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Mechanisms of non-classical multidrug resistance in cancer

癌症非经典多药耐药机制

基本信息

批准号：
10926078
负责人：
Michael Gottesman
金额：
$ 170.42万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/10926078
关键词：
3-Dimensional ABCB1 gene ABCC1 gene ABCG2 gene ATAC-seq Affect Agreement Amino Acid Transporter Animal Model Antineoplastic Agents Binding Bioreactors Blood capillaries Breast Cancer Cell CRISPR screen Cancer Cell Growth Cataloging Cell Death Cell Line Cell Survival Cell physiology Cell surface Cells Chickens Childhood Rhabdomyosarcoma Cisplatin Clinical Clustered Regularly Interspaced Short Palindromic Repeats Code Collaborations Colon Carcinoma Complex Cultured Cells Cutaneous Cytoplasm Cytotoxic agent Development Diffusion Disease Drug resistance Exposure to FDA approved Fishes Gene Expression Gene Expression Profile Genes Genomics Goals Growth Guide RNA HDAC3 gene Histone Deacetylase Histone Deacetylase Inhibitor Homologous Gene Hydrogels In Vitro KB Cells Knock-out Length MAP Kinase Gene MCF7 cell Malignant Neoplasms Malignant neoplasm of ovary Methylation Methyltransferase Microtubule Stabilization Microtubules Mitogen-Activated Protein Kinase Inhibitor Modeling Molecular Multi-Drug Resistance Multidrug Resistance-Associated Proteins Mus Mutation Oxygen PIK3CG gene Paclitaxel Patients Pattern Peripheral Pharmaceutical Preparations Phenotype Physiological Platinum Compounds Play Polymers Probability Protein Isoforms Proteins Rattus Resistance Rhabdomyosarcoma Role Signal Pathway Silicon Solid Neoplasm Specificity Specimen Sulfhydryl Compounds Suspensions System T-Cell Lymphoma Technology Testing Transfection Transgenic Organisms Tubulin Validation Verapamil Vertebrates Vorinostat Zebrafish Zinc cancer cell cancer drug resistance clinically relevant drug resistance development efficacy evaluation efflux pump in vivo inhibitor mutant neurotoxicity overexpression oxaliplatin paralogous gene patient derived xenograft model polymerization pre-clinical research prevent receptor resistance mechanism scale up transcriptome sequencing tumor uptake

