Overcoming chemoresistance in triple negative breast cancer

克服三阴性乳腺癌的化疗耐药性

基本信息

批准号：
10541879
负责人：
Ozgur Sahin
金额：
$ 36.34万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-12-16 至 2026-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10541879
关键词：
3-Dimensional Apoptosis Binding Biological Assay Breast Breast Cancer Cell Breast Cancer Model Breast Cancer Patient Breast Cancer cell line CRISPR/Cas technology Cell Survival Cells Cessation of life Chemoresistance Chemosensitization Clustered Regularly Interspaced Short Palindromic Repeats Collagen Communication Cryoultramicrotomy DNA DNA Damage Data Dose Doxorubicin Drug Kinetics Experimental Models Extracellular Matrix Fibronectins Generations Genes Genetic Transcription Goals HIF1A gene Hypoxia ITGA5 gene Immunofluorescence Immunologic Immunohistochemistry In Vitro Induction of Apoptosis Integrins Knock-out LOXL2 gene Mammary Neoplasms Maximum Tolerated Dose Measures Mediating Mediator Methods Microscopy Mission Nature Nuclear Organoids Output PTK2 gene Paper Pathway interactions Patient-derived xenograft models of breast cancer Penetration Pharmaceutical Preparations Pharmacodynamics Pharmacotherapy Phenotype Physiological Protein-Lysine 6-Oxidase Proteins Proteomics Public Health Refractory Relapse Resistance Role Signal Transduction Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Structure-Activity Relationship System Techniques Testing Therapeutic Toxic effect Transcriptional Regulation United States National Institutes of Health Western Blotting aggressive breast cancer cancer subtypes chemosensitizing agent chemotherapy clinically relevant crosslink disability experimental study in vivo in vivo Model inhibitor mRNA Expression malignant breast neoplasm mortality multi-photon mutant novel overexpression oxidation paralogous gene patient derived xenograft model patient subsets pharmacokinetics and pharmacodynamics pre-clinical protein expression prototype reconstitution screening second harmonic small molecule small molecule inhibitor standard of care success therapy resistant three dimensional cell culture trafficking transcriptome sequencing transcriptomics triple-negative invasive breast carcinoma tumor tumor xenograft

项目摘要

PROJECT SUMMARY Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. It accounts for ~15% of all breast cancer patients yet is responsible for 30% of breast cancer deaths. TNBC is treated primarily by conventional chemotherapy; however, resistance to therapy is common leading to high mortality rates. Recently, we identified hypoxia-induced ECM re-modeler, lysyl oxidase (LOX) as a key mediator of doxorubicin resistance in TNBC (Saatci et al, Nature Communications, 2020). LOX inhibition offers a unique opportunity to re-sensitize the most aggressive breast tumors to standard-of-care chemotherapeutics. The overall objectives of this project are to (i) delineate the roles of LOX in chemoresistance, (ii) determine the mechanisms through which LOX exerts these roles, (iii) and generate prototypes of potent and selective LOX inhibitors to overcome chemoresistance in TNBC. We hypothesize that (i) LOX induces resistance not only to doxorubicin but also to other chemotherapeutic drugs by its enzymatic activity; (ii) LOX exerts this effect both by increasing collagen cross-linking/fibronectin assembly (canonical LOX function) leading to reduced drug penetration and increased integrin-mediated signaling and by regulating transcription (non-canonical LOX function) via interacting and oxidizing its substrates, culminating in activation of FAK/Src signaling and cell survival; and (iii) targeting LOX activity with selective small-molecule inhibitors will overcome chemoresistance by blocking both canonical and non-canonical LOX functions in TNBC. These hypotheses will be tested by pursuing three specific aims: 1) To determine the role of canonical ECM cross-linking function of LOX in resistance to different chemotherapeutics in TNBC. We will test the general chemosensitizer role of LOX and necessity of its enzymatic activity by generating cells with CRISPR-mediated LOX knock-out and reconstitution and testing their effects on chemoresistance in vitro and in vivo. LOX-mediated ECM changes will be analyzed by advanced microscopy techniques, e.g. MP-SHG, and the resulting drug penetration will be studied by IF and MALDI-MSI. 2) To determine the role of non-canonical transcription-regulating functions of LOX in TNBC chemoresistance. We will determine if LOX controls global transcription and identify novel LOX substrates by combining transcriptomics (RNA-Seq) and proteomics (TurboID) approaches. We will generate oxidation-deficient LOX substrates and test their effects on LOX-mediated chemoresistance. 3) To characterize novel LOX enzymatic inhibitors and test their potential as chemosensitizers in TNBC. We will test the selectivity of our inhibitors in cells with LOX knock-out/reconstitution and their off-target profiles and test their chemosensitization ability in organoids. We will perform PK/PD and toxicity profiling studies and test the inhibitors for overcoming chemoresistance in TNBC PDXs. The proposed project is expected to provide key mechanistic and phenotypic pre-clinical data to show that targeting LOX will overcome chemoresistance in the most aggressive breast cancer subtype, with a potential to reduce mortality rates.

