The Beclin1‑Targeting stapled peptide suppresses colorectal cancer progression by disrupting EGFR/β-catenin axis.

BACKGROUND: Colorectal cancer (CRC) remains a leading global cause of cancer-related mortality, demanding innovative therapies. Beclin1, a key autophagy and endolysosomal trafficking regulator with tumor-suppressive properties, is frequently downregulated in CRC. Although Beclin1 downregulation correlates with poor clinical outcomes, its regulatory mechanism in CRC remains largely undefined, and effective Beclin1-targeting therapeutic strategies remain limited. Our group previously developed a Beclin1-binding stapled peptide i7-01s-20 for Beclin1 activation, yet its anti-tumor potential and underlying mechanism in CRC have not been elucidated.

METHODS: We analyzed Beclin1 and β-catenin expression and clinical correlations across public transcriptomic, proteomic, and human CRC patient tissue cohorts. A scrambled peptide (ScraP) was used as negative control. In vitro functional assays including autophagic flux detection, EGFR degradation assays, dual-luciferase reporter analysis, ubiquitination and cycloheximide chase tests were performed in CRC cell lines. Beclin1 knockdown rescue experiments were performed to verify target dependency. In vivo anti-tumor efficacy was investigated using DLD-1 subcutaneous xenograft tumor models with gradient-dose peptide treatment. Quantitative DIA proteomics of xenograft tumor tissues were conducted to profile global protein alterations induced by the peptide.

RESULTS: Beclin1 expression was significantly decreased in human CRC tissues and correlated with advanced tumor stage and unfavorable patient survival, while β-catenin was highly accumulated in tumors and negatively correlated with Beclin1 at protein level. In CRC cells, i7-01s-20 potently promoted EGFR endolysosomal degradation, suppressed its downstream AKT/GSK-3β pathway, and triggered ubiquitin-mediated proteasomal degradation of β-catenin. Silencing endogenous Beclin1 completely abrogated these effects triggered by i7-01s-20. Functionally, i7-01s-20 dose-dependently inhibited CRC cell proliferation, colony formation and migration in vitro, whereas its scrambled peptide ScraP exerted negligible biological activity at identical concentrations. In xenograft models, systemic i7-01s-20 administration produced robust tumor-suppressive effects without obvious systemic toxicity, accompanied by repressed AKT/β-catenin signaling in tumor tissues.

CONCLUSIONS: This work uncovers a previously uncharacterized Beclin1-dependent EGFR/β-catenin regulatory cascade in colorectal cancer. The stapled peptide i7-01s-20 achieves simultaneous blockade of two core oncogenic pathways by activating Beclin1. Our study provides mechanistic evidence supporting the Beclin1-targeted stapled peptide i7-01s-20 as a promising therapeutic candidate against CRC by disrupting EGFR/β-catenin signaling.