Research Article Loss of the Forkhead Transcription Factor FoxM1 Causes Centrosome Amplification and Mitotic Catastrophe Diane R. Wonsey and Maximillian T. Follettie Department of Discovery Medicine, Wyeth Research, Cambridge, Massachusetts Abstract with a targeted deletion of FoxM1 in the liver show decreased Expression of the forkhead transcription factor FoxM1 bromodeoxyuridine (BrdUrd) incorporation and fewer mitotic cells correlates with proliferative status in a variety of normal compared with wild-type controls following partial hepatectomy (6). and transformed cell types. Elevated expression of FoxM1 has Conversely, premature expression of FoxM1 in transgenic mice been noted in both hepatocellular carcinoma and basal cell accelerates hepatocyte DNA replication and the expression of cell carcinoma. However, whether FoxM1 expression is essential cycle regulatory proteins following partial hepatectomy (7). for the viability of transformed cells is unknown. We report Consistent with a role in proliferation, elevated expression of here that the expression of FoxM1 is significantly elevated in FoxM1 has been reported in both basal cell carcinoma (8) and in primary breast cancer. Microarray analysis shows that FoxM1 hepatocellular carcinoma (9). In addition, FoxM1 expression is re- regulates genes that are essential for faithful chromosome quired for the proliferative expansion of hepatocellular carcinoma segregation and mitosis, including Nek2, KIF20A, and CENP-A. in a mouse model of tumor induction (10). The observation that a p19ARF peptide fragment physically interacts with FoxM1, sup- Loss of FoxM1 expression generates mitotic spindle defects, delays cells in mitosis, and induces mitotic catastrophe. Time- presses FoxM1 transcriptional activity, and inhibits FoxM1- lapse microscopy indicates that depletion of FoxM1 generates enhanced anchorage-independent growth (10) suggests that cells that enter mitosis but are unable to complete cell FoxM1 may be an attractive target for cancer therapy. However, division, resulting in either mitotic catastrophe or endoredu- it has not been determined whether FoxM1 is essential for viability plication. These findings indicate that FoxM1 depletion causes in established cancer cells or whether specifically inhibiting cell death due to mitotic catastrophe and that inhibiting FoxM1 is an effective therapeutic strategy. FoxM1 represents a therapeutic strategy to target breast Centrosomes are the primary components of the microtubule organizing center in mammalian cells. Immediately after mitosis, cancer. (Cancer Res 2005; 65(12): 5181-9) each daughter cell contains a single centrosome. In most cell types, centrosome duplication begins in S phase, and the two daughter Introduction centrosomes form the poles of the mitotic spindle. The regulation The forkhead box (FOX) protein family consists of f50 of centrosome duplication in mammalian cells is tightly controlled proteins that are characterized by a conserved 100 amino acid in order to maintain genomic integrity and prevent aneuploidy winged-helix DNA binding domain. FoxM1 was originally (11). In addition to directing the formation of the mitotic spindle, identified in a screen designed to clone cDNAs encoding proteins recent evidence indicates that the centrosome participates in cell recognized by the MPM2 antibody, which recognizes an amino cycle regulation (12, 13), and this activity is independent of acid epitope found on M phase phosphoproteins (1). A partial microtubule nucleating activity. Cancer cells frequently contain cDNA sequence corresponding to FoxM1 was identified, indicating elevated numbers of centrosomes (14, 15), although whether that FoxM1 is phosphorylated in mitosis and is involved in the centrosome amplification contributes to transformation or is a G2-M phase of the cell cycle. Detailed cell cycle analysis revealed consequence of cancer progression remains to be determined. that expression of the FoxM1 protein increases during G1 and S Because centrosomes usually nucleate microtubules, cells with phase, reaching maximal levels in G2-M (2). Evidence suggesting supernumerary centrosomes form multipolar mitotic spindles and that FoxM1 is required for coupling DNA replication with mitosis may undergo mitotic catastrophe (16). is provided by the observation that hepatocytes and cardiomyo- Mitotic catastrophe is defined as a form of cell death that À/À cytes from FoxM1 mice are polyploid. Mice lacking FoxM1 die occurs during mitosis, often arising from aberrant G2 checkpoint in the perinatal period, with noticeable defects in the myocar- control (17). In normal cells, the G2 checkpoint is activated by dium, including irregular cellular orientation and enlarged nuclei DNA damage and involves a complex