Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Carnosine regulation of intracellular pH homeostasis promotes lysosome-dependent tumor immunoevasion

Nature Immunology volume 25, pages 483–495 (2024)Cite this article

Subjects

Abstract

Tumor cells and surrounding immune cells undergo metabolic reprogramming, leading to an acidic tumor microenvironment. However, it is unclear how tumor cells adapt to this acidic stress during tumor progression. Here we show that carnosine, a mobile buffering metabolite that accumulates under hypoxia in tumor cells, regulates intracellular pH homeostasis and drives lysosome-dependent tumor immune evasion. A previously unrecognized isoform of carnosine synthase, CARNS2, promotes carnosine synthesis under hypoxia. Carnosine maintains intracellular pH (pHi) homeostasis by functioning as a mobile proton carrier to accelerate cytosolic H+ mobility and release, which in turn controls lysosomal subcellular distribution, acidification and activity. Furthermore, by maintaining lysosomal activity, carnosine facilitates nuclear transcription factor X-box binding 1 (NFX1) degradation, triggering galectin-9 and T-cell-mediated immune escape and tumorigenesis. These findings indicate an unconventional mechanism for pHi regulation in cancer cells and demonstrate how lysosome contributes to immune evasion, thus providing a basis for development of combined therapeutic strategies against hepatocellular carcinoma that exploit disrupted pHi homeostasis with immune checkpoint blockade.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Carnosine facilitates cancer cell maintenance of pHi homeostasis under hypoxia.
Fig. 2: Alternative splicing by hnRNPA2B1/MBNL1 promotes the production of CARNS2 isoform in hypoxic HCC cells.
Fig. 3: Carnosine controls the expression of galectin-9 by modulating lysosome-dependent degradation of NFX1.
Fig. 4: The CARNS2/NFX1/galectin-9 axis suppresses CD8+ T-cell-mediated anti-tumor immunity.
Fig. 5: Aberrant activation of CARNS2/NFX1/galectin-9 signaling is predictive of poor clinical prognosis.
Fig. 6: Carnosine-mediated pHi homeostasis promotes lysosome-dependent tumor immune evasion.

Similar content being viewed by others

Data availability

RNA-seq data have been deposited in the Gene Expression Omnibus under accession code GSE198881. Proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE68 partner repository with the dataset identifier PXD047036. All other data are available in the article and Supplementary files. Source data are provided. Source data are provided with this paper.

References

  1. Lyssiotis, C. A. & Kimmelman, A. C. Metabolic interactions in the tumor microenvironment. Trends Cell Biol. 27, 863–875 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Vander Heiden, M. G., Cantley, L. C. & Thompson, C. B. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029–1033 (2009).

    Article  ADS  Google Scholar 

  3. Xu, K. et al. Glycolysis fuels phosphoinositide 3-kinase signaling to bolster T cell immunity. Science 371, 405–410 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. Faubert, B., Solmonson, A. & DeBerardinis, R. J. Metabolic reprogramming and cancer progression. Science 368, eaaw5473 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Webb, B. A., Chimenti, M., Jacobson, M. P. & Barber, D. L. Dysregulated pH: a perfect storm for cancer progression. Nat. Rev. Cancer 11, 671–677 (2011).

    Article  CAS  PubMed  Google Scholar 

  6. Casey, J. R., Grinstein, S. & Orlowski, J. Sensors and regulators of intracellular pH. Nat. Rev. Mol. Cell Biol. 11, 50–61 (2010).

