Mechanisms of non-classical multidrug resistance in cancer

癌症非经典多药耐药机制

• 批准号: 7965732
• 负责人: Michael Gottesman
• 金额: $ 86.48万
• 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/7965732
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; Affect; Antineoplastic Agents; Arsenites; Biological; Breast; Breast Cancer Cell; CCL21 gene; Cadmium; Cancer cell line; Cell Line; Cell model; Cell physiology; Cell surface; Cells; Characteristics; Cisplatin; Clinical; Collaborations; Collagen; Colon Carcinoma; Cytoplasm; Cytoskeleton; Defect; Doxorubicin; Drug resistance; Epithelial; Funding; Genes; Genetic Transcription; Glioblastoma; Goals; Growth; Head and Neck Cancer; Heat shock proteins; Hela Cells; Human; In Vitro; KB Cells; 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; Mitoxantrone; Molecular; Monomeric GTP-Binding Proteins; Multi-Drug Resistance; Multidrug Resistance Gene; National Institute of Child Health and Human Development; Nuclear; Operative Surgical Procedures; Organelles; P-Glycoprotein; P-Glycoproteins; Pathway interactions; Pattern; Pharmaceutical Preparations; Phenotype; Pigments; Play; Population; Primary carcinoma of the liver cells; Property; Recycling; Resistance; Ribosomal Proteins; Role; Sampling; Secretory Cell; Stem cells; Surface; System; Transfection; United States National Institutes of Health; Work; base; cDNA Library; cancer cell; cancer stem cell; chemotherapy; density; drug sensitivity; efflux pump; glucose uptake; human embryonic stem cell; in vitro Assay; mRNA Expression; malignant breast neoplasm; melanocyte; melanoma; migration; nucleoside analog; overexpression; receptor; resistance mechanism; response; selenoprotein; solute; tissue culture; tumor; uptake

Five 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 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 MBC3205 were identified from this selection and their role in cisplatin resistance has been demonstrated. Expression of MBC3205, a membrane protein expressed in normal secretory cells, in combination with the small GTPase Rab8, confers cisplatin resistance. There are also changes in specific micro RNAs (miRNAs) consistently seen in cisplatin-resistant KB cells, and their contribution to the drug resistance is being explored. A second approach was 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. A third approach is to determine the molecular basis of multidrug resistance in cancer stem cells. As part of an NIH Breast Cancer Consortium, supported in part by breast cancer stamp funds, we have begun to isolate breast cancer stem cells and normal breast epithelial stem cells from surgical samples. CD133 positive cells from these cell populations can be propagated in tissue culture using approaches previously developed for growing human ES cells. These cells have other characteristics of stem cells, such as growth as spheroids and expression ABCG2. Using an in vitro assay system for growth and migration of human breast cancer cells in collagen explants (in collaboration with Josh Zimmerberg, NICHD), we plan to evaluate the biological properties of these putative cancer stem cells. Our goal is to evaluate the expression of multidrug-resistance genes, including both classical ABC efflux transporters and uptake transporters, as well as non-classical mechanisms of multidrug resistance, in cancer stem cells derived from these surgical speciments. The ultimate level of drug accumulation in a cell depends on both the rate of drug uptake as well as the rate of efflux. We have begun to explore the role of known solute carrier (SLC and SLCO) transporters as contributors to patterns of drug sensitivity and resistance in cancer cells. Our initial approach has been to correlate expression of SLC and SLCO transporters with degree of drug sensitivity in the NCI-60 cell lines. Several hits were obtained, indicating that expression of uptake transporters is associated with drug sensitivity. Transfection of SLC22A4 into KB cells increases the uptake of cisplatin, doxorubicin and mitoxantrone and concomitantly confers increased sensitivity to these drugs. 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 is being 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 melanoma, on head and neck cancers, on hepatomas, on glioblastomas 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 <i>in vitro</i> cancer cell models to study multidrug resistance.

