Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Oct 1;29(1):76-88.e7.
doi: 10.1016/j.celrep.2019.08.076.

Liver-Type Glutaminase GLS2 Is a Druggable Metabolic Node in Luminal-Subtype Breast Cancer

Michael J Lukey  1 , Ahmad A Cluntun  2 , William P Katt  1 , Miao-Chong J Lin  1 , Joseph E Druso  1 , Sekar Ramachandran  3 , Jon W Erickson  3 , Henry H Le  4 , Zhihan-Emily Wang  1 , Bryant Blank  5 , Kai Su Greene  1 , Richard A Cerione  6
Affiliations

Liver-Type Glutaminase GLS2 Is a Druggable Metabolic Node in Luminal-Subtype Breast Cancer

Michael J Lukey et al. Cell Rep. .

Abstract

Efforts to target glutamine metabolism for cancer therapy have focused on the glutaminase isozyme GLS. The importance of the other isozyme, GLS2, in cancer has remained unclear, and it has been described as a tumor suppressor in some contexts. Here, we report that GLS2 is upregulated and essential in luminal-subtype breast tumors, which account for >70% of breast cancer incidence. We show that GLS2 expression is elevated by GATA3 in luminal-subtype cells but suppressed by promoter methylation in basal-subtype cells. Although luminal breast cancers resist GLS-selective inhibitors, we find that they can be targeted with a dual-GLS/GLS2 inhibitor. These results establish a critical role for GLS2 in mammary tumorigenesis and advance our understanding of how to target glutamine metabolism in cancer.

Keywords: 968; BPTES; CB-839; GLS; breast cancer; glutaminase; glutamine metabolism GLS2.

