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Regional glutamine deficiency in tumours promotes dedifferentiation through inhibition of histone demethylation

Nature Cell Biology volume 18, pages 1090–1101 (2016)Cite this article

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Abstract

Poorly organized tumour vasculature often results in areas of limited nutrient supply and hypoxia. Despite our understanding of solid tumour responses to hypoxia, how nutrient deprivation regionally affects tumour growth and therapeutic response is poorly understood. Here, we show that the core region of solid tumours displayed glutamine deficiency compared with other amino acids. Low glutamine in tumour core regions led to dramatic histone hypermethylation due to decreased α-ketoglutarate levels, a key cofactor for the Jumonji-domain-containing histone demethylases. Using patient-derived V600EBRAF melanoma cells, we found that low-glutamine-induced histone hypermethylation resulted in cancer cell dedifferentiation and resistance to BRAF inhibitor treatment, which was largely mediated by methylation on H3K27, as knockdown of the H3K27-specific demethylase KDM6B and the methyltransferase EZH2 respectively reproduced and attenuated the low-glutamine effects in vitro and in vivo. Thus, intratumoral regional variation in the nutritional microenvironment contributes to tumour heterogeneity and therapeutic response.

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Figure 1: Tumour core regions display low glutamine levels and hypermethylation of histone H3.
Figure 2: Low glutamine level leads to increased histone methylation in tumour cells.
Figure 3: Glutamine deficiency drives histone hypermethylation in tumour core regions.
Figure 4: Low glutamine leads to dedifferentiation in tumour cores.
Figure 5: Low-glutamine-induced dedifferentiation results in resistance to BRAF inhibitor treatment.
Figure 6: Low-glutamine-induced dedifferentiation is mediated by histone methylation on H3K27.
Figure 7: Low-glutamine-induced drug resistance is mediated by histone methylation on H3K27.

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Acknowledgements

We thank members of the Kong laboratory for helpful comments on the manuscript. This work was supported by National Institutes of Health (NIH)/National Cancer Institute (NCI) grant R01CA183989 (to M.K.), Caltech-City of Hope Biomedical Initiative Pilot Grant (to M.K. and V.G.), American Cancer Society Research Scholar RSG-16-085-01-TBE (to M.K.) and Stand up to Cancer Philip A. Sharp Innovation in Collaboration Award. M.K. is the Pew Scholar in the Biomedical Sciences and the V scholar in Cancer Research. X.H.L. is supported by the DNA Damage Response and Oncogenic Signaling (DDROS) Training Program at City of Hope. Research reported here includes work carried out in Core Facilities supported by the NIH/NCI under grant number P30CA33572.

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Authors and Affiliations

  1. Department of Cancer Biology, Beckman Research Institute of City of Hope Cancer Center, Duarte, California 91010, USA

    Min Pan, Michael A. Reid, Xazmin H. Lowman, Thai Q. Tran, Ying Yang, Jenny E. Hernandez-Davies, Kimberly K. Rosales & Mei Kong

  2. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA

    Rajan P. Kulkarni & Viviana Gradinaru

  3. Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90095, USA

    Rajan P. Kulkarni

  4. Department of Pharmacology and Cancer Biology, Duke University Medical School, Durham, North Carolina 27710, USA

    Xiaojing Liu & Jason W. Locasale

  5. Department of Information Sciences, Beckman Research Institute of City of Hope Cancer Center, Duarte, California 91010, USA

    Haiqing Li

  6. Division of Dermatology, Department of Medicine, David Geffen School of Medicine and Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California 90095, USA

    Willy Hugo, Chunying Song & Roger S. Lo

  7. Department of Pathology, University of California San Diego, La Jolla, California 92093, USA

    Xiangdong Xu

  8. Department of Diabetes and Metabolic Disease, Beckman Research Institute of City of Hope Cancer Center, Duarte, California 91010, USA

    Dustin E. Schones & David K. Ann

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Contributions

M.P. designed and performed most of the experiments, analysed and interpreted the data and wrote the manuscript. M.K. conceived and supervised this study, designed experiments and wrote the paper. M.A.R. and X.H.L. helped to measure metabolites and assisted with mouse experiments. R.P.K. and V.G. performed PACT experiments. T.Q.T. assisted with flow cytometry experiments. Y.Y. assisted with qPCR experiments. J.E.H.-D. and K.K.R. helped set up melanoma cell culture. W.H., C.S. and R.S.L. provided patient-derived melanoma cells and conceptual advice on melanoma dedifferentiation. X.X. assisted with IHC experiments. D.E.S. assisted with ChIP experiments and H.L. performed the bioinformatics analyses. D.K.A. provided conceptual advice on hypoxia and metabolism experiments. X.L. and J.W.L. performed and helped to analyse the metabolomics experiments.

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Integrated supplementary information

Supplementary Figure 1 Tumour core regions display heterogeneity.

(a) Schematic of tumour dissection. One unit equals 20% of the radius. (b) Schematic of PACT (defined as Passive CLARITY) Technique procedure. (c) 3D imaging of M229 xenograft tumour depicting H3K27me3 (red), Hif1α (orange) and KI67 (blue) staining. Periphery and core of tumour are as indicated. Scale bar = 250 μm. (d) Immunohistochemistry staining in xenograft tumours. Whole tumours were sliced and stained with indicated antibodies (cleaved caspase 3, HIF-1α). H&E staining is also shown. Scale bar = 80 μm. (e) Tissues from periphery and core regions of xenograft tumours were lysed to collect whole cell lysate. Proteins were assessed by western blotting with specified antibodies. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 2 Histone methylation in low glutamine is not due to low proliferation rate of cells.

