Differential Activation of NRF2 Signaling Pathway in Renal-Cell Carcinoma Caki Cell Lines.

Renal-cell carcinoma (RCC) is a heterogeneous disease consisting of several subtypes based on specific genomic profiles and histological and clinical characteristics. The subtype with the highest prevalence is clear-cell RCC (ccRCC), next is papillary RCC (pRCC), and then chromophobe RCC (chRCC). The ccRCC cell lines are further subdivided into prognostic expression-based subtypes ccA or ccB. This heterogeneity necessitates the development, availability, and utilization of cell line models with the correct disease phenotypic characteristics for RCC research. In this study, we focused on characterizing proteomic differences between the Caki-1 and Caki-2 cell lines that are commonly used in ccRCC research. Both cells are primarily defined as human ccRCC cell lines. Caki-1 cell lines are metastatic, harboring wild-type VHL, whereas Caki-2 are considered as the primary ccRCC cell lines expressing wild-type von Hippel-Lindau protein (pVHL). Here, we performed a comprehensive comparative proteomic analysis of Caki-1 and Caki-2 cells using tandem mass-tag reagents together with liquid chromatography mass spectrometry (LC/MS) for the identification and quantitation of proteins in the two cell lines. Differential regulation of a subset of the proteins identified was validated using orthogonal methods including western blot, q-PCR, and immunofluorescence assays. Integrative bioinformatic analysis identifies the activation/inhibition of specific molecular pathways, upstream regulators, and causal networks that are uniquely regulated and associated with the two cell lines and RCC subtypes, and potentially the disease stage. Altogether, we have identified multiple molecular pathways, including NRF2 signaling, which is the most significantly activated pathway in Caki-2 versus Caki-1 cells. Some of the differentially regulated molecules and signaling pathways could serve as potential diagnostic and prognostic biomarkers and therapeutic targets amongst ccRCC subtypes.

NEFL is overexpressed and it modulates invasion and migration in neuroendocrine-like PC3-ML2 prostate cancer cells.

Prostate cancer clinical outcomes are varied, from non-aggressive asymptomatic to lethal aggressive neuroendocrine forms which represent a critical challenge in the management of the disease. The neurofilament light ( NEFL ) is proposed to be a tumor suppressor gene. Studies have shown that expression of the gene is decreased in various cancers. We have used quantitative RT-PCR, immunoblotting, methylation specific PCR, siRNA knockdown followed by migration/invasion assays to determine associations between NEFL expression and disease phenotype in a panel of prostate cells. We demonstrate that NEFL is overexpressed and it modulates invasion and migration in PC3-ML2 prostate cancers cells which have an aggressive neuroendocrine-like phenotype.

Site-Specific Intact N-Linked Glycopeptide Characterization of Prostate-Specific Membrane Antigen from Metastatic Prostate Cancer Cells.

The composition of N-linked glycans that are conjugated to the prostate-specific membrane antigen (PSMA) and their functional significance in prostate cancer progression have not been fully characterized. PSMA was isolated from two metastatic prostate cancer cell lines, LNCaP and MDAPCa2b, which have different tissue tropism and localization. Isolated PSMA was trypsin-digested, and intact glycopeptides were subjected to LC-HCD-EThcD-MS/MS analysis on a Tribrid Orbitrap Fusion Lumos mass spectrometer. Differential qualitative and quantitative analysis of site-specific N-glycopeptides was performed using Byonic and Byologic software. Comparative quantitative analysis demonstrates that multiple glycopeptides at asparagine residues 51, 76, 121, 195, 336, 459, 476, and 638 were in significantly different abundance in the two cell lines (p < 0.05). Biochemical analysis using endoglycosidase treatment and lectin capture confirm the MS and site occupancy data. The data demonstrate the effectiveness of the strategy for comprehensive analysis of PSMA glycopeptides. This approach will form the basis of ongoing experiments to identify site-specific glycan changes in PSMA isolated from disease-stratified clinical samples to uncover targets that may be associated with disease progression and metastatic phenotypes.

Expression Changes in Mitochondrial Genes Affecting Mitochondrial Morphology, Transmembrane Potential, Fragmentation, Amyloidosis, and Neuronal Cell Death Found in Brains of Alzheimer's Disease Patients.

BACKGROUND: Alzheimer's disease (AD) is a neurological disease that has both a genetic and non-genetic origin. Mitochondrial dysfunction is a critical component in the pathogenesis of AD as deficits in oxidative capacity and energy production have been reported. OBJECTIVE: Nuclear-encoded mitochondrial genes were studied in order to understand the effects of mitochondrial expression changes on mitochondrial function in AD brains. These expression data were to be incorporated into a testable mathematical model for AD used to further assess the genes of interest as therapeutic targets for AD. METHODS: RT2-PCR arrays were used to assess expression of 84 genes involved in mitochondrial biogenesis in AD brains. A subset of mitochondrial genes of interest was identified after extensive Ingenuity Pathway Analysis (IPA) (Qiagen). Further filtering of this subset of genes of interest was achieved by individual qPCR analyses. Expression values from this group of genes were included in a mathematical model being developed to identify potential therapeutic targets. RESULTS: Nine genes involved in trafficking proteins to mitochondria, morphology of mitochondria, maintenance of mitochondrial transmembrane potential, fragmentation of mitochondria and mitochondrial dysfunction, amyloidosis, and neuronal cell death were identified as significant to the changes seen. These genes include TP53, SOD2, CDKN2A, MFN2, DNM1L, OPA1, FIS1, BNIP3, and GAPDH. CONCLUSION: Altered mitochondrial gene expression indicates that a subset of nuclear-encoded mitochondrial genes compromise multiple aspects of mitochondrial function in AD brains. A new mathematical modeling system may provide further insights into potential therapeutic targets.

Targeted Mass Spectrometry of a Clinically Relevant PSA Variant from Post-DRE Urines for Quantitation and Genotype Determination.

PURPOSE: The rs17632542 single nucleotide polymorphism (SNP) results in lower serum prostate specific antigen (PSA) levels which may further mitigate against its clinical utility as a prostate cancer biomarker. Post-digital rectal exam (post-DRE) urine is a minimally invasive fluid that is currently utilized in prostate cancer diagnosis. To detect and quantitate the variant protein in urine. EXPERIMENTAL DESIGN: Fifty-three post-DRE urines from rs17632542 genotyped individuals processed and analyzed by liquid chromatography/mass spectrometry (LC-MS) in a double-blinded randomized study. The ability to distinguish between homozygous wild-type, heterozygous, or homozygous variant is examined before unblinding. RESULTS: Stable-isotope labeled peptides are used in the detection and quantitation of three peptides of interest in each sample using parallel reaction monitoring (PRM). Using these data, groupings are predicted using hierarchical clustering in R. Accuracy of the predictions show 100% concordance across the 53 samples, including individuals homozygous and heterozygous for the SNP. CONCLUSIONS AND CLINICAL RELEVANCE: The study demonstrates that MS based peptide variant quantitation in urine could be useful in determining patient genotype expression. This assay provides a tool to evaluate the utility of PSA variant (rs17632542) in parallel with current and forthcoming urine biomarker panels.