Overcoming chemoresistance in triple negative breast cancer

项目摘要 三阴性乳腺癌（TNBC）是最具侵袭性的乳腺癌亚型。约占15%， 所有的乳腺癌患者仍然负责30%的乳腺癌死亡。TNBC主要通过以下方式治疗： 常规化疗;然而，对治疗的抗性是常见的，导致高死亡率。 最近，我们确定了缺氧诱导的ECM重塑剂赖氨酰氧化酶（LOX）作为缺氧诱导ECM重塑的关键介质。 在TNBC中的阿霉素抗性（Saatci等人，Nature Communications，2020）。LOX抑制提供了独特的 有机会使最具侵袭性的乳腺肿瘤对标准化疗药物重新敏感。的 该项目的总体目标是（i）描述LOX在化学抗性中的作用，（ii）确定 LOX发挥这些作用的机制，（iii）并产生有效和选择性LOX的原型 抑制剂以克服TNBC中的化学抗性。我们假设（i）LOX不仅诱导抗性， 通过其酶活性对多柔比星，但也对其他化疗药物;（ii）LOX发挥这种作用 两者都通过增加胶原蛋白交联/纤连蛋白组装（典型LOX功能）导致降低 药物渗透和增加整合素介导的信号传导，并通过调节转录（非经典 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， 细胞存活;和（iii）用选择性小分子抑制剂靶向LOX活性将克服 通过阻断TNBC中的典型和非典型LOX功能来抑制化疗抗性。这些假设 将通过追求三个具体目标进行测试：1）确定典型ECM交联功能的作用 LOX在TNBC中对不同化疗药物的抗性中的作用。我们将测试一般化疗增敏剂的作用 通过产生具有CRISPR介导的LOX敲除的细胞， 重组并测试它们在体外和体内对化学抗性的作用。LOX介导的ECM变化 将通过先进的显微镜技术（例如MP-SHG）进行分析，并且所产生的药物渗透将 通过IF和MALDI-MSI进行研究。2)确定非典型转录调节功能的作用 LOX在TNBC化疗耐药性中的作用。我们将确定LOX是否控制全局转录并鉴定新的 LOX底物通过结合转录组学（RNA-Seq）和蛋白质组学（TurboID）方法。我们将 产生氧化缺陷LOX底物并测试它们对LOX介导的化学抗性的影响。3)到 表征新型LOX酶抑制剂并测试其作为TNBC中的化学增敏剂的潜力。我们将 测试我们的抑制剂在具有LOX敲除/重建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， 测试抑制剂用于克服TNBC PDX中的化学抗性。拟议项目预计将 提供关键的机制和表型临床前数据，以表明靶向LOX将克服 最具侵袭性的乳腺癌亚型的化疗耐药性，具有降低死亡率的潜力。

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.