Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

肿瘤细胞多药耐药表型的遗传分析

• 批准号: 10702284
• 负责人: Michael Gottesman
• 金额: $ 60.75万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702284
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP Hydrolysis; ATP-Binding Cassette Transporters; Amino Acids; Binding; Binding Sites; Biological Models; Blood - brain barrier anatomy; Cell Line; Cells; Chemicals; Clinical; Collaborations; Cryoelectron Microscopy; Crystallography; Cytotoxic agent; Doxorubicin; Drug Efflux; Drug resistance; Failure; Family; Genes; Goals; Histone Deacetylase Inhibitor; Human; Laboratories; Malignant Neoplasms; Modeling; Molecular; Molecular Conformation; Molecular Profiling; Multi-Drug Resistance; Multidrug Resistance Gene; Mus; Natural Product Drug; Natural Products; Paclitaxel; Patients; Pattern; Pharmaceutical Preparations; Pharmacopoeias; Phenotype; Physiological; Population; Process; Property; Proteins; Refractory; Research Personnel; Resistance; Rhodamine; Role; Sampling; Site; Solubility; Specificity; Structure; Structure-Activity Relationship; System; Thiosemicarbazones; Transmembrane Domain; Vinca Alkaloids; Work; analog; cancer cell; chemotherapeutic agent; chemotherapy; genetic analysis; high throughput screening; human tissue; improved; inhibitor; insight; kinase inhibitor; member; multi drug transporter; multidrug resistant cancer; neoplastic cell; novel strategies; personalized approach; predictive signature; resistance mechanism; therapy resistant; tool; treatment response; uptake

Resistance to chemotherapy occurs in cancer cells because of intrinsic or acquired changes in expression of specific proteins. We have studied resistance to natural product chemotherapeutic agents such as doxorubicin, Vinca alkaloids, and taxol and more recently, histone deacetylase inhibitors and targeted kinase inhibitors. In most cases, cells become simultaneously resistant to multiple drugs because of reductions in intracellular drug concentrations. For the natural product drugs, this cross-resistance is frequently due to expression of an energy-dependent drug efflux system (ABC transporter) known as P-glycoprotein (P-gp), the product of the MDR1 or ABCB1 gene, or to other members of the ABC transporter family, including ABCG2 and ABCB5. Work from our laboratory and others has revealed that some drugs are more toxic to P-gp-expressing cells than to non-expressors, suggesting a novel approach to treatment of MDR cancers. Several different chemical classes with this property, including thiosemicarbazones (e.g., NSC73306), have been identified. A quantitative structure activity analysis of NSC73306 analogs, a further correlation analysis in the NCI-60 cell lines, and a high-throughput screen for compounds in the U.S. Pharmacopeia that kill P-gp-expressing cells have yielded many additional compounds with improved ability to kill selectively P-gp-expressing cells, but also with improved solubility properties. To understand how the structure of P-gp determines its polyspecificity and how specificity is altered with changes in folding, we have collaborated with other senior investigators in the LCB, including Di Xia, Suresh Ambudkar, and Sriram Subramaniam. Cryo-EM studies have demonstrated that apo P-gp has a dynamic structure in which the two ATP-binding sites are either separated or close together. Binding of ATP fixes the conformation of P-gp in the latter state and ATP hydrolysis results in separation of the ATP sites. Crystallography studies using mouse P-gp as a model show that the separation between the ATP sites determines the pitch of the transmembrane (TM) helices where substrates bind, suggesting the hypothesis that as the ATP sites move together and apart, the TM helices expose different residues that enable binding to many different substrates. Studies on mouse-human chimeric P-gps have revealed similar structure-function relationships for these two evolutionarily related transporters. In collaboration with the group of Suresh Ambudkar, we have examined the basis of directional transport of compounds out of cells by P-glycoprotein. These studies have revealed a set of amino acid residues in the transmembrane regions of P-glycoprotein which can be altered to change the direction of transport of certain rhodamine compounds from out of the cell to into the cell. This process is concentration- and ATP-dependent, and gives important insight into how directionality of transport is determined in P-glycoprotein. We have used AML as one model system to determine the clinical role of ABC transporters in drug resistance. In one study, samples from the same patients before and after chemotherapy were analyzed. In this case, resistance in each case shows a different pattern of expression of ABC genes and other MDR genes, suggesting that individualized approaches to resistance to therapy will be needed. A more detailed analysis of a large population of primary refractory AMLs has shown that there are 3 molecular signatures that predict poor response to therapy. One of these is associated with increased expression of ABCG2. These results argue that clinical samples must be stratified to facilitate effective targeting of inhibitors of ABC transporters to circumvent drug resistance.

