Dissecting mechanisms of anthracycline-induced cardiotoxicity

摘要 虽然癌症影响超过三分之一的人在他们的一生中，改善长期生存已导致 癌症治疗的不良心脏副作用的发生率增加。蒽环类，如 多柔比星（Dox）是各种癌症中化疗的基石，然而，它们的使用因以下因素而复杂化： 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DNA损伤， 细胞凋亡为了确定疾病过程中的新角色，我们首先在 Dox心脏毒性转录组学研究的生物医学数据库。筛选候选基因， 对Dox治疗的反应性，与涉及心脏修复的Notch和Hippo通路的相关性， 和控制细胞死亡。这种系统的方法导致了丝氨酸蛋白酶-3（serinc-3）的鉴定。 3）。W 我们假设在Dox诱导的心脏毒性的情况下，serinc-3通过以下方式表现出保护作用： 对抗Top-2β介导的心肌细胞凋亡，我们将在2个特定目的中探讨 .在目标1中，我们 确定serinc-3在心肌细胞和癌细胞中的Dox损伤中的作用。我们将（i）解剖保护性 serinc-3在Dox心脏毒性中涉及的凋亡途径中的作用，（ii）确定 serinc-3和心肌细胞中的心脏保护性Notch和Hippo通路，以及（iii）进行转录组 测序和定量，基因网络构建，并确定生化和功能途径 （iv）在丝氨酸蛋白酶-3表达调节后富集Dox处理， 使用3xFLAG标记策略、免疫共沉淀和质谱法测定serinc-3，和（v）评估 乳腺癌和淋巴瘤细胞中serinc-3的表达，并定量Dox后的癌细胞增殖 治疗和丝氨酸蛋白酶-3调节。在目标2中，我们将详细研究心肌细胞中serinc-3的体内功能 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Cre-Lox 具有增强的绿色荧光蛋白的小鼠用于成功的serinc-3Cre切除，（ii）定量整体（排出 分数，缩短分数）和节段性（应变，位移）心脏功能后serinc-3 Cre-Lox 在慢性Dox损伤的情况下敲低或腺相关病毒-9介导的过表达，（iii）评估 serinc-3过表达和敲低对体内Dox诱导细胞死亡途径的影响，和（iv）分离的 来自Dox处理的小鼠的心肌细胞，通过转录组测序建立相关矩阵， Serinc-3表达的调节。该研究的成功实施将提供新的 对蒽环类药物诱导的心脏毒性的病理生物学机制的见解，与翻译的影响。

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.