Renal cell carcinoma comprises 2 to 3% of malignancies in adults with the most prevalent subtype being clear-cell RCC (ccRCC). This type of cancer is well characterized at the genomic and ...transcriptomic level and is associated with a loss of VHL that results in stabilization of HIF1. The current study focused on evaluating ccRCC stage dependent changes at the proteome level to provide insight into the molecular pathogenesis of ccRCC progression. To accomplish this, label-free proteomics was used to characterize matched tumor and normal-adjacent tissues from 84 patients with stage I to IV ccRCC. Using pooled samples 1551 proteins were identified, of which 290 were differentially abundant, while 783 proteins were identified using individual samples, with 344 being differentially abundant. These 344 differentially abundant proteins were enriched in metabolic pathways and further examination revealed metabolic dysfunction consistent with the Warburg effect. Additionally, the protein data indicated activation of ESRRA and ESRRG, and HIF1A, as well as inhibition of FOXA1, MAPK1 and WISP2. A subset analysis of complementary gene expression array data on 47 pairs of these same tissues indicated similar upstream changes, such as increased HIF1A activation with stage, though ESRRA and ESRRG activation and FOXA1 inhibition were not predicted from the transcriptomic data. The activation of ESRRA and ESRRG implied that HIF2A may also be activated during later stages of ccRCC, which was confirmed in the transcriptional analysis. This combined analysis highlights the importance of HIF1A and HIF2A in developing the ccRCC molecular phenotype as well as the potential involvement of ESRRA and ESRRG in driving these changes. In addition, cofilin-1, profilin-1, nicotinamide N-methyltransferase, and fructose-bisphosphate aldolase A were identified as candidate markers of late stage ccRCC. Utilization of data collected from heterogeneous biological domains strengthened the findings from each domain, demonstrating the complementary nature of such an analysis. Together these results highlight the importance of the VHL/HIF1A/HIF2A axis and provide a foundation and therapeutic targets for future studies. (Data are available via ProteomeXchange with identifier PXD003271 and MassIVE with identifier MSV000079511.).
Biomarkers are rapidly gaining importance in personalized medicine. Although numerous molecular signatures have been developed over the past decade, there is a lack of overlap and many biomarkers ...fail to validate in independent patient cohorts and hence are not useful for clinical application. For these reasons, identification of novel and robust biomarkers remains a formidable challenge. We combine targeted proteomics with computational biology to discover robust proteomic signatures for prostate cancer. Quantitative proteomics conducted in expressed prostatic secretions from men with extraprostatic and organ-confined prostate cancers identified 133 differentially expressed proteins. Using synthetic peptides, we evaluate them by targeted proteomics in a 74-patient cohort of expressed prostatic secretions in urine. We quantify a panel of 34 candidates in an independent 207-patient cohort. We apply machine-learning approaches to develop clinical predictive models for prostate cancer diagnosis and prognosis. Our results demonstrate that computationally guided proteomics can discover highly accurate non-invasive biomarkers.
Abstract Urine is a complex biofluid that reflects both overall physiologic state and the state of the genitourinary tissues through which it passes. It contains both secreted proteins and proteins ...encapsulated in tissue-derived extracellular vesicles (EVs). To understand the population variability and clinical utility of urine, we quantified the secreted and EV proteomes from 190 men, including a subset with prostate cancer. We demonstrate that a simple protocol enriches prostatic proteins in urine. Secreted and EV proteins arise from different subcellular compartments. Urinary EVs are faithful surrogates of tissue proteomes, but secreted proteins in urine or cell line EVs are not. The urinary proteome is longitudinally stable over several years. It can accurately and non-invasively distinguish malignant from benign prostatic lesions and can risk-stratify prostate tumors. This resource quantifies the complexity of the urinary proteome and reveals the synergistic value of secreted and EV proteomes for translational and biomarker studies.
