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.

已经采用了三种主要方法来定义癌症中非经典的多药耐药性。首先，我们分离了抗抑制素水平（CP-R）的KB细胞（HELA的亚克隆），并证明了对砷和镉的多药耐药性，甲氨蝶呤和核苷类模拟。这种交叉耐药模式是由于每种药物的摄取减少所致，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这些转运蛋白的回收改变，这是由于影响顺铂耐药细胞中内吞回收室的细胞骨架的改变。阴性转录调节剂GCF2的过表达发生在抗铂耐药线中，这会降低RhoA的表达，从而导致细胞骨架的破坏，这是该回收缺陷的直接原因。细胞表面转运蛋白减少的另一个结果是葡萄糖摄取的降低和SIRT1介导的线粒体代谢改变。为了确定导致顺铂耐药性的其他分子缺陷，我们从抗性细胞中创建了一个cDNA库，并将其转染到敏感细胞中，以确定哪些基因赋予了多药耐药性，包括对顺铂的抗性。从这种选择中鉴定出了几种cDNA，包括那些编码金属蛋白，热休克蛋白，核糖体蛋白，硒蛋白和反膜蛋白TMEM205的cDNA，并且已经证明了它们在顺铂耐药性中的作用。 TMEM205的表达是一种在正常分泌细胞中表达的膜蛋白，结合了小的GTPase Rab8，赋予了顺铂的耐药性。在抗顺铂的KB细胞中，特定的microRNA（miRNA）也有变化，并且通过miRNA模拟物的表达来检查它们对耐药性的贡献。此外，在NIH高吞吐量siRNA中心正在进行对KB-CP-R细胞抗顺铂的抗性的高吞吐量分析。 第二种方法是评估有助于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色素体，这是一种溶酶体衍生的细胞器，发生色素形成。我们已经表明，顺铂在这种细胞器中被隔离，与黑色素形成的程度无关，并用黑色素体挤压到培养基中，从而减少了这种抗癌药物的核积累。证据表明II型和III型黑色素体，而不是I型或IV型黑色素体，对耐药性的贡献更大，这表明黑色素体成熟途径可能是使黑色素瘤对化学疗法敏感的靶标。正在进行的研究以确定ABCB5（与ABCB1同源的转运蛋白同源物在色素细胞（例如黑色素细胞和黑色素瘤）中以高水平表达的转运蛋白是否有助于黑色素瘤中的黑素体隔离。全长ABCB5已在KB细胞中表达，在该细胞中，它赋予了广泛的多药电阻表型。已经产生了ABCB5敲除小鼠，它们是可行的，但是色素沉着和神经系统疾病改变了。 在另一种方法中，我们开发了一个Taqman低密度阵列（TLDA）微流体芯片，以检测380种不同推定耐药性基因的mRNA表达，并证明它是一种敏感，准确，可再现和可靠的方法，可在肿瘤样品中测量mRNA水平。我们实验室的先前工作表明，对药物抗性基因水平的测量值是第一个近似值，可以预测药物耐药机制的功能表达。这种抗药性芯片已应用于对表现出对特定化学疗法反应或缺乏反应的人类癌症的分析。我们已经开始了关于卵巢癌的研究，癌症经常对化学疗法反应，然后变得抗性。在AML上；关于黑色素瘤；在头部和颈部癌症上；在肝瘤上；以及结肠癌。该分析的一个早期结果是，现有的癌细胞系并未模仿用于TLDA分析的380种推定耐药性基因的实际人类癌的表达模式，而3D培养中生长细胞的简单权宜之计并不能纠正此问题。这表明需要更好的体外癌细胞模型研究多药耐药性。另一个结论是，我们研究的11个MDR基因的签名预测了非效力卵巢癌的反应不佳，而18个MDR基因的不同亚群预测，卵巢癌的反应不佳。对于肝癌，两个不同的MDR基因表达特征与预后不良和预后更好有关。对于急性髓样白血病（AML），化学疗法诱导的缓解后疾病的复发与MDR基因表达的多种不同模式有关，这表明对于AML获得的抗性可能是多因素的。对这些结果的验证，表明MDR在临床癌症中是复杂而多因素的，将需要开发可靠的体外培养模型。