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

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.

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.

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.

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.