    Article  CAS  PubMed  Google Scholar 

  7. Parks, S. K., Chiche, J. & Pouyssegur, J. Disrupting proton dynamics and energy metabolism for cancer therapy. Nat. Rev. Cancer 13, 611–623 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. Bertholet, A. M. et al. H+ transport is an integral function of the mitochondrial ADP/ATP carrier. Nature 571, 515–520 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Liu, B. et al. STAT3 associates with vacuolar H+-ATPase and regulates cytosolic and lysosomal pH. Cell Res. 28, 996–1012 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Galenkamp, K. M. O. et al. Golgi acidification by NHE7 regulates cytosolic pH homeostasis in pancreatic cancer cells. Cancer Discov. 10, 822–835 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Schönichen, A., Webb, B. A., Jacobson, M. P. & Barber, D. L. Considering protonation as a posttranslational modification regulating protein structure and function. Annu. Rev. Biophys. 42, 289–314 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Vaughan-Jones, R. D., Peercy, B. E., Keener, J. P. & Spitzer, K. W. Intrinsic H+ ion mobility in the rabbit ventricular myocyte. J. Physiol. 541, 139–158 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hwang, J. Y. et al. Dual sensing of physiologic pH and calcium by EFCAB9 regulates sperm motility. Cell 177, 1480–1494 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Oginuma, M. et al. Intracellular pH controls WNT downstream of glycolysis in amniote embryos. Nature 584, 98–101 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. Reddy, A. et al. pH-Gated succinate secretion regulates muscle remodeling in response to exercise. Cell 183, 62–75 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Walton, Z. E. et al. Acid suspends the circadian clock in hypoxia through inhibition of mTOR. Cell 174, 72–87 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bohn, T. et al. Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages. Nat. Immunol. 19, 1319–1329 (2018).

    Article  CAS  PubMed  Google Scholar 

  18. Johnston, R. J. et al. VISTA is an acidic pH-selective ligand for PSGL-1. Nature 574, 565–570 (2019).

    Article  ADS  CAS  PubMed  Google Scholar 

  19. Boedtkjer, E. & Pedersen, S. F. The acidic tumor microenvironment as a driver of cancer. Annu. Rev. Physiol. 82, 103–126 (2020).

    Article  CAS  PubMed  Google Scholar 

  20. Corbet, C. & Feron, O. Tumour acidosis: from the passenger to the driver’s seat. Nat. Rev. Cancer 17, 577–593 (2017).

    Article  CAS  PubMed  Google Scholar 

  21. Savini, M., Zhao, Q. & Wang, M. C. Lysosomes: signaling hubs for metabolic sensing and longevity. Trends cell Biol. 29, 876–887 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cui, Y. et al. A COPII subunit acts with an autophagy receptor to target endoplasmic reticulum for degradation. Science 365, 53–60 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wyant, G. A. et al. NUFIP1 is a ribosome receptor for starvation-induced ribophagy. Science 360, 751–758 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yamamoto, K. et al. Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I. Nature 581, 100–105 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. Liu, X. et al. Inhibition of PCSK9 potentiates immune checkpoint therapy for cancer. Nature 588, 693–698 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Burr, M. L. et al. CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. Nature 549, 101–105 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Wang, H. et al. HIP1R targets PD-L1 to lysosomal degradation to alter T cell-mediated cytotoxicity. Nat. Chem. Biol. 15, 42–50 (2019).

    Article  CAS  PubMed  Google Scholar 

  28. Boldyrev, A. A., Aldini, G. & Derave, W. Physiology and pathophysiology of carnosine. Physiol. Rev. 93, 1803–1845 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Mahootchi, E. et al. GADL1 is a multifunctional decarboxylase with tissue-specific roles in β-alanine and carnosine production. Sci. Adv. 6, eabb3713 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sale, C., Saunders, B. & Harris, R. C. Effect of beta-alanine supplementation on muscle carnosine concentrations and exercise performance. Amino Acids 39, 321–333 (2010).

    Article  CAS  PubMed  Google Scholar 

  31. Everaert, I., De Naeyer, H., Taes, Y. & Derave, W. Gene expression of carnosine-related enzymes and transporters in skeletal muscle. Eur. J. Appl. Physiol. 113, 1169–1179 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Wang, N. et al. Structural basis of human monocarboxylate transporter 1 inhibition by anti-cancer drug candidates. Cell 184, 370–383 (2021).

    Article  CAS  PubMed  Google Scholar 

  33. Nakamura, N. et al. Endosomes are specialized platforms for bacterial sensing and NOD2 signalling. Nature 509, 240–244 (2014).

    Article  ADS  CAS  PubMed  Google Scholar 

  34. Saftig, P. & Klumperman, J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat. Rev. Mol. Cell Biol. 10, 623–635 (2009).

    Article  CAS  PubMed  Google Scholar 

  35. Smith, D. E., Clémençon, B. & Hediger, M. A. Proton-coupled oligopeptide transporter family SLC15: physiological, pharmacological and pathological implications. Mol. Asp. Med. 34, 323–336 (2013).

