Nicotinamide N-methyltransferase negatively regulates metastasis-promoting property of cancer-associated fibroblasts in lung adenocarcinoma

Abstract

Background: Recurrence and metastasis remain significant challenges in lung adenocarcinoma (LUAD) after radical resection. The mechanisms behind the recurrence and metastasis of LUAD remain elusive, and deregulated cellular metabolism is suspected to play a significant role. This study explores the metabolic and epigenetic regulation mediated by nicotinamide N-methyl transferase (NNMT) in LUAD.

Methods: Untargeted metabolomic analyses were performed to detect metabolism irregularities. Single-cell RNA sequencing (RNA-seq) databases and multiplex immunofluorescence analysis were used to identify the location of NNMT within the tumor microenvironment. The biological functions of NNMT were investigated both in vitro and in vivo, with RNA-seq and chromatin immunoprecipitation-PCR providing insights into underlying mechanisms. Finally, single-cell RNA-seq data and immunohistochemistry of primary tumors were analyzed to validate the main findings.

Results: Untargeted metabolomic analyses revealed metabolic aberrations in amino acids, organic acids, lipids, and nicotinamide pathways, which are linked to metastasis of non-small cell lung cancer. NNMT is a key enzyme in nicotinamide metabolism, and we found the bulk tissue mRNA level of NNMT gene was inversely associated with LUAD metastasis. NNMT was proved to be predominantly expressed in cancer-associated fibroblasts (CAFs) within the stromal regions of LUAD, and a low stromal NNMT expression was identified as a predictor of poor disease-free survival following radical resection of LUAD. The isolation and primary culture of CAFs from LUAD enabled in vitro and in vivo experiments, which confirmed that NNMT negatively regulated the metastasis-promoting properties of CAFs in LUAD. Mechanistically, the downregulation of NNMT led to an increase in intracellular methyl groups by reducing the activity of the methionine cycle, resulting in heightened methylation at H3K4me3. This alteration triggered the upregulation of genes involved in extracellular matrix remodeling in CAFs, including those encoding collagens, integrins, laminins, and matrix metalloproteinases, thereby facilitating cancer cell invasion and metastasis. Reanalysis of single-cell RNA-seq data and immunohistochemistry assays of primary LUAD tissues substantiated NNMT’s negative regulation of these genes in CAFs.

Conclusions: This study provides novel insights into the metabolic and epigenetic regulatory functions of NNMT in CAFs, expanding the current understanding of LUAD metastasis regulation and suggesting potential avenues for future research and therapeutic development.

Keywords

Nicotinamide N-methyl transferase / Lung cancer / Cancer-associated fibroblast / Extracellular matrix / Metastasis

References

[1] Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021; 71(3): 209-49.

[2] Bray F, Jemal A, Grey N, Ferlay J, Forman D. Global cancer transitions according to the Human Development Index (2008-2030): a population-based study. Lancet Oncol. 2012; 13(8): 790-801.

[3] Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024; 74(3): 229-263.

[4] Sullivan R, Alatise OI, Anderson BO, Audisio R, Autier P, Aggarwal A, et al. Global cancer surgery: delivering safe, affordable, and timely cancer surgery. Lancet Oncol. 2015; 16(11): 1193-224.

[5] Wang P, Wang S, Sun Z, Li H, Zhao Y, Li Y, et al. Systemic inflammation influences the prognosis of patients with radically resected non-small cell lung cancer and correlates with the immunosuppressive microenvironment. Int J Cancer. 2023; 153(4): 826-842.

[6] Chen P, Liu Y, Wen Y, Zhou C. Non-small cell lung cancer in China. Cancer Commun. 2022; 42(10): 937-70.

[7] Chen K, Wang X, Yang F, Li J, Jiang G, Liu J, et al. Propensity-matched comparison of video-assisted thoracoscopic with thoracotomy lobectomy for locally advanced non-small cell lung cancer. J Thorac Cardiovasc Surg. 2017; 153(4): 967-76 e2.

