Mechanisms of non-classical multidrug resistance in cancer

癌症非经典多药耐药机制

• 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
• 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

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.

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性。在某些情况下，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。我们使用 RNA-seq、ATAC-seq 和 Pro-seq 技术进行了完整的基因组分析，以确定顺铂选择的细胞系中伴随耐药性发展的基因表达的变化，并对顺铂耐药细胞中的变化进行了编目。有助于耐药性。比较顺铂敏感的 1A9 卵巢癌细胞、顺铂耐药的 1A9CP80 细胞和部分回复的 1A9CP80R 细胞中的基因表达，我们注意到耐药细胞中 TPPP3（微管蛋白聚合促进蛋白 3）的表达增加，回复细胞中的水平较低，并且没有在亲本系中观察到的表达。此外，我们注意到顺铂治疗会破坏微管末端的稳定性并减少微管长度，并假设 TPPP3 可能会减轻这些影响。有趣的是，TPPP3 抵消顺铂治疗效果的能力在从 1A9CP80 细胞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 敲除删除 TPPP3 使 1A9CP80 细胞对顺铂部分重新敏感。接受顺铂治疗的患者肿瘤中 TPPP3 的高表达与较差的生存概率相关，表明该蛋白可能具有临床作用。此外，暴露于顺铂（我们已证明其会破坏微管稳定）的细胞对紫杉醇（一种稳定微管的抗微生物药物）具有更强的抵抗力。这些结果对于顺铂的神经毒性以及采用顺铂和紫杉醇组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性的非经典机制，我们正在接触各种药物（包括顺铂和奥沙利铂）的细胞中进行 CRISPR 筛选。这些筛选涉及使用 gRNA 与 CRISPR-cas 构建体相结合，可以激活、抑制或敲除靶基因。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，这些gRNA会开启可以独立赋予耐药性的基因，或者表达不足的基因，这些基因的表达是对顺铂敏感所必需的。我们正在鉴定其过度表达或表达不足会影响耐药性的基因，目的是确定临床相关的分子变化。最近使用 CRISPR 筛选来确定结肠癌细胞奥沙利铂耐药性基础的研究发现，氨基酸转运蛋白在对该药物的敏感性中发挥着重要作用。组蛋白脱乙酰酶抑制剂 (HDI) 在临床上用于治疗皮肤和外周 T 细胞淋巴瘤，FDA 已批准 3 种 HDIs 作为单药疗法。对于实体瘤，HDI 并不有效，这表明这些药物具有内在的耐药机制。我们发现，在携带 Ras 突变的细胞中，HDI 以及 MAPK 和 PI3K 信号通路抑制剂可以实现协同杀伤。我们还发现，当与 HDI 组合时，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，BRD4/PI3K 双重抑制剂 SF2523 与 HDI 联合使用时，对 Ras 突变细胞具有协同毒性。我们与 Mari Yohe 博士合作，证明单独使用 SF2523 在儿童横纹肌肉瘤细胞系模型中特别有效，并且通过添加 HDI romidepsin 可以提高其疗效。高级临床前研究中心 (CAPR) 已同意检查 SF2523/罗米地辛组合在患者来源的横纹肌肉瘤异种移植模型中的疗效。由于 P-糖蛋白的过度表达，培养细胞中可能会出现对罗米地辛等 HDI 的耐药性，但在临床癌症中，耐药性似乎并不是由这种机制引起的。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，其对罗米地辛的耐药性比亲代细胞高约 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 HDIs（如伏立诺他、贝利司他或帕比司他）的耐药性仅高出 3 至 5 倍。 RNA Seq 分析比较了亲本系和抗性系，发现 METTL7A 基因是一种潜在的抗性机制，该基因编码一种描述甚少的甲基转移酶。 METTL7B 是 METTL7A 的旁系同源物，最近被确定为一种能够甲基化硫醇基团的烷基硫醇甲基转移酶。由于罗米地辛的活性形式在其活性形式中含有硫醇，并且硫醇基团的甲基化会阻止分子与 HDAC 结合袋中的锌的配位，因此我们假设 METTL7A 可能能够通过以下方式灭活罗米地辛或其他 HDI：硫醇作为锌结合基团。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基于硫醇的 HDI（例如 KD5170 和拉格唑）重新敏感。有趣的是，转染 METTL7A 的 HEK293 细胞对所有基于硫醇的 HDI 具有耐药性，但 METTL7B 过表达对拉格唑和 KD5170 的耐药性低于 METTL7A，并且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，通过灭活这些药物而赋予对基于硫醇的 HDI 的抗性。为了确定动物模型是否可用于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 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 细胞以表达小鼠、大鼠、鸡或斑马鱼 METTL7A。我们发现任何 METTL7A 同工型的表达都可以赋予对所有测试的含硫醇 HDAC 的抗性，这表明 METTL7A 的功能在物种间是保守的。这些结果使我们培育出 METTL7A 被删除的转基因斑马鱼。我们将对基因敲除鱼进行表征，希望找到 METTL7A 的生理作用。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。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（通过硅水凝胶和聚合物 PTMS）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。通过详细的 RNAseq 分析获得了氧梯度显着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，模拟了通过扩散输送的 20% 氧气的基因表达模式。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，以确定生长条件和氧输送模式是否在影响耐药性模式中发挥主要作用。

项目成果

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

对 DMSO 说不：二甲基亚砜会使顺铂、卡铂和其他铂络合物失活。

• 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

Acute myeloid leukemia : apoptotic signalling and gene expression associated with treatment response

急性髓系白血病：与治疗反应相关的细胞凋亡信号传导和基因表达

• DOI: 10.1038/leu.2016.287
• 发表时间: 2013-10-25
• 期刊: Leukemia
• 影响因子: 11.4
• 作者: M. Lindberg

The role of the NIH in nurturing clinician-scientists.

NIH 在培养临床医生科学家方面的作用。

• 发表时间: 2013-06-13
• 作者: Gottesman; Michael M
• 通讯作者: Michael M

Toward a Better Understanding of the Complexity of Cancer Drug Resistance.

更好地理解癌症耐药性的复杂性。

• DOI: 10.1146/annurev-pharmtox-010715-103111
• 发表时间: 2016-01-06
• 期刊: Annual review of pharmacology and toxicology
• 作者: M. Gottesman;Orit Lavi;M. Hall;J. Gillet
• 通讯作者: J. Gillet

Influence of melanosome dynamics on melanoma drug sensitivity.

黑素体动力学对黑色素瘤药物敏感性的影响。

• 发表时间: 2009-09-16
• 作者: Chen, Kevin G;Leapman, Richard D;Zhang, Guofeng;Lai, Barry;Valencia, Julio C;Cardarelli, Carol O;Vieira, Wilfred D;Hearing, Vincent J;Gottesman, Michael M
• 通讯作者: Gottesman, Michael M