PubMed Disclaimer

Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Luminal-Subtype Breast Cancer Cells Use Glutamine to Supply the TCA Cycle, but Resist GLS Inhibitors
(A) The effect of the GLS inhibitors BPTES and CB-839 on proliferation of basal-subtype (MDA-MB-231 and TSE) and luminal-subtype (MDA-MB-453 and T-47D) breast cancer cells over 6 days. Mean ± SD of triplicate assays. (B) Glutamine consumption rates, per milligram of total cellular protein, of breast cancer cell lines. Mean ± SD of biological triplicates. (C) Western blot showing relative levels of SLC1A5 in breast cancer cell lines. Note that SLC1A5 is an integral membrane protein subject to covalent posttranslational modifications including glycosylations, which cause it to run at a range of molecular weights on SDS-PAGE. (D) Abundance ratios of fully labeled intracellular glutamine, glutamate, and TCA cycle metabolites in breast cancer cells supplied with [U-13C]-glutamine for 10 h. Mean ± SEM of biological triplicate samples. See also Figure S1 and Tables S1–S3.
Figure 2.
Figure 2.. GLS2 Is Upregulated in Luminal-Subtype Breast Cancers
(A) Box and whisker plots showing transcript levels of GLS2 (left panel) and GLS (right panel) in the molecular subtypes of breast cancer. RNA-seq V2 RSEM data are from The Cancer Genome Atlas invasive breast cancer dataset. The mean expression in each group is indicated by a cross, and the box and whiskers indicate the minimum, first quartile, median, third quartile, and maximum values. **p ≤ 0.01. (B) Relative GLS2 protein levels in tissue microarray slices of normal mammary tissue, receptor-positive, and receptor-negative breast tumors. **p ≤ 0.01. (C) Microscopy images of breast tissue microarray slices stained brown for GLS2. Representative images are shown for normal breast tissue along with receptor-positive and receptor-negative breast tumors. Scale bars, 200 μm. (D) Quantitative real-time PCR data showing relative levels of GLS2 transcript in breast cancer cell lines. Reactions were carried out in triplicate, and error bars indicate the RQmax and RQmin values. (E) Quantitative real-time PCR data showing relative levels of GLS transcript in breast cancer cell lines. Reactions were carried out in triplicate, and error bars indicate the RQmax and RQmin values. (F) Western blots showing relative levels of GLS and GLS2 in breast cancer cell lines. A non-specific band from the GLS2 antibody, clearly visible for MDA-MB-231 and Hs 578T lysates, is labeled N/S. (G) Western blots of whole-cell lysates (WCLs), and cytosolic, mitochondrial, and nuclear fractions from MDA-MB-453 and MDA-MB-231 cells. VDAC, ASNS, and Lamin A serve as mitochondrial, cytosolic, and nuclear marker proteins, respectively. See also Figure S2.
Figure 3.
Figure 3.. GLS2 Gene Expression Is Regulated by GATA3 and Promoter Methylation
(A) Western blots showing relative levels of GLS2, and previously identified transcription factors for the GLS2 gene, in breast cancer cell lines. (B) Western blots showing relative levels of GLS2, the luminal-transcription factor GATA3, and the receptors ERα, PR, and HER2 in breast cancer cell lines. (C) Plot of GLS2 and GATA3 transcript levels in human breast tumors, with the Pearson correlation coefficient r indicated. RNA-seq V2 RSEM data from TCGA invasive breast cancer dataset. (D) Quantitative real-time PCR data showing relative levels of a 176-bp fragment of the GLS2 gene promoter, centered on the putative GATA3 binding site, in chromatin immunoprecipitations (ChIPs) using negative control IgG or a GATA3-targeted antibody. Mean ± SD of biological triplicate samples. (E) Western blots showing GLS2 and GATA3 levels in MDA-MB-453 and T-47D cells transfected with either a control siRNA (labeled C) or with two independent GATA3-targeted siRNAs. Bands in the GLS2 blot were quantified by densitometry using ImageJ, and relative band intensities are indicated above the blot. Since GATA3 regulates expression of Tubulin and several other cytoskeletal proteins, a non-specific (N/S) band is shown to demonstrate equal loading. (F) Heatmaps showing relative methylation levels at CpG sites within the CpG island of the GLS2 gene promoter in breast cancer cell lines. Labels for the CpG sites refer to the amplicon in which the sites are located (amplicons 0 to 9) and then the position of the site within that amplicon (i.e., first CpG site, second CpG site, etc.). Numbers missing in the sequence are for sites at which methylation ratios could not be determined. Although the amplicons were designed to overlap, only a single reading is shown for sites covered by more than one amplicon. (G) Western blot showing relative levels of GLS2 in TSE cells treated with different concentrations of the DNA hypomethylating agent azacitidine for 48 h. Bands in the GLS2 blot were quantified by densitometry using ImageJ, and relative band intensities are indicated above the blot. A non-specific band is labeled N/S. See also Figure S3.
