FOXM1 has been implicated in taxane resistance, but the molecular mechanism involved remains elusive. In here, we show that FOXM1 depletion can sensitize breast cancer cells and mouse embryonic fibroblasts into entering paclitaxel-induced senescence, with the loss of clonogenic ability, and the induction of senescence-associated β-galactosidase activity and flat cell morphology. We also demonstrate that FOXM1 regulates the expression of the microtubulin-associated kinesin KIF20A at the transcriptional level directly through a Forkhead response element (FHRE) in its promoter. Similar to FOXM1, KIF20A expression is downregulated by paclitaxel in the sensitive MCF-7 breast cancer cells and deregulated in the paclitaxel-resistant MCF-7TaxR cells. KIF20A depletion also renders MCF-7 and MCF-7TaxR cells more sensitive to paclitaxel-induced cellular senescence. Crucially, resembling paclitaxel treatment, silencing of FOXM1 and KIF20A similarly promotes abnormal mitotic spindle morphology and chromosome alignment, which have been shown to induce mitotic catastrophe-dependent senescence. The physiological relevance of the regulation of KIF20A by FOXM1 is further highlighted by the strong and significant correlations between FOXM1 and KIF20A expression in breast cancer patient samples. Statistical analysis reveals that both FOXM1 and KIF20A protein and mRNA expression significantly associates with poor survival, consistent with a role of FOXM1 and KIF20A in paclitaxel action and resistance. Collectively, our findings suggest that paclitaxel targets the FOXM1-KIF20A axis to drive abnormal mitotic spindle formation and mitotic catastrophe and that deregulated FOXM1 and KIF20A expression may confer paclitaxel resistance. These findings provide insights into the underlying mechanisms of paclitaxel resistance and have implications for the development of predictive biomarkers and novel chemotherapeutic strategies for paclitaxel 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.

In this study, we report the identification of a novel role of SIRT6 in both epirubicin and paclitaxel resistance in breast cancer. We found that SIRT6 protein levels are elevated in paclitaxel- and epirubicin-resistant MCF-7 cells compared with the parental sensitive cells. SIRT6 knockout and depletion sensitized cells to both paclitaxel and epirubicin treatment, whereas SIRT6 ectopic overexpression led to increased resistance to paclitaxel and epirubicin. Moreover, our data suggest that SIRT6 could be mediating epirubicin resistance through enhancing the DNA repair response to epirubicin-induced DNA damage. Clonogenic assays also revealed that mouse embryonic fibroblasts (MEFs) lacking SIRT6 have decreased long-term viability in response to epirubicin. The tumour suppressor FOXO3a increases its levels of acetylation in MEFs depleted of SIRT6, whereas its induction by epirubicin is attenuated in breast cancer cells overexpressing SIRT6. Further cell viability studies demonstrate that deletion of FOXO1/3/4 in MEFs can confer sensitivity to both paclitaxel and epirubicin, suggesting that SIRT6 reduces paclitaxel and epirubicin sensitivity, at least in part, through modulating FOXO acetylation and expression. Consistently, immunohistochemical analysis of 118 breast cancer patient samples revealed that high SIRT6 nuclear staining is significantly associated with poorer overall survival (P = 0.018; Kaplan-Meier analysis). Multivariate Cox analysis demonstrated that nuclear SIRT6 staining remained associated with death after correcting for tumour stage and lymph-node involvement (P = 0.033). Collectively, our data suggest that SIRT6 has a role in paclitaxel and epirubicin sensitivity via targeting FOXO proteins and that SIRT6 could be a useful biomarker and therapeutic target for paclitaxel- and epirubicin-resistant cancer.

FOXM1 is implicated in genotoxic drug resistance but its role and mechanism of action remain unclear. Here, we establish that γH2AX foci, indicative of DNA double strand breaks, accumulate in a time-dependent manner in the drug sensitive MCF-7 cells but not in the resistant counterparts in response to epirubicin. We find that FOXM1 expression is associated with epirubicin sensitivity and double strand break (DSB) repair. Ectopic expression of FOXM1 can increase cell viability and abrogate DSBs sustained by MCF-7 cells following epirubicin, owing to an enhancement in repair efficiency. Conversely, alkaline comet and γH2AX foci formation assays show that Foxm1-null cells are hypersensitive to DNA damage, epirubicin and γ-irradiation. Furthermore, we find that FOXM1 is required for DNA repair by homologous recombination (HR) but not non-homologous end joining (NHEJ), using HeLa cell lines habouring an integrated direct repeat green fluorescent protein (DR-GFP) reporter for DSB repair. We also identify BRIP1 as a direct transcription target of FOXM1 by promoter analysis and chromatin-immunoprecipitation assay. In agreement, depletion of FOXM1 expression by siRNA down-regulates BRIP1 expression at the protein and mRNA levels in MCF-7 and the epirubicin resistant MCF-7 EpiR cells. Remarkably, the requirement for FOXM1 for DSB repair can be circumvented by reintroduction of BRIP1, suggesting that BRIP1 is an important target of FOXM1 in DSB repair. Indeed, like FOXM1, BRIP1 is needed for HR. These data suggest that FOXM1 regulates BRIP1 expression to modulate epirubicin-induced DNA damage repair and drug resistance.

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.

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AimDeregulation of FOXM1 has been documented in various cancers. The aim of this study was to evaluate the role of FOXM1 in ovarian cancer tumorigenesis and paclitaxel resistance.Experimental DesignExpression of FOXM1 was examined in 119 clinical samples by immunohistochemistry and correlated with clinicopathological parameters. Effects of FOXM1 knockdown on ovarian cancer cell migration, invasion and mitotic catastrophe were also studied. qPCR and ChIP-qPCR were used to establish KIF2C as a novel FOXM1 target gene implicated in chemoresistance.ResultsHigh nuclear FOXM1 expression in ovarian cancer patient samples was significantly associated with advanced stages (P = 0.035), shorter overall (P = 0.019) and disease-free (P = 0.014) survival. Multivariate analysis confirmed FOXM1 expression as an independent prognostic factor for ovarian cancer. FOXM1 knockdown significantly inhibited migration and invasion of ovarian cancer cells and enhanced paclitaxel-mediated cell death and mitotic catastrophe in a p53-independent manner. Bioinformatics analysis suggested a number of potential transcription targets of FOXM1. One of the potential targets, KIF2C, exhibited similar expression pattern to FOXM1 in chemosensitive and chemoresistant cells in response to paclitaxel treatment. FOXM1 could be detected at the promoter of KIF2C and FOXM1 silencing significantly down-regulated KIF2C.ConclusionOur findings suggest that FOXM1 is associated with poor patient outcome and contributes to paclitaxel resistance by blocking mitotic catastrophe. KIF2C is identified as a novel FOXM1 transcriptional target that may be implicated in the acquisition of chemoresistance. FOXM1 should be further investigated as a potential prognostic marker and therapeutic target for ovarian cancer.