FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2912209648> ?p ?o ?g. }

• W2912209648 endingPage "5260" @default.
• W2912209648 startingPage "5246" @default.
• W2912209648 abstract "Cumulative evidence suggests that the heat shock protein 90 (Hsp90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-quantitative PCR analyses, revealed that UNC45A localizes to the cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types. Cumulative evidence suggests that the heat shock protein 90 (Hsp90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-quantitative PCR analyses, revealed that UNC45A localizes to the cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types. UNC45 3The abbreviations used are: UNC45uncoordinated 45/Cro1/She4pHspheat shock proteinNMIInonmuscle myosin IIChK1checkpoint kinase 1PRprogesteroneGRglucocorticoid receptorGREglucocorticoid response elementTSStranscription start siteqPCRquantitative PCRNEnuclear extractNEK7NIMA-related kinase 7MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideNOD-SCIDnon-obese diabetic/severe combined immunodeficiencyshRNAshort hairpin RNAHAhemagglutininTMAtissue microarrayDABdiaminobenzidineROIregions of interestMEMminimal essential mediumANOVAanalysis of varianceHOPHsp70/Hsp90 organizing proteinGFPgreen fluorescent protein. (UNCoordinated (1Brenner S. The genetics of Caenorhabditis elegans.Genetics. 1974; 77 (4366476): 71-94Crossref PubMed Google Scholar)) is a member of the UCS (UNC-45/Cro1/She4p) family of myosin-interacting proteins. In vertebrates, two genes encode two UNC45 isoforms that share ∼55% sequence identity (2Price M.G. Landsverk M.L. Barral J.M. Epstein H.F. Two mammalian UNC-45 isoforms are related to distinct cytoskeletal and muscle-specific functions.J. Cell Sci. 2002; 115 (12356907): 4013-402310.1242/jcs.00108Crossref PubMed Scopus (112) Google Scholar). Isoform A is ubiquitous, whereas isoform B is restricted to skeletal and cardiac muscle expression. The UCS domain of UNC45B is organized in helical armadillo repeats (3Lee C.F. Hauenstein A.V. Fleming J.K. Gasper W.C. Engelke V. Sankaran B. Bernstein S.I. Huxford T. X-ray crystal structure of the UCS domain-containing UNC-45 myosin chaperone from Drosophila melanogaster.Structure. 2011; 19 (21397190): 397-40810.1016/j.str.2011.01.002Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 4Shi H. Blobel G. UNC-45/CRO1/She4p (UCS) protein forms elongated dimer and joins two myosin heads near their actin binding region.Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (21115842): 21382-2138710.1073/pnas.1013038107Crossref PubMed Scopus (37) Google Scholar), which cooperate with the N-terminal tetratricopeptide domain to recruit the heat shock protein 90 (Hsp90) for myosin folding (5Barral J.M. Hutagalung A.H. Brinker A. Hartl F.U. Epstein H.F. Role of the myosin assembly protein UNC-45 as a molecular chaperone for myosin.Science. 2002; 295 (11809970): 669-67110.1126/science.1066648Crossref PubMed Scopus (201) Google Scholar6Landsverk M.L. Li S. Hutagalung A.H. Najafov A. Hoppe T. Barral J.M. Epstein H.F. The UNC-45 chaperone mediates sarcomere assembly through myosin degradation in Caenorhabditis elegans.J. Cell Biol. 2007; 177 (17438072): 205-21010.1083/jcb.200607084Crossref PubMed Scopus (75) Google Scholar, 7Etard C. Behra M. Fischer N. Hutcheson D. Geisler R. Strähle U. The UCS factor Steif/UNC-45b interacts with the heat shock protein Hsp90a during myofibrillogenesis.Dev. Biol. 2007; 308 (17586488): 133-14310.1016/j.ydbio.2007.05.014Crossref PubMed Scopus (90) Google Scholar, 8Wohlgemuth S.L. Crawford B.D. Pilgrim D.B. The myosin co-chaperone UNC-45 is required for skeletal and cardiac muscle function in zebrafish.Dev. Biol. 2007; 303 (17189627): 483-49210.1016/j.ydbio.2006.11.027Crossref PubMed Scopus (89) Google Scholar, 9Gazda L. Pokrzywa W. Hellerschmied D. Löwe T. Forné I. Mueller-Planitz F. Hoppe T. Clausen T. The myosin chaperone UNC-45 is organized in tandem modules to support myofilament