(a) For lane 1 and 2, 50% confluent M229 cells were cultured in complete (4 mM glutamine) or 0.1 mM glutamine medium for 4 days. For lane 3, 100% confluent M229 cells were cultured in complete medium for 4 days. Cells were lysed for histone extraction and histone lysine methylation levels were assessed by western blotting. Total histone H3 was used as loading control. Unprocessed original scans of blots are shown in Supplementary Fig. 8. (b) Cells were cultured under different conditions as indicated and cell number was determined by cell counting everyday (3T3 and Ras-3T3) or every other day (M229). Data represent mean ± S.D., n = 3 independent experiments. (c) Cells were cultured under different conditions as indicated. After PI staining, cell survival was assessed by flow cytometry. Data represent mean ± S.D., n = 3 independent experiments. Source data for b and c are shown in Supplementary Table 4.

Supplementary Figure 3 Low glutamine is the major driver of histone methylation.

(a,b) M229 cells were cultured under different conditions for 4 days as indicated. After that, cells were lysed for histone extraction and histone lysine methylation levels were assessed by western blotting. Total histone H3 was used as loading control. Unprocessed original scans of blots are shown in Supplementary Fig. 8. (c) M229 cell survival in low glucose medium. M229 cells were cultured in complete medium (25 mM glucose) or medium with different glucose concentrations for 4 days. Medium was changed twice everyday. Cells were stained with PI and cell survival was measured by Flow cytometry. Data represent mean ± S.D., n = 3 independent experiments.

Supplementary Figure 4 Low glutamine induces suppression of differentiation genes, which can be reversed by EPZ005687.

(a) M249 cells were cultured in complete or 0.1 mM glutamine medium for 12 days, then RNA was extracted and gene expression was assessed by qPCR. Data represent mean ± S.D. of three independent RNA extracts. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 by unpaired Student’s t-test. Source data can be found in Supplementary Table 4. (b) M229 cells were stained with Alexa647 conjugated CD271 antibody. Cells were then sorted by flow cytometry and CD271- cells were collected. (c) The CD271- cells were cultured for 2 days and then stained with APC conjugated CD133 antibody. The stained cells were sorted again and CD271-/CD133- cells were collected. (d) CD271-/CD133- cells were cultured in complete (4 mM Gln) or 0.1 mM Gln medium for 4 days, then cells were harvested for histone extraction or whole cell lysate collection. Histone lysine methylation and protein levels were assessed by western blotting. Total histone H3 and Actin were used as loading control. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 5 Low glutamine-induced differential gene expression is reversed by H3K27me3 inhibitor.

(a,b) M229 cells were cultured in complete medium or 0.1 mM glutamine medium with or without global histone methylation inhibitors (a) or H3K9 specific methylation inhibitors (b) for 4 days; histones were extracted and protein levels were assessed by western blotting. Total histone H3 was used as loading control. Unprocessed original scans of blots are shown in Supplementary Fig. 8. (c) M229 cells were cultured in complete medium or 0.1 mM glutamine medium with or without H3K27 methylation inhibitor EPZ005687 for 4 days, whole cell lysates were collected and protein levels were assessed by western blotting. Unprocessed original scans of blots are shown in Supplementary Fig. 8. (d) M229 and (e) M249 cells were cultured in complete medium or 0.1 mM glutamine medium with or without H3K27 specific methylation inhibitor EPZ005687 for 4 days, then RNA was extracted and gene expression was assessed by qPCR. Data represent mean ± S.D. of three independent RNA extracts. ∗∗P < .01, ∗∗∗P < .001 by Student’s t-test.

Supplementary Figure 6 Ectopic EZH2 expression rescues the EZH2 shRNA effects.

(a) EZH2 was knocked down with two different shRNAs in M229 cells, then the cells were transiently transfected with EZH2 cDNA. Cells were harvested after 4 days and EZH2 protein was measured by western blotting. Actin was used as loading control. (b) EZH2 was knocked down with two different shRNAs in M229 cells, then the cells were transiently transfected with EZH2 cDNA in the medium with 4 mM or 0.1 mM Gln. Cells were harvested after 4 days, EZH2 protein and H3K27me3 were assayed by western blotting. Total H3 and Actin were used as loading control. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 7 Neither HIF-1α nor DNA methylation is involved in low glutamine-induced epigenetic modification.

(a) M229 cells were transfected with HIF-1α siRNA in a 6-well plate at day 1. At day 6, cells were lysed to collect whole cell lysate and HIF-1α level was assessed by western blotting. Actin was used as loading control. Unprocessed original scans of blots are shown in Supplementary Fig. 8. (b) M229 cells were transfected with HIF-1α siRNA in a 6-well plate at day 1. From day 2, cells were cultured in complete (4 mM Gln) or 0.1 mM Gln medium for 4 days. At day 6, cells were harvested for histone extraction or whole cell lysate collection. Histone lysine methylation and protein levels were assessed by western blotting. Total histone H3 and Actin were used as loading control. Unprocessed original scans of blots are shown in Supplementary Fig. 8. (c) M229 cells were cultured in complete or 0.1 mM glutamine medium for 4 days, then DNA was extracted and used for whole genome DNA methylation sequencing. Methylation status of detected CpG sites (density) is shown. (d) M229 cells were cultured in complete medium, 0.1 mM glutamine medium with or without histone methylation inhibitor Adox, DNA methylation inhibitors 5-Azacytidine and 5-Aza-2′-deoxycytidine. RNA and protein were harvested after 4 days for qPCR (left) and western blotting (right). PCR data represent mean ± S.D.. of three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

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Pan, M., Reid, M., Lowman, X. et al. Regional glutamine deficiency in tumours promotes dedifferentiation through inhibition of histone demethylation. Nat Cell Biol 18, 1090–1101 (2016). https://doi.org/10.1038/ncb3410

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