项目摘要三重阴性乳腺癌（TNBC）是最具侵略性的乳腺癌亚型。它占约15％所有乳腺癌患者却造成30％的乳腺癌死亡。 TNBC主要由常规化学疗法；但是，对治疗的抵抗是普遍导致死亡率高的常见。最近，我们确定缺氧诱导的ECM重模型，赖氨酸氧化酶（LOX）作为关键介体 TNBC中的阿霉素耐药性（Saatci等人，自然通讯，2020年）。 LOX抑制提供了独特的将最具侵略性的乳腺肿瘤重新敏感到现有的标准化学治疗药的机会。这该项目的总体目标是（i）描述LOX在化学上的作用，（ii）确定 LOX发挥这些作用的机制（iii）并生成有效LOX的原型克服TNBC化学抗性的抑制剂。我们假设（i）lox不仅引起抗药性通过其酶活性来促进阿霉素，但也可以将其用于其他化学治疗药物；（ii）Lox发挥了这种效果通过增加胶原蛋白交联/纤连蛋白组件（典型LOX功能），导致降低药物渗透并增加整联蛋白介导的信号传导和调节转录（非典型的转录） LOX功能）通过相互作用和氧化其底物，最终激活FAK/SRC信号传导和细胞存活；（iii）用选择性小分子抑制剂靶向LOX活性通过阻断TNBC中的规范和非统计LOX功能，化学抗性。这些假设将通过追求三个特定目标来测试：1）确定规范ECM交联功能的作用在TNBC中对不同化学疗法的耐药性的LOX。我们将测试一般的化学敏化作用通过用CRISPR介导的LOX敲除和重建并测试其对体外和体内化学抗性的影响。 LOX介导的ECM变化将通过高级显微镜技术进行分析，例如MP-SHG，由此产生的药物渗透将由IF和MALDI-MSI研究。 2）确定非经典转录调节函数的作用 TNBC化学抗性中的LOX。我们将确定LOX是否控制全局转录并识别新颖 LOX底物通过结合转录组学（RNA-SEQ）和蛋白质组学（涡轮）方法。我们将产生氧化缺陷的LOX底物，并测试其对LOX介导的化学抗性的影响。 3）到表征了新型LOX酶抑制剂，并测试其作为TNBC中化学敏化剂的潜力。我们将通过LOX敲除/重构及其脱靶特征以及其脱靶特征，测试我们抑制剂在细胞中的选择性测试他们在器官中的化学敏化能力。我们将执行PK/PD和毒性分析研究以及测试抑制剂是否克服TNBC PDX中的化学耐药性。拟议的项目预计将提供关键的机械和表型前数据，以表明靶向LOX将克服最具侵略性的乳腺癌亚型的化学抗性，有可能降低死亡率。