formation in C. elegans.Cell. 2013; 152 (23332754): 183-19510.1016/j.cell.2012.12.025Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar10Ni W. Hutagalung A.H. Li S. Epstein H.F. The myosin-binding UCS domain but not the Hsp90-binding TPR domain of the UNC-45 chaperone is essential for function in Caenorhabditis elegans.J. Cell Sci. 2011; 124 (21914819): 3164-317310.1242/jcs.087320Crossref PubMed Scopus (31) Google Scholar). Mutations in UNC45B result in cardiomyopathies and defects in skeletal muscle (11Janiesch P.C. Kim J. Mouysset J. Barikbin R. Lochmüller H. Cassata G. Krause S. Hoppe T. The ubiquitin-selective chaperone CDC-48/p97 links myosin assembly to human myopathy.Nat. Cell Biol. 2007; 9 (17369820): 379-39010.1038/ncb1554Crossref PubMed Scopus (121) Google Scholar, 12Walker M.G. Pharmaceutical target identification by gene expression analysis.Mini. Rev. Med. Chem. 2001; 1 (12369984): 197-20510.2174/1389557013407034Crossref PubMed Scopus (29) Google Scholar). Although in vitro studies suggest that UNC45 isoforms share overlapping functions (e.g. myosin folding), zebrafish studies show that UNC45A is not required for myogenesis (13Comyn S.A. Pilgrim D. Lack of developmental redundancy between UNC45 proteins in zebrafish muscle development.PLoS ONE. 2012; 7 (23144999)e4886110.1371/journal.pone.0048861Crossref PubMed Scopus (19) Google Scholar). A recent study using the U2OS osteosarcoma cell line has shown that UNC45A promotes myosin folding and stress fibers assembly (14Lehtimäki J.I. Fenix A.M. Kotila T.M. Balistreri G. Paavolainen L. Varjosalo M. Burnette D.T. Lappalainen P. UNC-45a promotes myosin folding and stress fiber assembly.J. Cell Biol. 2017; 216 (29055011): 4053-407210.1083/jcb.201703107Crossref PubMed Scopus (22) Google Scholar). Biochemically, both isoforms interact with Hsp90 (5Barral J.M. Hutagalung A.H. Brinker A. Hartl F.U. Epstein H.F. Role of the myosin assembly protein UNC-45 as a molecular chaperone for myosin.Science. 2002; 295 (11809970): 669-67110.1126/science.1066648Crossref PubMed Scopus (201) Google Scholar, 15Chadli A. Graham J.D. Abel M.G. Jackson T.A. Gordon D.F. Wood W.M. Felts S.J. Horwitz K.B. Toft D. GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway.Mol. Cell. Biol. 2006; 26 (16478993): 1722-1730Crossref PubMed Scopus (51) Google Scholar), but UNC45A showed higher specificity toward Hsp90β (16Chadli A. Felts S.J. Toft D.O. GCUNC45 is the first Hsp90 co-chaperone to show α/β isoform specificity.J. Biol. Chem. 2008; 283 (18285346): 9509-951210.1074/jbc.C800017200Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). uncoordinated 45/Cro1/She4p heat shock protein nonmuscle myosin II checkpoint kinase 1 progesterone glucocorticoid receptor glucocorticoid response element transcription start site quantitative PCR nuclear extract NIMA-related kinase 7 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide non-obese diabetic/severe combined immunodeficiency short hairpin RNA hemagglutinin tissue microarray diaminobenzidine regions of interest minimal essential medium analysis of variance Hsp70/Hsp90 organizing protein green fluorescent protein. Cumulative evidence suggests that UNC45A contributes to tumorigenesis (15Chadli A. Graham J.D. Abel M.G. Jackson T.A. Gordon D.F. Wood W.M. Felts S.J. Horwitz K.B. Toft D. GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway.Mol. Cell. Biol. 2006; 26 (16478993): 1722-1730Crossref PubMed Scopus (51) Google Scholar, 17Bazzaro M. Santillan A. Lin Z. Tang T. Lee M.K. Bristow R.E. Shih IeM. Roden R.B. Myosin II co-chaperone general cell UNC-45 overexpression is associated with ovarian cancer, rapid proliferation, and motility.Am. J. Pathol. 