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MAPK signalling-induced phosphorylation and subcellular translocation of PDHE1α promotes tumour immune evasion

Nature Metabolism volume 4, pages 374–388 (2022)Cite this article

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Abstract

Tumour cells utilize multiple strategies to evade the immune system, but the underlying metabolic mechanisms remain poorly understood. The pyruvate dehydrogenase (PDH) complex converts pyruvate to acetyl-coenzyme A in mitochondria, thereby linking glycolysis to the ricarboxylic acid cycle. Here we show that the PDH complex E1 subunit α (PDHE1α) is also located in the cytosol. Cytosolic PDHE1α interacts with IKKβ and protein phosphatase 1B, thereby facilitating the inhibition of the NF-κB pathway. Cytosolic PDHE1α can be phosphorylated at S327 by ERK2 and translocated into mitochondria. Decreased cytosolic PDHE1α levels restore NF-κB signalling, whereas increased mitochondrial PDHE1α levels drive α-ketoglutarate production and promote reactive oxygen species detoxification. Synergistic activation of NF-κB and reactive oxygen species detoxification promotes tumour cell survival and enhances resistance to cytotoxic lymphocytes. Consistently, low levels of PDHE1α phosphorylation are associated with poor prognosis of patients with lung cancer. Our findings show a mechanism through which phosphorylation-dependent subcellular translocation of PDHE1α promotes tumour immune evasion.

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Fig. 1: Cytosolic PDHE1α impairs lung cancer development.
Fig. 2: ERK2 phosphorylates PDHE1α to promote its translocation.
Fig. 3: PDHE1α pS327 promotes tumour cell resistance to the killing of CTLs.
Fig. 4: PDHE1α pS327 activates NF-κB and sustains ROS homeostasis.
Fig. 5: Cytosolic PDHE1α facilitates IKKβ dephosphorylation via PPM1B.
Fig. 6: Clinical relevance of PDHE1α pS327 levels in lung cancer.
Fig. 7: The mechanism of PDHE1α pS327-promoted tumour immune evasion.

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Data availability

Source data are provided with this paper. The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the National Key Research and Development Programme of China (grant no. 2019YFA0802000) to W.Y.; the National Natural Science Foundation of China (grant nos 91857120 and 32025013) to W.Y.; the CAS Project for Young Scientists in Basic Research (grant no. YSBR-014) to W.Y.; the Chinese Academy of Sciences Interdisciplinary Innovation Team (grant no. JCTD-2018-14) to W.Y.; the Programme of Shanghai Academic/Technology Research Leader (grant no. 20XD1424400) to W.Y.; the CAS Facility-based Open Research Programme to W.Y.; the National Natural Science Foundation of China (grant no. 81902807) to Y. Zhang; the National Postdoctoral Programme for Innovative Talents (grant no. BX20190349) to Y. Zhang; the Shanghai Postdoctoral Excellence Programme (grant no. 2018244) to Y. Zhang; the Youth Innovation Promotion Association of the Chinese Academy of Sciences (grant no. 2022265) to Y. Zhang; and the China Postdoctoral Science Foundation (grant no. 2020M671258) to Y. Zhang. We gratefully acknowledge the support of the SA-SIBS Scholarship Programme and the Heye Scholarship Programme. We thank X. Liu for providing OVA-specific cytotoxic CD8 + T cells. We also thank the Cell Analysis Technology Platform and the Chemical Biology Technology Platform of SIBCB for technical support.

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Author notes

  1. These authors contributed equally: Yajuan Zhang, Ming Zhao.

Authors and Affiliations

  1. State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

    Yajuan Zhang, Ming Zhao, Hong Gao, Yun Zhao & Weiwei Yang

  2. Medical Artificial Intelligence Laboratory, Zhejiang Institute of Digital Media, Chinese Academy of Science, Shaoxing, China

    Guanzhen Yu

  3. School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

    Yun Zhao & Weiwei Yang

  4. Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China

    Feng Yao

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Contributions

This study was conceived by W.Y. W.Y. and Y. Zhang designed the study. Y. Zhang performed most of the experiments and also contributed to data analyses and figure editing. M.Z. constructed plasmids, purified proteins and provided experimental assistance. H.G. assisted in reviewing the manuscript. G.Y. and F.Y. provided reagents and pathological assistance. Y. Zhao provided reagents and constructive suggestions. W.Y. wrote the manuscript with comments from all authors.

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Correspondence to Yun Zhao, Feng Yao or Weiwei Yang.

