UNC-45A, a highly conserved member of the UCS (UNC45A/CRO1/SHE4P) protein family of cochaperones, plays an important role in regulating cytoskeletal-associated functions in invertebrates and mammalian cells, including cytokinesis, exocytosis, cell motility, and neuronal development. Here, for the first time, UNC-45A is demonstrated to function as a mitotic spindle-associated protein that destabilizes microtubules (MT) activity. Using in vitro biophysical reconstitution and total internal reflection fluorescence microscopy analysis, we reveal that UNC-45A directly binds to taxol-stabilized MTs in the absence of any additional cellular cofactors or other MT-associated proteins and acts as an ATP-independent MT destabilizer. In cells, UNC-45A binds to and destabilizes mitotic spindles, and its depletion causes severe defects in chromosome congression and segregation. UNC-45A is overexpressed in human clinical specimens from chemoresistant ovarian cancer and that UNC-45A–overexpressing cells resist chromosome missegregation and aneuploidy when treated with clinically relevant concentrations of paclitaxel. Lastly, UNC-45A depletion exacerbates paclitaxel-mediated stabilizing effects on mitotic spindles and restores sensitivity to paclitaxel.
These findings reveal novel and significant roles for UNC-45A in regulation of cytoskeletal dynamics, broadening our understanding of the basic mechanisms regulating MT stability and human cancer susceptibility to paclitaxel, one of the most widely used chemotherapy agents for the treatment of human cancers.
The uncoordinated protein 45 (UNC-45) is a member of the UCS protein family (UNC-45/CRO1/She4p) of myosin cochaperones highly conserved throughout evolution (1–7). Although UNC-45 conservation suggests it has critical importance, its functions are still largely unknown. We and others have contributed to the understanding of the role of UNC-45A in mammalian cells within and outside its regulation of myosin activity. UNC-45A has been shown to control myoblast cell proliferation and its levels to drop as differentiation occurs (2). Recently, UNC-45A has been shown to promote myosin folding and stress fiber assembly (8). We have shown that UNC-45A controls nonmuscle myosin II (NMII)–associated functions in ovarian cancer cells (9), immune cells (10), and neurons (11) via regulating NMII activation and its binding to actin. We and others have also shown that in breast and ovarian cancers, UNC-45A is a cell-cycle–associated protein whose expression pattern correlates with poor clinical outcome (9, 12).
In cervical cancer cells, UNC-45A has been shown to regulate the progesterone receptor/Hsp90 pathway (13). Furthermore, colocalization and cellular fractionation studies using cervical cancer cells have revealed that UNC-45A is a novel centrosomal-associated protein (14). This, along with the fact that UNC-45A overexpression correlates with poor outcome in human cancers (9, 12), and that a significant contributor to poor patient outcomes is chemoresistance to the microtubule (MT)-stabilizing chemotherapy agent paclitaxel (15, 16), suggests that UNC-45A may play a role in regulating MT stability.
In this study, we show for the first time that UNC-45A overexpression is associated with paclitaxel resistance but not carboplatin resistance in ovarian cancer cell lines and clinical specimens of human ovarian tumors. We also show that UNC-45A is a mitotic spindle-associated protein, and that UNC-45A–overexpressing cancer cells escape chromosomal missegregation and aneuploidy when exposed to paclitaxel. Furthermore, UNC-45A depletion results in mitotic defects characterized by improper chromosome congression, segregation, and presence of multipolar spindles caused by hyperstable MTs. Mechanistically, total internal reflection fluorescence (TIRF) microscopy analysis revealed that UNC-45A is a MT-destabilizing protein capable of depolymerizing otherwise stable, paclitaxel-treated MTs in the absence of any other cellular components. Lastly, we show that UNC-45A restores the sensitivity of cancer cells to clinically relevant concentrations of paclitaxel via exacerbating paclitaxel-mediated stabilizing effects on cancer cells’ mitotic spindles. Taken together, our studies support the role of UNC-45A as a novel member of the MT-destabilizing protein family and as a molecular target for paclitaxel-resistant human cancers.
Materials and Methods
The 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (WST-1) was purchased from Cayman Chemicals. Propidium iodide (PI) was purchased from Sigma. 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Invitrogen. Paclitaxel was purchased from Teva Pharmaceuticals, and carboplatin was purchased from Segent Pharmaceuticals. Ro 3306 was purchased from Abcam.
The ovarian cancer cell line COV362 was a generous gift from Dr. Panagiotis A. Konstantinopoulos (Dana-Farber Cancer Institute, Boston, MA). The ovarian cancer cell lines OVSAHO, Kuramochi, and JHOS2 were a generous gift from Dr. Douglas Levine (Memorial Sloan Kettering Cancer Center, New York, NY). The ovarian cancer cell line SKOV-3, the cervical cancer cell line HeLa, and the fibroblast cell line NIH3T3 were purchased from the ATCC. Cell lines were cultured in DMEM supplemented with 10% fetal bovine serum.
