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UNC-45A Is a Novel Microtubule-Associated Protein And Published OnlineFirst October 15, 2018; DOI: 10.1158/1541-7786.MCR-18-0670 Cell Cycle and Senescence Molecular Cancer Research UNC-45A Is a Novel Microtubule-Associated Protein and Regulator of Paclitaxel Sensitivity in Ovarian Cancer Cells Ashley Mooneyham1, Yoshie Iizuka1, Qing Yang2, Courtney Coombes2, Mark McClellan2, Vijayalakshmi Shridhar3, Edith Emmings1, Mihir Shetty1, Liqiang Chen4, Teng Ai4, Joyce Meints5, Michael K. Lee5, Melissa Gardner2, and Martina Bazzaro1 Abstract UNC-45A, a highly conserved member of the UCS congression and segregation. UNC-45A is overexpressed in (UNC45A/CRO1/SHE4P) protein family of cochaperones, human clinical specimens from chemoresistant ovarian plays an important role in regulating cytoskeletal-associat- cancer and that UNC-45A–overexpressing cells resist chro- ed functions in invertebrates and mammalian cells, includ- mosome missegregation and aneuploidy when treated with ing cytokinesis, exocytosis, cell motility, and neuronal clinically relevant concentrations of paclitaxel. Lastly, development. Here, for the first time, UNC-45A is demon- UNC-45A depletion exacerbates paclitaxel-mediated stabi- strated to function as a mitotic spindle-associated protein lizing effects on mitotic spindles and restores sensitivity to that destabilizes microtubules (MT) activity. Using in vitro paclitaxel. biophysicalreconstitutionandtotalinternalreflection fluo- rescence microscopy analysis, we reveal that UNC-45A Implications: These findings reveal novel and significant directly binds to taxol-stabilized MTs in the absence of any roles for UNC-45A in regulation of cytoskeletal dynamics, additional cellular cofactors or other MT-associated pro- broadening our understanding of the basic mechanisms teins and acts as an ATP-independent MT destabilizer. In regulating MT stability and human cancer susceptibility to cells, UNC-45A binds to and destabilizes mitotic spindles, paclitaxel, one of the most widely used chemotherapy and its depletion causes severe defects in chromosome agents for the treatment of human cancers. Introduction controls nonmuscle myosin II (NMII)–associated functions in ovarian cancer cells (9), immune cells (10), and neurons (11) The uncoordinated protein 45 (UNC-45) is a member of the via regulating NMII activation and its binding to actin. We and UCS protein family (UNC-45/CRO1/She4p) of myosin cocha- others have also shown that in breast and ovarian cancers, perones highly conserved throughout evolution (1–7). UNC-45A is a cell-cycle–associated protein whose expression Although UNC-45 conservation suggests it has critical impor- pattern correlates with poor clinical outcome (9, 12). tance, its functions are still largely unknown. We and others In cervical cancer cells, UNC-45A has been shown to regulate havecontributedtotheunderstanding of the role of UNC-45A the progesterone receptor/Hsp90 pathway (13). Furthermore, in mammalian cells within and outside its regulation of myosin colocalization and cellular fractionation studies using cervical activity. UNC-45A has been shown to control myoblast cell cancer cells have revealed that UNC-45A is a novel centrosomal- proliferation and its levels to drop as differentiation occurs (2). associated protein (14). This, along with the fact that UNC-45A Recently, UNC-45A has been shown to promote myosin fold- overexpression correlates with poor outcome in human cancers ing and stress fiber assembly (8). We have shown that UNC-45A (9, 12), and that a significant contributor to poor patient out- comes is chemoresistance to the microtubule (MT)-stabilizing 1Masonic Cancer Center and Department of Obstetrics, Gynecology and chemotherapy agent paclitaxel (15, 16), suggests that UNC-45A Women's Heath, University of Minnesota, Minneapolis, Minnesota. 2Department may play a role in regulating MT stability. of Genetics, Cell Biology, and Development, University of Minnesota, In this study, we show for the first time that UNC-45A over- Minneapolis, Minnesota. 3Department of Experimental Pathology, Mayo Clinic expression is associated with paclitaxel resistance but not carbo- College of Medicine, Rochester, Minnesota. 