FOXM1 is a transcription factor required for a wide spectrum of essential biological functions, including DNA damage repair, cell proliferation, cell cycle progression, cell renewal, cell differentiation and tissue homeostasis. Recent evidence suggests that FOXM1 also has a role in many aspects of the DNA damage response. Accordingly, FOXM1 drives the transcription of genes for DNA damage sensors, mediators, signal transducers and effectors. As a result of these functions, it plays an integral part in maintaining the integrity of the genome and so is key to the propagation of accurate genetic information to the next generation. Preserving the genetic code is a vital means of suppressing cancer and other genetic diseases. Conversely, FOXM1 is also a potent oncogenic factor that is essential for cancer initiation, progression and drug resistance. An enhanced FOXM1 DNA damage repair gene expression network can confer resistance to genotoxic agents. Developing a thorough understanding of the regulation and function of FOXM1 in DNA damage response will improve the diagnosis and treatment of diseases including cancer, neurodegenerative conditions and immunodeficiency disorders. It will also benefit cancer patients with acquired genotoxic agent resistance.

FOXM1 is implicated in genotoxic drug resistance but its mechanism of action remains elusive. We show here that FOXM1-depletion can sensitize breast cancer cells and MEFs into entering epirubicin-induced senescence, with the loss of long-term cell proliferation ability, the accumulation of γH2AX foci, and the induction of senescence-associated β-galactosidase activity and cell morphology. Conversely, reconstitution of FOXM1 in FOXM1-deficient MEFs alleviates the accumulation of senescence-associated γH2AX foci. We also demonstrate that FOXM1 regulates NBS1 at the transcriptional level through an FHRE on its promoter. Like FOXM1, NBS1 is overexpressed in the epirubicin-resistant MCF-7EpiR cells and its expression level is low but inducible by epirubicin in MCF-7 cells. Consistently, overexpression of FOXM1 augmented and FOXM1 depletion reduced NBS1 expression and epirubicin-induced ATM phosphorylation in breast cancer cells. Together these findings suggest that FOXM1 increases NBS1 expression and ATM phosphorylation, possibly through increasing the levels of the MRN(MRE11/RAD50/NBS1) complex. Consistent with this idea, the loss of P-ATM induction by epirubicin in the NBS1-deficient NBS1-LBI fibroblasts can be rescued by NBS1 reconstitution. Resembling FOXM1, NBS1 depletion also rendered MCF-7 and MCF-7EpiR cells more sensitive to epirubicin-induced cellular senescence. In agreement, the DNA repair-defective and senescence phenotypes in FOXM1-deficent cells can be effectively rescued by overexpression of NBS1. Moreover, overexpression of NBS1 and FOXM1 similarly enhanced and their depletion downregulated HR DNA repair activity. Crucially, overexpression of FOXM1 failed to augment HR activity in the background of NBS1 depletion, demonstrating that NBS1 is indispensable for the HR function of FOXM1. The physiological relevance of the regulation of NBS1 expression by FOXM1 is further underscored by the strong and significant correlation between nuclear FOXM1 and total NBS1 expression in breast cancer patient samples, further suggesting that NBS1 as a key FOXM1 target gene involved in DNA damage response, genotoxic drug resistance and DNA damage-induced senescence.

No abstract

The forkhead box transcription factor FOXM1 is an essential effector of G2/M-phase transition, mitosis and the DNA damage response. As such, it is frequently deregulated during tumorigenesis. Here we report that FOXM1 is dynamically modified by SUMO1 but not by SUMO2/3 at multiple sites. We show that FOXM1 SUMOylation is enhanced in MCF-7 breast cancer cells in response to treatment with epirubicin and mitotic inhibitors. Mutation of five consensus conjugation motifs yielded a SUMOylation-deficient mutant FOXM1. Conversely, fusion of the E2 ligase Ubc9 to FOXM1 generated an auto-SUMOylating mutant (FOXM1-Ubc9). Analysis of wild-type FOXM1 and mutants revealed that SUMOylation inhibits FOXM1 activity, promotes translocation to the cytoplasm and enhances APC/Cdh1-mediated ubiquitination and degradation. Further, expression of the SUMOylation-deficient mutant enhanced cell proliferation compared with wild-type FOXM1, whereas the FOXM1-Ubc9 fusion protein resulted in persistent cyclin B1 expression and slowed the time from mitotic entry to exit. In summary, our findings suggest that SUMOylation attenuates FOXM1 activity and causes mitotic delay in cytotoxic drug response.