Anti-UNC-45A (Enzo Life Sciences), anti-α-tubulin (Sigma), anti-acetylated-α-tubulin (Santa Cruz Biotechnology), anti-γ-tubulin (Sigma), anti-MCAK (GeneTex) were used. Peroxidase-linked anti-mouse immunoglobulin G and peroxidase-linked anti-rabbit immunoglobulin G were from Amersham. Texas Red–Goat anti-Mouse IgG, Texas Red–Goat anti-Rabbit IgG, FITC-Donkey and anti-Mouse IgG, peroxidase–goat anti-mouse IgG, peroxidase–goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories, Inc.
Modulation of UNC-45A expression levels in cells
For UNC-45A silencing or overexpression, scramble and UNC-45A shRNAs lentiviral supernatant or empty vector control and UNC-45A-GFP lentiviral supernatants were prepared and used to infect HeLa, fibroblasts, COV362, and SKOV-3 cell lines as we have previously described (10, 11). MCAK silencing was obtained via transfection with siRNA-MCAK (Ambion).
Immunofluorescence microscopy, image acquisition, and quantification
Cells were fixed in methanol for 5 minutes at −20°C. After blocking with 5% BSA in PBST, cells were stained with anti-UNC-45A, anti-α tubulin, anti-γ tubulin, or anti-acetylated α-tubulin primary antibodies followed by FITC- or Texas Red–conjugated secondary antibodies and analyzed via confocal fluorescence microscopy. Images were taken with an Olympus BX2 upright microscope equipped with a Fluoview 1000 confocal scan head. A UPlanApo N 60×/1.42 NA objective was used. FITC was excited with a 488-nm laser and emission collected between 505 and 525 nm. For Texas Red, a 543-nm laser was used for excitation and emission collected between 560 and 660 nm. Images were taken with sequential excitation. For all mitotic phenotype comparisons, analyzed cells were taken from same experiment dates with identical acquisition settings. Cells were synchronized using 5 μmol/L of Ro3306 for 20 hours followed by rescue with DMEM + 10% FBS. Images were analyzed using ImageJ software. Pole to pole distance was measured from the center of one pole, as identified by gamma tubulin staining, to the center of the next pole. Chromosome congression was measured from the widest metaphase plate area of each cell. Misaligned chromosomes were defined as chromosomes with a clear separation from the metaphase plate. Lagging chromosomes were defined as chromosomes trailing behind newly separated DNA in anaphase cells. Fluorescent intensity of the mitotic spindle was measured within a defined circular area containing the entirety of each spindle pole using ImageJ software.
Western blot analysis and immunoprecipitation
Total cellular protein (10–40 μg) from each sample was separated by SDS-PAGE, transferred to PVDF membranes and subjected to Western blot analysis. For coimmunoprecipitation, cells were lysed in lysis buffer (50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1% Nonidet P-40, 1 × protease inhibitor mixture, 1 × phosphatase inhibitor mixture), precleared and precipitated with primary antibody and protein A/G beads. Samples were subjected to Western blot analysis using the specified antibodies. Per each protein, Western blots were quantified within the linear range of detection (Supplementary Fig. S8).
MT cosedimentation assay in cells
Cells were lysed using 1% NP-40 lysis buffer containing 150 mmol/L NaCl, 50 mmol/L Tris-HCl pH 7.8, and protease inhibitor cocktail. The lysates were treated with either control DMSO or 1 μmol/L of taxol and incubated at 37°C for an hour. Lysates were spun at 15,000 rpm for 30 minutes at room temperature and the supernatant and pellet fractions were separated by SDS-PAGE and analyzed via Western blotting. Extent of UNC-45A association with polymerized MTs (pellet fraction) was determined by densitometric analysis using ImageJ software.
GFP-UNC-45A was cloned into pGEX-2TK to generate the GST-GFP-UNC-45A protein. The protein was expressed in Rosetta (DE3) pLysS, and following GST removal, it was affinity purified and dialyzed as we have previously described (17, 18).
Preparation of paclitaxel-stabilized MTs
The paclitaxel-stabilized MTs were prepared in a mixture that consisted of 8.3 μmol/L rhodamine-labeled tubulin, 24.7 μmol/L unlabeled tubulin, 1 mmol/L GTP, 4 mmol/L MgCl2, and 4% DMSO. The mixture was kept on ice for 5 minutes followed by 30-minute incubation at 37°C. After incubation, the mixture containing MTs was diluted into 390 μL warm Brb80 solution containing 10 μmol/L paclitaxel. The MTs were then spun down at 20 psi for 5 minutes. The MT pellet was then resuspended into 400 μL warm 10 μmol/L paclitaxel Brb80 solution and stored in 37°C incubator. All MTs were prepared on the day of experiment.
UNC-45A-GFP MT binding and depolymerization assay
The paclitaxel-stabilized MTs were prepared and flowed into the imaging chamber as we have previously described (17, 18). A final reaction mixture, containing 1× imaging buffer and 0.6 or 1.2 μmol/L final concentration of UNC-45A-GFP, was then introduced into the imaging chamber, and the interaction between UNC-45A-GFP and paclitaxel-stabilized MT was visualized via 488 nm and 561 nm lasers generated from Nikon TI-TIRF-PAU illuminator, which provided TIRF illumination. The images were collected from a Nikon CFI Apo TIRF 100× oil objective using an Andor iXon EMCCD camera. The time-lapse image collection was carried out automatically with 2.5-minute intervals for 1 hour at 10 preset locations on the imaging chamber. Laser power and exposure time were minimized while TIRF angle was maximized to avoid photobleaching and photodamage.
