Aberrant alternative splicing of purinergic receptor P2RX4 prevents sensitivity towards combinatorial treatment in colorectal and pancreatic cancer

Abstract

Recently, we suggested the combination of chemotherapy and P2RX4 inhibition as a promising novel therapeutic approach for P2RX4-expressing epithelial tumors to prevent paracrine resistance. Here, we aimed to assess whether determining P2RX4 expression status in colorectal and pancreatic cancer patients would allow stratification of potentially responsive patients. Therefore, P2RX4 expression levels were determined by RNA sequencing and immunohistochemistry. Subcellular localization of P2RX4 isoforms was analyzed in HeLa cells and patient-derived tumor organoids. In contrast to its RNA expression profile, P2RX4 protein levels exhibited differential regulation in human colorectal and pancreatic cancer epithelia due to alternative splicing. Interpatient heterogeneity was greater in colorectal cancer than in pancreatic cancer. Notably, these variations in expression did not correlate with overall patient survival. Alternative P2RX4 transcripts gave rise to functionally distinct protein isoforms that differed in subcellular localization and total protein abundance. Only the correctly spliced, canonical P2RX4 isoform was localized to the plasma membrane and was capable of mediating downstream signaling. Accordingly, P2RX4 inhibition in combination with chemotherapy was effective exclusively in patient-derived tumor organoids expressing the canonical P2RX4 transcript. In summary, immunohistochemical, but not transcriptomic, assessment of P2RX4 expression enabled the prediction of sensitivity to combinatorial treatment and facilitated the identification of patients who may benefit from P2RX4 inhibition during chemotherapy. Given the lower degree of heterogeneity observed in pancreatic cancer, this tumor entity may represent a promising candidate for early-phase clinical evaluation of chemotherapy combined with P2RX4 inhibition. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.