[8] Rami-Porta R, Asamura H, Travis WD, Rusch VW. Lung cancer — major changes in the American Joint Committee on Cancer eighth edition cancer staging manual. CA: A Cancer Journal for Clinicians. 2017; 67(2): 138-55.

[9] Martinez-Zayas G, Almeida FA, Yarmus L, Steinfort D, Lazarus DR, Simoff MJ, et al. Predicting Lymph Node Metastasis in Non-small Cell Lung Cancer. Chest. 2021; 160(3): 1108-20.

[10] Brock MV, Hooker CM, Ota-Machida E. Han Y, Guo M, Ames S, et al. DNA methylation markers and early recurrence in stage I lung cancer. N Engl J Med. 2008; 358(11): 1118-28.

[11] Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell. 2011; 144(5): 646-74.

[12] Ge T, Gu X, Jia R, Ge S, Chai P, Zhuang A, et al. Crosstalk between metabolic reprogramming and epigenetics in cancer: updates on mechanisms and therapeutic opportunities. Cancer Commun. 2022; 42(11): 1049-82.

[13] DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. The Biology of Cancer: Metabolic Reprogramming Fuels Cell Growth and Proliferation. Cell Metab. 2008; 7(1): 11-20.

[14] DeBerardinis RJ, Chandel NS. Fundamentals of cancer metabolism. Sci Adv. 2016; 2(5): e1600200.

[15] Zanotelli MR, Zhang J, Reinhart-King CA. Mechanoresponsive metabolism in cancer cell migration and metastasis. Cell Metab. 2021; 33(7): 1307-21.

[16] Gerstberger S, Jiang Q, Ganesh K. Metastasis. Cell. 2023; 186(8): 1564-79.

[17] Quail DF, Joyce JA. Microenvironmental regulation of tumor progression and metastasis. Nat Med. 2013; 19(11): 1423-37.

[18] Semenza GL. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene. 2009; 29(5): 625-34.

[19] Jones RG, Thompson CB. Tumor suppressors and cell metabolism: a recipe for cancer growth. Genes Dev. 2009; 23(5): 537-48.

[20] Bian Y, Li W, Kremer DM, Sajjakulnukit P, Li S, Crespo J, et al. Cancer SLC43A2 alters T cell methionine metabolism and histone methylation. Nature. 2020; 585(7824): 277-82.

[21] Kilgour MK, MacPherson S, Zacharias LG, Ellis AE, Sheldon RD, Liu EY, et al. 1-Methylnicotinamide is an immune regulatory metabolite in human ovarian cancer. Sci Adv. 2021; 7(4): eabe1174.

[22] Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009; 324(5930): 1029-33.

[23] Wang W, Yang C, Wang T, Deng H. Complex roles of nicotinamide N-methyltransferase in cancer progression. Cell Death Dis. 2022; 13(3): 267.

[24] Eckert MA, Coscia F, Chryplewicz A, Chang JW, Hernandez KM, Pan S, et al. Proteomics reveals NNMT as a master metabolic regulator of cancer-associated fibroblasts. Nature. 2019; 569(7758): 723-8.

[25] Cao L, Wu W, Deng X, Peng Y, Chen Y, Guo H, et al. Systematic pan-cancer analysis of the nicotinamide n-methyltransferase in human cancer. Front Genet. 2022; 13: 1000515.

[26] Cui Y, Zhang L, Wang W, Ma S, Liu H, Zang X, et al. Downregulation of nicotinamide N-methyltransferase inhibits migration and epithelial-mesenchymal transition of esophageal squamous cell carcinoma via Wnt/β-catenin pathway. Mol Cell Biochem. 2019; 460(1-2): 93-103.

[27] Wang Y, Zhou X, Lei Y, Chu Y, Yu X, Tong Q, et al. NNMT contributes to high metastasis of triple negative breast cancer by enhancing PP2A/MEK/ERK/c-Jun/ABCA1 pathway mediated membrane fluidity. Cancer Lett. 2022; 547: 215884.

[28] Campagna R, Salvolini E, Pompei V, Pozzi V, Salvucci A, Molinelli E, et al. Nicotinamide N-methyltransferase gene silencing enhances chemosensitivity of melanoma cell lines. Pigment Cell Melanoma Res. 2021; 34(6): 1039-48.