UNC-45A-GFP MT depolymerization image analysis
Individual paclitaxel-stabilized MT images were cropped from the 1-hour time-lapse image using ImageJ v1.49. The kymographs of individual MTs were then generated using ImageJ to illustrate the depolymerization process over time. The change in MT length was then calculated and divided by the total time span of the process to obtain the depolymerization rate. The units were converted from horizontal and vertical pixels on the kymograph into μm and minutes, respectively. The data points gathered at the three experimental conditions (control, 0.6 μmol/L UNC-45A and 1.2 μmol/L UNC-45A) were plotted separately in boxplots for comparison. Two-tailed Student t tests were performed to illustrate the significance.
Cell viability and proliferation rate assays
Cell viability was determined by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay as previously described (19). Briefly, cells were seeded at the concentration of 1,000 per well in 100 μL medium in 96-well plate and treated with the indicated concentrations of drugs. At the indicated time points, cells were incubated according to the manufacturer's protocol with the WST-1 labeling mixture for 2 hours. Formazan dye was quantified using a spectrophotometric plate reader to measure the absorbance at 450 nm (ELISA reader 190; Molecular Devices). Alternatively, cells were seeded in 6-well plates at a density of 30,000 cells per well and either mock treated or treated with 5 nmol/L of paclitaxel 24 hours after plating. Proliferation rate was determined by counting the cells over a period of 6 days via Trypan blue exclusion staining.
Measurements of intracellular levels of paclitaxel via high-performance liquid chromatography (HPLC)
COV362 ovarian cancer cells were grown in 10-cm dishes and treated with 100 nmol/L of paclitaxel for 18 hours, following which cells were trypsinized, pelleted, resuspended in 1 mL of ddH2O, and stored at −80°C. Thawed cells were homogenized in water, and the samples were applied to solid-phase extraction columns (Bond Elut C18, Agilent) and eluted with acetonitrile (1 mL). After the solvent was removed, the residue was lyophilized and reconstituted in the HPLC mobile phase (30% of 35 mmol/L acetic acid in ddH2O and 70% acetonitrile, 100 μL). A 50 μL injection was separated using isocratic 30% of 35 mmol/L acetic acid in ddH2O and 70% acetonitrile (2 mL/minute) on an Agilent 1200 Infinity series HPLC system with a Gemini 5μ C18 110A 4.6 Å–250 mm column. The paclitaxel signal (retention time 4.6 minutes) was monitored at 227 nm. Analyses were performed independently in triplicate. A standard curve was generated using untreated cell lysates that were spiked with paclitaxel in acetonitrile (250 nmol/L to 100 μmol/L, final concentrations) before solid-phase separation.
Archival tissues were used with the Institutional Review Board approval.
Five-micrometer-thick formalin-fixed, paraffin-embedded sections were deparaffinized and rehydrated by sequential washing with xylene, 100% ethanol, 95% ethanol, 80% ethanol, and PBS. For antigen retrieval, slides were immersed in Reveal Decloaker (Biocare Medical) and steamed for 30 minutes at 100°C. Endogenous peroxidase activity was blocked with 3% H2O2 for 10 minutes. After washing with PBS, slides were blocked with 10% normal goat serum in PBS for 10 minutes at room temperature, followed by incubation with rabbit anti-human polyclonal UNC-45 antibody (Proteintech Group Inc.) at a concentration of 1:200 in blocking solution overnight at 4°C. After washing twice with PBS, slides were incubated with a biotinylated anti-rabbit secondary antibody conjugated (10 minutes) and streptavidin/horseradish peroxidase (10 minutes; Dako), followed by 3,3-diaminobenzidine (Phoenix Biotechnologies) substrate for 3 minutes. Slides were lightly counterstained with Gill No. 3 hematoxylin (Sigma) for 60 seconds, dehydrated, and coverslipped. UNC-45A antibody validation was performed on COV-362 cell lines transduced with shRNA scramble or shRNA-UNC-45A and subject to IHC as above described. Immunostained slides were reviewed by a panel of five investigators blinded to the clinical outcome of the corresponding patients. The staining intensity was rated as follows: 0, no staining; 1+, weak intensity; 2+, moderate intensity; and 3+, high intensity.
Cell-cycle status following treatment with drug or vehicle alone was determined via flow cytometry analysis in cells harvested at indicated time points, fixed and stained with 0.1% (m/v) PI in PBST. Fluorescence was measured with a FACSCantoII flow cytometer (Becton Dickinson) and analyzed with FlowJo software.
Colony formation assay
The colony formation assay was performed in 6-well plates as we have previously described (20). Briefly, 10 days after plating, or when colonies of 30–50 cells were observed, colonies were either mock treated or treated with 5 nmol/L of paclitaxel continuously for 3 weeks by addition of paclitaxel to the feeder layer. Colony viability was assessed by phase contrast microscopy. Viable colonies appeared translucent and circular with regular borders. Viability was confirmed by isolation and mechanical dissociation of representative colonies in each condition followed by Trypan blue staining.