2007; 171 (17872978): 1640-164910.2353/ajpath.2007.070325Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 18Guo W. Chen D. Fan Z. Epstein H.F. Differential turnover of myosin chaperone UNC-45A isoforms increases in metastatic human breast cancer.J. Mol. Biol. 2011; 412 (21802425): 365-37810.1016/j.jmb.2011.07.012Crossref PubMed Scopus (20) Google Scholar19Epping M.T. Meijer L.A. Bos J.L. Bernards R. UNC45A confers resistance to histone deacetylase inhibitors and retinoic acid.Mol. Cancer Res. 2009; 7 (19843631): 1861-187010.1158/1541-7786.MCR-09-0187Crossref PubMed Scopus (22) Google Scholar); its expression in cancer cells is correlated with the stage and the grade of the disease (17Bazzaro M. Santillan A. Lin Z. Tang T. Lee M.K. Bristow R.E. Shih IeM. Roden R.B. Myosin II co-chaperone general cell UNC-45 overexpression is associated with ovarian cancer, rapid proliferation, and motility.Am. J. Pathol. 2007; 171 (17872978): 1640-164910.2353/ajpath.2007.070325Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 18Guo W. Chen D. Fan Z. Epstein H.F. Differential turnover of myosin chaperone UNC-45A isoforms increases in metastatic human breast cancer.J. Mol. Biol. 2011; 412 (21802425): 365-37810.1016/j.jmb.2011.07.012Crossref PubMed Scopus (20) Google Scholar). UNC45A co-localizes with nonmuscle myosin II (NMII) in the cleavage furrow during cytokinesis (17Bazzaro M. Santillan A. Lin Z. Tang T. Lee M.K. Bristow R.E. Shih IeM. Roden R.B. Myosin II co-chaperone general cell UNC-45 overexpression is associated with ovarian cancer, rapid proliferation, and motility.Am. J. Pathol. 2007; 171 (17872978): 1640-164910.2353/ajpath.2007.070325Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and to centrosomes, to which it helps recruit checkpoint kinase 1 (ChK1) (20Jilani Y. Lu S. Lei H. Karnitz L.M. Chadli A. UNC45A localizes to centrosomes and regulates cancer cell proliferation through ChK1 activation.Cancer Lett. 2015; 357 (25444911): 114-12010.1016/j.canlet.2014.11.009Crossref PubMed Scopus (10) Google Scholar). Recent work from Bazzaro’s group (21Mooneyham A. Iizuka Y. Yang Q. Coombes C. McClellan M. Shridhar V. Emmings E. Shetty M. Chen L. Ai T. Meints J. Lee M.K. Gardner M. Bazzaro M. UNC-45A is a novel microtubule-associated protein and regulator of paclitaxel sensitivity in ovarian cancer cells.Mol. Cancer Res. 2019; 17 (30322860): 370-383Crossref PubMed Scopus (11) Google Scholar) showed that UNC45A is a microtubule-associated protein that modulates the sensitivity of ovarian cancer cells to paclitaxel. UNC45A expression in reporter gene systems (chloramphenicol acetyltransferase and luciferase) has shown that UNC45A can regulate transcription of the progesterone (PR) (15Chadli A. Graham J.D. Abel M.G. Jackson T.A. Gordon D.F. Wood W.M. Felts S.J. Horwitz K.B. Toft D. GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway.Mol. Cell. Biol. 2006; 26 (16478993): 1722-1730Crossref PubMed Scopus (51) Google Scholar), retinoic acid α, and peroxisome proliferator-activated receptor-α and -γ (19Epping M.T. Meijer L.A. Bos J.L. Bernards R. UNC45A confers resistance to histone deacetylase inhibitors and retinoic acid.Mol. Cancer Res. 2009; 7 (19843631): 1861-187010.1158/1541-7786.MCR-09-0187Crossref PubMed Scopus (22) Google Scholar). The molecular mechanisms underlying the function and localization of UNC45A in cancer cells, however, has not been well studied. Here, we report that UNC45A is largely dispensable for the proliferation of immortalized, nontransformed mammary cell lines, but is essential for breast cancer cell proliferation in vitro and in vivo. In cancer cells, UNC45A localizes to the cell nucleus, where it promotes glucocorticoid receptor (GR) transcription of the mitotic kinase gene NEK7. Previous research established that NEK7 is required for cell cycle progression because of its ability to regulate aspects of mitotic spindle formation and cytokinesis (22Yissachar N. Salem H. Tennenbaum T. Motro B. Nek7 kinase is enriched at the centrosome, and is required for proper spindle assembly and mitotic progression.FEBS Lett. 2006; 580 (17101132): 6489-649510.1016/j.febslet.2006.10.069Crossref PubMed Scopus (69) Google Scholar23Kim S. Lee K. Rhee K. NEK7 is a centrosomal kinase critical for microtubule nucleation.Biochem. Biophys. Res. Commun. 