This study demonstrates that FoxM1, an essential transcription factor that controls the expression of many G2/M target genes, is downregulated by p53. Although ectopic p53 expression results in a reduction of FoxM1 mRNA levels, DNA damage cooperates with p53 (perhaps through modification of p53) to more potently repress FoxM1 mRNA. Nonetheless, DNA damage in the relative absence of p53 (shp53) gives rise to an increase in both FoxM1 mRNA and protein over basal levels, supporting previous work that identifies DNA damage as a positive regulator of FoxM1 protein stability (Tan et al., 2007
). Thus, DNA damage seems to cause the activation of multiple signaling pathways that converge to fine-tune FoxM1 levels.
As has been found for multiple targets of p53-mediated repression (Kannan et al., 2001
; Lohr et al., 2003
; Shats et al., 2004
), our data in MCF7, HepG2 and H1299 cells establish FoxM1 as an indirect repression target in which downregulation depends on p21. The Rb family (Rb, p130, p107) operates downstream of p21 and has been implicated in p53-mediated repression (Gottifredi et al., 2001
; Taylor et al., 2001
; Shats et al., 2004
; Jackson et al., 2005
). Similar to some FoxM1 target genes including Plk1 (Jackson et al., 2005
), our results indicate that the Rb family has a role in FoxM1 repression. Although it is unknown whether E2F1 directly activates FoxM1, two putative E2F1 sites have previously been identified in the FoxM1 promoter (Laoukili et al., 2007
). The finding that E2F1 contributes to FoxM1 expression further implicates Rb’s involvement in FoxM1 repression.
An intriguing possibility is that p21 may also function to inhibit other transcription factors that are responsible for FoxM1 activation. The FoxM1 promoter contains multiple putative transcription factor binding sites, and has been shown to be downstream of both Gli1 (Teh et al., 2002
) and c-Myc (Fernandez et al., 2003
; Blanco-Bose et al., 2008
) transcription factors. Interestingly, p21 is known to inhibit c-Myc-dependent transcription through direct interaction that disrupts the c-Myc–Max complex (Kitaura et al., 2000
). In addition to putative E2F1-binding sites, the FoxM1 promoter contains a B-Myb binding site and the cis
-regulatory module CHR-NF-Y (Linhart et al., 2005
). p21 could again participate here by impairing NF-Y function through inhibition of cyclin-dependent kinase 2 (Yun et al., 1999
). Furthermore, the presence of CDE/CHR elements in a gene’s promoter often correlates with an indirect repression by p53 (Badie et al., 2000
; St Clair et al., 2004
). In addition, our data reveal that a portion of FoxM1 repression can be considered p21 independent in both nutlin-treated HCT116 cells and after ectopic p53 expression in MCF7-24 cells. Although the mechanism is unexplored, p53-dependent inhibition of the factors listed above could effectuate this repression. In fact, a well-characterized interaction between p53 and NF-Y is known to directly inhibit NF-Y-dependent transcription (Imbriano et al., 2005
). Alternatively, the induction of microRNAs by p53 (reviewed in He et al. (2007)
and Vousden and Prives (2009)
) could lead to the observed p21-independent FoxM1 repression.
We observe that the basal levels of p53 contribute to FoxM1 regulation in HepG2 cells. This reflects the findings of a study in which p53 represses expression of the cell-surface molecule CD44 under basal conditions (Godar et al., 2008
), allowing cells to respond to apoptotic signals that would otherwise be blocked by CD44. Similarly, survivin (a FoxM1 target gene), has been shown to be regulated by the basal levels of p53 and Rb (Raj et al., 2008
). As FoxM1 is implicated in negative regulation of the cell cycle inhibitors, p21 and p27 (Wang et al., 2005
; Chan et al., 2008
; Xia et al., 2008
; Penzo et al., 2009
), repression of FoxM1 might be necessary for full p53-dependent responses to stress/treatments.