Results are reported as mean ± standard deviation of 3 or more independent experiments. Unless otherwise indicated, statistical significance of difference was assessed by two-tailed Student t test using Prism (V.4 GraphPad) and Excel. The level of significance was set at P < 0.05.
UNC-45A overexpression correlates with paclitaxel resistance in ovarian cancer
We and others have previously shown that UNC-45A overexpression correlates with patients' poor outcome in ovarian and breast cancers (9, 12). For ovarian cancer in particular, a significant contributor to poor outcome is recurrence after developing chemoresistance to the MT stabilizing agent paclitaxel (21–25). Thus, we set forth to determine whether UNC-45A overexpression is specifically associated with paclitaxel resistance in ovarian cancer. To this end, clinical specimens of ovarian cancer from patients who recurred and patients who did not recur were subjected to IHC for UNC-45A. Specifically, ovarian cancer chemoresistance is defined as the pathologic or radiologic evidence of tumor recurrence within 6 months from complete treatment. We found that clinical specimens derived from recurrent and chemoresistant patients had significantly higher UNC-45A levels as compared with chemosensitive patients who did not recur. Representative staining for UNC-45A expression in nonrecurrent and recurrent cancers is given in Fig. 1A. Quantification of staining intensity is given in Fig. 1B. The specificity of the UNC-45A antibody for IHC was validated using shRNA scramble and shRNA-UNC-45A in cancer cells (Supplementary Fig. S1). To directly assess whether UNC-45A overexpression was specifically associated with paclitaxel chemoresistance, we generated matched pairs of paclitaxel-sensitive and paclitaxel-resistant cells by exposing COV362 ovarian cancer cells to either mock (vehicle), or their IC50 concentration of paclitaxel for 3 cycles or continuously over a period of 3 weeks. At the end of the treatment, polyclonal cells were ∼5-fold less sensitive to paclitaxel than vehicle-treated cells, and clonal cells were between 15- and 20-fold less sensitive to paclitaxel than vehicle-treated cells (Fig. 1C). Importantly, chemoresistance was specific to paclitaxel, as sensitivity to a DNA-targeting chemotherapeutic agent, carboplatin, was not significantly different between these populations of cells (Fig. 1D). To exclude the possibility that the difference in IC50 values between the paclitaxel-sensitive and paclitaxel-resistant populations was the result of differential ability of paclitaxel to either enter or be retained by the cell, we measured the intracellular concentrations of the drug via HPLC. As shown in Fig. 1E, intracellular concentrations of paclitaxel were not significantly different. Next, we measured the expression levels of UNC-45A in paclitaxel-sensitive and paclitaxel-resistant (polyclonal, and clones #1 and #7) ovarian cancer cells and found that paclitaxel-resistant cell lines expressed greater levels of UNC-45A than their paclitaxel-sensitive counterparts (Fig. 1F). Next, we wanted to determine whether there was a correlation between intrinsic paclitaxel sensitivity and UNC-45A expression in a panel of ovarian cancer cell lines. To this end, UNC-45A levels and paclitaxel sensitivity were measured in Kuramochi, OVSAHO, JHOS2, and COV362 ovarian cancer cells. As shown in Fig. 1G, cells with lower (<5 nmol/L) IC50 to paclitaxel also had lower UNC-45A expression levels. Taken together, these data suggest that UNC-45A may be implicated in ovarian cancer cells' survival and resistance to the MT-stabilizing agent paclitaxel.
UNC-45A–overexpressing cells resist chromosome missegregation and aneuploidy when treated with clinically relevant concentrations of paclitaxel
Micromolar concentrations of paclitaxel kill mitotic cells by arresting them in the G2–M phase of the cell cycle (26–28). Recent evidence, however, indicates that clinically relevant concentrations of paclitaxel are in the low nanomolar range and cause chromosome missegregation on multipolar spindles resulting in aneuploidy, loss of essential chromosomal material, and cell death (3, 4). Thus, we initially determined the fate of ovarian cancer cells exposed to clinically relevant concentrations of paclitaxel. To this end, COV362 ovarian cancer cells were mock treated or treated with 5, 10, or 30 nmol/L of paclitaxel, and spindles per cell were counted following their visualization with α-tubulin antibody (Fig. 2A). We found that cells treated with paclitaxel accumulated multipolar spindles in a concentration-dependent manner (Fig. 2B). Next, we asked the question of whether UNC-45A–overexpressing cells that are paclitaxel resistant, resist chromosome missegregation and aneuploidy following paclitaxel treatment. To this end, sensitive and paclitaxel-resistant (clone #7) COV362 cells were treated with clinically relevant concentrations of paclitaxel, and the number of cells containing multipolar (>2) spindles was counted over time. As shown in Fig. 2C, COV362-sensitive cells accumulated multipolar spindles over time while resistant cells kept dividing on bipolar spindles. To further confirm these results, sensitive and resistant cells were exposed to 5 nmol/L paclitaxel and the mixoploid profile (<2n and >4n) was evaluated by FACS analysis. As shown in Fig. 2D, paclitaxel-sensitive cells accumulate a mixoploid profile over time while paclitaxel-resistant cells remain nearly diploid. Quantification of percentage of cells containing a mixoploid profile per condition is given in Fig. 2E. Similar results were obtained using paclitaxel-resistant polyclonal cells and clone #1 (Supplementary Fig. S2). Taken together, this suggests that paclitaxel resistance in UNC-45A–overexpressing ovarian cancer cells could be due to UNC-45A antagonizing the effects of paclitaxel by decreasing MT stability and avoiding multipolar mitotic spindles (29).
