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.

In invertebrates, UNC-45 regulates myosin stability and functions. Vertebrates have two distinct isoforms of the protein: UNC-45B, expressed in muscle cells only and UNC-45A, expressed in all cells and implicated in regulating both Non-Muscle Myosin II (NMII)- and microtubule (MT)-associated functions. Here we show that both, in vitro and in cells, UNC-45A binds to the MT lattice leading to MT bending, breakage and depolymerization. Furthermore, we show that UNC-45A destabilizes MTs independent of its NMII C-terminal binding domain and even in presence of the NMII inhibitor blebbistatin. These findings identified UNC-45A as a novel type of MT-severing protein with a not mutually exclusive but rather dual role in regulating NMII activity and MT stability. Because many human diseases, from cancer to neurodegenerative diseases, are caused by or associated with deregulation of MT stability our findings have profound implications in both, the biology of MTs as well as the biology of human diseases and possible therapeutic implications for their treatment.

UNC-45A is a novel regulator of neuronal differentiation. UNC-45A localizes at the growth cone, binds to NMIIA and NMIIB, and is disposable for neuronal survival but is required for neurite initiation and extension via regulating NMII activation. Thus UNC-45A is a potential master regulator of a number of NMII-mediated cellular processes.

Endometrial adenocarcinoma is the most common gynecologic malignancy in the United States. Most endometrial cancer cases are diagnosed at an early stage and have good prognosis. Unfortunately a subset of patients with early stage and low grade disease experience recurrence for reasons that remain unclear. Recurrence is often accompanied by chemoresistance and high mortality.Deubiquitinating enzymes (DUBs) are key components of the ubiquitin-dependent protein degradation pathway and act as master regulators in a number of metabolic processes including cell growth, differentiation, and apoptosis. DUBs have been shown to be upregulated in a number of human cancers and their aberrant activity has been linked to cancer progression, initiation and onset of chemoresistance. Thus, selective inhibition of DUBs has been proposed as a targeted therapy for cancer treatment.This study suggests the DUB USP14 as a promising biomarker for stratifying endometrial cancer patients at diagnosis based on their risk of recurrence. Further USP14 is expressed along with the marker of proliferation Ki67 in endometrial cancer cells in situ. Lastly, pharmacological targeting of USP14 with the FDA approved small-molecule inhibitor VLX1570, decreases cell viability in chemotherapy resistant endometrial cancer cells with a mechanism consistent with cell cycle arrest and caspase-3 mediated apoptosis.

UNC-45A is an ubiquitously expressed protein highly conserved throughout evolution. Most of what we currently know about UNC-45A pertains to its role as a regulator of the actomyosin system. However, emerging studies from both our and other laboratories support a role of UNC-45A outside of actomyosin regulation. This includes studies showing that UNC-45A: regulates gene transcription, co-localizes and biochemically co-fractionates with gamma tubulin and regulates centrosomal positioning, is found in the same subcellular fractions where MT-associated proteins are, and is a mitotic spindle-associated protein with MT-destabilizing activity in absence of the actomyosin system. Here, we extended our previous findings and show that UNC45A is variably expressed across a spectrum of cell lines with the highest level being found in HeLa cells and in ovarian cancer cells inherently paclitaxel-resistant. Furthermore, we show that UNC-45A is preferentially expressed in epithelial cells, localizes to mitotic spindles in clinical tumor specimens of cancer and co-localizes and co-fractionates with MTs in interphase cells independent of actin or myosin. In sum, we report alteration of UNC45A localization in the setting of chemotherapeutic treatment of cells with paclitaxel, and localization of UNC45A to MTs both in vitro and in vivo. These findings will be important to ongoing and future studies in the field that further identify the important role of UNC45A in cancer and other cellular processes.

max 250)UNC-45A is a ubiquitously expressed protein highly conserved throughout evolution.Most of what we currently know about UNC-45A pertains to its role as a regulator of the actomyosin system. However, emerging studies from both our and other laboratories support a role of UNC-45A outside of actomyosin regulation. This includes studies showing that UNC-45A:regulates gene transcription, co-localizes and biochemically co-fractionates with gamma tubulin and regulates centrosomal positioning, is found in the same subcellular fractions where MTassociated proteins are, and is a mitotic spindle-associated protein with MT destabilizing activity in absence of the actomyosin system.Here, we extended our previous findings and show that UNC45A is variably expressed across a spectrum of cell lines with the highest level being found in HeLa cells and in ovarian cancer cells inherently paclitaxel-resistant. Furthermore, we show that UNC-45A is preferentially expressed in epithelial cells, localizes to mitotic spindles in clinical tumor specimens of cancer and co-localizes and co-fractionates with MTs in interphase cells independent of actin or myosin.In sum, we report alteration of UNC45A localization in the setting of chemotherapeutic treatment of cells with paclitaxel, and localization of UNC45A to MTs both in vitro and in vivo.These findings will be important to ongoing and future studies in the field that further identify the important role of UNC45A in cancer and other cellular processes.

Despite advances in surgical technique and adjuvant treatment, endometrial cancer has recently seen an increase in incidence and mortality in the USA. The majority of endometrial cancers can be cured by surgery alone or in combination with adjuvant chemo- or radiotherapy; however, a subset of patients experience recurrence for reasons that remain unclear. Recurrence is associated with chemoresistance to carboplatin and paclitaxel and consequentially, high mortality. Understanding the pathways involved in endometrial cancer chemoresistance is paramount for the identification of biomarkers and novel molecular targets for this disease. Here, we generated the first matched pairs of carboplatin-sensitive/carboplatin-resistant and paclitaxel-sensitive/paclitaxel-resistant endometrial cancer cells and subjected them to bulk RNA sequencing analysis. We found that 45 genes are commonly upregulated in carboplatin- and paclitaxel-resistant cells as compared to controls. Of these, the leukemia inhibitory factor, (LIF), the protein tyrosine phosphatase type IVA, member 3 (PTP4A3), and the transforming growth factor beta 1 (TGFB1) showed a highly significant correlation between expression level and endometrial cancer overall survival (OS) and can stratify the 545 endometrial cancer patients in the TCGA cohort into a high-risk and low-risk-cohorts. Additionally, four genes within the 45 upregulated chemoresistance-associated genes are ADAMTS5, MICAL2, STAT5A, and PTP4A3 codes for proteins for which small-molecule inhibitors already exist. We identified these proteins as molecular targets for chemoresistant endometrial cancer and showed that treatment with their correspondent inhibitors effectively killed otherwise chemoresistant cells. Collectively, these findings underline the utility of matched pair of chemosensitive and chemoresistant cancer cells to identify markers for endometrial cancer risk stratification and to serve as a pharmacogenomics model for identification of alternative chemotherapy approaches for treatment of patients with recurrent disease.

Maintenance of proper cellular homeostasis requires constant surveillance and precise regulation of intracellular protein content. Protein monitoring and degradation is performed by two distinct pathways in a cell: the autophage-lysosome pathway and the ubiquitin-proteasome pathway. Protein degradation pathways are frequently dysregulated in multiple cancer types and can be both tumor suppressive and tumor promoting. This knowledge has presented the ubiquitin proteasome system (UPS) and autophagy as attractive cancer therapeutic targets. Deubiquitinating enzymes of the UPS have garnered recent attention in the field of cancer therapeutics due to their frequent dysregulation in multiple cancer types. The content of this chapter discusses reasoning behind and advances toward targeting autophagy and the deubiquitinating enzymes of the UPS in cancer therapy, as well as the compelling evidence suggesting that simultaneous targeting of these protein degradation systems may deliver the most effective, synergistic strategy to kill cancer cells.