Klotho is an anti-aging protein predominantly expressed in renal tubular epithelial cells. Although Klotho was recently identified as a tumor suppressor gene in a variety of cancers, the potential role and molecular events for Klotho in renal cell carcinoma (RCC) remain obscure. In the present study, immunohistochemical staining in tissue microarrays containing 125 RCC samples showed that intratumoral Klotho levels were negatively correlated with tumor size, TNM stage and nuclear grade. The overall survival rate of RCC patients with high Klotho expression was significantly higher than that of patients with low Klotho expression. Functional analysis after gain and loss of Klotho expression revealed that Klotho blunted epithelial-mesenchymal transition and cellular migration and invasion in RCC. Also, no alteration of α-2,6-sialidase activity was found after Klotho overexpression in RCC. The molecular signals for this phenomenon involved the Klotho-mediated inhibition of PI3K/Akt/GSK3β/Snail pathway. Importantly, compared to localized RCC tissues, advanced RCC tissues exhibited low Klotho expression accompanied with high pAkt and Snail expression. These results indicate Klotho acts as a tumor suppressor by inhibiting PI3K/Akt/GSK3β/Snail signaling, thus suppressing epithelial-mesenchymal transition and tumor migration and invasion during RCC progression. As a result, Klotho might be used as a potential therapy for advanced RCC.
Renal cell carcinoma (RCC) is the most common type of kidney cancer. It is the third most frequent malignancy within urological oncology, accounting for 2–3% of all cancers worldwide. Although early detection approaches are available for localized RCC, approximately 25–30% of patients have undiagnosed with metastatic diseases. Partial and radical nephrectomies remain the gold standard in treatment for localized RCC, and cytoreductive nephrectomy is often recommended before systemic treatment targeting invasiveness reduction and renal function preservation for metastatic diseases. Nevertheless, 20–40% of RCC patients will relapse after resection depending on the tumor stage and grade. Metastatic RCC (mRCC), which is uniquely resistant to chemotherapy and radiotherapy, has only a 26% 5-year survival. The therapeutic effectiveness of immunotherapy and anti-angiogenic intervention is still controversial. Moreover, present systemic therapies show limited overall survival benefit concomitant with substantial toxicities. Thus, explorations of molecular pathways for RCC tumorigenesis are likely to expand therapeutic options for mRCC.
Klotho is an aging suppressor gene that has been shown to extend lifespan when overexpressed in mice. Genetic ablation of the Klotho gene in mice leads to a syndrome closely resembling human aging that involves a shortened lifespan, growth arrest, premature thymic involution, arterial calcification, osteoporosis and emphysema.[7-9] The Klotho gene encodes a single-pass transmembrane protein of ~135 kDa expressed predominantly in distal renal tubular epithelial cells and shared amino acid sequence homology with family 1 glycosidases.[10, 11] The extracellular domain could be cleaved and secreted into the blood, urine, and cerebrospinal fluid, thus comprising membranous form and secreted form of Klotho. In the kidney, membranous Klotho functions as a co-receptor and relays signaling for fibroblast growth factor 23 (FGF23), an endocrine hormone that lowers blood phosphate and vitamin D levels.[13, 14] The secreted Klotho can act as α-2,6-sialidase and become involved in the regulating activities of multiple ion channels at the cell surface.[10, 15] Although the tumor suppressive function of Klotho has been indicated in breast cancer, pancreatic cancer, and melanoma, the functional roles and molecular events for Klotho in RCC remain poorly understood.
Epithelial-mesenchymal transition (EMT) is the process by which polarized epithelial cells undergo multiple biochemical changes and convert into motile mesenchymal cells that perform with migratory capacity, invasiveness and elevated resistance to apoptosis.[19, 20] This dramatic phenotype switch has emerged as a recognized mechanism for initiating RCC invasion and metastasis.[21, 22] Moreover, Klotho suppresses transforming growth factors-β1-induced EMT responses in renal fibrosis and cancer metastasis in mice.