    Article  CAS  Google Scholar 

  36. Derave, W., Everaert, I., Beeckman, S. & Baguet, A. Muscle carnosine metabolism and beta-alanine supplementation in relation to exercise and training. Sports Med. 40, 247–263 (2010).

    Article  PubMed  Google Scholar 

  37. Drozak, J., Veiga-da-Cunha, M., Vertommen, D., Stroobant, V. & Van Schaftingen, E. Molecular identification of carnosine synthase as ATP-grasp domain-containing protein 1 (ATPGD1). J. Biol. Chem. 285, 9346–9356 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Shen, S. et al. A miR-130a-YAP positive feedback loop promotes organ size and tumorigenesis. Cell Res. 25, 997–1012 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Heuser, J. Changes in lysosome shape and distribution correlated with changes in cytoplasmic pH. J. Cell Biol. 108, 855–864 (1989).

    Article  CAS  PubMed  Google Scholar 

  40. Johnson, D. E., Ostrowski, P., Jaumouillé, V. & Grinstein, S. The position of lysosomes within the cell determines their luminal pH. J. Cell Biol. 212, 677–692 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Behrends, C., Sowa, M. E., Gygi, S. P. & Harper, J. W. Network organization of the human autophagy system. Nature 466, 68–76 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lamb, C. A., Yoshimori, T. & Tooze, S. A. The autophagosome: origins unknown, biogenesis complex. Nat. Rev. Mol. Cell Biol. 14, 759–774 (2013).

    Article  CAS  PubMed  Google Scholar 

  43. Stolz, A., Ernst, A. & Dikic, I. Cargo recognition and trafficking in selective autophagy. Nat. Cell Biol. 16, 495–501 (2014).

    Article  CAS  PubMed  Google Scholar 

  44. Xia, H., Green, D. R. & Zou, W. Autophagy in tumour immunity and therapy. Nat. Rev. Cancer 21, 281–297 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Song, Z., Krishna, S., Thanos, D., Strominger, J. L. & Ono, S. J. A novel cysteine-rich sequence-specific DNA-binding protein interacts with the conserved X-box motif of the human major histocompatibility complex class II genes via a repeated Cys-His domain and functions as a transcriptional repressor. J. Exp. Med. 180, 1763–1774 (1994).

    Article  CAS  PubMed  Google Scholar 

  46. Wolf, Y., Anderson, A. C. & Kuchroo, V. K. TIM3 comes of age as an inhibitory receptor. Nat. Rev. Immunol. 20, 173–185 (2020).

    Article  CAS  PubMed  Google Scholar 

  47. Clayton, K. L. et al. T cell Ig and mucin domain-containing protein 3 is recruited to the immune synapse, disrupts stable synapse formation, and associates with receptor phosphatases. J. Immunol. 192, 782–791 (2014).

    Article  CAS  PubMed  Google Scholar 

  48. Johmura, Y. et al. Senolysis by glutaminolysis inhibition ameliorates various age-associated disorders. Science 371, 265–270 (2021).

    Article  ADS  CAS  PubMed  Google Scholar 

  49. Lu, P. et al. L-glutamine provides acid resistance for Escherichia coli through enzymatic release of ammonia. Cell Res. 23, 635–644 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  50. Sale, C. et al. Carnosine: from exercise performance to health. Amino Acids 44, 1477–1491 (2013).

    Article  CAS  PubMed  Google Scholar 

  51. Black, M. I. et al. The effects of β-alanine supplementation on muscle pH and the power-duration relationship during high-intensity exercise. Front. Physiol. 9, 111 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Hawley, J. A., Hargreaves, M., Joyner, M. J. & Zierath, J. R. Integrative biology of exercise. Cell 159, 738–749 (2014).

    Article  CAS  PubMed  Google Scholar 

  53. Kobayashi, T. et al. The histidine transporter SLC15A4 coordinates mTOR-dependent inflammatory responses and pathogenic antibody production. Immunity 41, 375–388 (2014).

