Klotho, an anti-aging gene, acts as a tumor suppressor and inhibitor of IGF-1R signaling in diffuse large B cell lymphoma

Abstract

Background: Klotho, is a transmembrane protein, performs as a circulating hormone and upstream modulator of the insulin-like growth factor-1 receptor (IGF-1R), fibroblast growth factor (FGF), and Wnt signaling pathways. These pathways are involved in the development and progression of B cell lymphoma. We aimed to explore the expression pattern and functional mechanism of Klotho in diffuse large B cell lymphoma (DLBCL).

Methods: Immunohistochemistry (IHC) and western blotting were performed to detect the expression level of Klotho in DLBCL patients and cell lines. Tumor suppressive effect of Klotho was determined by both in vitro and in vivo studies. Signaling pathway activity was assessed by western blotting.

Results: Remarkable lower expression levels of Klotho were observed in DLBCL patients and cell lines. Enforced expression of Klotho could significantly induce cell apoptosis and inhibit tumor growth in DLBCL. Upregulation of Klotho resulted in declined activation of IGF-1R signaling, accompanied with decreased phosphorylation of its downstream targets, including AKT and ERK1/2. Moreover, xenograft model treated with either Klotho overexpression vector or recombinant human Klotho administration presented restrained tumor growth and lower Ki67 staining.

Conclusions: Our findings establish that Klotho performs as a tumor suppressor and modulator of IGF-1R signaling in DLBCL. Targeting Klotho may provide novel strategies for future therapeutic intervention.

Keywords: Diffuse large B cell lymphoma; Insulin growth factor-1 receptor; Klotho; Tumor suppressor.

Figures

Fig. 1

Klotho was downregulated in DLBCL and related to tumor progression. a Compared with reactive hyperplasia, expression level of Klotho was significantly decreased in DLBCL tissues. Original magnification, ×100 (upper panel) and ×400 (lower panel). b Patients with Klotho negative expression showed shorter survival than those with Klotho positive expression. c As detected by real-time quantitative PCR, lower levels of Klotho mRNA expression were observed in DLBCL cell lines (LY1, LY8) than in CD19+ B cells (N1, N2, N3) (mean ± SD, n = 3, *p < 0.05). d Protein expression levels of Klotho were detected in DLBCL cells and normal PBMCs. The ratios of relative protein expression level of targets are indicated below the western blot

Fig. 2

Klotho suppressed DLBCL growth. a, b Relative expression levels of Klotho were confirmed by quantitative PCR (mean ± SD, n = 3, **p < 0.01) and western blot in stably transfected LY1 and LY8 cells compared to empty vectors. The ratios of relative protein expression level of targets are indicated below the bands. c DLBCL cells transfected with LV-KL presented significantly lower level of cell proliferation than those transfected with empty vector (mean ± SD, n = 3, **p < 0.01). d SCID mice with Klotho overexpression revealed significantly lower tumor volume than those with empty vector (n = 6 per group, **p < 0.01). e H&E staining and IHC staining of Ki67 and Klotho were performed in mice tumors. Original magnification, ×400

Fig. 3

Klotho promoted apoptosis of DLBCL. a, b Enforced expression of Klotho resulted in increased apoptosis rates in LY1 and LY8 cells assessed by flow cytometric analysis with Annexin V-PE/7AAD staining (mean ± SD, n = 3, **p < 0.01). c Declined expression levels of Mcl-1 and increased levels of activated Caspase-3 were observed in LV-KL-treated DLBCL cells. The ratios of relative protein expression level of targets are indicated below the bands

Fig. 4

Klotho modulated activation of IGF-1R pathway in DLBCL. a IGF-1-induced DLBCL cell (LY1 and LY8) proliferation is inhibited by Klotho overexpression. LY1 and LY8 cells were transfected with LV-KL or LV-Con, starved for 48 h and treated by IGF-1 (50 ng/ml) and analyzed by CCK-8 assay (mean ± SD, n = 3, *p < 0.05, **p < 0.01). b LY1 with stable transfection of LV-KL or LV-Con were serum starved for 48 h and treated with IGF-1 (50 ng/ml) for the indicated times or IGF-1 (30 min) for the indicated doses. After treatment, cells were harvested and analyzed by western blot. c LY1 and LY8 cells transfected with LV-KL or LV-Con, serum starved for 48 h, and treated with IGF-1 (50 ng/ml, 30 min). Western blot was conducted to assess the phosphorylated (p) and total (t) protein levels of IGF-1R, AKT, and ERK1/2. The ratios of relative protein expression level of targets are indicated below the bands. d Decreased activation of IGF1-R signaling was observed in LV-KL-treated mice. The ratios of relative protein expression level of targets are indicated below the bands. e Schematic description of Klotho mediated IGF1R signaling

Fig. 5

rhKL acted as an active form in vitro and vivo. a, b LY1 and LY8 cells were treated with rhKL, ADR, their combination, or a vehicle control. CCK-8 assay was conducted after 48 h (mean ± SD, n = 3, *p < 0.05 for comparison between treated cells and control, **p < 0.05 between Klotho and ADR combination versus ADR alone). c LY1 cells were injected subcutaneously into the left inferior legs of SCID Beige mice. The mice were treated with daily intraperitoneal injections of rhKL (7.5 μg/kg) or vehicle control (PBS) for 2 weeks. Tumor volumes were measured every 2 days (n = 6 per group, *p < 0.05). d H&E staining and IHC staining with Ki67 were performed. Original magnification, ×400. e Lower serum Klotho levels were detected by Elisa in DLBCL patients than the control subjects