Furthermore, as loss of p53 and Rb are frequent occurrences in tumors, their absence may contribute to FoxM1 misregulation and thereby adversely affect the ability to inhibit cellular proliferation. In fact, while this manuscript was being prepared, one report showed that p21 (which we have shown to be an important mediator of p53-dependent FoxM1 repression) is required for proper FoxM1 suppression during dextrose-mediated inhibition of liver regeneration after partial hepatectomy (Weymann et al., 2009
). This finding confirms the importance of p21 in FoxM1 regulation and also highlights a biological context in which precise regulation of FoxM1 levels is crucial to inhibition of proliferation. As such, it is likely that the inability to downregulate FoxM1 after loss of the tumor suppressor p53 will contribute greatly to biological processes such as carcinogenesis.
Although DNA damage facilitated FoxM1 repression in MCF7 and HepG2 cells, two other cell lines (U2OS and HCT116) were surprisingly unable to effectively repress FoxM1 mRNA and stabilize FoxM1 protein levels after daunorubicin treatment. This finding is reminiscent of a study in which doxorubicin caused p53/Rb-dependent downregulation of human telomerase reverse transcriptase in MCF7 but not in HCT116 cells (Shats et al., 2004
). However, we show that nutlin-3 does cause notable downregulation of FoxM1 mRNA and protein in both HCT116 and U2OS cells. Nutlin-3 activates p53 in the absence of DNA-damage signaling by disrupting the interaction between p53 and MDM2 (its major negative regulator). As disruption of the p53– MDM2 complex is thought to be complete after nutlin treatment, it is possible that residual MDM2–p53 complexes remaining after ectopic p53 expression or after DNA damage curb the ability of p53 to down-regulate FoxM1. Alternatively, these particular cell lines may require high p21 levels observed only after nutlin treatment () to repress FoxM1 (and possibly other indirect targets such as human telomerase reverse transcriptase).
As FoxM1 is often overexpressed in cancers, the biological outcome of p53-mediated repression of FoxM1 was an important goal of this study. In MCF7 cells, siRNA to FoxM1 causes a p53-independent G2 arrest. This underscores the importance of FoxM1 and supports a rich literature that depicts FoxM1 as a pro-proliferative transcription factor. As p53 is a major regulator of the cell cycle, it was of great interest to determine the contribution of FoxM1 repression to decisions about cell fate—namely, how does FoxM1 repression affect the cell cycle?
Although only cells that retain wild-type p53 repress FoxM1, this repression cannot be a requirement for initial G2 arrest, as both MCF7 wild-type and shp53-expressing cells arrest in G2 after DNA damage (). However, p53 is thought to be more important for maintenance of G2 arrest rather than its initiation (Taylor and Stark, 2001
), as cells without p53 aberrantly enter mitosis with damaged DNA (an event that could lead to chromosomal aberrations, anueploidy and potentially give rise to pro-tumorigenic cells). The Rb family members have also been shown to be important for a stable G2 arrest and cell cycle exit from G2 (Jackson et al., 2005
). On the contrary, FoxM1 has been shown to be important for entry into mitosis in both mouse cells and human osteosarcoma cells (U2OS cells) (Wang et al., 2005
The finding that FoxM1 siRNA rescues the aberrant mitotic entry ofMCF7 shp53-expressing cells reveals the role of p53-mediated repression of FoxM1. That is, for conditions in which p53-mediated repression is robust, the downregulation of a single target, FoxM1, could facilitate stable G2 arrest. Although it is likely that p53 uses several pathways to maintain a stable G2 arrest, the repression of FoxM1 provides an elegant way for p53 to achieve global cellular changes. Notably, many reported targets of p53-mediated repression (Jackson et al., 2005
; Spurgers et al., 2006
) overlap with the set of genes induced by FoxM1 (Laoukili et al., 2005
; Wang et al., 2005
; Fu et al., 2008
). As FoxM1 is a master regulator of factors that facilitate the G2/M transition and regulate mitotic events, p53-mediated repression of FoxM1 may cause indirect negative regulation of such factors, ultimately leading to stable arrest and maintenance of genomic integrity.