Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides (original) (raw)

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Acknowledgements

We thank G. Stephanopoulos for assistance with Nutrostat design, M. Holland for mtDNA sequencing consultation, L. Garraway for assistance identifying mtDNA mutant cell lines, T. Yagi for the NDI1 antibody, T. DiCesare for diagrams, and members of the Sabatini Laboratory for assistance (particularly A. Saucedo, C. Koch, O. Yilmaz, Y. Gultekin and A. Hutchins for technical assistance, and D. Lamming and W. Comb for critical reading of the manuscript). This research is supported by fellowships from The Leukemia and Lymphoma Society and The Jane Coffin Childs Fund to K.B., the Council of Higher Education Turkey and Karadeniz T. University Scholarships to B.Y. and grants from the David H. Koch Institute for Integrative Cancer Research at MIT, The Alexander and Margaret Stewart Trust Fund, the NIH (K99 CA168940 to R.P. and CA103866, CA129105, and AI07389 to D.M.S.) and the Starr Cancer Consortium. D.M.S. is an investigator of the Howard Hughes Medical Institute.

Author information

Author notes

  1. Kıvanç Birsoy and Richard Possemato: These authors contributed equally to this work.

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, 02142, Massachusetts, USA
    Kıvanç Birsoy, Richard Possemato, Franziska K. Lorbeer, Erol C. Bayraktar, Prathapan Thiru, Burcu Yucel, Tim Wang, Walter W. Chen & David M. Sabatini
  2. Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA
    Kıvanç Birsoy, Richard Possemato, Tim Wang, Walter W. Chen & David M. Sabatini
  3. Broad Institute of Harvard and MIT, Seven Cambridge Center, Cambridge, 02142, Massachusetts, USA
    Kıvanç Birsoy, Richard Possemato, Tim Wang, Walter W. Chen, Clary B. Clish & David M. Sabatini
  4. The David H. Koch Institute for Integrative Cancer Research at MIT, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA
    Kıvanç Birsoy, Richard Possemato, Tim Wang, Walter W. Chen & David M. Sabatini

Authors

  1. Kıvanç Birsoy
  2. Richard Possemato
  3. Franziska K. Lorbeer
  4. Erol C. Bayraktar
  5. Prathapan Thiru
  6. Burcu Yucel
  7. Tim Wang
  8. Walter W. Chen
  9. Clary B. Clish
  10. David M. Sabatini

Contributions

K.B., R.P. and D.M.S. conceived the project and designed the experiments. K.B. and R.P. designed and engineered the Nutrostat and performed the screening, knockdown, cell proliferation, extracellular flux, glucose consumption, and tumour formation experiments and processed and analysed sequencing and expression data. F.K.L., E.C.B., B.Y., and W.W.C. assisted with experiments. C.B.C. performed the metabolite profiling experiments. T.W. provided bioinformatic support for shRNA abundance deconvolution, P.T. assisted in identifying mtDNA mutations. K.B., R.L.P and D.M.S. wrote and all authors edited the manuscript.

Corresponding author

Correspondence toDavid M. Sabatini.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Model of the metabolic determinants of sensitivity to low glucose and biguanides.

This diagram outlines the interplay between reserve oxidative phosphorylation (OXPHOS) capacity, sensitivity to biguanides, and sensitivity to culture in low glucose. Most cancer cell lines and normal cells tested exhibited an ability to respond to glucose limitation by upregulating OXPHOS, rendering them less sensitive to biguanides and low-glucose conditions. In contrast, cell lines harbouring mutations in mtDNA-encoded complex I subunits or exhibiting impaired glucose utilization have a limited reserve OXPHOS capacity and are therefore unable to properly respond to biguanides and low glucose, rendering them sensitive to these perturbations. At the extreme, cells artificially engineered to have no OXPHOS (Rho cells) exhibit extreme low glucose sensitivity, but resistance to further inhibition of OXPHOS. Thus, mtDNA mutant cancer cells exist at an intermediate state of OXPHOS functionality that renders them sensitive to treatment with biguanides in vitro and in vivo. Similarly, cell lines with impaired glucose utilization exhibit biguanide sensitivity specifically under the low glucose conditions seen in the tumour microenvironment.

