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. 2019 May 14;21(1):61.
doi: 10.1186/s13058-019-1141-0.

Glutamine to proline conversion is associated with response to glutaminase inhibition in breast cancer

Maria T Grinde  1 , Bylgja Hilmarsdottir  2   3 , Hanna Maja Tunset  4 , Ida Marie Henriksen  5 , Jana Kim  4   6 , Mads H Haugen  2 , Morten Beck Rye  7   8 , Gunhild M Mælandsmo  2   9 , Siver A Moestue  10   11
Affiliations

Glutamine to proline conversion is associated with response to glutaminase inhibition in breast cancer

Maria T Grinde et al. Breast Cancer Res. .

Abstract

Introduction: Glutaminase inhibitors target cancer cells by blocking the conversion of glutamine to glutamate, thereby potentially interfering with anaplerosis and synthesis of amino acids and glutathione. The drug CB-839 has shown promising effects in preclinical experiments and is currently undergoing clinical trials in several human malignancies, including triple-negative breast cancer (TNBC). However, response to glutaminase inhibitors is variable and there is a need for identification of predictive response biomarkers. The aim of this study was to determine how glutamine is utilized in two patient-derived xenograft (PDX) models of breast cancer representing luminal-like/ER+ (MAS98.06) and basal-like/triple-negative (MAS98.12) breast cancer and to explore the metabolic effects of CB-839 treatment.

Experimental: MAS98.06 and MAS98.12 PDX mice received CB-839 (200 mg/kg) or drug vehicle two times daily p.o. for up to 28 days (n = 5 per group), and the effect on tumor growth was evaluated. Expression of 60 genes and seven glutaminolysis key enzymes were determined using gene expression microarray analysis and immunohistochemistry (IHC), respectively, in untreated tumors. Uptake and conversion of glutamine were determined in the PDX models using HR MAS MRS after i.v. infusion of [5-13C] glutamine when the models had received CB-839 (200 mg/kg) or vehicle for 2 days (n = 5 per group).

Results: Tumor growth measurements showed that CB-839 significantly inhibited tumor growth in MAS98.06 tumors, but not in MAS98.12 tumors. Gene expression and IHC analysis indicated a higher proline synthesis from glutamine in untreated MAS98.06 tumors. This was confirmed by HR MAS MRS of untreated tumors demonstrating that MAS98.06 used glutamine to produce proline, glutamate, and alanine, and MAS98.12 to produce glutamate and lactate. In both models, treatment with CB-839 resulted in accumulation of glutamine. In addition, CB-839 caused depletion of alanine, proline, and glutamate ([1-13C] glutamate) in the MAS98.06 model.

Conclusion: Our findings indicate that TNBCs may not be universally sensitive to glutaminase inhibitors. The major difference in the metabolic fate of glutamine between responding MAS98.06 xenografts and non-responding MAS98.12 xenografts is the utilization of glutamine for production of proline. We therefore suggest that addiction to proline synthesis from glutamine is associated with response to CB-839 in breast cancer. The effect of glutaminase inhibition in two breast cancer patient-derived xenograft (PDX) models. 13C HR MAS MRS analysis of tumor tissue from CB-839-treated and untreated models receiving 13C-labeled glutamine ([5-13C] Gln) shows that the glutaminase inhibitor CB-839 is causing an accumulation of glutamine (arrow up) in two PDX models representing luminal-like breast cancer (MAS98.06) and basal-like breast cancer (MAS98.12). In MAS98.06 tumors, CB-839 is in addition causing depletion of proline ([5-13C] Pro), alanine ([1-13C] Ala), and glutamate ([1-13C] Glu), which could explain why CB-839 causes tumor growth inhibition in MAS98.06 tumors, but not in MAS98.12 tumors.

Keywords: 13C MRS; Aldehyde dehydrogenase 18 family member A1 (ALDH18A1); CB-839; Cancer treatment; Gene expression analysis; Glutaminase; Glutaminase inhibitor; High-resolution magic angle spinning MR spectroscopy (HR MAS MRS); Immunohistochemistry; Patient-derived xenograft (PDX).