Figure 4.
Figure 4.. GLS2 Is Essential in Luminal-Subtype Breast Cancers
(A) Western blots showing GLS and GLS2 levels in MDA-MB-453 cells and MDA-MB-231 cells expressing either a control shRNA or two independent GLS2-targeted (MDA-MB-453 cells) or GLS-targeted (MDA-MB-231 cells) shRNAs. (B) Fold changes in fully labeled glutamate and TCA cycle metabolites (i.e., derived directly from [U-13C]-glutamine) in MDA-MB-453 cells following knockdown of GLS2, or in MDA-MB-231 cells following knockdown of GLS. Mean ± SEM of biological triplicates. (C) The effect of knocking down either GLS, GLS2, or both GLS and GLS2 simultaneously, on the proliferation of breast cancer cell lines over 6 days. For each condition, two independent siRNAs were used, and the effects were compared with those of a control siRNA (labeled C). Mean ± SD of triplicate assays. Lower panels show western blots for GLS and GLS2 in each sample. *p ≤ 0.05; **p ≤ 0.01; ns, not significant. (D) Western blot showing partial knockdown of GLS2 in MDA-MB-453 stably expressing two independent GLS2-targeted shRNAs, relative to cells stably expressing a control shRNA. (E) Inhibition of proliferation over 6 days for MDA-MB-453 cells stably expressing GLS2-targeted shRNAs, relative to cells stably expressing a control shRNA (labeled C). Experiments were run either without (left panel) or with (right panel) supplementation of the culture medium with 2 mM dimethyl α-ketoglutarate (dm-α-KG). Mean ± SD of triplicate assays. *p ≤ 0.05; **p ≤ 0.01; ns, not significant. (F) Growth of xenograft tumors in mice by MDA-MB-453 cells stably expressing either a control shRNA or two independent GLS2-targeted shRNAs. Mean ± SD (n = 6 tumors per condition). *p ≤ 0.05; **p ≤ 0.01. See also Figure S4.
Figure 5.
Figure 5.. GLS2 Expression Is Sufficient for Resistance to GLS Inhibitors
(A) Inhibition of glutaminase activity by 10 μM BPTES in mitochondria isolated from breast cancer cell lines. Plot shows activity in the presence of 10 μM BPTES relative to matched samples with no BPTES present. Mean ± SD of triplicate assays. (B) The effect of increasing inorganic phosphate concentrations on the catalytic activity of recombinant full-length human GLS (GAC splice variant) and GLS2. Mean ± SD of triplicate assays. (C) Fold changes in fully labeled intracellular metabolites (i.e., derived directly from [U-13C]-glutamine) in basal-subtype (top) and luminal-subtype (bottom) breast cancer cell lines, following treatment with 10 μM BPTES. Mean ± SEM of triplicates relative to the vehicle. (D) Western blots showing levels of endogenous GLS and ectopically expressed V5-tagged GLS2 in MDA-MB-231 and TSE cells. (E) Inhibition of proliferation over 6 days of MDA-MB-231 and TSE cells, stably expressing GLS2 (two clones for each cell line) or carrying the plasmid vector only, by treatment with different concentrations of BPTES. Mean ± SD of triplicate assays. (F) The effect of different concentrations of BPTES on proliferation of DU4475 cells over 6 days. Mean ± SD of triplicate assays. (G) Western blots showing relative levels of GLS and GLS2 in MDA-MB-453, MDA-MB-231, and DU4475 cells. A non-specific band from the GLS antibody is marked N/S. (H) The effect of knocking down either GLS or GLS2, or both GLS and GLS2 simultaneously, on the proliferation of DU4475 cells over 6 days. For each condition, two independent siRNAs were used, and the effects were compared with those of a control siRNA (labeled C). Mean ± SD of triplicate assays. **p ≤ 0.01; ns, not significant. See also Figure S5 and Table S4.
Figure 6.
Figure 6.. 968 Inhibits GLS2 and Suppresses BPTES-Resistant Breast Cancer Growth
(A) Inhibition of purified recombinant full-length human GLS (GAC splice variant) or GLS2 by different concentrations of 968. Mean ± SD of triplicate assays. (B) The effect of different concentrations of 968 or BPTES on proliferation of DU4475 cells over 6 days. Mean ± SD of triplicate assays. (C) Fold changes in fully labeled intracellular metabolites, derived directly from [U-13C]-glutamine, when breast cancer cells are treated with 10 μM 968 for the indicated periods of time. Mean ± SEM of triplicates relative to the vehicle. (D) Growth of MDA-MB-453 xenograft tumors in mice. Once palpable tumors were detected (at day 14), mice were divided into two groups, one of which received subcutaneous injections of 10 mg/kg 968 three times per week, while the other received carrier solution only. Mean ± SD (n = 6 tumors per condition). **p ≤ 0.01. (E) Plot showing the final size of MDA-MB-453 xenograft tumors following treatment of mice with 10 mg/kg 968, 10 mg/kg BPTES, or carrier solution only, three times per week from day 14 until day 35. Mice were sacrificed at day 35, and tumors were excised prior to measurement. Mean ± SD (n = 6 tumors per condition). **p ≤ 0.01; ns, not significant. See also Figure S6 and Table S4.
See this image and copyright information in PMC

