Liu Jian, Liu Zi, Hu Junfeng, Fan Binru, Zhang Shizhun, Chang Kaili, Mao Xiuping, Huang Guozheng, Liu Zhi, Ma Liang
Department of Chemical Biology and Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Anhui University of Technology, Ma'anshan, Anhui, 243002, China.
Department of Pathology, Ma'anshan Municipal People's Hospital, Ma'anshan, Anhui, 243000, China.
Med Oncol. 2025 Apr 9;42(5):155. doi: 10.1007/s12032-025-02707-3.
HER2-positive breast cancer is an aggressive subtype with unfavorable prognoses. Although HER2-targeted agents represented by monoclonal antibodies have achieved remarkable success in the clinic, there are still a substantial number of patients with disease relapse. Recently, multifunctional fusion proteins obtained via genetic engineering technology have received much attention in targeted tumor therapy, especially in breast cancer. In this study, we genetically engineered a novel recombinant fusion protein, named HMK, which was designed as a bifunctional construct including the HER2-specific affibody Z for targeted receptor recognition, and a proapoptotic module featuring a cell-penetrating octa-arginine (R8) motif conjugated to an antimicrobial peptide KLA. High-purity HMK proteins were successfully obtained using E. coli expression system and Ni-Nitrilotriacetic acid affinity purification method. HMK exhibited higher cytotoxicity in HER2-positive breast cancer cells SK-BR-3 (IC of 8.36 ± 0.62 μM) compared to normal breast epithelial cells MCF-10A (IC of 32.40 ± 2.93 μM), demonstrating favorable selectivity. HMK induced apoptosis in SK-BR-3 cells via activating both endogenous and exogenous apoptotic pathways, as evidenced by the cleavage of Caspase 8, Caspase 9, Caspase 3, and PARP. Caspase inhibitor Z-VAD significantly reversed the function of HMK in SK-BR-3 cells, suggesting that caspase-dependent apoptosis was crucial for the anti-breast cancer activity of HMK. Our results suggested that HMK protein may have the potential to become a candidate molecule for HER2-positive breast cancer treatment.
人表皮生长因子受体2（HER2）阳性乳腺癌是一种侵袭性亚型，预后不良。尽管以单克隆抗体为代表的HER2靶向药物在临床上取得了显著成功，但仍有相当数量的患者疾病复发。近年来，通过基因工程技术获得的多功能融合蛋白在肿瘤靶向治疗中备受关注，尤其是在乳腺癌治疗方面。在本研究中，我们通过基因工程构建了一种新型重组融合蛋白，命名为HMK，它被设计为一种双功能构建体，包括用于靶向受体识别的HER2特异性亲和体Z，以及一个具有细胞穿透性八聚精氨酸（R8）基序并与抗菌肽KLA偶联的促凋亡模块。使用大肠杆菌表达系统和镍-次氮基三乙酸亲和纯化方法成功获得了高纯度的HMK蛋白。与正常乳腺上皮细胞MCF-10A（IC50为32.40±2.93 μM）相比，HMK在HER2阳性乳腺癌细胞SK-BR-3中表现出更高的细胞毒性（IC50为8.36±0.62 μM），显示出良好的选择性。HMK通过激活内源性和外源性凋亡途径诱导SK-BR-3细胞凋亡，Caspase 8、Caspase 9、Caspase 3和PARP的裂解证明了这一点。Caspase抑制剂Z-VAD显著逆转了HMK在SK-BR-3细胞中的功能，表明caspase依赖性凋亡对于HMK的抗乳腺癌活性至关重要。我们的结果表明，HMK蛋白可能有潜力成为HER2阳性乳腺癌治疗的候选分子。
