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Hepatocyte growth factor upregulation promotes carcinogenesis and epithelial-mesenchymal transition in hepatocellular carcinoma via Akt and COX-2 pathways

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Abstract

Advanced hepatocellular carcinoma (HCC) is an important cause of cancer mortality. Epithelial-mesenchymal transition (EMT) has been shown to be an important biological process in cancer progression and metastasis. We have focused on elucidating factors that induce EMT to promote carcinogenesis and subsequent metastasis in HCC using the BNL CL.2 (BNL) and BNL 1ME A. 7R.1 (1MEA) cell lines. BNL cells are normal hepatocytes whereas the 1MEA cells are HCC cells derived from chemical transformation of the BNL cells. Their morphological characteristics were examined. Expression levels of hepatocyte growth factor (HGF), markers of EMT and mediators of HGF signaling were determined and functional characteristics were compared. BNL cells were treated with HGF and effects on EMT-marker and mediators of HGF signaling were analyzed. BNL cells display characteristic epithelial morphology whereas 1MEA cells display mesenchymal characteristics. 1MEA cells express and secrete more HGF than BNL cells. There was significantly decreased expression of E-cadherin, albumin, AAT and increased expression of fibronectin, collagen-1, vimentin, snail and slug in 1MEA cells. There was also increased expression of cyclooxygenase-2 (COX-2), Akt and phosphorylated Akt (pAkt) in 1MEA cells. Moreover, 1MEA cells had increased migratory capacity inhibited by inhibition of COX-2 and Akt but not extracellular signal regulated kinase (ERK). Molecular mesenchymal characteristics of 1MEA cells were reversed by inhibition of COX-2, Akt and ERK. Treatment of BNL cells with HGF led to decreased expression of E-cadherin and increased expression of fibronectin, vimentin, snail, slug, COX-2, Akt, pAkt and increased migration, invasiveness and clonogenicity. We conclude that development of HCC is associated with upregulation of HGF which promotes EMT and carcinogenesis via upregulation of COX-2 and Akt. Consequently, HGF signaling may be targeted for therapy in advanced and metastatic HCC.

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Abbreviations

COX-2:

Cyclooxygenase-2

ERK:

Extracellular signal regulated kinase

HGF:

Hepatocyte growth factor

DMSO:

Dimethyl sulfoxide

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Acknowledgments

This work was supported by the National Institutes of Health grant R01CA133086 to Chen Liu. Olorunseun O. Ogunwobi is a Postdoctoral Fellow on a NIH T32 grant.

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Authors and Affiliations

  1. Department of Pathology, Immunology and Laboratory Medicine and Shands Cancer Center, University of Florida, 1600 SW Archer Road, M651, PO 100275, Gainesville, FL, 32610, USA

    Olorunseun O. Ogunwobi & Chen Liu

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  1. Olorunseun O. Ogunwobi
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  2. Chen Liu
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Corresponding author

Correspondence to Chen Liu.

Electronic supplementary material

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10585_2011_9404_MOESM1_ESM.tif

Supplementary Figure 1 1MEA cells display molecular mesenchymal characteristics in comparison to BNL cells. A, B, D, E. Increased collagen I, vimentin, snail and slug expression respectively in 1MEA cells. C. Decreased AAT expression in 1MEA cells. *P < 0.05; **P < 0.01, N = 3 (TIFF 96 kb)

10585_2011_9404_MOESM2_ESM.tif

Supplementary Figure 2 Assessment of COX-2, ERK and Akt expression and effects of their inhibition on mesenchymal characteristics. Selective inhibitors of Akt (10 μM LY 294002), ERK (25 μM PD 98059) and COX-2 (1 μM celecoxib) inhibit Akt (A), ERK (B) and COX-2 (C). Inhibition of ERK but not Akt also leads to COX-2 inhibition (C). Inhibition of COX-2, Akt and ERK leads to reversal of vimentin (D), collagen I (E) and E-cadherin (F) by 1MEA cells. *P < 0.05, **P < 0.01, N = 3 (TIFF 109 kb)

10585_2011_9404_MOESM3_ESM.tif

Supplementary Figure 3 Migration, clonogenicity and in vivo tumorigenicity. A. 1MEA cells have greater migratory capacity than BNL cells. Selective inhibitors of Akt (10μM LY 294002) and COX-2 (1 μM celecoxib) but not ERK (25 μM PD 98059) inhibited the increased migratory capacity of 1MEA cells. B. 1MEA cells have greater clonogenicity than BNL cells. HGF stimulates increased clonogenicity of BNL cells. C. 1MEA cells but not BNL cells develop tumors in Balb/c mice. *P < 0.05, **P < 0.01, N = 3 (TIFF 876 kb)

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Ogunwobi, O.O., Liu, C. Hepatocyte growth factor upregulation promotes carcinogenesis and epithelial-mesenchymal transition in hepatocellular carcinoma via Akt and COX-2 pathways. Clin Exp Metastasis 28, 721–731 (2011). https://doi.org/10.1007/s10585-011-9404-x

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  • DOI: https://doi.org/10.1007/s10585-011-9404-x

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