The glutathione S-transferase GSTT1 mediates stemness and sensitivity to FGFR inhibitors in metastatic pancreatic cancer through regulation of PROM1

Pancreatic ductal adenocarcinoma (PDA) is among the deadliest cancers, primarily because most patients are diagnosed at metastatic stages, resulting in a five-year survival rate of only 8%. Our previous work identified Glutathione S-transferase theta 1 (GSTT1) as a key regulator of a slow-cycling, highly metastatic population, suggesting that the GSTT1 ^High subpopulation may be enriched in stem-like properties and thus contribute to chemoresistance. Using a mCherry-tagged Gstt1 reporter system in metastatic murine PDAC cells, we enriched for Gstt1 ^High subpopulations and characterized their tumor sphere-forming capacity and gene expression profiles. We compared these profiles with human pancreatic cancer datasets to identify conserved stemness-associated gene signatures. We found that Gstt1 ^High murine tumor spheres demonstrated increased size, number, and enrichment of stemness-related genes such as PROM1 (CD133) and components of Wnt/FGF signaling. We find that human CD133 ^High GSTT1 ^High cell lines formed significantly more and larger tumor spheres with spheroid morphologies linked to therapy resistance, suggesting that co-expression serves as a biomarker for a metastatic, stem-like subpopulation in PDA. These cells displayed selective sensitivity to FGFR inhibitors under tumor sphere conditions. FGFR3 expression correlated with CD133 and GSTT1 levels, and FGF signaling was required to sustain their expression. GSTT1 knockdown reduced CD133 protein, tumor sphere formation, and altered sensitivity to FGFR inhibition, suggesting a hierarchical regulatory relationship. Importantly, patient-derived PDA organoids recapitulated these findings, where co-expression of GSTT1 and PROM1 predicted larger tumor spheres and enhanced response to pan-receptor tyrosine kinase (RTK) inhibitor, Nintedanib. These results support GSTT1 as both a marker and mediator of CSC plasticity and identify FGFR inhibitors as a promising therapeutic strategy for targeting this stem-like metastatic population.