Exposure of benzo[a]pyrene induces HCC exosome-circular RNA to activate lung fibroblasts and trigger organotropic metastasis

Abstract

Background: Benzo[a]pyrene (B[a]P), a carcinogen pollutant produced by combustion processes, is present in the western diet with grilled meats. Chronic exposure of B[a]P in hepatocellular carcinoma (HCC) cells promotes metastasis rather than primary proliferation, implying an unknown mechanism of B[a]P-induced malignancy. Given that exosomes carry bioactive molecules to distant sites, we investigated whether and how exosomes mediate cancer-stroma communications for a toxicologically associated microenvironment.

Method: Exosomes were isolated from B[a]P stimulated BEL7404 HCC cells (7404-100Bap Exo) at an environmental relevant dose (100 nmol/L). Lung pre-education animal model was prepared via injection of exosomes and cytokines. The inflammatory genes of educated lungs were evaluated using quantitative reverse transcription PCR array. HCC LM3 cells transfected with firefly luciferase were next injected to monitor tumor burdens and organotropic metastasis. Profile of B[a]P-exposed exosomes were determined by ceRNA microarray. Interactions between circular RNA (circRNA) and microRNAs (miRNAs) were detected using RNA pull-down in target lung fibroblasts. Fluorescence in situ hybridization and RNA immunoprecipitation assay was used to evaluate the “on-off” interaction of circRNA-miRNA pairs. We further developed an adeno-associated virus inhalation model to examine mRNA expression specific in lung, thereby exploring the mRNA targets of B[a]P induced circRNA-miRNA cascade.

Results: Lung fibroblasts exert activation phenotypes, including focal adhesion and motility were altered by 7404-100Bap Exo. In the exosome-educated in vivo model, fibrosis factors and pro-inflammatory molecules of are up-regulated when injected with exosomes. Compared to non-exposed 7404 cells, circ_0011496 was up-regulated following B[a]P treatment and was mainly packaged into 7404-100Bap Exo. Exosomal circ_0011496 were delivered and competitively bound to miR-486-5p in recipient fibroblasts. The down-regulation of miR-486-5p converted fibroblast to cancer-associated fibroblast via regulating the downstream of Twinfilin-1 (TWF1) and matrix metalloproteinase-9 (MMP9) cascade. Additionally, increased TWF1, specifically in exosomal circ_0011496 educated lungs, could promote cancer-stroma crosstalk via activating vascular endothelial growth factor (VEGF). These modulated fibroblasts promoted endothelial cells angiogenesis and recruited primary HCC cells invasion, as a consequence of a pre-metastatic niche formation.

Conclusion: We demonstrated that B[a]P-induced tumor exosomes can deliver circ_0011496 to activate miR-486-5p/TWF1/MMP9 cascade in the lung fibroblasts, generating a feedback loop that promoted HCC metastasis.

Keywords

benzo(a)pyrene / cancer associate fibroblast / circular RNA / exosome / organotropic metastasis

References

[1] Lange NF, Radu P, Dufour JF. Prevention of NAFLD-associated HCC: role of lifestyle and chemoprevention. J Hepatol. 2021(5): 1217–1227.

[2] Huang DQ, El-Serag HB. Loomba R. Global epidemiology of NAFLD-related HCC: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol. 2020; 18(4): 223–238.

[3] Ma Y, Yang W, Li T, Liu Y, Simon TG, Sui J, et al. Meat intake and risk of hepatocellular carcinoma in two large US prospective cohorts of women and men. Int J Epidemiol. 2019; 48(6): 1863–1871.

[4] Wang Z, Yang P, Xie J, Lin HP, Yang C. Arsenic and benzo[a]pyrene co-exposure acts synergistically in inducing cancer stem cell-like property and tumorigenesis by epigenetically down-regulating SOCS3 expression. Environ Int. 2020; 137: 105560.

[5] Wu HC, Wang Q, Wang LW, Yang HI, Ahsan H, Tsai WY, et al. Polycyclic aromatic hydrocarbon-and aflatoxin-albumin adducts, hepatitis B virus infection and hepatocellular carcinoma in Taiwan. Cancer Lett. 2007; 252(1): 104–114.

[6] Aflatoxin and PAH exposure biomarkers in a U.S. population with a high incidence of hepatocellular carcinoma. Sci Total Environ. 2010; 408(23): 6027–6031.

[7] Zheng J, Zhao L, Dong J, Chen H, Li D, Zhang X, et al. The role of dietary factors in nonalcoholic fatty liver disease to hepatocellular carcinoma progression: A systematic review. Clin Nutr. 2022; 41(10): 2295–2307.

