C/EBPα Regulates FOXC1 to Modulate Tumor Growth by Interacting with PPARγ in Hepatocellular Carcinoma

Author(s): Zhuo Xu, Shao-Hua Meng, Jian-Guo Bai, Chao Sun*, Li-Li Zhao*, Rui-Feng Tang, Zhao-Lin Yin, Jun-Wei Ji, Wei Yang, Guang-Jun Ma.

Journal Name: Current Cancer Drug Targets

Volume 20 , Issue 1 , 2020

  Journal Home
Translate in Chinese
Become EABM
Become Reviewer

Graphical Abstract:


Abstract:

Background: Forkhead box C1 (FOXC1) is an important cancer-associated gene in tumor. PPAR-γ and C/EBPα are both transcriptional regulators involved in tumor development.

Objective: We aimed to clarify the function of PPAR-γ, C/EBPα in hepatocellular carcinoma (HCC) and the relationship of PPAR-γ, C/EBPα and FOXC1 in HCC.

Methods: Western blotting, immunofluorescent staining, and immunohistochemistry were used to evaluate protein expression. qRT-PCR was used to assess mRNA expression. Co-IP was performed to detect the protein interaction. And ChIP and fluorescent reporter detection were used to determine the binding between protein and FOXC1 promoter.

Results: C/EBPα could bind to FOXC1 promoter and PPAR-γ could strengthen C/EBPα’s function. Expressions of C/EBPα and PPAR-γ were both negatively related to FOXC1 in human HCC tissue. Confocal displayed that C/EBPα was co-located with FOXC1 in HepG2 cells. C/EBPα could bind to FOXC1 promoter by ChIP. Luciferase activity detection exhibited that C/EBPα could inhibit FOXC1 promoter activity, especially FOXC1 promoter from -600 to -300 was the critical binding site. Only PPAR-γ could not influence luciferase activity but strengthen inhibited effect of C/EBPα. Further, the Co-IP displayed that PPAR-γ could bind to C/EBPα. When C/EBPα and PPAR-γ were both high expressed, cell proliferation, migration, invasion, and colony information were inhibited enormously. C/EBPα plasmid combined with or without PPAR-γ agonist MDG548 treatment exhibited a strong tumor inhibition and FOXC1 suppression in mice.

Conclusion: Our data establish C/EBPα targeting FOXC1 as a potential determinant in the HCC, which supplies a new pathway to treat HCC. However, PPAR-γ has no effect on FOXC1 expression.

Keywords: HCC, FOXC1, PPAR-γ, C/EBPα, proliferation, hepatocellular carcinoma.

