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Targeting NAD+ metabolism of hepatocellular carcinoma cells by lenvatinib promotes M2 macrophages reverse polarization, suppressing the HCC progression

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Abstract

Background

Lowered nicotinamide adenine dinucleotide (NAD+) levels in tumor cells drive tumor hyperprogression during immunotherapy, and its restoration activates immune cells. However, the effect of lenvatinib, a first-line treatment for unresectable hepatocellular carcinoma (HCC), on NAD+ metabolism in HCC cells, and the metabolite crosstalk between HCC and immune cells after targeting NAD+ metabolism of HCC cells remain unelucidated.

Methods

Liquid chromatography-tandem mass spectrometry (LC–MS/MS) and ultra-high-performance liquid chromatography multiple reaction monitoring-mass spectrometry (UHPLC-MRM-MS) were used to detect and validate differential metabolites. RNA sequencing was used to explore mRNA expression in macrophages and HCC cells. HCC mouse models were used to validate the effects of lenvatinib on immune cells and NAD+ metabolism. The macrophage properties were elucidated using cell proliferation, apoptosis, and co-culture assays. In silico structural analysis and interaction assays were used to determine whether lenvatinib targets tet methylcytosine dioxygenase 2 (TET2). Flow cytometry was performed to assess changes in immune cells.

Results

Lenvatinib targeted TET2 to synthesize and increase NAD+ levels, thereby inhibiting decomposition in HCC cells. NAD+ salvage increased lenvatinib-induced apoptosis of HCC cells. Lenvatinib also induced CD8+ T cells and M1 macrophages infiltration in vivo. And lenvatinib suppressed niacinamide, 5-Hydroxy-L-tryptophan and quinoline secretion of HCC cells, and increased hypoxanthine secretion, which contributed to proliferation, migration and polarization function of macrophages. Consequently, lenvatinib targeted NAD+ metabolism and elevated HCC-derived hypoxanthine to enhance the macrophages polarization from M2 to M1. Glycosaminoglycan binding disorder and positive regulation of cytosolic calcium ion concentration were characteristic features of the reverse polarization.

Conclusions

Targeting HCC cells NAD+ metabolism by lenvatinib-TET2 pathway drives metabolite crosstalk, leading to M2 macrophages reverse polarization, thereby suppressing HCC progression. Collectively, these novel insights highlight the role of lenvatinib or its combination therapies as promising therapeutic alternatives for HCC patients with low NAD+ levels or high TET2 levels.

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Data availability

The RNA sequencing data and non-target metabolomics data can be shared if anyone interests and provides Email address.

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Acknowledgements

This work was supported by the National Nature Science Foundation of China (Grant No. 82172751), China Postdoctoral Science Foundation (Grant No. 2021M701625), President Foundation of Nanfang Hospital, Southern Medical University (Grant No. 2021C003), and Guangdong Natural Science Foundation (Grant No. 2022A1515110656). We would like to acknowledge Suzhou Panomix for providing help. We thank SHANGHAI BIOTREE BIOMEDICAL TECHNOLOGY CO., LTD for LC MS/MS and UHPLC-MRM-MS analysis. We also thank Novogene for assistance in RNAseq experiment.

Funding

This work was supported by the National Nature Science Foundation of China (Grant No. 82172751), China Postdoctoral Science Foundation (Grant No. 2021M701625), President Foundation of Nanfang Hospital, Southern Medical University (Grant No. 2021C003), and Guangdong Natural Science Foundation (Grant No. 2022A1515110656).

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Author notes

  1. Qingcan Sun and Mengying Shen contributed equally to this work.

Authors and Affiliations

  1. Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China

    Qingcan Sun, Mengying Shen, Subin Zhu, Yanxia Liao, Leyuan Wu, Lushan Xiao & Li Liu

  2. State Key Laboratory of Organ Failure Research, Guangzhou, 510515, China

    Qingcan Sun, Mengying Shen, Subin Zhu, Yanxia Liao, Leyuan Wu, Lushan Xiao & Li Liu

  3. Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Guangzhou, 510515, China

    Qingcan Sun, Mengying Shen, Subin Zhu, Yanxia Liao, Leyuan Wu, Lushan Xiao & Li Liu

  4. Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China

    Dongyan Zhang, Jingyuan Sun & Zeqin Guo

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  1. Qingcan Sun
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Contributions

QS and MS contributed equally to this work. QS, MS, SZ, LX and LL were responsible for the experimental design and data interpretation. QS, MS and SZ and YL performed the experiments. QS wrote the manuscript. DZ, JS, ZG and LW have verified the underlying data. All authors read and approved the final manuscript.

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Correspondence to Li Liu.

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Sun, Q., Shen, M., Zhu, S. et al. Targeting NAD+ metabolism of hepatocellular carcinoma cells by lenvatinib promotes M2 macrophages reverse polarization, suppressing the HCC progression. Hepatol Int 17, 1444–1460 (2023). https://doi.org/10.1007/s12072-023-10544-7

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