[8] Souza T, Jennen D, Delft JV, Herwijnen MV, Kyrtoupolos S, Kleinjans J. New insights into BaP-induced toxicity: role of major metabolites in transcriptomics and contribution to hepatocarcinogenesis. Arch Toxicol. 2016; 90(6): 1449–1458.

[9] Ba Q, Li J, Huang C, Qiu H, Li J, Chu R, et al. Effects of Benzo[a]pyrene Exposure on Human Hepatocellular Carcinoma Cell Angiogenesis, Metastasis, and NF-κB Signaling. Environ Health Perspect. 2015; 123(3): 246–254.

[10] Wang H, Chen L. Tumor microenviroment and hepatocellular carcinoma metastasis. J Gastroenterol Hepatol. 2013; 28(Suppl 1): 43–48.

[11] Uka K, Aikata H, Takaki S, Shirakawa H, Jeong SC, Yamashina K, et al. Clinical features and prognosis of patients with extrahepatic metastases from hepatocellular carcinoma. World J Gastroenterol. 2007; 13(3): 414–420.

[12] Ba Q, Huang C, Fu Y, Li J, Li J, Chu R, et al. Cumulative metabolic effects of low-dose benzo(a)pyrene exposure on human cells. Toxicol Res (Camb). 2016; 5(1): 107–115.

[13] Ba Q, Li J, Huang C, Qiu H, Li J, Chu R, et al. Effects of Benzo[a]pyrene Exposure on Human Hepatocellular Carcinoma Cell Angiogenesis, Metastasis, and NF-κB Signaling. Environ Health Perspect. 2014; 24(1): 135–157.

[14] Ge Y, Gu P, Wang W, Cao L, Zhang L, Li J, et al. Benzo[a]pyrene stimulates miR-650 expression to promote the pathogenesis of fatty liver disease and hepatocellular carcinoma via SOCS3/JAK/STAT3 cascades. J Mol Cell Biol. 2021; 13(8): 556–564.

[15] Zhan X, Wu R, Kong XH, You Y, He K, Sun XY, et al. Elevated neutrophil extracellular traps by HBV-mediated S100A9-TLR4/RAGE-ROS cascade facilitate the growth and metastasis of hepatocellular carcinoma. Cancer Commun. 2023; 43(2): 225–245.

[16] Song MJ, He JY, Pan QZ, Yang JY, Zhao JJ, Zhang YJ, et al. Cancer-Associated Fibroblast-Mediated Cellular Crosstalk Supports Hepatocellular Carcinoma Progression. Hepatology. 2021; 73(5): 1717–1735.

[17] Mu W, Rana S, Zoller M. Host matrix modulation by tumor exosomes promotes motility and invasiveness. Neoplasia. 2013; 15(8): 875–887.

[18] Lavie D, Ben-Shmuel A. Erez N, Scherz-Shouval R. Cancer-associated fibroblasts in the single-cell era. Nature Cancer. 2022; 3(7): 793–807.

[19] Hu DD, Li ZQ, Zheng B, Lin XX, Pan YH, Gong PR, et al. Cancer-associated fibroblasts in breast cancer: Challenges and opportunities. Cancer Commun. 2022; 42(5): 401–434.

[20] Lian XY, Zhang H, Liu Q, Lu X, Zhou P, He SQ, et al. Ovarian cancer-excreted exosomal miR-199a-5p suppresses tumor metastasis by targeting hypoxia-inducible factor-2alpha in hypoxia microenvironment. Cancer Commun (Lond). 2020; 40(8): 380–385.

[21] Gao Y, Huang K, Tang Y, Huang Y, Gao S. Exosome:A rising star in the era of precision oncology. Chin Sci Bull. 2018; 2(1): 452–498.

[22] Zhang H, Deng T, Ge S, Liu Y, Bai M, Zhu K, et al. Exosome circRNA secreted from adipocytes promotes the growth of hepatocellular carcinoma by targeting deubiquitination-related USP7. Oncogene. 2018; 38(15): 2884–2859.

[23] Zhang X, Xu YH, Ma LF, Yu KK, Niu YJ, Xu X, et al. Essential roles of exosome and circRNA_101093 on ferroptosis desensitization in lung adenocarcinoma. Cancer Commun. 2022; 42(4): 287–313.

[24] Wang Y, Liu J, Ma J, Sun T, Zhou Q, Wang W, et al. Exosomal circRNAs: biogenesis, effect and application in human diseases. Mol Cancer. 2019; 18(1): 116.

