Tanya C. Burch

Increased bisecting N-acetylglucosamine and decreased branched chain glycans of N-linked glycoproteins in expressed prostatic secretions associated with prostate cancer progression.

Using prostatic fluids rich in glycoproteins like prostate‐specific antigen and prostatic acid phosphatase (PAP), the goal of this study was to identify the structural types and relative abundance of glycans associated with prostate cancer status for subsequent use in emerging MS‐based glycopeptide analysis platforms.

Proteomic Analysis of Disease Stratified Human Pancreas Tissue Indicates Unique Signature of Type 1 Diabetes

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.

Relative sensitivity of immunohistochemistry, multiple reaction monitoring mass spectrometry, in situ hybridization and PCR to detect Coxsackievirus B1 in A549 cells

BACKGROUND Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples. OBJECTIVES This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 (CVB1), in acutely infected human A549 cells in vitro. STUDY DESIGN A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10(-1) to 10(-8). Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR. RESULTS RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10(-8)). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10(-7). The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10(-6), while ISH detected the virus at dilutions of 10(-4). CONCLUSIONS All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies.

Variable Metastatic Potentials Correlate with Differential Plectin and Vimentin Expression in Syngeneic Androgen Independent Prostate Cancer Cells

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.

Comparative Metabolomic and Lipidomic Analysis of Phenotype Stratified Prostate Cells

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.

Human endometrial milk fat globule-epidermal growth factor 8 (MFGE8) is up regulated by estradiol at the transcriptional level, and its secretion via microvesicles is stimulated by human chorionic gonadotropin (hCG)

Abstract Objective: We have recently showed that MFGE8, a novel epithelial cell protein in the human endometrium, upregulated during the window of implantation. We hypothesized that

Comparative quantitative proteomic analysis of disease stratified laser captured microdissected human islets identifies proteins and pathways potentially related to type 1 diabetes

Type 1 diabetes (T1D) is a chronic inflammatory disease that is characterized by autoimmune destruction of insulin-producing pancreatic beta cells. The goal of this study was to identify novel protein signatures that distinguish Islets from patients with T1D, patients who are autoantibody positive without symptoms of diabetes, and from individuals with no evidence of disease. High resolution high mass accuracy label free quantitative mass spectrometry analysis was applied to islets isolated by laser capture microdissection from disease stratified human pancreata from the Network for Pancreatic Organ Donors with Diabetes (nPOD), these included donors without diabetes, donors with T1D-associated autoantibodies in the absence of diabetes, and donors with T1D. Thirty-nine proteins were found to be differentially regulated in autoantibody positive cases compared to the no-disease group, with 25 upregulated and 14 downregulated proteins. For the T1D cases, 63 proteins were differentially expressed, with 24 upregulated and 39 downregulated, compared to the no disease controls. We have identified functional annotated enriched gene families and multiple protein-protein interaction clusters of proteins are involved in biological and molecular processes that may have a role in T1D. The proteins that are upregulated in T1D cases include S100A9, S100A8, REG1B, REG3A and C9 amongst others. These proteins have important biological functions, such as inflammation, metabolic regulation, and autoimmunity, all of which are pathways linked to the pathogenesis of T1D. The identified proteins may be involved in T1D development and pathogenesis. Our findings of novel proteins uniquely upregulated in T1D pancreas provides impetus for further investigations focusing on their expression profiles in beta cells/ islets to evaluate their role in the disease pathogenesis. Some of these molecules may be novel therapeutic targets T1D.

Mitochondria Biogenesis and Bioenergetics Gene Profiles in Isogenic Prostate Cells with Different Malignant Phenotypes.

Background. The most significant hallmarks of cancer are directly or indirectly linked to deregulated mitochondria. In this study, we sought to profile mitochondria associated genes in isogenic prostate cell lines with different tumorigenic phenotypes from the same patient. Results. Two isogenic human prostate cell lines RC77N/E (nonmalignant cells) and RC77T/E (malignant cells) were profiled for expression of mitochondrial biogenesis and energy metabolism genes by qRT-PCR using the Human Mitochondria and the Mitochondrial Energy Metabolism RT2 PCR arrays. Forty-seven genes were differentially regulated between the two cell lines. The interaction and regulatory networks of these genes were generated by Ingenuity Pathway Analysis. UCP2 was the most significantly upregulated gene in primary adenocarcinoma cells in the current study. The overexpression of UCP2 upon malignant transformation was further validated using human prostatectomy clinical specimens. Conclusions. This study demonstrates the overexpression of multiple genes that are involved in mitochondria biogenesis, bioenergetics, and modulation of apoptosis. These genes may play a role in malignant transformation and disease progression. The upregulation of some of these genes in clinical samples indicates that some of the differentially transcribed genes could be the potential targets for therapeutic interventions.