Similar articles

  • Two Faces of Glutaminase GLS2 in Carcinogenesis.
    Buczkowska J, Szeliga M. Buczkowska J, et al. Cancers (Basel). 2023 Nov 24;15(23):5566. doi: 10.3390/cancers15235566. Cancers (Basel). 2023. PMID: 38067269 Free PMC article. Review.
  • Structure and activation mechanism of the human liver-type glutaminase GLS2.
    Ferreira IM, Quesñay JEN, Bastos AC, Rodrigues CT, Vollmar M, Krojer T, Strain-Damerell C, Burgess-Brown NA, von Delft F, Yue WW, Dias SM, Ambrosio AL. Ferreira IM, et al. Biochimie. 2021 Jun;185:96-104. doi: 10.1016/j.biochi.2021.03.009. Epub 2021 Mar 18. Biochimie. 2021. PMID: 33746066 Free PMC article.
  • The role of glutaminase in cancer.
    Masisi BK, El Ansari R, Alfarsi L, Rakha EA, Green AR, Craze ML. Masisi BK, et al. Histopathology. 2020 Mar;76(4):498-508. doi: 10.1111/his.14014. Epub 2020 Feb 18. Histopathology. 2020. PMID: 31596504 Review.
  • GLS2 is protumorigenic in breast cancers.
    Dias MM, Adamoski D, Dos Reis LM, Ascenção CFR, de Oliveira KRS, Mafra ACP, da Silva Bastos AC, Quintero M, de G Cassago C, Ferreira IM, Fidelis CHV, Rocco SA, Bajgelman MC, Stine Z, Berindan-Neagoe I, Calin GA, Ambrosio ALB, Dias SMG. Dias MM, et al. Oncogene. 2020 Jan;39(3):690-702. doi: 10.1038/s41388-019-1007-z. Epub 2019 Sep 20. Oncogene. 2020. PMID: 31541193
  • Glutamine to proline conversion is associated with response to glutaminase inhibition in breast cancer.
    Grinde MT, Hilmarsdottir B, Tunset HM, Henriksen IM, Kim J, Haugen MH, Rye MB, Mælandsmo GM, Moestue SA. Grinde MT, et al. Breast Cancer Res. 2019 May 14;21(1):61. doi: 10.1186/s13058-019-1141-0. Breast Cancer Res. 2019. PMID: 31088535 Free PMC article.
See all similar articles

Cited by

See all "Cited by" articles

References

    1. Altman BJ, Stine ZE, and Dang CV (2016). From Krebs to clinic: glutamine metabolism to cancer therapy. Nat. Rev. Cancer 16, 619–634. - PMC - PubMed
    1. Asselin-Labat ML, Sutherland KD, Barker H, Thomas R, Shackleton M, Forrest NC, Hartley L, Robb L, Grosveld FG, van der Wees J, et al. (2007). Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nat. Cell Biol 9, 201–209. - PubMed
    1. Biancur DE, Paulo JA, Ma1achowska B, Quiles Del Rey M, Sousa CM, Wang X, Sohn ASW, Chu GC, Gygi SP, Harper JW, et al. (2017). Compensatory metabolic networks in pancreatic cancers upon perturbation of glutamine metabolism. Nat. Commun 8, 15965. - PMC - PubMed
    1. Cardona C, Sánchez-Mejías E, Dávila JC, Martín-Rufián M, Campos-Sandoval JA, Vitorica J, Alonso FJ, Matés JM, Segura JA, Norenberg MD, et al. (2015). Expression of Gls and Gls2 glutaminase isoforms in astrocytes. Glia 63, 365–382. - PubMed
    1. Cluntun AA, Huang H, Dai L, Liu X, Zhao Y, and Locasale JW (2015). The rate of glycolysis quantitatively mediates specific histone acetylation sites. Cancer Metab 3, 10. - PMC - PubMed

Publication types

-