Dissecting mechanisms of anthracycline-induced cardiotoxicity

摘要 虽然癌症在一生中影响着三分之一以上的人，但长期存活率的提高导致了 癌症治疗的心脏不良反应发生率增加。蒽环类药物 阿霉素(Dox)是各种癌症化疗的基石，然而，它们的使用因 蒽环类药物引起的心脏毒性，最常见的是心肌病。在Dox中发挥作用的机制 心脏毒性是多因素的，包括拓扑异构酶(TOP)-2β介导的最终可能导致的DNA损伤 在细胞凋亡中。为了确定疾病过程中的新因素，我们首先在 Dox心脏毒性转录研究生物医学数据库。对候选基因进行筛选 对Dox治疗的反应性，与心脏修复中牵涉的Notch和Hippo通路有关， 以及对细胞死亡的控制。这一系统的方法导致了丝氨酸结合蛋白-3(丝氨酸-3)的鉴定。 3)。W E假设在Dox诱导的心脏毒性的背景下，Serinc3通过以下方式显示保护作用 对抗TOP-2β介导的心肌细胞凋亡--我们将从两个特定目的来探讨 。在目标1中，我们将 确定丝氨酸氨基转移酶-3在Dox损伤心肌细胞和癌细胞中的作用。我们将(I)剖析保护性 Serinc3在Dox心脏毒性中涉及的细胞凋亡途径中的作用，(Ii)确定 SERINC-3和心肌细胞中保护心脏的Notch和Hippo通路，以及(Iii)转录组 测序和量化，基因网络构建，并确定生化和功能途径 在Serinc3表达调控后的Dox处理中的富集物，(Iv)建立与 使用3xFLAG标记策略、免疫共沉淀和质谱学的血清-3，以及(V)评估 SERINC-3在乳腺癌和淋巴瘤细胞中的表达，并定量检测Dox作用后癌细胞的增殖 治疗和丝裂原-3调节。在目标2中，我们将仔细研究在体心肌细胞中血清-3的功能。 在成年小鼠DOX治疗后。我们将(I)开发他莫昔芬诱导的、心肌细胞特异性的Cre-Lox 用增强的绿色荧光蛋白成功切除SERINC-3Cre的小鼠，(Ii)定量全局(射血) SERINC-3Cre-Lox后的心功能(分数、分数缩短)和节段性(应变、移位) 敲除或腺相关病毒-9介导的过度表达在慢性Dox损伤的背景下，(Iii)评估 丝氨酸氨基转移酶-3过表达和基因敲除对Dox诱导的细胞死亡途径的影响 Dox处理的小鼠心肌细胞通过以下转录组测序建立相关矩阵 丝氨酸蛋白-3表达的调控。拟议研究的成功实施将提供新的 对蒽环类药物引起的心脏毒性的病理生物学的机械洞察，具有翻译含义。

ABSTRACT While cancer affects more than one in three people over their lifetime, improved long-term survival has led to an increase in the incidence of adverse cardiac side-effects of cancer treatments. Anthracyclines such as doxorubicin (Dox) are a cornerstone of chemotherapy in various cancers, however, their use is complicated by anthracycline-induced cardiotoxicity, most commonly cardiomyopathy. The mechanisms at play in Dox cardiotoxicity are multifactorial and include Topoisomerase (Top)-2β-mediated DNA damage that may culminate in apoptosis. To identify novel actors in the disease process, we first conducted a comprehensive search in biomedical databases of Dox cardiotoxicity transcriptomic studies. Candidate genes were screened for responsiveness to Dox treatment, association with the Notch and Hippo pathways implicated in cardiac repair, and the control of cell death. This systematic approach led to the identification of serine incorporator-3 (serinc- 3). W e hypothesize that in the setting of Dox-induced cardiotoxicity, serinc-3 exhibits protective effects by opposing Top-2β-mediated cardiomyocyte apoptosis that we will explore in 2 Specific Aims . In Aim 1, we will determine the role of serinc-3 in Dox injury in cardiomyocytes and cancer cells. We will (i) dissect the protective role of serinc-3 on apoptosis pathways implicated in Dox cardiotoxicity, (ii) determine an association between serinc-3 and the cardioprotective Notch and Hippo pathways in cardiomyocytes, and (iii) perform transcriptome sequencing and quantification, gene network construction, and determine biochemical and functional pathway enrichment in Dox treatment following serinc-3 expression modulation, (iv) establish protein interactions with serinc-3 using a 3xFLAG tagging strategy, co-immunoprecipitation, and mass spectrometry, and (v) evaluate serinc-3 expression in breast cancer and lymphoma cells, and quantify cancer cell proliferation following Dox treatment and serinc-3 modulation. In Aim 2, we will scrutinize in vivo serinc-3 function in cardiomyocytes following adult mouse Dox treatment. We will (i) develop tamoxifen-inducible, cardiomyocyte-specific Cre-Lox mice with enhanced green fluorescent protein for successful serinc-3 Cre excision, (ii) quantify global (ejection fraction, fractional shortening) and segmental (strain, displacement) cardiac function following serinc-3 Cre-Lox knockdown or adeno-associated virus-9-mediated overexpression in the setting of chronic Dox injury, (iii) assess the effect of serinc-3 overexpression and knockdown on Dox-induced cell death pathways in vivo, and (iv) isolate cardiomyocytes from Dox treated mice to establish correlation matrices by transcriptome sequencing following modulation of serinc-3 expression. The successful implementation of the proposed research will provide novel mechanistic insights into the pathobiology of anthracycline-induced cardiotoxicity, with translational implications.