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.

已采用五种主要方法来定义癌症的非经典多药耐药性。首先，我们分离了对顺铂水平升高具有耐药性的 KB 细胞（HeLa 的亚克隆），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 MBC3205 的 cDNA，并且它们在顺铂耐药性中的作用已得到证明。 MBC3205（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 细胞中也经常观察到特定的微小 RNA (miRNA) 的变化，并且正在探索它们对耐药性的贡献。 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。第三种方法是确定癌症干细胞多药耐药性的分子基础。作为 NIH 乳腺癌联盟的一部分，在乳腺癌邮票基金的部分支持下，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开发的用于培养人类 ES 细胞的方法在组织培养物中繁殖。这些细胞具有干细胞的其他特征，例如球状生长和表达 ABCG2。我们计划使用胶原外植体中人类乳腺癌细胞生长和迁移的体外测定系统（与 NICHD 的 Josh Zimmerberg 合作），计划评估这些假定的癌症干细胞的生物学特性。我们的目标是评估源自这些手术样本的癌症干细胞中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。细胞中药物积累的最终水平取决于药物摄取速率和外排速率。我们已经开始探索已知溶质载体（SLC 和 SLCO）转运蛋白对癌细胞药物敏感性和耐药性模式的影响。我们最初的方法是将 SLC 和 SLCO 转运蛋白的表达与 NCI-60 细胞系中的药物敏感性程度相关联。获得了几次命中，表明摄取转运蛋白的表达与药物敏感性相关。将 SLC22A4 转染到 KB 细胞中会增加顺铂、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 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。我们已经启动了对卵巢癌（此类癌症经常对化疗产生反应，然后产生耐药性）、黑色素瘤、头颈癌、肝癌、胶质母细胞瘤和结肠癌的研究。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<i>体外</i>癌细胞模型来研究多药耐药性。