Elevated Expression of A-Raf and FA2H in Hepatocellular Carcinoma is Associated with Lipid Metabolism Dysregulation and Cancer Progression

Author(s): Maryam Ranjpour, Saima Wajid, Swatantra K. Jain*.

Journal Name: Anti-Cancer Agents in Medicinal Chemistry
(Formerly Current Medicinal Chemistry - Anti-Cancer Agents)

Volume 19 , Issue 2 , 2019

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Graphical Abstract:


Abstract:

Background: Identification of events leading to hepatocellular carcinoma (HCC) progression is essential for understanding its pathophysiology. The aims of this study are to identify and characterize differentially expressed proteins in serum of HCC-bearing rats and the corresponding controls during cancer initiation, progression and tumorigenesis.

Methods: Chemical carcinogens, N-Nitrosodiethylamine and 2-aminoacetylfluorine are administered to induce HCC to male Wistar rats. The 2D-Electrophoresis and PD-Quest analyses are performed to identify several differentially expressed proteins in serum of HCC-bearing animals. These proteins are further characterized by MALDI-TOF-MS/MS analyses. Using pathwaylinker a HCC-specific network is analyzed among the MALDITOF- MS/MS characterized proteins and their interactors.

Results: Carcinogen administration caused inflammation leading to liver injury and HCC development. Liver inflammation was confirmed by increase in the levels of TNF-α and IL-6 in carcinogen treated rats. We report significant increase in expression of two differentially expressed proteins, namely, A-Raf and Fatty Acid 2- Hydroxylase (FA2H), at early stage of HCC initiation, during its progression and at tumor stage. Real-time PCR analysis of mRNA for these proteins confirmed up-regulation of their transcripts. Further, we validated our experimental data with sera of clinically confirmed liver cancer patients.

Conclusion: The study suggests that FA2H and A-Raf play a major role in the progression of HCC.

Keywords: Cancer, initiation, tumors, western blot, HCC-specific network, pathwaylinker.

