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FoxM1 insufficiency hyperactivates Ect2–RhoA–mDia1 signaling to drive cancer

Nature Cancer volume 1, pages 1010–1024 (2020)Cite this article

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Abstract

FoxM1 activates genes that regulate S–G2–M cell-cycle progression and, when overexpressed, is associated with poor clinical outcome in multiple cancers. Here we identify FoxM1 as a tumor suppressor in mice that, through its N-terminal domain, binds to and inhibits Ect2 to limit the activity of RhoA GTPase and its effector mDia1, a catalyst of cortical actin nucleation. FoxM1 insufficiency impedes centrosome movement through excessive cortical actin polymerization, thereby causing the formation of nonperpendicular mitotic spindles that missegregate chromosomes and drive tumorigenesis in mice. Importantly, low FOXM1 expression correlates with RhoA GTPase hyperactivity in multiple human cancer types, indicating that suppression of the newly discovered Ect2–RhoA–mDia1 oncogenic axis by FoxM1 is clinically relevant. Furthermore, by dissecting the domain requirements through which FoxM1 inhibits Ect2 guanine nucleotide-exchange factor activity, we provide mechanistic insight for the development of pharmacological approaches that target protumorigenic RhoA activity.

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Fig. 1: FoxM1 insufficiency causes tumor formation.
Fig. 2: FoxM1 controls centrosome movement and perpendicular spindle assembly.
Fig. 3: The FoxM1 NTD nontranscriptionally regulates centrosome movement.
Fig. 4: The FoxM1 NTD acts to inhibit cortical actin nucleation.
Fig. 5: Cytoplasmic FoxM1 binds to and inhibits the GEF activity of Ect2 towards RhoA.
Fig. 6: Excessive cortical actin rigidity slows centrosome movement regardless of cause.
Fig. 7: Low FOXM1 expression correlates with high RhoA activity in several human cancer types.
Fig. 8: Correction of cortical actin hypernucleation by NTD overexpression inhibits tumorigenesis.

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Data availability

RNA-sequencing data have been deposited in the Gene Expression Omnibus under the accession number GSE130410. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank B. Childs, R. Naylor and C. Sieben for helpful discussions, and G. Nelson for managing the mouse colony. We thank the transgenic and gene knockout core at Mayo Clinic for generation of all mutant mouse strains, D. Billadeau (Mayo Clinic, Rochester) for the Cofilin 1 antibody, G. Razidlo (Mayo Clinic, Rochester) for Cytochalasin D and GST-PBD construct, S. Kaufmann (Mayo Clinic, Rochester) for H1299 and A549 cell lines and R. Thaler for assistance with the in vitro GEF activity assay. The human tumor results shown here are in whole or part based upon data generated by TCGA Research Network: https://www.cancer.gov/tcga. This work was supported by NIH grant nos. R01 CA096985, CA126828 and CA168709 to J.M.v.D. J.F.L is supported by Mayo Clinic Graduate School of Biomedical Sciences and J.A.K. by NIH grant no. T32 GM65841.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA

    Jazeel F. Limzerwala, Jake A. Kloeber, Brian A. Davies, Darren J. Baker, David J. Katzmann & Jan M. van Deursen

  2. Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN, USA

    Karthik B. Jeganathan, Ines Sturmlechner, Jian Zhong, Raul Fierro Velasco, Darren J. Baker & Jan M. van Deursen

  3. Mayo Clinic Medical Scientist Training Program, Mayo Clinic, Rochester, MN, USA

    Jake A. Kloeber

  4. Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA

    Cheng Zhang & Hu Li

  5. Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, USA

    Alan P. Fields

  6. Department of Pharmacodynamics, University of Florida, Gainesville, FL, USA

    Yaxia Yuan & Daohong Zhou

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  1. Jazeel F. Limzerwala
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Contributions

J.F.L. and J.M.v.D. designed experiments, interpreted data and wrote the manuscript with input from all authors. I.S., C.Z. and H.L. performed RNA sequencing and systems biology analyses. J.A.K. performed experiments pertaining to ROCK-myosin signaling with J.F.L. J.Z. generated the Foxm1 mutant mouse model. B.A.D. and D.J.K. assisted with recombinant protein production and conducted in vitro binding experiments in collaboration with J.F.L. K.B.J. and J.F.L. performed biochemical experiments, and R.F.V. immunofluorescence and histological assessments on mouse tissues with J.F.L. A.P.F., Y.Y. and D.Z. collaborated with J.F.L. on FoxM1-Ect2 binding studies. D.J.B. assisted with data interpretation and statistical analyses. J.M.v.D. supervised and directed all aspects of the study.

