A RANKL-derived Peptide Inhibits RSPO3-LGR4-Wnt Signaling and Lung Adenocarcinoma in Mice

Despite advances in therapy, lung cancer remains the leading cause of cancer-related mortality. R-spondin (RSPO) 3 and its receptor, leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4), drive tumor progression in a subset of lung adenocarcinomas by potentiating Wnt signaling. In addition to RSPO3, LGR4 is also a receptor for receptor activator of nuclear factor-кB ligand (RANKL). Here, we investigated whether MHP1-AcN, our previously developed RANKL-derived peptide, acts on LGR4 and examined its effects on RSPO3-LGR4 signaling in A549 lung cancer cells.

An A549 xenograft model was established by subcutaneous inoculation in BALB/c nude mice, followed by daily intraperitoneal administration of MHP1-AcN. Tumor growth was assessed by volume and weight. In vitro, the effect of MHP1-AcN on A549 cell proliferation, cytotoxicity, migration, and invasion were evaluated. Molecular interactions and signaling pathways were analyzed using immunoprecipitation and immunoblotting.

Intraperitoneal administration of MHP1-AcN significantly reduced tumor volume and weight in the subcutaneous A549 xenograft model. Mechanistically, MHP1-AcN directly interacted with LGR4 and disrupted RSPO3-induced LGR4-IQ motif-containing GTPase activating protein 1 complex formation. MHP1-AcN inhibited A549 cell proliferation without inducing cytotoxicity and suppressed RSPO3-enhaced phosphorylation of LRP6 and accumulation of β-catenin. Furthermore, MHP1-AcN dose-dependently inhibited A549 cell migration and invasion by reducing focal adhesion kinase phosphorylation and disrupting F-actin organization.

These findings demonstrate MHP1-AcN as a novel LGR4 antagonist that inhibits RSPO3-LGR4-Wnt signaling, tumor growth, and metastatic potential in lung adenocarcinoma, highlighting its potential as a therapeutic agent targeting this pathway.