项目摘要

Three major approaches have been taken to define non-classical multidrug resistance in cancer. In the first, we isolate KB cells and ovarian cancer cells resistant to increasing levels of cisplatin (CP-r) and demonstrate multidrug resistance to many other cytotoxic agents. In some cases, 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. We have undertaken a complete genomic analysis using RNA-seq, ATAC-seq and Pro-seq technologies to define the alterations in gene expression that accompany the development of drug resistance in cisplatin-selected cell lines and one cataloguing alterations in cisplatin-resistant cells that contribute to drug resistance. Comparing gene expression in cisplatin-sensitive 1A9 ovarian cancer cells, cisplatin-resistant 1A9CP80 cells and partially revertant 1A9CP80R cells, we noted increased expression of TPPP3 (tubulin polymerization promoting protein 3) in the resistant cells, with lower levels in the revertant cells and no expression observed in the parental line. Additionally, we noted that cisplatin treatment destabilizes microtubule ends and reduces microtubule length and hypothesized that TPPP3 might mitigate these effects. Interestingly, the ability of TPPP3 to counteract the effects of cisplatin treatment were most effective in tubulin purified from 1A9CP80 cells and least effective in 1A9 cells and appeared to correlate with changes in expression of tubulin isoforms in the cell lines. Deletion of TPPP3 via CRISPR knockout partially resensitized the 1A9CP80 cells to cisplatin. High expression of TPPP3 in tumors from patients treated with cisplatin correlated with worse survival probability, suggesting a possible clinical role for this protein. In addition, cells exposed to cisplatin (which we have shown destabilizes microtubules) are more resistant to paclitaxel, an antimicrobule drug that stabilizes microtubules. These results have implications both for the neurotoxicity of cisplatin, and for strategies that employ combinations of cisplatin and paclitaxel to treat ovarian cancer and other cancers. To understand more about non-classical mechanisms of multidrug resistance in cancer, we are undertaking CRISPR screens in cells exposed to various drugs including cisplatin and oxaliplatin. These screens involve using gRNAs in combination with CRISPR-cas constructs that can activate, inhibit, or knock out target genes. Cells exposed to platinum compounds or other drugs undergo cell death and surviving cells overexpress gRNAs which turn on genes which can independently confer resistance, or underexpress genes whose expression is needed for sensitivity to cisplatin. We are identifying genes whose over- or under-expression affects drug resistance with the goal of defining clinically relevant molecular changes. Recent studies using CRISPR screens to determine the basis of oxaliplatin resistance in colon cancer cells has identified amino acid transporters as playing an important role in sensitivity to this drug. Histone deacetylase inhibitors (HDIs) are used clinically to treat cutaneous and peripheral T-cell lymphomas, diseases for which 3 HDIs have been FDA approved as single-agent therapies. In the case of solid tumors, the HDIs have not been effective, suggesting intrinsic resistance mechanisms to these drugs. We found that synergistic killing can be achieved with HDIs and inhibitors of the MAPK and PI3K signaling pathways in cells that harbor Ras mutations. We also found that a dual ERK/PI3K inhibitor could take the place of separate MAPK and PI3K inhibitors when combined with an HDI. Further studies have shown that the dual BRD4/PI3K inhibitor SF2523 is synergistically toxic to Ras mutant cells when combined with an HDI. In collaboration with Dr. Mari Yohe, we demonstrated that SF2523 alone is particularly effective in childhood rhabdomyosarcoma cell line models and its efficacy can be increased by the addition of the HDI romidepsin. The Center for Advanced Preclinical Research (CAPR) has agreed to examine the efficacy of the SF2523/romidepsin combination in patient-derived xenograft models of rhabdomyosarcoma. Resistance to HDI's such as romidepsin can occur in cultured cells owing to overexpression of P-glycoprotein, but in clinical cancers resistance does not appear to be due to this mechanism. To identify non-P-gp mechanisms of resistance, we selected MCF-7 breast cancer cells with romidepsin and verapamil to yield the MCF-7 DpVp300 line which is about 200-fold more resistant to romidepsin than the parental cells. The cells are uniquely resistant to romidepsin, as the resistant line was only 3- to 5-fold more resistant to other HDIs such as vorinostat, belinostat, or panobinostat. RNA Seq analysis comparing the parental and resistant line identified the gene METTL7A, which codes for a poorly-described methyltransferase, as a potential resistance mechanism. METTL7B, a paralog of METTL7A, was recently determined to be an alkly thiol methyltransferase that is capable of methylating thiol groups. As the active form of romidepsin has a thiol in its active form, and as methylation of the thiol group would prevent coordination of the molecule with zinc in the HDAC binding pocket, we hypothesized that METTL7A might be able to inactivate romidepsin or other HDIs with a thiol as the zinc-binding group. In support of this hypothesis, knockout of METTL7A from DpVp300 cells resensitized the cells to romidepsin as well as other thiol-based HDIs such as KD5170 and largazole. Interestingly, HEK293 cells transfected with METTL7A were resistant to all of the thiol-based HDIs, but METTL7B overexpression conferred less resistance to largazole and KD5170 than METTL7A and no resistance to romidepsin. METTL7A and METTL7B thus appear to be methyltransferases with somewhat different specificity that confer resistance to thiol-based HDIs by inactivating these drugs. To determine if animal models could be used to elucidate the normal function of these methyltransferases, we examined homologs of METTL7A from different species. We found that METTL7A is conserved across vertebrates while METTL7B is not. To determine if the ability of METTL7A to methylate thiols is conserved, we transfected HEK293 cells to express mouse, rat, chicken, or zebrafish METTL7A. We found that expression of any of the METTL7A isoforms could confer resistance to all of the thiol-containing HDACis tested, suggesting that the function of METTL7A is conserved across species. These results have led us to create a transgenic zebrafish where METTL7A is deleted. We will characterize the knockout fish in hopes of finding a physiological role for METTL7A. Validation of these results, indicating that MDR is complex and multifactorial in clinical cancers, will require the development of reliable in vitro culture models. Towards this goal, we have developed a bioreactor that mimics capillary delivery (through silicon hydrogels and the polymer PTMS) 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. Evidence that oxygen gradients substantially change gene expression patterns has been obtained by detailed RNAseq analysis. Delivery of physiological concentrations of 3% oxygen directly to cells via artificial capillaries mimics the gene expression patterns of 20% oxygen delivered via diffusion. The bioreactor can be scaled up for growth of multiple cultures of primary cancer cells or cultured cancer cells to determine whether growth conditions and mode of oxygen delivery play a primary role in affecting patterns of drug resistance.