Prostate cancer is a clinically heterogeneous disease, ranging from indolent asymptomatic disease to very aggressive metastatic and life threatening forms of the disease. Distant metastasis ...represents the major lethal cause of prostate cancer. The most critical clinical challenge in the management of the patients is identifying those individuals at risk of developing metastatic disease. To understand the molecular mechanisms of prostate cancer metastasis and identify markers with metastatic potential, we have analyzed protein expression in two syngeneic prostate cancer cells lines PC3-N2 and PC3-ML2 using isobaric tags for relative and absolute quantitation labeling and multi-dimensional protein identification technology liquid chromatography matrix assisted laser desorption ionization tandem mass spectrometry. PC3-N2 is lowly metastatic while PC3-ML2 highly metastatic. A total of 1,756 proteins were identified in the analyses with 130 proteins showing different expression levels (p<0.01) in the two cell lines. Out of these, 68 proteins were found to be significantly up-regulated while 62 are significantly down-regulated in PC3-ML2 cells compared with PC3-N2 cells. The upregulation of plectin and vimentin which were the most significantly differentially expressed were validated by Western blot and their functional relevance with respect to invasion and migration was determined by siRNA gene silencing. To our knowledge, this study is the first to demonstrate that up-regulation of vimentin and plectin expression positively correlates with the invasion and metastasis of androgen-independent PCA.
Similar to other highly successful invasive bacterial pathogens, Staphylococcus aureus recruits the complement regulatory protein factor H (fH) to its surface to inhibit the alternative pathway of ...complement. Here, we report the identification of the surface-associated protein SdrE as a fH-binding protein using purified fH overlay of S. aureus fractionated cell wall proteins and fH cross-linking to S. aureus followed by mass spectrometry. Studies using recombinant SdrE revealed that rSdrE bound significant fH whether from serum or as a purified form, in both a time- and dose-dependent manner. Furthermore, rSdrE-bound fH exhibited cofactor functionality for factor I (fI)-mediated cleavage of C3b to iC3b which correlated positively with increasing amounts of fH. Expression of SdrE on the surface of the surrogate bacterium Lactococcus lactis enhanced recruitment of fH which resulted in increased iC3b generation. Moreover, surface expression of SdrE led to a reduction in C3-fragment deposition, less C5a generation, and reduced killing by polymorphonuclear cells. Thus, we report the first identification of a S. aureus protein associated with the staphylococcal surface that binds factor H as an immune evasion mechanism.
Histopathology is the standard approach for tissue diagnostics and centerpiece of pathology. Although the current system provides prognostic information, there is need for molecular markers that ...enhance diagnosis and better predict clinical prognosis. The ability to localize disease-specific molecular changes in biopsy tissue would help improve critical pathology decision making. Direct profiling of proteins from tissue using matrix-assisted laser desorption/ionization imaging mass spectrometry has the potential to supplement morphology with underlying molecular detail.
A discovery set of 11 prostate cancer (PCa)-containing and 10 benign prostate tissue sections was evaluated for protein expression differences. A separate validation set of 54 tissue sections (23 PCa and 31 benign) was used to verify the results. Cryosectioning was done to yield tissue sections analyzed by a pathologist to determine tissue morphology and mirror sections for imaging mass spectrometry. Spectra were acquired and the intensity of signals was plotted as a function of the location within the tissue.
An expression profile was found that discriminates between PCa and normal tissue. The overexpression of a single ion at m/z 4,355 was able to discriminate cancer from uninvolved tissue. Tandem mass spectrometry identified this marker as a fragment of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 2 (MEKK2). The ability of MEKK2 to discriminate tumor from normal cells was orthogonally confirmed.
This study highlights the potential of this approach to uncover molecular detail that can be correlated with pathology decision making. In addition, the identification of MEKK2 shows the ability to discover proteins of relevance to PCa biology.
Type 1 diabetes (T1D) and type 2 diabetes (T2D) are associated with functional beta cell loss due to ongoing inflammation. Despite shared similarities, T1D is an autoimmune disease with evidence of ...autoantibody production, as well as a role for exocrine pancreas involvement. Our hypothesis is that differential protein expression occurs in disease stratified pancreas tissues and regulated proteins from endocrine and exocrine tissues are potential markers of disease and potential therapeutic targets. The study objective was to identify novel proteins that distinguish the pancreas from donors with T1D from the pancreas from patients with T2D, or autoantibody positive non-diabetic donors. Detailed quantitative comprehensive proteomic analysis was applied to snap frozen human pancreatic tissue lysates from organ donors without diabetes, with T1D-associated autoantibodies in the absence of diabetes, with T1D, or with T2D. These disease-stratified human pancreas tissues contain exocrine and endocrine tissues (with dysfunctional islets) in the same microenvironment. The expression profiles of several of the proteins were further verified by western blot. We identified protein panels that are significantly and uniquely upregulated in the three disease-stratified pancreas tissues compared to non-disease control tissues. These proteins are involved in inflammation, metabolic regulation, and autoimmunity, all of which are pathways linked to, and likely involved in, T1 and T2 diabetes pathogenesis. Several new proteins were differentially upregulated in prediabetic, T1D, and T2D pancreas. The results identify proteins that could serve as novel prognostic, diagnostic, and therapeutic tools to preserve functional islet mass in Type 1 Diabetes.
