Reynolds Brobey

Klotho Depletion Contributes to Increased Inflammation in Kidney of the db/db Mouse Model of Diabetes Via RelA (Serine)536 Phosphorylation

OBJECTIVE Klotho is an antiaging hormone present in the kidney that extends the lifespan, regulates kidney function, and modulates cellular responses to oxidative stress. We investigated whether Klotho levels and signaling modulate inflammation in diabetic kidneys. RESEARCH DESIGN AND METHODS Renal Klotho expression was determined by quantitative real-time PCR and immunoblot analysis. Primary mouse tubular epithelial cells were treated with methylglyoxalated albumin, and Klotho expression and inflammatory cytokines were measured. Nuclear factor (NF)-κB activation was assessed by treating human embryonic kidney (HEK) 293 and HK-2 cells with tumor necrosis factor (TNF)-α in the presence or absence of Klotho, followed by immunoblot analysis to evaluate inhibitor of κB (IκB)α degradation, IκB kinase (IKK) and p38 activation, RelA nuclear translocation, and phosphorylation. A chromatin immunoprecipitation assay was performed to analyze the effects of Klotho signaling on interleukin-8 and monocyte chemoattractant protein-1 promoter recruitment of RelA and RelA serine (Ser)536. RESULTS Renal Klotho mRNA and protein were significantly decreased in db/db mice, and a similar decline was observed in the primary cultures of mouse tubule epithelial cells treated with methylglyoxal-modified albumin. The exogenous addition of soluble Klotho or overexpression of membranous Klotho in tissue culture suppressed NF-κB activation and subsequent production of inflammatory cytokines in response to TNF-α stimulation. Klotho specifically inhibited RelA Ser536 phosphorylation as well as promoter DNA binding of this phosphorylated form of RelA without affecting IKK-mediated IκBα degradation, total RelA nuclear translocation, and total RelA DNA binding. CONCLUSIONS These findings suggest that Klotho serves as an anti-inflammatory modulator, negatively regulating the production of NF-κB–linked inflammatory proteins via a mechanism that involves phosphorylation of Ser536 in the transactivation domain of RelA.

Klotho Protects Dopaminergic Neuron Oxidant-Induced Degeneration by Modulating ASK1 and p38 MAPK Signaling Pathways.

Klotho transgenic mice exhibit resistance to oxidative stress as measured by their urinal levels of 8-hydroxy-2-deoxyguanosine, albeit this anti-oxidant defense mechanism has not been locally investigated in the brain. Here, we tested the hypothesis that the reactive oxygen species (ROS)-sensitive apoptosis signal-regulating kinase 1 (ASK1)/p38 MAPK pathway regulates stress levels in the brain of these mice and showed that: 1) the ratio of free ASK1 to thioredoxin (Trx)-bound ASK1 is relatively lower in the transgenic brain whereas the reverse is true for the Klotho knockout mice; 2) the reduced p38 activation level in the transgene corresponds to higher level of ASK1-bound Trx, while the KO mice showed elevated p38 activation and lower level of–bound Trx; and 3) that 14-3-3ζ is hyper phosphorylated (Ser-58) in the transgene which correlated with increased monomer forms. In addition, we evaluated the in vivo robustness of the protection by challenging the brains of Klotho transgenic mice with a neurotoxin, MPTP and analyzed for residual neuron numbers and integrity in the substantia nigra pars compacta. Our results show that Klotho overexpression significantly protects dopaminergic neurons against oxidative damage, partly by modulating p38 MAPK activation level. Our data highlight the importance of ASK1/p38 MAPK pathway in the brain and identify Klotho as a possible anti-oxidant effector.

