Currently prostate-specific antigen is used for prostate cancer (PCa) screening, however it lacks the necessary specificity for differentiating PCa from other diseases of the prostate such as benign prostatic hyperplasia (BPH), presenting a clinical need to distinguish these cases at the molecular level. Protein glycosylation plays an important role in a number of cellular processes involved in neoplastic progression and is aberrant in PCa. In this study, we systematically interrogate the alterations in the circulating levels of hundreds of serum proteins and their glycoforms in PCa and BPH samples using multi-lectin affinity chromatography and quantitative mass spectrometry-based proteomics. Specific lectins (AAL, PHA-L and PHA-E) were used to target and chromatographically separate core-fucosylated and highly-branched protein glycoforms for analysis, as differential expression of these glycan types have been previously associated with PCa. Global levels of CD5L, CFP, C8A, BST1, and C7 were significantly increased in the PCa samples. Notable glycoform-specific alterations between BPH and PCa were identified among proteins CD163, C4A, and ATRN in the PHA-L/E fraction and among C4BPB and AZGP1 glycoforms in the AAL fraction. Despite these modest differences, substantial similarities in glycoproteomic profiles were observed between PCa and BPH sera.

Background Distinguishing men with aggressive from indolent prostate cancer is critical to decisions in the management of clinically localized prostate cancer. Molecular signatures of aggressive disease could help men overcome this major clinical challenge by reducing unnecessary treatment and allowing more appropriate treatment of aggressive disease. Methods We performed a mass spectrometry‐based proteomic analysis of normal and malignant prostate tissues from 22 men who underwent surgery for prostate cancer. Prostate cancer samples included Grade Groups (3–5), with 8 patients experiencing recurrence and 14 without evidence of recurrence with a mean of 6.8 years of follow‐up. To better understand the biological pathways underlying prostate cancer aggressiveness, we performed a systems biology analysis and gene enrichment analysis. Proteins that distinguished recurrent from nonrecurrent cancer were chosen for validation by immunohistochemical analysis on tissue microarrays containing samples from a larger cohort of patients with recurrent and nonrecurrent prostate cancer. Results In all, 24,037 unique peptides (false discovery rate < 1%) corresponding to 3,313 distinct proteins were identified with absolute abundance ranges spanning seven orders of magnitude. Of these proteins, 115 showed significantly (p < 0.01) different levels in tissues from recurrent versus nonrecurrent cancers. Analysis of all differentially expressed proteins in recurrent and nonrecurrent cases identified several protein networks, most prominently one in which approximately 24% of the proteins in the network were regulated by the YY1 transcription factor (adjusted p < 0.001). Strong immunohistochemical staining levels of three differentially expressed proteins, POSTN, CALR, and CTSD, on a tissue microarray validated their association with shorter patient survival. Conclusions The protein signatures identified could improve understanding of the molecular drivers of aggressive prostate cancer and be used as candidate prognostic biomarkers.

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA–protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)—for which RNA–protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA–protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA–protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA–protein interactions, while the growth process is driven less by RNA–protein interactions and more by protein–protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.

Positive-sense RNA viruses often have a cytotoxic effect on their host cell because of the strength of their RNA replicase proteins, even though only one copy of their genome begins the viral life cycle in each cell. Noninfectious forms of them—replicons—which include just their RNA replication-related genes, are also strongly self-amplifying and cytotoxic.

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The primary aim of this study is to identify and quantify aberrancies in glycosylation patterns and glycoprotein levels expressed in high grade prostate cancer tissue from men with and without recurrence following radical prostatectomy using multi-dimensional chromatography and tandem mass spectrometry techniques. Glycosylation is a common and highly complex posttranslational modification, and is linked to protein function. Elucidating specific glycan structures is key to understanding the underlying role glycosylation plays in regulating cellular activity, including tumorigenesis, invasion, and metastasis. Aberrant glycosylation is an emerging hallmark of various cancers, demonstrating that alterations in glycosylation disrupt cellular behavior in key pathways. Furthermore, comprehensive glycan characterization can lead to more effective biomarkers with increased clinical utility for distinguishing indolent disease from cancers that are more likely to become metastatic, recur and/or pose higher risk. In this study, glycoproteomic analysis was performed on prostate cancer tissue and matched normal prostate tissue from ten men with high grade prostate cancer (Gleason 7/8) - five of which experienced recurrence. Proteins were extracted from tissue lysates, denatured, reduced and alkylated. Isotopic labels for quantitation were incorporated during alkylation - normal and cancer tissue was labeled with 12C and 13C acrylamide respectively. Paired normal/cancer tissues were combined. Glycosylated proteins from each pair were separated by multi-lectin chromatography designed to capture sialylated, core-fucosylated, and highly branched complex glycans, fractionating the complex mixture into four discrete fractions containing specific glycoforms. LC-MS/MS was used to analyze the tryptic digest from each fraction for protein identification and quantitation. This experimental design reveals differences between the glycosylation patterns and protein levels of cancerous and normal tissue in ten men, but also allows for the comparison between those with recurrent cancers. The glycosylation patterns across hundreds of prostate tissue proteins were systematically screened, enabling the detection and relative quantitation of specific glycoforms of proteins that may be dysregulated in prostate cancer. Preliminary results reveal the identification of 6,202 unique proteins, and quantitation of 2,894 proteins, most of which contained sialylated glycoforms. In all patients (regardless of treatment outcome), several hundred proteins had elevated total protein levels in the 13C-labeled cancer tissue compared to the 12C-labeled normal tissue proteins, such as TACSTD2 and LPP, while other proteins demonstrated upregulation only among certain glycoforms. Other glycoproteins were up-regulated in cancer only in recurrent patients, including CPNE1, TXND5, and CASC4. Citation Format: Sarah M. Totten, Cheylene Tanimoto, Abel Bermudez, Amy Hembree, James D. Brooks, Sharon J. Pitteri. Aberrant glycoprotein expression in recurrent and non-recurrent prostate cancer tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2213. doi:10.1158/1538-7445.AM2017-2213

