【作者】 郭丽娟；

【导师】 张国淳；

【作者基本信息】 华南理工大学 ， 临床医学， 2024， 硕士

【摘要】 研究背景和目的:乳腺癌是当今世界女性最多发的恶性肿瘤之一,人表皮生长因子受体2(Human epidermal growth factor receptor 2,HER2)的高表达常与侵袭性更强的乳腺癌表型相关。以德喜曲妥珠单抗(Trastuzumab Deruxtecan,T-DXd)为代表的新型抗体偶联药物(Antibody-drug conjugate,ADC)显著改善了HER2阳性乳腺癌患者的预后,但HER2低表达患者的整体客观有效率欠佳,且部分患者对ADC存在原发性或继发性耐药,其中HER2内吞抵抗特性和药物进入溶酶体降解途径的不足是重要的耐药机制。若能增强HER2的内吞作用,提高ADC内化入胞的效率,增加其到达酸性的晚期内体或溶酶体的量,有助于增敏ADC药物的抗肿瘤效应。研究方法:1)Western blot实验检测HER2蛋白表达量,加入蛋白质合成抑制剂和溶酶体抑制剂,与对照组相比,观察溶酶体途径在HER2蛋白降解中的作用;使用瞬时质粒转染实验、实时荧光聚合酶链式反应和western blot实验检测溶酶体相关膜蛋白2A(Lysosome-associated membrane protein type 2A,LAMP2A)和HER2的蛋白表达量之间的关系。2)慢病毒构建稳定过表达HER2的MDA-MB-231细胞模型(野生型、突变型HER2K753Q和突变型HER2 K753R),使用免疫荧光、western blot和活细胞成像实验,检测K753位点乙酰化修饰后HER2在胞膜分布和胞内运输的改变,以及HER2在胞浆中分别与热休克蛋白家族A成员8(Heat shock protein family A member 8,HSPA8)和LAMP2A的共定位关系。3)使用western blot、免疫沉淀、活细胞成像和细胞增殖实验,探究去乙酰化酶(Histone deacetylase,HDAC)抑制剂TMP195与HER2乙酰化修饰的关系,及联用TMP195对T-DXd的内化和细胞毒效应的影响。研究结果:1)溶酶体通路是HER2重要的蛋白降解途径,LAMP2A可负性调控HER2的蛋白水平。2)HER2的K753位点乙酰化修饰能改变其在胞膜上的分布模式,促进HER2内吞并加速其在胞浆内的运输,增强HER2与HSPA8和LAMP2A的共定位,并促进ADC入胞。3)HDAC抑制剂TMP195能使HER2的乙酰化水平升高,提高T-DXd入胞的效率而增加其细胞毒性。结论:溶酶体是HER2重要的蛋白降解场所,其中LAMP2A能负性调控HER2的蛋白量,而HER2 K753位点的乙酰化修饰可能是这种负相关关系的潜在机制;HER2的K753位点乙酰化修饰能够改变胞膜形态并驱动HER2发生快速内吞,这种内吞作用并不依赖于缢断蛋白。同时,HER2在胞浆内运输的效率得到明显提高,与HSPA8、LAMP2A的共定位均显著增加,提示该位点的乙酰化修饰可促进HSPA8参与的HER2胞内运输,并驱动HER2蛋白进入LAMP2A相关溶酶体降解途径。HER2的乙酰化修饰能促进T-DXd高效入胞,而HDAC抑制剂TMP195能够提高HER2的乙酰化水平,二者联用能显著增加T-DXd入胞的量并协同杀伤乳腺癌细胞。HDAC抑制剂与T-DXd药物联用的治疗策略,有望为临床上增敏ADC的疗效提供新思路。更多还原

【Abstract】 Background and object:Breast cancer is the most prevalent malignancy among global women.HER2 amplification is associated with a more aggressive behavior in breast cancer.Although antibody-drug conjugates(ADCs)such as Trastuzumab Deruxtecan(T-DXd)have significantly prolonged the overall survival and decreased the risk of recurrence in patients with HER2 positive breast cancer,the low overall objective response rate still calls for persistent studies.Moreover,some patients are initially resistant or acquired resistance to ADCs.Resistance characteristics of HER2 endocytosis and insufficient drug entry into lysosomal degradation pathways are important mechanisms of ADC drugs resistance.Rapid internalization of HER2 can promote the endocytosis of ADCs,enabling the release of cytotoxicity in late endosomal or lysosomal and enhance the anti-cancer effect of ADC.Methods:1)Western blot was used to detect the expression level of HER2 protein,protein synthesis inhibitors and lysosomal inhibitors were added to observe the role of lysosomal degradation pathway in HER2 protein compared to the control group;The relationship between lysosome-associated membrane protein type 2A(LAMP2A)and HER2 protein expression levels was determined using transient plasmid transfection experiments,real-time quantitative PCR,and western blot.2)We generated stable MDA-MB-231 cell line expressing HER2 via lentivirus transduction,and generated glutamine and arginine substitution mutants at the K753 site(K753Q and K753R).Immunofluorescence,western blot,and live cell imaging were used to detect the membrane distribution and intracellular trafficking of HER2,as well as the colocalization relationships between HER2,HSPA8,and LAMP2 A.3)Western blot,immunoprecipitation,live cell imaging,and cell proliferation experiments were used to detect the effect of histone deacetylase(HDAC)inhibitor TMP195 on HER2 acetylation,and the relationship between TMP195 and the cytotoxicity of ADCs.Results:1)Lysosomal pathway degradation is important for HER2,there is a negative correlation between the protein levels of LAMP2 A and HER2.2)Acetylation at K753 of HER2 could alter the distribution pattern of HER2 on the membrane and promote its trafficking within the cytoplasm,enhance the colocalization of HER2 with HSPA8 and LAMP2 A respectively,and promote the internalization of ADCs.3)HDAC inhibitor TMP195 may increase HER2 acetylation,thereby improving ADC endocytosis efficiency and increasing cytotoxicity.Conclusion:Lysosomal is an important degradation site for HER2,and there is a negative correlation between the expression level of LAMP2 A and HER2,HER2 acetylated at K753 might be the potential molecular mechanism;Acetylation modification at K753 of HER2 can alter its distribution on the membrane,not only promote its rapid dynamin-independent endocytosis,but also significantly improve the trafficking efficiency in the cytoplasm.More importantly,the colocalizations of HER2 with HSPA8 and LAMP2 A were significantly enhanced,suggesting that acetylation at K753 of HER2 may promote the lysosomal degradation associated with LAMP2 A,which might be mediated by HSPA8.Acetylation modification at K753 may also promote efficient internalization of ADCs in breast cancer cells.HDAC inhibitor TMP195 could increase the level of HER2 acetylation and promote the internalization of ADCs into breast cancer cells and synergistically kill them.The strategy of TMP195 in combination with T-DXd is expected to be an improved strategy for patients with HER2 positive breast cancer.更多还原