PrP-Neutralizing Antibody Confers an Additive Benefit in Combination with 5-Fluorouracil in KRAS-Mutant Colorectal Cancer Models, Associated with Reduced RAS-GTP and AKT/ERK Phosphorylation.

Colorectal cancer (CRC) remains a major cause of cancer-related deaths in advanced disease, and activating KRAS/NRAS mutations limit the use of anti-EGFR antibodies to RAS-wild-type tumors. The cellular prion protein (PrP) has been linked to aggressive and chemoresistant CRC, but its extracellular partners and functional relevance in KRAS-mutant disease are not fully defined. Here, we examined extracellular PrP complexes and PrP-associated signaling in CRC cell lines and xenografts using a neutralizing PrP monoclonal antibody. Across a CRC panel that included SNU-C5/WT and its 5-fluorouracil- and oxaliplatin-resistant derivatives, HT-29 (KRAS-wild-type), and HCT-8 and LoVo (KRAS-mutant), co-immunoprecipitation showed that PrP forms complexes with the 37/67 kDa laminin receptor (RPSA), with PrP-RPSA association particularly increased in KRAS-mutant HCT-8 and LoVo cells. PrP protein levels were higher in KRAS-mutant HCT-8, SW620, and SNU-407 cells than in HT-29, and PrP neutralization reduced viability in all four lines. Accordingly, we assessed upstream RAS activity and found that active RAS (RAS-GTP) was higher in KRAS-mutant cells than in HT-29, and PrP treatment was associated with reduced RAS-GTP levels. In the same KRAS-mutant setting, basal AKT phosphorylation exceeded that in HT-29, and PrP treatment lowered AKT phosphorylation without changing total AKT. Moreover, PrP treatment was associated with reduced ERK1/2 phosphorylation in KRAS-mutant cells, suggesting attenuation of downstream RAS pathway output. These signaling changes coincided with a decrease in the S-phase fraction and an increase in G1. In an HCT-8 (KRAS G13D) xenograft model, PrP monotherapy inhibited tumor growth in a dose-dependent manner, and 5-fluorouracil (5-FU) monotherapy produced an intermediate effect. The combination of PrP (10 mg/kg) and 5-FU (20 mg/kg) yielded the greatest tumor growth inhibition among the tested regimens. Consistent with this enhanced tumor control, immunofluorescence of xenograft tissues showed that PrP, particularly with 5-FU, reduced intratumoral PrP and PCNA and decreased CD31-positive microvessels and α-SMA-positive vessel structures. Taken together, these findings suggest that extracellular PrP supports RAS-AKT signaling, proliferation, and tumor-associated angiogenesis in KRAS-mutant colorectal cancer, and that PrP neutralization additively enhances 5-fluorouracil activity in KRAS-mutant models. The data provide a preclinical basis for evaluating PrP antibodies in combination with fluoropyrimidine-based regimens in patients with KRAS-mutant CRC.