The molecular signaling pathways orchestrating the biology of cancer stem-like cells (CSLCs), including glioblastoma, remain to be elucidated. We investigated in this study the role of the MEK/extracellular signal-regulated kinase (ERK) pathway in the control of self-renewal and tumorigenicity of glioblastoma CSLCs, particularly in relation to the PI3K/mTOR (mammalian target of rapamycin) pathway. Targeted inactivation of MEK alone using pharmacological inhibitors or siRNAs resulted in reduced sphere formation of both cell line- and patient-derived glioblastoma CSLCs, accompanied by their differentiation into neuronal and glial lineages. Interestingly, this effect of MEK inactivation was apparently augmented in the presence of NVP-BEZ235, a dual inhibitor of PI3K and mTOR. As a potential explanation for this observed synergy, we found that inactivation of either the MEK/ERK or PI3K/mTOR pathway triggered activation of the other, suggesting that there may be mutually inhibitory crosstalk between these two pathways. Significantly, inactivation of either pathway led to the reduced activation of p70S6K, and siRNA-mediated knockdown of p70S6K resulted in the activation of both pathways, which no longer maintained the cross-inhibitory relationship. Finally, combinational blockade of both pathways in glioblastoma CSLCs suppressed their tumorigenicity, whether transplanted subcutaneously or intracranially, more efficiently than blockade of either alone. Our findings suggest that there is p70S6K-mediated, cross-inhibitory regulation between the MEK/ERK and PI3K/mTOR pathways, in which each contribute to the maintenance of the self-renewal and tumorigenic capacity of glioblastoma CSLCs. Thus, combinational disruption of these pathways would be a rational and effective strategy in the treatment of glioblastoma.

Glioblastoma is one of the most aggressive types of human cancer, with invariable and fatal recurrence even after multimodal intervention, for which cancer stem-like cells (CSLCs) are now being held responsible. Our recent findings indicated that combinational inhibition of phosphoinositide-3-kinase/Akt/mammalian target of rapamycin (mTOR) and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways effectively promotes the commitment of glioblastoma CSLCs to differentiation and thereby suppresses their tumorigenicity. However, the mechanism by which these two signaling pathways are coordinated to regulate differentiation and tumorigenicity remains unknown. Here, we identified FoxO3a, a common phosphorylation target for Akt and ERK, as a key transcription factor that integrates the signals from these pathways. Combinational blockade of both the pathways caused nuclear accumulation and activation of FoxO3a more efficiently than blockade of either alone, and promoted differentiation of glioblastoma CSLCs in a FoxO3a expression-dependent manner. Furthermore, the expression of a constitutively active FoxO3a mutant lacking phosphorylation sites for both Akt and ERK was sufficient to induce differentiation and reduce tumorigenicity of glioblastoma CSLCs. These findings suggest that FoxO3a may play a pivotal role in the control of differentiation and tumorigenicity of glioblastoma CSLCs by the PI3K/Akt/mTOR and MEK/ERK signaling pathways, and also imply that developing methods targeting effective FoxO3a activation could be a potential approach to the treatment of glioblastoma.

Purpose: Molecular targeted therapy using BRAF and/or MEK inhibitors has been applied to BRAFV600E-mutant high-grade gliomas (HGG); however, the therapeutic effect is limited by the emergence of drug resistance. Experimental Design: We established multiple paired BRAFV600E-mutant HGG patient-derived xenograft models based on tissues collected prior to and at relapse after molecular targeted therapy. Using these models, we dissected treatment-resistant mechanisms for molecular targeted therapy and explored therapeutic targets to overcome resistance in BRAFV600E HGG models in vitro and in vivo. Results: We found that, despite causing no major genetic and epigenetic changes, BRAF and/or MEK inhibitor treatment deregulated multiple negative feedback mechanisms, which led to the reactivation of the MAPK pathway through c-Raf and AKT signaling. This altered oncogenic signaling primarily mediated resistance to molecular targeted therapy in BRAFV600E-mutant HGG. To overcome this resistance mechanism, we performed a high-throughput drug screening to identify therapeutic agents that potently induce additive cytotoxicity with BRAF and MEK inhibitors. We discovered that HSP90 inhibition combined with BRAF/MEK inhibition coordinately deactivated the MAPK and AKT/mTOR pathways, and subsequently induced apoptosis via dephosphorylation of GSK3β (Ser9) and inhibition of Bcl-2 family proteins. This mediated potent cytotoxicity in vitro and in vivo in refractory models with acquired resistance to molecular targeted therapy. Conclusions: The combination of an HSP90 inhibitor with BRAF or MEK inhibitors can overcome the limitations of the current therapeutic strategies for BRAFV600E-mutant HGG.