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of over 500 individual particles of brome mosaic virus (BMV)—for which RNA-protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA-protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA-protein interactions increases, the nucleation time becomes shorter and more narrowly distributed until the assembly kinetics become indistinguishable from diffusion-limited adsorption. In contrast, the time to grow a capsid after nucleation varies weakly with both salt and protein concentration. These results show that the nucleation rate is controlled by RNA-protein interactions, while the growth process is driven less by RNA-protein interactions and more by protein-protein interactions and intra-protein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.RNA viruses first inspired the term “self-assembly.” Yet much is still not understood about how even the simplest such viruses assemble or if different viruses assemble in similar ways. Theoretical models suggest many possible assembly pathways, with many different roles for the RNA, but until recently measuring these pathways has not been possible. We use a sensitive microscopy technique to follow the assembly of individual particles of BMV, a plant virus. We find evidence of an RNA-mediated nucleation-and-growth pathway that is strikingly similar to that of MS2, a bacterial virus. The last common ancestor of BMV and MS2 existed only in ancient times, suggesting that their assembly pathway might be evolutionarily conserved and other viruses might follow a similar pathway.

In this study, a novel analytical workflow was used to interrogate differences in the expressed levels of proteins and glycoproteins detectable in plasma in men with benign prostate hyperplasia versus those diagnosed with low- to high-grade prostate cancer. Currently, the use of early detection techniques such as measuring the level of prostate-specific antigen (PSA) in the blood lead to over-diagnosis and over-treatment in men with slow-growing or none-threatening prostate tumors, making evident the need for a biomarker that is indicative of cancer aggressiveness. Using the in-depth chromatographic separations and LC-MS/MS methods described below, circulating levels of moderate- to low-abundant proteins and their glycoforms were measured, providing the opportunity to observe alterations in the patterns of glycosylation - a posttranslational modification known to be aberrant with tumorigenesis - in those who developed more aggressive prostate cancers. We hypothesize that alterations in protein glycosylation with the development of prostate cancer are measurable in the blood, and, once identified, quantified, and verified, can be used as early molecular indicators of prostate cancer grade, aggressiveness, and overall risk. Twenty blinded plasma samples collected from men with benign, low-, or high-grade prostate cancer biopsies, along with a pooled plasma sample from healthy men for reference, were first depleted of the fourteen most abundant proteins, as these have been extensively studied. The depleted prostate samples and the reference pooled plasma were respectively isotopically labeled with 13C and 12C acrylamide for quantitation, and combined. Multi-lectin affinity chromatography was used to target various glycoforms, specifically those containing core-fucosylated (Aleuria aurantia lectin or AAL), or branched complex-type glycans (Phaseolus vulgaris Leucoagglutinin and Phaseolus vulgaris Erythroagglutinin lectin, or PHA-L/E). For further sample decomplexing, each lectin fraction was subjected to reversed-phase fractionation prior to tryptic digestion. Peptides were analyzed by nanoflow liquid chromatography on a C18 column coupled to a LTQ-Orbitrap Elite mass spectrometer. Alterations in the glycoprotein profiles were then correlated to disease status as determined by prostate biopsies. Using this approach, an average of 270 proteins were identified in the protein fraction bound to AAL, and 285 proteins were identified in the fraction bound to PHA-L/E, as well as the fraction of proteins unbound to either lectin. The preliminary data demonstrate significant differences between case and control samples in the types and levels of the targeted glycoforms present in each lectin fraction in comparison to the reference plasma, suggesting there are measurable alterations in the glycosylation of several plasma proteins between men with benign, indolent and aggressive prostate tumors. Citation Format: Sarah M. Totten, Majlinda Kulloli, Cheylene Tanimoto, James D. Brooks, Sharon J. Pitteri. In-depth quantitative analysis of protein glycoforms in human prostate cancer plasma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1835. doi:10.1158/1538-7445.AM2015-1835