In this study, we examined whether Klotho could suppress RCC invasion and metastasis through EMT inhibition. Intratumoral Klotho levels were inversely correlated with tumor size, stage, nuclear grade, and overall survival in RCC patients. Moreover, Klotho expression could blunt RCC migration and invasion by repressing EMT mediated by PI3K/Akt/GSK3β/Snail signal activation in vitro and in vivo. These data identify the potential tumor suppressive role of Klotho in RCC tumorigenesis and open a new avenue for therapeutic intervention with Klotho protein administration for mRCC patients.
Materials and Methods
Cell lines and human samples
Four human RCC cell lines 786-O, OS-RC-2, ACHN, Caki-1 and one murine RCC cell line, Renca, were obtained from the Shanghai Cell Bank (Shanghai, China). One human renal proximal tubular epithelial cell line, HKC, was provided by Dr Donghai Wen (Fudan University, Shanghai, China). Cells were cultured in DMEM or RPMI 1640 supplemented with 10% FBS at 37°C in a humidified 5% CO2 incubator. These cell lines have been characterized at the bank by DNA fingerprinting analysis using short tandem repeat markers. Information on human RCC tissues is described in detail in the Supporting Information.
The plasmid containing full-length Klotho (pcDNA3.1/V5-His-TOPO-Klotho) was kindly provided by Dr Ci-Di Chen (Boston University School of Medicine, Boston, MA, USA). The Snail WT and Snail (8SA) constitutively active plasmids were kindly provided by Dr Mien-Chie Hung (MD Anderson Cancer Center, Houston, TX, USA). All constructs were confirmed by DNA sequencing.
Plasmid transfection and RNA interference
Transient and stable transfections with various plasmids and corresponding empty vectors as control were performed as before. These are described in detail in the Supporting Information. The shRNA against Klotho gene, Klotho shRNA (h), and control shRNA (h) lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for RNA interference as described previously. The gene-silencing effect was confirmed by Western blot and RT-PCR at 72 h post-transfection.
Western blot and qRT-PCR
Western blot and qRT-PCR were carried out as previously described. Primary antibodies used in Western blot included those against Klotho (Abcam, Cambridge, MA, USA), E-cadherin, N-cadherin, vimentin, GAPDH (Santa Cruz Biotechnology), and pAkt, Akt, pGSK3β, GSK3β, Snail (Cell Signaling Technologies, Beverly, MA, USA). The PCR primers used in qRT-PCR were as follows: Klotho (5′-ACTCCCCCAGTCAGGTGGCGGTA-3′ and 5′- TGGGCCC GGGAAACCATTGCTGTC-3′) and GAPDH (5′-CGCGGGG CTCTCCAGAACATCATCC-3′ and 5′-CTCCGACGCCTGCT TCACCACCTTCTT-3′).
All abovementioned experiments were performed as before or according to the manufacturer's instructions. Details are described in the Supporting Information.
Immunohistochemistry staining and evaluation
Tumor sections from patients with RCC were immunohistochemically analyzed as before and are described in detail in the Supporting Information. Primary antibodies against Klotho (Abcam), pAkt, Snail (Cell Signaling Technologies) were used in the procedure.
Experimental data were presented as mean ± SD or SEM using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Values and percentages between groups were compared using Student's t-test and χ2 test, respectively. Survival was calculated from the date of surgery to date of death or last follow-up. Survival curves and univariate analysis were estimated using the Kaplan–Meier method and the log-rank test. Those parameters demonstrated a statistically significant effect on overall survival in the univariate analysis were included in a Cox multivariate proportional hazards regression model. P < 0.05 was considered statistically significant.