[1]
Jemal, A.; Bray, F.; Center, M.M.; Ferlay, J.; Ward, E.; Forman, D. Global cancer statistics. CA Cancer J. Clin., 2011, 61(2), 69-90.
[http://dx.doi.org/10.3322/caac.20107] [PMID: 21296855]
[2]
Ahn, S.M.; Jang, S.J.; Shim, J.H.; Kim, D.; Hong, S.M.; Sung, C.O.; Baek, D.; Haq, F.; Ansari, A.A.; Lee, S.Y.; Chun, S.M.; Choi, S.; Choi, H.J.; Kim, J.; Kim, S.; Hwang, S.; Lee, Y.J.; Lee, J.E.; Jung, W.R.; Jang, H.Y.; Yang, E.; Sung, W.K.; Lee, N.P.; Mao, M.; Lee, C.; Zucman-Rossi, J.; Yu, E.; Lee, H.C.; Kong, G. Genomic portrait of resectable hepatocellular carcinomas: Implications of RB1 and FGF19 aberrations for patient stratification. Hepatology, 2014, 60(6), 1972-1982.
[http://dx.doi.org/10.1002/hep.27198] [PMID: 24798001]
[3]
Myatt, S.S.; Lam, E.W. The emerging roles of forkhead box (Fox) proteins in cancer. Nat. Rev. Cancer, 2007, 7(11), 847-859.
[http://dx.doi.org/10.1038/nrc2223] [PMID: 17943136]
[4]
Seo, S.; Singh, H.P.; Lacal, P.M.; Sasman, A.; Fatima, A.; Liu, T.; Schultz, K.M.; Losordo, D.W.; Lehmann, O.J.; Kume, T. Forkhead box transcription factor FoxC1 preserves corneal transparency by regulating vascular growth. Proc. Natl. Acad. Sci. USA, 2012, 109(6), 2015-2020.
[http://dx.doi.org/10.1073/pnas.1109540109] [PMID: 22171010]
[5]
Seo, S.; Fujita, H.; Nakano, A.; Kang, M.; Duarte, A.; Kume, T. The forkhead transcription factors, Foxc1 and Foxc2, are required for arterial specification and lymphatic sprouting during vascular development. Dev. Biol., 2006, 294(2), 458-470.
[http://dx.doi.org/10.1016/j.ydbio.2006.03.035] [PMID: 16678147]
[6]
Bloushtain-Qimron, N.; Yao, J.; Snyder, E.L.; Shipitsin, M.; Campbell, L.L.; Mani, S.A.; Hu, M.; Chen, H.; Ustyansky, V.; Antosiewicz, J.E.; Argani, P.; Halushka, M.K.; Thomson, J.A.; Pharoah, P.; Porgador, A.; Sukumar, S.; Parsons, R.; Richardson, A.L.; Stampfer, M.R.; Gelman, R.S.; Nikolskaya, T.; Nikolsky, Y.; Polyak, K. Cell type-specific DNA methylation patterns in the human breast. Proc. Natl. Acad. Sci. USA, 2008, 105(37), 14076-14081.
[http://dx.doi.org/10.1073/pnas.0805206105] [PMID: 18780791]
[7]
Wang, L.; Gu, F.; Liu, C.Y.; Wang, R.J.; Li, J.; Xu, J.Y. High level of FOXC1 expression is associated with poor prognosis in pancreatic ductal adenocarcinoma. Tumour Biol., 2013, 34(2), 853-858.
[http://dx.doi.org/10.1007/s13277-012-0617-7] [PMID: 23242609]
[8]
Ray, P.S.; Wang, J.; Qu, Y.; Sim, M.S.; Shamonki, J.; Bagaria, S.P.; Ye, X.; Liu, B.; Elashoff, D.; Hoon, D.S.; Walter, M.A.; Martens, J.W.; Richardson, A.L.; Giuliano, A.E.; Cui, X. FOXC1 is a potential prognostic biomarker with functional significance in basal-like breast cancer. Cancer Res., 2010, 70(10), 3870-3876.
[http://dx.doi.org/10.1158/0008-5472.CAN-09-4120] [PMID: 20406990]
[9]
Taube, J.H.; Herschkowitz, J.I.; Komurov, K.; Zhou, A.Y.; Gupta, S.; Yang, J.; Hartwell, K.; Onder, T.T.; Gupta, P.B.; Evans, K.W.; Hollier, B.G.; Ram, P.T.; Lander, E.S.; Rosen, J.M.; Weinberg, R.A.; Mani, S.A. Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes. Proc. Natl. Acad. Sci. USA, 2010, 107(35), 15449-15454.
