Abstract
Aerobic glycolysis in cancer cells, also known as the ‘Warburg effect’, is driven by hyperactivity of lactate dehydrogenase A (LDHA). LDHA is thought to be a substrate-regulated enzyme, but it is unclear whether a dedicated intracellular protein also regulates its activity. Here, we identify the human tumor suppressor folliculin (FLCN) as a binding partner and uncompetitive inhibitor of LDHA. A flexible loop within the amino terminus of FLCN controls movement of the LDHA active-site loop, tightly regulating its enzyme activity and, consequently, metabolic homeostasis in normal cells. Cancer cells that experience the Warburg effect show FLCN dissociation from LDHA. Treatment of these cells with a decapeptide derived from the FLCN loop region causes cell death. Our data suggest that the glycolytic shift of cancer cells is the result of FLCN inactivation or dissociation from LDHA. Together, FLCN-mediated inhibition of LDHA provides a new paradigm for the regulation of glycolysis.
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Data availability
The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE51 partner repository with the dataset identifier PXD018410 (www.ebi.ac.uk/pride/). Source data are provided with this paper.
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Acknowledgements
We thank B. A. Knutson, R. A. Oot, S. Wilkens and L. Neckers for assistance and scientific discussions as well as D. E. Post of the Upstate Cancer Center Biorepository. This work was partly supported with federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract no. HHSN26120080001E (L.S.S.) and with funds from SUNY Upstate Medical University, Upstate Cancer Center and the Upstate Foundation (M.M.).
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M.R.W., A.J.B.-W., R.A.S., S.J.B., A.R.B., F.H., P.K., A.G., D.R.L., M.C., S.A.S., S.M.J., A.B. and M.M. performed experiments. M.R.W., A.J.B.-W., G.C., T.A.H., W.G.S.-S., S.N.L., L.S.S., W.M.L., A.B., D.B., G.B. and M.M. designed experiments. M.R.W., R.A.S., A.B., D.B., G.B. and M.M. wrote the manuscript. M.R.W. and M.M. conceived the project.
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Extended data
Extended Data Fig. 1 The tumor suppressor FLCN mediated binding and inhibition of LDHA.
a) Gene Ontology term analysis of FLCN proteomic data using Panther v15.0 (pantherdb.org). b) LDHA-FLAG IP from HEK293 cells immunoblotted with anti-FLCN antibody. c) Titration of FLCN-FLAG expression in HEK293 cells immunoblotted with anti-pTyr10-LDHA antibody. d) Titration of siRNA targeting FLCN in HEK293 cells immunoblotted with anti-pTyr10-LDHA antibody. Bar chart contains densitometry of pTyr10-LDHA:FLCN ratio. Unpaired Student’s t-test, (p < 2.9x10−10, n=3). Data shown as mean ± s.d.
Extended Data Fig. 2 Inherent specificity of FLCN for binding to dimeric LDHA.
a) IP of endogenous FLCN from whole cell lysates of LDHA or LDHB knockout HAP1 cells. Immunoblotted with anti-FLCN antibody. b) Western blot of siRNA knockdown of FLCN in Fig. 1d. c) Tagged LDHA and LDHB subunits were consecutively immunoprecipitated and immunoblotted with anti-FLCN. d) Lysates from Extended Data Fig. 1c subjected to Native PAGE. Bar chart contains ratio of densitometry of largest LDHA oligomer:GAPDH (see Extended Data Fig. 1c; Unpaired Student’s t-test, n=3). Data shown as mean ± s.d. e) IP of FLCN from HEK293 cells subjected to Native PAGE. SE = short exposure of immunoblot, LE = long exposure of immunoblot.
Extended Data Fig. 3 The tumor suppressor FLCN specifically binds to the amino and carboxy domain of LDHA.
a) Sequence alignment of human LDHA and human LDHB using https://clustalw.ddbj.nig.ac.jp/. The red N- and blue C-terminal residues that were swapped to generate the N, C and NC chimeric constructs used in Extended Data Fig. 2c are highlighted. The active site loop (99–110) is colored cyan. b) LDHA tetramer (PDB ID: 1i10) in ribbon (top) and surface (bottom) models represented in the standard view (left) and 90° rotated view (right). The N-terminus (residues 1–22) is highlighted in red and the C-terminus (residues 294–332) is highlighted in blue. Note the proximity of the C-terminal helix (blue) to the active site loop (residues 99–110; cyan) within each subunit. In the tetrameric arrangement, the N- and C-terminal residues of adjacent subunits form a continuous surface. Structure rendered using PyMOL 2.3. c) LDHA/LDHB chimeric constructs were transfected into HEK293 cells and immunoprecipitated. Co-IP of FLCN was detected by immunoblot.
Extended Data Fig. 4 FLCN interference with cofactor binding to LDHA.
a) Immunoblot of anti-FLCN IPs and whole cell extracts following exogenous addition of pyruvate to HEK293s (6 h treatment duration; n=3). SE = short exposure of immunoblot, LE = long exposure of immunoblot. b) Immunoblot of anti-FLCN IPs and whole cell extracts following exogenous addition of lactate to HEK293s (6 h treatment durations; n=3). c) Pulldown of LDHA from cell lysate expressing EV or FLCN-FLAG using Cibacron blue agarose was evaluated by immunoblot. Bar chart represents the ratio of Pulldown:Input normalized to EV. Unpaired Student’s t-test, (p < 3.3x10-8; n=3). Data shown as mean ± s.d.