Intratumoral Klotho levels correlate with RCC progression and prognosis
To explore the clinical significance of Klotho in RCC patients, we analyzed Klotho levels in a cohort of 125 RCC specimens with immunohistochemical staining. Klotho staining, located predominantly on cell surfaces and in cytoplasm, was much lower in tumor tissues compared with surrounding non-tumor tissues (Fig. 1a). Additionally, intratumoral Klotho levels declined gradually with disease progression from TNM stage I to IV (Fig. 1b). As shown in Table 1, low intratumoral Klotho levels strongly correlated with primary tumor size (P < 0.001), tumor thrombus (P = 0.005), primary tumor classification (P < 0.001), regional lymph node involvement (P = 0.011), distant metastasis (P = 0.005), TNM stage grouping (P < 0.001), and nuclear grade (P < 0.001). Univariate analysis revealed that age at surgery (P = 0.024), primary tumor size (P < 0.001), tumor thrombus (P < 0.001), primary tumor classification (P < 0.001), regional lymph node involvement (P < 0.001), distant metastasis (P < 0.001), TNM stage group (P < 0.001), nuclear grade (P < 0.001), and Klotho expression (P < 0.001) all significantly influenced overall survival (Table 2). More importantly, the overall survival of RCC patients with low intratumoral Klotho expression was significantly poorer than those patients with high intratumoral Klotho expression (P < 0.001) (Fig. 1c). Furthermore, Cox multivariate regression analysis suggested that Klotho expression (P = 0.022), primary tumor size (P = 0.044) and nuclear grade (P = 0.022) are independent prognostic factors in RCC patients. These data indicate that low intratumoral Klotho expression represents a novel marker for RCC progression and poor prognosis.
Table 1. Clinicopathological features of the 125 clear cell renal cell carcinoma patients and their correlations with Klotho expression
Klotho expression suppresses RCC cellular migration and invasion
To elucidate the functional relevance of Klotho in RCC progression, we performed functional analysis in RCC cells after Klotho expression or treatment. As shown in Figure 2(a–c), RCC cells, including 786-O, OS-RC-2, ACHN, Caki-1 and Renca, exhibit lower protein and mRNA levels of Klotho compared with the renal proximal tubular epithelial cell HKC. We next evaluated the effect of Klotho on RCC cellular EMT, a fundamental step of tumor invasion and metastasis. We found that RCC cells with Klotho overexpression or recombinant Klotho (rKlotho, 250 ng/mL) administration displayed increased expression of the epithelial marker E-cadherin and decreased levels of the mesenchymal markers N-cadherin and vimentin (Fig. 2d). Moreover, an in vitro scratch wound healing assay, a transwell cell migration assay, and a collagen-based cell invasion assay showed that RCC cells with Klotho overexpression or rKlotho administration had decreased cell migratory and invasive capabilities (Fig. 2e–g). These results collectively suggest that Klotho expression blunts RCC cellular migration and invasion in vitro.
Klotho ablation improves RCC cellular migration and invasion
To further confirm the crucial role of Klotho in RCC cellular EMT, we examined functional alterations in ACHN cells, which have relatively high intrinsic Klotho levels, after shRNA-mediated Klotho ablation. Consistent with the results in RCC cells undergoing Klotho overexpression or rKlotho administration, ACHN cells with Klotho ablation displayed decreased expression of the epithelial marker E-cadherin and increased levels of the mesenchymal markers N-cadherin and vimentin (Fig. 3a). Additionally, an in vitro scratch wound healing assay, a transwell cell migration assay, and a collagen-based cell invasion assay showed that ACHN cells with Klotho knockdown had increased cell migratory and invasive capabilities (Fig. 3b–d). These data suggest that Klotho ablation accelerates RCC cellular migration and invasion in vitro.