[http://dx.doi.org/10.1073/pnas.1004900107] [PMID: 20713713]
[10]
Sizemore, S.T.; Keri, R.A. The forkhead box transcription factor FOXC1 promotes breast cancer invasion by inducing matrix metalloprotease 7 (MMP7) expression. J. Biol. Chem., 2012, 287(29), 24631-24640.
[http://dx.doi.org/10.1074/jbc.M112.375865] [PMID: 22645147]
[11]
Huang, W.; Chen, Z.; Zhang, L.; Tian, D.; Wang, D.; Fan, D.; Wu, K.; Xia, L. Interleukin-8 induces expression of FOXC1 to promote transactivation of CXCR1 and CCL2 in hepatocellular carcinoma cell lines and formation of metastases in mice. Gastroenterology, 2015, 149(4), 1053-67. e14
[12]
Novak, P.; Stampfer, M.R.; Munoz-Rodriguez, J.L.; Garbe, J.C.; Ehrich, M.; Futscher, B.W.; Jensen, T.J. Cell-type specific DNA methylation patterns define human breast cellular identity. PLoS One, 2012, 7(12)e52299
[http://dx.doi.org/10.1371/journal.pone.0052299] [PMID: 23284978]
[13]
Dong, Y.W.; Wang, X.P.; Wu, K. Suppression of pancreatic carcinoma growth by activating peroxisome proliferator-activated receptor gamma involves angiogenesis inhibition. World J. Gastroenterol., 2009, 15(4), 441-448.
[http://dx.doi.org/10.3748/wjg.15.441] [PMID: 19152448]
[14]
Shigeto, T.; Yokoyama, Y.; Xin, B.; Mizunuma, H. Peroxisome proliferator-activated receptor alpha and gamma ligands inhibit the growth of human ovarian cancer. Oncol. Rep., 2007, 18(4), 833-840.
[PMID: 17786343]
[15]
Cao, L.Q.; Shao, Z.L.; Liang, H.H.; Zhang, D.W.; Yang, X.W.; Jiang, X.F.; Xue, P. Activation of peroxisome proliferator-activated receptor-γ (PPARγ) inhibits hepatoma cell growth via downregulation of SEPT2 expression. Cancer Lett., 2015, 359(1), 127-135.
[http://dx.doi.org/10.1016/j.canlet.2015.01.004] [PMID: 25592041]
[16]
Hsu, H.T.; Sung, M.T.; Lee, C.C.; Kuo, Y.J.; Chi, C.W.; Lee, H.C.; Hsia, C.Y. Peroxisome proliferator-activated receptor γ expression is inversely associated with macroscopic vascular invasion in human hepatocellular carcinoma. Int. J. Mol. Sci., 2016, 17(8)E1226
[http://dx.doi.org/10.3390/ijms17081226] [PMID: 27483249]
[17]
Shen, B.; Chu, E.S.; Zhao, G.; Man, K.; Wu, C.W.; Cheng, J.T.; Li, G.; Nie, Y.; Lo, C.M.; Teoh, N.; Farrell, G.C.; Sung, J.J.; Yu, J. PPARgamma inhibits hepatocellular carcinoma metastases in vitro and in mice. Br. J. Cancer, 2012, 106(9), 1486-1494.
[http://dx.doi.org/10.1038/bjc.2012.130] [PMID: 22472882]
[18]
Yu, J.; Shen, B.; Chu, E.S.; Teoh, N.; Cheung, K.F.; Wu, C.W.; Wang, S.; Lam, C.N.; Feng, H.; Zhao, J.; Cheng, A.S.; To, K.F.; Chan, H.L.; Sung, J.J. Inhibitory role of peroxisome proliferator-activated receptor gamma in hepatocarcinogenesis in mice and in vitro. Hepatology, 2010, 51(6), 2008-2019.
[http://dx.doi.org/10.1002/hep.23550] [PMID: 20512989]
[19]
Pang, X.; Wei, Y.; Zhang, Y.; Zhang, M.; Lu, Y.; Shen, P. Peroxisome proliferator-activated receptor-γ activation inhibits hepatocellular carcinoma cell invasion by upregulating plasminogen activator inhibitor-1. Cancer Sci., 2013, 104(6), 672-680.
[http://dx.doi.org/10.1111/cas.12143] [PMID: 23461356]
[20]
