Mechanisms of non-classical multidrug resistance in cancer

三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（CP-r）水平升高具有耐药性，并证明对亚砷酸盐和镉、甲氨蝶呤和核苷类似物具有多药耐药性。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。从这种选择中鉴定出几种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TMEM205，并证实了它们在顺铂耐药中的作用。TMEM205（一种在正常分泌细胞中表达的膜蛋白）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microrna （miRNA）的变化，它们对耐药的贡献正在通过miRNA模拟物的表达进行研究。此外，NIH高通量siRNA中心正在对逆转KB-CP-r细胞顺铂耐药的mirna进行高通量分析。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。ABCB5基因敲除的小鼠已经被培育出来，它们可以存活，但色素沉着发生了变化，并出现了神经紊乱。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的研究，这种癌症经常对化疗有反应，然后变得耐药；AML;在黑色素瘤;头颈癌；肝癌;还有结肠癌。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。另一个结论是，我们研究的11个MDR基因的特征可以预测非积液性卵巢癌的不良反应，18个MDR基因的不同亚群可以预测积液性卵巢癌的不良反应。对于肝癌，两种不同的MDR基因表达特征与预后较差和预后较好的肝癌相关。对于急性髓性白血病（AML），化疗缓解后的疾病复发与多种不同的MDR基因表达模式相关，提示AML获得性耐药可能是多因素的。验证这些结果，表明耐多药在临床癌症中是复杂和多因素的，将需要开发可靠的体外培养模型。

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.