Advertisement

Applied Microbiology and Biotechnology

, Volume 101, Issue 21, pp 7823–7835 | Cite as

Expression and purification of an FGF9 fusion protein in E. coli, and the effects of the FGF9 subfamily on human hepatocellular carcinoma cell proliferation and migration

  • Shen Wang
  • Haipeng Lin
  • Tiantian Zhao
  • Sisi Huang
  • David G. Fernig
  • Nuo Xu
  • Fenfang Wu
  • Mi Zhou
  • Chao JiangEmail author
  • Haishan TianEmail author
Biotechnological products and process engineering

Abstract

Fibroblast growth factor (FGF) 9 has oncogenic activity and plays an important role in the development of ovarian, lung, prostate, and gastric cancers. In the present study, with the aim of reducing the cost of utilizing growth factors in cancer research, a simple and efficient method for the preparation of recombinant human (rh)FGF9 in Escherichia coli was established. The rhFGF9 fusion protein (6 × His-TEV-rhFGF9) and the native protein released by tobacco etch virus (TEV) protease were obtained using a Ni-NTA system, with > 95% purity. Both purified forms of rhFGF9, with and without fusion tags, significantly stimulated the proliferation of NIH3T3 cells. The FGF9 subfamily, including FGF9, FGF16, and FGF20, in addition to rhFGF16, rhFGF9, and rhFGF20, were shown to stimulate the proliferation and migration of HuH7 human hepatocellular carcinoma (HCC) cells. Mechanistic studies revealed that the stimulation of HuH7 cell proliferation and migration with rhFGF9 and rhFGF20 were associated with the activation of the extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) pathways and matrix metalloproteinase-26 (MMP26). Inhibition of the ERK and NF-κB pathways blocked cell migration, and NF-κB was demonstrated to be regulated by ERK. Therefore, the present study demonstrates a simple method for the preparation of biologically active rhFGF9 protein. Furthermore, the results indicate that exogenous rhFGF9- and rhFGF20-activated ERK/NF-κB signal transduction pathways play important roles in the regulation of HCC cell proliferation and migration, and this discovery helps to find the potential for new solutions of the treatment of liver cancer.

Keywords

Recombinant human FGF9 Fusion expression TEV protease cleavage Mitogen activity HuH7 cells Signal transduction mechanism 

Notes

Acknowledgments

This work was supported by grants of National Natural Science Foundation of China (No. 81471075) and granted by the Opening Project of Zhejiang Provincial TOP Key Discipline of Pharmaceutical Sciences (No.201720).

Compliance with ethical standards

This article does not contain any studies with animals or human participants. All authors confirm that ethical principles have been followed in the experiments.

Conflicts of interest

The authors declare that they have no conflict of interest.