Klotho Regulates 14-3-3ζ Monomerization and Binding to the ASK1 Signaling Complex in Response to Oxidative Stress

The reactive oxygen species (ROS)-sensitive apoptosis signal-regulating kinase 1 (ASK1) signaling complex is a key regulator of p38 MAPK activity, a major modulator of stress-associated with aging disorders. We recently reported that the ratio of free ASK1 to the complex-bound ASK1 is significantly decreased in Klotho-responsive manner and that Klotho-deficient tissues have elevated levels of free ASK1 which coincides with increased oxidative stress. Here, we tested the hypothesis that: 1) covalent interactions exist among three identified proteins constituting the ASK1 signaling complex; 2) in normal unstressed cells the ASK1, 14-3-3ζ and thioredoxin (Trx) proteins simultaneously engage in a tripartite complex formation; 3) Klotho’s stabilizing effect on the complex relied solely on 14-3-3ζ expression and its apparent phosphorylation and dimerization changes. To verify the hypothesis, we performed 14-3-3ζ siRNA knock-down experiments in conjunction with cell-based assays to measure ASK1-client protein interactions in the presence and absence of Klotho, and with or without an oxidant such as rotenone. Our results show that Klotho activity induces posttranslational modifications in the complex targeting 14-3-3ζ monomer/dimer changes to effectively protect against ASK1 oxidation and dissociation. This is the first observation implicating all three proteins constituting the ASK1 signaling complex in close proximity.

Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

Klotho is a single-pass transmembrane protein with documented anti-cancer properties. Recent reports have implicated Klotho as an inhibitor of transforming growth factor β1 induced cell migration in renal fibrosis. Overexpression of epidermal growth factor receptor (EGFR) is known to promote tumor initiation and progression in clear-cell renal cell carcinoma (cRCC). We tested our hypothesis that Klotho inhibits EGF-mediated cell migration in cRCC by interfering with the EGFR signaling complex and mitogen-activated protein kinase (MAPK) pathways. We performed cell adhesion, migration, and biochemical studies in vitro using Caki-1 cell line. In addition, we validated the cell culture studies with expression analysis of six de-identified FFPE tissues from primary and metastatic cRCC patients. Our studies show that Klotho inhibited EGF-induced Caki-1 de-adhesion and decreased spreading on collagen type 1. Klotho also inhibited EGF-induced α2β1 integrin-dependent cell migration on collagen type 1. To test the involvement of MAPK pathways in EGF-induced Caki-1 cell motility, the cells were pretreated with either SB203580, a specific p38 MAPK inhibitor, or Klotho. SB203580 blocked the EGF-induced Caki-1 cell migration. Klotho had a comparable inhibitory effect. Our FFPE clinical specimens revealed decreased Klotho mRNA expression compared to a control, non-cancer kidney tissue. The decrease in Klotho mRNA levels correlated with increased c-Src expression, while E-Cadherin was relatively reduced in metastatic FFPE specimens where Klotho was least expressed. Taken together, these results suggest that secreted Klotho inhibits EGF-induced pro-migratory cell morphological changes and migration in Caki-1 cells. Our data additionally suggest that decreased Klotho expression may be involved in cRCC metastasis.

Isolation and analysis of circulating tumor cell samples from both spiked analytical sources and patients with renal cell cancer and prostate cancer.

584 Background: Tumor genotyping using fluid samples such as blood can potentially allow tracking of dynamic changes in mutational profiles over time and allow better access than biopsies. We present a method to detect somatic mutations from a blood draw, where circulating tumor cell (CTC) enrichment above 10% of total cell numbers allows the use of standard gene panels typically used to analyze tissue-based biopsies. Methods: Analytical samples were obtained from 9 prostate cancer (PC) patients and 6 renal cell cancer (RCC) patients, followed by CTC enrichment using the IsoFlux System. Cells were lysed and DNA amplified by whole genome amplification (WGA) using the NGS Kit (Fluxion Biosciences) and quantified via qPCR. Samples were enumerated to determine CTC load, with CTCs defined as CK+, CD45- nucleated cells (DAPI+). Next-generation sequencing was performed using 3 targeted cancer panels on the Ion torrent PGM platform: the Ion ampliseq cancer hotspot panel (50 genes; 6 PC samples), the Oncomine (143...

