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Glucocorticoid Receptor Regulates Accurate Chromosome Segregation and Is Associated with Malignancy

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Glucocorticoid Receptor Regulates Accurate Chromosome Segregation and Is Associated with Malignancy Glucocorticoid receptor regulates accurate chromosome segregation and is associated with malignancy Laura C. Matthewsa, Andrew A. Berrya, David J. Morgana, Toryn M. Poolmana, Kerstin Bauerb, Frederike Kramerc, David G. Spillerd, Rachel V. Richardsone, Karen E. Chapmane, Stuart N. Farrowa,f, Michael R. Normang, Andrew J. K. Williamsonh, Anthony D. Whettonh, Stephen S. Taylord, Jan P. Tuckermannb,c, Michael R. H. Whited, and David W. Raya,1 aManchester Centre for Nuclear Hormone Research in Disease and Manchester Academic Health Sciences Centre, Institute of Human Development, Faculty of Medical and Human Sciences, dFaculty of Life Sciences, and hFaculty Institute of Cancer Sciences, University University of Manchester, Manchester M13 9PT, United Kingdom; bInstitute for Comparative Molecular Endocrinology, University of Ulm, 89081 Ulm, Germany; cLeibniz Institute for Age Research, Fritz Lipmann Institute, 07745 Jena, Germany; eUniversity of Edinburgh, Edinburgh EH16 4TJ, United Kingdom; fGlaxoSmithKline, Stevenage SG1 2NY, United Kingdom; and gDivision of Medicine, University of Bristol, Bristol BS1 3NY, United Kingdom Edited by Bert W. O’Malley, Baylor College of Medicine, Houston, TX, and approved February 26, 2015 (received for review June 27, 2014) The glucocorticoid receptor (GR) is a member of the nuclear receptor have not been defined. The role of GR in cancer has received superfamily, which controls programs regulating cell proliferation, little attention beyond acute lymphoblastic leukemia, in which differentiation, and apoptosis. We have identified an unexpected role GR is powerfully proapoptotic (15). However, GR inhibits ex- for GR in mitosis. We discovered that specifically modified GR species pression of cyclin D and augments expression of cyclin-dependent accumulate at the mitotic spindle during mitosis in a distribution that protein kinase inhibitors p21 and p27 to effect cell cycle slowing or overlaps with Aurora kinases. We found that Aurora A was required arrest in a cell type-specific manner (16). to mediate mitosis-driven GR phosphorylation, but not recruitment of We now define a role for GR in regulating mitosis using, at GR to the spindle. GR was necessary for mitotic progression, with least in part, nontranscriptional mechanisms. Loss of GR results increased time to complete mitosis, frequency of mitotic aberrations, in a robust mitotic phenotype, with aberrant chromosome seg- MEDICAL SCIENCES and death in mitosis observed following GR knockdown. Complemen- regation, accumulation of chromosome complement defects, and tation studies revealed an essential role for the GR ligand-binding death in mitosis, with the extent of cell survival or death varying domain, but no clear requirement for ligand binding in regulating by cell type and model. Moreover, GR haploinsufficient mice chromosome segregation. The GR N-terminal domain, and specifically develop tumors with age. Analysis of common human cancers phosphosites S203 and S211, were not required. Reduced GR expres- revealed frequent cell type-specific loss of GR expression, im- sion results in a cell cycle phenotype, with isolated cells from mouse plying an important causal relationship between GR and ma- and human subjects showing changes in chromosome content over lignant transformation or progression. prolonged passage. Furthermore, GR haploinsufficient mice have an increased incidence of tumor formation, and, strikingly, these tumors Results are further depleted for GR, implying additional GR loss as a conse- We profiled the GR interactome (1,696 proteins) using immune quence of cell transformation. We identified reduced GR expression in enrichment and MS methods, and compared the list of GR a panel of human liver, lung, prostate, colon, and breast cancers. We interacting proteins with the mitotic spindle proteome (17). This therefore reveal an unexpected role for the GR in promoting accurate chromosome segregation during mitosis, which is causally linked to Significance tumorigenesis, making GR an authentic tumor suppressor gene. We have discovered a role for the glucocorticoid receptor (GR) glucocorticoid receptor | mitosis | aneuploidy | DNA damage | cancer in coordinating cell division. We find enrichment of GR to mi- totic spindles and demonstrate that GR knockdown causes ac- lucocorticoids (Gcs) act through the glucocorticoid receptor cumulation of mitotic defects, including delayed anaphase, G(GR), a member of the nuclear hormone receptor super- ternary chromosome segregation, and death in mitosis. Mitotic family, and a ligand-activated transcription factor (1–4). The GR is GR