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Correspondence to Jan M. van Deursen.

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Extended data

Extended Data Fig. 1 Generation of Foxm1 knockout and hypomorphic alleles.

a, Copy number variability (CNV) of the FOXM1 gene in the indicated TCGA cohort. b, Schematic representation of the gene targeting strategy used to generate Foxm1 hypomorph (H) and knockout (–) alleles. c, Quantification of FoxM1 protein levels in MEFs and lung tissue. PonS staining of blotted proteins was used as loading control. d, Western blot analysis of FoxM1 in lysates of the indicated tissues. Western blots are representative of at least 3 independent MEF lines or mice per genotype. e, Overall survival analysis of human colorectal cancer patients from the TCGA COADREAD cohort with indicated FOXM1 gene expression (n = 214 > 10.51, n = 216 < 10.51). Significance determined by Log Rank Test. See source file for uncropped immunoblots.

Source data

Extended Data Fig. 2 FoxM1 insufficiency does not perturb common CIN causing mechanisms.

a, MEFs in metaphase with misaligned chromosomes after monastrol washout assay. (See methods). b, Time taken from nuclear envelope breakdown (NEBD) to anaphase onset in MEFs expressing H2B-mRFP. c, Quantification of MEFs with indicated spindle defect. d, Quantification of G2-phase cells with premature centrosome disjunction. (n = 3 independent MEF lines in (a-d). e, Time taken for centrosomes to separate after disjunction to NEBD in MEFs expressing H2B-YFP and γTubulin-tdTomato. (n = 3 + /+ and 4 –/H independent MEF lines). Data represent mean ± s.e.m. None of the analyses were statistically significant after performing one-way ANOVA with Tukey’s correction (a–d) or two-tailed unpaired t-test (e). Scale bar, 10 μm. See source file for original data.

Source data

Extended Data Fig. 3 FoxM1 non-transcriptionally regulates centrosome movement independent of Eg5.

a, Left: Images of MEFs in prophase immunostained for Eg5 and γtubulin. Right: Quantification of Eg5 signal at centrosomes. b, Schematic representation and western blot analysis of tdTomato(tdT)-tagged FoxM1 cDNA constructs. N-terminal domain (NTD), Forkhead domain (FHD), Transactivation domain (TAD). Western blots are representative of 3 independent MEF lines per group. c, Quantification of prophases with slow centrosome movement and d, metaphases with non-perpendicular spindles in Foxm1–/H MEFs expressing indicated cDNA constructs. e, Chromosome segregation analysis of MEFs expressing H2B-mRFP as in c. f, Chromosome segregation analysis of Foxm1+/+ MEFs stably expressing FoxM11–232. (n = 3 independent MEF lines per genotype in (a, c–f). g, Representative cell cycle profiles of the indicated propidium iodide-stained MEFs. h, Quantification of cells in the indicated stage of the cell cycle as in g. (n = 6 independent MEF lines per genotype in (g, h). Data represent mean ± s.e.m. Differences are not statistically significant in a, f. Statistics: a, c–e, h one-way ANOVA with Tukey’s correction; f, two-tailed paired t-test. Scale bar, 5 μm. See source file for original data and uncropped immunoblots.

Source data

Extended Data Fig. 4 FoxM1 insufficiency does not hyperactivate ROCK-Myosin-II signaling.

a, Left: Images of the indicated MEFs stained for TRITC-Phalloidin and phospho-histoneH3Ser10. Right: Quantification of cortical actin intensity (n = 3 independent MEF lines). b, Left: Images of the indicated MEFs stained with TRITC-Phalloidin. Right: Quantification of cortical actin intensity in telophase (n = 3 independent MEF lines) c, Left: Images of MEFs stained for myosin light chain (MLC2). Right: Quantification of cortical MLC2 intensity (n = 5 independent MEF lines). d, Quantification of cortical actin intensity and e, non-perpendicular spindles in mitotic Foxm1–/– MEFs treated with the indicated drugs for 4 h (n = 3 independent MEF lines). f, Chromosome segregation analysis of MEFs as in d. g, Western blot analysis of MEFs of the indicated genotypes. Western blots are representative of 3 independent experiments. h, Densitometric quantification of band intensity of indicated proteins (n = 9 MEF lines analyzed across 3 independent experiments). i, Left: Images of MEFs stained with phospho-myosin light chain (pMLC2Ser19). Right: Quantification of cortical pMLC2 intensity (n = 7 independent MEF lines). Data represent mean ± s.e.m. Statistics: a–e, h, i one-way ANOVA with Tukey’s correction; f, two-tailed paired t-test. Scale bars, 10 μm. See source file for original data and uncropped immunoblots.

