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Adaptive stimulation of macropinocytosis overcomes aspartate limitation in cancer cells under hypoxia

Nature Metabolism volume 4, pages 724–738 (2022)Cite this article

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Abstract

Stress-adaptive mechanisms enable tumour cells to overcome metabolic constraints under nutrient and oxygen shortage. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumour growth are poorly understood. Here we identify GOT2-catalysed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumour cells under hypoxic culture conditions. In contrast, GOT2-catalysed aspartate synthesis is dispensable for pancreatic tumour formation in vivo. The dependence of pancreatic tumour cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRAS dependent, and is mediated by hypoxia-inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9). Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRAS tumours can overcome nutrient deprivation under hypoxic conditions.

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Fig. 1: The metabolic route for aspartate synthesis in hypoxic pancreatic cancer cells.
Fig. 2: The plasticity of aspartate metabolism in PDAC tumours.
Fig. 3: Macropinocytosis provides sufficient aspartate to enable PDAC cell growth under hypoxic culture conditions.
Fig. 4: Hypoxia upregulates macropinocytosis in mutant KRAS cells in vivo and in vitro.
Fig. 5: HIF1A is necessary and sufficient for hypoxia-dependent upregulation of micropinocytosis.
Fig. 6: The HIF1A target CA9 is required for the upregulation of macropinocytosis in response to hypoxia.
Fig. 7: Loss of CA9 inhibits hypoxic cancer cell growth under aspartate limitation.

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Data availability

All data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank all members of the Bar-Sagi and Birsoy laboratories for helpful suggestions. The KP mouse cancer cell lines are gifts from N. Bardeesy and T. Papagiannakopoulos. We thank the New York University Langone Medical Center Experimental Pathology Research Laboratory, which is partially supported by NIH/National Cancer Institute (NCI) 5370 P30CA16087. This research is supported by an NIH K99/R00 to J.G.-B. (grant no. 1K99CA248838-01/4R00CA248838-02). K.B. was supported by NCI (grant no. DP2 OD024174-01), AACR NextGen grant, Pershing Square Sohn foundation and the Breast Cancer Research Foundation, and is a Searle, Pew-Stewart and Sidney Kimmel Scholar. M.A.B was supported by an NIH T32 (grant no. T32 CA009161). This work was supported by NIH/NCI grant no. CA210263 (D.B.-S).

Author information

Author notes

  1. Javier Garcia-Bermudez

    Present address: Children’s Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

  2. These authors contributed equally: Javier Garcia-Bermudez, Michael A. Badgley, Sheela Prasad.

Authors and Affiliations

  1. Laboratory of Metabolic Regulation and Genetics, The Rockefeller University, New York, NY, USA

    Javier Garcia-Bermudez, Lou Baudrier, Yuyang Liu, Konnor La, Mariluz Soula, Robert T. Williams & Kıvanç Birsoy

  2. Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY, USA

    Michael A. Badgley, Sheela Prasad, Laura J. Taylor & Dafna Bar-Sagi

  3. Laboratory of Systems Cancer Biology, The Rockefeller University, New York, NY, USA

    Norihiro Yamaguchi

  4. Department of Breast Surgical Oncology, Division of Surgery, MD Anderson Cancer Center, University of Texas, Houston, TX, USA

    Rosa F. Hwang

  5. Antitumor Assessment Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Elisa de Stanchina

  6. The Proteomics Resource Center, The Rockefeller University, New York, NY, USA

    Bety Rostandy, Hanan Alwaseem & Henrik Molina

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Contributions

D.B.-S., M.A.B., S.P., K.B. and J.G.-B. conceived the project and designed the experiments. J.G.-B. and S.P. performed most of the experiments with assistance of M.A.B., L.B., Y.L. and M.S. Macropinocytosis experiments and tumour immunohistochemistry were done by M.A.B. and S.P. K.L. performed computational analysis. H.A., B.R. and H.M. performed analysis of metabolite profiling experiments. R.T.W. performed real-time PCR experiments. R.F.H. provided hPSCs. E.S. provided PDAC PDXs and N.Y. performed experiments with them. D.B.-S., M.A.B., K.B. and J.G.-B wrote and edited the manuscript with help from L.J.T.

Corresponding authors

Correspondence to Dafna Bar-Sagi or Kıvanç Birsoy.

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Competing interests

K.B. is scientific adviser to Nanocare Pharmaceuticals and a consultant to Barer Institute. The remaining authors declare no competing interests.

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Nature Metabolism thanks Marcia Haigis, Nabeel Bardeesy and Nicholas Denko for their contribution to the peer review of this work. Primary handling editors: Alfredo Giménez-Cassina and George Caputa in collaboration with the Nature Metabolism team

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Extended data

Extended Data Fig. 1 Hypoxia and ETC inhibition trigger similar metabolic signatures in PDAC cells.