Extended Data Fig. 5 FLCN-10 peptide is able to effectively inhibit dimeric but not tetrameric LDHA.
a–d) Immunoprecipitation of transiently expressed short segments of FLCN-FLAG protein implies the critical interacting region with LDHA. e) Ka of LDHA with FLCN-1 peptide (See Fig. 2c), measured by fluorescence polarization anisotropy (n=3). f) Ka plotted against LDHA activity in the presence of peptides. FLCN-1 peptide (blue) and FLCN-10 peptide (red) are highlighted. g) LDHA activity at 10 ng/µl (predominantly dimeric) and 500 ng/µl (predominantly tetrameric) in the presence of increasing amounts of recombinant FLCN protein or FLCN-10 peptide (n=3). Data shown as mean ± s.d. h) Data from Extended Data Fig. 5g presented as percent activity. Unpaired Student’s t-test, n=3. Data shown as mean ± s.d.
Extended Data Fig. 6 FLCN-10 peptide uncompetitively inhibits LDHA.
a) Michaelis-Menten kinetics of LDHA in the presence of FLCN protein, FLCN-10 peptide, or FX11 (n=3). Data shown as mean ± s.d. b) FLCN-10 is an uncompetitive inhibitor of LDHA based on Lineweaver-Burke plot (n=3). c–e) IC50 measurements for LDHA in the presence of FLCN protein, FLCN-10 peptide, or FX-11 (n=3). Data shown as mean ± s.d. f) Summary of LDHA enzyme kinetics. g) LDHA-R106-FLAG mutants were transiently transfected and immunoprecipitated from HEK293 cells. FLCN interaction assessed by immunoblotting. Binding to FLCN-10-Biotin peptide was assessed by Streptavidin pulldown.
Extended Data Fig. 7 inhibitory function of FLCN toward LDHA and its physiological outcome.
a) Western blot confirming transfection of FLCN-WT-FLAG and FLCN-M222A-FLAG or b) FLCN-WT-FLAG and FLCN-F226A-FLAG in UOK257 cells in Fig. 3d. c) Western blot confirming transfection of FLCN-WT-FLAG, FLCN-M222A-FLAG, or FLCN-F226A-FLAG in Fig. 3f. d) Representative images of UOK257 colonies in soft agar from Fig. 3f. Scale bar = 100 µM. e) LDHA activity in UOK257 cells transfected with EV, WT FLCN, FLCN-F118D-FLAG and FLCN-R164A-FLAG. Unpaired Student’s t-test, n=3. Data shown as mean ± s.d. *P < 0.05, **P < 0.005, ***P < 0.001.
Extended Data Fig. 8 FLCN-10 peptide is cell-permeable and inhibits LDHA.
a) Fluorescence microscopy of HEK293 cells treated with 1 µM Rhodamine B-labeled peptides from Fig. 2b. Peptides that are resident after two hours treatment have been denoted with a red number and box. NT = No treatment. Scale bar = 10 µM. b) HEK293 cells treated with peptides from Extended Data Fig. 8a. Bar chart represents fold change of pTyr10-LDHA normalized to NT. Data shown as mean ± s.d. (n=3).
Extended Data Fig. 9 FLCN-10 peptide inhibits LDHA activity and induces cell death in ccRCC cell lines.
a) Caki-1 cells treated for 2 h with FLCN-10 or FLCN-13 were blotted using anti-cleaved caspase-3 and anti-pTyr10-LDHA. b) LDHA activity in HEK293 cells following 2 h treatment with FLCN-10 peptide. c) d) Flow cytometric assessment of cell death in renal cell lines following 1 µM FLCN-10 for 1 or 2 h (Extended Data Fig. 9c) or 0.1 µM or 1 µM FLCN-10 treatment for 2 h (Extended Data Fig. 9d) as determined by Annexin V/Propidium iodide staining. Representative of three independent experiments. e) Immunoblot of whole cell lysates following overexpression of FLAG-tagged FLCN-WT, FLCN-M222A, or FLCN-F226A in normal HEK293 cells or the ccRCC cell lines 786-O, Caki-1 and Caki-2. f) HEK293 cells were treated with and without 1 µM Rhodamine B-labeled FLCN-10A. Scale bar = 20 µM. g) LDHA activity in Caki-1 cells following 2 h treatment with FLCN-10A peptide.
Extended Data Fig. 10 Heterocyclic FLCN-10 peptide induces cell death in ccRCC cells.
a) Structural representation of head-to-tail cyclic-FLCN-10 peptide, created using MAESTRO (Schrodinger.com). b) Flow cytometric assessment of cell death in renal cell lines following 1 µM or 10 µM cyclic-FLCN-10 treatment as determined by propidium iodide staining. Representative of three independent experiments. c) Representative flow cytometry gating strategy. Cells were first stratified by size, followed by staining intensity with Annexin V-FITC and Propidium Iodide (Extended Data Fig. 9c, d). The PI intensity signal was converted into a histogram for presentation in Fig. 5a and Extended Data Fig. 10b.
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Supplementary Table 1.
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Woodford, M.R., Baker-Williams, A.J., Sager, R.A. et al. The tumor suppressor folliculin inhibits lactate dehydrogenase A and regulates the Warburg effect. Nat Struct Mol Biol 28, 662–670 (2021). https://doi.org/10.1038/s41594-021-00633-2
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DOI: https://doi.org/10.1038/s41594-021-00633-2
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