Abstract
Glioblastoma (GBM) resistance to therapy is the most common cause of tumor recurrence, which is ultimately fatal in 90% of the patients 5 years after initial diagnosis. A sub-population of tumor cells with stem-like properties, glioma stem cells (GSCs), is specifically endowed to resist or adapt to the standard therapies, leading to therapeutic resistance. Several anticancer agents, collectively termed redox therapeutics, act by increasing intracellular levels of reactive oxygen species (ROS). In this study, we investigated mechanisms underlying GSC response and resistance to cannabidiol (CBD), a non-toxic, non-psychoactive cannabinoid and redox modulator. Using primary GSCs, we showed that CBD induced a robust increase in ROS, which led to the inhibition of cell survival, phosphorylated (p)-AKT, self-renewal and a significant increase in the survival of GSC-bearing mice. Inhibition of self-renewal was mediated by the activation of the p-p38 pathway and downregulation of key stem cell regulators Sox2, Id1 and p-STAT3. Following CBD treatment, a subset of GSC successfully adapted, leading to tumor regrowth. Microarray, Taqman and functional assays revealed that therapeutic resistance was mediated by enhanced expression of the antioxidant response system Xc catalytic subunit xCT (SLC7A11 (solute carrier family 7 (anionic amino-acid transporter light chain), member 11)) and ROS-dependent upregulation of mesenchymal (MES) markers with concomitant downregulation of proneural (PN) markers, also known as PN–MES transition. This ‘reprogramming' of GSCs occurred in culture and in vivo and was partially due to activation of the NFE2L2 (NRF2 (nuclear factor, erythroid 2-like)) transcriptional network. Using genetic knockdown and pharmacological inhibitors of SLC7A11, we demonstrated that combining CBD treatment with the inhibition of system Xc resulted in synergistic ROS increase leading to robust antitumor effects, that is, decreased GSC survival, self-renewal, and invasion. Our investigation provides novel mechanistic insights into the antitumor activity of redox therapeutics and suggests that combinatorial approaches using small molecule modulators of ROS offer therapeutic benefits in GBM.
Highlights
Subset of GBM cells with stem-like characteristics, termed glioma stem cells (GSCs), have been shown to underlie the therapeutic resistance and tumor recurrence in GBM.[6,7]
We show that cotargeting GSCs using Reactive oxygen species (ROS) modulators (CBD) and inhibitors of the antioxidant response genes together is more effective than either approach alone in halting GBM growth
CBD inhibited the viability of 3832 and 387 GSCs with an IC50 (50% inhibitory concentration) value of 3.5 μM (3.4–3.6) and 2.6 μM (2.5–2.7), respectively (Figures 1a and b). In both GSC cultures, CBD increased the production of ROS (Figures 1c and d), which was reversed in the presence of vitamin E (VitE)
Summary
Subset of GBM cells with stem-like characteristics, termed glioma stem cells (GSCs), have been shown to underlie the therapeutic resistance and tumor recurrence in GBM.[6,7]. The high basal metabolic rate of cancer cells makes them more susceptible to redoxdirected therapeutics in comparison with non-transformed cells.[10] Redox-directed therapeutics have been developed to act as direct inhibitors of cancer and to sensitize tumors to firstline agents; they are associated with significant toxicity.[9] The discovery of non-toxic molecules that selectively upregulate ROS in malignant cells would be beneficial. Our recent study demonstrated CBD-produced robust antitumor activity against a human-derived GBM in an intracranial xenograft model;[13] no investigations to date have interrogated the therapeutic effects of CBD on GSCs. One of the major systems used by both normal and cancerous cells to counteract oxidative insult is the NRF2 ( known as NFE2L2) transcriptionally regulated program.[9] The role of NRF2 transcriptional regulator and SLC7A11 (solute carrier family 7 (anionic amino-acid transporter light chain), member 11) in mediating GBM response and resistance to redox-directed therapeutics has not been evaluated. We show that cotargeting GSCs using ROS modulators (CBD) and inhibitors of the antioxidant response genes together is more effective than either approach alone in halting GBM growth
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