UNC-45A is a mitotic spindle-associated protein whose depletion results in impaired mitotic progression
We and others have previously shown that UNC-45A is overexpressed in human ovarian and breast cancers and that its loss results in reduced cell proliferation (9, 12). In osteosarcoma and cervical cancer cells, UNC-45A has recently been shown to colocalize with γ-tubulin at the MT organizing center (MTOC) and to control cell proliferation via regulating centrosomes positioning during mitosis (14). To gain further insight into the role of UNC-45A during cell division, we first asked whether, in addition to localization with γ-tubulin at the MTOC, UNC-45A could also colocalize with MTs during mitosis. To this end, we determined the subcellular localization of endogenous UNC-45A in both HeLa cancer cells and NIH3T3 fibroblasts during mitosis. As shown in Fig. 3, we found that UNC-45A colocalizes with mitotic MTs throughout cell division from prophase to telophase in both cancer cells (Fig. 3A) and fibroblasts (Fig. 3B). The same colocalization of UNC-45A with the mitotic spindle was found in COV362 ovarian cancer cells (Fig. 3C). The specificity of the UNC-45A antibody used in immunofluorescence experiments is given in Supplementary Fig. S3. Because mitotic spindles regulate cell division and because loss of UNC-45A has been shown to hinder cell proliferation in several human cancer cells, we evaluated the effect of UNC-45A depletion on mitotic progression.
To this end, UNC-45A was knocked down via lentiviral-mediated delivery of scramble or UNC-45A shRNA in HeLa cells, and the efficiency of the knock down was evaluated with Western blot analysis. As shown in Fig. 3D, we obtained approximately 80% of protein knockdown. Scramble and UNC-45A knockdown cells were stained for DAPI and analyzed for their mitotic index and for the percentage of metaphase versus mitotic cells (Fig. 3E). Loss of UNC-45A resulted in almost double the mitotic index (Fig. 3F) and almost a 30% increase in the percentage of metaphase cells over mitotic cells (Fig. 3G). Similar results were obtained using a second shRNA-UNC-45A (KD#2) and are shown in Supplementary Fig. S4A and S4B. Taken together, this suggests that UNC-45A is a novel mitotic spindle-associated protein that plays a crucial role during mitotic progression in both cancer and fibroblasts cells.
Loss of UNC-45A results in mitotic defects consistent with overly stable MTs
The effect of UNC-45A loss on the mitotic index and progression together with its localization with mitotic spindle suggests that UNC-45A may be a key regulator of spindle-associated functions. In this scenario, loss of UNC-45A would result in defects during chromosome congression and segregation (30, 31). To determine the effects of UNC-45A loss on chromosome congression, we measured pole-to-pole distance and metaphase plate width in scramble and UNC-45A knockdown HeLa cells subjected to γ-tubulin and DAPI staining (Fig. 4A). As shown in Fig. 4B, loss of UNC-45A resulted in approximately 50% increase in the distance between centrosomes as compared with control. Loss of UNC-45A also resulted in an almost double the metaphase plate width (Fig. 4C) as compared with control. To determine the effects of UNC-45A loss on chromosome alignment, scramble and UNC-45A knockdown HeLa cells were stained with DAPI (Fig. 4D), and the effects of UNC-45A depletion on chromosome alignment was evaluated by counting the number of cells whose chromosomes were clearly separated from the metaphase plate. As shown in Fig. 4E, loss of UNC-45A resulted in a 3-fold increase in the percentage of cells that had misaligned chromosomes as compared with control. To determine the effects of UNC-45A loss on chromosome segregation, scramble and UNC-45A knockdown HeLa cells were stained with DAPI (Fig. 4F) and the effect of UNC-45A depletion on segregation was evaluated by counting the number of cells whose chromosomes trailed behind newly separated DNA in anaphase. As shown in Fig. 4G, loss of UNC-45A resulted in nearly 3 times the percentage of cells having lagging chromosomes as compared with controls. Importantly, loss of UNC-45A also resulted in an increase in the number of cells having multipolar spindles (Fig. 4H). Specifically, the percentage of metaphase HeLa cells having multipolar spindles in the UNC-45A knockdown condition was 4-fold as compared with controls (Fig. 4I). Similar results were obtained using a second shRNA-UNC-45A (KD#2) and are shown in Supplementary Fig. S4C, S4D, and S4E. Importantly, we found that UNC-45A–overexpressing clinical specimens (from recurrent and chemoresistant patients) have less mitotic abnormalities as compared with specimens derived from patients who are nonrecurrent (Fig. 4J and K). This finding is consistent with a role for UNC-45A in counteracting the effects of paclitaxel on MTs in vivo.
Taken together, the loss of UNC-45A caused a phenotype that is similar to the one caused by clinically relevant doses of paclitaxel in cancer cells (30, 31), which is consistent with hyperstabilization of mitotic spindles.
