Investigation of the ABC Half-Transporter ABCG2

ABC 半转运蛋白 ABCG2 的研究

• 批准号: 7965472
• 负责人: susan bates
• 金额: $ 51.92万
• 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/7965472
• 关键词: 3' Untranslated Regions; ABCG2 gene; ATP-Binding Cassette Transporters; Acetylation; Amino Acids; Antineoplastic Agents; Binding; Binding Sites; Biological Assay; Biological Availability; Blood - brain barrier anatomy; Brain; Camptothecin; Cancer cell line; Cell Line; Cell surface; Cells; Chemicals; Clinic; Clinical; Collaborations; Communication; Depsipeptides; Detection; Development; Dimerization; Drosophila genus; Drug Exposure; Drug resistance; Endothelial Cells; Epigenetic Process; Exhibits; Exposure to; Gene Expression; Genes; Glutamine; Histone Acetylation; Histone Deacetylase; Histone Deacetylase Inhibitor; Histones; Human; Imatinib; Induced Mutation; Insecta; Intestinal Absorption; Investigation; Label; Laboratories; Laboratory Study; Learning; Lysine; Mammalian Cell; Mediating; Messenger RNA; Metastatic Neoplasm to the Central Nervous System; Methods; Methylation; MicroRNAs; Mitoxantrone; Modeling; Molecular Target; Mutation; Names; New Agents; Nomenclature; Normal tissue morphology; Oral; P-Glycoprotein; P-Glycoproteins; Patients; Pharmaceutical Preparations; Pharmacotherapy; Play; Preclinical Drug Evaluation; Program Development; Proteins; Regulation; Renal Cell Carcinoma; Reporting; Research Personnel; Resistance; Role; SN-38; Sampling; Sequence Analysis; Single Nucleotide Polymorphism; Site; Slide; Specificity; Stem cells; Structure-Activity Relationship; Study models; System; Tail; Tertiary Protein Structure; Tissue Microarray; Topotecan; Translations; Transmembrane Domain; Tyrosine Kinase Inhibitor; Untranslated Regions; Up-Regulation; Work; absorption; base; cancer cell; cancer stem cell; cancer therapy; cell type; chemotherapy; chromatin immunoprecipitation; colon cancer cell line; crosslink; human ABCG2 protein; improved; inhibitor/antagonist; interest; intestinal epithelium; irinotecan; malignant breast neoplasm; monomer; overexpression; pre-clinical; prevent; promoter; protein expression; small molecule; therapeutic target; tumor; uptake

Our laboratory has a long-standing interest in non-Pgp mediated mechanisms of drug resistance, having established several cell line models of resistance focusing on the ABC half-transporter ABCG2. We successfully cloned ABCG2 from a mitoxantrone-resistant colon cancer cell line, S1-M1-80, that exhibited an ATP-dependent reduction in drug accumulation. Comprising 6 transmembrane domains and a single ATP binding domain, the gene encodes a half-transporter molecule, and it is thought that dimerization is required for activity. Overexpression of ABCG2 renders cells resistant to mitoxantrone and to the camptothecins, topotecan and SN-38 (the active metabolite of irinotecan). Both substrates and inhibitors of ABCG2 have been discovered at an accelerating pace, and the variety of both substrates and inhibitors rivals that described for P-glycoprotein. There is increasing evidence supporting a role for ABCG2 in limiting the oral absorption of pharmacologic agents and in limiting the brain uptake through expression in the blood brain barrier. We have worked on structure and function relationships in the protein. We identified a drug-induced mutation in ABCG2 (R482T; R482G) that alters substrate and inhibitor specificity; and then carried out a sequence analysis of ABCG2, identifying single nucleotide polymorphisms. We and others reported impaired transport in cells bearing a single nucleotide polymorphism at amino acid 141 that changes glutamine to lysine. Since gastrointestinal absorption of topotecan has been related to ABCG2 expression in the intestinal epithelium, one implication of this work is that the Q141K SNP could be associated with increased exposure to substrate drugs in patients. To the extent that ABCG2 is involved in drug exposure, this SNP could increase exposure to substrates such as imatinib, irinotecan or topotecan. Further, ABCG2 is expressed in the endothelial cells in the brain and another important role for the protein is in protection of the CNS as a component of the blood-brain barrier. An implication of this finding is that compounds that circumvent ABCG2 and thus, the blood-brain barrier, could have increased efficacy in treating or preventing CNS metastases. The importance of localization of ABCG2 in the blood brain barrier will increase as we