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，我们分离出对顺铂（CP-r）耐药的KB细胞和卵巢癌细胞，并证明了对许多其他细胞毒性药物的多药耐药。在某些情况下，这种交叉抗性模式是由于每种药物的摄取减少，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。我们使用RNA-seq、ATAC-seq和Pro-seq技术进行了完整的基因组分析，以确定顺铂选择细胞系中伴随耐药发展的基因表达改变，并对顺铂耐药细胞中导致耐药的改变进行了编目录。比较顺铂敏感的1A9卵巢癌细胞、顺铂耐药的1A9CP80细胞和部分逆转的1A9CP80R细胞的基因表达，我们发现耐药细胞中TPPP3（微管蛋白聚合促进蛋白3）的表达增加，而逆转细胞中TPPP3的表达水平较低，亲本系中未见表达。此外，我们注意到顺铂治疗破坏微管末端的稳定性并减少微管长度，并假设TPPP3可能减轻这些影响。有趣的是，TPPP3对抗顺铂治疗效果的能力在从1A9CP80细胞中纯化的微管蛋白中最有效，而在1A9细胞中效果最差，并且似乎与细胞系中微管蛋白亚型表达的变化相关。通过CRISPR敲除TPPP3使1A9CP80细胞对顺铂部分重敏。顺铂治疗患者肿瘤中TPPP3的高表达与较差的生存概率相关，提示该蛋白可能具有临床作用。此外，暴露于顺铂的细胞（我们已经证明顺铂会破坏微管的稳定性）对紫杉醇（一种稳定微管的抗微生物药物）更有抵抗力。这些结果对顺铂的神经毒性以及采用顺铂和紫杉醇联合治疗卵巢癌和其他癌症的策略都有影响。为了更多地了解癌症多药耐药的非经典机制，我们正在对暴露于包括顺铂和奥沙利铂在内的各种药物的细胞进行CRISPR筛选。这些筛选包括使用grna与CRISPR-cas结构相结合，可以激活、抑制或敲除目标基因。暴露于铂化合物或其他药物的细胞会发生细胞死亡，存活的细胞会过度表达grna，从而开启能够独立赋予耐药性的基因，或者表达对顺铂敏感所需的基因。我们正在鉴定过度或过低表达影响耐药性的基因，目的是确定临床相关的分子变化。最近的研究使用CRISPR筛选来确定结肠癌细胞对奥沙利铂耐药的基础，发现氨基酸转运蛋白在对这种药物的敏感性中起着重要作用。组蛋白去乙酰化酶抑制剂（hdi）在临床上用于治疗皮肤和周围t细胞淋巴瘤，其中3种hdi已被FDA批准为单药治疗。在实体瘤的情况下，hdi没有效果，这表明对这些药物的内在耐药机制。我们发现hdi和MAPK和PI3K信号通路抑制剂可以在含有Ras突变的细胞中实现协同杀伤。我们还发现，当与HDI联合使用时，双重ERK/PI3K抑制剂可以取代单独的MAPK和PI3K抑制剂。进一步的研究表明，双BRD4/PI3K抑制剂SF2523与HDI联合使用时对Ras突变细胞具有协同毒性。与Mari Yohe博士合作，我们证明SF2523单独对儿童横纹肌肉瘤细胞系模型特别有效，并且可以通过添加HDI罗米地新来提高其疗效。美国高级临床前研究中心（CAPR）已同意研究SF2523/罗米地辛联合疗法在横纹肌肉瘤患者来源异种移植模型中的疗效。由于p -糖蛋白的过度表达，在培养细胞中可能发生对罗米地辛等HDI的耐药性，但在临床癌症中，耐药性似乎不是由于这种机制。为了确定非p -gp耐药机制，我们选择了含有罗米地辛和维拉帕米的MCF-7乳腺癌细胞，以产生MCF-7 DpVp300系，该系对罗米地辛的耐药性比亲本细胞高约200倍。这些细胞对罗米地辛具有独特的耐药性，因为耐药系对其他hdi（如伏立诺他、贝利诺他或帕比诺他）的耐药性仅为3- 5倍。通过比较亲本和抗性品系的RNA Seq分析，鉴定出METTL7A基因是一种潜在的抗性机制，该基因编码一种鲜为人知的甲基转移酶。METTL7B是METTL7A的类似物，最近被确定为一种碱性硫醇甲基转移酶，能够甲基化硫醇基团。由于罗米地辛的活性形式在其活性形式中含有巯基，并且巯基的甲基化会阻止HDAC结合袋中分子与锌的配合，我们假设METTL7A可能能够灭活罗米地辛或其他以巯基为锌结合基的hdi。为了支持这一假设，从DpVp300细胞中敲除METTL7A使细胞对罗米地辛以及其他硫醇基hdi（如KD5170和largazole）重新敏感。有趣的是，转染METTL7A的HEK293细胞对所有巯基hdi都有耐药性，但METTL7B过表达对largazole和KD5170的耐药性低于METTL7A，对罗米地辛没有耐药性。因此，METTL7A和METTL7B似乎是具有不同特异性的甲基转移酶，通过使这些药物失活而赋予对巯基hdi的抗性。为了确定动物模型是否可以用来阐明这些甲基转移酶的正常功能，我们检查了来自不同物种的METTL7A的同源物。我们发现METTL7A在脊椎动物中是保守的，而METTL7B则不是。为了确定METTL7A甲基化硫醇的能力是否保守，我们转染HEK293细胞表达小鼠、大鼠、鸡或斑马鱼的METTL7A。我们发现任何一种METTL7A亚型的表达都可以赋予对所有含硫醇的HDACis的抗性，这表明METTL7A的功能在物种间是保守的。这些结果使我们创造了一种转基因斑马鱼，其中METTL7A被删除。我们将对敲除鱼进行表征，希望找到METTL7A的生理作用。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。为了实现这一目标，我们开发了一种生物反应器，可以模拟在3D悬浮液中生长的细胞中氧气的毛细管输送（通过硅水凝胶和聚合物PTMS）。我们已经证明了生理氧梯度和癌细胞的改变生长更接近体内表型。通过详细的RNAseq分析获得了氧气梯度实质上改变基因表达模式的证据。通过人工毛细血管将生理浓度的3%氧气直接输送到细胞中，模拟了通过扩散输送20%氧气的基因表达模式。该生物反应器可按比例放大，用于原发癌细胞或培养癌细胞的多种培养物的生长，以确定生长条件和氧气输送模式是否在影响耐药模式中起主要作用。

