Hypoxia-mediated upregulation of MCT1 expression supports the glycolytic phenotype of glioblastomas

Abstract

Background: Glioblastomas (GBM) present a high cellular heterogeneity with conspicuous necrotic regions associated with hypoxia, which is related to tumor aggressiveness. GBM tumors exhibit high glycolytic metabolism with increased lactate production that is extruded to the tumor microenvironment through monocarboxylate transporters (MCTs). While hypoxia-mediated regulation of MCT4 has been characterized, the role of MCT1 is still controversial. Thus, we aimed to understand the role of hypoxia in the regulation of MCT expression and function in GBM, MCT1 in particular.

Methods: Expression of hypoxia- and glycolytic-related markers, as well as MCT1 and MCT4 isoforms was assessed in in vitro and in vivo orthotopic glioma models, and also in human GBM tissues by immunofluorescence/immunohistochemistry and Western blot. Following MCT1 inhibition, either pharmacologically with CHC (α-cyano-4-hydroxynnamic acid) or genetically with siRNAs, we assessed GBM cell viability, proliferation, metabolism, migration and invasion, under normoxia and hypoxia conditions.

Results: Hypoxia induced an increase in MCT1 plasma membrane expression in glioma cells, both in in vitro and in vivo models. Additionally, treatment with CHC and downregulation of MCT1 in glioma cells decreased lactate production, cell proliferation and invasion under hypoxia. Moreover, in the in vivo orthotopic model and in human GBM tissues, there was extensive co-expression of MCT1, but not MCT4, with the GBM hypoxia marker CAIX.

Conclusion: Hypoxia-induced MCT1 supports GBM glycolytic phenotype, being responsible for lactate efflux and an important mediator of cell survival and aggressiveness. Therefore, MCT1 constitutes a promising therapeutic target in GBM.

Keywords: Warburg effect; glioblastomas; lactate; monocarboxylate transporters (MCTs); tumor hypoxia.

Figure 1. Protein levels, cellular localization and glycolytic metabolism of glioma cells under hypoxia

A. Cellular localization of the different metabolic markers and monocarboxylate transporters in normoxic and hypoxic regions, by immunofluorescence; pictures were taken using the microscope Olympus BX16 at 400x. B. Protein levels under normoxia and hypoxia in glioma cells by Western blot analysis; MCT1 50kDa, MCT4 44kDa, CD147, high glycosylated (HG) 52-42kDa and low glycosylated (LG) 34kDa; GLUT1 52kDa, HKII 95kDa, CAIX 52kDa, HIF-1α 110kDa and β-Actin 42kDa. C. Protein levels of cytoplasm and plasma membrane fractions for MCT1, MCT4 and CD147; β-actin and ATPase (90kDa) were used as loading controls for cytoplasm and plasma membrane fractions, respectively. D. Glucose consumption and lactate secretion in glioma cells under normoxia and hypoxia conditions; results are the mean±SEM of at least three independent experiments, each one in triplicate; *** p≤0.001 normoxia vs hypoxia.

Figure 2. Lactate secretion upon MCT inhibition

Extracellular lactate levels upon MCT pharmacological (CHC) and genetic inhibition (siRNA) in SW1088 and U251 cells under hypoxia conditions; results are the mean±SEM of at least three independent experiments, each one in triplicate; *p<0.05 compared CHC or downregulation vs control condition.

Figure 3. Effect of MCT1 inhibition on cell proliferation and cell death of glioma cells under hypoxia

Cell proliferation in SW1088 and U251 cells under hypoxia by A. trypan blue and B. BrdU assay, respectively. C. Cell death in SW1088 and U251 cells under hypoxia by AnnexinV/PI flow cytometry analysis; results are the mean±SEM of at least three independent experiments, each one in triplicate; To MCT downregulation graphs the results were compared to scramble condition which was normalized to 100% represented as grey line; *p<0.05, **p≤0.01 *** p≤0.001; # p<0.05 compared normoxia vs hypoxia at respective conditions.

Figure 4. Cell migration and invasion behavior upon MCT1 under hypoxia

Cell migration A. and cell invasion B. of glioma cells under normoxia and hypoxia after MCT1 downregulation and MCT activity inhibition with CHC. Pictures were taken at 40x magnification (migration) and 200x magnification (invasion) in an Olympus BX16 microscope. Results represent the mean±SEM of three independent experiments. The different conditions of silencing were compared to scramble conditions that was normalized to 100% (represented as grey line); *p<0.05, **p≤0.01 *** p≤0.001; # p<0.05 compares normoxia vs hypoxia at respective conditions.

Figure 5. MCT1 and MCT4 expression distribution in 3D culture and in in vivo glioma models

A. Immunohistochemical expression of MCT1, MCT4 and GLUT1 in SW1088 spheroids. Pictures are representative of n=10 spheroids and taken using the Olympus BX16 microscope at 40x, 100x and 400x magnification; B. MCT1, MCT4 and CAIX expression in U251 microtumors by immunohistochemistry. Pictures are representative of n=8 eggs and taken using the Olympus BX16 microscope, at 40x, 200x and 400x magnification; C. MCT1 and MCT4 expression in in vivo U87-MG intracranial tumors by immunofluorescence. Pictures are taken at 100x and highlighted at 400x for MCT1, MCT4 and CAIX in normoxic (close to blood vessels) and hypoxic regions (distant from blood vessels, positive for the hypoxia marker CAIX).

Figure 6. Immunohistochemical expression of monocarboxylate transporters, CD147, GLUT1, HKII, CAIX and HIF-1α in GBM and normal adjacent tissues

Expression of MCT1, MCT4, CD147 (MCT1/4 chaperone), glycolytic markers (HKII, GLUT1) and hypoxia markers (CAIX, HIF-1α) in neoplastic and non-neoplastic of GBM patient tissues, by immunohistochemistry. Pictures were obtained using the Olympus BX61 microscope, at 100x and the inserts at 400x magnification.

Figure 7. Association of MCTs and CD147 expression with hypoxia markers (GLUT-1 and CAIX) in GBMs

A. Heat map representation of the monocarboxylate transporter and chaperone expression, and hypoxic markers for the 45 GBM tissues evaluated under normoxia and hypoxia regions. Blue color corresponds to negative or cytoplasm expression and red corresponds to plasma membrane expression; B. Plasma membrane expression of the hypoxia marker CAIX (in green) and MCT1 (in red) in GBM tissue. Co-localization of MCT1 and CAIX at the plasma membrane (orange). Pictures were taken using the microscope Olympus BX16 at 100x (I), 200x (II) and 400x (III).

Figure 8. MCT1 as a mediator of aggressiveness at hypoxic regions in glioblastomas

In normoxic tumor cells, MCT1 is expressed at the plasma membrane, whereas MCT4 remains at the cytoplasm. Hypoxia induces upregulation of MCT1 plasma membrane expression, which is responsible for lactate efflux. Inhibition of MCT1 expression or activity impairs lactate secretion, contributing to a decrease in cell proliferation, cell migration and invasion.