[29] Zhao H, Li R, Chen Y, Yang X, Shang Z. Stromal nicotinamide N-methyltransferase orchestrates the crosstalk between fibroblasts and tumour cells in oral squamous cell carcinoma: evidence from patient-derived assembled organoids. Oncogene. 2023; 42(15): 1166-80.

[30] Pang Z, Chong J, Zhou G, de Lima Morais DA, Chang L, Barrette M, et al. MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional insights. Nucleic Acids Res. 2021; 49(W1): W388-W96.

[31] Kanehisa M, Furumichi M, Tanabe M, Sato Y, Morishima K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 2017; 45(D1): D353-D61.

[32] Zhang B, Hu S, Baskin E, Patt A, Siddiqui J, Mathé E. RaMP: A Comprehensive Relational Database of Metabolomics Pathways for Pathway Enrichment Analysis of Genes and Metabolites. Metabolites. 2018; 8(1): 16.

[33] Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics. 2015; 31(2): 166-9.

[34] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12): 550.

[35] The Gene Ontology Consortium. The Gene Ontology Resource: 20 years and still GOing strong. Nucleic Acids Res. 2019; 47(D1): D330-D8.

[36] Yu, G, Wang, LG, Han, Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012; 16(5): 284-7.

[37] Zhang B, Schmoyer D, Kirov S, Snoddy J. GOTree Machine (GOTM): a web-based platform for interpreting sets of interesting genes using Gene Ontology hierarchies. BMC Bioinf. 2004; 5: 16.

[38] Shannon P MA, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498-504.

[39] Peng H, Wu X, Liu S, He M, Xie C, Zhong R, et al. Multiplex immunofluorescence and single-cell transcriptomic profiling reveal the spatial cell interaction networks in the non-small cell lung cancer microenvironment. Clin Transl Med. 2023; 13(1): e1155.

[40] Arrieta O, Aviles-Salas A. Orozco-Morales M, Hernández-Pedro N, Cardona AF, Cabrera-Miranda L. et al. Association between CD47 expression, clinical characteristics and prognosis in patients with advanced non-small cell lung cancer. Cancer Med. 2020; 9(7): 2390-402.

[41] Xu Y, Li J, Tong B, Chen M, Liu X, Zhong W, et al. Positive tumour CD47 expression is an independent prognostic factor for recurrence in resected non-small cell lung cancer. ESMO Open. 2020; 5(4): e000823.

[42] Merchant FA, Shah SK, Castleman KR. Average Optical Intensity. In: Merchant FA and Castleman KR. Chief editors, 2nd ed. Microscope Image Processing. Press: Mara Conner. 2023; 8(4): 168.

[43] Griffin KH, Fok SW, Kent Leach J. Strategies to capitalize on cell spheroid therapeutic potential for tissue repair and disease modeling. npj Regenerative Medicine. 2022; 7(1): 70.

[44] Białkowska K, Komorowski P, Bryszewska M, Miłowska K. Spheroids as a Type of Three-Dimensional Cell Cultures—Examples of Methods of Preparation and the Most Important Application. Int J Mol Sci. 2020; 21(17): 6225.

[45] Berens EB, Holy JM, Riegel AT, Wellstein A. A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting. Journal of Visualized Experiments. 2015; (105):53409.

[46] Slyper M, Porter CBM, Ashenberg O, Waldman J, Drokhlyansky E, Wakiro I, et al. A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors. Nat Med. 2020; 26(5): 792-802.

[47] Waise S, Parker R, Rose-Zerilli MJJ. Layfield DM, Wood O, West J, et al. An optimised tissue disaggregation and data processing pipeline for characterising fibroblast phenotypes using single-cell RNA sequencing. Sci Rep. 2019; 9(1): 9580.

[48] Lambrechts D, Wauters E, Boeckx B, Aibar S, Nittner D, Burton O, et al. Phenotype molding of stromal cells in the lung tumor microenvironment. Nat Med. 2018; 24(8): 1277-89.

