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. 2012 Mar 23:11:14.
doi: 10.1186/1476-4598-11-14.

Klotho-beta overexpression as a novel target for suppressing proliferation and fibroblast growth factor receptor-4 signaling in hepatocellular carcinoma

Weijie Poh  1 , Winnie Wong, Huimin Ong, Myat Oo Aung, Seng Gee Lim, Boon Tin Chua, Han Kiat Ho
Affiliations

Klotho-beta overexpression as a novel target for suppressing proliferation and fibroblast growth factor receptor-4 signaling in hepatocellular carcinoma

Weijie Poh et al. Mol Cancer. .

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

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Figures

Figure 1
Figure 1
KLB expression is upregulated in HCC tumors and cell lines. (A) Analysis of KLB gene expression using qRT-PCR in tumor tissue relative to its matched normal tissue after normalizing to GPADH mRNA. Fold change of < 0.5 indicates down-regulation of KLB expression while > 2-fold change indicates KLB up-regulation in tumors. (B) Wilcoxon signed rank test was used to analyze the difference of KLB gene expression in paired normal (Δ) and tumor liver tissues (○). (C) KLB protein expression was determined in Huh7 cells and an independent set of paired HCC lysates. The ratio of KLB between tumor and normal tissue was calculated as (tumor KLB/normal KLB) after normalizing the protein band density to β-actin. (D) The Wilcoxon signed rank test was used to analyze the difference in KLB/β-actin expression between the two groups. (E) KLB mRNA expression in HCC cell lines are expressed as fold changes relative to THLE2 with error bars representing standard deviation (SD) converted to fold changes. (***, P < 0.001 using Student's t-test).
Figure 2
Figure 2
Silencing of KLB with siRNA suppresses cell viability and proliferation. Huh7 cells were transfected with 100 nM scrambled control or KLB-targeting siRNA. (A) After 48 h, qRT-PCR was performed to confirm KLB knockdown after normalizing to GAPDH. Error bars represent SD converted to fold changes. (B) At 48 h post transfection, Huh7 cells were re-seeded in a 96-well plate. After additional 24 h incubation, cell viability was measured using CellTiter-Glo and evaluated relative to cells transfected with scrambled siRNA. (C) At the indicated time points after transfection, Huh7 cells were harvested for counting. (D) Immunoblotting with PCNA antibody was performed 72 h post siRNA transfection using tubulin as loading control. For A-C, means of three independent experiments are indicated with SD as error bars. (***, P < 0.001; *, P < 0.05 using Student's t-test).
Figure 3
Figure 3
KLB mediates FGFR4 downstream signaling and phosphorylation of pro-survival proteins. 72 h post-transfection with siRNA, Huh7 cells were lysed, resolved with 7.5% SDS-PAGE and immunoblotted using the indicated antibodies. Results are indicative of two independent experiments.
Figure 4
Figure 4
Silencing of KLB or FGFR4 increases expression of liver stemness genes. (A) 48 h post siRNA silencing of KLB or FGFR4 in Huh7 cells, AFP gene expression was determined by qRT-PCR. Data indicates fold change in gene expression normalized to scrambled siRNA. Error bars represent SD converted to fold changes. (B) After 72 h, AFP protein expression was also determined using polyclonal anti-AFP antibodies, normalized to β-actin as loading control. (C) CD133 and CD44 expression after KLB-silencing was subsequently determined as described for (A). (D) The effect of the silencing on CD133 protein expression was performed using anti-CD133 antibody. (E) CD133 and CD44 mRNA levels were measured in Huh7 stable clones expressing control shRNA and FGFR4 shRNA after normalizing to GAPDH.
Figure 5
Figure 5
CD133 and CD44 expression are upregulated in PD173074-resistant cells. Resistant Huh7 cells were generated by culturing in sub-lethal concentrations of PD173074 (0.1 μM - 1.0 μM). After eight weeks, (A) CD133 and (B) CD44 mRNA expression were analyzed by qRT-PCR. Results are indicated with SD converted to fold changes as error bars. The differences were statistically significant (P < 0.001 by one-way ANOVA, P < 0.05 by Tukey's multiple comparison test). (C) CD133 protein expression was determined by immunoblotting and normalized to β-actin. (D) Long term effect of PD173074 treatment on FGFR4 and KLB protein expression were determined by immunoblot assays. For (C) and (D), the respective relative protein levels were calculated relative to DMSO control after normalizing the protein band density to that of the loading control.
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