Mechanisms of non-classical multidrug resistance in cancer

癌症非经典多药耐药机制

• 批准号: 9153686
• 负责人: Michael Gottesman
• 金额: $ 121.52万
• 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/9153686
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; Accounting; Acute Myelocytic Leukemia; Adjuvant; Affect; Antineoplastic Agents; Apoptosis; Arsenites; Biological; Bioreactors; Blood capillaries; CHES1 gene; Cadmium; Cancer Cell Growth; Cancer cell line; Cell Death; Cell model; Cell physiology; Cell surface; Cells; Cisplatin; Clinical; Collaborations; Complex; Cultured Cells; Cytoplasm; Cytoskeleton; DNA Damage; DNA Repair; Data; Defect; Development; Dimethyl Sulfoxide; Disease; Disease remission; Drug resistance; Elements; Family member; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genetic Transcription; Goals; Growth; Heat shock proteins; Hela Cells; Heterogeneity; Human; Hydrogels; In Vitro; KB Cells; Laboratories; Link; Literature; Luciferases; Lysosomes; Malignant Neoplasms; Malignant neoplasm of ovary; Maryland; Measurement; Measures; Melanins; Melanoma Cell; Melanosomes; Membrane Proteins; Messenger RNA; Methotrexate; MicroRNAs; Microfluidics; Mitosis; Modeling; Molecular; Monomeric GTP-Binding Proteins; Multi-Drug Resistance; Multidrug Resistance Gene; Mus; Mutation; Nuclear; Organelles; Oxygen; P-Glycoprotein; P-Glycoproteins; Paper; Pathway interactions; Pattern; Pharmaceutical Preparations; Phase I Clinical Trials; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Pigments; Platinum; Play; Primary carcinoma of the liver cells; Protein phosphatase; Proteins; RNA Interference; Recurrence; Recycling; Research; Resistance; Ribosomal Proteins; Role; SKOV3 cells; Sampling; Secretory Cell; Silicon; Solvents; Specimen; Surface; Suspension substance; Suspensions; System; Testing; Universities; Validation; Work; base; cancer cell; capillary; chemotherapy; density; efflux pump; effusion; high throughput analysis; in vivo; inhibitor/antagonist; interest; killings; mRNA Expression; malignant breast neoplasm; mathematical model; melanocyte; melanoma; nucleoside analog; outcome forecast; overexpression; prevent; receptor; resistance gene; resistance mechanism; response; scale up; screening; selenoprotein; theories; 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. The protein metallotheinein, heat shock proteins, ribosomal proteins, a selenoprotein, and the trans-membrane protein TMEM205 have also been shown to play a role in cisplatin resistance. Expression of TMEM205, a membrane protein expressed in normal secretory cells, in combination with the small GTPase Rab8, confers cisplatin resistance. We have demonstrated changes in specific microRNAs (miRNAs), such as miRNA-181, consistently seen in cisplatin-resistant KB cells, and their contribution to drug resistance has been demonstrated by expression of miRNA mimics and inhibitors. In addition, a high throughput analysis of miRNAs that reverse the cisplatin resistance of KB-CP-r cells has identified WEE1 and CHK1 as essential elements of resistance to cisplatin. miRNA155 and miR-15 family members are miRNAs whose expression affects cisplatin resistance through WEE1 and CHK1. Our interest in the checkpoint kinases and their role in resistance led us to the protein phosphatase 2A (PP2A) inhibitor LB100, currently in Phase I clinical trials for breast cancer. As PP2A controls the phosphorylation status of a number of DNA-damage response (DDR) genes, we hypothesized that LB100 would sensitize ovarian cancer cells to cisplatin. We demonstrated that inhibition of PP2A by LB100 sensitized cells (OVCAR8 and SKOV3) to cisplatin, and that LB100 induces hyperphosphorylation of Chk1 and other genes in the DNA-damage response pathway, preventing cisplatin-induced G2 arrest and forcing cells into mitosis, resulting in apoptosis. We showed that mice injected intraperitoneally with SKOV3-luciferase cells were sensitized to cisplatin (3 mg/kg) when treated with LB100 compared with control. We recently completed an RNAi screen in cells exposed to cisplatin, in order to identify genes associated with cisplatin sensitivity. If cells exposed to sub-toxic cisplatin undergo cell death when a particular gene is deleted, one can hypothesize that inhibition of this gene target might prove to be a useful adjuvant for platinum chemotherapy. The strongest sensitizing effects were observed when DNA damage repair genes (including a phosphoprotein phosphatase) were silenced, and several of these are now being investigated for their role in cisplatin tolerance. In this screening context, we found a need to identify a solvent appropriate for dissolving cisplatin for screening. We recently showed that DMSO inactivated the biological activity of all clinical and experimental platinum complexes tested. Furthermore, a review of the cisplatin literature revealed that about a third of all research papers have used cisplatin dissolved in DMSO, calling into question the data and conclusions of those papers. This has important implications for the reliability of a significant portion of the literature and points the way for appropriate use of platinum drugs in research. 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. 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. Approximately 15% of human melanomas carry mutations in ABCB5, suggesting that defects in ABCB5 are linked to melanoma progression. 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. One 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, and interpretation of these data using mathematical models based on network theory is proceeding. Towards this goal, we have developed a bioreactor that mimics capillary delivery (through silicon hydrogels) of oxygen to cells grown in 3D suspension. We have demonstrated physiological oxygen gradients and altered growth of cancer cells more closely approximating in vivo phenotypes. This bioreactor can be scaled up for growth of multiple cultures of primary cancer cells or cultured cancer cells to determine whether growth conditions play a primary role in affecting patterns of drug resistance. Because of the apparent complexity of drug resistance mechanisms in vivo, we have re-examined existing mathematical models that predict the development of drug resistance based on more homogeneous systems. In collaboration with Doron Levy (University of Maryland), we have formulated a new mathematical model that takes into account tumor heterogeneity and other features associated with in vivo systems. This model allows a prediction of how chemotherapy should be targeted to optimize killing of drug-resistant cancer cells.

