RRM1 stabilizes USP19 by blocking K387 ubiquitination, suppressing autophagy-mediated 5-FU chemoresistance in CRC; peptide GAGGVGKSAL disrupts this axis.
Background
Resistance to 5-fluorouracil (5-FU) remains a critical challenge in treating colorectal cancer (CRC), significantly limiting therapeutic efficacy and patient prognosis. A key mechanism contributing to this resistance is the dysregulation of autophagy, a cellular recycling process that, when hyperactivated, can protect cancer cells from chemotherapy-induced death. Current standard-of-care often fails to overcome this autophagy-driven chemoresistance, necessitating the identification of novel molecular targets and therapeutic strategies. This study investigates the non-canonical role of ribonucleotide reductase subunit M1 (RRM1) in modulating autophagy-mediated 5-FU resistance.
Study Design
Researchers employed a multi-omics approach, including metabolomics, transcriptomics, and proteomics, on 5-FU-resistant CRC patient tissues and matched drug-resistant cell models. Protein-protein interactions and ubiquitination events were validated using Co-IP, GST pull-down, in vivo ubiquitination assays, and structure-guided molecular docking. Site-directed mutagenesis was used to pinpoint critical residues. Functional consequences were assessed in subcutaneous xenograft mouse models. A structure-based virtual screening identified the peptide GAGGVGKSAL as a candidate compound, with its anti-tumor activity verified in 5-FU-resistant CRC xenografts.
Results
RRM1 was significantly downregulated in 5-FU-resistant CRC tissues, with low RRM1 expression correlating with poor clinical prognosis. Mechanistically, RRM1 directly binds to the deubiquitinase USP19 via conserved E647/R648 residues. This binding competitively blocks NEDD4-mediated USP19 K387 ubiquitination, thereby preventing its proteasomal degradation and stabilizing USP19. Loss of RRM1 destabilizes USP19, which in turn impairs autophagic substrate deubiquitination, leading to hyperactivated autophagic flux and subsequent 5-FU resistance. Restoration of RRM1 or expression of the ubiquitination-defective USP19-K387R mutant re-sensitized resistant CRC cells to 5-FU.
The peptide GAGGVGKSAL, identified through virtual screening, specifically disrupted the RRM1-USP19 interface, inhibited excessive autophagy, and potently suppressed tumor progression in 5-FU-resistant CRC xenograft models.
Key Findings
- RRM1 is significantly downregulated in 5-FU-resistant CRC tissues, correlating with poor clinical prognosis.
- RRM1 directly binds USP19 via
E647/R648residues, competitively blocking NEDD4-mediated USP19K387ubiquitination. - Loss of RRM1 destabilizes USP19, hyperactivates autophagic flux, and induces 5-FU resistance.
- Restoration of RRM1 or expression of
USP19-K387Rmutant re-sensitizes resistant CRC cells to 5-FU. - The peptide GAGGVGKSAL disrupts the RRM1-USP19 interface, inhibits excessive autophagy, and suppresses tumor progression in 5-FU-resistant CRC xenografts.
Why It Matters
This research unveils a novel regulatory axis, RRM1-USP19-NEDD4, as a core mediator of autophagy-driven 5-FU resistance in colorectal cancer. This discovery provides a critical understanding of how cancer cells evade chemotherapy and offers a new therapeutic vulnerability. Targeting the RRM1-USP19 interaction, potentially with agents like the identified peptide GAGGVGKSAL, could re-sensitize resistant CRC tumors to 5-FU, significantly improving treatment outcomes. This opens avenues for developing small molecule or peptide-based interventions that specifically modulate autophagy to overcome chemoresistance, moving closer to a usable protocol for patients with refractory CRC.
rrm1
usp19
nedd4
colorectal-cancer
5-fluorouracil
chemoresistance