Somatic mutation detection from liquid biopsy-derived cellular aggregates formed by magnetic 3D bioprinting.

291 Background: A challenge in the analysis of circulating tumor cells (CTC) is their scarcity, and the inability to expand them for further analysis. To overcome this obstacle, we used magnetic 3D bioprinting to form CTC spheroids that could grow. The principle of magnetic 3D bioprinting is the magnetization of cells with nanoparticles and their subsequent printing into spheroids. For this project, CTCs can be aggregated into close contact to interact and grow in culture. In this study, we demonstrated the ability to aggregate CTCs and perform next generation sequencing (NGS) to detect somatic mutations from renal and prostate cancers. Methods: Blood samples from prostate and kidney cancer patients were enriched for CTCs (Isoflux, Fluxion), from a starting blood volume of 7.5-14 mL. CTCs were isolated immunomagnetically for EpCAM+ EGFR+ cells, then enumerated for CK+ CD45-. The cells were then removed of microbeads, then magnetized by incubation with NanoShuttle (NS, Nano3D), a magnetic nanoparticle asse...

Abstract 4251: Development of spheroids derived from tumor biopsies and patient-derived xenografts using magnetic 3D bioprinting

Precision medicine holds the promise of designing patient-specific therapies to improve therapeutic efficiency. However, the scarcity of tumor and biopsy tissue is a limiting factor in the development of diagnostic assays. Cells isolated from these tissues could be used to overcome these issues, while serving as the basis for assays to diagnose and guide treatment. It is critical that the in vitro culture of these cells be performed in three-dimensional (3D) environments that can better replicate the native tumor microenvironment. However, currently available 3D cell culture platforms, like Matrigel, suffer from technical limitations in reproducibility and handling that make the development of such assays difficult. Towards that end, this study isolates cells from human prostate cancer (PC) and renal cell carcinoma (RCC) tumor biopsies and patient-derived xenografts (PDX) and prints them into spheroids using magnetic 3D bioprinting. The core principle of magnetic 3D bioprinting is the magnetization of cells and their aggregation using mild magnetic forces. Once aggregated, these cells form spheroids that mimic native tumor environments in extracellular matrix and cell-cell and cell-ECM interactions. This technique can be used to actively magnetize cells and generate spheroids from a scarce cell source, while overcoming the limitations of other 3D cell culture platforms. In this study, we demonstrated our ability to print spheroids from cells isolated from human tumor biopsies and PDX. Isolation techniques ranging from simple mincing and filtration to enzymatic digestion were employed. Next, these cells were magnetized by incubation with a biocompatible magnetic nanoparticle assembly, NanoShuttle. Once magnetized, these cells were printed into spheroids of varying sizes, from 1,000-20,000 cells, in 384-well plates. These cells were cultured for days, after which viability was measured using CellTiter-Glo. Our preliminary studies demonstrated our ability to isolate cells and print them into spheroids. Isolation was best with either mincing and filtration alone or collagenase II (400 U/mL) digestion for 1 h. These cells were then successfully magnetized and printed into spheroids, which remained viable after 72 h. Spheroids of 10,000-20,000 cells were the most successful, and further optimization is needed to reduce the size needed for viable spheroids to take full advantage of scarce resources such as tumor biopsies. We also demonstrated the ability to assay compound toxicity, showing a dose-dependent toxicity on spheroids derived from PDX tumors. In all, we demonstrated our ability to isolate cells from human tumor biopsies and PDX models and print them into spheroids with high throughput. These preliminary results will serve as a platform for the further development of precision medicine assays to optimize PC and RCC treatment. Citation Format: Hubert Tseng, Jacob A. Gage, Pujan K. Desai, Reynolds Brobey, Sheri Skinner, Mehdi Dehghani, Kevin P. Rosenblatt, Wenliang Li, Robert J. Amato, Glauco R. Souza. Development of spheroids derived from tumor biopsies and patient-derived xenografts using magnetic 3D bioprinting. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4251.