recognize ABCG2 substrates in new agents such as small molecule tyrosine kinase inhibitors. To evaluate dimerization of ABCG2, our laboratory studied a GXXXG dimerization motif in transmembrane helix 1. We found this motif critical for normal transport activity in ABCG2, but could not prove that it mediated dimerization. We recently discovered that mutation of a nearby residue, T402, completely destabilized the protein. Mutation of the highly conserved amino acid residue 553 (the homologous residue in Drosophila is said to drive dimerization) results in loss of protein expression on the mammalian cell surface, and expression of a nonfunctional protein on the insect cell surface. In both of these systems, chemical cross-linking is preserved, suggesting a proximity of the two monomers even when the protein fails to fold properly. We also identified a critical residue in amino acid 383 that may promote communication between the major domains of the protein. We recently studied mechanisms of regulation of ABCG2, discovering that the ABCG2 promoter is methylated in some renal cell cancer cell lines, resulting in reduced expression of the gene. We also determined that the promoter is regulated by histone acetylation and that depsipeptide is able to upregulate expression in some cell types. Interestingly, there are also cell types where the gene is repressed in a non-HDAC, non-methylation dependent manner. This study may help us understand how normal stem cells turn off ABCG2 as they differentiate and how some cancer cells re-express the transporter; eventually leading to strategies to target ABCG2-expressing cells. We have also learned using chromatin immunoprecipitation that permissive epigenetic marks are evident in the ABCG2 promoter in cells that respond to HDAC inhibition with upregulation of ABCG2 mRNA. However, repressive epigenetic marks persist in the ABCG2 promoter in cells that do not respond to HDAC inhibition with ABCG2 mRNA upregulation. These repressive marks persist in the promoter despite acetylation of the lysine tails of nearby histone proteins, and despite upregulation of other genes in the same cells. This constitutes a model for studying resistance to HDAC inhibitors, an important facet of project #2: II. Clinical and Laboratory Studies of the Histone Deacetylase Inhibitor Depsipeptide. We recently recognized that cells expressing high levels of ABCG2 have truncation at the 3'untranslated region of the gene, which removes a microRNA binding site. The microRNA, hsa-miR 519c, was shown to bind at this site and reduce gene expression and protein translation. New studies have shown that a variety of drug resistant cell lines use the short 3' UTR, suggesting it as a general mechanism of ABCG2 upregulation. Our next aim is to evaluate other micro RNAs involved in drug resistance. In order to evaluate clinical samples for ABCG2 expression, we have developed, in collaboration with Stephen Hewitt and Patty Fetsch, an immunohistochemical assay that has been used to evaluate slides bearing tissue arrays. Using these slides we hope to discover specific tumor types overexpressing ABCG2. In collaboration with Dr. Michael Dean and the Molecular Targets Development Program, led by Dr. James McMahon, ABCG2-overexpressing cells have been used to screen for inhibitors of ABCG2. This is an important undertaking since the potential ability to modulate oral drug absorption and CNS uptake will be important whether or not ABCG2 proves important in oncologic drug resistance. Dr. Curtis Henrich, of the Molecular Targets Development Program, has identified a number of hits that we have now confirmed as ABCG2 inhibitors. These compounds have been evaluated in secondary screens in our laboratory and in that of Dr. Suresh Ambudkar, to prioritize for further preclinical development. In another strategy to identify ABCG2 substrates and inhibitors, we characterized ABCG2 expression in the 60 cell lines of the NCI drug screen. This profile allowed us to identify potential substrates and inhibitors using the COMPARE analysis. These compounds have also been secondarily screened. We have concluded that the drug screen cannot be used to specifically define whether or not a compound is an ABCG2 substrate -- however, a set of compounds can be enriched for substrates by this method. Collectively, this work is best characterized as an effort to both understand and exploit ABCG2 as a therapeutic target to improve anticancer therapy.

