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.

项目概要三阴性乳腺癌（TNBC）是最具侵袭性的乳腺癌亚型。约占15% 所有乳腺癌患者仍占乳腺癌死亡人数的 30%。 TNBC 的治疗主要是常规化疗；然而，对治疗的耐药性很常见，导致高死亡率。最近，我们发现缺氧诱导的 ECM 重塑因子赖氨酰氧化酶 (LOX) 是缺氧诱导的 ECM 重塑的关键介质。 TNBC 中的阿霉素耐药性（Saatci 等人，Nature Communications，2020）。 LOX 抑制提供了独特的使最具侵袭性的乳腺肿瘤对标准化疗药物重新敏感的机会。这该项目的总体目标是 (i) 描述 LOX 在化学耐药性中的作用，(ii) 确定 LOX 发挥这些作用的机制，(iii) 并生成有效且选择性的 LOX 原型抑制剂克服 TNBC 的化疗耐药性。我们假设 (i) LOX 不仅会诱导耐药性通过其酶活性，不仅适用于阿霉素，还适用于其他化疗药物； (ii) LOX发挥这种作用两者都是通过增加胶原蛋白交联/纤连蛋白组装（典型的 LOX 功能）来减少药物渗透和增加整合素介导的信号传导以及通过调节转录（非规范 LOX 功能）通过与其底物相互作用和氧化，最终激活 FAK/Src 信号传导，细胞存活； (iii) 使用选择性小分子抑制剂靶向 LOX 活性将克服通过阻断 TNBC 中的经典和非经典 LOX 功能来实现化学耐药性。这些假设将通过追求三个具体目标进行测试：1）确定规范 ECM 交联功能的作用 LOX 在 TNBC 中对不同化疗药物的耐药性。我们将测试一般化学增敏剂的作用通过生成具有 CRISPR 介导的 LOX 敲除的细胞来了解 LOX 及其酶活性的必要性重构并测试它们对体外和体内化疗耐药性的影响。 LOX介导的ECM变化将通过先进的显微镜技术进行分析，例如MP-SHG，由此产生的药物渗透将通过 IF 和 MALDI-MSI 进行研究。 2) 确定非规范转录调节功能的作用 LOX 在 TNBC 化疗耐药中的作用。我们将确定 LOX 是否控制全局转录并识别新的通过结合转录组学 (RNA-Seq) 和蛋白质组学 (TurboID) 方法来研究 LOX 底物。我们将生成氧化缺陷的 LOX 底物并测试它们对 LOX 介导的化学耐药性的影响。 3) 至表征新型 LOX 酶抑制剂并测试其作为 TNBC 化学增敏剂的潜力。我们将测试我们的抑制剂在具有 LOX 敲除/重构的细胞中的选择性及其脱靶特征，测试它们在类器官中的化学增敏能力。我们将进行 PK/PD 和毒性分析研究，测试抑制剂克服 TNBC PDX 的化疗耐药性。拟议项目预计提供关键的机制和表型临床前数据，表明靶向 LOX 将克服最具侵袭性的乳腺癌亚型的化疗耐药性，有可能降低死亡率。