Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL), accounts for nearly 40% of all newly diagnosed cases [
]. This disease presents as an aggressive process and exhibits high heterogeneity in gene expression and clinical outcomes [
]. Although majority of DLBCL patients could be cured by anthracycline-based chemotherapies combined with rituximab, one third of them presented refractory or relapsed process [
]. Therefore, more effective treatment strategies based on novel therapeutic targets and molecular oncogenic pathways are still needed.
Klotho is an anti-aging gene originally identified in 1997 [
]. Kuro-o et al. [
] found that Klotho-deficient mice developed multiple premature aging syndromes, whereas overexpression of Klotho extended the lifespan of mice. The Klotho gene is located in chromosome 13q12 in human with the size of 50 kb [
]. It encodes a single-pass transmembrane protein, which consists of an extracellular domain, a single transmembrane domain, and an intracellular domain. The intracellular domain is very short and has no clear functions. Membrane Klotho functions as an obligate co-receptor of fibroblast growth factor 23 (FGF23) to regulate phosphate homeostasis [
]. The extracellular domain (secreted Klotho) could be released into the serum and functions as a circulating hormone to regulate the activity of oxidative stress, multiple growth factor receptors, and ion channels [
The tumor suppressive activity of Klotho was first identified in breast cancer in 2008 [
]. Recent investigations have implicated that Klotho is extensively downregulated in several solid tumors, including cervical cancer, pancreatic cancer, melanoma, and several digestive neoplasm [
]. In these malignancies, Klotho was elucidated to be a modulator of several signaling pathways, including the FGF signaling, insulin-like growth factor-1 receptor (IGF-1R), and Wnt pathways, which are also involved in the pathogenesis of hematological malignancies [
]. However, the role of Klotho in hematological malignancies has not been reported.
A large number of aberrant receptor tyrosine kinases (RTKs) have been found in hematological malignancies [
], but they are still indefinite in DLBCL. IGF-1R is a RTK primarily activated by its cognate ligands, IGF-1, and IGF-2. It plays a crucial role in the establishment and progression of tumors by regulating proliferation, self-renewal, apoptosis, and drug resistance of cancer cells [
]. Activation of IGF-1R following IGF-1 treatment results in phosphorylation of downstream signaling cascades, including PI3K/AKT and MAPK/ERK [
]. Blockade of PI3K/AKT signaling could restrain cell survival and function of lymphocytes [
]. Activation of MAPK/ERK signaling promotes cell proliferation and metastasis of cancer cells [
]. Strategies to block IGF-1R pathway in solid malignancies are being tested in clinical trials [
], whereas the function of IGF-1R signaling in DLBCL has been less studied [
In this present study, we aimed to assess the expression level and functional mechanism of Klotho in DLBCL. We identified reduced expression of Klotho in DLBCL for the first time. Noted inhibition of cell growth and induction of apoptosis were observed in DLBCL with Klotho overexpression. Tumor growth was restrained by administration of Klotho protein in xenograft model of DLBCL. Our findings demonstrated that Klotho was a tumor suppressor and modulator of IGF-1R signaling in DLBCL, indicating that targeting Klotho may provide novel therapeutic strategy in DLBCL.
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 [
]. 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.
Cell lines and reagents
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).
Quantitative real-time PCR
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
Immunohistochemistry (IHC) and hematoxylin-eosin staining
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 assay
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).
Flow cytometry analysis
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
−), dead cells (Annexin V-PE
+), apoptotic cells (Annexin V-PE
−), and necrotic cells (Annexin V-PE
+). 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.
In vivo xenograft study
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 × 10
7 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 × 10
7 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 = (
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.
In this study, our observations identified for the first time that Klotho, an anti-aging gene, as a potential tumor suppressor in DLBCL tumorigenesis. Klotho was downregulated in human DLBCL and inhibited the growth of DLBCL both in vitro and in vivo. Upregulation of Klotho resulted in declined activation of IGF-1R signaling pathway in DLBCL.
We identified remarkable reduced expression of Klotho in DLBCL tissues and cell lines, whereas higher expression in reactive hyperplasia and CD19+ B cells from normal donors. Decreased level of Klotho was associated with advanced stage and more aggressive disease process in DLBCL. Tumor suppressive activity of Klotho has been reported in several human solid malignancies, but never in hematological cancers [
]. Recent investigations elucidated that epigenetic mechanisms, including promoter methylation and histone deacetylation, contributed to the reduced Klotho expression in human breast cancer, cervical cancer, and hepatocellular carcinomas [
]. The similar mechanism may be involved in DLBCL. At present, the diagnosis of DLBCL is mainly based on the biopsy histopathology and IHC [
]. With the deepening of additional studies, lower expression of Klotho may serve as a potential marker for the pathological diagnosis of DLBCL.