我们的实验室对非PGP耐药性机制具有长期的兴趣，该机制建立了以ABC半转运蛋白ABCG2为重点的几个细胞系模型。我们成功地从抗米托酮的结肠癌细胞系S1-M1-80克隆了ABCG2，该细胞表现出ATP依赖性降低药物积累的降低。 该基因由6个跨膜结构域和一个单个ATP结合结构域编码半转运蛋白分子，并认为活性需要二聚化。 ABCG2的过表达使细胞具有对Mitoxantrone的抗性，以及对甲瓜蛋白，拓扑替克和SN-38（伊立替康的活性代谢产物）。 ABCG2的底物和抑制剂均以加速速度发现，以及用于P-糖蛋白所描述的底物和抑制剂竞争对手。有越来越多的证据支持ABCG2在限制药理学剂的口服吸收以及通过血液脑屏障中的表达限制脑摄取方面的作用。我们已经从事蛋白质中的结构和功能关系。我们确定了在ABCG2（R482T; R482G）中的药物诱导的突变，从而改变了底物和抑制剂的特异性。然后对ABCG2进行了序列分析，以鉴定单核苷酸多态性。我们和其他人报告说，在氨基酸141处带有单个核苷酸多态性的细胞中的运输受损，该细胞将谷氨酰胺变成赖氨酸。由于拓扑替克的胃肠道吸收与肠上皮中的ABCG2表达有关，因此这项工作的一种含义是Q141K SNP可能与患者中底物药物的接触增加有关。在某种程度上，ABCG2参与药物暴露，该SNP可能会增加对伊马替尼，伊立替康或拓扑替康等底物的暴露。此外，ABCG2在大脑的内皮细胞中表达，蛋白质的另一个重要作用是保护中枢神经系统作为血脑屏障的组成部分。这一发现的含义是，绕过ABCG2的化合物，因此，血脑屏障可能会增加治疗或预防CNS转移的功效。随着我们认识到新的药物（例如小分子酪氨酸激酶抑制剂）中ABCG2底物的ABCG2定位的重要性将增加。为了评估ABCG2的二聚化，我们的实验室研究了跨膜螺旋1中的GXXXG二聚基序。我们发现，ABCG2中正常运输活性至关重要，但无法证明它介导了二聚体。我们最近发现，附近残基T402的突变使该蛋白质完全不稳定。高度保守的氨基酸残基553的突变（据说果蝇中的同源残基会导致二聚化）导致哺乳动物细胞表面上蛋白质表达的丧失，以及在昆虫细胞表面上的非功能性蛋白质的表达。在这两个系统中，都保留了化学交联，即使蛋白质无法正确折叠，这两个单体也邻近。我们还确定了氨基酸383中的关键残基，该残基可能促进蛋白质主要结构域之间的通信。我们最近研究了ABCG2调节的机制，发现ABCG2启动子在某些肾细胞癌细胞系中被甲基化，从而导致基因表达降低。我们还确定启动子受组蛋白乙酰化调节，而深肽能够上调某些细胞类型的表达。有趣的是，在某些细胞类型中，基因以非HDAC，非甲基化依赖性方式抑制。这项研究可能有助于我们了解正常干细胞在分化时如何关闭ABCG2以及某些癌细胞如何重新表达转运蛋白。最终导致靶向表达ABCG2细胞的策略。我们还了解到，使用染色质免疫沉淀，在ABCG2启动子中，允许的表观遗传标记在细胞中很明显，这些细胞会响应HDAC抑制，并上调ABCG2 mRNA。然而，在ABCG2 mRNA上调对HDAC抑制不反应HDAC的细胞中，ABCG2启动子的抑制性表观遗传标记持续存在。尽管附近组蛋白的赖氨酸尾巴乙酰化，但这些抑制性标记仍然存在于启动子中，尽管同一细胞中其他基因的上调。这构成了研究对HDAC抑制剂的抗性的模型，这是项目＃2：II的重要方面。组蛋白脱乙酰基酶抑制剂深度肽的临床和实验室研究。我们最近认识到，表达高水平ABCG2的细胞在基因的3'untranslated区域具有截断，该区域去除了去除microRNA结合位点。 MicroRNA，HSA-MIR 519C，显示在该位点结合并减少基因表达和蛋白质翻译。新的研究表明，多种耐药细胞系使用短3'UTR，这表明它是ABCG2上调的一般机制。我们的下一个目的是评估与耐药性有关的其他微RNA。为了评估ABCG2表达的临床样品，我们与Stephen Hewitt和Patty Fetsch合作开发了一种免疫组织化学测定，该测定已用于评估带有载重的载玻片组织阵列。使用这些幻灯片，我们希望发现过表达ABCG2的特定肿瘤类型。由James McMahon博士领导的Michael Dean博士和分子目标发展计划，ABCG2过表达的细胞已用于筛选ABCG2抑制剂。这是一项重要的事业，因为是否可以证明ABCG2在肿瘤药物耐药性中调节口服药物吸收的潜在能力和CNS摄取至关重要。分子目标开发计划的柯蒂斯·亨里希（Curtis Henrich）博士已经确定了我们现在已确认为ABCG2抑制剂的许多打击。这些化合物已在我们的实验室和Suresh Ambudkar博士的二级筛查中进行了评估，以优先考虑进一步的临床前开发。在鉴定ABCG2底物和抑制剂的另一种策略中，我们在NCI药物筛选的60个细胞系中表征了ABCG2表达。该概况使我们能够使用比较分析识别潜在的底物和抑制剂。这些化合物也已进行了次要筛选。我们得出的结论是，药物筛选不能专门定义化合物是否是ABCG2底物 - 但是，通过这种方法可以富集一组化合物。总的来说，这项工作的最佳特征是理解和利用ABCG2作为改善抗癌疗法的治疗靶点的努力。