FoxM1 regulates transcription of JNK1 to promote the G1/S transition and tumor cell invasiveness

Abstract

The Forkhead box M1 (FoxM1) protein is a proliferation-specific transcription factor that plays a key role in controlling both the G(1)/S and G(2)/M transitions through the cell cycle and is essential for the development of various cancers. We show here that FoxM1 directly activates the transcription of the c-Jun N-terminal kinase (JNK1) gene in U2OS osteosarcoma cells. Expression of JNK1, which regulates the expression of genes important for the G(1)/S transition, rescues the G(1)/S but not the G(2)/M cell cycle block in FoxM1-deficient cells. Knockdown of either FoxM1 or JNK1 inhibits tumor cell migration, invasion, and anchorage-independent growth. However, expression of JNK1 in FoxM1-depleted cells does not rescue these defects, indicating that JNK1 is a necessary but insufficient downstream mediator of FoxM1 in these processes. Consistent with this interpretation, FoxM1 regulates the expression of the matrix metalloproteinases MMP-2 and MMP-9, which play a role in tumor cell invasion, through JNK1-independent and -dependent mechanisms in U2OS cells, respectively. Taken together, these findings identify JNK1 as a critical transcriptional target of FoxM1 that contributes to FoxM1-regulated cell cycle progression, tumor cell migration, invasiveness, and anchorage-independent growth.

FIGURE 1.

FoxM1 and JNK1 expression in response to serum stimulation. A, MEFs immortalized by dominant negative p53 were serum-starved for 72 h and restimulated by the addition of FBS to 10%. FoxM1 and JNK1 mRNA levels were detected by qRT-PCR. B, diminished levels of endogenous JNK1, ATF-2, and FoxM1 mRNAs were found in U2OS cells transfected with FoxM1 siRNA as determined by qRT-PCR. No significant change in JNK2 mRNA was detected. C, U2OS cells transfected with siFoxM1 exhibited diminished JNK1, ATF-2, cyclin A2 protein levels, and reduced phosphorylation of c-Jun and ATF2 as determined by immunoblotting. D, RNA preparations and total cell extracts from early passage of FoxM1^+/+, FoxM1^+/–, and FoxM1^–/– MEFs were used for qRT-PCR (upper) and immunoblot (lower) analysis. FoxM1^–/– MEFs expressed reduced levels of JNK1 mRNA and protein compared with WT MEFs. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 2.

FoxM1 transcriptionally activates JNK1. A, EMSA was performed using U2OS cell nuclear lysate to bind ³²P-labeled double-stranded oligonucleotide probe made to human JNK1 promoter (–1275/–1248 bp) (lane 2). For DNA competitions, 100-fold molar excess of cold double-stranded oligonucleotide probe was added to the binding reaction (lane 3). Radioactivity-labeled probe only was used as negative control (lane 1). B, U2OS cells were transfected with the CMV-FoxM1b expression vector, a reporter construct with the –1308 bp human JNK1 promoter linked to firefly luciferase, and CMV-Renilla luciferase as the internal control. Cells were cotransfected with siFoxM1 where indicated. Cell extracts were prepared 24 h after transfection; dual luciferase enzyme activity was measured and expressed as fold induction of transcription. C, binding of FoxM1 to JNK1 and ATF2 promoters as determined by quantitative promoter ChIP assays. Untreated or FoxM1-depleted U2OS cells were cross-linked and sonicated, and chromatin fragments were immunoprecipitated with antibodies specific to FoxM1, CBP, or RNA polymerase II. The promoter DNA associated with the IP chromatin was quantified by qRT-PCR with primers for the human JNK1 (–1770/–1670) or ATF-2 (–4340/–4151) promoter, and normalized to the amount of DNA bound to FoxM1 in untreated cells. Error bars represent ± S.D. from experiments done in triplicate. *, p < 0.05, and ***, p < 0.001.

FIGURE 3.

