Overcoming chemoresistance in triple negative breast cancer

项目总结 三阴性乳腺癌(TNBC)是最具侵袭性的乳腺癌亚型。它占到约15%的 然而，所有乳腺癌患者都要为30%的乳腺癌死亡负责。TNBC的主要治疗方法是 常规化疗；然而，对治疗的抗药性普遍导致高死亡率。 最近，我们发现低氧诱导的细胞外基质重塑因子赖氨酰氧化酶(LOX)是一种关键的细胞外基质调节因子。 TNBC对阿霉素的耐药性(Saatci等人，自然通讯，2020年)。LOX抑制提供了一种独特的 使最具侵袭性的乳腺肿瘤对标准护理化疗药物重新敏感的机会。这个 该项目的总体目标是(I)阐明LOX在化疗耐药中的作用，(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 克服TNBC化疗耐药的抑制剂。我们假设(I)LOX不仅诱导抗性 对阿霉素，但也对其他化疗药物的酶活性；(Ii)LOX发挥这一作用 两者都通过增加胶原交联/纤维连接蛋白组装(典型的LOX功能)导致减少 药物渗透和增加整合素介导的信号和通过调节转录(非规范 LOX功能)通过相互作用和氧化其底物，最终激活FAK/Src信号和 细胞存活；以及(Iii)用选择性小分子抑制剂靶向LOX活性将克服 阻断TNBC中典型和非典型LOX功能的化疗耐药。这些假设 将通过追求三个具体目标进行测试：1)确定规范的ECM交联剂的作用功能 LOX在TNBC对不同化疗药物耐药中的作用我们将测试一般的化学增敏剂的作用 CRISPR介导的LOX基因敲除细胞及其酶活性的必要性 重组和检测它们在体内外对化疗耐药的影响。LOX介导的细胞外基质改变 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 被IF和MALDI-MSI研究。2)确定非规范转录调节功能的作用 LOX在TNBC耐药中的作用。我们将确定LOX是否控制全球转录并识别新的 转录组学(RNA-Seq)和蛋白质组学(TurboID)相结合的方法。我们会 产生氧化缺陷的LOX底物，并测试它们对LOX介导的化疗耐药性的影响。3)至 表征新的LOX酶抑制剂，并测试它们作为TNBC化学增敏剂的潜力。我们会 在LOX基因敲除/重组的细胞中测试我们的抑制剂的选择性及其脱靶曲线和 检测它们对有机物的化学增敏能力。我们将进行PK/PD和毒性分析研究，并 测试这些抑制剂以克服TNBC PDX中的化疗耐药性。拟议中的项目预计将 提供关键的机制和表型临床前数据，以表明靶向LOX将克服 在最具侵袭性的乳腺癌亚型中存在化疗耐药性，有可能降低死亡率。

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.