    Article  CAS  PubMed  Google Scholar 

  54. Deng, J. et al. ULK1 inhibition overcomes compromised antigen presentation and restores antitumor immunity in LKB1 mutant lung cancer. Nat. Cancer 2, 503–514 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Lawson, K. A. et al. Functional genomic landscape of cancer-intrinsic evasion of killing by T cells. Nature 586, 120–126 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  56. Cheung, P. F. et al. Progranulin mediates immune evasion of pancreatic ductal adenocarcinoma through regulation of MHCI expression. Nat. Commun. 13, 156 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  57. Noman, M. Z. et al. Inhibition of Vps34 reprograms cold into hot inflamed tumors and improves anti-PD-1/PD-L1 immunotherapy. Sci. Adv. 6, eaax7881 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  58. Gupta, S. et al. Lysosomal retargeting of Myoferlin mitigates membrane stress to enable pancreatic cancer growth. Nat. Cell Biol. 23, 232–242 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Perera, R. M. et al. Transcriptional control of autophagy-lysosome function drives pancreatic cancer metabolism. Nature 524, 361–365 (2015).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  60. Wang, Y. et al. CLN7 is an organellar chloride channel regulating lysosomal function. Sci. Adv. 7, eabj9608 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  61. Swietach, P. et al. Hydrogen ion dynamics in human red blood cells. J. Physiol. 588, 4995–5014 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Long, C. P. & Antoniewicz, M. R. High-resolution (13)C metabolic flux analysis. Nat. Protoc. 14, 2856–2877 (2019).

    Article  CAS  PubMed  Google Scholar 

  63. Wiśniewski, J. R., Vildhede, A., Norén, A. & Artursson, P. In-depth quantitative analysis and comparison of the human hepatocyte and hepatoma cell line HepG2 proteomes. J. Proteom. 136, 234–247 (2016).

    Article  Google Scholar 

  64. Wang, T. et al. Secreted protease PRSS35 suppresses hepatocellular carcinoma by disabling CXCL2-mediated neutrophil extracellular traps. Nat. Commun. 14, 1513 (2023).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  65. Hu, M. et al. Parkinson’s disease-risk protein TMEM175 is a proton-activated proton channel in lysosomes. Cell 185, 2292–2308 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Li, J. et al. Co-inhibitory molecule B7 superfamily member 1 expressed by tumor-infiltrating myeloid cells induces dysfunction of anti-tumor CD8+ T cells. Immunity 48, 773–786 (2018).

    Article  CAS  PubMed  Google Scholar 

  67. Zhang, T. et al. ENO1 suppresses cancer cell ferroptosis by degrading the mRNA of iron regulatory protein 1. Nat. Cancer 3, 75–89 (2022).

    Article  CAS  PubMed  Google Scholar 

  68. Perez-Riverol, Y. et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 50, D543–D552 (2022).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work is supported in part by the National Natural Science Foundation of China (81930083, 82130087, 82192893, 81821001, 82341013, 91957203 and 82303217), the National Key R&D Program of China (2022YFA1304504), the Chinese Academy of Sciences (XDB39000000), the Global Select Project (DJK-LX-2022001) of the Institute of Health and Medicine and Hefei Comprehensive National Science Center and the Fundamental Research Funds for the Central Universities (WK9100000051). Please address all correspondence and requests for materials to P.G. (pgao2@ustc.edu.cn).

Author information

Author notes

  1. These authors contributed equally: Ronghui Yan, Pinggen Zhang.

Authors and Affiliations

  1. Anhui Key Laboratory of Hepatopancreatobiliary Surgery, Department of General Surgery, Anhui Provincial Hospital, the First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, China

    Ronghui Yan, Pinggen Zhang, Ting Wang, Zhaolin Chen, Wenhao Ma, Junru Feng, Zetan Jiang, Weidong Jia & Huafeng Zhang

  2. The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, China

    Ronghui Yan, Pinggen Zhang, Yu Zeng, Ting Wang, Wenhao Ma, Junru Feng, Zetan Jiang, Chunlei Cang & Huafeng Zhang

  3. Anhui Province Key Laboratory of Biomedical Aging Research, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, China

    Pinggen Zhang & Huafeng Zhang

  4. Insitute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China

    Pinggen Zhang & Huafeng Zhang

  5. Medical Research Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Southern Medical University, Guangzhou, China

    Shengqi Shen, Caixia Suo, Tong Zhang, Haoran Wei, Rui Chen, Shi-ting Li, Xiuying Zhong, Linchong Sun & Ping Gao

