SUMOylation inhibits FOXM1 activity and delays mitotic transition

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.

Figure 1

FOXM1 is SUMOylated by SUMO1. (a) Protein lysates were prepared from MCF-7 cells with or without transfection of the SUMO E2-ligase Ubc-9. FOXM1 was detected by western blot analysis and a higher molecular weight form was observed consistent with SUMOylation of FOXM1. (b) MCF-7 cells were transfected with FOXM1, Ubc9 or SUMO1, -2 or -3, and FOXM1 was detected by western blot analysis. Multiple higher molecular weight forms of FOXM1 were observed, consistent with poly-SUMOylation. (c) MCF-7 cells were co-transfected with His-SUMO1, FOXM1 and Ubc9. SUMOylated proteins were purified using Ni²⁺-column affinity pulldown under denaturing conditions (8 M urea). Input and His-tagged proteins were probed for FOXM1, and poly-SUMOylated FOXM1 was detectable demonstrating the covalent linkage of His-SUMO1 to FOXM1. (d) IPTG-inducible recombinant His-tagged FOXM1 was expressed in E. coli and isolated using Ni²⁺-column affinity pulldown. Cell lysates from induced and non-induced E. coli were incubated with Ubc9 and SUMO1 in the presence and absence of Mg²⁺-ATP and reaction buffer, and resolved using SDS-PAGE. SUMO1 was detected by western blot analysis. (e) MCF-7 cells were transfected with constructs encoding FOXM1 fused to mouse Ubc9 (FOXM1-Ubc9) with or without eGFP-tagged SUMO1. The higher molecular weight band detected by FOXM1 and Ubc9 antibodies, which is consistent with the auto-SUMOylation of FOXM1-Ubc9, is indicated. SUMOylation was enhanced and increased in molecular weight by co-transfection of eGFP-SUMO1 (indicated by the multiple arrows). (f) MCF-7 cells were co-transfected with FOXM1, SUMO1, Ubc9, SENP-1 and the dominant-negative SENP-1 construct (SENP1-C630S), and lyssates were analysed by western blot analysis.

Figure 2

FOXM1 is SUMOylated in response to epirubicin treatment. (a) MCF-7 cells were treated with epirubicin (1 μM) for 0, 6 and 24 h. Co-immunoprecipitation (co-IP) was performed with a FOXM1 antibody and was probed for SUMO1 and vice versa; inputs (1/10 of IP) and IP products with IgG and specific antibodies were resolved on western blot analysis and were probed for FOXM1 and SUMO1. High molecular weights of FOXM1- and SUMO1-containing species are highlighted ‘*’. (b) MCF-7 cells co-transfected with 0.05 μg of eGFP-FOXM1 and 0.025 μg per well of either tRFP-SUMO1 (FRET acceptor) or empty expression plasmids were treated with 0.1 μM epirubicin for 0, 2, 4, 6 and 24 h. Intensity-merged fluorescence lifetime images of doxorubicin treatment time course are shown. The reduction in donor fluorescence lifetime indicates the occurrence of FRET, implying that eGFP-FOXM1 and tRFP-SUMO1 are within 10 nm of each other. (c) Analysis of fluorescence lifetime data, showing mean lifetimes ±s.e.m. of eGFP-FOXM1 fluorescence,was determined by fitting the fluorescence decay as described. Each measurement is based on >100 cells. The results show that eGFP-FOXM1 and tRFP-SUMO1 interactions significantly increase after 6 or 8 h epirubicin treatment (Tukey’s HSD test: http://www.amazon.co.uk/Biostatistics-Methodology-Sciences-Probability-Statistics/dp/0471031852: 0 versus 3, 6, 8 or 24 h; *significant P<0.05; NS, not significant).

Figure 3

SUMOylation of FOXM1 occurs during mitotic arrest. (a) MCF-7 cells were treated with dimethyl sulfoxide (DMSO) (0.001% (v/v); 16 h) or nocodazole (0.0001 mg/ml; 16 h), and immunoprecipitation (IP) was performed with a FOXM1 antibody; inputs and IP products were resolved on western blot analysis and were probed for SUMO1. The membrane was then reprobed with a FOXM1 antibody. Arrows indicate higher molecular weight forms of FOXM1. (b) MCF-7 cells were treated with paclitaxel (100 nM) for 0, 6 and 24 h. Co-IP was performed with a FOXM1 antibody and was probed for SUMO1 and vice versa; inputs (1/10 of IP) and IP products with IgG and specific antibodies were resolved on western blot analysis and were probed for FOXM1 and SUMO1. * indicates higher molecular weight FOXM1 complex.

