Characterizing Regulatory Mechanisms Underlying Drug Resistance in Breast Cancer Using Keratin 19

项目总结 抗药性仍然是癌症患者治愈的主要限制因素。在胸前 最近，细胞周期蛋白依赖性激酶4和6(CDK4/6)抑制剂已被批准用于治疗癌症患者 患有晚期雌激素受体阳性的肿瘤。然而，大多数患者表现出抵抗力，因为缺乏 预测性生物标记物。需要了解耐药的分子基础才能提供 在确定预测性生物标记物和开发有效治疗策略方面取得重大突破。 根据我们以前的工作和初步数据，我们提出了一个细胞骨架蛋白K19(K19)， 它目前是最可靠的诊断和预后标志物之一，调节信号转导 对CDK4/6抑制剂产生抗药性的事件。我们发现K19结合并抑制了一个 多功能激酶葛兰素史克稳定乳腺癌细胞中CDK4/6伴侣细胞周期蛋白D3。K19-GSK3 相互作用与Gsk3在细胞核中的积累减少有关，而Gsk3是Gsk3 DNA的关键 功能。K19还能促进细胞增殖，并维持细胞对CDK4/6抑制剂的敏感性。我们 假设K19依赖抑制GSK3稳定的细胞周期蛋白D3导致肿瘤 依赖于CDK4/6途径的生长，并使它们对CDK4/6抑制剂敏感。在分子水平上， 我们推测K19丝作为Gsk3的细胞质支架，阻止其进入细胞核。 在那里它使细胞周期蛋白D3磷酸化以进行降解。为了解决我们的假设，我们建议1)确定 K19对Gsk3信令网络的影响；2)K19与Gsk3的相互作用；3) 确定K19对耐药和肿瘤生长的影响。为此，在目标1中，我们将确定 K19-Gsk3相互作用的上游调节因子以及其活动影响K19-Gsk3的靶标 依赖表型。在目标2中，GSK3结合缺陷的K19突变体将被表征以确定如何 K19与Gsk3相互作用，影响Gsk3的核进入。在目标3中，我们将评估K19如何影响 不同乳腺癌亚型对CDK4/6抑制剂的敏感性及检测K19和GSK3在乳腺癌发生发展中的作用 体内肿瘤对CDK4/6抑制剂的敏感性。在成功完成后，从 研究可以帮助设计针对K19依赖的信号通路的新药，建立K19作为预测因素 CDK4/6抑制剂的生物标志物，并开发有效的治疗策略来对抗药物 抵抗。

PROJECT SUMMARY Drug resistance continues to be the major limiting factor in achieving cures for cancer patients. In breast cancer, cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors have been approved recently to treat patients with advanced estrogen receptor-positive tumors. However, most patients exhibit resistance due to a lack of predictive biomarker. Understanding the molecular basis underlying drug resistance is required to provide a critical breakthrough in identifying a predictive biomarker and developing effective therapeutic strategies. Based on our previous work and preliminary data, we propose that a cytoskeletal protein keratin 19 (K19), which currently serves as one of the most reliable diagnostic and prognostic markers, regulates signaling events to impact resistance against CDK4/6 inhibitors. We found that K19 binds to and inhibits a multifunctional kinase GSK to stabilize a CDK4/6 partner cyclin D3 in breast cancer cells. K19-GSK3 interaction was associated with decreased accumulation of GSK3 in the nucleus that is crucial to GSK3 function. Moreover, K19 promoted proliferation and maintained the sensitivity of cells to CDK4/6 inhibitors. We hypothesize that cyclin D3 stabilized by K19-dependent inhibition of GSK3 causes tumors to become dependent on the CDK4/6 pathway for growth and sensitizes them to CDK4/6 inhibitors. At the molecular level, we surmise that K19 filaments serve as cytoplasmic scaffolds for GSK3 to prevent its entry into the nucleus where it phosphorylates cyclin D3 for degradation. To address our hypotheses, we propose to 1) determine K19 effects on the GSK3 signaling network, 2) characterize the interaction between K19 and GSK3, and 3) determine the impact of K19 on drug resistance and tumor growth. To this end, in Aim 1, we will identify upstream regulators of K19-GSK3 interaction as well as GSK3 targets whose activities affect K19- dependent phenotypes. In Aim 2, GSK3-binding deficient K19 mutant will be characterized to determine how K19 interacts with GSK3 and affects the nuclear entry of GSK3. In Aim 3, we will assess how K19 affects sensitivities of various breast cancer subtypes to CDK4/6 inhibitors and test the role of K19 and GSK3 on the sensitivity of tumors to a CDK4/6 inhibitor in vivo. Upon successful completion, the knowledge gained from the study can help design new drugs targeting K19-dependent signaling pathways, establish K19 as a predictive biomarker for response to CDK4/6 inhibitors, and develop effective therapeutic strategies to combat drug resistance.