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.

由于特定蛋白质表达的内在或获得的变化，癌细胞中对化疗的抗性发生在癌细胞中。我们研究了对天然产物化学治疗剂的耐药性，例如阿霉素，芬卡生物碱和紫杉醇，以及最近的组蛋白脱乙酰基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的降低，细胞同时对多种药物具有抗性。对于天然产物药物，这种交叉耐药通常是由于能量依赖性药物外排系统（ABC转运蛋白）称为P-糖蛋白（P-GP），MDR1或ABCB1基因的乘积，或其他ABC Transporter家族的成员，包括ABCG2和ABCB5。我们实验室和其他实验室的工作表明，某些药物对表达P-gp的细胞的毒性比对非表达者的毒性更具毒性，这表明一种新型的MDR癌症治疗方法。已经确定了与该特性的几种不同的化学类别，包括硫代性氨基酮（例如NSC73306）。 NSC73306类似物的定量结构活性分析，NCI-60细胞系中的进一步相关性分析以及在美国Pharmacopeia的化合物的高通量屏幕，可杀死P-gp表达P-gp的细胞，从而产生了许多其他化合物，具有改进的能力，可杀死选择性地表达P-gp-gp-gp-gp-gp-Exparts-Exparts-Exparts-Express-Express-Express-Sexpress-Selubility Propties。为了了解P-gp的结构如何确定其多元特异性以及如何随着折叠的变化而改变特异性，我们已经与LCB的其他高级研究人员合作，包括Di Xia，Suresh Ambudkar和Sriram Subramaniam。冷冻EM研究表明，APO P-GP具有动态结构，其中两个ATP结合位点要么分开或靠近。 ATP的结合固定了后一种状态和ATP水解的P-gp构象会导致ATP位点分离。使用小鼠P-gp作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）螺旋的间隔，这表明当ATP位点一起移动并与TM螺旋分开时，TM螺旋揭露了不同的残基，使不同的底物与许多不同的底物结合。小鼠人类嵌合P-GP的研究揭示了这两个相关的转运蛋白的相似结构 - 功能关系。在与Suresh Ambudkar组合作的情况下，我们研究了P-糖蛋白从细胞中从细胞中取出的定向转运的基础。这些研究揭示了P-糖蛋白跨膜区域中的一组氨基酸残基，可以改变这些氨基酸残基，以改变某些若丹明化合物的转运方向，从细胞外到细胞。该过程依赖浓度和ATP依赖性，并且对如何在P-糖蛋白中确定转运的方向性有重要见解。我们已经使用AML作为一种模型系统来确定ABC转运蛋白在耐药性中的临床作用。在一项研究中，分析了化学疗法之前和之后的同一患者的样本。在这种情况下，在每种情况下，耐药性都显示出ABC基因和其他MDR基因的表达方式不同，这表明需要进行个性化治疗的方法。对大量原发性难治性AML的更详细分析表明，有3种分子特征可以预测对治疗的反应不佳。其中之一与ABCG2的表达增加有关。这些结果认为，必须对临床样本进行分层以促进ABC转运蛋白抑制剂的有效靶向以绕过耐药性。