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Nature Metabolism thanks David Barbie, Jiyeon Kim and Kathryn O’Donnell for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina

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Extended data

Extended Data Fig. 1 Cytosolic PDHE1α enhances TNFα-induced apoptosis.

a, H1299 cells stably expressing shNT or shPDHE1α were subcutaneously implanted into BALB/c nude mice. IF analyses were performed with an anti-PDHE1α antibody in dissected tumour tissues. b, H1299 or A549 cells were infected with the lentivirus expressing shNT or shPDHE1α. IF analysis with anti-PDHE1α and anti-COX4 antibodies were performed. c, H1299 or A549 cells were harvested and subcellular fractionation assay was performed. d,e, HEK293T cells were infected with the lentivirus expressing PDHE1α D1-30-Flag. Subcellular fractionation assay (d) and IF staining (e) were performed. f-h, H1299, A549 or HEK293T cells were infected with the lentivirus stably expressing EV or PDHE1α-Flag WT or D1-30. PDHE1α-Flag expression was examined by immunoblotting analyses (f). Cells were treated with or without 50 ng/ml TNFα for 24 h. Cells were harvested and stained with FITC-annexin V, followed by flow cytometry analysis (g). Cell proliferation was also examined (h). i-k, H1299 or A549 cells were infected with the lentivirus stably expressing EV or PDHE1α-Flag D1-30. Cells were treated with or without 50 ng/ml IFNγ for 24 h, followed by apoptosis analysis (i). Cells were treated with or without 50 ng/ml TNFα and 50 ng/ml IFNγ for 24 h, followed by apoptosis analysis (j). Cells were treated with or without 20 μM cisplatin for 24 h, followed by apoptosis analysis (k). Rel, relative. Immunoblots are representative of three independent experiments. (g,h) 1-way ANOVA with Tukey’s multiple comparison test. (i-k) Unpaired, two-tailed t-test.

Source data

Extended Data Fig. 2 Cytosolic PDHE1α impairs lung cancer development.

a-e, LLC cells stably expressing luciferase were infected with the lentivirus stably expressing EV, mPDHE1α-Flag WT or D1-30. mPDHE1α-Flag expression was examined by immunoblotting analyses (a). Subcellular fractionation assay was performed (b). Cells were subcutaneously implanted into C57BL/6J mice (6 mice per group), tumor growth over time was measured (c). Tumor weight was measured at day 11 (d). TUNEL assay of dissected tumors was performed and the percentage of apoptotic tumor cells was shown (e). Data represent the means ± SD of six mice. f-h, Cells in (a) were injected into lung of randomized C57BL/6J mice (6 mice per group). 14 days after inoculation, mice were euthanized. IHC staining of dissected tumors using anti-F4/80, anti-CD8 or anti-TNFα antibody was performed. Representative images (top) and semiquantitative analysis (bottom) of IHC staining were shown. Data represent the means ± SD of six mice. Immunoblots are representative of three independent experiments. (c-h) 1-way ANOVA with Tukey’s multiple comparison test.

Source data

Extended Data Fig. 3 ERK2 phosphorylates PDHE1α to promote its translocation.

a, A549 cells were treated with or without 20 ng/ml EGF for 30 min. Subcellular fractionation assay was performed. b, H1299 cells were treated with or without no glucose DMEM or 200 μM CoCl2 for 24 h. Subcellular fractionation assay was performed. c, H1299 or A549 cells were depleted of endogenous PDHE1α and rescued with rPDHE1α WT, S327A, S327D, S331A or S331D. d, A549 cells were depleted of endogenous PDHE1α and rescued with rPDHE1α WT, S327A, S327D, S331A or S331D. Cells were treated with or without 20 ng/ml EGF for 30 min and subcellular fractionation assay was performed. e, Mass spectrometry analysis of PDHE1α D1-30-associated proteins were performed in H1299 cells. The top 10 protein kinase that showed strong interactions with PDHE1α D1-30 were listed. f, H1299 cells were infected with the lentivirus stably expressing shNT or shERK2. g, H1299 cells were depleted of endogenous PDHE1α and rescued with rPDHE1α-Flag WT or S327A. PDHE1α-Flag proteins were immunoprecipitated using anti-Flag agarose beads. IP, immunoprecipitation. h, H1299 or A549 cells stably expressing PDHE1α-Flag were infected with the lentivirus stably expressing shNT or shERK2. i, A549 cells stably expressing PDHE1α-Flag were infected with the lentivirus stably expressing shNT or shERK2. PDHE1α-Flag proteins were immunoprecipitated using anti-Flag agarose beads. Immunoblots are representative of three independent experiments.

