Targeting osteopontin suppresses glioblastoma stem-like cell character and tumorigenicity in vivo

Osteopontin (OPN) is a secreted protein involved in most aspects of tumor progression and metastasis development. Elevated OPN expression has been reported in multiple types of cancer including glioblastoma (GBM), the highest grade and most aggressive brain tumor. GBMs contain a subpopulation of glioma-initiating cells (GICs) implicated in progression, therapeutic resistance and recurrence. We have previously demonstrated that OPN silencing inhibited GBM cell growth in vitro and in vivo. Moreover, activation of CD44 signaling upon OPN ligation has been recently implicated in the acquisition of a stem cell phenotype by GBM cells. The present study is aimed to explore OPN autocrine function using shRNA silencing strategy in GICs enriched from GBM cell lines and a human primary GBM grown in EGF and bFGF defined medium. The removal of these growth factors and addition of serum induced a significant loss of OPN expression in GICs. We showed that OPN-silenced GICs were unable to grow as spheres and this capacity was restored by exogenous OPN. Importantly, the expression of Sox2, Oct3/4 and Nanog, key stemness transcription factors, was significantly decreased in GICs upon OPN targeting. We identified Akt/mTOR/p70S6K as the main signaling pathway triggered following OPN-mediated EGFR activation in GICs. Finally, in an orthotopic xenograft mouse model, the tumorigenic potential of U87-MG sphere cells was completely abrogated upon OPN silencing. Our demonstration of endogenous OPN major regulatory effects on GICs stemness phenotype and tumorigenicity implies a greater role than anticipated for OPN in GBM pathogenesis from initiation and progression to probable recurrence. What's new? Glioblastoma (GBM) is a highly malignant and aggressive brain tumor, in which poor prognosis is associated with elevated osteopontin (OPN) expression. GBM progression also is linked to the existence of glioma-initiating cells (GICs). In this study, the loss of OPN expression in GICs was associated with reduced expression of the Sox-2, Nanog, and Oct-4 transcription factors. OPN-silenced GICs also experienced a reduction in sphere growth potential in vitro and a loss of tumorigenicity in vivo. In OPN-secreting cells, the secreted protein was found to activate EGFR, thereby stimulating the Akt/mTOR/p70S6K signaling cascade, the primary pathway implicated in GBM progression.