[1]
Kim, J.U.; Shariff, M.I.F.; Crossey, M.M.E.; Gomez-Romero, M.; Holmes, E.; Cox, I.J.; Fye, H.K.S.; Njie, R.; Taylor-Robinson, S.D. Hepatocellular carcinoma: Review of disease and tumor biomarkers. World J. Hepatol., 2016, 8(10), 471-484.
[2]
Zhen, C.; Zhu, C.; Chen, H.; Xiong, Y.; Tan, J.; Chen, D.; Li, J. Systematic analysis of molecular mechanisms for HCC metastasis via text mining approach. Oncotarget, 2017, 8(8), 13909-13916.
[3]
Varnholt, H.; Drebber, U.; Schulze, F.; Wedemeyer, I.; Schirmacher, P.; Dienes, H.P.; Odenthal, M. MicroRNA gene expression profile of hepatitis C virus-associated hepatocellular carcinoma. Hepatology, 2008, 47(4), 1223-1232.
[4]
Tsai, C.C.; Huang, K.W.; Chen, H.F.; Zhan, B.W.; Lai, Y.H.; Lee, F.H.; Lin, C.Y.; Ho, Y.C.; Chao, Y.W.; Su, Y.C.; Jane, S.W.; Chen, Y.C.; Hsu, C.I.; Li, P.H.; Hsu, H.C.; Suzuki, Y.; Sugano, S.; Lin, J.Y. Gene expression analysis of human hepatocellular carcinoma by using full-length cDNA library. J. Biomed. Sci., 2006, 13(2), 241-249.
[5]
Wee, P.; Wang, Z. Epidermal growth factor receptor cell proliferation signaling pathways. Cancers., 2017, 9(5), 52.
[6]
Steelman, L.S.; Chappell, W.H.; Abrams, S.L.; Kempf, C.R.; Long, J.; Laidler, P.; Mijatovic, S.; Maksimovic-Ivanic, D.; Stivala, F.; Mazzarino, M.C.; Donia, M.; Fagone, P.; Malaponte, G.; Nicoletti, F.; Libra, M.; Milella, M.; Tafuri, A.; Bonati, A.; Bäsecke, J.; Cocco, L.; Evangelisti, C.; Martelli, A.M.; Montalto, G.; Cervello, M.; McCubrey, J.A. Roles of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways in controlling growth and sensitivity to therapy-implications for cancer and aging. Aging (Albany N.Y.), 2011, 3(3), 192-222.
[7]
Li, L.; Zhao, G.D.; Shi, Z.; Qi, L.L.; Zhou, L.Y.; Fu, Z.X. The Ras/Raf/MEK/ERK signaling pathway and its role in the occurrence and development of HCC. Oncol. Lett., 2016, 12(5), 3045-3050.
[8]
Huerta-Yepez, S.; Yoon, N.K.; Hernandez-Cueto, A.; Mah, V.; Rivera-Pazos, C.M.; Chatterjee, D.; Vega, M.I.; Maresh, E.L.; Horvath, S.; Chia, D.; Bonavida, B.; Goodglick, L. Expression of phosphorylated Raf Kinase Inhibitor Protein (pRKIP) is a predictor of lung cancer survival. BMC Cancer, 2011, 11(1), 259.
[9]
Akyol, G.; Yilmaz, G. Stem cell expression profile in hepatocellular carcinoma, small cell dysplasia, and cirrhosis. Stem Cell Transl. Investig., 2014, 1(1), 14232.
[10]
Guo, L.; Zhou, D.; Pryse, K.M.; Okunade, A.L.; Su, X. Fatty acid 2-hydroxylase mediates diffusional mobility of Raft-associated lipids, GLUT4 level, and lipogenesis in 3T3-L1 adipocytes. J. Biol. Chem., 2010, 285(33), 25438-25447.
[11]
Daniels, V.W.; Smans, K.; Royaux, I.; Chypre, M.; Swinnen, J.V.; Zaidi, N. Cancer cells differentially activate and thrive on de novo lipid synthesis pathways in a low-lipid environment. PLoS One, 2014, 9(9), e106913.
[12]
Beloribi-Djefaflia, S.; Vasseur, S.; Guillaumond, F. Lipid metabolic reprogramming in cancer cells. Oncogenesis, 2016, 5, e189.
[13]
Malik, S.; Bhatnagar, S.; Chaudhary, N.; Katare, D.P.; Jain, S. DEN+ 2-AAF-induced multistep hepatotumorigenesis in wistar rats: Supportive evidence and insights. Protoplasma, 2013, 250(1), 175-183.
[14]
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72(1), 248-254.
[15]
Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001, 25(4), 402-408.
[16]
Chatr-aryamontri, A.; Oughtred, R.; Boucher, L.; Rust, J.; Chang, C.; Kolas, N.K.; O’Donnell, L.; Oster, S.; Theesfeld, C.; Sellam, A.; Stark, C.; Breitkreutz, B.J.; Dolinski, K.; Tyers, M. The BioGRID interaction database: 2017 update. Nucleic Acids Res., 2017, 45(Database issue), D369-D379.
[17]
Ranjpour, M.; Katare, D.P.; Wajid, S.; Jain, S.K. HCC specific protein network involving interactions of EGFR with A-Raf and transthyretin: Experimental analysis and computational biology correlates. Anticancer. Agents Med. Chem., 2018, 18(8), 1163-1176.
[18]
Schmidt-Arras, D.; Rose-John, S. IL-6 pathway in the liver: From physiopathology to therapy. J. Hepatol., 2016, 64(6), 1403-1415.
[19]
Tian, G.; Mi, J.; Wei, X.; Zhao, D.; Qiao, L.; Yang, C.; Li, X.; Zhang, S.; Li, X.; Wang, B. Circulating interleukin-6 and cancer: A meta-analysis using Mendelian randomization. Sci. Rep., 2015, 5, 11394.
[20]
Naugler, W.E.; Karin, M. The wolf in sheep’s clothing: the role of interleukin-6 in immunity, inflammation and cancer. Trends Mol. Med., 2008, 14(3), 109-119.