Source data

Extended Data Fig. 4 Oncogenic signaling induces PDHE1α pS327 and promotes its translocation.

a, EGFR WT (HTB182, H1299, H292 and A549) or EGFR mutant/active (PC9, H3255, HCC827 and H1650) lung cancer cells were collected. Cell fraction assay was performed. b, EGFR and KRAS mutation status were shown. c-e, H1299 cells were transfected with EV, KRAS G12V (c), MET-H1094R (d) or ERBB2 G776VC (e) with or without PDHE1α-HA. PDHE1α-HA proteins were immunoprecipitated using anti-HA agarose beads (left panel). Cell fraction assay was performed (right panel). Immunoblots are representative of three independent experiments.

Source data

Extended Data Fig. 5 PDHE1α pS327 promotes tumor cell resistance to CTLs’ killing.

a, A549 cells were depleted of endogenous PDHE1α and rescued with rPDHE1α WT, S327A or S327D. Cells were treated with or without 50 ng/ml TNFα for 24 h. Cells were harvested for apoptosis analysis. b-d, H1299 cells were depleted of endogenous PDHE1α and rescued with rPDHE1α WT, S327A or S327D. Cells were treated with or without 50 ng/ml TNFα and 50 ng/ml IFNγ for 24 h and then cells were harvested for apoptosis analysis (b). Proliferation was examined with these cells (c). Cells were harvested for PDH activity assay (d). e, LLC cells stably expressing ovalbumin (OVA) were depleted of endogenous mPDHE1α and rescued with rmPDHE1α WT, S327A or S327D. f-i, LLC cells stably expressing luciferase were depleted of endogenous mPDHE1α and rescued with rmPDHE1α WT, S327A or S327D. PDHE1α expression was examined by immunoblotting analyses (f). Cells were injected into lung of randomized C57BL/6J mice (6 mice per group). 14 days after inoculation, mice were euthanized. IF analysis with anti-Flag and COX4 antibodies were performed in lung sections from these mice. The levels of cytosolic PDHE1α in tumors were compared (g). IHC staining of dissected tumors using anti-F4/80 or anti-CD8 antibodies was performed. Semiquantitative analysis of IHC staining were shown (h,i). Data represent the means ± SD of six mice. (a-d, g-i) 1-way ANOVA with Tukey’s multiple comparison test.

Source data

Extended Data Fig. 6 Inhibiting PDHE1α pS327 improves the efficacy of ICB therapy.

a, LLC cells stably expressing luciferase were depleted of endogenous mPDHE1α and rescued with rmPDHE1α WT, S327A or S327D. Cells were injected into lung of randomized C57BL/6J mice (6 mice per group). These mice were treated with control IgG2a or anti-PD-1 antibody by intraperitoneal injection. 14 days after inoculation, mice were euthanized. IHC staining of dissected tumors using anti-CD8 antibody were performed. Representative images of IHC staining were presented (left). Semi-quantitative analyses were performed (right). Data represent the means ± SD of six mice. b,c, LLC cells stably expressing luciferase were depleted of endogenous mPDHE1α and rescued with rmPDHE1α WT, S327A or S327D. Cells were subcutaneously implanted into C57BL/6J mice (6 mice per group). These mice were treated with control IgG2a or anti-PD-1 antibody by intraperitoneal injection. Tumor growth over time was measured (b). Tumor weight was measured at day 11 (c). Data represent the means ± SD of six mice. (a-c) Unpaired, two-tailed t-test.

Source data

Extended Data Fig. 7 PDHE1α pS327 activates NF-κB and sustains ROS homeostasis.

a, H1299 cells were infected with the lentivirus expressing EV or PDHE1α D1-30-Flag. Cells were treated with 20 ng/ml TNFα for indicated time. b, A549 cells were depleted of endogenous PDHE1α and rescued with rPDHE1α WT, S327A or S327D. Cells were treated with or without 20 ng/ml TNFα for 10 min. c, DNA sequencing of genomic DNA from H1299 cells with or without PDHE1α S327A knockin. d, HEK293T cells were co-transfected with EV or PDHE1α D1-30-HA and NF-κB reporter. Cells were treated with 10 ng/ml TNFα for 3 h. The relative levels of luciferase activity of Firefly were normalized to the levels of luciferase activity of control Renilla. e, H1299 cells were depleted of endogenous PDHE1α and rescued with rPDHE1α WT or S327A. Cells were then transfected with EV or HA-p65. f,g, H1299 cells stably expressing EV or PDHE1α D1-30-Flag were transfected with EV or HA-p65. Immunoblotting analysis (f) and apoptosis analysis (g) were performed. h, LLC cells stably expressing luciferase were depleted of endogenous mPDHE1α and rescued with rmPDHE1α WT or S327A. Cells were infected with the lentivirus expressing EV or HA-mp65. i, H1299 cells were depleted of endogenous PDHE1α and rescued with rPDHE1α WT or S327A. Cells were treated with or without 50 ng/ml TNFα for 24 h. Cells were harvested and subjected to real-time qPCR. j, H1299 cells were depleted of endogenous PDHE1α and rescued with rPDHE1α WT or S327A. Cells were then transfected with EV or HA-BCL2A1. k, H1299 cells were depleted of endogenous PDHE1α and rescued with rPDHE1α WT, S327A or S327D. Cells were treated with 20 ng/ml TNFα for 1 h and mitochondria ROS was measured using MitoSOX. Immunoblots are representative of three independent experiments. (d,g) Unpaired, two-tailed t-test. (k) 1-way ANOVA with Tukey’s multiple comparison test.

