Mechanisms of non-classical multidrug resistance in cancer

癌症非经典多药耐药机制

• 批准号: 8349191
• 负责人: Michael Gottesman
• 金额: $ 74.18万
• 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/8349191
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; Acute Myelocytic Leukemia; Affect; Antineoplastic Agents; Arsenites; Cadmium; Cancer cell line; Cell model; Cell physiology; Cell surface; Cells; Cisplatin; Clinical; Colon Carcinoma; Complementary DNA; Complex; Cytoplasm; Cytoskeleton; Defect; Development; Disease; Disease remission; Drug resistance; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Goals; Head and Neck Cancer; Heat shock proteins; Hela Cells; Human; In Vitro; KB Cells; Knockout Mice; Laboratories; Lead; Length; Lysosomes; Malignant Neoplasms; Malignant neoplasm of ovary; Measurement; Measures; Mediating; Melanins; Melanoma Cell; Melanosomes; Membrane Proteins; Messenger RNA; Metabolism; Methotrexate; MicroRNAs; Microfluidics; Mitochondria; Modeling; Molecular; Monomeric GTP-Binding Proteins; Multi-Drug Resistance; Multidrug Resistance Gene; Nuclear; Organelles; P-Glycoprotein; P-Glycoproteins; Pathway interactions; Pattern; Phenotype; Pigmentation physiologic function; Pigments; Play; Primary carcinoma of the liver cells; Recurrence; Recycling; Resistance; Ribosomal Proteins; Role; Sampling; Secretory Cell; Small Interfering RNA; Specimen; Surface; United States National Institutes of Health; Validation; Work; base; cDNA Library; cancer cell; chemotherapy; density; efflux pump; effusion; glucose uptake; high throughput analysis; mRNA Expression; melanocyte; melanoma; nervous system disorder; nucleoside analog; outcome forecast; overexpression; receptor; resistance mechanism; response; selenoprotein; tissue culture; tumor; uptake

Three major approaches have been taken to define non-classical multidrug resistance in cancer. In the first, we isolated KB cells (a subclone of HeLa) resistant to increasing levels of cisplatin (CP-r) and demonstrated multidrug resistance to arsenite and cadmium, to methotrexate, and to nucleoside analogs. This cross-resistance pattern is due to reduced uptake of each of these agents because their receptors have been relocalized from the cell surface into the cytoplasm of the cell. This relocalization of surface transporters appears to be due to altered recycling of these transporters due to alterations in the cytoskeleton that affect endocytic recycling compartments in cisplatin-resistant cells. Overexpression of the negative transcription regulator GCF2 occurs in cisplatin-resistant lines, which reduces expression of rhoA, causing disruption of the cytoskeleton as a proximate cause of this recycling defect. One additional consequence of reduced cell surface transporters is a reduction in glucose uptake and altered mitochondrial metabolism mediated by SIRT1. To determine additional molecular defects that lead to cisplatin resistance, we created a cDNA library from resistant cells and transfected it into sensitive cells to determine which genes confer multidrug resistance, including resistance to cisplatin. Several cDNAs, including those encoding metallotheinein, heat shock proteins, ribosomal proteins, a selenoprotein, and the trans-membrane protein TMEM205 were identified from this selection and their role in cisplatin resistance has been demonstrated. Expression of TMEM205, a membrane protein expressed in normal secretory cells, in combination with the small GTPase Rab8, confers cisplatin resistance. There are also changes in specific microRNAs (miRNAs) consistently seen in cisplatin-resistant KB cells, and their contribution to drug resistance is being examined by expression of miRNA mimics. In addition, a high throughput analysis of miRNAs that reverse the cisplatin resistance of KB-CP-r cells is underway at the NIH high throughput siRNA center. A second approach is to evaluate the unique features of melanoma cells that contribute to multidrug-resistance. One obvious feature of melanoma cells is the melanosome, a lysosome-derived organelle in which pigment formation takes place. We have shown that cisplatin is sequestered in this organelle, independent of extent of melanin formation, and extruded with melanosomes into the medium, reducing nuclear accumulation of this anti-cancer drug. Evidence indicating that type II and III melanosomes, and not type I or type IV melanosomes, contribute more to drug resistance suggests that the melanosomal maturation pathway could be a target for sensitizing melanomas to chemotherapy. Studies are underway to determine whether ABCB5, a transporter homologous to ABCB1, expressed at high levels in pigmented cells such as melanocytes and melanomas, contributes to the melanosomal sequestration seen in melanomas. Full-length ABCB5 has been expressed in KB cells, where it confers a broad multidrug resistance phenotype. ABCB5 knock-out mice have been generated, and they are viable, but have altered pigmentation and a neurological disorder. In another approach, we have developed a Taqman Low Density Array (TLDA) microfluidic chip to detect mRNA expression of 380 different putative drug resistance genes and demonstrated that it is a sensitive, accurate, reproducible, and robust way to measure mRNA levels in tumor samples. Previous work from our laboratory indicates that mRNA measurements of levels of drug-resistance genes are, to a first approximation, predictive of functional expression of drug-resistance mechanisms. This drug-resistance chip has been applied to analysis of human cancers that show either response or lack of response to specific chemotherapy. We have initiated our studies on ovarian cancer, where cancers frequently respond to chemotherapy and then become resistant; on AML; on melanoma; on head and neck cancers; on hepatomas; and on colon cancer. One early result from this analysis is that existing cancer cell lines do not mimic the expression patterns of actual human cancers for the 380 putative drug resistance genes chosen for the TLDA analysis and the simple expedient of growing cells in 3D culture does not correct this problem. This suggests the need for better in vitro cancer cell models to study multidrug resistance. Another conclusion is that a signature of eleven MDR genes we have studied predicts poor response in non-effusion ovarian cancer, and different subsets of 18 MDR genes predict poor response in ovarian cancer with effusions. For hepatoma, two different MDR gene expression signatures are associated with poor prognosis and better prognosis hepatoma. For acute myeloid leukemia (AML), recurrence of disease after remission induced by chemotherapy is associated with multiple different patterns of MDR gene expression, suggesting that for AML acquired resistance may be multifactorial. Validation of these results, indicating that MDR is complex and multifactorial in clinical cancers, will require the development of reliable in vitro culture models.