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Conflict of interest statement

Ethics approval and consent to participate

All procedures and experiments involving animals were approved by the Norwegian Animal Research Authority (FOTS ID: 7713 and 9126) and carried out according to the European Convention for the Protection of Vertebrates used for Scientific Purposes.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Effect of CB-839 on tumor growth in luminal-like (MAS98.06) and basal-like (MAS98.12) PDX models. CB-839 significantly (p < 0.001) reduces tumor growth in MAS98.06 tumors. Tumor growth of MAS98.12 breast cancer xenografts is not affected by CB-839. Mean ± SEM values are plotted. N = 5 for each group
Fig. 2
Fig. 2
Expression of genes involved in glutaminolysis from untreated MAS98.06 and MAS98.12 tumors. a Heatmap of expression of the 60 selected genes involved in glutaminolysis in untreated MAS98.06 and MAS98.12 tumors. b Differences in gene expression between untreated MAS98.06 and MAS98.12 xenografts. Blue, the gene is significantly higher expressed with at least a two-fold higher expression in MAS98.06; red, the gene is significantly higher expressed with at least two-fold higher expression in MAS98.12; white, the gene is not significantly different expressed between the models. Log2-fold change (log2FC) between the two models is indicated by color intensity. Dotted arrows from Myc indicate some of the most important genes that are positively (+) and negatively (−) regulated by Myc, as shown by others [, , –36]. Full gene names, enzyme commission numbers (EC numbers), gene expression levels, q values, log2 fold change, and fold changes are specified in Additional file 5
Fig. 3
Fig. 3
H-scores and representative images from IHC of untreated MAS98.06 and MAS98.12 tumors. The tumors were stained with antibodies against aldehyde dehydrogenase 18 family member A1 (ALDH18A1), cMYC, glutamate dehydrogenase 1 (GLUD1), glutaminase (GLS1), glutamine synthetase (GS), pyrroline-5-carboxylate reductase 1 (PYCR1), and solute carrier family 1 member 5 (SLC1A5). Left columns present the percentage areas with different intensity levels (DAB: 3′-diaminobenzidine) for MAS98.06 (blue shades), and MAS98.12 (red shades) tumors. Neg, no labeling; 1+, light label; 2+, moderate label; 3+, dark label. Right columns show representative IHC images for each antibody for both MAS98.06 tumors (left) and MAS98.12 tumors (right) in two different magnifications of × 4 and × 20. Scale bar is 200 μm for × 4 magnification and 50 μm for × 20 magnification. *p < 0.05, ***p < 0.001, *****p < 0.00001
Fig. 4
Fig. 4
Utilization of [5-13C] Gln in untreated luminal-like (MAS98.06) and basal-like (MAS98.12) tumor xenografts. a 13C labeling patterns in MAS98.06 and MAS98.12 tumors after administration of [5-13C] Gln. MAS98.06 tumors (blue) use [5-13C] Gln to produce [5-13C] Glu, [5-13C] Pro, [1-13C] Ala, [1-13C] Glu, and [1-13C] Lac (gray, only borderline significant). They also store a significant amount of [5-13C] Gln in the tumors. MAS98.12 tumors (pink) take up [5-13C] Gln and use it for production of [5-13C] Glu, [1-13C] Lac and [5-13C] Ala (gray, only borderline significant). b Amount of 13C-labeled metabolites in the tumors, calculated by subtracting natural abundance spectra from 13C-enriched spectra. Stars (*) indicate that there is a significantly higher amount of the metabolite in 13C-enriched samples compared to natural abundance samples, while up arrowheads (^) indicate borderline significance. The total amount of 13C-labeled metabolites were not significantly different between the two models. ^p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: Ala, alanine; Gln, glutamine; GLS1, glutaminase; Glu, glutamate; Lac, lactate; Pro, proline; Pyr, pyruvate; TCA, tricarboxylic acid
Fig. 5
Fig. 5
Effect of CB-839 on glutamine metabolism in MAS98.06 and MAS98.12 PDX tumors. a Average 13C NMR spectra (173.5-185.5 ppm and 75-13 ppm) for CB-839-treated and untreated MAS98.06 (blue) and MAS98.12 (red) tumors receiving 13C-labeled glutamine. The contribution to the average from each individual NMR spectrum is scaled to the sample mass. b Quantified amounts of selected 13C-labeled metabolites in each experimental group: 13C glutamine ([5-13C] Gln), glutamate to glutamine ratio ([5-13C] Glu/[5-13C] Gln), alanine ([1-13C] Ala), and proline ([5-13C] Pro) in the experimental groups. c MAS98.06 tumors take up and store glutamine (Gln) and use glutamine to produce glutamate (Glu), proline (Pro), alanine (Ala), lactate (Lac), and glutamate (Glu) through one turn in TCA cycle, as indicated by the filled blue circles (gray circle for Lac, only borderline significant). CB-839 causes an accumulation of Gln (arrow up) and reduced amounts of Pro, Al, and Glu (arrows down) in the tumors (only [1-13C] Glu, which is created after one turn in TCA cycle, is reduced). MAS98.12 tumors use glutamine (Gln) to produce Glu, Lac, and Ala as indicated by filled pink circles (gray circle for Ala, only borderline significant). CB-839 causes accumulation of Gln in MAS98.12 tumors, but does not significantly change the amount of any other 13C-enriched metabolites *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
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