PIWIL2 contributes to glioblastoma stem cell phenotypes

Abstract PIWI proteins, a conserved Argonaute subfamily of proteins that interact with PIWI-interacting RNAs (piRNAs), have been implicated in cancer development. However, their roles in glioblastoma (GBM), particularly in glioblastoma stem cells (GSCs), a therapy-resistant subpopulation of malignant cells that drives tumor progression, remain insufficiently defined. We integrated bulk RNA sequencing, single-nuclei RNA sequencing, and RT-qPCR across large and well-defined GBM cohorts (146 patients) to generate the first comprehensive transcriptomic, tumor-, and cell-state-resolved analysis of PIWIL1–4 expression. PIWIL2 and PIWIL4 were consistently expressed in GBM, whereas PIWIL1 and PIWIL3 were largely absent. Single-nuclei RNA sequencing showed that PIWIL2 was enriched in the mesenchymal-like 2 (MES2) state associated with hypoxia, a condition linked to cancer stemness. Using 24 paired patient-derived GSC and non-GSC cultures, we found that PIWIL2 transcript expression was consistently enriched in GSCs and decreased upon differentiation. Transient siRNA-mediated silencing of PIWIL2 reduced GSC viability and was associated with reduced sphere-forming capacity, decreased total normalized piRNA counts, and altered expression of specific piRNAs. PIWIL4 was expressed in both GSC and non-GSC cultures and was enriched in MES1-, AC-, and NPC2-like states. Together, these findings provide a comprehensive characterization of PIWI family expression in GBM and, through integrated transcriptomic and functional analyses, identify PIWIL2 as the PIWI family member most consistently linked to the GSC phenotype.