Hepatocarcinogenesis commonly involves the gradual progression from hepatitis to fibrosis and cirrhosis, and ultimately to hepatocellular carcinoma (HCC). Endothelin 1 (Edn1) has been identified as a gene that is significantly up-regulated in HBx-induced HCC in mice. In this study, we further investigated the role of edn1 in hepatocarcinogenesis using a transgenic zebrafish model and a cell culture system. Liver-specific edn1 expression caused steatosis, fibrosis, glycogen accumulation, bile duct dilation, hyperplasia, and HCC in zebrafish. Overexpression of EDN1 in 293T cells enhanced cell proliferation and cell migration in in vitro and xenotransplantation assays and was accompanied with up-regulation of several cell cycle/proliferation- and migration-specific genes. Furthermore, expression of the unfolded protein response (UPR) pathway-related mediators, such as spliced XBP1, ATF6, IRE1, and PERK, was also up-regulated at both the RNA and protein levels. In the presence of an EDN1 inhibitor or an AKT inhibitor, these increases were diminished and the EDN1-induced migration ability also was disappeared, suggesting that the EDN1 effects act through activation of the AKT pathway to enhance the UPR and subsequently activate the expression of downstream genes. Additionally, p-AKT is enhanced in the edn1 transgenic fish compared to the GFP-mCherry control. The micro RNA miR-1 was found to inhibit the expression of EDN1. We also observed an inverse correlation between EDN1 and miR-1 expression in HCC patients. In conclusion, our data suggest that EDN1 plays an important role in HCC progression by activating the PI3K/AKT pathway and is regulated by miR-1.
Marrero CR, Marrero JA (2007) Viral hepatitis and hepatocellular carcinoma. Arch Med Res 38: 612–620.
[4]
McGlynn KA, London WT (2005) Epidemiology and natural history of hepatocellular carcinoma. Best Pract Res Clin Gastroenterol 19: 3–23.
[5]
Morgan TR, Mandayam S, Jamal MM (2004) Alcohol and hepatocellular carcinoma. Gastroenterology 127: S87–96.
[6]
Seeff LB, Hoofnagle JH (2006) Epidemiology of hepatocellular carcinoma in areas of low hepatitis B and hepatitis C endemicity. Oncogene 25: 3771–3777.
[7]
Jain S, Singhal S, Lee P, Xu R (2010) Molecular genetics of hepatocellular neoplasia. Am J Transl Res 2: 105–118.
[8]
Lin TY, Lee CS, Chen KM, Chen CC (1987) Role of surgery in the treatment of primary carcinoma of the liver: a 31-year experience. Br J Surg 74: 839–842.
Kim CM, Koike K, Saito I, Miyamura T, Jay G (1991) HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature 351: 317–320.
[11]
van Malenstein H, Verslype C, Windmolders P, van Eijsden R, Nevens F, et al. (2012) Characterization of a cell culture model for clinically aggressive hepatocellular carcinoma induced by chronic hypoxia. Cancer Lett 315: 178–188.
[12]
Pfab T, Stoltenburg-Didinger G, Trautner C, Godes M, Bauer C, et al. (2004) The endothelin system in Morris hepatoma-7777: an endothelin receptor antagonist inhibits growth in vitro and in vivo. Br J Pharmacol 141: 215–222.
[13]
Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, et al. (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411–415.
[14]
Komuro I, Kurihara H, Sugiyama T, Yoshizumi M, Takaku F, et al. (1988) Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells. FEBS Lett 238: 249–252.
[15]
Pedram A, Razandi M, Hu RM, Levin ER (1997) Vasoactive peptides modulate vascular endothelial cell growth factor production and endothelial cell proliferation and invasion. J Biol Chem 272: 17097–17103.
[16]
Matsuura A, Yamochi W, Hirata K, Kawashima S, Yokoyama M (1998) Stimulatory interaction between vascular endothelial growth factor and endothelin-1 on each gene expression. Hypertension 32: 89–95.
[17]
Rosano L, Varmi M, Salani D, Di Castro V, Spinella F, et al. (2001) Endothelin-1 induces tumor proteinase activation and invasiveness of ovarian carcinoma cells. Cancer Res 61: 8340–8346.