2007; 360 (17586473): 56-6210.1016/j.bbrc.2007.05.206Crossref PubMed Scopus (56) Google Scholar, 24Kim S. Kim S. Kim S. Rhee K. NEK7 is essential for centriole duplication and centrosomal accumulation of pericentriolar material proteins in interphase cells.J. Cell Sci. 2011; 124 (22100915): 3760-377010.1242/jcs.078089Crossref PubMed Scopus (31) Google Scholar, 25Salem H. Rachmin I. Yissachar N. Cohen S. Amiel A. Haffner R. Lavi L. Motro B. Nek7 kinase targeting leads to early mortality, cytokinesis disturbance and polyploidy.Oncogene. 2010; 29 (20473324): 4046-405710.1038/onc.2010.162Crossref PubMed Scopus (56) Google Scholar, 26O'Regan L. Fry A.M. The Nek6 and Nek7 protein kinases are required for robust mitotic spindle formation and cytokinesis.Mol. Cell. Biol. 2009; 29 (19414596): 3975-399010.1128/MCB.01867-08Crossref PubMed Scopus (123) Google Scholar, 27Bertran M.T. Sdelci S. Regué L. Avruch J. Caelles C. Roig J. Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5.EMBO J. 2011; 30 (21642957): 2634-264710.1038/emboj.2011.179Crossref PubMed Scopus (116) Google Scholar28Cullati S.N. Kabeche L. Kettenbach A.N. Gerber S.A. A bifurcated signaling cascade of NIMA-related kinases controls distinct kinesins in anaphase.J. Cell Biol. 2017; 216 (28630147): 2339-235410.1083/jcb.201512055Crossref PubMed Scopus (22) Google Scholar). Cancer cells lacking UNC45A expression exhibit a strikingly similar phenotype to NEK7 knockout cells, suggesting that UNC45A control of the cell cycle in cancer cells is mediated by NEK7 signaling. We investigated the role of UNC45A in the proliferation of breast cancer cell lines in vitro. Microscopic analysis indicates that silencing UNC45A using siRNA causes cell proliferation arrest and ultimately cell death in Hs578T cells (Fig. 1, A and B). In contrast, loss of UNC45A expression had no noticeable effect on the shape or proliferation of Hs578Bst cells; these results were confirmed using an MTT cell proliferation assay. Fig. 1C shows that silencing UNC45A did not affect the proliferation of any nontransformed cell line tested, including the Hs578Bst, HME, and MCF-10A mammary epithelial lines. In contrast, loss of UNC45A significantly reduced the proliferation of all transformed cell lines tested, including Hs578T and MDA-MB-231 (both triple negative), (MCF-7 (ER/PR-positive), and the metastatic ZR-75-1, further supporting the concept that UNC45A expression may be required for the growth of various breast cancer subtypes. To investigate the specific role of UNC45A in cancer cell proliferation in vivo, we used the non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mouse model (Fig. 1, E–G), surgically implanted with 105 MDA-MB-231 cells stably harboring lentivirus-based doxycycline-inducible UNC45A shRNA (or nontargeting shRNA control) into the mammary fat pads. Two days later, mice were fed doxycycline-containing food for 6 weeks. UNC45A was efficiently silenced in tumors harboring UNC45A versus control shRNA (Fig. 1G). Loss of UNC45A expression inhibited tumor growth, as illustrated by ex vivo images of tumors in control versus knockdown animals (Fig. 1E, left and right panels, respectively), as well as by the average tumor weights (Fig. 1F). Together, these findings demonstrate that UNC45A is essential for cancer cell proliferation in vitro and in vivo. To better understand how UNC45A might regulate cancer cell proliferation, we performed microarray analysis using mRNAs from Hs578T cells treated with either UNC45A siRNA or control nontargeting siRNA. As predicted, UNC45A mRNA was significantly reduced in UNC45A siRNA-treated Hs578T cells, which correlated with changes in the expression of 121 other genes, of which 62 exhibit at least a 1.64-fold or greater change (Fig. 2A, Table S1). Ingenuity Pathway Analysis indicates that these differentially expressed genes are involved in different pathologies, including: cancer, endocrine disorders, and inflammatory and cardiovascular diseases (Fig. 2B). Further analysis indicated that these genes are also connected to molecular networks involved in: cell-to-cell signaling and proliferation, cellular movement, molecular transport, and lipid metabolism (Fig. 2C). Some of the genes identified are also associated with control of cell cycle, cell morphology, multicellular organization, and development of the nervous system and its function (Fig. S1A). Based on its relevance to cell division, we noted that loss of UNC45A was associated with a 2-fold decrease in the mRNA encoding the mitotic kinase NEK7 (Fig. 2A, Table S1). Loss of NEK7 was validated by RT-qPCR and immunoblot analyses of a panel of cancer-derived cell lines. Silencing UNC45A drastically reduced mRNA and protein expression of NEK7 in Hsp578T, MDA-MB-453, MCF-7, and T47D breast cancer cells (Fig. 2D). Loss of NEK7 was also observed in HeLa cells and MDA-MB-231 (Fig. S1, C and E). Remarkably, whereas loss of UNC45A in nontransformed, immortalized breast epithelial cells (Hs578Bst and HME) significantly reduced NEK7 mRNA expression, this resulted in only a 50–70% decrease in NEK7 protein expression (Fig. 2D), suggesting that in normal cells, NEK7 expression is not totally dependent on UNC45A, whereas in cancer cells, UNC45A appears to be required for NEK7 expression. Indeed, the remaining NEK7 protein (30–50%) observed in normal cells might contribute to the relative insensitivity of these cells to the requirement of UNC45A for cell proliferation that we observed in cancer cells (Fig. 1C). Although NEK7 is one of 11 NEK family members (29Fry A.M. O'Regan L. Sabir S.R. Bayliss R. Cell cycle regulation by the NEK family of protein kinases.J. Cell Sci. 2012; 125 (23132929): 4423-443310.1242/jcs.111195Crossref PubMed Scopus (257) Google Scholar), our microarray data showed that only NEK7 expression is altered when UNC45A is silenced (Fig. 2A, Table S1). Loss of UNC45A did not significantly affect the transcription of other key components of the NEK signaling axis. This was validated by RT-qPCR showing that silencing UNC45A did not alter NEK6 mRNA levels in Hs578T, MCF-7, or MDA-MB-453 cells (Fig. 3A). Similarly, NEK9 patterns of expression were unchanged after UNC45A silencing in both MCF-7 and MDA-MB-453 cells. In Hs578T cells, however, loss of UNC45A significantly increased NEK9 mRNA expression, but it did not alter NEK9 protein expression (Fig. 3B). Similar results were obtained when HeLa cells were used (Fig. 3B). We next examined the impact of UNC45A loss on NEK7 signaling. We tested whether loss of UNC45A alters the phosphorylation/activation of the mitotic kinesin Eg5, an established downstream target of NEK6 and NEK7. Eg5 is a member of molecular motor proteins that play essential roles in mitosis through regulation of spindle assembly and function. Eg5 contributes to the establishment and balance of forces in the mitotic spindles and drives the sliding of microtubules (30Kapitein L.C. Peterman E.J. Kwok B.H. Kim J.H. Kapoor T.M. Schmidt C.F. The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks.Nature. 2005; 435 (15875026): 114-11810.1038/nature03503Crossref PubMed Scopus (508) Google Scholar, 31Ferenz N.P. Gable A. Wadsworth P. Mitotic functions of kinesin-5.Semin. Cell Dev. Biol. 2010; 21 (20109572): 255-25910.1016/j.semcdb.2010.01.019Crossref PubMed Scopus (119) Google Scholar). NEK6/7 phosphorylation of Eg5 at Ser-1033 is essential for Eg5 accumulation at the centrosomes, which is necessary for centrosomal separation and normal mitotic spindle formation (27Bertran M.T. Sdelci S. Regué L. Avruch J. Caelles C. Roig J. Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5.EMBO J. 2011; 30 (21642957): 2634-264710.1038/emboj.2011.179Crossref PubMed Scopus (116) Google Scholar, 32Sdelci S. Bertran M.T. Roig J. Nek9, Nek6, Nek7 and the separation of centrosomes.Cell Cycle. 