying therapeutic targets. Because they are key signal integrators connecting cellular processes to clinical outcome, we aimed to identify transcriptional nodes regulating cancer cell metastasis. Using rodent xenograft models that we previously developed, we identified the transcription factor Fos-related antigen-1 (Fra-1) as a key coordinator of metastasis. Because Fra-1 often is overexpressed in human metastatic breast cancers and has been shown to control their invasive potential in vitro, we aimed to assess the implication and prognostic significance of the Fra-1-dependent genetic program in breast cancer metastasis and to identify potential Fra-1-dependent therapeutic targets. In several in vivo assays in mice, we demonstrate that stable RNAi depletion of Fra-1 from human breast cancer cells strongly suppresses their ability to metastasize. These results support a clinically important role for Fra-1 and the genetic program it controls. We show that a Fra-1-dependent gene-expression signature accurately predicts recurrence of breast cancer. Furthermore, a synthetic lethal drug screen revealed that antagonists of the adenosine receptor A 2B (ADORA2B) are preferentially toxic to breast tumor cells expressing Fra-1. Both RNAi silencing and pharmacologic blockade of ADORA2B inhibited filopodia formation and invasive activity of breast cancer cells and correspondingly reduced tumor outgrowth in the lungs. These data show that Fra-1 activity is causally involved in and is a prognostic indicator of breast cancer metastasis. They suggest that Fra-1 activity predicts responsiveness to inhibition of pharmacologically tractable targets, such as ADORA2B, which may be used for clinical interference of metastatic breast cancer.epithelial-mesenchymal transition | invasion T he path toward improved management of metastatic tumors, the major cause of death among cancer patients, involves tackling of two major challenges: the development of therapies that combat the patient's metastatic disease and of means of reliably assessing the individual patient's risk of developing metastasis. In line with the second objective, patient stratification has received increasing attention as a way to improve the therapeutic management of cancer patients. In recent years, for example, gene-expression profiling of primary breast cancer has uncovered gene signatures that assist clinicians in classifying breast cancer subtypes more accurately (1, 2) and that provide predictions of tumor recurrence and clinical outcome (3-5). Such classifiers have proven useful for formulating prognosis and adjusting therapeutic management of breast cancer patients, complementing conventional histopathological criteria such as tumor size, nodal status, and estrogen receptor (ER) status (6-8) to predict clinical outcome better.The most important challenge posed by metastatic cancer, blocking metastatic spread, has proven more troublesome. Indeed, most ...

SUMMARY Retinoids play key roles in differentiation, growth arrest and apoptosis and are increasingly used in the clinic for the treatment of a variety of cancers, including neuroblastoma. Using a large-scale RNA interference-based genetic screen we identify ZNF423 (also known as Ebfaz, OAZ or Zfp423) as a component critically required for retinoic acid (RA)-induced differentiation. ZNF423 associates with the RARα/RXRα nuclear receptor complex and is essential for transactivation in response to retinoids. Down-regulation of ZNF423 expression by RNA interference in neuroblastoma cells results in a growth advantage and resistance to RA-induced differentiation, whereas overexpression of ZNF423 leads to growth inhibition and enhanced differentiation. Finally, we show that low ZNF423 expression is associated with poor disease outcome of neuroblastoma patients. SIGNIFICANCE Cancer biomarkers make it possible to foretell cancer outcome (prognosis) or responses to therapy (prediction). Human neuroblastoma is the most common childhood solid tumor with a broad range of clinical outcomes, ranging from spontaneous regression to extremely aggressive disease. We show here that ZNF423 is a prognostic biomarker for human neuroblastoma independent of MYCN amplification. We also establish here a causal role of ZNF423 in RA-induced differentiation and proliferation of neuroblastoma cells. Therefore, ZNF423 may also predict responses to RA-based therapies in the clinic. More generally, our results underscore that the identification of novel components of key signaling pathways using genetic screens can yield biomarkers having clinical utility.

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The Forkhead transcription factor FoxM1 is required for the timely expression of many mitotic regulators, such as Cyclin B, Plk1, Aurora B and Cdc25B.(1-3) For this, FoxM1 is specifically activated in G(2) phase through Cyclin A/cdk2-dependent phosphorylation.(4-6) However, it is currently unclear how FoxM1 activity is removed as cells complete mitosis, and need to shut down expression of the mitotic regulators that are transcriptional targets of FoxM1. Here, we demonstrate that FoxM1 is actively degraded during exit from mitosis by the APC/C. We find that FoxM1 degradation requires Cdh1, a known co-factor for APC/C that is responsible for degradation of many mitotic regulators from anaphase until early G(1). FoxM1 binds to Cdh1, and FoxM1 degradation involves both D- and KEN-boxes present in the N-terminal part of FoxM1. Based on these data we propose that Cdh1-dependent degradation of FoxM1 is required to shut down transcriptional activation of mitotic regulators during exit from mitosis.