Authors
  1. Ronghui Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pinggen Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shengqi Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yu Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ting Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhaolin Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wenhao Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Junru Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Caixia Suo
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Tong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Haoran Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Zetan Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Rui Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Shi-ting Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Xiuying Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Weidong Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Linchong Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Chunlei Cang
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Huafeng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Ping Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.G. and H.Z. conceived the study and supervised the experiments. R.Y., P.Z., P.G. and H.Z. designed the experiments. R.Y., P.Z., Y.Z., T.W., W.M., C.S., T.Z., H.W., S.L., Z.J. and R.C. performed experiments. S.S. and J.F analyzed RNA-seq data. W.J. and Z.C. provided clinical specimens. C.C., L.S. and X.Z. provided constructive guidance and advice. P.G., H.Z., R.Y. and P.Z. wrote the paper. All the authors read and approved the manuscript.

Corresponding authors

Correspondence to Huafeng Zhang or Ping Gao.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. N. Bernard was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Carnosine promotes cancer cell to maintain intracellular pH homeostasis under hypoxia.

a, The extracellular acidification rate (ECAR) was measured from HepG2 cells with hypoxia and normoxia treatment for 48 h (n = 3 biological replicates). b, List of intracellular mobile buffering power. c, Heat map analysis showing the levels of mobile buffers in PLC cells cultured under hypoxia for 48 h as compared with normoxia (n = 3 samples). d, Carnosine levels were measured in paired YAP-5SA induced HCC tumor and para of mice (n = 3 samples). e, Carnosine levels were measured in HepG2, PLC and Hepa 1-6 cells. Cells were cultured under normoxia or hypoxia for 48 h (n = 3 biological replicates). f, Schematic of the carnosine metabolic pathway (left). CARNS mRNA expression was measured in HepG2 cells expressing NTC and shCARNS with hypoxia and normoxia treatment for 36 h (n = 3 biological replicates) (right). g, pHi was detected in HepG2 cells expressing NTC or shCARNS. Cells were cultured under normoxia or hypoxia for 48 h in the presence of vehicle or 5 mM carnosine (n = 3 biological replicates). h, Transporters of carnosine in cells (upper). A model for H+-coupled release of the protonated form of carnosine (lower). i, Carnosine levels in the culture medium (left), endosome/lysosome (right) were respectively detected in HepG2 cells expressing NTC, shSLC15A1/A2 or shSLC15A3/A4 with normoxia or hypoxia treatment for 48 h (n = 3 biological replicates). shA1/A2: shSLC15A1 and shSLC15A2. shA3/A4: shSLC15A3 and shSLC15A4. j, Immunoblotting analysis confirmed knockdown of SLC15A1, SLC15A2, SLC15A3 and SLC15A4. k, pHi was detected in HepG2 cells expressing NTC, shCARNS, shCARNS plus shSLC15A1/2 (left) or shSLC15A3/4 (right). Cells were cultured under normoxia or hypoxia for 48 h, and treated with or without 1.5 mM carnosine (n = 3 biological replicates). Data presented as mean ± s.e.m (a) or s.d. (d-g,l,k). Statistical significance was determined by two-tailed unpaired Student’s t-test (a,d-g,I,k).

Source data

Extended Data Fig. 2 Hypoxia promotes the expression of CARNS2, which can facilitate tumor progression.