化疗耐药发生在癌细胞中，是因为特定蛋白质表达的内在或获得性变化。我们已经研究了对天然产物化疗药物的耐药性，如阿霉素、长春花生物碱和紫杉醇，以及最近的组蛋白去乙酰化酶抑制剂和靶向激酶抑制剂。在大多数情况下，由于细胞内药物浓度降低，细胞同时对多种药物产生耐药性。对于天然产物药物，这种交叉耐药通常是由于被称为p -糖蛋白（P-gp）的能量依赖性药物外排系统（ABC转运蛋白）的表达，p -糖蛋白是MDR1或ABCB1基因的产物，或ABC转运蛋白家族的其他成员，包括ABCG2和ABCB5。我们的实验室和其他实验室的研究表明，一些药物对表达p- gp的细胞的毒性比不表达p- gp的细胞更大，这提示了一种治疗耐多药癌症的新方法。已经确定了几种具有这种性质的不同化学类别，包括硫代氨基脲（例如NSC73306）。通过对NSC73306类似物的定量结构活性分析，对NCI-60细胞系的进一步相关性分析，以及对美国药书中杀死表达p- gp细胞的化合物的高通量筛选，我们发现了许多额外的化合物，它们具有更好的选择性杀死表达p- gp细胞的能力，而且还具有更好的溶解度。为了了解P-gp的结构如何决定其多特异性，以及特异性如何随着折叠的变化而改变，我们与LCB的其他高级研究人员（包括Di Xia， Suresh Ambudkar和Sriram Subramaniam）合作。Cryo-EM研究表明载脂蛋白P-gp具有两个atp结合位点分离或靠近的动态结构。ATP的结合使P-gp的构象处于后一种状态，ATP的水解导致ATP位点的分离。以小鼠P-gp为模型的晶体学研究表明，ATP位点之间的分离决定了底物结合的跨膜（TM）螺旋的间距，这表明，当ATP位点一起或分开移动时，TM螺旋暴露出不同的残基，使其能够与许多不同的底物结合。对小鼠-人嵌合P-gps的研究揭示了这两种进化相关转运体的相似结构-功能关系。在与Suresh Ambudkar小组的合作中，我们研究了p -糖蛋白将化合物定向运输出细胞的基础。这些研究揭示了p糖蛋白跨膜区域的一组氨基酸残基，可以改变某些罗丹明化合物从细胞外到细胞内的运输方向。这个过程是浓度和atp依赖的，并提供了重要的洞察如何在p糖蛋白中确定运输的方向性。我们使用AML作为一个模型系统来确定ABC转运蛋白在耐药性中的临床作用。在一项研究中，对同一患者化疗前后的样本进行了分析。在这种情况下，每个病例的耐药表现出ABC基因和其他耐多药基因的不同表达模式，这表明需要个性化的治疗耐药方法。对大量原发性难治性aml的更详细分析表明，有3个分子特征可以预测对治疗的不良反应。其中之一与ABCG2的表达增加有关。这些结果表明，临床样本必须分层，以促进ABC转运蛋白抑制剂的有效靶向，以避免耐药性。