2011; 10 (22064517): 3816-381710.4161/cc.10.22.18226Crossref PubMed Scopus (27) Google Scholar). As expected, UNC45A deletion drastically reduced NEK7 protein expression in Hs578T and HeLa cells (Fig. 3B). Accordingly, we also observed a drastic loss of phosphorylation as determined by the only available phosphospecific antibody recognizing active forms of both NEK7 and -6 (Fig. 3B). The loss of NEK7 translates into a significant decrease in phosphorylation of Eg5 at Ser-1033, previously shown to be the target site of NEK6/7 kinases (27Bertran M.T. Sdelci S. Regué L. Avruch J. Caelles C. Roig J. Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5.EMBO J. 2011; 30 (21642957): 2634-264710.1038/emboj.2011.179Crossref PubMed Scopus (116) Google Scholar). These findings suggest that UNC45A's function lies upstream of NEK7 and the signaling pathway controlling Eg5. Supporting this conclusion, knocking down NEK7 in Hs578T and HeLa cells did not affect UNC45A protein levels (Fig. S2A), and had no noticeable decrease in total Eg5 or NEK9 protein levels (Fig. 3C). To test the hypothesis that NEK7 might play a key role in UNC45A function during cell proliferation, we assessed whether expression of heterologous NEK7 could rescue the growth of cancer cells lacking UNC45A. pcDNA3 vector expressing mouse HA-tagged NEK7 (HA-mNEK7) was transfected into HeLa and MDA-MB-23 cells harboring doxycycline-inducible UNC45A shRNA. We initially treated cells with doxycycline for 5 days to completely silence endogenous UNC45A expression before transfecting them with the heterologous HA-mNEK7 construct. As shown in Fig. S1, B–E), transient expression of HA-mNEK7 in HeLa and MDA-MB-231 cells treated with doxycycline resulted in a significant rescue of cell proliferative capacity. In fact, when heterologous HA-mNEK7 is introduced earlier (48 h), before complete loss of UNC45A and NEK7 has occurred, cell proliferation capacity is even further enhanced (Fig. 3, D–G). These results strongly suggest that NEK7 is a major mediator of the cancer cell proliferation defect we observed in UNC45A-silenced cells. Because silencing UNC45A results in a significant decrease in NEK7 mRNA and protein levels selectively in cancer cells, as well as reduced phosphorylation of NEK7’s downstream target Eg5 in cancer cells, we reasoned that lack of UNC45A might lead to centrosomal separation defects and mitotic abnormalities, two phenotypes previously associated with NEK7 deletion in various cell lines (22Yissachar N. Salem H. Tennenbaum T. Motro B. Nek7 kinase is enriched at the centrosome, and is required for proper spindle assembly and mitotic progression.FEBS Lett. 2006; 580 (17101132): 6489-649510.1016/j.febslet.2006.10.069Crossref PubMed Scopus (69) Google Scholar23Kim S. Lee K. Rhee K. NEK7 is a centrosomal kinase critical for microtubule nucleation.Biochem. Biophys. Res. Commun. 2007; 360 (17586473): 56-6210.1016/j.bbrc.2007.05.206Crossref PubMed Scopus (56) Google Scholar, 24Kim S. Kim S. Kim S. Rhee K. NEK7 is essential for centriole duplication and centrosomal accumulation of pericentriolar material proteins in interphase cells.J. Cell Sci. 2011; 124 (22100915): 3760-377010.1242/jcs.078089Crossref PubMed Scopus (31) Google Scholar, 25Salem H. Rachmin I. Yissachar N. Cohen S. Amiel A. Haffner R. Lavi L. Motro B. Nek7 kinase targeting leads to early mortality, cytokinesis disturbance and polyploidy.Oncogene. 2010; 29 (20473324): 4046-405710.1038/onc.2010.162Crossref PubMed Scopus (56) Google Scholar, 26O'Regan L. Fry A.M. The Nek6 and Nek7 protein kinases are required for robust mitotic spindle formation and cytokinesis.Mol. Cell. Biol. 2009; 29 (19414596): 3975-399010.1128/MCB.01867-08Crossref PubMed Scopus (123) Google Scholar27Bertran M.T. Sdelci S. Regué L. Avruch J. Caelles C. Roig J. Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5.EMBO J. 2011; 30 (21642957): 2634-264710.1038/emboj.2011.179Crossref PubMed Scopus (116) Google Scholar). To test this hypothesis, we used a pericentrin antibody and immunofluorescence microscopy to examine the number and pattern of centrosomes in cells lacking UNC45A expression. As illustrated in Fig. 4A (lower panels), loss of UNC45A expression results in centrosome amplification (cells with more than 2 centrosomes), as well as defects in centrosome separation in Hs578T cells compared with control cells (Fig. 4A, upper panels). The overall average distance between centrosomes is significantly smaller in Hs578T, MCF-7, and HeLa cells lacking UNC45A (Fig. 4B). In contrast, centrosome number and separation in the nontransformed Hs578Bst cells is not significantly affected (Fig. 4B), consistent with the hypothesis that “residual” NEK7 expression in UNC45A-silenced normal cells might be protective (Fig. 1C). Further immunofluorescence analysis showed a strikingly disorganized pattern of pericentrosomal material in Hs578T cells lacking UNC45A compared with control cells (Fig. 4, C–E). The observed centrosomal abnormalities in UNC45A-deficient cancer cells correlated with loss of mitotic activity and an accumulation of polynucleated cells (Fig. 4C). As expected, silencing NEK7 in Hs578T (Fig. S2B) also induced accumulation of polynucleated cells (Fig. S2C, white arrowheads, and D) with micronuclei (Fig. S2C, red arrowheads). Further analysis showed that loss of NEK7 caused a drastic disorganization of centrosomal material in Hs578T cells (Fig. S2, E and F). Importantly, the abnormal centrosome phenotype induced by loss of UNC45A was significantly rescued when exogenous HA-mNEK7 was overexpressed in Hs578T cells lacking UNC45A (Fig. 4, F and G, and Fig. S3). Together, these findings suggest that UNC45A is essential for normal centrosome homeostasis, and provide further support for the notion that these functions are mediated by NEK7, a known centrosome regulator, in cancer cells. Similar results were obtained when HeLa cells were used. Indeed, loss of UNC45A causes drastic centrosomal abnormalities in HeLa cells leading to accumulation of multinucleated cells (Fig. 5, A–C). Additional insight into the specific role that UNC45A might play during cell division was obtained by time-lapse microscopy using HeLa cells expressing the fusion protein histone H2B-GFP (33Kanda T. Sullivan K.F. Wahl G.M. Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells.Curr. Biol. 1998; 8 (9545195): 377-38510.1016/S0960-9822(98)70156-3Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar). HeLa cells were transiently transfected with UNC45A siRNA (or nontargeting siRNA control), followed by time-lapse fluorescence microscopy after 72 h post-transfection (Movies S1 and S2). Cells treated with control siRNA progress to anaphase within 20–30 min of initiating metaphase, completing mitosis within 60–90 min (Movie S1, Fig. 5D, upper panel, and E, Fig. S4). In contrast, cells lacking UNC45A are delayed in mitosis and form multinucleated cells, which frequently undergo apoptosis (Movie S2, Fig. 5D, middle panels, and E, Fig. S4). During this aberrant, prolonged metaphase (∼120 min), chromosomes failed to align (Fig. 5D, middle panels, 30–165 min). In some cases, cells were unable to enter anaphase and instead retrogress to form an early metaphase-like state trying unsuccessfully to align their chromosomes. Ultimately, a few chromosomes remained lagging (Fig. 5D, middle panel, 105–165 min) and formed extra micronuclei (Fig" @default.
• W2912209648 created "2019-02-21" @default.
• W2912209648 creator A5000074630 @default.
• W2912209648 creator A5002135150 @default.
• W2912209648 creator A5002730078 @default.
• W2912209648 creator A5004084013 @default.
• W2912209648 creator A5006845225 @default.
• W2912209648 creator A5014614503 @default.
• W2912209648 creator A5016580028 @default.
• W2912209648 creator A5018945844 @default.
• W2912209648 creator A5020219621 @default.
• W2912209648 creator A5037279132 @default.
• W2912209648 creator A5037782182 @default.
• W2912209648 creator A5040217158 @default.
• W2912209648 creator A5046760570 @default.
• W2912209648 creator A5047770529 @default.
• W2912209648 creator A5048579921 @default.