(a) Individual sgRNA scores for GOT2 under indicated conditions. (b) Heat map showing fold changes (log2) of polar metabolites significantly changed when comparing normoxic to hypoxic culture conditions. (c) Immunoblot analysis of GOT2. ACTB is a loading control. (d) Fold change in cell number (log2) of MIA PaCa-2 GOT2KO cell line expressing indicated constructs grown under indicated conditions for 5 days. (e) Common metabolite changes across three PDAC cell lines upon ETC inhibition. (f) Principal component analysis (PCA) of metabolic changes in indicated PDAC cell lines under indicated conditions. (g) Ranks of metabolites most significantly changed (p value < 0.01, dotted lines) across 3 PDAC cell lines under indicated conditions. Metabolites significantly accumulated (blue) or depleted (pink) under both conditions are in the lower left quadrant (right). (h) Top scoring genes and their differential gene scores in HY15549 cells treated with piericidin (Pier.) compared to untreated cells. (i) Individual sgRNA scores for GOT1 in HY15549 cells in indicated conditions. (j) Immunoblot analysis of GOT1 in indicated cell lines expressing indicated sgRNAs. ACTB is a loading control. (k) Fold change in cell number (log2) of indicated HY15549 cell lines expressing indicated constructs for 5 days in the presence or absence of Pier., 20 nM. (l) Fold change in cell number (log2) of indicated cell lines expressing indicated cDNAs grown for 5 days in the presence or absence of Pier., 20 nM. (m) Relative fold change in cell number of indicated parental PDAC cell lines transduced with indicated cDNAs grown for 5 days under indicated conditions; Pier., 20-100. (n) Fold change in cell number (log2) of indicated HY15549 cell lines transduced with indicated vectors grown for 5 days under indicated conditions; Pier. 50 nM. a, i, bars represent individual sgRNA scores; d, k, l, m, n, Bars represent mean ± s.d. d, k, l, m, n, n = 3 biologically independent samples. Statistical significance was determined by two-tailed unpaired t-test.

Source data

Extended Data Fig. 2 Mitochondrial NAD+ is limiting for PDAC cell proliferation under hypoxia.

(a) Immunoblot analysis of FLAG-tagged cytoplasmic (cyto-) and mitochondrial (mito-) LbNOX heterologous expression in the indicated cell lines. ACTB was used as a loading control. (b) Fraction of labelled aspartate (left) and citrate (right) derived from labelled glutamine in control and cyto- or mito-LbNOX expressing HY15549 cells cultured for 8 h with [U-13C]-Glutamine (500 μM) with piericidin treatment (50 nM) or under 0.5% oxygen. Colours indicate mass isotopomers. (c) Expression of LbNOX enzymes rescues proliferation of MIA PaCa-2 cells under piericidin treatment (50 nM), whereas only mito-LbNOX expression rescues proliferation under 0.5% O2. Data is shown as fold change in cell number in the indicated cell lines grown for 5 days relative to untreated cells cultured at 21% O2. (d) Fraction of 13C-labeled aspartate (m + 4) and malate (m + 4) in HY15549 parental and GOT2-knockout cells transduced with a control vector or an sgRNA-resistant GOT2 cDNA cultured for 24 hours under hypoxia in the presence of [U-13C]-L-glutamine (1 mM). (e) Relative fold change in cell number of HY15549 GOT2-knockout cells transduced with a control vector or the indicated cDNAs grown for 5 days under hypoxia (0.5% O2) to those cultured under normoxia (21% O2). b-e, Bars represent mean ± s.d. b-e, n = 3 biologically independent samples. Statistical significance was determined by two-tailed unpaired t-test.

Source data

Extended Data Fig. 3 Aspartate synthesis by GOTs is essential in culture but redundant for in vivo tumor growth.

