UNC-45A breaks the microtubule lattice independently of its effects on non-muscle myosin II.

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, in vitro and in human and rat 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 C-terminal NMII-binding domain and even in the presence of the NMII inhibitor blebbistatin. These findings identified UNC-45A as a novel type of MT-severing protein with a dual non-mutually exclusive 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 the biology of MTs, as well as the biology of human diseases and possible therapeutic implications for their treatment.This article has an associated First Person interview with the joint first authors of the paper.

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

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.

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

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.

A Cell-Based Method for Identification of Chemotherapy Resistance Cancer Genes.

Here we describe a method for identifying genes and genetic pathways responsible for chemoresistance in cancer cells. The method is based on generation and characterization of matched pairs of chemotherapy-sensitive/chemotherapy-resistant cancer cell lines. In this protocol we are using endometrial cancer cell lines treated with carboplatin and paclitaxel, which are first-line chemotherapies for gynecologic malignancies. The chemoresistant cells and their chemosensitive counterparts are used for downstream applications including bulk RNA-sequencing analysis to identify a set of genes and pathways that are associated with chemoresistance. Identification of pathways responsible for innate or acquired chemoresistance is of paramount importance for the identification of biomarkers for cancer risk stratification and prognosis, and as a pharmacogenomics model for identification of alternative chemotherapy approaches for treatment of patients with recurrent and chemoresistant disease.

RNA Sequencing of Carboplatin- and Paclitaxel-Resistant Endometrial Cancer Cells Reveals New Stratification Markers and Molecular Targets for Cancer Treatment.

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.

Cavity Ringdown Spectrum of 2-Cyclohexen-1-one in the CO/Alkenyl CC Stretch Region of the S1(n, π*)-S0 Vibronic Band System.

The 2-cyclohexen-1-one (2CHO) molecule serves as a prototype for understanding the photochemical properties of conjugated enones. We have recorded the cavity ringdown (CRD) absorption spectrum of 2CHO vapor at room temperature over the 360-380 nm range. This portion of the spectrum encompasses the S1(n,π*) ← S0 vibronic band system in the region of the C═C and C═O stretch fundamentals. We have assigned about 40 vibronically resolved features in the spectrum, affording fundamental frequencies for 7 different vibrational modes in the S1(n,π*) state, including the C═C (1554 cm-1) and OC-CH (1449 cm-1) stretch modes. The C═O stretch character is spread over at least four different vibrational modes in the S1(n,π*) state, with fundamentals spanning the 1340-1430 cm-1 interval. This finding stems from a significant reduction in C═O bond order upon excitation, which leads to near-coincidence of the C═O stretch and several CH2 wag frequencies. Such complexities make 2CHO an ideal candidate for testing excited-state computational methods. We have used the present spectroscopic results to test EOM-EE-CCSD harmonic-frequency predictions for the S1(n,π*) state. We have also benchmarked the performance of less costly computational methods, including CIS(D) and TDDFT. For certain density functionals (e.g., B3LYP and PBE0), we find that the accuracy of TDDFT frequency predictions can approach but not meet that of EOM-EE-CCSD.

UNC-45A is required for neurite extension via controlling NMII activation.

UNC-45A is a highly conserved member of the UNC-45/CRO1/She4p family of proteins, which act as chaperones for conventional and nonconventional myosins. NMII mediates contractility and actin-based motility, which are fundamental for proper growth cone motility and neurite extension. The presence and role of UNC-45A in neuronal differentiation have been largely unknown. Here we demonstrate that UNC-45A is a novel growth cone--localized, NMII-associated component of the multiprotein complex regulating growth cone dynamics. We show that UNC-45A is dispensable for neuron survival but required for neurite elongation. Mechanistically, loss of UNC-45A results in increased levels of NMII activation. Collectively our results provide novel insights into the molecular mechanisms of neurite growth and define UNC-45A as a novel and master regulator of NMII-mediated cellular processes in neurons.

Targeting Deubiquitinating Enzymes and Autophagy in Cancer.

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.

USP14 is a predictor of recurrence in endometrial cancer and a molecular target for endometrial cancer treatment.

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.