Abstract 3483: Inhibition of Monocarboxylate Transporter 4 (MCT4) targets stem-like cells in glioblastoma

Abstract

Glioblastomas (GBM) are the most common adult malignant brain tumors and contain a hypoxic/necrotic core surrounded by proliferative cells. To unmask genes important in hypoxia, we exposed 2 GBM neurosphere lines, HSR-GBM1 and JHH-GBM10, to 1% and 21% oxygen levels for 24 hours and compared gene expression using Agilent oligonucleotide microarrays. We identified SLC16A3 (Monocarboxylate transporter-4, MCT4) as one of the most upregulated genes in response to hypoxia. To investigate the clinical importance of MCT4 in GBM, we examined the Kaplan-Meier survival curves of glioma patients using public databases. We found that patients with upregulation of MCT4 (≥2.0X) have a significantly shorter survival (p<0.0001) than patients with intermediate expression. Consistent with this, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM (p<0.0001). We next examined MCT4 protein levels using immunohistochemical analysis of tissue microarrays, confirming that MCT4 protein levels were increased in high-grade as compared to lower grade astrocytomas (p<0.0001). These data clearly demonstrated that MCT4 is a clinically relevant target. We next tested the requirement of MCT4 in vitro. We found that when neurospheres were transduced with viruses encoding short hairpin RNA (shRNA) against MCT4, cell growth was inhibited by 30-69% in hypoxia. Interestingly, similar results were observed in normoxia, suggesting that MCT4 may be critical to tumor growth and survival independent of oxygen levels. We next explored the role that MCT4 may play in the maintenance of the stem-like population in our neurosphere lines. We found that MCT4 is overexpressed in sorted CD133-positive cells compared to CD133-negative cells in both HSR-GBM1 and JHH-GBM10. Furthermore, flow cytometric analysis of shMCT4-expressing neurospheres showed a decrease in the percentage of CD133-positive cells as compared to controls. This suggested to us that MCT4 silencing might inhibit stem-like cell proliferation/survival, offering a potential explanation for growth inhibition following MCT4 knockdown in normoxia. To test this hypothesis, we examined the effect of MCT4 inhibition functionally using a clonogenic assay. Cells were exposed to normoxia or hypoxia for 48 hours followed by recovery in normoxia. We found that MCT4 silencing resulted in significant reduction in sphere number and size in both normoxic and hypoxic cells, consistent with a loss of clonogenicity. In agreement with what we found in vitro, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo (p=0.009). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3483. doi:1538-7445.AM2012-3483

Intratumoral Distribution of Lactate and the Monocarboxylate Transporters 1 and 4 in Human Glioblastoma Multiforme and Their Relationships to Tumor Progression-Associated Markers

“…This finding is supported by a study in which an increased lactate transport via monocarboxylate transporters (MCT1-4) was found in glioma stem-like cells [39]. Moreover, previous studies showed an upregulation of MCT4 in GBM neurospheres [5].…”

“…In particular, GBM cells located at the resection margin were shown to proliferate rapidly, and to be more invasive than GBM cells at the center of the tumor [31,32]. With regards to the dormancy phenomenon and the slightly increased expression of the dormancy markers EPHA5 and IGFBP5 at the edge of the tumor observed in our study, the only connection published to date is an induction of MCT4 and dormancy markers by hypoxia [5,33]. The current understanding of GBM suggests that up to 50% of the tumor mass is generated by the tumor stroma.…”

“…MCT1 and MCT4, in particular, are overexpressed in GBM cells [4]. Previous studies have shown a positive correlation between the expression of both transporters, with tumor cell proliferation, angiogenesis, and the degree of malignancy of gliomas [5]. Initial findings from studies focusing on the function of the transporters in animal experiments suggest that MCT4 is responsible for the exportation of lactate.…”

“…On the other hand, the exported lactate could be absorbed again via MCT1 and then be metabolized for further energy production [4]. Lactate transporters also seem to play a role in the context of the epithelial to mesenchymal transition (EMT), a process for generating a highly invasive GBM phenotype [5]. Cells with a stem cell-like character, which are associated with the high invasiveness of GBM cells and their resistance to radio-and chemotherapy [6], showed an increased expression of MCT1 and MCT4 [5].…”

“…Lactate transporters also seem to play a role in the context of the epithelial to mesenchymal transition (EMT), a process for generating a highly invasive GBM phenotype [5]. Cells with a stem cell-like character, which are associated with the high invasiveness of GBM cells and their resistance to radio-and chemotherapy [6], showed an increased expression of MCT1 and MCT4 [5]. Interestingly, a close relationship between the tumor stem cell theory and the dormancy phenomenon seems to be obvious [7].…”