References

  1. Ahokas K, Karjalainen-Lindsberg ML, Sihvo E, Isaka K, Salo J, Saarialho-Kere U (2006) Matrix metalloproteinases 21 and 26 are differentially expressed in esophageal squarnous cell cancer. Tumour Biol 27(3):133–141.  https://doi.org/10.1159/000092774 CrossRefPubMedGoogle Scholar
  2. Asada M, Shinomiya M, Suzuki M, Honda E, Sugimoto R, Ikekita M, Imamura T (2009) Glycosaminoglycan affinity of the complete fibroblast growth factor family. Biochim Biophys Acta 1790(1):40–48.  https://doi.org/10.1016/j.bbagen.2008.09.001 CrossRefPubMedGoogle Scholar
  3. Basu M, Mukhopadhyay S, Chatterjee U, Roy SS (2014) FGF16 Promotes invasive behavior of SKOV-3 ovarian cancer cells through activation of mitogen-activated protein kinase (MAPK) signaling pathway. J Biol Chem 289(3):1415–1428.  https://doi.org/10.1074/jbc.M113.535427 CrossRefPubMedGoogle Scholar
  4. Bruix J, Sherman M, Practice Guidelines Committee, American Association for the Study of Liver Diseases (2005) Management of hepatocellular carcinoma. Hepatology 42(5):1208–1236.  https://doi.org/10.1002/hep.20933 CrossRefPubMedGoogle Scholar
  5. Chamorro MN, Schwartz DR, Vonica A, Brivanlou AH, Cho KR, Varmus HE (2005) FGF-20 and DKK1 are transcriptional targets of beta-catenin and FGF-20 is implicated in cancer and development. EMBO J 24(1):73–84.  https://doi.org/10.1038/sj.emboj.7600460 CrossRefPubMedGoogle Scholar
  6. Di Bisceglie AM (2004) Issues in screening and surveillance for hepatocellular carcinoma. Gastroenterology 127(5 Suppl 1):S104–S107CrossRefPubMedGoogle Scholar
  7. Hendrix ND, Wu R, Kuick R, Schwartz DR, Fearon ER, Cho KR (2006) Fibroblast growth factor 9 has oncogenic activity and is a downstream target of Wnt signaling in ovarian endometrioid adenocarcinomas. Cancer Res 66(3):1354–1362.  https://doi.org/10.1158/0008-5472.CAN-05-3694 CrossRefPubMedGoogle Scholar
  8. Hsu FT, Liu YC, Chiang IT, Liu RS, Wang HE, Lin WJ, Hwang JJ (2014) Sorafenib increases efficacy of vorinostat against human hepatocellular carcinoma through transduction inhibition of vorinostat-induced ERK/NF-kappa B signaling. Int J Oncol 45(1):177–188.  https://doi.org/10.3892/ijo.2014.2423 CrossRefPubMedGoogle Scholar
  9. Imamura H, Matsuyama Y, Tanaka E, Ohkubo T, Hasegawa K, Miyagawa S, Sugawara Y, Minagawa M, Takayama T, Kawasaki S, Makuuchi M (2003) Risk factors contributing to early and late phase intrahepatic recurrence of hepatocellular carcinoma after hepatectomy. J Hepatol 38(2):200–207CrossRefPubMedGoogle Scholar
  10. Itoh N, Ornitz DM (2008) Functional evolutionary history of the mouse Fgf gene family. Dev Dyn 237(1):18–27.  https://doi.org/10.1002/dvdy.21388 CrossRefPubMedGoogle Scholar
  11. Koyama N, Ohmae H, Tsuji S, Tanaka Y, Kurokawa T, Nishimura O (2001) Improved preparation and crystallization of 25 kDa human fibroblast growth factor-9. Biotechnol Appl Biochem 33(Pt 2):117–121CrossRefPubMedGoogle Scholar
  12. Li Y, Basilico C, Mansukhani A (1994) Cell transformation by fibroblast growth factors can be suppressed by truncated fibroblast growth factor receptors. Mol Cell Biol 14(11):7660–7669CrossRefPubMedPubMedCentralGoogle Scholar
  13. Li J, Lau GK, Chen L, Dong SS, Lan HY, Huang XR, Li Y, Luk JM, Yuan YF, Guan XY (2011) Interleukin 17A promotes hepatocellular carcinoma metastasis via NF-kB induced matrix metalloproteinases 2 and 9 expression. PLoS One 6(7):e21816.  https://doi.org/10.1371/journal.pone.0021816 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Llovet JM, Villanueva A, Lachenmayer A, Finn RS (2015) Advances in targeted therapies for hepatocellular carcinoma in the genomic era. Nat Rev Clin Oncol 12(8):436.  https://doi.org/10.1038/nrclinonc.2015.121 CrossRefPubMedGoogle Scholar
  15. Luedde T, Schwabe RF (2011) NF-kappa B in the liver-linking injury, fibrosis and hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 8(2):108–118.  https://doi.org/10.1038/nrgastro.2010.213 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Marchenko GN, Marchenko ND, Leng J, Strongin AY (2002) Promoter characterization of the novel human matrix metalloproteinase-26 gene: regulation by the T-cell factor-4 implies specific expression of the gene in cancer cells of epithelial origin. Biochem J 363(Pt2):253–262CrossRefPubMedPubMedCentralGoogle Scholar
  17. Ornitz DM, Itoh N (2015) The fibroblast growth factor signaling pathway. Wiley Interdiscip Rev Dev Biol 4(3):215–266.  https://doi.org/10.1002/wdev.176 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Pathak A, Kumar S (2011) Biophysical regulation of tumor cell invasion: moving beyond matrix stiffness. Integr Biol (Camb) 3(4):267–278.  https://doi.org/10.1039/c0ib00095g CrossRefGoogle Scholar