[21]
Alderson, N.L.; Hama, H. Fatty acid 2-hydroxylase regulates cAMP-induced cell cycle exit in D6P2T schwannoma cells. J. Lipid Res., 2009, 50(6), 1203-1208.
[22]
Alderson, N.L.; Rembiesa, B.M.; Walla, M.D.; Bielawska, A.; Bielawski, J.; Hama, H. The human FA2H gene encodes a fatty acid 2-hydroxylase. J. Biol. Chem., 2004, 279(47), 48562-48568.
[23]
Colombino, M.; Sperlongano, P.; Izzo, F.; Tatangelo, F.; Botti, G.; Lombardi, A.; Accardo, M.; Tarantino, L.; Sordelli, I.; Agresti, M.; Abbruzzese, A.; Caraglia, M.; Palmieri, G. BRAF and PIK3CA genes are somatically mutated in hepatocellular carcinoma among patients from south Italy. Cell Death Dis., 2012, 3(1), e259.
[24]
Tannapfel, A.; Sommerer, F.; Benicke, M.; Katalinic, A.; Uhlmann, D.; Witzigmann, H.; Hauss, J.; Wittekind, C. Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma. Gut, 2003, 52(5), 706-712.
[25]
Lee, J.W.; Soung, Y.H.; Kim, S.Y.; Park, W.S.; Nam, S.W.; Min, W.S.; Kim, S.H.; Lee, J.Y.; Yoo, N.J.; Lee, S.H. Mutational analysis of the ARAF gene in human cancers. APMIS, 2005, 113(1), 54-57.
[26]
Moretti, L.; Tchernin, L.; Scapozza, L. Tyrosine kinase drug discovery: What can be learned from solved crystal structures? ARKIVOC, 2006, 8, 38-49.
[27]
Rauch, J.; O’Neill, E.; Mack, B.; Matthias, C.; Munz, M.; Kolch, W.; Gires, O. Heterogeneous nuclear ribonucleoprotein H blocks MST2-mediated apoptosis in cancer cells by regulating A-Raf transcription. Cancer Res., 2010, 70(4), 1679-1688.
[28]
Rauch, J.; Kolch, W. Spatial regulation of ARAF controls the MST2-Hippo pathway. Small GTPases, 2017, 1-6.
[29]
Matallanas, D.; Birtwistle, M.; Romano, D.; Zebisch, A.; Rauch, J.; von Kriegsheim, A.; Kolch, W. Raf family kinases: Old dogs have learned new tricks. Genes Cancer, 2011, 2(3), 232-260.
[30]
Mazurek, S.; Drexler, H.C.; Troppmair, J.; Eigenbrodt, E.; Rapp, U.R. Regulation of pyruvate kinase type M2 by A-Raf: A possible glycolytic stop or go mechanism. Anticancer Res., 2007, 27(6b), 3963-3971.
[31]
Borisov, N.; Aksamitiene, E.; Kiyatkin, A.; Legewie, S.; Berkhout, J.; Maiwald, T.; Kaimachnikov, N.P.; Timmer, J.; Hoek, J.B.; Kholodenko, B.N. Systems-level interactions between insulin–EGF networks amplify mitogenic signaling. Mol. Syst. Biol., 2009, 5, 256-256.
[32]
Ferreira, I.G.; Pucci, M.; Venturi, G.; Malagolini, N.; Chiricolo, M.; Dall’Olio, F. Glycosylation as a main regulator of growth and death factor receptors signaling. Int. J. Mol. Sci., 2018, 19(2), E580.
[33]
Knowlden, J.M.; Jones, H.E.; Barrow, D.; Gee, J.M.W.; Nicholson, R.I.; Hutcheson, I.R. Insulin receptor substrate-1 involvement in epidermal growth factor receptor and insulin-like growth factor receptor signalling: implication for Gefitinib (‘Iressa’) response and resistance. Breast Cancer Res. Treat., 2008, 111(1), 79-91.
[34]
Laplante, M.; Sabatini, D.M. An emerging role of mTOR in lipid biosynthesis. Curr. Biol., 2009, 19(22), R1046-R1052.
[35]
Caillot, F.; Hiron, M.; Goria, O.; Gueudin, M.; Francois, A.; Scotte, M.; Daveau, M.; Salier, J.P. Novel serum markers of fibrosis progression for the follow-up of hepatitis C virus-infected patients. Am. J. Pathol., 2009, 175(1), 46-53.
[36]
Sousa, M.M.; Berglund, L.; Saraiva, M.J. Transthyretin in high density lipoproteins: Association with apolipoprotein A-I. J. Lipid Res., 2000, 41(1), 58-65.
[37]
Liz, M.A.; Gomes, C.M.; Saraiva, M.J.; Sousa, M.M. ApoA-I cleaved by transthyretin has reduced ability to promote cholesterol efflux and increased amyloidogenicity. J. Lipid Res., 2007, 48(11), 2385-2395.
[38]
Katare, D.P.; Malik, S.; Mani, R.J.; Ranjpour, M.; Jain, S.K. Novel mutations in transthyretin gene associated with hepatocellular carcinoma. Mol. Carcinog., 2017, 57(1), 70-77.
[39]
Sethakorn, N.; Dulin, N.O. RGS expression in cancer: Oncomining the cancer microarray data. J. Recep. Sig. Trans., 2013, 33(3), 166-171.
[40]
Bjarnadottir, O.; Kimbung, S.; Johansson, I.; Veerla, S.; Jonsson, M.; Bendahl, P.O.; Grabau, D.; Hedenfalk, I.; Borgquist, S. Global transcriptional changes following statin treatment in breast cancer. Clin. Cancer Res., 2015, 21(15), 3402-3411.


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Article Details

VOLUME: 19
ISSUE: 2
Year: 2019
Page: [236 - 247]
Pages: 12
DOI: 10.2174/1871520618666181015142810
Price: $58

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