III. Investigation of the ABC Half-Transporter ABCG2

我们的实验室对非pgp介导的耐药机制有着长期的兴趣，已经建立了几种以ABC半转运体ABCG2为重点的耐药细胞系模型。我们成功地从耐米托蒽醌结肠癌细胞系S1-M1-80中克隆了ABCG2，该细胞系表现出atp依赖性的药物积累减少。该基因由6个跨膜结构域和一个ATP结合结构域组成，编码一个半转运蛋白分子，据认为二聚化是其活性所必需的。ABCG2的过度表达使细胞对米托蒽醌、喜树碱、拓扑替康和SN-38（伊立替康的活性代谢物）产生耐药性。ABCG2的底物和抑制剂的发现速度正在加快，并且各种底物和抑制剂都与p -糖蛋白相竞争。越来越多的证据支持ABCG2在药物口服吸收中的作用。我们研究了蛋白质的结构和功能关系。我们在ABCG2 （R482T; R482G）中发现了药物诱导的突变，改变了底物和抑制剂的特异性；然后对ABCG2进行序列分析，确定单核苷酸多态性。我们和其他人报道了在氨基酸141上携带单核苷酸多态性的细胞中运输受损，该多态性将谷氨酰胺转变为赖氨酸。由于拓扑替康的胃肠道吸收与肠上皮中ABCG2的表达有关，这项工作的一个含义是Q141K SNP可能与患者底物药物暴露增加有关。就ABCG2参与药物暴露的程度而言，该SNP可能增加对伊马替尼、伊立替康或拓扑替康等底物的暴露。此外，ABCG2在脑内皮细胞中表达，该蛋白的另一个重要作用是作为血脑屏障的组成部分保护中枢神经系统。这一发现的一个含义是，绕开ABCG2和血脑屏障的化合物可能在治疗或预防中枢神经系统转移方面具有更高的功效。随着我们在新药物如小分子酪氨酸激酶抑制剂中识别ABCG2底物，ABCG2在血脑屏障中定位的重要性将会增加。为了评估ABCG2的二聚化作用，我们的实验室研究了跨膜螺旋1中的gxxx二聚化基序。我们发现该基序对ABCG2的正常运输活性至关重要，但无法证明它介导二聚化。我们最近发现，附近残基T402的突变完全破坏了这种蛋白质的稳定性。高度保守的氨基酸残基553（果蝇的同源残基被认为可以驱动二聚化）的突变导致哺乳动物细胞表面蛋白质表达缺失，昆虫细胞表面表达无功能蛋白。在这两种系统中，化学交联被保留下来，这表明即使蛋白质不能正确折叠，两个单体也会接近。与Balasz Sarkadi博士合作，我们已经生成了Sf9昆虫载体，允许在高表达昆虫系统中转染。该系统对错误折叠的蛋白质具有更强的耐受性，并且已经证明可以产生功能性的ABCG2分子。该系统将允许共同免疫沉淀研究，需要提供我们的突变对二聚化的影响的确认。我们最近研究了ABCG2的调控机制，发现在一些肾细胞癌细胞系中，ABCG2启动子甲基化，导致该基因的表达减少。我们还确定启动子受组蛋白乙酰化的调节，在某些细胞类型中，抑郁肽能够上调表达。有趣的是，也有一些细胞类型的基因以非hdac、非甲基化依赖的方式被抑制。这项研究可能有助于我们了解正常干细胞在分化过程中如何关闭ABCG2，以及一些癌细胞如何重新表达转运蛋白；最终导致针对abcg2表达细胞的策略。我们还通过染色质免疫沉淀了解到，在对HDAC抑制有反应的细胞中，允许的表观遗传标记在ABCG2启动子中是明显的，ABCG2 mRNA上调。然而，在不响应HDAC抑制ABCG2 mRNA上调的细胞中，抑制表观遗传标记持续存在于ABCG2启动子中。尽管附近组蛋白赖氨酸尾部乙酰化，尽管同一细胞中其他基因上调，但这些抑制标记仍然存在于启动子中。这构成了研究对HDAC抑制剂耐药性的模型，这是项目#2:II的一个重要方面。组蛋白去乙酰化酶抑制剂抑郁肽的临床和实验室研究。一个目标是开发一种针对ABCG2的特异性功能检测方法。由于米托蒽醌也是p -糖蛋白的底物，所以当Schinkel等人首次将磷合物a描述为在ABCG2基因敲除小鼠中产生光毒性的药物时，我们对它很感兴趣。我们推断，由于在Pgp基因敲除小鼠中未观察到光毒性，因此嗜磷素a可能是abcg2特异性底物。这可以使临床检测ABCG2更加准确。我们用编码ABCG2的pcDNA载体转染了HEK 293细胞系和HEK 293克隆，用野生型、突变型和SNP序列对其进行了测试，发现5D3抗体测量的细胞表面表达与FTC抑制测量的pheophbide外排之间存在密切的相关性。在表达Pgp或MRP的细胞中未观察到转运。Hoechst 33342 DNA染色也被描述为ABCG2的底物。在骨髓细胞（称为侧群）中鉴定的Hoechst dim细胞在其他实验室中被证明为干细胞富集，被认为是富含abcg2的。我们目前正在参与一项多实验室的研究，旨在表征这些表达abcg2的细胞，并评估癌症干细胞假说。为了评估临床样本中ABCG2的表达，我们与Andrea Abati和Patty Fetsch合作开发了一种使用我们生成的多克隆抗体的免疫组织化学分析。通过这项检测，以及米托蒽醌或磷酸铁苷a的流式细胞检测，我们有能力评估ABCG2在临床耐药中的潜在作用。在Michael Dean博士和James McMahon博士领导的分子靶标开发项目的合作下，ABCG2过表达细胞被用于筛选ABCG2抑制剂。这是一项重要的工作，因为无论ABCG2在肿瘤耐药中是否重要，其调节口服药物吸收和中枢神经系统摄取的潜在能力都将是重要的。分子靶标开发项目的柯蒂斯·亨里奇博士，已经确定了一些靶点，我们现在已经确认这些靶点是ABCG2抑制剂。这些化合物正在我们的实验室和Suresh Ambudkar博士的实验室进行二次筛选评估，以优先进行进一步的临床前开发。在另一种鉴定ABCG2底物和抑制剂的策略中，我们在NCI药物筛选的60个细胞系中表征了ABCG2的表达。该概况使我们能够识别潜在的底物和抑制剂[摘要截断为7800个字符]