项目成果

期刊论文数量（25）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Say no to DMSO: dimethylsulfoxide inactivates cisplatin, carboplatin, and other platinum complexes.

DOI：
10.1158/0008-5472.can-14-0247
发表时间：
2014-07-15
期刊：
Cancer research
影响因子：
11.2
作者：
Hall MD;Telma KA;Chang KE;Lee TD;Madigan JP;Lloyd JR;Goldlust IS;Hoeschele JD;Gottesman MM
通讯作者：
Gottesman MM

Involvement of ABC transporters in melanogenesis and the development of multidrug resistance of melanoma.

DOI：
10.1111/j.1755-148x.2009.00630.x
发表时间：
2009-12
期刊：
Pigment cell & melanoma research
影响因子：
4.3
作者：
Chen KG;Valencia JC;Gillet JP;Hearing VJ;Gottesman MM
通讯作者：
Gottesman MM

SIRT1 contributes in part to cisplatin resistance in cancer cells by altering mitochondrial metabolism.

DOI：
10.1158/1541-7786.mcr-07-2130
发表时间：
2008-09
期刊：
MOLECULAR CANCER RESEARCH
影响因子：
5.2
作者：
Liang, Xing-Jie;Finkel, Toren;Shen, Ding-Wu;Yin, Jun-Jie;Aszalos, Adorjan;Gottesman, Michael M.
通讯作者：
Gottesman, Michael M.

Evaluation of fluorophore-tethered platinum complexes to monitor the fate of cisplatin analogs.

DOI：
10.1007/s00775-015-1290-2
发表时间：
2015-10
期刊：
JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY
影响因子：
3
作者：
Jagodinsky, Justin C.;Sulima, Agnieszka;Cao, Yiqi;Poprawski, Joanna E.;Blackman, Burchelle N.;Lloyd, John R.;Swenson, Rolf E.;Gottesman, Michael M.;Hall, Matthew D.
通讯作者：
Hall, Matthew D.

Cross-resistance of cisplatin selected cells to anti-microtubule agents: Role of general survival mechanisms.