Walter, I.; Schulz, U.; Vogelhuber, M.; Wiedmann, K.; Endlicher, E.; Klebl, F.; Andreesen, R.; Herr, W.; Ghibelli, L.; Hackl, C.; Wiest, R.; Reichle, A. Communicative reprogramming non-curative hepatocellular carcinoma with low-dose metronomic chemotherapy, COX-2 inhibitor and PPAR-gamma agonist: a phase II trial. Med. Oncol., 2017, 34(12), 192.
[http://dx.doi.org/10.1007/s12032-017-1040-0] [PMID: 29098441]
[21]
Diezko, R.; Suske, G. Ligand binding reduces SUMOylation of the peroxisome proliferator-activated receptor γ (PPARγ) activation function 1 (AF1) domain. PLoS One, 2013, 8(6) e66947
[http://dx.doi.org/10.1371/journal.pone.0066947] [PMID: 23826177]
[22]
Schmidt, S.F.; Jørgensen, M.; Chen, Y.; Nielsen, R.; Sandelin, A.; Mandrup, S. Cross species comparison of C/EBPα and PPARγ profiles in mouse and human adipocytes reveals interdependent retention of binding sites. BMC Genomics, 2011, 12, 152.
[http://dx.doi.org/10.1186/1471-2164-12-152] [PMID: 21410980]
[23]
Koschmieder, S.; Halmos, B.; Levantini, E.; Tenen, D.G. Dysregulation of the C/EBPalpha differentiation pathway in human cancer. J. Clin. Oncol., 2009, 27(4), 619-628.
[http://dx.doi.org/10.1200/JCO.2008.17.9812] [PMID: 19075268]
[24]
Lu, G. D.; Leung, C. H.; Yan, B.; Tan, C. M.; Low, S. Y.; Aung, M. O.; Salto-Tellez, M.; Lim, S. G.; Hooi, S. C. C/EBPalpha is upregulated in a subset of hepatocellular carcinomas and plays a role in cell growth and proliferation. Gastroenterology., 2010, 139(2), 632-43, 643.
[25]
Lu, G.D.; Ang, Y.H.; Zhou, J.; Tamilarasi, J.; Yan, B.; Lim, Y.C.; Srivastava, S.; Salto-Tellez, M.; Hui, K.M.; Shen, H.M.; Nguyen, L.N.; Tan, B.C.; Silver, D.L.; Hooi, S.C. CCAAT/enhancer binding protein α predicts poorer prognosis and prevents energy starvation-induced cell death in hepatocellular carcinoma. Hepatology, 2015, 61(3), 965-978.
[http://dx.doi.org/10.1002/hep.27593] [PMID: 25363290]
[26]
Pabst, T.; Mueller, B.U.; Zhang, P.; Radomska, H.S.; Narravula, S.; Schnittger, S.; Behre, G.; Hiddemann, W.; Tenen, D.G. Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia. Nat. Genet., 2001, 27(3), 263-270.
[http://dx.doi.org/10.1038/85820] [PMID: 11242107]
[27]
Wouters, B.J.; Jordà, M.A.; Keeshan, K.; Louwers, I.; Erpelinck-Verschueren, C.A.; Tielemans, D.; Langerak, A.W.; He, Y.; Yashiro-Ohtani, Y.; Zhang, P.; Hetherington, C.J.; Verhaak, R.G.; Valk, P.J.; Löwenberg, B.; Tenen, D.G.; Pear, W.S.; Delwel, R. Distinct gene expression profiles of acute myeloid/T-lymphoid leukemia with silenced CEBPA and mutations in NOTCH1. Blood, 2007, 110(10), 3706-3714.
[http://dx.doi.org/10.1182/blood-2007-02-073486] [PMID: 17671232]
[28]
Cast, A.; Valanejad, L.; Wright, M.; Nguyen, P.; Gupta, A.; Zhu, L.; Shin, S.; Timchenko, N. C/EBPα-dependent preneoplastic tumor foci are the origin of hepatocellular carcinoma and aggressive pediatric liver cancer. Hepatology, 2018, 67(5), 1857-1871.
[http://dx.doi.org/10.1002/hep.29677] [PMID: 29159818]
[29]
Xu, J.D.; Jiang, H.S.; Wei, T.D.; Zhang, K.Y.; Wang, X.W.; Zhao, X.F.; Wang, J.X. Interaction of the small GTPase Cdc42 with arginine kinase restricts white spot syndrome virus in shrimp. J. Virol., 2017, 91(5), e01916-e16.
