The online version of this article (doi:10.1186/1476-4598-11-14) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
WP carried out the molecular genetic studies and drafted the manuscript. WW and HO assisted with in vitro experiments and data analysis. MO and SL processed the patient liver samples and collected clinical information. HH and BC conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
We had previously demonstrated overexpression of fibroblast growth factor receptor-4 (FGFR4) in hepatocellular carcinoma (HCC). However, additional molecular mechanisms resulting in amplified FGFR4 signaling in HCC remain under-studied. Here, we studied the mechanistic role of its co-receptor klotho-beta (KLB) in driving elevated FGFR4 activity in HCC progression.
Quantitative real-time PCR analysis identified frequent elevation of KLB gene expression in HCC tumors relative to matched non-tumor tissue, with a more than two-fold increase correlating with development of multiple tumors in patients. KLB-silencing in Huh7 cells decreased cell proliferation and suppressed FGFR4 downstream signaling. While transient repression of KLB-FGFR4 signaling decreased protein expression of alpha-fetoprotein (AFP), a HCC diagnostic marker, prolonged inhibition enriched for resistant HCC cells exhibiting increased liver stemness.
Elevated KLB expression in HCC tissues provides further credence to the oncogenic role of increased FGFR4 signaling in HCC progression and represents a novel biomarker to identify additional patients amenable to anti-FGFR4 therapy. The restricted tissue expression profile of KLB, together with the anti-proliferative effect observed with KLB-silencing, also qualifies it as a specific and potent therapeutic target for HCC patients. The enrichment of a liver stem cell-like population in response to extended KLB-FGFR4 repression necessitates further investigation to target the development of drug resistance.
Additional file 1: Table S1 Clinicopathological parameters of HCC patients. (DOC 67 KB)12943_2012_1006_MOESM1_ESM.DOC
Additional file 2: Figure S1 KLB mRNA is overexpressed in HCC (Oncomine expression array data). Expression of KLB mRNA in HCC tissue relative to (A) adjacent non-tumor liver tissues [ 34] and (B) normal liver tissues from normal patients [ 35]. (DOC 59 KB)
Additional file 3: Figure S2 Upregulation of CYP7A1 mRNA in KLB-silenced Huh7 cells. 48 h post siRNA transfection in Huh7 cells, CYP7A1 gene expression was measured by qRT-PCR and normalized to GAPDH. Results are indicated with SD converted to fold changes as error bars. (DOC 87 KB)12943_2012_1006_MOESM3_ESM.DOC
Additional file 4: Figure S3 Decreased KLB and FGFR4 expression with RNAi silencing. 48 h post siRNA silencing of (A) KLB and (B) FGFR4 in Huh7 cells, gene expression of KLB and FGFR4 were measured by qRT-PCR and normalized to GAPDH. Results are indicated with SD converted to fold changes as error bars. (DOC 55 KB)12943_2012_1006_MOESM4_ESM.DOC
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Ye QH, Qin LX, Forgues M, He P, Kim JW, Peng AC, Simon R, Li Y, Robles AI, Chen Y: Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. NatMed. 2003, 9 (4): 416-423.
Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A: Sorafenib in advanced hepatocellular carcinoma. NEnglJMed. 2008, 359 (4): 378-390. 10.1056/NEJMoa0708857. CrossRef
Roberts LR, Gores GJ: Hepatocellular carcinoma: molecular pathways and new therapeutic targets. SeminLiver Dis. 2005, 25 (2): 212-225. CrossRef
Kan M, Wu X, Wang F, McKeehan WL: Specificity for fibroblast growth factors determined by heparan sulfate in a binary complex with the receptor kinase. JBiolChem. 1999, 274 (22): 15947-15952.
Streit S, Bange J, Fichtner A, Ihrler S, Issing W, Ullrich A: Involvement of the FGFR4 Arg388 allele in head and neck squamous cell carcinoma. IntJCancer. 2004, 111 (2): 213-217.