Abstract 54: Differential plectin isoform expression correlates with aggressive prostate cancer phenotypes

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Prostate cancer (PCa) is the most prevalent cancer amongst men and the second most common cause of cancer related-deaths in the US. Although PCa has a long latent period of development, clinically, the disease has very heterogeneous phenotypes ranging from indolent asymptomatic cases to very aggressive metastatic life threatening and lethal forms. The most critical clinical challenge in the management of the patients is identifying those individuals at risk of developing aggressive metastatic disease. Currently there are no biomarkers that accurately predict the aggressiveness of PCa based solely on standard clinicopathologic features. Our long term objective is to identify the molecules and characterize the mechanisms that are involved in PCa metastasis. We recently demonstrated that plectin is upregulated in aggressive versus non-aggressive forms of syngeneic prostate cancer cell lines. In this study, we show that PCa aggressive phenotype correlates with the over expression of specific plectin isoforms. Understanding the mechanism and roles of different plectin isoforms in the progression of PCa will potentially better discriminate the more aggressive metastatic forms and provide better treatment and clinical management opportunities of the disease. Methods: We have compared the mRNA and protein expression profiles of plectin isoforms in prostate cancer cell lines and prostatectomy tissues with different ethnic backgrounds and malignancy phenotypes. Validations of differentially expressed isoforms have been done by Western blot, qRT-PCR, siRNA knockdowns, proliferation assays and invasion assays, flow cytometry, IHC and IFA. Results: We demonstrate the correlation between the expression of specific isoforms with aggressive PCa. This was validated by Western blot, confocal microscopy and immunohistochemistry. SiRNA knockdown of specific plectin isoforms suppresses the invasive and migratory capacity of PCa cells. To our knowledge, this study is the first to demonstrate that differential expression of plectin isoforms correlates with the invasion and metastasis in PCa. Conclusions: We have identified plectin isoforms that are differentially expressed in PCa cells lines and prostatectomy tissues with different malignancy phenotypes. These findings are currently being validated using disease stratified TMAs. Further studies are focusing on uncovering the mechanisms responsible for progression and metastasis of PCa that are modulated by specific plectin isoforms. These could assist in the development of novel diagnostic and therapeutic strategies for the disease. Citation Format: Tanya C. Burch, Johng S. Rhim, Julius O. Nyalwidhe. Differential plectin isoform expression correlates with aggressive prostate cancer phenotypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 54. doi:10.1158/1538-7445.AM2014-54

Abstract C20: Comparative proteomics analysis of ethnically stratified prostate cancer cells reveal potential markers of disparities

Introduction: Prostate cancer (PCa) is the most prevalent cancer among men and the third most common cause of cancer related-deaths in the USA. Clinically, PCa is a heterogeneous disease ranging from indolent asymptomatic cases to very aggressive life threatening forms. The most critical clinical challenge in the management of the patients is identifying those individuals at risk of developing metastatic disease. Positive family history and African American ancestry are two of the most important recognized risk factors for PCa. African American (AA) men have disproportionately high incidence and mortality rates of PCa when compared to Caucasian American (CA) men and other ethnic groups in the USA. The molecular basis of this disparity is unknown and our long term objective is to identify the molecules and characterize the mechanisms that are involved in PCa aggressiveness in African American populations. Potential biomarkers for progression of PCa from the precursor lesion to organ confined primary tumor and finally to distant metastasis may include genes, proteins and metabolites. The identification of biomarkers that contribute to this disparity could potentially improve diagnosis, prognosis and allow for better clinical management decisions in PCa. Methods: We have analyzed protein expression in prostate cancer cells lines with different ethnic backgrounds using high throughput liquid chromatography tandem quantitative mass spectrometry. Validations of the most significantly differentially expressed proteins were done by Western blot and qRT-PCR. Characterization of the target proteins was carried out by performing siRNA knockdowns, proliferation assays and invasion assays, flow cytometry, Western blot and immunofluorescence assays. Results: We demonstrate for the first time differential expression of specific classes of proteins between Caucasian and African American derived prostate cancer cells with the same tumorigenic phenotypes. The most significantly differentially upregulated proteins in the proteomics experiments were validated by Western blot, confocal microscopy and immunohistochemistry. SiRNA knockdown of target proteins suppress the growth, invasive and migratory capacity of the malignant cells and results in the modulation of various signal transduction pathways. Conclusions: We demonstrate the differential expression of specific classes of proteins in prostate cancer cell lines with different ethnic and tumorigenic phenotypes. Some of these proteins may play a role in the progression, aggression and metastasis of PCa. These findings are currently being validated using disease stratified tissue microarrays to determine the expression patterns of these proteins in PCa patients. We are currently focusing on uncovering the potential mechanisms responsible for progression and aggression of PCa that are modulated by these proteins. These studies may provide novel avenues for the development of new personalized cancer therapies, cancer diagnosis, prognosis, and early detection in African American populations that are currently disproportionally impacted by aggressive forms of prostate cancer. Citation Format: Tanya C. Burch, Julius O. Nyalwidhe. Comparative proteomics analysis of ethnically stratified prostate cancer cells reveal potential markers of disparities. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C20. doi:10.1158/1538-7755.DISP13-C20