4Center for Drug Design, Academic 5 platin resistance in ovarian cancer cell lines and clinical specimens Health Center, University of Minnesota, Minneapolis, Minnesota. Department of of human ovarian tumors. We also show that UNC-45A is a Neuroscience, University of Minnesota Minneapolis, Minnesota. mitotic spindle-associated protein, and that UNC-45A–overex- Note: Supplementary data for this article are available at Molecular Cancer pressing cancer cells escape chromosomal missegregation and Research Online (http://mcr.aacrjournals.org/). aneuploidy when exposed to paclitaxel. Furthermore, UNC-45A Corresponding Author: Martina Bazzaro, University of Minnesota, 515 Delaware depletion results in mitotic defects characterized by improper Street SE, Minneapolis, MN 55455. Phone: 612-625-2889; Fax: 612-626-0665; chromosome congression, segregation, and presence of multipo- E-mail: [email protected] lar spindles caused by hyperstable MTs. Mechanistically, total doi: 10.1158/1541-7786.MCR-18-0670 internal reflection fluorescence (TIRF) microscopy analysis Ó2018 American Association for Cancer Research. revealed that UNC-45A is a MT-destabilizing protein capable of 370 Mol Cancer Res; 17(2) February 2019 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst October 15, 2018; DOI: 10.1158/1541-7786.MCR-18-0670 Paclitaxel Sensitivity Is Regulated by UNC-45A depolymerizing otherwise stable, paclitaxel-treated MTs in the collected between 505 and 525 nm. For Texas Red, a 543-nm absence of any other cellular components. Lastly, we show that laser was used for excitation and emission collected between UNC-45A restores the sensitivity of cancer cells to clinically 560 and 660 nm. Images were taken with sequential excitation. relevant concentrations of paclitaxel via exacerbating paclitaxel- For all mitotic phenotype comparisons, analyzed cells were mediated stabilizing effects on cancer cells' mitotic spindles. taken from same experiment dates with identical acquisition Taken together, our studies support the role of UNC-45A as a settings. Cells were synchronized using 5 mmol/L of Ro3306 for novel member of the MT-destabilizing protein family and as a 20 hours followed by rescue with DMEM þ 10% FBS. Images molecular target for paclitaxel-resistant human cancers. were analyzed using ImageJ software. Pole to pole distance was measured from the center of one pole, as identified by gamma Materials and Methods tubulin staining, to the center of the next pole. Chromosome congression was measured from the widest metaphase plate Chemicals area of each cell. Misaligned chromosomes were defined as The 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetra- chromosomes with a clear separation from the metaphase zolium-5-carboxanilide inner salt (WST-1) was purchased from plate. Lagging chromosomes were defined as chromosomes Cayman Chemicals. Propidium iodide (PI) was purchased from 0 trailing behind newly separated DNA in anaphase cells. Fluo- Sigma. 4 ,6-diamidino-2-phenylindole (DAPI) was purchased rescent intensity of the mitotic spindle was measured within a from Invitrogen. Paclitaxel was purchased from Teva Pharmaceu- defined circular area containing the entirety of each spindle ticals, and carboplatin was purchased from Segent Pharmaceu- pole using ImageJ software. ticals. Ro 3306 was purchased from Abcam. Cell lines Western blot analysis and immunoprecipitation – The ovarian cancer cell line COV362 was a generous gift from Total cellular protein (10 40 mg) from each sample was sep- Dr. Panagiotis A. Konstantinopoulos (Dana-Farber Cancer Insti- arated by SDS-PAGE, transferred to PVDF membranes and sub- tute, Boston, MA). The ovarian cancer cell lines OVSAHO, Kur- jected to Western blot analysis. For coimmunoprecipitation, cells amochi, and JHOS2 were a generous gift from Dr. Douglas Levine were lysed in lysis buffer (50 mmol/L Tris, pH 7.4, 150 mmol/L Â Â (Memorial Sloan Kettering Cancer Center, New York, NY). The NaCl, 1% Nonidet P-40, 1 protease inhibitor mixture, 1 ovarian cancer cell line SKOV-3, the cervical cancer cell line HeLa, phosphatase inhibitor mixture), precleared and precipitated with and the fibroblast cell line NIH3T3 were purchased from the primary antibody and protein A/G beads. Samples were subjected fi ATCC. Cell lines were cultured in DMEM supplemented with 10% to Western blot analysis using the speci ed antibodies. Per each fi fetal bovine serum. protein, Western blots were quanti ed within the linear range of detection (Supplementary Fig. S8). Antibodies Anti-UNC-45A (Enzo Life Sciences), anti-a-tubulin (Sigma), MT cosedimentation assay in cells anti-acetylated-a-tubulin (Santa Cruz Biotechnology), anti- Cells were lysed using 1% NP-40 lysis buffer containing g-tubulin (Sigma), anti-MCAK (GeneTex) were used. Peroxi- 150 mmol/L NaCl, 50 mmol/L Tris-HCl pH 7.8, and protease dase-linked anti-mouse immunoglobulin G and peroxidase- inhibitor cocktail. The lysates were treated with either control linked anti-rabbit immunoglobulin G were from Amersham. DMSO or 1 mmol/L of taxol and incubated at 37 C for an hour. Texas Red–Goat anti-Mouse IgG, Texas Red–Goat anti-Rabbit Lysates were spun at 15,000 rpm for 30 minutes at room tem- IgG, FITC-Donkey and anti-Mouse IgG, peroxidase–goat anti- perature and the supernatant and pellet fractions were separated mouse IgG, peroxidase–goat
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