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.

有三种主要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)，耐受顺铂(CP-r)水平的增加，并显示出对亚砷酸盐和镉、甲氨蝶呤和核苷类似物的多重耐药性。这种交叉耐药模式是由于这些药物的摄取减少，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。这种表面转运蛋白的重新定位似乎是由于细胞骨架的改变而改变了这些转运蛋白的循环，从而影响了顺铂耐药细胞中的细胞内循环间隔。负转录调节因子GCF2的过度表达发生在顺铂耐药株系中，这会减少RhoA的表达，导致细胞骨架的破坏，这是这种循环缺陷的一个直接原因。细胞表面转运蛋白减少的另一个后果是葡萄糖摄取减少，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一个cDNA文库，并将其导入敏感细胞，以确定哪些基因具有多药耐药性，包括对顺铂的耐药性。这些基因包括金属硫蛋白、热休克蛋白、核糖体蛋白、硒蛋白和跨膜蛋白TMEM205，它们在顺铂耐药中的作用已被证实。TMEM205是一种在正常分泌细胞中表达的膜蛋白，它的表达与小的GTP酶Rab8结合，使人对顺铂产生耐药。在顺铂耐药的KB细胞中也有持续存在的特定microRNAs(MiRNAs)的变化，它们对耐药性的贡献正在通过miRNA模拟物的表达来检验。此外，NIH高通量siRNA中心正在对逆转KB-CP-r细胞顺铂耐药的miRNAs进行高通量分析。第二种方法是评估导致多药耐药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小体，这是一个溶酶体衍生的细胞器，色素在其中形成。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，并与黑素小体一起挤出到介质中，减少了这种抗癌药物的核积聚。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，对耐药有更大的贡献，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ABCB5，一种与ABCB1同源的转运蛋白，在黑素细胞和黑色素瘤等色素细胞中高水平表达，是否与黑色素瘤的黑素小体隔离有关。全长ABCB5已经在KB细胞中表达，在那里它赋予了广泛的多药耐药表型。已经产生了ABCB5基因敲除小鼠，它们是可以存活的，但改变了色素沉着和神经障碍。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种不同的耐药基因的mRNA表达，并证明了它是一种灵敏、准确、可重复性和健壮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，对耐药基因水平的信使核糖核酸测量初步预测了耐药机制的功能表达。这种耐药芯片已被应用于分析对特定化疗有反应或无反应的人类癌症。我们已经开始了对卵巢癌、急性髓系白血病、黑色素瘤、头颈癌、肝癌和结肠癌的研究，卵巢癌经常对化疗有反应，然后产生耐药性。这项分析的一个早期结果是，现有的癌细胞株不能模仿实际人类癌症的表达模式，TLDA分析选择了380个可能的耐药基因，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性。另一个结论是，我们研究的11个MDR基因的一个特征预示着非积液性卵巢癌的不良反应，而18个MDR基因的不同亚型预示着卵巢癌伴积液的不良反应。对于肝癌来说，两种不同的MDR基因表达特征与预后差和预后好相关。急性髓系白血病(AML)化疗缓解后复发与多种不同的MDR基因表达模式有关，提示AML获得性耐药可能是多因素的。要验证这些结果，表明临床癌症的MDR是复杂的和多因素的，将需要开发可靠的体外培养模型。