Effect of a small molecule inhibitor of the JAK2/STAT3 pathway on self-renewal of glioblastoma stem cells

Abstract

2003 Background: Recent data indicate that initiation and maintenance of several cancers including glioblastoma (GBM) may be driven by a small subset of cells called tumor stem cells. A key feature necessary to maintain stem cells is self-renewal. However, the mechanisms underlying GBM stem cell (GSC) self-renewal remain unknown. We identified STAT3 as a key transcription factor associated with poor outcome and treatment resistance in GBM. STAT3 is also an important regulator of self-renewal in several stem cell types. We thus tested the hypothesis that targeting STAT3 with a novel small molecule inhibitor would block GSC self-renewal. Methods: GSC neurosphere cultures from 12 human GBM tumors were expanded under serum free conditions. Self-renewal of GSCs was quantified by neurosphere formation efficiency. Synthesis and analysis of over 150 caffeic acid analogs was performed to identify the novel and potent STAT3 inhibitor WP1193. Results: Western blot analysis demonstrated that STAT3 was activated and phosphorylated (ser727 and tyr705) at high levels in 10 out of 12 GSC neurosphere cultures undergoing self-renewal. Treatment of GSCs with WP1193 at increasing concentrations from 0 to 10uM resulted in a dose dependent decrease in cell proliferation and neurosphere formation in all GSC lines tested, with an associated decrease in both total and phosphorylated STAT3. Treatment with WP1193 at 5uM for 48 hrs was sufficient to completely eliminate self-renewal and neurosphere formation at 14 days under limiting dilution conditions versus untreated controls (control neurosphere formation efficiency 12–40%). Quantitative RT-PCR and FACS analysis of CD133, a putative marker of GBM stem cells, showed a significant depletion of CD133 positive cells after WP1193 treatment (>5uM). Cell cycle analysis demonstrated that WP1193 treatment also significantly reduced the number of cells in S-Phase and significantly increased G1 arrest compared to matched controls of untreated or temozolomide treated GSCs. Conclusions: These data indicate that STAT3 is potentially important regulator of GSC self-renewal that is also associated treatment resistance in GBM. WP1193 is a potent inhibitor of GSC self-renewal and is a promising agent for therapeutically targeting GSCs. No significant financial relationships to disclose.