[49] Camp RL, Dolled-Filhart M. Rimm DL. X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization. Clin Cancer Res. 2004; 10(21): 7252-9.

[50] Wang J, Liu X, Huang Y, Li P, Yang M, Zeng S, et al. Targeting nicotinamide N-methyltransferase overcomes resistance to EGFR-TKI in non-small cell lung cancer cells. Cell Death Discovery. 2022; 8(1): 170.

[51] Grout JA, Sirven P, Leader AM, Maskey S, Hector E, Puisieux I, et al. Spatial Positioning and Matrix Programs of Cancer-Associated Fibroblasts Promote T-cell Exclusion in Human Lung Tumors. Cancer Discov. 2022; 12(11): 2606-25.

[52] Pellinen T, Paavolainen L, Martín-Bernabé A, Papatella Araujo R, Strell C, Mezheyeuski A, et al. Fibroblast subsets in non-small cell lung cancer: Associations with survival, mutations, and immune features. J Natl Cancer Inst. 2023; 115(1): 71-82.

[53] Hanley CJ, Waise S, Ellis MJ, Lopez MA, Pun WY, Taylor J, et al. Single-cell analysis reveals prognostic fibroblast subpopulations linked to molecular and immunological subtypes of lung cancer. Nat Commun. 2023; 14(1): 387.

[54] Sun J, Wu S, Jin Z, Ren S, Cho WC, Zhu C, et al. Lymph node micrometastasis in non–small cell lung cancer. Biomed Pharmacother. 2022; 149: 112817.

[55] Wu DM, Deng SH, Zhou J, Han R, Liu T, Zhang T, et al. PLEK2 mediates metastasis and vascular invasion via the ubiquitin-dependent degradation of SHIP2 in non-small cell lung cancer. Int J Cancer. 2019; 146(9): 2563-75.

[56] Shih AR, Mino-Kenudson M. Updates on spread through air spaces (STAS) in lung cancer. Histopathology. 2020; 77(2): 173-80.

[57] Hamidi H, Ivaska J. Every step of the way: integrins in cancer progression and metastasis. Nat Rev Cancer. 2018; 18(9): 533-48.

[58] Xu S, Xu H, Wang W, Li S, Li H, Li T, et al. The role of collagen in cancer: from bench to bedside. J Transl Med. 2019; 17(1): 309.

[59] Kessenbrock K, Plaks V, Werb Z. Matrix Metalloproteinases: Regulators of the Tumor Microenvironment. Cell. 2010; 141(1): 52-67.

[60] Zheng B, Qu J, Ohuchida K, Feng H, Chong SJF, Yan Z, et al. LAMA4 upregulation is associated with high liver metastasis potential and poor survival outcome of Pancreatic Cancer. Theranostics. 2020; 10(22): 10274-89.

[61] Pham VN, Bruemmer KJ, Toh JDW, Ge EJ, Tenney L, Ward CC, et al. Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism. Science. 2023; 382(6670): eabp9201.

[62] He L, Huang H, Bradai M, Zhao C, You Y, Ma J, et al. DNA methylation-free Arabidopsis reveals crucial roles of DNA methylation in regulating gene expression and development. Nat Commun. 2022; 13(1): 1335.

[63] Meyer KD, Jaffrey SR. The dynamic epitranscriptome: N6-methyladenosine and gene expression control. Nat Rev Mol Cell Biol. 2014; 15(5): 313-26.

[64] Hyun K, Jeon J, Park K, Kim J. Writing, erasing and reading histone lysine methylations. Exp Mol Med. 2017; 49(4):e324-e.

[65] Millán-Zambrano G, Burton A, Bannister AJ, Schneider R. Histone post-translational modifications -cause and consequence of genome function. Nat Rev Genet. 2022; 23(9): 563-80.

[66] Cao Q, Wang W, Williams JB, Yang F, Wang ZJ, Yan Z. Targeting histone K4 trimethylation for treatment of cognitive and synaptic deficits in mouse models of Alzheimer’s disease. Sci Adv. 2020; 6(50): eabc8096.

