Glioblastomas (GBM) contain a hypoxic core surrounded by proliferative cells. Our group and others have shown that GBM stem cells favor a hypoxic microenvironment, and it is believed that many reside in the tumor core. Conventional therapies target the tumor bulk, but may spare stem cells in the hypoxic niche. To patch this therapeutic loophole, we sought to target the GBM stem cell microenvironment by identifying genes that are important for survival in hypoxia. We identified monocarboxylate transporter 4 (MCT4) as one of the most upregulated genes in our GBM neurosphere lines in response to hypoxia. Clinically, GBM patients with a two-fold or more upregulation of MCT4 have a significantly shorter survival (p = 0.036) than patients with intermediate expression. Consistent with this data, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM (p<0.0001). Using immunohistochemical analysis, we also found that MCT4 protein levels are increased in high-grade as compared to lower grade astrocytomas (p<0.0001), confirming the clinical importance of MCT4. To test the requirement for MCT4 under hypoxia in vitro, we silenced MCT4 in neurospheres using lentiviruses encoding short hairpin RNA (shRNA) specific for MCT4. Cell growth was inhibited in hypoxia by ∼60% in HSR-GBM1 and ∼70% in JHH-GBM10 in these neurosphere lines following MCT4 knockdown. Interestingly, while MCT4 was expressed at lower levels in normoxia, silencing it in 21% oxygen also significantly inhibited growth. CD133-positive stem-like cells expressed a significantly higher amount of MCT4 compared to CD133-negative cells, and decreased proliferation in vitro following MCT4 silencing was associated with reduced CD133-positive stem-like cells and increased apoptosis. This suggests that MCT4 is critical for the survival of CD133-positive stem-like cells under hypoxia, and its inhibition targets the stem cell pool in the hypoxic niche in tumors. Importantly, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo (p=0.009). Interestingly, while multiple earlier studies had identified and characterized MCT4 as a lactate exporter, we found that both extracellular and intracellular lactate levels did not change following MCT4 silencing. Instead, MCT4 inhibition led to downregulation of the HIF (Hypoxia-Inducible Factor) response, reducing HIF target gene expression and pointing to a potential novel lactate-independent function of MCT4 and a unique mechanism for growth inhibition in malignant gliomas.

Citation Format: Kah Suan Lim, Kah Jing Lim, Brent A. Orr, Antoinette C. Price, Charles G. Eberhart, Eli E. Bar. Silencing MCT4 inhibits GBM growth, HIF response, and CD133-positive fraction in a lactate-independent fashion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2930. doi:10.1158/1538-7445.AM2013-2930