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 the oral absorption of pharmacologic agents. 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. In collaboration with Dr. Balasz Sarkadi, we have generated Sf9 insect vectors to allow transfection in the high expression insect system. This system is more tolerant of misfolded protein and has already been shown to generate functional ABCG2 molecules. This system will allow co-immunoprecipitation studies that are needed to provide confirmation of the impact of our mutations on dimerization. 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 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. One goal has been to develop a specific, functional assay for ABCG2. Since mitoxantrone is also a substrate for P-glycoprotein, we were interested in pheophorbide a when it was first described by Schinkel et al as the agent that produced phototoxicity in ABCG2 knockout mice. We reasoned that since phototoxicity had not been observed in the intensively studied Pgp knockout mice, pheophorbide a might be an ABCG2-specific substrate. This could allow more accurate clinical detection of ABCG2. We tested pheophorbide a selected cell lines and in the HEK 293 clones transfected with pcDNA vectors encoding ABCG2 with wild type, mutant and SNP sequences and found a tight correlation between cell surface expression as measured by 5D3 antibody and pheophorbide a efflux as measured by inhibition with FTC. No transport was observed in cells expressing Pgp or MRP. Hoechst 33342 DNA stain has also been described as a substrate for ABCG2. Hoechst dim cells identified among bone marrow cells (known as the side population) were shown in other laboratories to be enriched for stem cells, which were considered to be ABCG2-rich. We are currently involved in a multi-laboratory effort designed to characterize these ABCG2-expressing cells and to evaluate the cancer stem cell hypothesis. In order to evaluate clinical samples for ABCG2 expression, we have developed, in collaboration with Andrea Abati and Patty Fetsch, an immunohistochemical assay using a polyclonal antibody that we generated. With this assay, and with mitoxantrone or pheophorbide a in a flow cytometric assay, we have the ability to evaluate the potential role of ABCG2 in clinical drug resistance. 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 are being 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 us [summary truncated at 7800 characters]