[18]
Zhao Y, Liao Q, Zhu Y, Long H (2011) Endothelin-1 promotes osteosarcoma cell invasion and survival against cisplatin-induced apoptosis. Clin Orthop Relat Res 469: 3190–3199.
[19]
Ali H, Loizidou M, Dashwood M, Savage F, Sheard C, et al. (2000) Stimulation of colorectal cancer cell line growth by ET-1 and its inhibition by ET(A) antagonists. Gut 47: 685–688.
[20]
Kojima K, Nihei Z (1995) Expression of endothelin-1 immunoreactivity in breast cancer. Surg Oncol 4: 309–315.
[21]
Nelson JB, Hedican SP, George DJ, Reddi AH, Piantadosi S, et al. (1995) Identification of endothelin-1 in the pathophysiology of metastatic adenocarcinoma of the prostate. Nat Med 1: 944–949.
[22]
Nakamuta M, Ohashi M, Tabata S, Tanabe Y, Goto K, et al. (1993) High plasma concentrations of endothelin-like immunoreactivities in patients with hepatocellular carcinoma. Am J Gastroenterol 88: 248–252.
[23]
Oikawa T, Kushuhara M, Ishikawa S, Hitomi J, Kono A, et al. (1994) Production of endothelin-1 and thrombomodulin by human pancreatic cancer cells. Br J Cancer 69: 1059–1064.
[24]
Asham E, Shankar A, Loizidou M, Fredericks S, Miller K, et al. (2001) Increased endothelin-1 in colorectal cancer and reduction of tumour growth by ET(A) receptor antagonism. Br J Cancer 85: 1759–1763.
[25]
Grant K, Knowles J, Dawas K, Burnstock G, Taylor I, et al. (2007) Mechanisms of endothelin 1-stimulated proliferation in colorectal cancer cell lines. Br J Surg 94: 106–112.
[26]
Kar S, Yousem SA, Carr BI (1995) Endothelin-1 expression by human hepatocellular carcinoma. Biochem Biophys Res Commun 216: 514–519.
[27]
Ishibashi M, Fujita M, Nagai K, Kako M, Furue H, et al. (1993) Production and secretion of endothelin by hepatocellular carcinoma. J Clin Endocrinol Metab 76: 378–383.
[28]
Lu JW, Hsia Y, Yang WY, Lin YI, Li CC, et al. (2012) Identification of the common regulators for hepatocellular carcinoma induced by hepatitis B virus X antigen in a mouse model. Carcinogenesis 33: 209–219.
[29]
Datta J, Kutay H, Nasser MW, Nuovo GJ, Wang B, et al. (2008) Methylation mediated silencing of MicroRNA-1 gene and its role in hepatocellular carcinogenesis. Cancer Res 68: 5049–5058.
[30]
Lam SH, Wu YL, Vega VB, Miller LD, Spitsbergen J, et al. (2006) Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression. Nat Biotechnol 24: 73–75.
[31]
Lieschke GJ, Currie PD (2007) Animal models of human disease: zebrafish swim into view. Nat Rev Genet 8: 353–367.
[32]
Lu JW, Yang WY, Tsai SM, Lin YM, Chang PH, et al. (2013) Liver-Specific Expressions of HBx and src in the p53 Mutant Trigger Hepatocarcinogenesis in Zebrafish. PLoS One 8: e76951.
[33]
Liu W, Chen JR, Hsu CH, Li YH, Chen YM, et al. (2012) A zebrafish model of intrahepatic cholangiocarcinoma by dual expression of hepatitis B virus X and hepatitis C virus core protein in liver. Hepatology 56: 2268–2276.
[34]
Nguyen AT, Emelyanov A, Koh CH, Spitsbergen JM, Lam SH, et al. (2011) A high level of liver-specific expression of oncogenic Kras(V12) drives robust liver tumorigenesis in transgenic zebrafish. Dis Model Mech 4: 801–813.
[35]
Nguyen AT, Emelyanov A, Koh CH, Spitsbergen JM, Parinov S, et al. (2012) An inducible kras(V12) transgenic zebrafish model for liver tumorigenesis and chemical drug screening. Dis Model Mech 5: 63–72.