已采用三种主要方法来定义癌症的非经典多药耐药性。首先，我们分离了对顺铂 (CP-r) 水平升高具有耐药性的 KB 细胞（HeLa 的亚克隆），并表现出对亚砷酸盐和镉、甲氨蝶呤和核苷类似物的多药耐药性。这种交叉耐药模式是由于这些药物中的每一种的摄取减少所致，因为它们的受体已从细胞表面重新定位到细胞的细胞质中。表面转运蛋白的这种重新定位似乎是由于影响顺铂抗性细胞中的内吞回收区室的细胞骨架的改变而改变了这些转运蛋白的回收。负转录调节因子 GCF2 的过度表达发生在顺铂耐药株系中，这会降低 rhoA 的表达，导致细胞骨架破坏，这是造成这种再循环缺陷的直接原因。细胞表面转运蛋白减少的另一个后果是葡萄糖摄取减少以及 SIRT1 介导的线粒体代谢改变。为了确定导致顺铂耐药的其他分子缺陷，我们从耐药细胞中创建了 cDNA 文库，并将其转染到敏感细胞中，以确定哪些基因赋予多药耐药性，包括对顺铂的耐药性。从该选择中鉴定了几种 cDNA，包括编码金属酪蛋白、热休克蛋白、核糖体蛋白、硒蛋白和跨膜蛋白 TMEM205 的 cDNA，并且它们在顺铂耐药性中的作用已得到证明。 TMEM205（一种在正常分泌细胞中表达的膜蛋白）的表达与小 GTPase Rab8 结合，赋予顺铂耐药性。在顺铂耐药的 KB 细胞中也始终观察到特定 microRNA (miRNA) 的变化，并且正在通过 miRNA 模拟物的表达来检查它们对耐药性的贡献。此外，NIH 高通量 siRNA 中心正在对逆转 KB-CP-r 细胞顺铂耐药性的 miRNA 进行高通量分析。 第二种方法是评估黑色素瘤细胞导致多重耐药性的独特特征。黑色素瘤细胞的一个明显特征是黑色素体，这是一种溶酶体衍生的细胞器，其中发生色素形成。我们已经证明，顺铂被隔离在该细胞器中，与黑色素形成的程度无关，并与黑素体一起被挤出到培养基中，减少了这种抗癌药物的核积累。有证据表明，II 型和 III 型黑素体，而不是 I 型或 IV 型黑素体，对耐药性的贡献更大，这表明黑素体成熟途径可能是使黑色素瘤对化疗敏感的目标。正在进行研究以确定 ABCB5（一种与 ABCB1 同源的转运蛋白，在黑色素细胞和黑色素瘤等色素细胞中高水平表达）是否有助于黑色素瘤中的黑色素体隔离。全长 ABCB5 已在 KB 细胞中表达，赋予广泛的多药耐药表型。 ABCB5 基因敲除小鼠已经诞生，它们可以存活，但色素沉着发生改变并出现神经系统疾病。 在另一种方法中，我们开发了 Taqman 低密度阵列 (TLDA) 微流控芯片来检测 380 种不同的假定耐药基因的 mRNA 表达，并证明这是一种灵敏、准确、可重复且稳健的测量肿瘤样本中 mRNA 水平的方法。我们实验室之前的工作表明，耐药基因水平的 mRNA 测量可以初步近似地预测耐药机制的功能表达。这种耐药芯片已应用于分析对特定化疗有反应或无反应的人类癌症。我们已经启动了对卵巢癌的研究，这种癌症经常对化疗产生反应，然后产生耐药性。关于反洗钱；关于黑色素瘤；头颈癌；关于肝癌；和结肠癌。该分析的一个早期结果是，对于为 TLDA 分析选择的 380 种推定耐药基因，现有癌细胞系并不模仿实际人类癌症的表达模式，并且在 3D 培养中生长细胞的简单方法并不能解决这一问题。这表明需要更好的体外癌细胞模型来研究多药耐药性。另一个结论是，我们研究的 11 个 MDR 基因的特征预测非积液性卵巢癌的不良反应，而 18 个 MDR 基因的不同子集预测有积液的卵巢癌的不良反应。对于肝癌，两种不同的MDR基因表达特征与肝癌的不良预后和较好预后相关。对于急性髓系白血病 (AML)，化疗诱导缓解后疾病复发与多种不同的 MDR 基因表达模式相关，这表明 AML 获得性耐药可能是多因素的。这些结果的验证表明临床癌症中的 MDR 是复杂且多因素的，需要开发可靠的体外培养模型。