PO.ET09.03 · 实验与分子治疗
基于Brigatinib的降解剂作为三阴性乳腺癌的治疗策略
Brigatinib based degraders as a therapeutic strategy for triple negative breast cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：三阴性乳腺癌（TNBC）患者的治疗一直极具挑战性，原因在于缺乏明确定义的分子靶点，且这些细胞具有高度侵袭和增殖能力。因此，迫切需要针对TNBC的新型治疗策略。蛋白酶靶向嵌合体（PROTAC）是一类新兴的治疗性抑制剂，通过招募泛素E3连接酶来选择性降解蛋白质。PROTAC降解剂技术在抑制TNBC进展方面已展现出稳健的效果。
方法：进行了RNA测序及分析。样本提交至PamGene进行激酶组分析。采用Western blot检测蛋白表达。通过MTT实验评估细胞活力。集落形成实验评估克隆形成能力的变化。数据采用GraphPad Prism软件8进行分析，使用单因素方差分析（ANOVA）和非配对双尾Student t检验。所有数据均以三复孔对照细胞进行评估。
结果：对经我们的靶向降解剂处理的LM2-4175 TNBC细胞进行RNA测序分析显示，与凋亡相关的基因增加，而与细胞周期检查点、DNA复制以及P53信号转导通路调控相关的基因减少。对经我们降解剂处理的MDAMB231-LM2-4175 TNBC细胞进行激酶活性谱分析显示，与丝裂原活化蛋白激酶（MAPK）和细胞周期蛋白依赖性激酶（CDK）家族相关的丝氨酸/苏氨酸激酶（STK）显著减少。此外，我们注意到与ephrin受体蛋白相关的蛋白酪氨酸激酶（PTK）显著减少。Western blot分析证实，在多种TNBC细胞系（MDAMB-231-LM2-4175、MDAMB-468和4QXTB）中局部黏着斑激酶（Focal Adhesion Kinase）发生靶向降解。细胞增殖MTT分析显示，我们的降解剂在纳摩尔范围内选择性靶向MDAMB231、MDAMB231-LM2-4175、MDAMB-468和4QXTB（原代）TNBC细胞，而对我们的非肿瘤细胞系MCF10A影响较小。集落形成实验也显示，我们的降解剂能够降低TNBC细胞系MDAMB231-LM2-4175、MDAMB-468和4QXTB的克隆形成能力。
结论：乳腺癌是一种多样且复杂的疾病，已知具有独特的肿瘤间和肿瘤内特征。我们已鉴定出一种候选PROTAC，可在体外以纳摩尔范围选择性靶向TNBC细胞，同时对正常旁观细胞影响极小。该降解剂的进一步开发可作为TNBC新型治疗方法的基础。
查看英文原文 English abstract
Background: Treatment of patients with triple‐negative breast cancer (TNBC) has been challenging due to the absence of well‐defined molecular targets and high invasive and proliferative capabilities of these cells. Therefore, new therapeutic strategies for treatment of TNBC are urgently needed. Protease-targeted chimeras (PROTACs) are a class of emerging therapeutic inhibitors that recruit the ubiquitin E3 ligase to selectively degrade proteins. PROTAC degrader technology has shown robust results in inhibiting TNBC progression.
Methods: RNA sequencing and analysis were done. Samples were submitted to PamGene for kinome profiling and analysis. Western blotting was performed to visualize protein expression. MTT assays were carried out to evaluate cell viability. Colony formation assays assessed changes in clonogenicity. Data were analyzed using Graph Pad Prism Software 8 using one-way ANOVA and the unpaired two-tailed Students t-test. All data were evaluated in triplicate against control cells.
Results: Analysis of RNA sequencing in LM2-4175 TNBC cells treated with our target degrader showed increased genes associated with apoptosis and a decrease in genes associated with cell cycle checkpoint, DNA replication, and regulation of P53 signal transduction pathways. Kinase activity profiling of MDAMB231-LM2-4175 TNBC cells treated with our degrader showed a significant reduction in serine/threonine kinases (STKs) associated with Mitogen-activated protein kinase (MAPK) and cyclin-dependent kinase (CDK) families. In addition, we noticed a significant decrease in protein tyrosine kinases (PTK) associated with ephrin receptor proteins. Western blot analysis confirmed targeted degradation of Focal Adhesion Kinase in multiple TNBC cell lines (MDAMB-231-LM2-4175, MDAMB-468, and 4QXTB). MTT analysis of cell proliferation showed our degrader selectively targeted
MDAMB231, MDAMB231-LM2-4175, MDAMB-468, and 4QXTB (Primary)TNBC cells over our non-neoplastic cell line, MCF10A, in a nanomolar range. Colony formation assays also showed our degrader was able to reduce clonogenicity in TNBC cell lines MDAMB231-LM2-4175, MDAMB-468, and 4QXTB.
Conclusions:Breast cancer is a diverse and intricate disease which is known to have unique inter- and intra-tumoral characteristics. We have identified a candidate PROTAC which can selectively target TNBC cells while minimally affecting normal bystander cells at nanomolar ranges in vitro. The further development of this degrader can serve as the fundamental basis for a novel therapeutic treatment in TNBC.
利益披露 Disclosure
M. Khosla, None..
S. Alahari, None..
G. Wang, None.