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

This study was approved by the Medical Ethical Committee of Shandong Provincial Hospital affiliated to Shandong University. The paraffin-embedded archived samples from 50 newly diagnosed DLBCL patients and 20 reactive hyperplasia patients were collected. Samples of patients with reactive hyperplasia were referred as control. Histological diagnoses were established according to the WHO classification [26]. Peripheral blood mononuclear cells (PBMCs) from the whole blood of healthy donors were isolated using Ficoll-Hypaque density gradient centrifugation (TBD science, Tianjin, China). Normal peripheral blood CD19+ B cells were purified from freshly isolated PBMCs using CD19+ magnetic microbeads kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were incubated with beads for 15 min at 4 °C while rotating. Purified CD19+ B cells were selected according to the manufacturer’s protocols. The purity of isolated populations was assessed by FACS analysis, and cells with >90% purity were collected. All samples were obtained with informed consent in accordance with the Declaration of Helsinki.

Human DLBCL cell lines LY1 and LY8 were cultured in Iscove modified Dulbecco medium with 10% heat-inactivated fetal bovine serum. The medium contains 1% penicillin/streptomycin mixture and 2 mmol/l glutamine. CD19+ B cells and PBMCs obtained from three healthy donors were used as controls (N1, N2, and N3 cells). Recombinant human Klotho (rhKL) and recombinant human IGF-1 were obtained from PeproTech (Rocky Hill, NJ, USA), and adriamycin (ADR) was bought from Actavis (S.p.A, Italy).

Lentivirus vectors either encoding Klotho or an empty lentiviral vector were from Genechem (Shanghai, China). Lentivirus transfection was carried out according to the manufacturers’ instruction. Infection efficiencies were assessed by green fluorescent protein (GFP) through flow cytometry. The stably transfected cells were selected 48 h later with 5 μg/ml puromycin (Sigma-Aldrich, USA).

Total RNA was extracted using RNAiso Plus (TaKaRa, Dalian, China). Reverse transcription reaction was conducted with the reverse transcription reagents (TaKaRa, Dalian, China). Amplification reactions were performed with SyberGreen (TaKaRa, Dalian, China) in LightCycler 480II (Roche, Basel, Swizerland). Klotho-specific primers were as follows: forward, 5′-AGCAATCTGGTCTGAATAACACTGG; reverse, 5′-CATGTTTCAGCGTGAAAGTTCAAAG. Relative quantification was calculated using the ^△△CT method.

The 4-μm-thick formalin-fixed, paraffin-embedded tissue sections were deparaffinized and hydrate. Antigen retrieval was performed using 0.01 mol/l sodium citrate buffer (pH 6.0) under high pressure followed by a 1-h cool-down and rinses in phosphate buffer solution (PBS). Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 15 min, followed by incubation with normal serum to block non-specific binding. The slides were then incubated overnight at 4 °C with primary antibodies, anti-Klotho (1:150) or anti-Ki67 (1:100). After washing, the tissue sections were treated with the second antibody from SP reagent kit (Zhongshan Goldenbridge, Beijing, China) for 30 min at room temperature, followed by further treated with strept avidin-horseradish peroxidase complex (SABC) for 30 min at room temperature. After treated with diaminobenzidine (DAB) Kit (ZhongshanGoldenbridge, Beijing, China), the stained slides were counterstained with hematoxylin and mounted. Negative control was carried out with the primary antibody replaced by PBS. IHC staining was scored by the proportion of positive tumor cells. Five microscopic fields with the highest immunoreactivity at ×400 magnification were evaluated by two independent observers who were blinded to the patients’ clinical data. Cases with at least 10% of tumor cells with Klotho staining were considered as positive. Fresh mice subcutaneous tumors were fixed in 4% paraformaldehyde (PFA) and embedded with paraffin for histological examinations. Sections with 4-μm thickness were cut and stained with hematoxylin-eosin (H&E).

