Investigation of the role of UNC-45A in Paclitaxel-mediated microtubule stability

项目摘要 微管(MT)是紫杉醇的靶点，紫杉醇是治疗乳腺癌和卵巢癌最广泛的处方药。 紫杉醇通过稳定导致细胞死亡的微管起作用。无法预测和影响 癌症患者对紫杉醇的反应是一个主要的临床问题。UNC-45A是UCS的成员 肌球蛋白II辅助伴侣蛋白家族。最近的证据，包括我们最近公布的数据表明，在 UNC-45A除了调节肌球蛋白的收缩能力外，还是一种MT不稳定蛋白。在人类癌症中 UNC-45A过度表达与预后不良相关。导致患者预后不佳的一个重要因素是 紫杉醇的化疗耐药性。在这里，我们想要研究UNC-45A是否以及如何赋予癌细胞 紫杉醇治疗的细胞存活优势。目标1.确定UNC-45A在 对紫杉醇耐药的人类癌症。我们将(A)确定UNC-45A过度表达是否可以预测 卵巢癌和乳腺癌临床标本中紫杉醇的耐药性，以及(B)确定癌细胞中是否存在紫杉醇耐药 来自卵巢癌和乳腺癌患者的UNC-45A调节细胞对紫杉醇的敏感性。目标2. 测定UNC-45A调节MT的生物物理和细胞生物学参数。我们将(A) 测定UNC-45A在MTS上的结合、定位特性和MT不稳定效应 和没有紫杉醇的情况下，(B)进行计算建模以评估 UNC-45A的定位和MT的稳定性，以及(C)确定UNC-45A对活细胞中MT动态的影响。 目的3.确定UNC-45A对MTS影响的生化基础。我们将(A)确定北卡罗来纳大学是否- 45A在体外进行寡聚并作为寡聚体与MTS结合(B)，和(C)确定UNC-45A的结构域(S)，该结构域 参与MT结合、MT失稳和寡聚。 相关性：我们的研究将为紫杉醇耐药提供一个新的生物标志物和分子靶点 人类癌症，深刻理解这种新的不依赖于ATP的机制相互作用 蛋白质和MTS，以及开发包括小分子抑制剂在内的新治疗靶点的平台 蛋白质-蛋白质(低聚物)相互作用。

PROJECT ABSTRACT Microtubules (MT) are the target of paclitaxel, the most widely prescribed drug for the breast and ovarian cancers. Paclitaxel acts by stabilizing microtubules which results with cell death. The inability to predict and influence how cancer patients will respond to paclitaxel represents a major clinical problem. UNC-45A is a member of the UCS protein family of myosin II co-chaperones. Recent evidences including our recently published data show that, in addition to regulating actomyosin contractility, UNC-45A is also a MT destabilizing protein. In human cancers UNC-45A overexpression correlates with poor outcome. A significant contributor to poor patient outcomes is chemoresistance to paclitaxel. Here we want to investigate whether and how UNC-45A confers cancer cells’ a cellular survival advantage upon paclitaxel treatment. Aim 1. Determine the contribution of UNC-45A in paclitaxel resistant human cancers. We will (a) Determine whether UNC-45A overexpression is a predictor of paclitaxel resistance in ovarian and breast cancer clinical specimens, and (b) Determine whether in cancer cells derived from ovarian and breast cancer patients, UNC-45A regulates cells’ sensitivity to paclitaxel. Aim 2. Determine the biophysical and cell biological parameters of MT regulation by UNC-45A. We will (a) Determine the binding, localization properties and MT destabilizing effects of UNC-45A on MTs in the presence and absence of paclitaxel, (b) Perform computational modeling to evaluate a mechanistic connection between UNC-45A localization and MT stability and, (c) Determine the effects of UNC-45A on MT dynamics in living cells. Aim 3. Determine the biochemical basis of UNC-45A’s effect on MTs. We will (a) Determine whether UNC- 45A oligomerizes in vitro and binds to MTs as an oligomer (b), and (c) Determine the domain(s) of UNC-45A that are involved in MT-binding, MT-destabilization and oligomerization. RELEVANCE: Our studies will provide us with a new biomarker and molecular target for paclitaxel-resistant human cancers, a deep understanding of the mechanistic interaction between this novel ATP-independent protein and MTs, and a platform for development of novel therapeutic targets including small molecule inhibitors of protein-protein (oligomer) interactions.