network of genes involved with up to a 50-fold increase in DNA content (3). Although FoxM1 in cell cycle arrest, DNA repair, and apoptosis (18). Failure of the is widely expressed in embryonic tissues (4), the defects in G2 checkpoint allows premature progression through mitosis, FoxM1À/À mice are restricted to tissues that normally become resulting in mitotic catastrophe. Although the molecular details polyploid in adult mice. Terminally differentiated, nonproliferating of mitotic catastrophe remain to be defined, several genes tissues display relatively low levels of FoxM1 expression (5). Several involved in the G2 checkpoint induce mitotic catastrophe when studies indicate that FoxM1 participates in cell cycle control. Mice disrupted, including 14-3-3r (19), ATR (20), and the CHK1 kinase (21). In addition, defects in proteins required for mitotic spin- dle assembly also induce catastrophe. Depletion of hNuf2, a Note: Supplementary data for this article are available at Cancer Research Online kinetochore protein involved in microtubule attachment, arrests (http://cancerres.aacrjournals.org/). cells in prometaphase and induces mitotic cell death (22). Requests for reprints: Diane R. Wonsey, Dana Farber Cancer Institute, 44 Binney Because mitotic catastrophe, by definition, is only induced in Street, Mayer 649, Boston, MA 02115. Phone: 617-582-7944; E-mail: Diane_Wonsey@ dfci.harvard.edu. proliferating cells, and also occurs following DNA damage in cells I2005 American Association for Cancer Research. with mutations in checkpoint proteins, induction of catastrophe www.aacrjournals.org 5181 Cancer Res 2005; 65: (12). June 15, 2005 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2005 American Association for Cancer Research. Cancer Research presents a promising opportunity for specifically targeting can- transfections, cells were plated at f50% confluence and transfected with cer cells. 200 nmol/L oligo using siPORT (Ambion, Austin, TX) for 5 hours according Analysis of microarray data from primary breast cancers to the manufacturer’s instructions. Stable expression of the FoxM1 short revealed that FoxM1 expression is increased in infiltrating ductal hairpin RNA (shRNA) was achieved using the pSilencer 3.1 H1 puro vector (Ambion), with a FoxM1 hairpin sequence based on the FoxM1 siRNA carcinoma, the most commonly diagnosed form of breast cancer. indicated above, according to the manufacturer’s instructions for hairpin In order to evaluate whether FoxM1 expression is essential for design. The negative control vector is pSilencer 3.1 H1 puro, containing a cancer cell growth or is secondary to an increase in the number of 66-bp hairpin with limited homology to known sequences in the human proliferating cells, we used short interfering RNA (siRNA) to genome, supplied by the manufacturer (Ambion). Cells were transfected evaluate the effect of directly inhibiting FoxM1 in breast cancer cell with Effectene transfection reagent (Qiagen, Valencia, CA) and selected for 6 lines. Microarray data from cells treated with FoxM1 siRNA days in the presence of 0.5 Ag/mL puromycin. identified several previously unreported genes that are regulated Cell proliferation assays. The cell proliferation reagent WST-1 (Roche, by FoxM1, including CENP-A, Nek2, and KIF20A. Using a stable Indianapolis, IN) was used to assay cell number. Cells were trypsinized FoxM1 hairpin construct, we found that continuous depletion 24 hours after transfection and plated in 96-well plates. Absorbance was of FoxM1 results in mitotic catastrophe. Our results indicate that assayed 30 minutes after adding WST-1 according to the manufacturer’s instructions. FoxM1 is required for cancer cell viability and show that loss of Immunoblot analysis. Culture cells were lysed in buffer containing FoxM1 causes aberrant spindle formation, ultimately leading 10 mmol/L Tris (pH 7.4), 1% SDS, 1 mmol/L sodium orthovanadate, and to mitotic catastrophe. complete protease inhibitors (Roche). Protein concentrations were analyzed using the bicinchoninic acid assay (Pierce, Rockford, IL), and equivalent Materials and Methods amounts of protein were loaded on 12% SDS-PAGE gels. Proteins were transferred to Optitran nitrocellulose (Schleicher & Schuell, Keene, NH) Primary tissue expression profiling data. Expression profiling data and blotted with antibodies against FoxM1 (MPP2 K-19, Santa Cruz, Santa from primary human cancer samples was obtained from the GeneLogic Cruz, CA) or h-actin (A-5441, Sigma, St. Louis, MO). BioExpress Database (Gaithersburg, MD), a commercially available repos- Microarray analysis. BT-20 cells were transfected in triplicate with itory of transcriptional profiling data from thousands of diseased and a mock transfection (no siRNA), GFP siRNA, or