[http://dx.doi.org/10.1128/JVI.01916-16] [PMID: 28031362]
[30]
Chen, Y.J.; Wu, H.; Shen, X.Z. The ubiquitin-proteasome system and its potential application in hepatocellular carcinoma therapy. Cancer Lett., 2016, 379(2), 245-252.
[http://dx.doi.org/10.1016/j.canlet.2015.06.023] [PMID: 26193663]
[31]
Dawson, S.P. Hepatocellular carcinoma and the ubiquitin-proteasome system. Biochim. Biophys. Acta, 2008, 1782(12), 775-784.
[http://dx.doi.org/10.1016/j.bbadis.2008.08.003] [PMID: 18778769]
[32]
Bosch, F.X.; Ribes, J.; Díaz, M.; Cléries, R. Primary liver cancer: Worldwide incidence and trends. Gastroenterology, 2004, 127(5)(Suppl. 1), S5-S16.
[http://dx.doi.org/10.1053/j.gastro.2004.09.011] [PMID: 15508102]
[33]
Frau, M.; Feo, C.F.; Feo, F.; Pascale, R.M. New insights on the role of epigenetic alterations in hepatocellular carcinoma. J. Hepatocell. Carcinoma, 2014, 1, 65-83.
[PMID: 27508177]
[34]
Tomizawa, M.; Watanabe, K.; Saisho, H.; Nakagawara, A.; Tagawa, M. Down-regulated expression of the CCAAT/enhancer binding protein alpha and beta genes in human hepatocellular carcinoma: A possible prognostic marker. Anticancer Res., 2003, 23(1A), 351-354.
[PMID: 12680236]
[35]
Misra, S.K.; Ray, T.; Ostadhossein, F.; Kim, B.; Ray, P.S.; Pan, D. Carotenoid nanovector for efficient therapeutic gene knockdown of transcription factor FOXC1 in liver cancer. Bioconjug. Chem., 2016, 27(3), 594-603.
[http://dx.doi.org/10.1021/acs.bioconjchem.5b00601] [PMID: 26720420]
[36]
Zeng, C.; Wang, R.; Li, D.; Lin, X.J.; Wei, Q.K.; Yuan, Y.; Wang, Q.; Chen, W.; Zhuang, S.M. A novel GSK-3 beta-C/EBP alpha-miR-122-insulin-like growth factor 1 receptor regulatory circuitry in human hepatocellular carcinoma. Hepatology, 2010, 52(5), 1702-1712.
[http://dx.doi.org/10.1002/hep.23875] [PMID: 21038412]
[37]
Lan, Y.; Han, J.; Wang, Y.; Wang, J.; Yang, G.; Li, K.; Song, R.; Zheng, T.; Liang, Y.; Pan, S.; Liu, X.; Zhu, M.; Liu, Y.; Meng, F.; Mohsin, M.; Cui, Y.; Zhang, B.; Subash, S.; Liu, L. STK17B promotes carcinogenesis and metastasis via AKT/GSK-3β/Snail signaling in hepatocellular carcinoma. Cell Death Dis., 2018, 9(2), 236.
[http://dx.doi.org/10.1038/s41419-018-0262-1] [PMID: 29445189]
[38]
Yu, J.; Qiao, L.; Zimmermann, L.; Ebert, M.P.; Zhang, H.; Lin, W.; Röcken, C.; Malfertheiner, P.; Farrell, G.C. Troglitazone inhibits tumor growth in hepatocellular carcinoma in vitro and in vivo. Hepatology, 2006, 43(1), 134-143.
[http://dx.doi.org/10.1002/hep.20994] [PMID: 16374840]
[39]
Medina-Trillo, C.; Aroca-Aguilar, J.D.; Ferre-Fernández, J.J.; Méndez-Hernández, C.D.; Morales, L.; García-Feijoo, J.; Escribano, J. The role of hsa-miR-548l dysregulation as a putative modifier factor for glaucoma-associated FOXC1 mutations. MicroRNA, 2015, 4(1), 50-56.
[http://dx.doi.org/10.2174/2211536604666150320234654] [PMID: 25809640]


Rights & PermissionsPrintExport Cite as


Article Details

VOLUME: 20
ISSUE: 1
Year: 2020
Page: [59 - 66]
Pages: 8
DOI: 10.2174/1568009619666190912161003
Price: $65

Article Metrics

PDF: 34
HTML: 2