Colorectal cancer (CRC) remains a significant cause of global cancer mortality. While aspirin demonstrates therapeutic benefits, the molecular determinants underlying patient response are poorly understood, hindering personalised treatment strategies. This study aimed to identify molecular subtypes in CRC based on prostaglandin pathway markers and assess their association with clinical outcomes and aspirin use.

We analysed paired tumour and adjacent normal tissues from 86 CRC patients (43 regular aspirin users, 43 aspirin-free) via qPCR for mRNA expression (PTGS1, PTGS2, PTGER2, PTGER3) and immunohistochemistry for protein levels (COX-2, Caspase-8, MMP-9). Normalised expression data were correlated with clinicopathological features and survival. Unsupervised clustering algorithms (K-means, hierarchical, and hybrid) were applied independently to each cohort to identify distinct molecular subtypes using Z-score-normalised data across all seven markers.

Aspirin-treated patients showed significantly higher PTGS2 and PTGER3 mRNA, elevated Caspase-8 protein, and reduced MMP-9 protein compared to the aspirin-free group. Unsupervised clustering identified distinct molecular subtypes within both cohorts. In the Aspirin-Treated group, clinical stratification yielded a two-subtype solution (AT-1, AT-2), primarily driven by differential protein expression (Caspase-8, COX-2, MMP-9), which demonstrated a trend towards distinct survival outcomes (P = 0.073). In the Aspirin-Free group, a clinically relevant two-subtype solution (AF-1, AF-2) demonstrated significant survival differences (47.4% vs 16.7%, P = 0.016); the poor-prognosis subtype (AF-2) was characterised by higher PTGS2 mRNA, higher COX-2 protein, lower Caspase-8, and higher MMP-9 levels. Notably, strong correlations between PTGS2 mRNA and adverse clinical outcomes observed in the aspirin-free cohort were absent in the aspirin-treated group.

Integrated prostaglandin pathway gene and protein expression analysis identifies distinct CRC molecular subtypes with prognostic significance, particularly related to aspirin use. Key protein markers (Caspase-8, COX-2, MMP-9) and PTGS2 mRNA levels differentiate these subtypes. Aspirin treatment disrupts specific molecular correlations linked to prognosis, suggesting modulation of post-transcriptional networks. These findings support the use of multi-marker molecular profiles to improve patient stratification and personalised aspirin therapy in CRC.