Cells were lysed in radio-immunoprecipitation assay buffer (Shenergy Biocolor, Shanghai, China) together with 1× phosphatase inhibitor cocktail (PhosSTOP; Roche, Mannheim, Germany). The BCA assay (Shenergy Biocolor, Shanghai, China) was performed to detect protein concentration. Protein extracts (30 μg) were then electrophoresed on SDS-polyacrylamide gels and blotted from the gel onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were incubated with the blocking solution (5% skim milk in Tris-buffered saline containing 0.05% Tween-20) for 1 h at room temperature and then immunoblotted with the indicated antibodies (1:1000 dilution) overnight at 4 °C. After which, the membranes were washed with TBS-T and then probed with the HRP-conjugated secondary antibodies (Zhongshan Goldenbridge, Beijing, China) and the electro-chemi-luminescence kit (Millipore, Billerica, MA, USA). Chemiluminescent signals were detected using the Amersham Imager 600 imaging system (General Electric, USA). ImageJ software (ImageJ, NIH) was used to quantify the protein bands normalized to control. Primary antibodies used were Klotho (Abcam, Cambridge, MA, USA), phospho-IGF-1R (Tyr1135/1136), IGF-1R, phospho-AKT(Ser473), total pan-AKT, Mcl-1, Caspase-3, diphosphorylated and total ERK1/2 (Cell Signaling Technologies, Beverly, MA, USA), β-actin, and GAPDH (Zhongshan Goldenbridge, Beijing, China). The experiments were performed in triplicate with GAPDH or β-actin (Zhongshan Goldenbridge, Beijing, China) as endogenous control.

Cell proliferation was assessed by performing triplicate assays with the Cell Counting Kit-8 (CCK-8) assay (Enogene, Nanjing, China). DLBCL cells with designed treatment were seeded in 96-well plates at a density of 5000 cells/well for 48 or 24~96 h later. Thereafter, the cells were incubated with 10 μl/well CCK-8 for 4 h according to the manufacturer’s proposal. Cell proliferation was detected by light absorption at 450 nm by Multiskan GO Microplate Reader (Thermo Scientific, Rockford, IL, USA).

Apoptosis of transfected DLBCL cells were detected by Annexin V-PE/7-aminoactinomycin (7AAD) (BD Biosciences, Bedford, MA, USA) assay according to the manufacturers’ instructions. DLBCL cells with designed treatments were harvested and washed twice with ice-cold PBS and incubated in 1× binding buffer (containing 5 μl Annexin V-PE and 5 μl 7AAD). After incubation in the dark for 15 min, cells were subjected to the flow cytometry. At least 10,000 events per sample were acquired. Cells were discriminated into viable cells (AnnexinV-PE⁻/7AAD⁻), dead cells (Annexin V-PE⁻/7AAD⁺), apoptotic cells (Annexin V-PE⁺/7AAD⁻), and necrotic cells (Annexin V-PE⁺/7AAD⁺). The rates of apoptotic cells were acquired on a FACS-Navios Flow Cytometer (Beckman Coulter, CA, USA). Data were analyzed using FlowJo Version 7.6 software (Tree Star Inc., OR, USA).

Serum soluble Klotho levels were measured using the Elisa kit (Immuno-Biological Laboratories, Gunma, Japan), with a lower limit of assay of 6.15 pg/ml.

All animal experiments were performed in accordance with the principles of the Institutional Animal Care. Severe combined immunodeficiency (SCID) Beige female mice of 5-week old were bought (Weitong Lihua Laboratory Animal Center, Beijing, China) and maintained in a pathogen-free environment under controlled condition of light and humidity. 1 × 10⁷ LY1 cells (untransfected, stably Klotho-overexpressing vector tranfected, or empty control vector transfected, respectively), mixed with 100 μl Matrigel (BD Biosciences, Bedford, MA, USA), were subcutaneously injected into the right inferior limb of mice. Tumor size was measured by the digital caliper. For the experiment with rhKL, SCID Beige mice were injected subcutaneously with 1 × 10⁷ LY1 cells into the left inferior limbs. Mice were treated with daily intraperitoneal injections of rhKL (7.5 μg/kg) or PBS control (six mice per group) for 2 weeks. The volume of tumor was estimated using the equation V = (a × b ²) × 0.5236, where a is the largest dimension and b is the perpendicular diameter.

