Mutant IDH1 expression is associated with down-regulation of monocarboxylate transporters

Abstract

Mutations in isocitrate dehydrogenase 1 (IDH1) are characteristic of low-grade gliomas. We recently showed that mutant IDH1 cells reprogram cellular metabolism by down-regulating pyruvate dehydrogenase (PDH) activity. Reduced pyruvate metabolism via PDH could lead to increased pyruvate conversion to lactate. The goal of this study was therefore to investigate the impact of the IDH1 mutation on the pyruvate-to-lactate flux. We used 13C magnetic resonance spectroscopy and compared the conversion of hyperpolarized [1-13C]-pyruvate to [1-13C]-lactate in immortalized normal human astrocytes expressing mutant or wild-type IDH1 (NHAIDHmut and NHAIDHwt). Our results indicate that hyperpolarized lactate production is reduced in NHAIDHmut cells compared to NHAIDHwt. This reduction was associated with lower expression of the monocarboxylate transporters MCT1 and MCT4 in NHAIDHmut cells. Furthermore, hyperpolarized lactate production was comparable in lysates of NHAIDHmut and NHAIDHwt cells, wherein MCTs do not impact hyperpolarized pyruvate delivery and lactate production. Collectively, our findings indicated that lower MCT expression was a key contributor to lower hyperpolarized lactate production in NHAIDHmut cells. The SLC16A3 (MCT4) promoter but not SLC16A1 (MCT1) promoter was hypermethylated in NHAIDHmut cells, pointing to possibly different mechanisms mediating reduced MCT expression. Finally analysis of low-grade glioma patient biopsy data from The Cancer Genome Atlas revealed that MCT1 and MCT4 expression was significantly reduced in mutant IDH1 tumors compared to wild-type. Taken together, our study shows that reduced MCT expression is part of the metabolic reprogramming of mutant IDH1 gliomas. This finding could impact treatment and has important implications for metabolic imaging of mutant IDH1 gliomas.

Keywords: MCT1; MCT4; magnetic resonance spectroscopy; metabolic reprogramming; mutant IDH1.

Figure 1

A. ¹³C MR spectral array showing hyperpolarized [1-¹³C]-lactate production from hyperpolarized [1-¹³C]-pyruvate in live NHAIDHwt cells. B. Build-up of hyperpolarized [1-¹³C]-lactate following injection of hyperpolarized [1-¹³C]-pyruvate in live NHAIDHwt and NHAIDHmut cells. C. Ratio of the AUC of hyperpolarized lactate to the AUC of hyperpolarized pyruvate normalized to cell number for live NHAIDHwt and NHAIDHmut cells. * represents statistically significant difference (p<0.05).

Figure 2

A. K_m for pyruvate of LDHA from NHAIDHwt and NHAIDHmut cells. B. V_max of LDHA from NHAIDHwt and NHAIDHmut cells. C. Western blots for LDHB in NHAIDHwt and NHAIDHmut cells. D. Quantification of LDHB expression in NHAIDHwt and NHAIDHmut cells. E. NADH concentration in NHAIDHwt and NHAIDHmut cells. F. NAD⁺/NADH ratio in NHAIDHwt and NHAIDHmut cells. NS indicates no statistically significant difference.

Figure 3

A. Representative spectra showing a comparison of [1-¹³C]-glucose consumption and [3-¹³C]-lactate production at 0 and 24 h in NHAIDHwt and NHAIDHmut cells (the portion of the spectrum containing [1-¹³C]-glucose (90-102 ppm) is scaled differently than that containing [3-¹³C]-lactate (16-36 ppm) in order to illustrate the difference in glucose uptake between NHAIDHwt and NHAIDHmut cells). B. Quantification of [1-¹³C]-glucose uptake over the course of 40 h in NHAIDHwt and NHAIDHmut cells. C. Quantification of [3-¹³C]-lactate production over the course of 40 h in NHAIDHwt and NHAIDHmut cells. D. Representative ³¹P MR spectra for NHAIDHwt and NHAIDHmut cells. The chemical shift difference (A) between the P_i int and PCr peaks was used to calculate the intracellular pH in NHAIDHwt and NHAIDHmut cells. PE=phosphoethanolamine, PC=phosphocholine, P_i ext=extracellular inorganic phosphate, P_i int= intracellular inorganic phosphate, PCr=phosphocreatine, ATPY=Y phosphate of ATP, ATPα= α phosphate of ATP and ATPβ= β phosphate of ATP.

Figure 4

A. MCT1 expression in NHAIDHwt and NHAIDHmut cells determined by western blotting. B. Quantification of MCT1 expression in NHAIDHwt and NHAIDHmut cells. C. MCT4 expression in NHAIDHwt and NHAIDHmut cells determined by western blotting. D. Quantification of MCT4 expression in NHAIDHwt and NHAIDHmut cells. * represents statistically significant difference (p<0.05).

Figure 5

A. Representative ¹³C MR spectra comparing maximum hyperpolarized [1-¹³C]-lactate produced from hyperpolarized [1-¹³C]-pyruvate in NHAIDHwt and NHAIDHmut lysates. B. Ratio of the AUC of hyperpolarized lactate to the AUC of hyperpolarized pyruvate normalized to cell number for NHAIDHwt and NHAIDHmut lysates. NS indicates no significant difference.

Figure 6. Comparison of normalized expression z-scores for MCT1 A. and MCT4 B. mRNA in low-grade glioma patient samples

Boxes denote the mean z-score and whiskers denote 1-99 percentile for tumors within the group (n=68 for IDH1 wild-type and n=218 for IDH1 mutant). A negative z-score represents expression value below the population mean. * indicates statistically significant effect (p<0.05).