Prostate cancer (PCa) is the most prevalent cancer amongst men and the second most common cause of cancer related-deaths in the USA. Prostate cancer is a heterogeneous disease ranging from indolent ...asymptomatic cases to very aggressive life threatening forms. The goal of this study was to identify differentially expressed metabolites and lipids in prostate cells with different tumorigenic phenotypes. We have used mass spectrometry metabolomic profiling, lipidomic profiling, bioinformatic and statistical methods to identify, quantify and characterize differentially regulated molecules in five prostate derived cell lines. We have identified potentially interesting species of different lipid subclasses including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), glycerophosphoinositols (PIs) and other metabolites that are significantly upregulated in prostate cancer cells derived from distant metastatic sites. Transcriptomic and biochemical analysis of key enzymes that are involved in lipid metabolism demonstrate the significant upregulation of choline kinase alpha in the metastatic cells compared to the non-malignant and non-metastatic cells. This suggests that different de novo lipogenesis and other specific signal transduction pathways are activated in aggressive metastatic cells as compared to normal and non-metastatic cells.
Prostate cancer is annually the most common newly diagnosed cancer in men. The prostate functions as a major secretory gland for the production of glycoproteins critical to sperm activation and ...reproduction. Prostate-specific antigen (PSA), produced by the prostate, is one of the most commonly assayed glycoproteins in blood, serving as a biomarker for early detection and progression of prostate cancer. The single site of N-glycosylation on PSA has been the target of multiple glycan characterization studies. In this review, the extensive number of studies that have characterized the changes in O-linked and N-linked glycosylations associated with prostate cancer development and progression will be summarized. This includes analysis of the glycosylation of PSA, and other prostate glycoproteins, in tissues, clinical biofluids, and cell line models. Other studies are summarized in the context of understanding the complexities of these glycan changes in order to address the many confounding questions associated with prostate cancer, as well as efforts to improve prostate cancer biomarker assays using targeted glycomic-based strategies.
Introduction
Aberrant glycosylation of proteins is an important hallmark in multiple cancers. Prostate‐specific membrane antigen (PSMA), a highly glycosylated protein with 10 N‐linked glycosylation ...sites, is an Food and Drug Administration approved theranostic for prostate cancer. However, glycosylation changes in PSMA that are associated with prostate cancer disease progression have not been fully characterized.
Methods
We investigated whether urinary PSMA sialylation correlate with high‐grade prostate cancer. Urine samples were collected from men after digital rectal examination (DRE) before prostate biopsy. Lectin‐antibody enzyme‐linked immunoassay was used to quantify α2,3‐sialyl PSMA in post‐DRE urine samples from subjects with benign prostate tumors, Grade Group 1 prostate cancer and those with Grade Group ≥2 disease.
Results
There are significant increases in α2,3‐sialylated PSMA in patients with Grade Group ≥2 disease compared to benign (p = 0.0009) and those with Grade Group 1 disease (p = 0.0063). There were no significant differences in α2,3‐sialyl PSMA levels between Grade Group 1 and benign prostate tumors (p = 0.7947).
Conclusions
Our study shows that there are significant differences in the abundance of α2,3‐sialylated PSMA in post‐DRE urines from disease stratified prostate cancer patients, and the increase is correlated with progression and disease severity. The detection of increased PSMA sialyation in post‐DRE urines from patients with higher Grade Group ≥2 disease states provides novel untapped potential for the development of prognostic biomarkers for prostate cancer. Specifically, quantitation of α2,3‐sialylated PSMA shows potential for discriminating between benign to intermediate grade disease, which is a significant clinical challenge in staging and risk stratification of prostate cancer.
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