• W2912209648 creator A5048793499 @default.
• W2912209648 creator A5071698177 @default.
• W2912209648 creator A5074041292 @default.
• W2912209648 creator A5079808476 @default.
• W2912209648 creator A5084951623 @default.
• W2912209648 creator A5088797284 @default.
• W2912209648 date "2019-04-01" @default.
• W2912209648 modified "2023-10-15" @default.
• W2912209648 title "The co-chaperone UNC45A is essential for the expression of mitotic kinase NEK7 and tumorigenesis" @default.
• W2912209648 cites W1944127002 @default.
• W2912209648 cites W1964718989 @default.
• W2912209648 cites W1975250295 @default.
• W2912209648 cites W1983689793 @default.
• W2912209648 cites W1986815595 @default.
• W2912209648 cites W1988139565 @default.
• W2912209648 cites W1990943500 @default.
• W2912209648 cites W1992925096 @default.
• W2912209648 cites W1998342895 @default.
• W2912209648 cites W1999722591 @default.
• W2912209648 cites W2009012143 @default.
• W2912209648 cites W2019312150 @default.
• W2912209648 cites W2021468144 @default.
• W2912209648 cites W2023253519 @default.
• W2912209648 cites W2028385416 @default.
• W2912209648 cites W2031337671 @default.
• W2912209648 cites W2031638259 @default.
• W2912209648 cites W2037812046 @default.
• W2912209648 cites W2045382123 @default.
• W2912209648 cites W2046038671 @default.
• W2912209648 cites W2053998644 @default.
• W2912209648 cites W2057400222 @default.
• W2912209648 cites W2070026305 @default.
• W2912209648 cites W2074338059 @default.
• W2912209648 cites W2074834552 @default.
• W2912209648 cites W2084858513 @default.
• W2912209648 cites W2094646101 @default.
• W2912209648 cites W2102444112 @default.
• W2912209648 cites W2103678187 @default.
• W2912209648 cites W2105779261 @default.
• W2912209648 cites W2114843025 @default.
• W2912209648 cites W2118639465 @default.
• W2912209648 cites W2124749085 @default.
• W2912209648 cites W2134539748 @default.
• W2912209648 cites W2134815855 @default.
• W2912209648 cites W2146314877 @default.
• W2912209648 cites W2148153922 @default.
• W2912209648 cites W2149441684 @default.
• W2912209648 cites W2160582391 @default.
• W2912209648 cites W2161270866 @default.
• W2912209648 cites W2162904618 @default.
• W2912209648 cites W2244650649 @default.
• W2912209648 cites W2602224300 @default.
• W2912209648 cites W2618454343 @default.
• W2912209648 cites W2720405934 @default.
• W2912209648 cites W2736733526 @default.
• W2912209648 cites W2766092740 @default.
• W2912209648 cites W2896150982 @default.
• W2912209648 doi "https://doi.org/10.1074/jbc.ra118.006597" @default.
• W2912209648 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6462532" @default.
• W2912209648 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30737284" @default.
• W2912209648 hasPublicationYear "2019" @default.
• W2912209648 type Work @default.
• W2912209648 sameAs 2912209648 @default.
• W2912209648 citedByCount "25" @default.
• W2912209648 countsByYear W29122096482019 @default.
• W2912209648 countsByYear W29122096482020 @default.
• W2912209648 countsByYear W29122096482021 @default.
• W2912209648 countsByYear W29122096482022 @default.
• W2912209648 countsByYear W29122096482023 @default.
• W2912209648 crossrefType "journal-article" @default.
• W2912209648 hasAuthorship W2912209648A5000074630 @default.
• W2912209648 hasAuthorship W2912209648A5002135150 @default.
• W2912209648 hasAuthorship W2912209648A5002730078 @default.
• W2912209648 hasAuthorship W2912209648A5004084013 @default.
• W2912209648 hasAuthorship W2912209648A5006845225 @default.
• W2912209648 hasAuthorship W2912209648A5014614503 @default.
• W2912209648 hasAuthorship W2912209648A5016580028 @default.
• W2912209648 hasAuthorship W2912209648A5018945844 @default.
• W2912209648 hasAuthorship W2912209648A5020219621 @default.
• W2912209648 hasAuthorship W2912209648A5037279132 @default.
• W2912209648 hasAuthorship W2912209648A5037782182 @default.

• next