No alterations of α-2,6-sialidase activity involves in RCC cellular Klotho overexpression
Klotho participates in the specific removal of α-2,6-linked sialic acids and regulates multiple ion channels through this activity.[10, 15] Plant lectin Sambucus nigra agglutinin specifically bind to α-2,6-sialylated glyco-conjugates. To determine the potential involvement of α-2,6-sialidase activity in the Klotho-mediated RCC cellular phenotype, we investigated alterations of α-2,6-sialidase activity by probing with Sambucus nigra agglutinin in RCC cells after Klotho overexpression. Immunofluorescence staining, flow cytometry analysis and lectin blot analysis showed that HKC, ACHN and 786-O cells exhibited lower α-2,6-sialidase activity compared with OS-RC-2 and Renca cells, which is mostly consistent with the earlier findings of Klotho expression (Fig. 4a–c). However, no alterations of α-2,6-sialidase activity were found in RCC cells after Klotho overexpression with immunofluorescence staining, flow cytometry analysis, and lectin blot (Fig. 4d–g). These findings indicate that Klotho overexpression might regulate RCC cellular phenotype independent on α-2,6-sialidase activity.
Klotho blunts RCC cellular EMT by inhibiting PI3K/Akt/GSK3β/Snail signaling
Our previous study indicated the crucial role of the PI3K/Akt signal in RCC tumorigenesis. Recent investigations have implicated Klotho molecular events in mammalian PI3K/Akt pathway inhibition, which could lead to GSK3β activity for Snail degradation.[29-32] Another work has suggested that Snail has a pivotal role in RCC progression. To dissect the molecular signals underlying a Klotho-induced functional phenotype, we examined the potential link between Klotho and Snail, a recognized extensive EMT inducer relaying signals from the PI3K/Akt/GSK3β pathway, in RCC cells. 786-O and OS-RC-2 cells displayed decreased pAkt, Snail and pGSK3β levels after Klotho overexpression (Fig. 5a). Consistent with this finding, ACHN cells showed increased pAkt and Snail levels concomitant with increased pGSK3β after shRNA-mediated Klotho knockdown (Fig. 5b). Moreover, PI3K inhibition with LY294002 and GSK3β knockdown with LiCl imitate the phenomenon displayed in OS-RC-2 cells after Klotho overexpression and knockdown, respectively (Fig. 5c). Also, ACHN cellular EMT phenotype induced by shRNA-mediated Klotho knockdown, which including decreased expression of the epithelial marker E-cadherin, increased levels of the mesenchymal markers N-cadherin and vimentin levels, and enhanced invasion ability, could be alleviated upon PI3K inhibition with LY294002 (Fig. 5d,e). Consistent with this finding, the OS-RC-2 cellular mesenchymal-epithelial transition phenotype induced by Klotho overexpression, which included increased expression of the epithelial marker E-cadherin, decreased levels of mesenchymal markers N-cadherin and vimentin, and reduced invasion ability, could be reversed by Snail activity (Fig. 5f,g). These data elucidate that Klotho blunts RCC cellular EMT by suppressing the PI3K/Akt/GSK3β/Snail pathway.
Klotho levels negatively correlate with pAkt and Snail expression in RCC samples
To ascertain PI3K/Akt/GSK3β/Snail molecular pathway relays signaling downstream Klotho in clinical RCC tumorigenesis, we analyzed Klotho, pAkt and Snail immunohistochemical staining in a cohort of 125 RCC specimens. Relative to localized RCC tissues, advanced RCC tissues displayed lower Klotho staining, concomitant with higher cytoplasmic pAkt staining and higher nuclear Snail staining (Fig. 6). Correlation analysis indicated that Klotho immunostaining in the 125 RCC specimens showed significant negative correlations with pAkt (P = 0.005, r = −0.251) and Snail staining (P = 0.017, r = −0.213) (Table 3). This supports our in vitro data indicating that the PI3K/Akt/GSK3β/Snail molecular pathway relays oncogenic signals resulting from decreased Klotho in RCC tumorigenesis.
Table 3. Klotho immunostaining negatively correlates with pAkt and Snail in clear cell renal cell carcinoma samples (n = 125)
Akt; r, Spearman correlation coefficient.
