Anti-breast cancer activity of a novel genetically engineered fusion protein composed of HER2 affibody and proapoptotic peptide R8-KLA.

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.