UNC-45A is an MT-destabilizing protein
Because the loss of UNC-45A in cells results in a phenotype consistent with excessive mitotic spindle stabilization, we aimed to determine MT stability of spindles in scramble and UNC-45A knockdown cells. To this end, HeLa cancer cells (Fig. 5A) or NIHT3T3 fibroblasts (Fig. 5B) were transduced with either scramble or UNC-45A shRNA, and the MT stability was evaluated following staining for acetylated α-tubulin. Importantly, acetylation is an MT posttranslational modification associated with long-lived MTs and is therefore used as a marker for stable and long-lived MTs (32). Quantification of florescence intensity in each condition revealed that loss of UNC-45A resulted in nearly a 2-fold increase in spindle stability for both cancer cells (Fig. 5C) and fibroblasts (Fig. 5D), suggesting that the loss of UNC-45A may act as an MT-destabilizing protein and its loss results in excessive MT stabilization. Complementary experiments of UNC-45A overexpression revealed that an increase in UNC-45A expression is associated with significant reduction in the levels of acetylated α-tubulin, here used as a marker of spindle stability (Supplementary Fig. S5).
To directly test whether UNC-45A modulates MT stability and whether UNC-45A binds MTs in the absence of any other cellular factors, we used TIRF microscopy to evaluate the binding of purified UNC-45A-GFP (Supplementary Fig. S6) to paclitaxel-stabilized, rhodamine-labeled bovine brain MTs (Fig. 5E). We observed robust binding of green UNC-45A-GFP to the red MTs (Fig. 5F), suggesting that UNC45A binds MTs directly, without the requirement for any additional cellular factors. To determine whether UNC-45A acted to destabilize paclitaxel-stabilized MTs in our cell-free system, we collected time-lapse movies of paclitaxel-stabilized MTs, in the presence and absence of UNC-45A-GFP. We found that while taxol-stabilized MTs in the control movies without UNC-45A-GFP showed little or no depolymerization over 60 minutes of collecting data, addition of UNC-45A-GFP to the imaging chamber resulted in depolymerization of the MT over time (Fig. 5G). Because it appeared that higher concentrations of UNC-45A-GFP led to more rapid MT depolymerization, we quantified the average MT depolymerization rate for increasing concentrations of UNC-45A-GFP. We found that 0.6 μmol/L of UNC-45-GFP depolymerized MTs ∼5-fold faster than the controls, and 1.2 μmol/L of UNC-45A-GFP depolymerized MT ends ∼10-fold faster than the controls (Fig. 5H). Next, we determined whether endogenous UNC-45A binds to MTs in living cells. To this end, lysates from COV362 cells were either mock treated or treated with 1 μmol/L of taxol for 1 hour, and polymerized tubulin and associated proteins were separated in pellet and supernatant fractions via ultracentrifugation. As shown in Fig. 5I, after taxol treatment and tubulin polymerization, both UNC-45A and tubulin were found in the pellet fractions. Quantification of the percentage of UNC-45A bound to MTs showed that nearly 100% of UNC-45A was bound to the available polymerized MTs (Fig. 5J). Importantly, while UNC-45A was found to immunoprecipitate with Hsp90 (here used as an immunoprecipitation positive control; ref. 33), it did not precipitate with α-tubulin (Fig. 5K), suggesting that in vivo UNC-45A preferentially binds to polymerized MTs. Taken together, this strongly suggests that UNC-45A is an MT-destabilizing protein that directly destabilizes MTs in living cells and in vitro.
UNC-45A depletion exacerbates paclitaxel-mediated stabilizing effects on mitotic spindles and increases sensitivity to paclitaxel
Our data indicate that UNC-45A–overexpressing ovarian cancer cells resist cell death when exposed to the MT-stabilizing agent paclitaxel. Our data also indicate that UNC-45A is an MT-associated protein that acts as an MT destabilizer in cells and in vitro. Thus, we asked the question of whether UNC-45A–overexpressing, paclitaxel-resistant cells are characterized by endogenously less stable MTs. To this end, we measured the expression levels of acetylated-tubulin in paclitaxel-sensitive and paclitaxel-resistant (polyclonal, and clones #1 and #7) ovarian cancer cells and found that UNC-45A overexpression and paclitaxel chemoresistance was associated with reduction in MT stability as determined by Western blot analysis (Fig. 6A). To confirm that this was due to UNC-45A, we performed complementary experiments of UNC-45A knockdown and overexpression in COV362 ovarian cancer cells and showed that reduction of UNC-45A expression results in approximate 80% increase in acetylated-tubulin expression levels (Fig. 6B, left) while UNC-45A overexpression results in an over 60% decrease of acetylated-tubulin expression levels (Fig. 6B, right). Next, we asked the question of whether loss of UNC-45A would result in increased cancer cell sensitivity to paclitaxel. To this end, we first tested the effect of drug treatment on incidence of cells containing multipolar spindles in the presence and absence of UNC-45A. As shown in Fig. 6C and D, paclitaxel treatment following UNC-45A knockdown and γ-tubulin and DAPI staining resulted in a significantly higher percentage of cells containing multipolar spindles as compared with controls. Complementary experiments of UNC-45A overexpression (Fig. 6E and F) show that overexpressing UNC-45A cells remain mostly bipolar when exposed to increasing