Figure 4

FOXM1 is SUMOylated at multiple sites. (a) Schematic showing consensus SUMOylation sites in FOXM1 identified using online computational prediction software Abgent SUMOplot (Abgent, Maidenhead, UK) and SUMOsp 2.0 (The Cuckoo Workgroup, Hefei, China). NRD, N-terminal regulatory domain; FKH, forkhead domain; TAD, transactivation domain. (b) Site-directed mutagenesis was performed using WT-FOXM1 expression vector at the indicated sites (lysine to arginine). In all cases, mutants were confirmed by sequencing. MCF-7 cells were then co-transfected with WT- or indicated mutant-FOXM1, SUMO1 and Ubc9 constructs, and 24 h later protein lysates were prepared and FOXM1 SUMOylation was determined by western blot analysis. (c) FOXM1 mutants were generated, containing multiple lysine-to-arginine mutations, from FOXM11X(K>R) (K210R) to FOXM17X(K>R) (K201R/K218R/K356R/K440R/K460R/K478R/K495R). Mutational order was based on site location. FOXM1 mutant SUMOylation was determined as above. (d) FOXM17X(K>R) was subjected to reversal of individual mutations (R>K) to examine site redundancy. Mutants were assessed for SUMOylation as above and K356R and K440R were identified as redundant mutations. (e) MCF-7 cells were transfected with WT-FOXM1 or FOXM15X(K>R) (K201R/K218R/K460R/K478R/K495R) with or without Ubc9 and SUMO1. SUMOylation of FOXM1 was determined as above. (f) MCF-7 cells were transfected with either FOXM1-Ubc9 or FOXM15X(K>R)-Ubc9 with or without eGFP-tagged SUMO1. Loss of the auto-SUMOylated form of FOXM1 was observed in FOXM15X(K>R)-Ubc9. SUMOylation of FOXM1 was determined as above.

Figure 5

SUMOylation of FOXM1 promotes its cytoplasmic localization and occurs preferentially on phosphorylated FOXM1. (a) MCF-7 cells were co-transfected with FOXM1, SUMO1 and Ubc9 constructs and cytoplasmic and nuclear fractions were isolated. FOXM1 SUMOylation and cell fractionation were confirmed by western blot analysis against FOXM1, SUMO1, lamin and β-tubulin. (b) MCF-7 cells were transfected with FOXM1-Ubc9 or FOXM15X(K>R)-Ubc9 and were fractionated as above. The cytoplasmically localized auto-SUMOylation event is indicated. (c) MCF-7 cells were transfected with FOXM1-Ubc9 or FOXM15X(K>R)-Ubc9 and stained for FOXM1, β-tubulin and 4′,6-diamidino-2-phenylindole before analysis by confocal microscopy. Images are representative of three independent experiments. (d) MCF-7 cells were transfected with FOXM1 or the N-terminal truncated FOXM1 (Δ-FOXM1) and cells were fractionated as above. (e) MCF-7 cells were co-transfected with WT-FOXM1 or mutant-FOXM15X(K>R) with or without SUMO1 and Ubc9 constructs, and 24 h later protein lysates were subjected to immunoprecipitation (IP) with a FOXM1 antibody. Precipitated proteins were separated by SDS-PAGE and FOXM1 phosphorylation was detected using a phosphorylated-M-phase-associated protein-specific antibody (MPM-2). The membrane was then reprobed with a FOXM1 antibody, demonstrating that the free FOXM1 displayed no MPM-2 detectable phosphorylation following SUMOylation in the WT-FOXM1, whereas the SUMOylated form of FOXM1 was detectable by MPM-2. These are indicated by boxes.