The forkhead transcription factor FOXM1 has a key role in DNA damage response, and its deregulated overexpression is associated with genotoxic drug resistance in breast cancer. However, little is known about the posttranslational mechanisms by which FOXM1 expression is regulated by genotoxic agents and how they are deregulated in resistant cells. Initial co-immunoprecipitation studies verified previous proteomic analysis finding that the OTUB1 is a novel FOXM1-interacting protein. Western blot analysis showed that both OTUB1 and FOXM1 expression reduced upon genotoxic agent treatment in MCF-7 cells, but remained relatively constant in resistant cells. FOXM1 expression reduced upon OTUB1 depletion by siRNA and increased with OTUB1 overexpression in MCF-7 cells, arguing that OTUB1 positively regulates FOXM1 expression. In agreement, co-immunoprecipitation experiments demonstrated that FOXM1 expression is associated with OTUB1 binding but inversely correlates with conjugation to the protein degradation-associated Lys-48-linked ubiquitin-chains. Overexpression of wild-type (WT) OTUB1, but not the OTUB1(C91S) mutant, disrupted the formation of Lys48-linked ubiquitin-conjugates on FOXM1. Importantly, knockdown of OTUB1 by siRNA resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide, whereas overexpression of WT OTUB1, but not the OTUB1(C91S) mutant, significantly enhances the half-life of FOXM1. In addition, proliferative and clonogenic assays also show that OTUB1 can enhance the proliferative rate and epirubicin resistance through targeting FOXM1, as OTUB1 has little effect on FOXM1-deficient cells. The physiological relevance of the regulation of FOXM1 by OTUB1 is further underscored by the significant correlations between FOXM1 and OTUB1 expression in breast cancer patient samples. Cox-regression survival analysis indicates that OTUB1 overexpression is linked to poorer outcome in particular in patients treated with chemotherapy. Collectively, these data suggest that OTUB1 limits the ubiquitination and degradation of FOXM1 in breast cancer and has a key role in genotoxic agent resistance.

No abstract

Substantial evidence suggests that breast cancer initiation, recurrence and drug resistance is supported by breast cancer stem cells (BCSCs). Recently, we reported a novel role of Aurora kinase A (AURKA) in BCSCs, as a transactivating co-factor in the induction of the c-Myc oncoprotein. However, the mode of action and transcriptional network of nuclear AURKA in BCSCs remain unknown. Here, we report that nuclear AURKA can be recruited by Forkhead box subclass M1 (FOXM1) as a co-factor to transactivate FOXM1 target genes in a kinase-independent manner. In addition, we show that AURKA and FOXM1 participate in a tightly coupled positive feedback loop to enhance BCSC phenotype. Indeed, kinase-dead AURKA can effectively transactivate the FOXM1 promoter through a Forkhead response element, whereas FOXM1 can activate AURKA expression at the transcriptional level in a similar manner. Consistently, breast cancer patient samples portrayed a strong and significant correlation between the expression levels of FOXM1 and AURKA. Moreover, both FOXM1 and AURKA were essential for maintaining the BCSC population. Finally, we demonstrated that the AURKA inhibitor AKI603 and FOXM1 inhibitor thiostrepton acted synergistically to inhibit cytoplasmic AURKA activity and disrupt the nuclear AURKA/FOXM1-positive feedback loop, respectively, resulting in a more effective inhibition of the tumorigenicity and self-renewal ability of BCSCs. Collectively, our study uncovers a previously unknown tightly coupled positive feedback signalling loop between AURKA and FOXM1, crucial for BCSC self-renewal. Remarkably, our data reveal a novel potential therapeutic strategy for targeting both the cytoplasmic and nuclear AURKA function to effectively eliminate BCSCs, so as to overcome both breast cancer and drug resistance.

FOXO3a and FOXM1 are two forkhead transcription factors with antagonistic roles in cancer and DNA damage response. FOXO3a functions like a typical tumour suppressor, whereas FOXM1 is a potent oncogene aberrantly overexpressed in genotoxic resistant cancers. FOXO3a not only represses FOXM1 expression but also its transcriptional output. Recent research has provided novel insights into a central role for FOXO3a and FOXM1 in DNA damage response. The FOXO3a-FOXM1 axis plays a pivotal role in DNA damage repair and the accompanied cellular response through regulating the expression of genes essential for DNA damage sensing, mediating, signalling and repair as well as for senescence, cell cycle and cell death control. In this manner, the FOXO3a-FOXM1 axis also holds the key to cell fate decision in response to genotoxic therapeutic agents and controls the equilibrium between DNA repair and cell termination by cell death or senescence. As a consequence, inhibition of FOXM1 or reactivation of FOXO3a in cancer cells could enhance the efficacy of DNA damaging cancer therapies by decreasing the rate of DNA repair and cell survival while increasing senescence and cell death. Conceptually, targeting FOXO3a and FOXM1 may represent a promising molecular therapeutic option for improving the efficacy and selectivity of DNA damage agents, particularly in genotoxic agent resistant cancer. In addition, FOXO3a, FOXM1 and their downstream transcriptional targets may also be reliable diagnostic biomarkers for predicting outcome, for selecting therapeutic options, and for monitoring treatments in DNA-damaging agent therapy.