(a) Immunoblot analysis of indicated proteins and cell lines with vinculin loading control. (b) Weights of subcutaneous tumor xenografts from MIA PaCa-2 cells transduced with indicated vectors. (c) In vivo sgRNA competition assay scheme (left). Gene scores for indicated conditions (right). Each dot represents one sgRNA (n = 5 tumours). (d) Gene scores for in vivo sgRNA competition assay performed in KRAS-mutant PDAC patient-derived xenografts (PDX). Each dot represents an sgRNA (n = 5 subcutaneous tumors). (e, f) Fold change cell number of GOT2-KO HY15549 cells transduced with indicated vectors grown 5 days under indicated conditions. Pyruvate (Pyr). (g) Fold change in cell number of HY15549 cells grown 5 days in regular or conditioned media from hPSCs (hPSC CM) with or without monocarboxylate transporter inhibitor AR-C155858 (5 μM). (h) Immunoblot analysis of indicated cells under indicated conditions with vinculin as loading control. (i). Representative bright-field micrographs of indicated cells grown 6 days in the indicated media. Scale bar = 50 μm (left). Quantification graph (right); aspartate (20 mM). (j) Weights, images, and immunoblot analysis of indicated tumors with vinculin loading control (right). (k) Relative aspartate abundance normalized to lysine levels of established xenografts from indicated cells transduced with indicated vectors. (l) Immunoblot of SLC1A3 in indicated cell lines with b-actin loading control. (m) SLC1A2 and SLC1A3 mRNA expression data from the Cancer Cell Line Encyclopedia (CCLE) (log transformed) in KP PDAC cell lines and a glioma cell line (KNS-42). (n) Relative abundance of indicated metabolites in the culture media of indicated MIA PaCa-2 cells grown under indicated conditions for 24 hours in the presence of pyruvate (100 μM). c, d, e, f, g, i, k, n Bars represent mean ± s.d. b, j, boxes represent the median, first, and third quartiles; whiskers represent the minima and maxima. c, d, e, f, g, i, k, n, n = 3; b, n = 10; j, n = 5; k, n = 4. All replicates are biologically independent. Statistical significance determined by two-tailed unpaired t-test.

Source data

Extended Data Fig. 4 Macropinocytosis enables proliferation of KRAS-mutant cells under conditions where aspartate is limited.

(a) Fold change in cell number (log2) of HeLa (left) or BxPC-3 tet-KRASV12 (right) cells cultured under indicated concentrations of oxygen for 5-6 days in the presence or absence of 1% BSA. BxPC-3 tet-KRASV12 cells were cultured in the presence or absence of doxycycline (0.1μg/mL) to activate KRASV12 expression. (b) Immunoblot analysis of several members of the ETC complex in parental MIA PaCa-2 cells or Rho(0) counterparts. ACTB was used as a loading control. (c) Representative bright-field micrographs of HY15549 cells cultured for 5 days in media with or without 2% BSA. Where indicated, cells were treated with bafilomycin A1 (BafA1, 10 nM), ferric ammonium citrate (FAC, 0.1 μg/mL) and complex III inhibitor antimycin A (Anti. A, 100 nM). Scale bar = 50 μm. (d) Immunoblot analysis of GOT1 and GOT2 in GOT1/2- double knockout BxPC-3 tet-KRASV12 cells compared to parental controls. ACTB was used as a loading control. (e) Fold change in cell number (log2) of GOT1/2- double knockout BxPC-3 tet-KRASV12 cells cultured in the indicated media conditions for 7 days in the presence or absence of doxycycline (0.1 μg/mL) (top). Representative bright-field micrographs of GOT1/2- double knockout BxPC-3 tet-KRASV12 cells in media supplemented with 1% BSA in the presence and absence of doxycycline (0.1 μg/mL) (bottom). a, e, Bars represent mean ± s.d. a, e, n = 3 biologically independent samples. Statistical significance was determined by two-tailed unpaired t-test.

Source data

Extended Data Fig. 5 Hypoxia-induced macropinocytosis depends on oncogenic KRAS in PDACs.

(a) Immunoblot analysis of FLAG and T7, showing inducible expression of stably transduced FLAG-tagged KRASG12V BxPC-3 cells and T7-tagged KRASG12V HeLa cells after addition of doxycycline (1 μg/mL for 2 days). ACTB or vinculin were used as a loading control. (b-e) Representative images (b, d) and quantification (c, e) of DQ-BSA fluorescence (green) in PANC-1 (b, c) and MIA PaCa-2 (d, e) cells under normoxia (21% O2) and hypoxia (0.5% O2). Nuclei are labeled with DAPI (blue). Scale bar, 10 μm. In c, e, data are presented relative to normoxia. c, e, Bars represent mean ± s.e.m. At least 500 (c, d) cells were quantified in each biological replicate (n = 3). Statistical significance was determined by two-tailed unpaired t-test.

Source data

Extended Data Fig. 6 HIF1A stabilization stimulates macropinocytosis in PDAC cells and tumors.

(a) Immunoblot analysis indicated proteins in the indicated sgRNA-transduced PANC-1 cells treated with FG-4592 as shown. Vinculin is a loading control. (b, c) Representative images (b) and quantification (c) of TMR-dextran (red) uptake in control or sgHIF1A- or sgARNT-transduced PANC-1 cells cultured under indicated conditions. Nuclei are blue. Scale bar, 10 μm. In c, data are values normalized to control normoxic cells. (d) Relative mRNA levels of the HIF1A-target lactate dehydrogenase A (LDHA) in the indicated MIA PaCa-2 cell lines expressing the indicated vectors and treated with the prolyl-hydroxylase (PHD)-inhibitor FG-4592 (100 μM, 72 hrs) as shown. (e, f) Representative images (e) and quantification (f) of TMR-dextran (red) uptake in PANC-1 cells expressing indicated constructs cultured under indicated conditions. Nuclei are blue. Scale bar, 10 μm. In f, data are normalized to values for control normoxic cells. (g) Immunoblot analysis of HIF1A in MIA PaCa-2 and BxPC-3 cells treated with 0, 100, and 500 μM of the prolyl-hydroxylase inhibitor DMOG under normoxia (21% O2). Tubulin was used as a loading control. (h) Representative images of TMR-dextran (red) uptake in MIA PaCa-2 cells in the absence or presence of DMOG (500 μM) under normoxia (21% O2). Nuclei are labeled with DAPI (blue). Scale bar, 10 μm. (i) Quantification of macropinocytic uptake in MIA PaCa-2, PANC-1 and BxPC-3 cells treated in the absence or presence of DMOG (500 μM) under normoxia (21% O2). Data are presented relative to values obtained for untreated cells. (j) Representative images of TMR-dextran (red) uptake from sections of xenograft tumors arising from MIA PaCa-2 cells transduced with sgRNAs targeting HIF1A or ARNT with tumor cells immunostained with anti-CK8 (green), and pimonidazole detected with anti-pimonidazole (purple). Nuclei are labeled with DAPI (blue). Scale bar = 50 μm, inset = 10 μm c, d, f, i, Bars represent mean ± s.e.m. d, n = 3 independent, biological replicates. At least 500 (c, f, i) cells were quantified in each biological replicate (n = 3). Statistical significance for all experiments was determined by two-tailed unpaired t-test.

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Extended Data Fig. 7 Bicarbonate generation by CA9 mediates the effect of HIF1A on PDAC macropinocytosis under hypoxia.

(a) Representative images of CA9 (red) from sections of indicated tumor xenografts with +CK8 tumor cells (green), and hypoxic areas (purple). Nuclei are blue. Scale bar = 10 uM. (b) Immunofluorescence of CA9 relative to CK8 in sections of MIA PaCa-2 xenograft tumors. Data are normalized to values from CK8 + /pimo- areas. (c) Immunoblot analysis of CA9 in MIA PaCa-2 cells grown under hypoxia (0.5% O2) for the indicated times (hours). (d) Immunoblot analysis of indicated proteins in the indicated cell lines expressing indicated constructs. (e) Immunoblot analysis of CA9 in the indicated cell lines transduced with the indicated sgRNAs. (f, g) Representative images (f) and quantification (g) of TMR-dextran (red) uptake in PANC-1 cells transduced with indicated constructs and cultured under the indicated conditions. Nuclei are blue. Scale bar, 10 μm. In g, data are normalized to values from normoxic control cells. (h) Immunoblot analysis of CA9 in indicated cells transduced with indicated vectors. (i) Representative images of TMR-dextran (red) uptake in indicated PANC-1 cells cultured under indicated conditions. Nuclei are blue. Scale bar = 10uM. (j) Quantification of TMR-dextran uptake in indicated PANC-1 cells under indicated conditions. Data are normalized to values from control cells. (k) Quantification of TMR-dextran uptake in the indicated cell lines transfected with indicated constructs and cultured under indicated conditions. Data are normalized to values from control normoxic cells. (l) Quantification of TMR-dextran uptake in MIA PaCa-2 cells treated with soluble adenylate cyclase inhibitor KH7 under indicated conditions. Data normalized to values obtained from control normoxic cells. (m) Quantification of TMR-dextran uptake in MIA PaCa-2 cells treated with the PKA inhibitor H89 (15 μM) as shown under indicated conditions. Data are normalized to values obtained for control normoxic cells. b, g, j, k, l, m, Bars represent mean ± s.e.m. At least 300 (b) and 500 (g, j, k, l, m) cells were quantified in each biological replicate (n = 3). Vinculin is a loading control in all immunoblots shown. Statistical significance was determined by two-tailed unpaired t-test.

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Supplementary information

Reporting Summary

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Oligos and DNA sequences.

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Reagents/antibody information.

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Garcia-Bermudez, J., Badgley, M.A., Prasad, S. et al. Adaptive stimulation of macropinocytosis overcomes aspartate limitation in cancer cells under hypoxia. Nat Metab 4, 724–738 (2022). https://doi.org/10.1038/s42255-022-00583-z

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