Extended Data Figure 2 Proliferation and media glucose levels in standard culture conditions.

a, Jurkat cell proliferation under 10 mM (black) versus 1 mM (blue) glucose in standard culture conditions. b, Media glucose concentrations over time from cultures in a. Error bars are s.e.m., n = 3. Replicates are biological, means reported. *P < 0.05 by two-sided student’s _t_-test.

Extended Data Figure 3 Additional data supporting the RNAi screen.

a, Genes scoring as preferentially required for growth in 10 mM glucose compared to 0.75 mM glucose (top). shRNAs scores (given in per cent) and pathway classifications are indicated. Immunoblot analyses depict suppression of PKM by shRNAs (PKM1, PKM2) compared to control (RFP). Proliferation of cells in 0.75 mM (blue) relative to 10 mM glucose (black) harbouring shRNAs targeting PKM or control is also shown (PKM1). *P < 0.05 relative to RFP 0.75 mM glucose. b, Nuclear-encoded core complex I genes are shown in the grey box, with those that scored as differentially required under 0.75 mM glucose in red. The dot plot reports the degree of differential requirement for growth in 0.75 mM glucose (as the log2 difference in shRNA abundance in 0.75 mM glucose versus 10 mM glucose) of individual shRNAs targeting non-core complex I genes, core complex I genes, or non-targeting controls. Red bar, population median. c, Top, mRNA levels of the non-scoring OXPHOS genes (black) and the scoring OXPHOS gene (blue) indicated upon suppression, with the shRNAs indicated as measured by qPCR, relative to a non-targeting shRNA (RFP). Bottom, cell number from 7-day proliferation assay of cells in 0.75 mM glucose relative to 10 mM glucose (not shown) harbouring the indicated shRNAs. shRFP control normalized to 1. Error bars are s.e.m., n = 3. Replicates are biological, means reported. *P < 0.05 by two-sided student’s _t_-test.

Extended Data Figure 4 Additional data characterizing mitochondrial dysfunction and impaired glucose utilization in cancer cell lines.

a, Ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) (left) or OCR normalized to protein content (right) for glucose-limitation-resistant (black) or glucose-limitation-sensitive (blue) cell lines. b, Left, mtDNA content for indicated cell lines by qPCR using primers targeting ND1 (black) or ND2 (grey) normalized to gDNA repetitive element (Alu) relative to KMS-12BM. Right, mitochondrial mass measured by fluorescence intensity of Mitotracker Green dye for indicated cell lines. c, Per cent change from baseline (second measurement) of ECAR or OCR in Jurkat cells in conditions in which glucose concentration was maintained at 0.75 mM (blue) or increased to indicated concentrations (black). d, Uptake of 3H-labelled 2-DG (counts per min per ng protein) in 0.75 mM glucose at indicated time points in GLUT3-high (grey) or GLUT3-low (blue) cell lines. e, Heatmap of gene expression values for genes indicated at top and cell lines (left). Genes organized by P value with lowest expressed genes in NCI-H929 and KMS-26 to the left, those with significantly lower expression are in red. Expression values reported as log2-transformed fold difference from the median (scale colour bar shown to the right). f, Immunoblot analyses for GLUT3 and NDI1 expression in indicated cell lines (β-actin loading control). g, i, Proliferation of cell number in cells overexpressing GLUT3 or NDI1 relative to control vector (4 days). h, OCR of permeabilized cells indicated upon addition of indicated metabolic toxins and substrates. j, Fold change in OCR in indicated cells expressing NDI1 relative to control vector. k, l, Proliferation for 4 days of control (Vector) or NDI1 expressing cell lines indicated (NDI1) under 10 mM (black) and 0.75 mM glucose (blue). Error bars are s.e.m. n = 4 for a–c, h, j; n = 3 for d, g, i, k, l. Replicates are biological, means reported. *P < 0.05 by two-sided student’s _t_-test.

Extended Data Figure 5 Gene expression signature for identifying cell lines with impaired glucose utilization.

Heatmap of gene expression values for the genes indicated on the right for the cell lines in the CCLE set. Gene expression values are reported as the difference from the median across the entire sample set according to the scale colour bar to the top right. Genes 1–8 comprised the gene expression signature used to identify samples with impaired glucose utilization. Samples are sorted based upon this signature, with those predicted to exhibit impaired glucose utilization at the top. The order of samples and all values are reported in Supplementary Table 4.