a, Immunoblotting analysis of different isoforms of CARNS in mouse tissues (left). Western blot analysis of different isoforms of CARNS in human brain, liver and breast tissues. Adjacent non-cancerous tissues (N), tumor tissues (T) (right). b, Immunoblotting analysis of CARNS in HepG2 cells expressing shCARNS1 or sgCARNS1. c, qPCR analysis of CARNS expression in HepG2 and PLC cells. Cells were treated under normoxia or hypoxia for 48 h (n = 3 biological replicates). d, ChIP experiments were performed in HepG2 and PLC cells expressing Flag-HIF-2a or EV using anti-Flag antibody. The occupancy of potential binding sites in CARNS by HIF-2a was determined by qRT-PCR (n = 3 biological replicates). e, Dual-luciferase analysis in HEK293 cells transfected with pGL3-HRE and HIF-1α or HIF-2α plasmids (n = 4 biological replicates). f, View of aligned reads at 20 kb resolution for a matched hypoxia/normoxia pair of CARNS. Paired arrows represent semi-RT-PCR primers. g, Schematic diagram of isoforms of CARNS. h, i, Intracellular carnosine levels were detected in HepG2 cells stably expressing EV, CARNS1-V1, CARNS1-V2 and CARNS2 (n = 3 biological replicates) (h). Immunoblotting analysis confirmed overexpression of CARNS1-V1, CARNS1-V2 and CARNS2 in HepG2 cells (i). j, Plasmids expressing RFP or YAP-5SA together with PB transposase plasmids were delivered into WT, Carns1−/− or Carns1/2−/− mice by hydrodynamic injection. Liver tumors were analyzed at 100 days after injection. Red fluorescent protein (RFP) served as a control (n = 5 mice). Data presented as mean ± s.d. (c, d, e, h). Statistical significance was determined by two-tailed unpaired Student’s t-test (c, d, e, h).

Source data

Extended Data Fig. 3 hnRNPA2B1 and MBNL1 mediate the alternative splicing of CARNS2 under hypoxia.

a, Immunoblotting analysis of MBNL1 expression in whole-cell lysates (WCL) and nucleus lysates from PLC cells with normoxia or hypoxia treatment for 48 h. b, RNA immunoprecipitation analysis of the binding of endogenous CARNS mRNAs by MBNL in PLC cells. Cells were treated under normoxia or hypoxia for 24 h (n = 3 biological replicates). c, Venn diagram showing overlapping proteins bound to both MBNL1 protein and CARNS pre-mRNA in PLC cells. d, qPCR and Immunoblotting analysis of hnRNPA2B1 mRNA levels (n = 3 biological replicates) (left panel) and protein levels (right panel) in PLC cells expressing NTC, shHIF-1α or shHIF-2α, cultured under normoxia or hypoxia for 24 h. e, Binding of the endogenous CARNS mRNAs by HnRNPA2B1 in PLC cells expressing NTC and shMBNL1 was determined by RNA immunoprecipitation. Cells were treated under normoxia or hypoxia for 24 h (n = 3 biological replicates). Data presented as mean ± s.d. (b, d, e). Statistical significance was determined by two-tailed unpaired Student’s t-test (b, d, e).

Source data

Extended Data Fig. 4 Carnosine-mediated pHi homeostasis regulates lysosomal distribution and function.

a, LysoSensor staining in PLC cells expressing NTC and shCARNS2 cultured under normoxia or hypoxia for 48 h in the presence or absence of 1.5 mM carnosine as indicated. The yellow arrows indicate perinuclear lysosomes with low luminal pH. The blue arrows indicate peripheral lysosomes with high luminal pH. Representative LysoSensor staining images were show (upper panel). Averaged fluorescent intensities of lysosomes in cells wad measured, n represents the total number of lysosomes we counted (lower panel). b, Representative images display red and green fluorescence for microtubule-associated proteins 1 A/1B light chain 3 A (LC3) expression using mCherry-GFP-LC3 fusion protein stably expressed in PLC cells expressing NTC, shCARNS2 or shCARNS2 treated with 5 mM carnosine cultured under normoxia or hypoxia for 48 h. Quantitative fluorescence results were shown in the right panel. NII: Normalized Integrated Intensity. c,e, Distribution of lysosome labeled with TMEM192-RFP was detected in PLC cells expressing NTC, shCARNS2, shCARNS2 together with shSLC15A1/2 (c) or shSLC15A3/4 (e) cultured under normoxia or hypoxia for 48 h. Cells were treated with or without 1.5 mM carnosine. d, f, Lysosomal pH was respectively measured with OG-514 dye in HepG2 cells expressing NTC, shCARNS2, shCARNS2 together with shSLC15A1/2 (d) or shSLC15A3/4 (f) cultured under normoxia or hypoxia for 48 h in the presence or absence of carnosine as indicated (n = 3 biological replicates). Data presented as mean ± s.e.m.(a) or s.d.(d, f). Statistical significance was determined by two-tailed unpaired Student’s t-test (a, d, f).