Source data

Extended Data Fig. 8 NF-κB and α-KG are required for PDHE1α pS327-inhibited apoptosis.

a-f, H1299 cells were depleted of endogenous PDHE1α with another shRNA (shPDHE1α-2) and rescued with rPDHE1α WT, S327A or S327D (a). Cells were treated with or without 20 ng/ml TNFα for 10 min and immunoblotting analysis was performed (b). Cells were transfected with EV or HA-p65 and treated with 50 ng/ml TNFα for 24 h. Cell apoptosis was measured (c,d). Cells were harvested for the measurement of α-KG levels (e). Cells were treated with or without 0.5 mM octyl-α-KG and 50 ng/ml TNFα for 24 h. Then cells were harvested for apoptosis analysis (f). (d-f) Unpaired, two-tailed t-test.

Source data

Extended Data Fig. 9 Cytosolic PDHE1α facilitates IKKβ dephosphorylation via PPM1B.

a, H1299 cells were co-transfected with EV or SFB-IKKβ, HA-PPM1B and PDHE1α-Flag. Cells were treated with 10 ng/ml TNFα for 10 min. b, H1299 or A549 cells depleted of endogenous PDHE1α and rescued with rPDHE1α WT or S327A were infected with the lentivirus expressing shNT or shPPM1B. c, H1299 or A549 cells stably expressing EV or PDHE1α D1-30-Flag were infected with the lentivirus expressing shNT or shPPM1B. d, Cells in (c) were treated with 20 ng/ml TNFα for 10 min and immunoblotting analysis was performed. e, H1299 cells depleted of endogenous PDHE1α and rescued with rPDHE1α WT or S327A were infected with the lentivirus expressing shNT or shPPM1B. Cells were treated with 20 ng/ml TNFα for 10 min. Cell fraction assay was performed. f, A549 cells were depleted of endogenous PDHE1α and rescued with rPDHE1α WT or S327A. Cells were then infected with the lentivirus expressing shNT or shPPM1B. Cells were treated with 50 ng/ml TNFα for 24 h. Cells were harvested for apoptosis analysis. g, H1299 or A549 cells stably expressing EV or PDHE1α D1-30-Flag were infected with the lentivirus expressing shNT or shPPM1B. Cells were treated with 50 ng/ml TNFα for 24 h. Cells were harvested for apoptosis analysis. Immunoblots are representative of three independent experiments. (f,g) Unpaired, two-tailed t-test.

Source data

Extended Data Fig. 10 PPM1B is required for cytosolic PDHE1α-inhibited tumor growth.

a-c, LLC cells stably expressing luciferase were infected with the lentivirus expressing EV or mPDHE1α D1-30-Flag and shNT or shmPPM1B (a). Cells were subcutaneously implanted into C57BL/6J mice (6 mice per group), tumor growth over time was measured (b). Tumor weight was measured at day 11 (c). Data represent the means ± SD of six mice. d, H1299 cells were depleted of endogenous PDHE1α and rescued with rPDHE1α WT, S327A or S327D. Cells were co-transfected with EV or SFB-IKKβ and HA-PPM1B. Cells were treated with 10 ng/ml TNFα for 10 min. Cell fraction assay was performed and SFB-IKKβ proteins were pulldown using streptavidin agarose beads. Immunoblots are representative of three independent experiments. (b,c) Unpaired, two-tailed t-test.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Reporting Summary

Supplementary Data 1

PDHE1α-Flag was immunoprecipitated from H1299 cells treated with or without 20 ng ml−1 EGF for 30 min. The S327 and S331 residues in PDHE1α were identified to be phosphorylated after EGF treatment by mass spectrometry analysis.

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Zhang, Y., Zhao, M., Gao, H. et al. MAPK signalling-induced phosphorylation and subcellular translocation of PDHE1α promotes tumour immune evasion. Nat Metab 4, 374–388 (2022). https://doi.org/10.1038/s42255-022-00543-7

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