  19. Riddick AC, Shukla CJ, Pennington CJ, Bass R, Nuttall RK, Hogan A, Sethia KK, Ellis V, Collins AT, Maitland NJ, Ball RY, Edwards DR (2005) Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues. Br J Cancer 92(12):2171–2180.  https://doi.org/10.1038/sj.bjc.6602630 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Roberts PJ, Der CJ (2007) Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 26(22):3291–3210.  https://doi.org/10.1038/sj.onc.1210422 CrossRefPubMedGoogle Scholar
  21. Santos-Ocampo S, Colvin JS, Chellaiah A, Ornitz DM (1996) Expression and biological activity of mouse fibroblast growth factor-9. J Biol Chem 271(3):1726–1731CrossRefPubMedGoogle Scholar
  22. Structural Genomics Consortium, China Structural Genomics Consortium, Northeast Structural Genomics Consortium, Graslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schutz A, Heinemann U, Yokoyama S, Bussow K, Gunsalus KC (2008) Protein production and purification. Nat Methods 5(2):135–146.  https://doi.org/10.1038/nmeth.f.202 CrossRefPubMedCentralGoogle Scholar
  23. Sun C, Li Y, Taylor SE, Mao XQ, Wilkinson MC, Fernig DG (2015) HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors. PeerJ 3:e1060.  https://doi.org/10.7717/peerj.1060 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Suyama K, Shapiro I, Guttman M, Hazan RB (2002) A signaling pathway leading to metastasis is controlled by N-cadherin and the FGF receptor. Cancer Cell 2(4):301–314.  https://doi.org/10.1016/S1535-6108(02)00150-2 CrossRefPubMedGoogle Scholar
  25. Tanner Y, Grose RP (2016) Dysregulated FGF signalling in neoplastic disorders. Semin Cell Dev Biol 53:126–135.  https://doi.org/10.1016/j.semcdb.2015.10.012 CrossRefPubMedGoogle Scholar
  26. Turner N, Grose R (2010) Fibroblast growth factor signalling: from development to cancer. Nat Rev Cancer 10(2):116–129.  https://doi.org/10.1038/nrc2780 CrossRefPubMedGoogle Scholar
  27. Uria JA, Lopez-Otin C (2000) Matrilysin-2, a new matrix metalloproteinase expressed in human tumors and showing the minimal domain organization required for secretion, latency, and activity. Cancer Res 60(17):4745–4751PubMedGoogle Scholar
  28. Wang J, Su H, Han X, Xu K (2014) Inhibition of fibroblast growth factor receptor signaling impairs metastasis of hepatocellular carcinoma. Tumour Biol 35(11):11005–11011.  https://doi.org/10.1007/s13277-014-2384-0 CrossRefPubMedGoogle Scholar
  29. Whittaker S, Marais R, Zhu AX (2010) The role of signaling pathways in the development and treatment of hepatocellular carcinoma. Oncogene 29(36):4989–5005.  https://doi.org/10.1038/onc.2010.236 CrossRefPubMedGoogle Scholar
  30. Xu J, Zhu X, Wu L, Yang R, Yang Z, Wang QF, Wu F (2012) MicroRNA-122 suppresses cell proliferation and induces cell apoptosis in hepatocellular carcinoma by directly targeting Wnt/ss-catenin pathway. Liver Int 32(5):752–760.  https://doi.org/10.1111/j.1478-3231.2011.02750.x CrossRefPubMedGoogle Scholar
  31. Yang H, Fang F, Chang R, Yang L (2013) MicroRNA-140-5p suppresses tumor growth and metastasis by targeting transforming growth factor beta receptor 1 and fibroblast growth factor 9 in hepatocellular carcinoma. Hepatology 58(1):205–217.  https://doi.org/10.1002/hep.26315 CrossRefPubMedGoogle Scholar
  32. Yi S, Yang J, Huang J, Guan L, Du L, Guo Y, Zhai F, Wang Y, Lu Z, Wang L, Li H, Li X, Jiang C (2016) Expression of bioactive recombinant human fibroblast growth factor 9 in oil bodies of Arabidopsis thaliana. Protein Expr Purif 116:127–132.  https://doi.org/10.1016/j.pep.2015.08.006 CrossRefGoogle Scholar
  33. Yu C, Wang Z, Xu X, Xiang W, Huang X (2015) Circulating hepatocellular carcinoma cells are characterized by CXCR4 and MMP26. Cell Physiol Biochem 36(6):2393–2402.  https://doi.org/10.1159/000430201 CrossRefPubMedGoogle Scholar
  34. Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, Ornitz DM (2006) Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem 281(23):15694–15700.  https://doi.org/10.1074/jbc.M601252200 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Zhao YG, Xiao AZ, Park HI, Newcomer RG, Yan M, Man YG, Heffelfinger SC, Sang QXA (2004) Endometase/matrilysin-2 in human breast ductal carcinoma in situ and its inhibition by tissue inhibitors of metalloproteinases-2 and -4: a putative role in the initiation of breast cancer invasion. Cancer Res 64(2):590–598CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.School of Pharmaceutical ScienceWenzhou Medical UniversityWenzhouChina
  2. 2.Department of Biochemistry, Institute of Integrative BiologyUniversity of LiverpoolLiverpoolUK
  3. 3.Biomedicine Collaborative Innovation CenterWenzhou UniversityWenzhouChina

Personalised recommendations