All statistical analyses were performed by using the statistic software SPSS17.0 (SPSS Inc., Chicago, IL, USA) for Windows. In vitro experimental results were presented as mean ± SD of data obtained from three separate experiments. Overall survival time was measured from the date of diagnosis to the date of death or the last follow-up. Kaplan-Meier analysis was performed to estimate the survival functions. A log-rank test was used to assess survival differences. Chi-square test was used to analyze the clinical characteristics of patients. One-way analysis of variance (ANOVA) or t tests were used to assess the differences between groups. p < 0.05 was considered to be statistically significant.

Compared with reactive hyperplasia, expression level of Klotho was significantly lower in DLBCL tissues (Fig. 1a). Klotho positive rate was 14% (7 of 50) in DLBCL whereas 80% (16 of 20) in reactive hyperplasia. To evaluate the clinical significance of Klotho in DLBCL, clinical and pathological characteristics of DLBCL patients were analyzed. The expression level of Klotho was associated with the Ann Arbor stage of DLBCL patients. Patients with stage III or IV presented markedly lower Klotho level than those with stage I or II (p = 0.002, Table 1). In addition, Kaplan-Meier survival analysis were performed based on the expression levels of Klotho (n = 50). The median survival of patients in Klotho-positive group was 48.46 months, significantly longer than those in Klotho-negative group (29.27 months, p = 0.045, Fig. 1b). We next confirmed the expression of Klotho in DLBCL cell lines. Klotho mRNA expression was detected in DLBCL cell lines and normal CD19+ B cells. Significantly decreased levels of Klotho mRNA expression were observed in DLBCL cell lines (LY1 0.024 ± 0.037, LY8 0.002 ± 0001, Fig. 1c). Reduced expression levels of Klotho protein were also noted in DLBCL cell lines compared to human PBMCs (Fig. 1d).

To further establish the biological function of Klotho, human DLBCL cells were stably transfected with either negative control lentivirus vectors (LV-Con) or Klotho-overexpression lentivirus vectors (LV-KL). The upregulation of Klotho levels were confirmed by quantitative PCR and western blot (Fig. 2a, b). Cell proliferation rates were evaluated by CCK-8 assay. Significant reduction of cell proliferation was observed in LY1 and LY8 cells transfected with LV-KL, compared with those transfected with empty vector (Fig. 2c). Furthermore, we investigated the anti-tumor effect of Klotho on DLBCL xenografts in vivo. LY1 cells, stably expressing Klotho, or the vector control cells were injected subcutaneously into SCID Beige mice, respectively. Consistent with the in vitro results, mice treated with Klotho overexpression cells revealed remarkable reduction in tumor volume compared to that transfected with empty vector (Fig. 2d). Higher expression level of Klotho was identified in mice treated with LV-KL-transfected LY1 cells (Fig. 2e). In addition, we estimated the expression level of proliferative marker Ki67 [27, 28] in xenograft mice by IHC. Lower level of Ki67 positive rate were observed in LV-KL group (Fig. 2e).

To investigate whether Klotho could promote the apoptosis of DLBCL cells, Annexin-V-based apoptotic assays were performed. Flow cytometry analysis indicated that Klotho overexpression resulted in enforced apoptosis rates in both LY1 (6.56 ± 0.71% in LV-Con vs. 16.41 ± 2.16% in LV-KL group, p = 0.002) and LY8 cells (7.23 ± 0.65% in LV-Con vs. 16.03 ± 1.85% in LV-KL group, p = 0.001, Fig. 3a, b). In addition, western blot analysis confirmed the pro-apoptotic effects of Klotho. Dramatically reduction of anti-apoptotic protein Mcl-1 and increased cleaved forms of Caspase-3 were observed in DLBCL cell lines (Fig. 3c). Altogether, these results indicate that upregulation of Klotho could promote the apoptosis of DLBCL cells.