[67] Nissen NI, Karsdal M, Willumsen N. Collagens and Cancer associated fibroblasts in the reactive stroma and its relation to Cancer biology. J Exp Clin Cancer Res. 2019; 38(1): 115.

[68] Bian X, Liu R, Meng Y, Xing D, Xu D, Lu Z. Lipid metabolism and cancer. J Exp Med. 2021; 218(1): e20201606.

[69] Sivanand S, Vander Heiden MG. Emerging Roles for Branched-Chain Amino Acid Metabolism in Cancer. Cancer Cell. 2020; 37(2): 147-56.

[70] Wang X, Liu R, Zhu W, Chu H, Yu H, Wei P, et al. UDP-glucose accelerates SNAI1 mRNA decay and impairs lung cancer metastasis. Nature. 2019; 571(7763): 127-31.

[71] Wang Y, Fu L, Cui M, Wang Y, Xu Y, Li M, et al. Amino acid transporter SLC38A3 promotes metastasis of non-small cell lung cancer cells by activating PDK1. Cancer Lett. 2017; 393: 8-15.

[72] Merino Salvador M, Gómez de Cedrón M, Moreno Rubio J, Falagán Martínez S, Sánchez Martínez R, Casado E, et al. Lipid metabolism and lung cancer. Critical Reviews in Oncology/Hematology. 2017; 112: 31-40.

[73] Seferbekova Z, Lomakin A, Yates LR, Gerstung M. Spatial biology of cancer evolution. Nat Rev Genet. 2022; 24(5): 295-313.

[74] Wang G, Qiu M, Xing X, Zhou J, Yao H, Li M, et al. Lung cancer scRNA-seq and lipidomics reveal aberrant lipid metabolism for early-stage diagnosis. Sci Transl Med. 2022; 14(630): eabk2756.

[75] Roberti A, Fernández AF, Fraga MF. Nicotinamide N-methyltransferase: At the crossroads between cellular metabolism and epigenetic regulation. Molecular Metabolism. 2021; 45: 101165.

[76] Qu Y, Feng J, Wu X, Bai L, Xu W, Zhu L, et al. A proteogenomic analysis of clear cell renal cell carcinoma in a Chinese population. Nat Commun. 2022; 13(1): 2052.

[77] Yang C, Wang T, Zhu S, Zong Z, Luo C, Zhao Y, et al. Nicotinamide N-Methyltransferase Remodeled Cell Metabolism and Aggravated Proinflammatory Responses by Activating STAT3/IL1beta/PGE(2) Pathway. ACS Omega. 2022; 7(42): 37509-19.

[78] Wang Z, Wang Q, Chen C, Zhao X, Wang H, Xu L, et al. NNMT enriches for AQP5+ cancer stem cells to drive malignant progression in early gastric cardia adenocarcinoma. Gut. 2023; 73(1): 63-77.

[79] Couto JP, Vulin M, Jehanno C, Coissieux MM, Hamelin B, Schmidt A, et al. Nicotinamide N-methyltransferase sustains a core epigenetic program that promotes metastatic colonization in breast cancer. EMBO J. 2023; 42(13): e112559.

[80] Lu S, Ke S, Wang C, Xu Y, Li Z, Song K, et al. NNMT promotes the progression of intrahepatic cholangiocarcinoma by regulating aerobic glycolysis via the EGFR-STAT3 axis. Oncogenesis. 2022; 11(1): 39.

[81] Tahara S, Nojima S, Ohshima K, Hori Y, Sato K, Kurashige M, et al. Nicotinamide N-methyltransferase is related to MELF pattern invasion in endometrioid carcinoma. Cancer Med. 2021; 10(23): 8630-40.

[82] Ulanovskaya OA, Zuhl AM, Cravatt BF. NNMT promotes epigenetic remodeling in cancer by creating a metabolic methylation sink. Nat Chem Biol. 2013; 9(5): 300-6.

[83] Wong KY, Cheung AH, Chen B, Chan WN, Yu J, Lo KW, et al. Cancer-associated fibroblasts in nonsmall cell lung cancer: From molecular mechanisms to clinical implications. Int J Cancer. 2022; 151(8): 1195-215.

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