Source data

Extended Data Fig. 5 FoxM1 insufficiency does not alter Rac1 and Cdc42 Rho GTPases.

a, Western blot analysis of Foxm1–/– MEFs lentivirally transduced with the indicated shRNAs. PonS staining was used as loading control. b–d, Foxm1–/– MEFs stably expressing indicated shRNAs analyzed for cortical actin intensity (b), incidence of slow centrosome movement in prophase (c), and non-perpendicular spindles in metaphase (d) (n = 5 independent MEF lines in (b–d). e, Left: Western blot analysis of the indicated MEF lysates. β-actin served as a loading control. Right: Densitometric quantification of signals from indicated proteins in the indicated MEFs (n = 9 MEF lines analyzed across 3 independent experiments). f, Left: Western blot analysis of the indicated MEFs. PonS staining was used as loading control. Right: Densitometric quantification of signals from the indicated proteins (n = 9 MEF lines analyzed across 3 independent experiments). All western blots are representative of at least 3 independent experiments. Data represent mean ± s.e.m. Statistics: b–d, one-way ANOVA with Sidak’s correction; e, f, two-tailed unpaired t-test. See source file for original data and uncropped immunoblots.

Source data

Extended Data Fig. 6 Ect2 overexpression phenocopies FoxM1 insufficiency.

a, FoxM1 immunoprecipitation from A549 cells, H1299 cells and primary HSFs. b, HA immunoprecipitation from whole-cell mitotic MEF extracts stably expressing indicated FoxM1 cDNA constructs. c, HA immunoprecipitation from extracts of wildtype-MEFs stably expressing HA-FoxM11–232 subject to sub-cellular fractionation: C, cytoplasmic fraction; and N, nuclear fraction. d, Quantification of cortical actin intensity in indicated MEFs stably expressing HA-FoxM11–1232 or empty vector (EV) (n = 3 independent MEF lines). e, Western blot analysis of wildtype (WT) MEFs stably expressing empty-vector (EV) or HA-Ect2. PonS served as loading control. f, Left: Images of MEFs stained with TRITC-Phalloidin Right: Quantification of cortical actin intensity of MEFs as in e (n = 3 independent MEF lines). g, Quantification of prophases with slow centrosome movement and h) metaphases with non-perpendicular spindles as in e. (n = 3 independent MEF lines). i, Chromosome counts and j, chromosome segregation analysis of P5 MEFs as in e. (n = 3 independent MEF lines). All western blots are representative of at least 3 independent experiments or MEF lines. Data represent mean ± s.e.m. Statistics: d, one-way ANOVA with Tukey’s correction; f–j, two-tailed paired t-test. Scale bar, 5 μm. See source file for original data and uncropped immunoblots.

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Extended Data Fig. 7 FoxM1-independent cortical actin hypernucleation slows centrosome movement and yields non-perpendicular spindles.

a, Western blot analysis of primary HSFs lentivirally transduced with scramble (shScr) or two independent FOXM1 shRNAs (shFOXM1). PonS staining of blotted proteins served as loading control. b, Left: Images of the indicated metaphases stained with TRITC-Phalloidin as in a. Right: Quantification of cortical actin intensity (n = 3 independent fibroblast lines for shFOXM1 groups and 6 independent lines for shScr group). c, Left: Images of prophases immunostained with γtubulin as in a. Right: Quantification of prophases with slow centrosome movement. (n = 3 independent fibroblast lines for shFOXM1 groups and 6 independent lines for shScr group). d, Quantification of metaphases with non-perpendicular spindles as in a. (n = 3 independent fibroblast lines for shFOXM1 groups and 6 independent lines for shScr group). e, Chromosome segregation analysis of HSFs expressing H2B-mRFP as in a. (n = 3 independent fibroblast lines for shScr and shFOXM1 #2 groups and 4 independent lines for shFOXM1 #1 group). f, Western blot analysis of wildtype MEFs stably expressing the indicated shRNAs. g, Left: Images of wildtype MEFs stably expressing the indicated shRNAs stained with TRITC-Phalloidin. Right: Quantification of cortical actin intensity in wildtype MEFs stably expressing the indicated shRNAs (n = 3 independent MEF lines). h–k, Wildtype MEFs stably expressing the indicated shRNAs analyzed for slow centrosome movement in prophase (h), non-perpendicular spindles in metaphase (i), aneuploidy rates (chromosome counts on metaphase spreads) (j), and chromosome segregation defects (k). (n = 3 independent MEF lines). l, Western blot analysis of the indicated MEFs. m–p, Quantification of cortical actin intensity (m), quantification of prophases with slow centrosome movement (n), metaphases with non-perpendicular spindles (o), aneuploidy rates (chromosome counts on metaphase spreads) on the indicated P5 MEFs (p) (n = 5 independent MEF lines per genotype). q, Quantification of cortical actin and cortical phospho-MLC2Ser19 intensity in Foxm1–/– MEFs after the indicated treatments for 4 h. (n = 3 independent MEF lines). r, Quantification of prophases with slow centrosome movement of wildtype MEFs stably transduced with scr or Capzb shRNA, after treatment with 10 μM ROCK inhibitor Y-27632 for 4 h (n = 5 independent MEF lines). All western blots are representative of at least 3 HSF or MEF lines. Data represent mean ± s.e.m. Statistics: b–k, q, r, one-way ANOVA with Sidak’s correction, m–p, two-tailed unpaired t-test. Scale bar, 5 μm. See source file for original data and uncropped immunoblots.