Figure 6

SUMOylation of FOXM1 promotes its degradation and a SUMOylation mutant is resistant to Cdh1-mediated degradation. (a) MCF-7 cells were transfected with FOXM1 with or without Ubc9 and SUMO1, and 16 h later cells were treated with cycloheximide (CHX) or vehicle (0.001% (v/v) dimethyl sulfoxide (DMSO)) and protein lysates were prepared from 0 to 8 h following CHX treatment. Densitometry was used to quantify the unconjugated FOXM1 relative to the SUMOylated-FOXM1, for which independent background readings were taken. Western blot analysis was representative of three independent experiments; densitometry is mean±s.e.m. Densitometry was used to quantify FOXM1 levels and were normalized to β-tubulin. Western blot analysis was representative of three independent experiments; densitometry is mean±s.e.m. (t-test: *significant P<0.05; otherwise not significant). (b) Asynchronous MCF-7 cells were co-transfected with FOXM-1 or FOXM15X(K>R) with or without increasing levels of Cdh1(4a). At 24 h after transfection, protein lysates were prepared and FOXM1 was examined by western blot analysis. The arrow indicates the higher molecular weight form of FOXM1 suggesting protein ubiquitination. (c) MCF-7 cells were transfected with FOXM1 or FOXM1(5XK>R) with or without Cdh1(4a), and 16 h later cells were treated with cycloheximide or vehicle (0.001% (v/v) DMSO) and protein lysates were prepared from 0 to 8 h following cycloheximide treatment. Densitometry was used to quantify FOXM1 levels and were normalized to β-tubulin. Western blot analysis was representative of three independent experiments; densitometry is mean±s.e.m. (t-test: *significant P<0.05; otherwise not significant). (d) MCF-7 cells were co-transfected with FOXM1 or FOXM15X(K>R) with Flag-Cdh1(4a), and 24 h later protein lysates were subjected to co-immunoprecipitation (co-IP) with a FOXM1 antibody. Precipitated proteins were separated by SDS–PAGE and Flag-Cdh1(4a) binding was detected using an anti-Flag antibody. The membrane was then reprobed with a FOXM1 antibody. Ubiquitinated-FOXM1 (Ubq-FOXM1) and IgG are indicated by arrows. (e) MCF-7 cells were co-transfected with FOXM1 or FOXM15X(K>R) with or without haemagglutinin-ubiquitin (HA-Ubq). After 24 h, protein lysates were subjected to IP with a FOXM1 antibody. Precipitated proteins were separated by SDS–PAGE and ubiquitination of FOXM1 was detected by anti-HA antibody. The membrane was then reprobed with a FOXM1 antibody.

Figure 7

SUMOylation of FOXM1 represses its transcriptional activity and a SUMOylation-deficient FOXM1 mutant can enhance cell proliferation. MCF-7 cells were co-transfected with FOXM1-Ubc9 or FOXM1(5XK>R)-Ubc9 (0–20 ng) and luciferase reporters were driven by (a) the FOXM1 6X-DNA-binding element (6XDBE), (b) the wild-type cyclin B1 promoter (cyclin B1-pGL2) or the cyclin B1 promoter containing mutations in the three consensus forkhead binding sites (cyclin B1-mut3-pGL2), or (c) the GADD45 promoter (GADD45-pGL2). After 8 h, cells were placed in 0.5% fetal calf serum for 24 h before the luciferase assay was performed. All DNA concentrations were normalized using empty vector. Reporter gene activity was expressed as a ratio of firefly luciferase activity to control Renilla luciferase activity. (d) MCF-7 cells were transfected with FOXM1 with or without SUMO1. After 20 h, cells were treated with BrdU (10 μmol/l) for 4 h and then harvested and stained with propidium iodide and fluorescein isothiocyanate-conjugated anti-BrdU antibody and analysed by fluorescence-activated cell sorter (FACS) (20 000 gated events were counted; data are representative of three independent experiments; graph shows mean of three experiments±s.e.m.). (e) MCF-7 cells were transfected with empty vector, FOXM1 or FOXM15X(K>R), and 20 h later they were treated with BrdU for 4 h before being harvested and stained with propidium iodide- and fluorescein isothiocyanate-conjugated anti-BrdU. Fluoresence was determined by FACS analysis (20 000 gated events were counted; data are representative of three independent experiments; graph shows mean of three experiment ±s.e.m.) (t-test: *significant P<0.05; otherwise not significant).

Figure 8

SUMOylation of FOXM1 delays mitotic progression and deregulates cyclin B1 expression. (a) RPE-hTERT-GFP-α-tubulin cells were transfected with empty vector, pmCherry plus FOXM1-Ubc9 or pmCherry plus FOXM15X(K>R)-Ubc9 and synchronized in the G1 phase by double thymidine block. Following removal of thymidine, progression through mitosis was monitored by time-lapse phase-contrast and fluorescent microscopy. (b) Graphical representation of mitotic progression. Time from mitotic entry to exit, mitotic entry to metaphase alignment and metaphase alignment to anaphase onset were calculated on a total of 200 cells per condition from three independent experiments (mean±s.e.m.). Only cells that were transfected with the pmCherry-tagged FOXM1 constructs were included. (c) HeLa cells were transfected with empty vector, FOXM1-Ubc9 or FOXM15X(K>R)-Ubc9, and 24 h later they were synchronised by double thymidine block. Following release from thymidine block, protein lysates were prepared from 0 to 13 h; lysates were resolved by gel electrophoresis and western blot analysis was performed for mitotic and S-phase-associated cyclins and checkpoint protein. β-Actin was used as a loading control; data are representative of three independent experiments.