Extended Data Figure 6 GLUT3 overexpression increases tumour xenograft growth and cell proliferation in low-glucose media.

a, KMS-26 cell lines infected with GLUT3-overexpressing vector or infected with control vector were mixed in equal proportions and cultured under different glucose concentrations. In addition, these mixed cell lines were injected into NOD/SCID mice subcutaneously. After 2.5 weeks, genomic DNA was isolated from tumours as well as cells grown in vitro under the indicated glucose concentrations. Using qPCR, relative abundance of control vector and GLUT3 vector were determined and plotted relative to 10 mM glucose in culture (n = 9). b, Average volume of unmixed tumour xenografts from KMS-26 cell lines infected with GLUT3-overexpressing vector relative to control vector (2.5 weeks) (n = 6). Replicates are biological, means reported. *P < 0.05 by two-sided student’s _t_-test.

Extended Data Figure 7 Sanger sequencing traces validating mtDNA mutations.

The table summarizing mtDNA mutations in complex I subunits from Fig. 3j is reproduced (bottom right). Traces for each cell line (left) are shown in the order indicated by the table. In cases in which the sequence shown is in the reverse orientation to the revised Cambridge Reference Sequence, these are indicated by ‘reverse str’. For each trace, the gene sequenced is at the bottom left, the DNA sequence is at the top, and the nucleotide alteration is in red text.

Extended Data Figure 8 Additional data supporting the hypersensitivity of cell lines with the identified biomarkers to biguanides.

a, b, Viability (a, 10 mM glucose) or relative change in cell number (b, 4 days, glucose concentration indicated in key) of indicated cell lines at phenformin concentrations indicated. Viability measured by ATP levels on day 3 at phenformin concentrations indicated by the black–blue scale, compared to ATP levels on day 0. Value of 1 indicates fully viable cells (untreated). Value of 0 indicates no change in ATP level compared to day 0 (cytostatic). Negative values indicate decrease in ATP levels (−1 indicates no ATP). c, Viability as in a of indicated cell lines under 0.75 mM and 10 mM glucose at indicated phenformin concentrations. d, Left, relative change in cell number in 0.75 mM glucose, 2 mM metformin relative to untreated in glucose limitation resistant (black) and sensitive (blue) cell lines. Right, relative size of tumour xenografts derived from the indicated cell lines in mice injected with PBS or metformin (intraperitoneal, 300 mg kg−1 day−1). e, Viability as in a of NCI-H929 cells at the indicated concentrations of phenformin and glucose. f, Relative size of indicated cell line xenografts in mice treated with PBS or phenformin (1.7 mg ml−1 in drinking water). g, Per cent change in OCR of control (Vector) or NDI1-expressing lines (NDI1) relative to the second basal measurement and at indicated phenformin concentrations. h, Proliferation of 143B wild-type or 143B rho (no mtDNA) cell lines under 0.75 mM or 10 mM glucose with or without phenformin treatment. Error bars are s.e.m. n = 4 for a, c, e, g; n = 3 for b, d, h (left); n = 5 for d (right), f. Replicates are biological, means reported. *P < 0.05 by two-sided student’s _t_-test.

Extended Data Figure 9 Long-term treatment of mtDNA mutant cells with phenformin.

a, Sanger sequencing traces of mtDNA-encoded ND1 and ND4 genes from Cal-62 cells expressing NDI1 or control vector cultured under 5 to 20 µM phenformin or no phenformin for 1.5 months. Regions containing mutant sequence are indicated by the red box. b, Heteroplasmy levels for mutation in ND1 or ND4 were assessed by measuring the relative areas under the curve from Sanger-sequencing and plotted. c, Cal-62 cell lines cultured with or without phenformin for 1.5 months assessed for their ability to proliferate in 0.75 mM glucose (blue) relative to 10 mM glucose (black). The proliferation assay was for 4 days in the absence of phenformin. d, Heteroplasmy levels of ND1 and ND4 as in b of Cal-62 tumour xenografts in mice treated with or without phenformin for 28 days. Error bars are s.e.m., n = 3. Replicates are biological (c) or technical (b, d), means reported. *P < 0.05 by two-sided student’s _t_-test.

Extended Data Figure 10 Schematic of Nutrostat setup.

Part numbers, sizes and dimensions for the Nutrostat assembly are indicated (see Methods for additional details).

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Birsoy, K., Possemato, R., Lorbeer, F. et al. Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides.Nature 508, 108–112 (2014). https://doi.org/10.1038/nature13110

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