Our present study identifies Klotho as a potential tumor suppressor in RCC tumorigenesis. Klotho declination during disease progression initiates an EMT phenotypic switch through PI3K/Akt/GSK3β/Snail signal activation in vitro and in vivo. Additionally, intratumoral Klotho declination, which correlates with RCC progression and a poor prognosis, is indicated as an independent prognosticator for overall survival in clinical patients. This work illuminates a crucial molecular pathway and a functional phenotype downstream of Klotho declination in RCC progression, highlighting potential applications for intratumoral Klotho immunostaining and rKlotho administration in future clinical prognostic strati?cation and therapeutic intervention, respectively.
Klotho is a type-I transmembrane protein predominantly expressed in renal tubules, parathyroid gland, and choroid plexus epithelium. The secreted form of Klotho is generated either when the extracellular domain of the transmembrane protein is shed or as a product of alternative splicing. Klotho exerts diverse effects on the physiological regulation of mineral ions, especially calcium and phosphate, and energy metabolism by influencing the endocrine activities of fibroblast growth factors.[13, 36] Recently, the tumor suppressive function of Klotho has been extensively identified in various cancers. Functional loss of Klotho due to epigenetic silencing promotes tumor progression in cervical, colorectal, and gastric cancers. Klotho can also inhibit tumor growth, promote apoptosis, and predict clinical outcome in human lung cancers.[40, 41] Decreased Klotho is associated with invasiveness of breast cancers and melanomas.[16, 18] Consistent with previous findings, our present study indicates that Klotho is a potential tumor suppressor that works by blunting the Snail-induced EMT phenotype mediated by PI3K/Akt/GSK3β signal activation. This extends our understanding of molecular mechanisms underlying RCC progression. However, upstream events for Klotho declination during RCC tumorigenesis are not addressed here and merit further investigation.
Klotho exhibits α-2,6-sialidase activity and regulates cell-surface expression of transient receptor potential type 5 and renal outer medullary potassium channels.[10, 15] However, no alterations of α-2,6-sialidase activity were found in RCC cells undergoing Klotho overexpression, which excludes the potential effects of Klotho on cancer sialylation during RCC development. Further molecular analysis of the increased or decreased function of Klotho indicates that Klotho blunts the RCC cellular EMT phenotypic switch through PI3K/Akt/GSK3β/Snail signal suppression. In addition to previous findings implicating this pathway's pivotal role in RCC tumorigenesis,[27, 42] our current data interlink Klotho declination and Snail-induced EMT with PI3K/Akt/GSK3β signal activation in vitro and in vivo. Interestingly, we found negative correlation between Klotho with pAkt and Snail staining in human specimens, strengthening the clinical significance of the Klotho/Akt/Snail pathway in RCC tumorigenesis. However, molecular mechanisms for Klotho-mediated PI3K/Akt inhibition during RCC development remain to be addressed in future investigation.
Klotho is essential for endogenous FGF23 function, the newest member of FGF family. Although studies regarding the role of FGF in renal cancer remain sparse, increased basic FGF serum levels have been found to be associated with poor survival and high frequency of pulmonary metastases in RCC. Furthermore, abnormal regulation of FGF23–Klotho axis in renal cancer could severely disrupt mineral ion homeostasis and vitamin D metabolism and induce disease pathology. The expression of FGF23 is probably one of the major causes of tumor-induced osteomalacia. Thus, the potential relevance of the FGF23 signal in tumor suppressive Klotho during renal carcinogenesis requires clarification.
In summary, this study elucidated the crucial role of PI3K/Akt/GSK3β/Snail signal activation in EMT-driving RCC progression mediated by Klotho declination. Furthermore, being an endogenous circulating hormone, Klotho administration in mice is safe and inhibits tumor growth effectively.[8, 17] Our data presented here reveal a novel molecular mechanism underlying RCC progression, leading to a potential therapeutic intervention with rKlotho administration for mRCC treatment.
We thank Dr Ci-Di Chen (Boston University School of Medicine, Boston, MA, USA) and Dr Mien-Chie Hung (MD Anderson Cancer Center, Houston, TX, USA) for the generous gifts of plasmids. This work was supported by grants from the National Natural Science Foundation of China (grant numbers 31100629, 31270863).