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Extended Data Fig. 8 NTDT corrects mitotic defects caused by Foxm1 insufficiency by inhibiting Ect2-RhoA signaling.

a, Schematic for the generation of NTDT mice. b, Chromosome counts performed on MEFs of indicated genotypes (n = 3 independent MEF lines). c-f, MEFs of indicated genotypes analyzed for cortical actin intensity in metaphase (c), slow centrosome movement in prophase (d), non-perpendicular spindles in metaphase (e), and chromosome segregation errors (f). (n = 3 independent MEF lines). g, Top: Western blot analysis of GTP-bound (active) and total RhoA in indicated mammary tumor extracts. Bottom: Densitometric quantification of RhoA signals. (n = 16 PyVT and 15 PyVT; NTDT tumors analyzed across 3 independent experiments). h–j, Epithelial cells derived from MMTV-PyVT primary tumors, treated with vehicle (Veh) or 3 μg/ml RhoA inhibitor (C3) for 4 h and quantified for non-perpendicular spindles (h), lagging chromosome incidence (i), and cortical actin intensity (j), (n = 3 independent tumor derived lines). k, Western blot analysis of mammary epithelial tumor cells derived from MMTV-PyVT primary tumors lentivirally transduced with the indicated shRNAs. l–n, MMTV-PyVT epithelial tumor cells quantified for non-perpendicular spindles (l), lagging chromosome incidence (m), and cortical actin intensity (n). (n = 7 independent tumor derived lines). All western blots are representative of at least 3 independent experiments or tumor lines. Data represent mean ± s.e.m. Statistics: b–f, one-way ANOVA with Tukey’s correction; g, two-tailed unpaired t-test h–n, two-tailed paired t-test. See source file for original data and uncropped immunoblots.

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Extended Data Fig. 9 Hypothetical model for how FoxM1 controls cortical actin nucleation.

Ect2 hyperactivity has been linked to overactive Rho GTPases in human cancers. How Ect2 can become hyperactive remains incompletely understood. Here we identify FoxM1 as a key inhibitor of Ect2 activity and that its complete or partial loss results in increased signaling through the Ect2-RhoA-mDia- signaling axis. We find that FoxM1 binds to Ect2 via its NTD, thereby inhibiting Ect2-mediated activation of RhoA without impacting the activities of two other Rho GTPases, Rac1 and Cdc42 activity. RhoA regulates the actomyosin network through two effectors mDia1, which stimulates actin polymerization, and ROCK which stimulates contractility through MLC activation and F-actin stabilization through LIMK (not shown). We find that FoxM1 selectively inhibits Ect2-mediated activation of mDia1, implying that RhoA activity toward ROCK is independently controlled. Cortical actin hypernucleation resulting from FoxM1 insufficiency slows movement of centrosomes along the cortex, yielding non-perpendicular spindles enriched for merotelic MT-kinetochore attachments that promote aneuploidization and providing a rate of genetic heterogeneity that stimulates tumor formation. FoxM1 independent mechanisms of cortical actin hypernucleation produce the same phenotype, which can be ameliorated by inhibiting myosin activity, indicating that rigidity of the actomyosin cortex is a key determinant of centrosome movement and spindle symmetry.

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Limzerwala, J.F., Jeganathan, K.B., Kloeber, J.A. et al. FoxM1 insufficiency hyperactivates Ect2–RhoA–mDia1 signaling to drive cancer. Nat Cancer 1, 1010–1024 (2020). https://doi.org/10.1038/s43018-020-00116-1

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