UNC-45A Is a Novel Microtubule-Associated Protein and Regulator of Paclitaxel Sensitivity in Ovarian Cancer Cells

Abstract

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. Implications: 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.

UNC-45A is preferentially expressed in epithelial cells and binds to and co-localizes with interphase MTs

“…We and others have previously shown that UNC-45A is a mitotic spindle-associated protein in a number of cell types including cancer cells and fibroblasts and that it has a predominantly cytoplasmic subcellular localization 5,[14][15][16] . We and others have also previously shown that UNC-45A is overexpressed in cancer versus benign tissue and in cancer cell lines versus normal cells.…”

“…10,11 This is consistent with our recently published report suggesting that UNC-45A could be a marker of chemoresistance. 5 We had previously shown that endogenous UNC-45A binds to polymerized MTs and not to free tubulin in COV-362 ovarian cancer cells. 5 Here we used a standard cosedimentation assay, which is one of the most commonly used approaches for characterizing and quantifying the ability of a protein to bind MTs, 12 to determine whether endogenous UNC-45A cofractionates with MTs in living cells.…”

“…5 We had previously shown that endogenous UNC-45A binds to polymerized MTs and not to free tubulin in COV-362 ovarian cancer cells. 5 Here we used a standard cosedimentation assay, which is one of the most commonly used approaches for characterizing and quantifying the ability of a protein to bind MTs, 12 to determine whether endogenous UNC-45A cofractionates with MTs in living cells. In this assay, cells are treated (or not, vehicle) with taxol so to increase the fraction of MTs and microtubule-associatedproteins (MAPs), and the NP-40-resistant cytoskeletal ghosts are separated from the soluble fraction via ultracentrifugation.…”

“…In our work to date, we have used several anti-UNC45A antibodies, from different source companies. We identified the need to streamline use of anti-UNC45A antibodies testing for future studies, and thus sought to provide a biochemical validation of two of the most commonly used commercially available UNC-45A antibodies: the rabbit polyclonal anti-UNC-45A from Proteintech, which we have previously used to show that UNC-45A is a mitotic spindle-associated protein, 5 and the mouse polyclonal anti-UNC-45A from Abnova, which was used to show that UNC-45A is required for stress fibers formation . 6 As shown in Figure 1a, full membrane Western blot revealed that the Proteintech antibody detects a single band corresponding to UNC-45A in lysates of HeLa (human cervical cancer) and RFL-6 (rat lung fibroblast) cell lines.…”

“…Proteomic analysis of HeLa cells shows that not only is UNC-45A in the subcellular fractions where other well-known MTassociated and destabilizing proteins are found but that it is approximately 20-fold more abundant (0.4 uM) than other MT-destabilizing proteins. 7 In light of our recent report that UNC-45A is an MT-destabilizing protein, 5 this suggests that UNC-45A may have a dominant role in the regulation of cellular MTs and that UNC-45A levels may vary across multiple cell types. Here we set forth to determine endogenous UNC-45A protein levels by Western blot analysis in commonly used cell lines including the cervical cancer cell line HeLa, the ovarian cancer cell line SKOV-3, the rat fibroblast RFL-6 cell line and in a panel of ovarian cancer cell lines: ES-2, TOV-21G, JHOC5, OVTOKO, RMGI and ES-2.…”