concentration of paclitaxel. This suggests that, similar to paclitaxel, loss of UNC-45A stabilizes MTs predisposing cancer cells to chromosome missegregation on multipolar spindles. Thus, we next tested whether depletion of UNC-45A would increase cancer cells' sensitivity to paclitaxel by exacerbating its effects on mitotic spindles. To this end, we measured the residual cell viability of scramble and UNC-45A knockdown COV362 ovarian cancer cells exposed to 5 nmol/L paclitaxel over a period of 6 days. As shown in Fig. 6G, depletion of UNC-45A increased cells' sensitivity to paclitaxel as compared with the control counterpart. Because tumors in vivo are tridimensional, we next evaluated the effect of UNC-45A loss on paclitaxel sensitivity in cancer cells grown as tridimensional structures. For these experiments, we used SKOV-3 ovarian cancer cells because they are known to form spheroids (34). Specifically, UNC-45A was knocked down in SKOV-3 ovarian cancer cells (Supplementary Fig. S7A) and scramble and UNC-45A knockdown cells were grown in soft agar for 10 days prior to being exposed to 5 nmol/L paclitaxel over a period of 3 weeks. At the end of this period, colonies in each condition were biopsied, and cell viability was evaluated via Trypan blue exclusion assay. As shown in Fig. 6H, UNC-45A depletion resulted in smaller colonies of cells that were significantly more sensitive to paclitaxel treatment as compared with control cells. Quantification of viable versus nonviable colonies in each condition is given in Fig. 6I. Because we and others have previously shown that loss of UNC-45A can affect the proliferation rate in some cancer cells (9, 12), we measured the daily proliferation rate of shRNA scramble and shRNA-UNC-45A COV362 and SKOV-3 cells. As shown in Supplementary Fig. S7B, reduction of UNC-45A levels resulted in a mild, yet significant decrease in cells' proliferation rate in both COV362 (left) and SKOV-3 (right) cells. In this context, the increased sensitivity to paclitaxel in UNC-45A knockdown cells is particularly striking considering that paclitaxel targets rapidly dividing cells.
Because our data show that UNC-45A is a novel MT-destabilizing protein, we tested whether the loss of one of the most well-known MT-destabilizing proteins, MCAK, would cause similar effects in terms of increasing paclitaxel sensitivity in ovarian cancer cells. For this purpose, control siRNA- and siMCAK-treated COV362 cells were subjected to Western blot analysis for MCAK and acetylated-tubulin expression. As shown in Fig. 6J and consistent with its effect as an MT-destabilizing protein, a less than 50% reduction in MCAK expression resulted in an almost 2-fold increase in the levels of acetylated tubulin, here used as a marker of MT stability. Importantly, further reduction of MCAK expression levels resulted in severe cell toxicity and death (data not shown). Next, siRNA scramble- and siRNA-MCAK–treated cells were exposed to 5 nmol/L paclitaxel, and residual cell viability was measured over a period of 3 days. As shown in Fig. 6K, partial loss of MCAK resulted in increased cell sensitivity to paclitaxel as compared with the control counterpart. Although UNC-45A knockdown in SKOV-3 cells did not result in reduction in the levels of the well-known MT-destabilizing protein MCAK (Supplementary Fig. S9A), it was accompanied by an increase in the acetylation levels of α-tubulin (Supplementary Fig. S9A) and an increase in the mitotic index and cells containing multipolar spindles (Supplementary Fig. S9B, left and right, respectively). Taken together, this suggests that UNC-45A plays a direct role in modulating ovarian cancer cell sensitivity to paclitaxel, its depletion restores cancer cell sensitivity to the drug, and the effect is mediated by perturbation of MT stability (Fig. 7).
The results of this study demonstrate that UNC-45A is a mitotic spindle-associated protein that acts as a regulator of mitotic progression via destabilizing MTs in vitro and in vivo in human cells. Our data also demonstrate that human cancer cells resistant to the MT stabilizer paclitaxel upregulate UNC-45A and that the abnormally hypostable MT levels conferred by UNC-45A to cancer cells allow them to escape the otherwise lethal effect of paclitaxel. Most importantly, we demonstrate that depletion of UNC-45A increases human cancer cell sensitivity to paclitaxel by exacerbating its mitotic spindle hyperstabilizing effects.
A number of studies, including studies from our laboratory, have revealed that in human cancer cells, UNC-45A is an NMII binding and colocalizing protein that plays a role in regulation of NMII folding, activity, and binding to actin (7–10, 12). Furthermore, we recently proposed that in neurons, UNC-45A is required for neurite extension (11). In this scenario, UNC-45A acts like an inhibitor of NMII activation, and its loss increases NMII contractility and F-actin retrograde flow at the growth cone (11, 26, 27, 35). Independent of NMII regulation, UNC-45A has been shown to be a modulator of the progesterone receptor/Hsp90 complex (13) and a centromosomal-associated protein in human cancer cells (14). Notably, one of the most well-known cell-cycle regulators, the cyclin-dependent kinase inhibitor 1B (p27), is both an MT- and NMII-associated protein and has been shown to regulate both NMII activity and MT stability in normal and cancer cells (26–29, 35–37).