FoxM1 depletion inhibits cell cycle reentry and progression in response to serum stimulation. A, U2OS cells were untreated or transfected with siFoxM1 and maintained in 0.1% FBS for 48 h. Cells were then stimulated with 10% FBS and harvested at various times (0–24 h) thereafter for flow cytometry analysis. Percentages of cells in the G₁ phase were counted. B, cells were treated as in A, and percentages of cells in S phase were quantified. C, U2OS cells were either untreated, transfected with siFoxM1 or siJNK1, or treated with SP600125 (10 μm) and cultured in 10% FBS. Numbers of viable cells at indicated days were counted. D, cells were either untreated, transfected with siFoxM1, cotransfected with siFoxM1 and pHA-JNK1, or treated with SP600125. Percentages of cells in G₁, S, and G₂/M phases were analyzed by flow cytometry 72 h after transfection or 24 h after JNK inhibitor treatment. E, cells were treated as in D and serum-starved for 48 h, followed by the addition of 10% FBS. Cells were pulse-labeled with BrdU for 1 h following 16 h of serum stimulation, and the percentages of cells with BrdU-positive staining were quantified. F, Western blot analysis demonstrated diminished JNK1 protein level in siJNK1-transfected U2OS cells for 72 h. Error bars represent ± S.D. from experiments done in triplicate. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

FIGURE 4.

FoxM1- or JNK1-depletion inhibits anchorage-independent growth in soft agar. U2OS cells were either left untreated (A), or transfected with siFoxM1 (B), siFoxM1 with pHA-JNK1 (C), siJNK1 (D), sip27 (F), or treated with SP600125 (E) as indicated. One day after treatment, cells were trypsinized and replated on soft agar. Microphotographs showed colonies grown in soft agar for 2 weeks. G, numbers of colonies per microscopic field were counted; error bars represent ± S.D. from experiments done in triplicate. H, total cell lysates harvested in cells treated in A–F as indicated were immunoblotted to determine the levels of expression of FoxM1, JNK1, HA-JNK1, and α-tubulin. ***, p < 0.001.

FIGURE 5.

FoxM1- or JNK1-depletion compromises in vitro wound closure. U2OS cells were plated and grown to confluence before scratch wounds were inflicted. A–F, phase contrast micrographs of scratch wounds at the time of wounding (t = 0 h) and 5-h postwounding (t = 5 h) are shown. G, wound closure rates were calculated and presented in the bar graph. Error bars represent ± S.D. from experiments done in triplicate. H, total cell lysates harvested in cells treated in A–F as indicated were immunoblotted to determine the levels of expression of FoxM1, JNK1, HA-JNK1, and α-tubulin. **, p < 0.01 and ***, p < 0.001.

FIGURE 6.

Impaired invasion in cells depleted of FoxM1 or JNK1. A–F, cells were treated as above, and their migration through Matrigel invasion chambers toward 5% FBS was monitored. Migrated cells were stained with Giemsa and photographed. G, numbers of migrated cells were counted in five fields per well. The error bar represent results ± S.D. in experiments done in triplicate. H, total cell lysates harvested in cells treated in A–F as indicated were immunoblotted to determine the levels of expression of FoxM1, JNK1, HA-JNK1, and α-tubulin. ***, p < 0.001.

FIGURE 7.

Diminished expression of MMP-9 and MMP-2 in FoxM1-deficient U2OS cells. A, expression of MMP-9 in U2OS cells transfected with siFoxM1 or treated with SP600125 was compared with that in untreated control cells as determined by qTR-PCR. Re-expression of JNK1 in FoxM1-deficient U2OS cells significantly increased MMP-9 expression. B, expression of MMP-2 cells treated as above was determined by qRT-PCR and calculated as a percentage relative to untreated cells. Error bars represent ± S.D. in triplicate experiments. C, total cell lysates harvested in cells treated in A and B as indicated were immunoblotted to determine the levels of expression of FoxM1, JNK1, HA-JNK1, and α-tubulin. **, p ≤ 0.01 and ***, p ≤ 0.001.

FIGURE 8.

Model for the roles of FoxM1 and JNK1 in cell proliferation, migration, and invasion. FoxM1 transcriptionally activates JNK1, which regulates the expression of a set of genes critical for G₁/S progression, including cyclin A. JNK1 also mediates the regulation of MMP-9 by FoxM1, thus contributing to cell migration and invasion. FoxM1 directly regulates MMP2 expression (59) to enhance cell migration and invasion.