Having shown that Klotho could impair cell proliferation and induce apoptosis in DLBCL, we next investigated the molecular mechanisms responsible for the function of Klotho. The IGF-1R pathway plays a vital role in the development of hematological malignancies [14, 25, 29]. CCK-8 assay was conducted to assess the effect of Klotho on IGF-1-induced cell proliferation. DLBCL cells transfected with either LV-KL or LV-Con were treated with IGF-1 or vehicle control in 0.5% FBS culture medium for 24–96 h. In the groups untreated with IGF-1, LV-KL transfection resulted in declined proliferation of LY1 and LY8 cells compared to empty-vector group. In the IGF-1-treated groups, we observed that cell proliferation was less restored by IGF-1 in cells transfected with LV-KL compared to that transfected with LV-Con. Following addition of IGF-1, cell proliferation of LV-Con-treated cells increased by up to 60%, whereas the only up to 40% enhancement of cell proliferation was found in LV-KL transfected cells (Fig. 4a).

As the IGF-1R signaling could be activated by IGF-1, we explored the optimal activation dose and time of IGF-1 in DLBCL cell lines. LY1 cells were transfected with either LV-KL or LV-Con, serum starved for 48 h and treated with IGF-1 for the indicated times and doses. Western blot was carried out to evaluate the phosphorylation level of the IGF-1R. As shown in Fig. 4b, the maximum activation occurred at IGF-1 concentration of 50 ng/ml and at the time of 30 min after treatment. Then, we studied the ability of Klotho to modulate activation of IGF-1R signaling in DLBCL cells. Cells transfected with either LV-KL or LV-Con were serum starved for 48 h, treated with IGF-1 (50 ng/ml for 30 min) or vehicle control. Following treatment, cells were harvested and immunoblotting was conducted. Decreased phosphorylation level of IGF-1R and its downstream targets, including AKT and ERK1/2, were observed in cells transfected with LV-KL (Fig. 4c). Furthermore, we evaluated the modulation of Klotho on IGF-1R signaling in DLBCL xenograft model. Enhanced expression of Klotho in LV-KL-treated mice was confirmed by immunoblotting (Fig. 5d). Decreased phosphorylation of IGF1-R as well as its downstream targets were observed in mice treated with LV-KL compared to the control group (Fig. 4d). These results demonstrated that Klotho may act as a modulator of IGF-1R signaling contributed to the tumorigenesis of DLBCL (Fig. 4e).

We further investigated the activity of secreted Klotho protein on DLBCL. To explore the effect of secreted Klotho on proliferation of DLBCL cells, LY1 and LY8 cells were seeded and treated with rhKL. The rhKL was found to be active and reduced the proliferative ability of DLBCL cells by 30~40% at the concentration of 2 μg/ml (Fig. 5a, b). ADR is one of the most commonly used drugs in the chemotherapeutic strategy of DLBCL. We detected the function of Klotho on cell responses to ADR. LY1 and LY8 cells were treated for 48 h with rhKL, ADR, or their combination, and cell proliferation was then evaluated. Combination of rhKL (2 μg/ml) increased the sensitivity of DLBCL cells to ADR (Fig. 5a, b).

The in vivo activity of rhKL in DLBCL xenograft model was also detected. LY1 cells were injected subcutaneously into the SCID Beige mice to build a DLBCL xenograft model. After 15 days, the mice were treated with daily intraperitoneal injection of either rhKL (7.5 μg/kg, n = 6) or a control vehicle (n = 6) for 2 weeks. Significant decreased tumor volumes were observed in the mice treated with rhKL compared with those treated with vehicle control (Fig. 5c). Moreover, reductive expression level of proliferative marker Ki67 was found in rhKL-treated mice (Fig. 5d).

In addition, we employed ELISA assays to test the soluble Klotho levels in the serum of 30 initially diagnosed DLBCL patients and 15 healthy volunteers. Lower serum Klotho levels were noted in DLBCL patients (628.54 ± 219.39 pg/ml) than the control subjects (818.87 ± 241.51 pg/ml, p = 0.045, Fig. 5e).