[36]
Li Z, Zheng W, Wang Z, Zeng Z, Zhan H, et al. (2013) A transgenic zebrafish liver tumor model with inducible Myc expression reveals conserved Myc signatures with mammalian liver tumors. Dis Model Mech 6: 414–423.
[37]
Tseng WF, Jang TH, Huang CB, Yuh CH (2011) An evolutionarily conserved kernel of gata5, gata6, otx2 and prdm1a operates in the formation of endoderm in zebrafish. Dev Biol 357: 541–557.
[38]
Kwan KM, Fujimoto E, Grabher C, Mangum BD, Hardy ME, et al. (2007) The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn 236: 3088–3099.
[39]
Zhu S, Lee JS, Guo F, Shin J, Perez-Atayde AR, et al. (2012) Activated ALK collaborates with MYCN in neuroblastoma pathogenesis. Cancer Cell 21: 362–373.
[40]
Lu JW, Lin YM, Chang JG, Yeh KT, Chen RM, et al. (2013) Clinical implications of deregulated CDK4 and Cyclin D1 expression in patients with human hepatocellular carcinoma. Med Oncol 30: 379.
[41]
Boorman GA, Botts S, Bunton TE, Fournie JW, Harshbarger JC, et al. (1997) Diagnostic criteria for degenerative, inflammatory, proliferative nonneoplastic and neoplastic liver lesions in medaka (Oryzias latipes): consensus of a National Toxicology Program Pathology Working Group. Toxicol Pathol 25: 202–210.
[42]
Hobbie KR, DeAngelo AB, George MH, Law JM (2012) Neoplastic and nonneoplastic liver lesions induced by dimethylnitrosamine in Japanese medaka fish. Vet Pathol 49: 372–385.
[43]
Lu JW, Yang WY, Lin YM, Jin SL, Yuh CH (2013) Hepatitis B virus X antigen and aflatoxin B1 synergistically cause hepatitis, steatosis and liver hyperplasia in transgenic zebrafish. Acta Histochem 115: 728–739.
[44]
Shen C, Gu M, Song C, Miao L, Hu L, et al. (2008) The tumorigenicity diversification in human embryonic kidney 293 cell line cultured in vitro. Biologicals 36: 263–268.
[45]
Kulasekaran P, Scavone CA, Rogers DS, Arenberg DA, Thannickal VJ, et al. (2009) Endothelin-1 and transforming growth factor-beta1 independently induce fibroblast resistance to apoptosis via AKT activation. Am J Respir Cell Mol Biol 41: 484–493.
[46]
Wu MH, Lo JF, Kuo CH, Lin JA, Lin YM, et al. (2012) Endothelin-1 promotes MMP-13 production and migration in human chondrosarcoma cells through FAK/PI3K/Akt/mTOR pathways. J Cell Physiol 227: 3016–3026.
[47]
Her GM, Pai WY, Lai CY, Hsieh YW, Pang HW (2013) Ubiquitous transcription factor YY1 promotes zebrafish liver steatosis and lipotoxicity by inhibiting CHOP-10 expression. Biochim Biophys Acta 1831: 1037–1051.
[48]
Her GM, Hsu CC, Hong JR, Lai CY, Hsu MC, et al. (2011) Overexpression of gankyrin induces liver steatosis in zebrafish (Danio rerio). Biochim Biophys Acta 1811: 536–548.
[49]
Shieh YS, Chang YS, Hong JR, Chen LJ, Jou LK, et al. (2010) Increase of hepatic fat accumulation by liver specific expression of Hepatitis B virus X protein in zebrafish. Biochim Biophys Acta 1801: 721–730.
[50]
Duan Q, Wang X, Gong W, Ni L, Chen C, et al. (2012) ER stress negatively modulates the expression of the miR-199a/214 cluster to regulates tumor survival and progression in human hepatocellular cancer. PLoS One 7: e31518.
[51]
Arai M, Kondoh N, Imazeki N, Hada A, Hatsuse K, et al. (2006) Transformation-associated gene regulation by ATF6alpha during hepatocarcinogenesis. FEBS Lett 580: 184–190.
