Klotho: a tumor suppressor and modulator of the Wnt/β-catenin pathway in human hepatocellular carcinoma

Abstract

Klotho, an anti-aging gene, has recently been shown to contribute to human hepatic tumorigenesis. In addition, it is known that Wnt signaling is antagonized by the protein klotho. Because augmented Wnt signaling has an important role in tumorigenesis of human hepatocellular carcinoma (HCC), we studied the relationship of klotho expression and activity to the Wnt pathway in this malignancy. Immunohistochemical analysis performed on tissue arrays revealed that klotho expression levels were significantly lower in HCC than in adjacent noncancerous tissues, while klotho staining was inversely correlated with clinical stage and histologic grade. Patients with klotho-expressing tumors had longer survival periods than did those with klotho-negative tumors. Overexpression of klotho as well as treatment with soluble klotho protein reduced hepatoma cell growth in vitro and in vivo, whereas klotho silencing enhanced cellular proliferation. Moreover, forced expression of klotho inhibited Wnt/β-catenin signaling, as confirmed by reduced expression of β-catenin, inhibition of translocation of β-catenin from the cytoplasm to the nucleus, and reduced expression of c-myc and cyclin D1, two known target genes of the Wnt/β-catenin pathway. In contrast, activation of the Wnt/β-catenin pathway was enhanced when klotho was silenced by inhibitory RNAs. Furthermore, serum levels of soluble klotho in patients with malignant tumors were studied, and results suggested a significant increase in these levels in HCC patients. These data suggest that klotho acts as a tumor suppressor and an inhibitor of the Wnt/β-catenin pathway in HCC, and moreover, that soluble klotho is a potential serum biomarker for HCC.

Conflict of interest statement

Disclosure/Conflict of Interest: The authors declare no conflict of interest.

Figures

Figure 1

Reduced klotho expression in HCC tissues is associated with reduced survival of HCC patients, (a) Immunohistochemical staining reveals low-klotho expression in HCC tissues (left panels, ×200 and ×400 magnification) and high-klotho expression in adjacent non-tumor liver parenchyma (right panels, ×200 and ×400 magnification), (b) Kaplan-Meier survival analyses show worse survival in HCC patients with klotho-negative (n = 20) than those with klotho-positive tumors (n = 32); P<0.05 by log-rank test. HCC, hepatocellular carcinoma.

Figure 2

Forced overexpression of klotho inhibits HCC cell growth, (a) Western blotting analysis of native klotho protein expression levels in L02, HepG1, and HepG2 cell lines, (b) Western blotting analysis of klotho protein expression levels in HepG2 and SMMC-7721 cell lines, (c) Klotho protein level measured by western blot, (d) cellular viability by MTT, and (e) apoptosis by flow cytometry, respectively, in HepG2 cells transiently transfected with empty vector (pCMV6) or a klotho overexpression vector (pCMV6-KL) for 48 h. Western blot bands are representative of three independent experiments. The bars represent means of three independent replicates per group, with s.e. bars (*t-test P<0.05). HCC, hepatocellular carcinoma.

Figure 3

Knockdown of klotho enhances HCC cell growth. Klotho protein expression measured by western blotting (a), cellular viability by MTT (b), and apoptosis by flow cytometry (c), respectively, in HepG2 cells transfected with a nonspecific siRNA (siRNAc) or a klotho-specific siRNA (siRNAc-kl) for 48 h, respectively. Western blotting bands are representative of three independent experiments. Bars represent means of three independent replicates per group, with s.e. bars (*t-test P<0.05). HCC, hepatocellular carcinoma.

Figure 4

Blockage of Wnt signaling by klotho in HCC cells. Western blots of whole-cell (a and d), cytosolic (b and e), and endonuclear (b and e) fractions reveals the expression levels of β-catenin, c-Myc, and cyclin D1 in HepG2 cells transfected with klotho plasmid (a and b) or klotho-specific siRNA (d and e) for 48 h, respectively. β-actin was used as a whole-cell and cytoplasmic internal standard, while histone H3 was used as a nuclear internal standard to exclude cross-contamination during subcellular fraction isolation. Bands are representative of three independent experiments, (c) Representative immunofluorescence images show inhibition of translocation of β-catenin from the cytoplasm to the nucleus in HepG2 cells transfected with klotho plasmid vs empty vector for 48 h, respectively. β-catenin is labeled green, while nuclei were blue counterstained with DAPI and membranes were red with anti-klotho antibody. Magnification 400 ×. HCC, hepatocellular carcinoma.

Figure 5

sKL inhibits tumor growth in vitro and in vivo, (a) Diminished viability of HepG2 cells treated with sKL. MTT assays were conducted at 48 h. The bars represent means of three Independent replicates per group, with s.e. bars. *P<0.05 for treated cells vs control, (b) Diminished levels of β-catenin, c-Myc, and cyclin D1 protein in HepG2 cells treated with sKL (1.00μg/ml) for 48 h. Western blotting bands are representative of three independent experiments, (c) Whole-animal weight in HCC-bearing mice treated by intratumoral injections of sKL (7 μg/kg) or saline control twice weekly for 4 weeks (n = 5 per group). Weight was measured weekly, (d) Diminished tumor volume in sKL-treated vs control mice, monitored weekly. (e) Diminished tumor weight and (f) grossly visible tumor size in sKL-treated vs control mice, measured on the day of sacrifice (*P<0.05). HCC, hepatocellular carcinoma; sKL, soluble human klotho protein.

Figure 6

Serum levels of soluble klotho in HCC patients, (a) Klotho serum levels in HCC patients (n=20) were significantly higher than in control subjects (n = 29). (b) Receiver operating characteristic (ROC) curve analysis of serum klotho levels in 20 HCC patients vs 29 controls (*P<0.05). HCC, hepatocellular carcinoma.