function requires the ligand-binding domain but not ligand ubiquitously expressed and regulates energy metabolism, immunity, binding, revealing a nontranscriptional and ligand-independent and cell fate decisions. The quiescent GR resides in the cytoplasm in mechanism of action. Analysis of GR haploinsufficient cells and a complex with heat shock proteins and immunophilins, attached to tissues reveals increased aneuploidy and DNA damage, and the microtubule architecture of the cell in a heat shock protein 90- mice show an increased incidence of tumors in vivo, with fur- dependent manner (5). Ligand binding drives GR transformation, ther GR loss within those incident tumors. We also identify involving N-terminal phosphorylation on S203 and S211 and rapid reduced GR expression in several common human cancers, translocation to the nucleus requiring attachment to dynein by heat thereby implicating GR as a novel tumor suppressor gene. shock protein 90, immunophilins, and dynamitin (6). Once in the Author contributions: L.C.M., K.E.C., S.N.F., M.R.N., A.D.W., S.S.T., J.P.T., M.R.H.W., and nucleus, GR binds directly to DNA to regulate transcription, or D.W.R. designed research; L.C.M., A.A.B., D.J.M., T.M.P., K.B., F.K., D.G.S., R.V.R., and A.J.K.W. tethers to other DNA-bound transcription factors, such as nuclear performed research; S.S.T. contributed new reagents/analytic tools; L.C.M., A.A.B., D.J.M., factor kappa-light-chain-enhancer of activated B cells (NF-κB) and T.M.P., K.B., F.K., D.G.S., R.V.R., and A.J.K.W. analyzed data; and L.C.M., K.E.C., S.N.F., M.R.N., activator protein 1 (AP1), to regulate their function (7–10). A.D.W., J.P.T., M.R.H.W., and D.W.R. wrote the paper. In mitosis, GR is phosphorylated on both S203 and S211, but The authors declare no conflict of interest. in a ligand-independent manner (11), and more comprehensive This article is a PNAS Direct Submission. phosphoproteomic analyses identify the presence of multiple Freely available online through the PNAS open access option. N-terminal GR phosphoforms in purified mitotic spindle fractions 1To whom correspondence should be addressed. Email: [email protected]. (12). Although altered GR function in mitosis has been shown This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (11, 13, 14), the kinases responsible and cellular consequences 1073/pnas.1411356112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1411356112 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 approach identified GR binding to complexes involving 478 of which confirmed localization of total GR, and both PS203GR the 795 known protein components of the mitotic spindle (Fig. and PS211GR at the centrosome (SI Appendix, Figs. S5B,S7B, 1A and SI Appendix, Fig. S1), including both CENP-E and and S8B). Although GR is known to interact with the kineto- BUB3, which are required for spindle checkpoint function (18, chore protein Ska2 (23, 24), the distribution of PS211GR over- 19). Analysis of purified mitotic spindle fractions by immuno- lying the condensed chromosomes and kinetochores was not blotting using specific phospho-GR and pan-GR antibodies consistently lost with both GR siRNAs (SI Appendix, Figs. S8B revealed PS203 and PS211 modified GR species (Fig. 1B). The and S9), suggesting that this immunoreactivity may be due to presence of multiple GR protein species on immunoblots is cross-reaction with another phosphorylated protein. Aligned frequently seen, and may result from alternate protein isoforms surface plots of the three GR antibodies suggest robust enrich- or posttranslational modification. Phosphatase treatment did not ment of total GR and both GR phosphoforms to centrosomes result in loss of the additional bands, indicating that the mobility (SI Appendix, Fig. S9). shift is not due to differential phosphorylation (SI Appendix, The distribution of GR therefore overlaps with components of Fig. S2). the microtubule organizing center, which is a site of Aurora ki- P Immunofluorescence analysis confirmed GR localization to the nase activity. The distribution of phospho-GR (both S203GR P mitotic spindle, with particular enrichment at the centrosomes in and S211GR) on the centrosome overlaps with Aurora A (SI metaphase and anaphase cells (Fig. 1C and SI Appendix,Fig.S3). Appendix, Fig. S10A). Therefore, we tested Aurora kinase in- The mitotic distribution of GR to the spindle was also demon- hibition, initially with tozasertib (which inhibits both Aurora A strated using another antibody raised against a different GR and Aurora B with a similar IC50). Tozasertib virtually abolished epitope (SI Appendix, Fig. S4), was sensitive to knockdown with mitotic GR phosphorylation on both S203 and S211 (SI Appen- dix B GR-specific siRNA (SI Appendix,Fig.S5), and was also seen , Fig. S10 ). D E Immunofluorescence of tozasertib-treated cells demonstrates using an epitope-tagged
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