Source data

Extended Data Fig. 5 CARNS2 promotes the expression of galectin-9 by lysosome-dependent degradation of NFX1.

a, Immunoblotting shows the indicated proteins levels in whole-cell lysates or lysosomes purified from HepG2 cells expressing NTC, shCARNS2 cultured under normoxia or hypoxia for 48 h. b, Immunoblotting analysis of NFX1 expression in HepG2 cells expressing NTC, shSLC15A1/2 and SLC15A3/4 cultured under normoxia or hypoxia for 48 h. P62 served as positive control. c, Co-IP experiment was performed in PLC cells with normoxia or hypoxia treatment for 48 h (left). The working model depicts that NFX1 was recognized by p62 and LC3II for targeted degradation, especially under hypoxia (right). d, List of the 20 most strongly downregulated genes under NFX1 overexpression during hypoxia in PLC cells. e, ChIP experiments were performed in PLC cells expressing Flag-NFX1 using IgG or anti-Flag antibody. The occupancy of potential binding sites in galectin-9 by NFX1 was determined by qRT-PCR (left). Dual-luciferase analysis in HEK293 cells transfected with pGL3 plasmids containing different regions and EV or NFX1 plasmids (right) (n = 3 biological replicates). f, qPCR analysis of galectin-9 mRNA levels in PLC cells with overexpression NFX1 cultured under normoxia or hypoxia for 36 h (n = 3 biological replicates). g, galectin-9 expression in the whole-cell lysates and conditional medium were detected by Immunoblotting in HepG2 (left) and Hepa 1-6 (right) cells with NFX1 knockdown or overexpression with normoxia or hypoxia treatment for 48 h. h, Immunoblotting analyzing the expression of CARNS2, NFX1, galectin-9 in whole-cell lysates and secreted galectin-9 in culture medium of PLC cells expressing NTC, shCARNS2, shNFX1 or shCARNS2 together with shNFX1 with normoxia or hypoxia treatment for 48 h. i, Immunoblotting verified that the blot of galectin-9 showed in this manuscript is correct. These experiments were performed in Hepa1-6 cells. Data presented as mean ± s.d.(e, f). Statistical significance was determined by two-tailed unpaired Student’s t-test (e, f).

Source data

Extended Data Fig. 6 NFX1 is involved in CARNS2 and CD8+ T-cell-mediated anti-tumor immunity.

a-d, At the end of the experiment in Fig. 4a, tumors were collected and tumor masses were calculated (n = 5 mice) (a). Immunoblotting verified CARNS2, NFX1, galectin-9 expression in the extracted tumors (b). Representative flow staining (c) and frequency of exhaustion and effector function markers (d) of CD8+ T cells in NTC and shCARNS2 Hepa 1-6 tumors (n = 3 samples). e, Corresponding to the experiment in Fig. 4c, tumors were collected (n = 5 mice) (left). Immunoblotting verified CARNS2, NFX1 and galectin-9 expression in the extracted tumors (right). f, Plasmids expressing human YAP-5SA alone or human YAP-5SA plus shNFX1 plasmids together with PB transposase plasmids were delivered by hydrodynamic injection into WT or Carns1&2-/- mice. Liver/body weight ratios were measured approximately at 100 days after injection (n = 5 mice). CD8+ T cells numbers, exhaustion and effector function markers were determined by flow cytometry (n = 3 samples). g, Flow cytometry analysis of Ki67, TIM-3, TNFα, INFγ expression on in vitro differentiated CD8+ T cells cultured with CM from Hepa 1-6 cells expressing EV or NFX1. Hepa 1-6 cells were treated under 20% O2 or 1% O2 for 48 h (n = 3 biological replicates). Data presented as mean ± s.d.(a, d, f, g). Statistical significance was determined by two-tailed unpaired Student’s t-test (a, d, f, g).

