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.

三种主要方法已被用来定义癌症中的非经典多药耐药。在第一，我们分离KB细胞（HeLa的亚克隆）耐顺铂（CP-r）的水平增加，并表现出多药耐药性亚砷酸盐和镉，甲氨蝶呤，核苷类似物。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，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这些转运蛋白的再循环改变所致，这些转运蛋白的再循环改变是由于细胞骨架的改变影响顺铂耐药细胞中的内吞再循环隔室。负转录调节因子GCF 2的过表达发生在顺铂耐药株系中，其降低rhoA的表达，导致细胞骨架的破坏，作为这种再循环缺陷的近因。细胞表面转运蛋白减少的另一个后果是葡萄糖摄取减少和SIRT 1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一个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。几个cDNA，包括那些编码金属蛋白酶，热休克蛋白，核糖体蛋白，硒蛋白，和跨膜蛋白TMEM 205从这个选择和它们的作用，顺铂耐药性已被证明。TMEM 205（一种在正常分泌细胞中表达的膜蛋白）的表达与小GTMERab 8的组合赋予顺铂抗性。在顺铂耐药的KB细胞中也一直观察到特异性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中心正在进行逆转KB-CP-r细胞顺铂耐药性的miRNA的高通量分析。 第二种方法是评估导致多药耐药性的黑色素瘤细胞的独特特征。黑色素瘤细胞的一个明显特征是黑素体，这是一种溶酶体衍生的细胞器，其中发生色素形成。我们已经表明，顺铂被隔离在这个细胞器中，独立于黑色素形成的程度，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靶点。研究正在进行中，以确定ABCB 5，一种与ABCB 1同源的转运蛋白，在色素细胞如黑色素细胞和黑色素瘤中高水平表达，是否有助于黑色素瘤中观察到的黑色素体隔离。全长ABCB 5已在KB细胞中表达，它赋予广泛的多药耐药表型。ABCB 5基因敲除小鼠已经产生，它们是可行的，但改变了色素沉着和神经系统疾病。 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种不同的推定耐药基因的mRNA表达，并证明它是一种灵敏、准确、可重复和可靠的方法来测量肿瘤样品中的mRNA水平。我们实验室以前的工作表明，耐药基因水平的mRNA测量，在第一近似值，预测耐药机制的功能表达。这种耐药芯片已被应用于分析人类癌症，这些癌症对特定的化疗有反应或没有反应。我们已经开始了对卵巢癌的研究，这种癌症经常对化疗有反应，然后变得耐药; AML;黑色素瘤;头颈癌;肝癌;结肠癌。这项分析的一个早期结果是，现有的癌细胞系不能模拟实际人类癌症的380个假定耐药基因的表达模式，这些基因被选择用于TLDA分析，并且在3D培养中生长细胞的简单方法不能纠正这个问题。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基因的特征预测了非渗出性卵巢癌的低反应，18个MDR基因的不同子集预测了有渗出性卵巢癌的低反应。对于肝癌，两种不同的MDR基因表达特征与肝癌的预后不良和预后较好相关。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，表明AML获得性耐药可能是多因素的。这些结果表明MDR在临床癌症中是复杂和多因素的，验证这些结果需要开发可靠的体外培养模型。