Here, we show that UNC-45A is a novel MT- and spindle-associated protein with MT-destabilizing effects in vivo. Mechanistically, we show that UNC-45A binds directly to paclitaxel-stabilized MTs in the absence of any other cellular component and that it depolymerizes paclitaxel-stabilized MTs in a concentration-dependent manner, thus identifying UNC-45A as a novel MT depolymerase. Unlike many of the so far identified MTs depolymerizing or severing proteins, UNC-45A does not have an identified ATP-ase domain and has activity in the absence of ATP (38–40). This suggests that UNC-45A-mediated MT destabilization is ATP-independent and puts UNC-45A in a unique class of MT-destabilizing proteins.
Here, we also show that ovarian cancer patients who have developed chemoresistance to paclitaxel present with significantly higher levels of UNC-45A in their tumors as compared with patients who are sensitive to the drug. This holds true for matched pairs of paclitaxel-sensitive and paclitaxel-resistant ovarian cancer cells that were originated in the laboratory via exposure to paclitaxel over the period of several weeks. In these cells, paclitaxel, but not carboplatin resistance, is associated with higher UNC-45A levels. Importantly, the different sensitivity to paclitaxel was not due to differences in intracellular levels of the drug. This suggests that UNC-45A confers a selective survival advantage to cancer cells following paclitaxel exposure.
The role of paclitaxel in stabilizing MTs is well established. The effect of paclitaxel-mediated stabilization on cancer cells' cell cycle and ultimately cell death is less clear. High doses (in the micromolar range) of paclitaxel have been shown to stabilize MTs and kill cancer cells by causing a mitotic arrest in G2 phase of the cell cycle (26, 41, 42). On the other hand, lower doses (in the nanomolar range) of paclitaxel have been proposed to stabilize MTs and kill cancer cells due to chromosome missegregation on multipolar spindles (43, 44). Importantly, because paclitaxel is known to accumulate in cancer cells in vitro and in vivo (intratumorally, in patients), it has been recently proposed that therapeutically relevant concentrations of paclitaxel are in the nanomolar range (24, 45).
Here, we show that ovarian cancer cells die in a dose-dependent manner when exposed to therapeutically relevant concentrations of paclitaxel, and that cell death following treatment is preceded by the appearance of cells with multipolar spindles. This is consistent with what has been shown in breast cancer cells in vitro and in tumor samples in vivo (45). Because supernumerary centrosomes can lead to multipolar spindles (46), it has been proposed that an increase in MT stability via either loss of MT-destabilizing proteins or treatment with MT-stabilizing agent can lead to abnormal spindle pole formation, supernumerary centrosomes, and aneuploidy (27, 44, 47). We also show that paclitaxel-resistant, UNC-45A–overexpressing ovarian cancer cells maintain bipolar spindles and diploidy when exposed to paclitaxel. Furthermore, genetic silencing of UNC-45A restores cancer cells' sensitivity to paclitaxel via a mechanism that is consistent with exacerbation of the morphologic defects caused by paclitaxel on spindle MTs. This is consistent with what is observed in cancer cells following combination of anti-MT agents and kinesins (48). Importantly, additional MT-destabilizing proteins including the spleen tyrosine kinase (SYK; ref. 49) pathways have been shown to be aberrantly expressed in cancer cells resistant to paclitaxel, and SYK's pharmacologic inhibition has been shown to restore cancer cells' sensitivity to mitotic poisons.
To conclude, our results put UNC-45A as a key protein involved in regulating cell cycle, mitotic progression, MT stability, and human cancer behavior with regard to chemotherapy.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: A. Mooneyham, M. Bazzaro
Development of methodology: A. Mooneyham, Q. Yang, M. McClellan, M. Shetty, M. Gardner, M. Bazzaro
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Mooneyham, Q. Yang, C. Coombes, E. Emmings, M. Shetty, L. Chen, T. Ai, M.K. Lee, M. Bazzaro
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Mooneyham, Q. Yang, C. Coombes, E. Emmings, L. Chen, M. Gardner, M. Bazzaro
Writing, review, and/or revision of the manuscript: A. Mooneyham, Q. Yang, V. Shridhar, M.K. Lee, M. Gardner, M. Bazzaro
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Iizuka, M. Shetty, M. Gardner, M. Bazzaro
Study supervision: M. Bazzaro
Other (IHC staining of tissue): J. Meints
The authors are grateful to Dr. Henry Epstein (1944–2013) for his friendship and helpful discussions and to Dr. David Odde for providing helpful insights. We thank Guillermo Marques (University of Minnesota Imaging Center) for assistance with image analysis. This work was supported by Department of Defense Ovarian Cancer Research Program Grant OC160377, the Minnesota Ovarian Cancer Alliance, and the Randy Shaver Cancer Research Funds to M. Bazzaro. M. Gardner was supported by NIH grant NIGMS R01 GM-103833. A. Mooneyham was funded by the NIH T32 CA009138 training grant. The funders had no role in study design, data collection and analysis, and decision to publish or preparation of the manuscript.
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