有五种主要的方法被用来定义癌症中的非经典多药耐药。首先，我们分离了KB细胞（HeLa的一个亚克隆），对顺铂水平的增加具有耐药性，并证明了对亚砷酸盐和镉、甲氨蝶呤和核苷类似物的多药耐药性。这种交叉抗性模式是由于每一种药物的摄取减少，因为它们的受体已经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顺铂耐药细胞中细胞骨架的改变影响了内吞循环区，从而改变了这些转运蛋白的再循环。负转录调控因子GCF2的过表达发生在顺铂耐药品系中，这降低了rhoA的表达，导致细胞骨架破坏，这是导致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由SIRT1介导的葡萄糖摄取减少和线粒体代谢改变。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，包括对顺铂的耐药。几种cdna，包括编码金属茶蛋白、热休克蛋白、核糖体蛋白、硒蛋白和跨膜蛋白MBC3205的cdna，已从这一选择中鉴定出来，并证实了它们在顺铂耐药中的作用。正常分泌细胞中表达的膜蛋白MBC3205与小GTPase Rab8联合表达，赋予顺铂耐药性。在顺铂耐药的KB细胞中也有特异性微rna （miRNAs）的变化，它们对耐药的贡献正在探索中。第二种方法是评估黑色素瘤细胞导致多药耐药的独特特征。黑色素瘤细胞的一个明显特征是黑色素小体，这是一种溶酶体衍生的细胞器，色素的形成发生在其中。我们已经证明，顺铂被隔离在这个细胞器中，与黑色素形成的程度无关，并与黑色素小体一起挤进培养基，减少了这种抗癌药物的核积累。有证据表明，II型和III型黑素小体，而不是I型或IV型黑素小体，对耐药的贡献更大，这表明黑素小体成熟途径可能是使黑色素瘤对化疗敏感的靶点。ABCB5是一种与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，目前正在进行研究，以确定ABCB5是否有助于黑色素瘤中所见的黑素体隔离。全长ABCB5已经在KB细胞中表达，它赋予了广泛的多药耐药表型。第三种方法是确定癌症干细胞多药耐药的分子基础。作为部分由乳腺癌印章基金支持的NIH乳腺癌联盟的一部分，我们已经开始从手术样本中分离乳腺癌干细胞和正常乳腺上皮干细胞。来自这些细胞群的CD133阳性细胞可以使用先前用于培养人类胚胎干细胞的方法在组织培养中繁殖。这些细胞具有干细胞的其他特征，如球形生长和表达ABCG2。利用人乳腺癌细胞在胶原外植体中生长和迁移的体外测定系统（与NICHD的Josh Zimmerberg合作），我们计划评估这些假定的癌症干细胞的生物学特性。我们的目标是评估多药耐药基因的表达，包括经典的ABC外排转运蛋白和摄取转运蛋白，以及多药耐药的非经典机制，这些肿瘤干细胞来源于这些手术标本。细胞内药物积累的最终水平既取决于药物摄取的速率，也取决于药物排出的速率。我们已经开始探索已知的溶质载体（SLC和SLCO）转运体在癌细胞药物敏感性和耐药性模式中的作用。我们最初的方法是将NCI-60细胞系中SLC和SLCO转运体的表达与药物敏感性程度联系起来。获得了几个命中，表明摄取转运蛋白的表达与药物敏感性有关。将SLC22A4转染到KB细胞中增加顺铂、阿霉素和米托蒽醌的摄取，同时增加对这些药物的敏感性。在另一种方法中，我们开发了一种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，并证明它是一种敏感、准确、可重复和可靠的方法来测量肿瘤样本中的mRNA水平。我们实验室以前的工作表明，耐药基因水平的mRNA测量可以初步预测耐药机制的功能表达。这种耐药芯片正被应用于分析对特定化疗有反应或无反应的人类癌症。我们已经开始了卵巢癌的研究，卵巢癌的癌症通常对化疗有反应，然后变得耐药，还有黑色素瘤、头颈癌、肝癌、胶质母细胞瘤和结肠癌。该分析的一个早期结果是，对于选择用于TLDA分析的380个假定的耐药基因，现有的癌细胞系并不能模拟实际人类癌症的表达模式，而在3D培养中培养细胞的简单权宜之计并不能纠正这个问题。这表明需要更好的体外癌细胞模型来研究多药耐药。