Source data

Extended Data Fig. 7 CARNS2/NFX1 facilitated tumor immune evasion is mediated by galectin-9 and CD8+ T cell.

a, b, Corresponding to the experiment in Fig. 4e. Tumors were collected (n = 5 mice) (a). Immunoblotting verified CARNS2, NFX1, galectin-9 expression in the extracted tumors (b). c, Flow cytometry analysis of Ki67 (left), INFγ, TNFα, TIM-3 (right) expression on in vitro differentiated CD8+ T cells cultured with CM from Hepa 1-6 cells expressing NTC, shCARNS2, OE-Gal-9, or shCARNS2 plus OE-Gal-9. Hepa 1-6 cells were treated under 20% O2 or 1% O2 for 48 h (n = 3 biological replicates). d, Hepa 1-6 cells expressing NTC, shNFX1, shgalectin-9, or shNFX1 plus shgalectin-9 were injected subcutaneously into C57BL/6 mice. Tumors were collected and tumor masses were calculated at the end of the experiment. Tumor sizes were measured starting at 12 days after inoculation (n = 5 mice). Immunoblotting verified NFX1, galectin-9 expression in the extracted tumors. e, Corresponding to the experiment in Fig. 4f. Immunoblotting verified CARNS2 expression in the extracted tumors (upper). Representative flow staining indicated the proportion of CD8+ T cells in tumors and spleens (lower). f, Hepa 1-6 cells expressing NTC or shCARNS2 were injected subcutaneously into WT or Rag1-/- C57BL/6 mice. Tumors were collected and tumor masses were calculated at the end of the experiment. Tumor sizes were measured starting at 12 days after inoculation (n = 5 mice). CD8+ T cells numbers were determined by flow cytometry (n = 3 samples). Immunoblotting verified CARNS2, NFX1, galectin-9 expression in the extracted tumors. g-i, C57BL/6 mice injected subcutaneously with Hepa 1-6 cells expressing NTC and shCARNS2 treated with PBS or CQ. Tumors were collected and tumor masses were calculated at the end of the experiment. Tumor sizes were measured starting at 10 days after inoculation (n = 5 mice) (g). Immunoblotting verified NFX1 expression in the extracted tumors (h). CD8+ T cells numbers, exhaustion and effector function markers were determined by flow cytometry (n = 3 samples) (i). Data presented as mean ± s.d.(c, d, f, g, i). Statistical significance was determined by two-tailed unpaired Student’s t-test (c, d, f, g, i) or two-way ANOVA (d, f, g).

Source data

Extended Data Fig. 8 Gating strategies used in FACS analysis.

a, Gating strategy to identify the proportion of infiltrated immune cells in the tumor. b, Gating strategy to analyse the exhaustion markers of CD8+ T cells in the tumor. c, Gating strategy to analyse the effector function of CD8+ T cells in the tumor.

Supplementary information

Supplementary Information

Supplementary Tables 1-10

Reporting Summary

Peer Review File

Supplementary Video 1

Supplementary Video 1 CARNS2 regulates lysosomal subcellular distribution under hypoxia CARNS2 suppression led to a striking abnormal distribution of lysosome, in which they shifted from perinuclear localization to an evenly dispersed cytosolic and peripheral distribution under hypoxia.

Source data

Source Data Fig. 2

Unprocessed Western Blots

Source Data Fig. 3

Unprocessed Western Blots

Source Data Fig. 5

Unprocessed Western Blots

Source Data Extended Data Fig. 1

Unprocessed Western Blots

Source Data Extended Data Fig. 2

Unprocessed Western Blots

Source Data Extended Data Fig. 3

Unprocessed Western Blots

Source Data Extended Data Fig. 5

Unprocessed Western Blots

Source Data Extended Data Fig. 6

Unprocessed Western Blots

Source Data Extended Data Fig. 7

Unprocessed Western Blots

Source Data Fig. 1

Statistical Source Data

Source Data Fig. 2

Statistical Source Data

Source Data Fig. 3

Statistical Source Data

Source Data Fig. 4

Statistical Source Data

Source Data Fig. 5

Statistical Source Data

Source Data Extended Data Fig. 1

Statistical Source Data

Source Data Extended Data Fig. 2

Statistical Source Data

Source Data Extended Data Fig. 3

Statistical Source Data

Source Data Extended Data Fig. 4

Statistical Source Data

Source Data Extended Data Fig. 5

Statistical Source Data

Source Data Extended Data Fig. 6

Statistical Source Data

Source Data Extended Data Fig. 7

Statistical Source Data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yan, R., Zhang, P., Shen, S. et al. Carnosine regulation of intracellular pH homeostasis promotes lysosome-dependent tumor immunoevasion. Nat Immunol 25, 483–495 (2024). https://doi.org/10.1038/s41590-023-01719-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41590-023-01719-3

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer