Decreased expression of the mitochondrial BCAT protein correlates with improved patient survival in IDH‐WT gliomas

Gliomas represent 43% of all solid intracranial tumors, of which glioblastomas have the poorest prognosis. For the first time, we report increased expression of the human mitochondrial branched‐chain aminotransferase protein (hBCATm) (P = 0.036) in glioblastomas carrying wild‐type isocitrate dehydrogenase (IDH‐WT) genes. Moreover, in IDH‐WT gliomas we identify low BCATm as a positive prognostic marker linked to improved patient overall survival.

Recently, Tönjes et al showed that increased gene expression of the branched chain amino‐acid transaminase 1 (BCAT1—gene encoding cytoplasmic protein hBCATc) promotes cell proliferation through amino acid catabolism in gliomas carrying wild‐type IDH1 and implicated this pathway as a potential novel therapeutic target in these poor prognosis tumors 7. BCAT1 encoding the hBCATc protein is however one of two isoforms (the other is BCAT2, encoding mitochondrial protein hBCATm) that can catalyze the transamination of the branched‐chain amino acids (BCAA) followed by complete oxidation by the branched‐chain α‐keto acid dehydrogenase complex (BCKDC) 1. hBCATm and BCKDC are key players in BCAA metabolism and would affect the efficacy of a potential BCAT1 or hBCATc inhibitor. We examined hBCATc, hBCATm, BCKDC; IDH1 and MGMT status compared with Overall Survival in a cohort of gliomas [(n = 58) 33 glioblastomas WHO Grade IV, 9 anaplastic astrocytomas WHO Grade III, 3 diffuse astrocytomas WHO Grade II, 1 oligodendroglioma WHO Grade II, 7 oligoastrocytoma WHO Grade II and 5 anaplastic oliogodendroglioma WHO Grade III] to better delineate the prognostic and predictive value of these novel potential biomarkers.

In our study, all IDH‐WT gliomas examined have increased expression of the hBCATc and hBCATm proteins relative to normal brain tissue 3. Western blot analysis showed a significant increase in the expression of hBCATm (P = 0.036) and hBCATc (P = 0.007) in IDH–WT compared with IDH1 mutant glioblastomas (Figure 1A,B). This increase in hBCAT expression was supported using IHC analysis (Figure 1C). Statistically, hBCATm and hBCATc have a high positive correlation coefficient (r = 0.625, P < 0.001). By contrast, the overall protein expression of BCKDC between IDH‐WT and IDH1 mutant glioblastomas did not show a significant difference (Figure 1A,B,C). Interestingly several banding patterns for BCKDC are evident in these gliomas, where combinations of 50 and 70 kDa proteins exist (Figure 1A).

Figure 1.

Increased hBCAT expression in IDH‐WT gliomas relative to IDH1 gliomas. Western blot analysis and immunohistochemistry was used to evaluate the expression of BCKDC, hBCATm and hBCATc in IDH‐WT and IDH1 mutation gliomas. A. Patient details and Western blot analysis of glioma homogenates. The density of each band was measured using ImageJ™ software and analyzed for significance using a one‐way ANOVA test in Minitab™ (8 representative samples of a cohort of 55 are depicted). B. Panels show interquartile range (box) sample variability (whiskers) and the median (horizontal line within the interquartile range) of Western blot analysis for BCKDC, hBCATm and hBCATc relative to α‐tubulin. Statistical analysis reveals a significant increase in hBCATm (P = 0.036) and hBCATc (P = 0.007) protein expression in IDH‐WT gliomas compared to IDH mutation gliomas. C. BCKDC, hBCATm, hBCATc staining of glioma sections, where increased staining was observed for hBCATm and hBCATc for IDH‐WT relative to IDH mutation gliomas (n = 51). Magnification for all sections, X20. F = female; M = male; Me = methylated; MGMT = methylguanine‐DNA methytransferase methylation; U = unmethylated.

Kaplan‐Meier survival curves (Figure 2 A,B,C) and log‐rank tests demonstrated that the predominant factor effecting overall survival is IDH status (Figure 2A), with IDH1 mutant gliomas associated with significantly better survival (P = 0.005) as expected. For the first time, we show that IDH‐WT gliomas (Grade II, III, IV) expressing low hBCATm (median split) are associated with better overall survival (Figure 2B, P = 0.003). This result is reflected across all grades in the TCGA dataset as demonstrated in Figure 3, and further stratified analysis will further elucidate the potential clinical role of the BCAT2 gene. Moreover, in the Grade IV IDH‐WT glioblastomas, Kaplan‐Meier curve (Figure 2C) and log‐rank analysis shows that low hBCATm is significantly associated with better overall survival (P = 0.043). hBCATc nor BCKDC had a statistical effect on patient survival (P = 0.971 and 0.642, respectively). In addition, multiple banding patterns for hBCATm were identified in oligodendrogliomas (oligodendrogliomas/anaplastic oligodendrogliomas) indicating that different isoforms of hBCATm exist in these tumor types (Figure 2D).

Figure 2.

Analysis of hBCATm expression and survival. A. Cumulative survival of IDH status demonstrates significantly increased rate of survival in IDH mutation gliomas compared to IDH‐WT (P = 0.005) (n = 47). Kaplan Meier analysis for IDH status and hBCATm expression. Statistics were calculated from Log rank Mantel‐Cox analysis. B. Kaplan Meier analysis shows that IDH‐WT gliomas (Grade II, III, IV) expressing low hBCATm (median split) are associated with better overall survival (P = 0.003). Statistics were calculated from Log rank Mantel‐Cox analysis. C. Cumulative survival of hBCATm expression in IDH‐WT patients (grade IV only) demonstrates significantly increased rate of survival in low (median split) hBCATm expressing gliomas (P = 0.043) (n = 24). D. Western blot analysis of BCKD, hBCATm and hBCATc in IDH‐WT and IDH mutant glioma samples (n = 55). Patient details and Western blot analysis of glioma homogenates. It is observed that patient number 20 has negligible hBCATc expression despite being IDH‐WT, conversely patient 15 expresses hBCATc despite being IDH mutant. This is contrary to what has previously been noted. BCKD = branched chain ketoacid dehydrogenase; F = female; hBCATc = cytosolic human branched chain aminotransferase protein; hBCATm = mitochondrial human branched chain aminotransferase protein; IDH = isocitrate dehydrogenase; M = male; Me = methylated; MGMT = methylguanine‐DNA methytransferase methylation; O1 = oligodendroglioma; O2 = oligo‐astrocytoma; U = unmethylated.

Figure 3.

Survival analysis of BCAT2 levels in the TCGA dataset. Cumulative survival of hBCATm expression across all grades in the TCGA dataset (n = 667) demonstrates significantly increased rate of survival in low (median split) hBCATm expressing gliomas. The results shown here are in whole or part based on data generated by the TCGA Research Network: http://cancergenome.nih.gov/.

BCAA metabolism, produces glutamate which is a precursor for glutamine synthesis, a major nutrient for tumorigenesis. Increased concentrations of glutamate has already been shown to occur in gliomas, providing multiple benefits to tumor mass, including the increase in the growth rate of tumor cells 6, and an increased ability to invade the surrounding tissue by increasing cellular motility 4. Glutamate release also destroys neuronal cells creating a path for tumor growth 5. Release of glutamate in exchange for cysteine is controlled by the system x_c‐cysteine glutamine exchanger. The exchanger functions to support tumor survival by generating glutathione (GSH) with increased glutamate production leading to a rise in GSH concentration which, in tumors, is protective against reactive oxygen species 2.

Clearly, the work of Tönjes et al presents the BCAT proteins as an attractive novel therapeutic target for gliomas. Our data indicates that different BCAT isoforms (BCATc, BCATm) and BCKDC expression may affect the efficacy of potential inhibitors. Moreover, for the first time in the poor prognosis group of IDH‐WT gliomas we identify low BCATm as a positive prognostic marker linked to improved patient Overall Survival.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

Supporting information

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Table S1. Glioma cases used in immunohistochemical and Western blot analysis of BCKD, hBCATc and hBCATm. All cases were from the South West Brain Tumour Bank (Southmead Hospital) and had no significant comorbidities (such as Vascular dementia or Parkinson's disease). Abbreviations: M – methylated; MGMT – methylguanine‐DNA methytransferase methylation; U – unmethylated; UK ‐ unknown.