Peptide L1H9 derived from the interaction of structural human rhomboid family 1 and 80-kDa subunit of Ku antigen sensitizes breast cancer to chemoradiotherapy

Chemoradio-resistance remains a major challenge in breast cancer (BC). Human rhomboid family 1 (RHBDF1) is known as an oncoprotein, but its function on BC chemoradio-resistance is unclear. Here, we reported that peptide LHRHBDF1KWQRKSIRH9 (L1H9) derived from the structural RHBDF1-80-kDa subunit of Ku antigen (Ku80) interaction sensitized BC to chemoradiotherapy. RHBDF1 pulldown assay in combination with liquid chromatography-mass spectrometry analysis (LC–MS/MS) and protein–protein interaction simulation demonstrated that Ku80 was a key interacting partner for RHBDF1-regulated BC chemoradio-resistance. Activities of Ku80 and downstream signaling factors DNA-dependent protein kinase (DNA-PK), gamma-histone H2AX (γH2AX), and tumor suppressor p53 (P53) were markedly suppressed by RHBDF1 gene-silencing. Moreover, RHBDF1 Ser105 phosphorylation was first characterized by LC–MS/MS, and it was a key active site in RHBDF1-Ku80 interaction. Importantly, a bioactive peptide L1H9 based on the structural RHBDF1-Ku80 interaction and key active site RHBDF1 Ser105 was designed, and its target protein Ku80 was identified by a photocrosslinking reaction in living cells, molecular docking, and molecular dynamic simulation. Peptide L1H9 was able to sensitize BC cells and mouse tumors to doxorubicin (DOX), ultraviolet (UV), and ionizing radiation (IR) treatments. Overall, peptide L1H9 can be an adjuvant sensitizer with a potential value for BC chemoradiotherapy and can be used to form bio-nanostructures through supramolecular self-assembly to enhance the chemoradiotherapeutic efficacy of BC in the future. RHBDF1 interacted with Ku80 to facilitate Ku80 stability, while peptide L1H9 or RHBDF1 gene-silencing impaired Ku80 stability, attenuated downstream DNA-PK activity, and sensitized BC to chemoradiotherapy.