[25] Huang K, Lu Z, Li L, Peng G, Zhou W, Ye Q. Construction of a ceRNA network and a genomic-clinicopathologic nomogram to predict survival for HBV-related HCC. Hum Cell. 2021; 34(6): 1830–1842.

[26] Liang C, Ge S. CircRNA in cancer: Fundamental mechanism and clinical potential. Cancer Lett. 2021; 505(11): 49–57.

[27] Wang F, Niu Y, Chen K, Yuan X, Qin Y, Zheng F, et al. Extracellular Vesicle-Packaged circATP2B4 Mediates M2 Macrophage Polarization via miR-532-3p/SREBF1 Axis to Promote Epithelial Ovarian Cancer Metastasis. Cancer Immunol Res. 2023; 11(2): 199–216.

[28] Huang RX, Zhou PK. Double-edged effects of noncoding RNAs in responses to environmental genotoxic insults: Perspectives with regards to molecule-ecology network. Environ Pollut. 2019; 247: 64–71.

[29] Monti P, Solazzo G, Bollati V. Effect of environmental exposures on cancer risk: Emerging role of non-coding RNA shuttled by extracellular vesicles. Environ Int. 2023; 181: 108255.

[30] Zhang Q, Wang W, Zhou Q, Chen C, Yuan W, Liu J, et al. Roles of circRNAs in the tumour microenvironment. Mol Cancer. 2020; 19(1): 14.

[31] Chen Y, Cai KZ, Tu ZH, Nie W, Ji T, Hu B, et al. Prediction of benzo[a]pyrene content of smoked sausage using back-propagation artificial neural network. J Sci Food Agric. 2018; 98(8): 3022–3230.

[32] Caligiuri G, Tuveson DA. Activated fibroblasts in cancer: Perspectives and challenges. Cancer Cell. 2023; 41(3): 434–449.

[33] Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B. Moreno-Bueno G, et al. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat Med. 2012; 18(6): 883–891.

[34] Chen G, Huang AC, Zhang W, Zhang G, Wu M, Xu W, et al. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature. 2018; 560(7718): 382–386.

[35] Shang A, Gu C, Wang W, Wang X, Li D. Exosomal circPACRGL promotes progression of colorectal cancer via the miR-142-3p/miR-506-3p-TGF-β1 axis. Mol Cancer. 2020; 19(1): 117.

[36] Chen L, Wang C, Sun H, Wang J, Liang Y, Wang Y, et al. The bioinformatics toolbox for circRNA discovery and analysis. Brief Bioinform. 2021; 22(2): 1706–1728.

[37] Ulrichs H, Gaska I, Shekhar S. Multicomponent regulation of actin barbed end assembly by twinfilin, formin and capping protein. Nat Commun. 2023; 14(1): 3981.

[38] Houthuijzen JM, Jonkers J. Cancer-associated fibroblasts as key regulators of the breast cancer tumor microenvironment. Cancer Metastasis Rev. 2018; 37(4): 577–597.

[39] Olatunde , Olatunji, Olalekan, Fatoki, Beatrice, Opeolu, et al. Benzo[a]pyrene and Benzo[k]fluoranthene in some processed fish and fish products. Int J Environ Res Public Health. 2015; 12(1): 940–951.

[40] Souz T, Jennen D, Delft JV, Herwijnen MV, Kyrtoupolos S, Kleinjans J. New insights into BaP-induced toxicity: role of major metabolites in transcriptomics and contribution to hepatocarcinogenesis. Arch Toxicol. 2015; 90(6): 1449–1458.

[41] Guerreiro C, Horalek J, Leeuw FD, Couvidat F. Benzo(a)pyrene in Europe: Ambient air concentrations, population exposure and health effects. Environ Pollut. 2016; 214(jul.): 657–667.

[42] Jin Y, Qi G, Shou Y, Li D, Liu Y, Guan H, et al. High throughput data-based, toxicity pathway-oriente. development of a quantitative adverse outcome pathway network linking AHR activation to lung damages. J Hazard Mater. 2022; 425: 128041.

[43] Liu Z, Li Y, Sepulveda MS, Jiang Q, Jiao Y, Chen Q, et al. Development of an adverse outcome pathway for nanoplastic toxicity in Daphnia pulex using proteomics. Sci Total Environ. 2021; 766: 144249.

[44] Huo G, Wang Y, Chen J, Song Y, Chen P. A Pan-Cancer Analysis of the Oncogenic Role of Twinfilin Actin Binding Protein 1 in Human Tumors. Front Oncol.2021; 11:692136.

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2024 2024 The Author(s). Cancer Communications published by John Wiley & Sons Australia, Ltd on behalf of Sun Yat-sen University Cancer Center.