Bange J, Prechtl D, Cheburkin Y, Specht K, Harbeck N, Schmitt M, Knyazeva T, Muller S, Gartner S, Sures I: Cancer progression and tumor cell motility are associated with the FGFR4 Arg(388) allele. Cancer Res. 2002, 62 (3): 840-847. PubMed
Ho HK, Pok S, Streit S, Ruhe JE, Hart S, Lim KS, Loo HL, Aung MO, Lim SG, Ullrich A: Fibroblast growth factor receptor 4 regulates proliferation, anti-apoptosis and alpha-fetoprotein secretion during hepatocellular carcinoma progression and represents a potential target for therapeutic intervention. J Hepatol. 2009, 50 (1): 118-127. 10.1016/j.jhep.2008.08.015 CrossRefPubMed
Kurosu H, Choi M, Ogawa Y, Dickson AS, Goetz R, Eliseenkova AV, Mohammadi M, Rosenblatt KP, Kliewer SA, Kuro-o M: Tissue-specific expression of betaKlotho and fibroblast growth factor (FGF) receptor isoforms determines metabolic activity of FGF19 and FGF21. J Biol Chem. 2007, 282 (37): 26687- 10.1074/jbc.M704165200 PubMedCentralCrossRefPubMed
Ito S, Fujimori T, Furuya A, Satoh J, Nabeshima Y: Impaired negative feedback suppression of bile acid synthesis in mice lacking betaKlotho. JClinInvest. 2005, 115 (8): 2202-2208.
Ogawa Y, Kurosu H, Yamamoto M, Nandi A, Rosenblatt KP, Goetz R, Eliseenkova AV, Mohammadi M, Kuro-o M: BetaKlotho is required for metabolic activity of fibroblast growth factor 21. ProcNatlAcadSciUSA. 2007, 104 (18): 7432-7437. CrossRef
Schmelzer E, Zhang L, Bruce A, Wauthier E, Ludlow J, Yao HL, Moss N, Melhem A, McClelland R, Turner W: Human hepatic stem cells from fetal and postnatal donors. JExpMed. 2007, 204 (8): 1973-1987. 10.1084/jem.20061603. CrossRef
Zhu Z, Hao X, Yan M, Yao M, Ge C, Gu J, Li J: Cancer stem/progenitor cells are highly enriched in CD133(+)CD44(+) population in hepatocellular carcinoma. Int J Cancer. 2009,
Huang X, Yang C, Jin C, Luo Y, Wang F, McKeehan WL: Resident hepatocyte fibroblast growth factor receptor 4 limits hepatocarcinogenesis. MolCarcinog. 2009, 48 (6): 553-562. CrossRef
Huang X, Yu C, Jin C, Yang C, Xie R, Cao D, Wang F, McKeehan WL: Forced expression of hepatocyte-specific fibroblast growth factor 21 delays initiation of chemically induced hepatocarcinogenesis. MolCarcinog. 2006, 45 (12): 934-942. CrossRef
Luo Y, Yang C, Lu W, Xie R, Jin C, Huang P, Wang F, McKeehan WL: Metabolic regulator betaKlotho interacts with fibroblast growth factor receptor 4 (FGFR4) to induce apoptosis and inhibit tumor cell proliferation. J Biol Chem. 2010, 285 (39): 30069-30078. 10.1074/jbc.M110.148288 PubMedCentralCrossRefPubMed
Nicholes K, Guillet S, Tomlinson E, Hillan K, Wright B, Frantz GD, Pham TA, llard-Telm L, Tsai SP, Stephan JP: A mouse model of hepatocellular carcinoma: ectopic expression of fibroblast growth factor 19 in skeletal muscle of transgenic mice. AmJPathol. 2002, 160 (6): 2295-2307.
Schmitz KJ, Wohlschlaeger J, Lang H, Sotiropoulos GC, Malago M, Steveling K, Reis H, Cicinnati VR, Schmid KW, Baba HA: Activation of the ERK and AKT signalling pathway predicts poor prognosis in hepatocellular carcinoma and ERK activation in cancer tissue is associated with hepatitis C virus infection. JHepatol. 2008, 48 (1): 83-90. 10.1016/j.jhep.2007.08.018. CrossRef
Llovet JM, Chen Y, Wurmbach E, Roayaie S, Fiel MI, Schwartz M, Thung SN, Khitrov G, Zhang W, Villanueva A: A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis. Gastroenterology. 2006, 131 (6): 1758-1767. 10.1053/j.gastro.2006.09.014 CrossRefPubMed
- Klotho-beta overexpression as a novel target for suppressing proliferation and fibroblast growth factor receptor-4 signaling in hepatocellular carcinoma
Myat Oo Aung
Seng Gee Lim
Boon Tin Chua
Han Kiat Ho
- BioMed Central
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