DLBCL is a high-grade heterogeneous disorder defined by heterogeneity in clinical and biological characteristics [
]. Personalized prognostic stratification and targeted therapeutic strategies are urgently needed to improve the outcomes of DLBCL patient [
]. Ki67 as a proliferative marker performed as a poor prognostic marker in DLBCL [
]. In this study, we discovered that DLBCL xenograft mice with Klotho overexpression exhibited significantly lower Ki67 staining positive rate than that without Klotho upregulation.
As Klotho exists in both membrane-bound form and secreted form, the secreted Klotho could be shed and released into the circulation [
]. Recent investigation elucidated the low serum level of Klotho in renal cell carcinoma [
]. Low serum Klotho level was an independent adverse prognostic factor for cancer-specific and progression-free survival in RCC [
]. However, the level of serum soluble Klotho was unchanged in multiple myeloma [
]. Thus, the diagnostic role of secreted Klotho in human cancer remains controversial. In this study, we identified the decreased level of serum Klotho in DLBCL patients. The correlation of serum Klotho with disease diagnosis and progression still needs further exploration. Larger number of the included patients will better confirm the role of serum Klotho in DLBCL.
Importantly, our study illuminated that Klotho effectively inhibited the growth of DLBCL cells. Overexpression of Klotho significantly inhibited cell proliferation and induced cell apoptosis in DLBCL. We also discovered that efficacy of ADR could be enhanced by combination with rhKL in DLBCL cells. This finding suggested the potential of Klotho in therapeutic intervention of DLBCL. Our in vivo investigations demonstrated that upregulation of Klotho, either by LV-KL transfection or rhKL administration, congruously led to inhibitory effect in the tumor growth of xenograft model of DLBCL. This will pave the way for rational design of Klotho-based molecular products in DLBCL. Nevertheless, pharmacokinetic investigations are still required to explore the optimal dose and adverse reaction of rhKL in the use of either single agent treatment or drug combination.
Significantly, we also found that Klotho could inhibit the activation of IGF-1R signaling in DLBCL. Induction of IGF-1R signaling was involved in the pathogenesis of hematological malignancies [
]. Recently, Stromberg et al. reported the involvement of IGF-1R and the antitumor effects of specific IGF-1R inhibitors in DLBCL tumorigenesis [
]. Structure-function analysis of Klotho indicated that Klotho could interact with the IGF-1R [
]. Klotho-induced inhibition of IGF-1R signaling may act as a novel mechanism involved in the development of DLBCL. Apart from IGF-1R signaling, many other pathways have been confirmed to be modulated by Klotho in tumorigenesis. The most studied is the FGFR-Klotho axis, and Klotho could act as cofactor of endocrine FGFs to bind and induce the activation of FGFRs in breast, pancreatic, and prostate cancers [
]. Deregulation of Wnt signaling pathway plays a critical role in the pathogenesis of cancers [
]. It was proved that Klotho participated in tumorigenesis partly through restraining the Wnt signaling pathway [
]. The modulation of Klotho on these pathways in DLBCL is poorly understood. In spite of the important discoveries revealed by these studies, there are also limitations on the detailed mechanisms and the cross-talks of them involved in Klotho deregulation.
This study was supported by National Natural Science Foundation (No. 81473486 and No. 81270598), National Public Health Grand Research Foundation (No. 201202017), Natural Science Foundations of Shandong Province (No. ZR2012HZ003 and No. 2009ZRB14176), Technology Development Projects of Shandong Province (No. 2014GSF118021, No. 2010GSF10250, and No. 2008GG2NS02018), Program of Shandong Medical Leading Talent, and Taishan Scholar Foundation of Shandong Province.
Availability of data and materials
All data generated and analyzed during this study are included in this published article.
XXZ and XW conceived and designed the experiments. XXZ, XSF, YJJ, and LYG performed the experiments. XYL, YL, KL, PPL, and XL assisted with the performance of experiments. XXZ, XSF and YJJ analyzed the data. XXZ and XW wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study was approved by the Medical Ethical Committee of Shandong Provincial Hospital affiliated to Shandong University. All samples of patients were obtained with informed consent in accordance with the Declaration of Helsinki. All animal procedures described in this article have been approved by the Animal Care and Use Committee of Shandong Provincial Hospital affiliated to Shandong University.