已采用三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 (CP-r) 水平升高具有耐药性的 KB 细胞（HeLa 的亚克隆），并表现出对亚砷酸盐和镉、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。金属茶蛋白、热休克蛋白、核糖体蛋白、硒蛋白和跨膜蛋白 TMEM205 也已被证明在顺铂耐药性中发挥作用。 TMEM205（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。我们已经证明了特定 microRNA (miRNA) 的变化，例如 miRNA-181，这些变化在顺铂耐药的 KB 细胞中始终可见，并且它们对耐药性的贡献已通过 miRNA 模拟物和抑制剂的表达得到证明。此外，对逆转 KB-CP-r 细胞顺铂耐药性的 miRNA 进行的高通量分析已确定 WEE1 和 CHK1 是顺铂耐药性的重要元件。 miRNA155和miR-15家族成员是其表达通过WEE1和CHK1影响顺铂耐药性的miRNA。我们对检查点激酶及其在耐药性中的作用的兴趣促使我们开发出了蛋白磷酸酶 2A (PP2A) 抑制剂 LB100，该抑制剂目前正处于乳腺癌的 I 期临床试验中。由于 PP2A 控制许多 DNA 损伤反应 (DDR) 基因的磷酸化状态，我们假设 LB100 会使卵巢癌细胞对顺铂敏感。我们证明，LB100 对 PP2A 的抑制使细胞（OVCAR8 和 SKOV3）对顺铂敏感，并且 LB100 诱导 Chk1 和 DNA 损伤反应途径中其他基因的过度磷酸化，防止顺铂诱导的 G2 停滞并迫使细胞进入有丝分裂，从而导致细胞凋亡。我们发现，与对照组相比，腹腔注射 SKOV3-荧光素酶细胞的小鼠在用 LB100 治疗时对顺铂 (3 mg/kg) 敏感。我们最近在暴露于顺铂的细胞中完成了 RNAi 筛选，以鉴定与顺铂敏感性相关的基因。如果当特定基因被删除时，暴露于亚毒性顺铂的细胞会发生细胞死亡，那么我们可以推测，对该基因靶标的抑制可能被证明是铂类化疗的有用佐剂。当 DNA 损伤修复基因（包括磷蛋白磷酸酶）被沉默时，观察到最强的致敏作用，其中一些基因现在正在研究它们在顺铂耐受性中的作用。在此筛选背景下，我们发现需要确定适合溶解顺铂的溶剂以进行筛选。我们最近表明，DMSO 使所有测试的临床和实验铂络合物的生物活性失活。此外，对顺铂文献的回顾显示，大约三分之一的研究论文使用了溶解在 DMSO 中的顺铂，这对这些论文的数据和结论提出了质疑。这对于很大一部分文献的可靠性具有重要意义，并为研究中适当使用铂类药物指明了道路。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。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。大约 15% 的人类黑色素瘤携带 ABCB5 突变，表明 ABCB5 缺陷与黑色素瘤进展有关。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。这种耐药芯片已应用于人类癌症的分析。该分析的一个结果是，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 基因的特征预测非积液性卵巢癌的不良反应，而 18 个 MDR 基因的不同子集预测有积液的卵巢癌的不良反应。对于肝癌，两种不同的MDR基因表达特征与肝癌的不良预后和较好预后相关。对于急性髓系白血病 (AML)，化疗诱导缓解后疾病复发与多种不同的 MDR 基因表达模式相关，这表明 AML 获得性耐药可能是多因素的。这些结果的验证表明临床癌症中的 MDR 是复杂且多因素的，需要开发可靠的体外培养模型，并且正在使用基于网络理论的数学模型解释这些数据。为了实现这一目标，我们开发了一种生物反应器，可以模拟毛细管（通过硅水凝胶）向 3D 悬浮液中生长的细胞输送氧气。我们已经证明了生理氧梯度和癌细胞生长的改变更接近体内表型。该生物反应器可以扩大规模，用于原代癌细胞或培养癌细胞的多种培养物的生长，以确定生长条件是否在影响耐药性模式中发挥主要作用。由于体内耐药机制明显复杂，我们重新审视了基于更均质系统预测耐药发展的现有数学模型。我们与 Doron Levy（马里兰大学）合作，制定了一个新的数学模型，该模型考虑了肿瘤异质性和与体内系统相关的其他特征。该模型可以预测化疗应如何靶向以优化对耐药癌细胞的杀伤。