AZGP1 deficiency promotes angiogenesis in prostate cancer.

BACKGROUND: Loss of AZGP1 expression is a biomarker associated with progression to castration resistance, development of metastasis, and poor disease-specific survival in prostate cancer. However, high expression of AZGP1 cells in prostate cancer has been reported to increase proliferation and invasion. The exact role of AZGP1 in prostate cancer progression remains elusive. METHOD: AZGP1 knockout and overexpressing prostate cancer cells were generated using a lentiviral system. The effects of AZGP1 under- or over-expression in prostate cancer cells were evaluated by in vitro cell proliferation, migration, and invasion assays. Heterozygous AZGP1± mice were obtained from European Mouse Mutant Archive (EMMA), and prostate tissues from homozygous knockout male mice were collected at 2, 6 and 10 months for histological analysis. In vivo xenografts generated from AZGP1 under- or over-expressing prostate cancer cells were used to determine the role of AZGP1 in prostate cancer tumor growth, and subsequent proteomics analysis was conducted to elucidate the mechanisms of AZGP1 action in prostate cancer progression. AZGP1 expression and microvessel density were measured in human prostate cancer samples on a tissue microarray of 215 independent patient samples. RESULT: Neither the knockout nor overexpression of AZGP1 exhibited significant effects on prostate cancer cell proliferation, clonal growth, migration, or invasion in vitro. The prostates of AZGP1-/- mice initially appeared to have grossly normal morphology; however, we observed fibrosis in the periglandular stroma and higher blood vessel density in the mouse prostate by 6 months. In PC3 and DU145 mouse xenografts, over-expression of AZGP1 did not affect tumor growth. Instead, these tumors displayed decreased microvessel density compared to xenografts derived from PC3 and DU145 control cells, suggesting that AZGP1 functions to inhibit angiogenesis in prostate cancer. Proteomics profiling further indicated that, compared to control xenografts, AZGP1 overexpressing PC3 xenografts are enriched with angiogenesis pathway proteins, including YWHAZ, EPHA2, SERPINE1, and PDCD6, MMP9, GPX1, HSPB1, COL18A1, RNH1, and ANXA1. In vitro functional studies show that AZGP1 inhibits human umbilical vein endothelial cell proliferation, migration, tubular formation and branching. Additionally, tumor microarray analysis shows that AZGP1 expression is negatively correlated with blood vessel density in human prostate cancer tissues. CONCLUSION: AZGP1 is a negative regulator of angiogenesis, such that loss of AZGP1 promotes angiogenesis in prostate cancer. AZGP1 likely exerts heterotypical effects on cells in the tumor microenvironment, such as stromal and endothelial cells. This study sheds light on the anti-angiogenic characteristics of AZGP1 in the prostate and provides a rationale to target AZGP1 to inhibit prostate cancer progression.

Identification and characterization of intact glycopeptides in human urine.

Glycoproteins in urine have the potential to provide a rich class of informative molecules for studying human health and disease. Despite this promise, the urine glycoproteome has been largely uncharacterized. Here, we present the analysis of glycoproteins in human urine using LC-MS/MS-based intact glycopeptide analysis, providing both the identification of protein glycosites and characterization of the glycan composition at specific glycosites. Gene enrichment analysis reveals differences in biological processes, cellular components, and molecular functions in the urine glycoproteome versus the urine proteome, as well as differences based on the major glycan class observed on proteins. Meta-heterogeneity of glycosylation is examined on proteins to determine the variation in glycosylation across multiple sites of a given protein with specific examples of individual sites differing from the glycosylation trends in the overall protein. Taken together, this dataset represents a potentially valuable resource as a baseline characterization of glycoproteins in human urine for future urine glycoproteomics studies.

UCHL1 is a potential molecular indicator and therapeutic target for neuroendocrine carcinomas.

Neuroendocrine carcinomas, such as neuroendocrine prostate cancer and small-cell lung cancer, commonly have a poor prognosis and limited therapeutic options. We report that ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, is elevated in tissues and plasma from patients with neuroendocrine carcinomas. Loss of UCHL1 decreases tumor growth and inhibits metastasis of these malignancies. UCHL1 maintains neuroendocrine differentiation and promotes cancer progression by regulating nucleoporin, POM121, and p53. UCHL1 binds, deubiquitinates, and stabilizes POM121 to regulate POM121-associated nuclear transport of E2F1 and c-MYC. Treatment with the UCHL1 inhibitor LDN-57444 slows tumor growth and metastasis across neuroendocrine carcinomas. The combination of UCHL1 inhibitors with cisplatin, the standard of care used for neuroendocrine carcinomas, significantly delays tumor growth in pre-clinical settings. Our study reveals mechanisms of UCHL1 function in regulating the progression of neuroendocrine carcinomas and identifies UCHL1 as a therapeutic target and potential molecular indicator for diagnosing and monitoring treatment responses in these malignancies.

Guanylate-binding protein 1 modulates proteasomal machinery in ovarian cancer.

Guanylate-binding protein 1 (GBP1) is known as an interferon-γ-induced GTPase. Here, we used genetically modified ovarian cancer (OC) cells to study the role of GBP1. The data generated show that GBP1 inhibition constrains the clonogenic potential of cancer cells. In vivo studies revealed that GBP1 overexpression in tumors promotes tumor progression and reduces median survival, whereas GBP1 inhibition delayed tumor progression with longer median survival. We employed proteomics-based thermal stability assay (CETSA) on GBP1 knockdown and overexpressed OC cells to study its molecular functions. CETSA results show that GBP1 interacts with many members of the proteasome. Furthermore, GBP1 inhibition sensitizes OC cells to paclitaxel treatment via accumulated ubiquitinylated proteins where GBP1 inhibition decreases the overall proteasomal activity. In contrast, GBP1-overexpressing cells acquired paclitaxel resistance via boosted cellular proteasomal activity. Overall, these studies expand the role of GBP1 in the activation of proteasomal machinery to acquire chemoresistance.

Proteomics analysis of urine and catheter-associated biofilms in spinal cord injury patients.

After spinal cord injury (SCI), use chronic urinary catheters for bladder management is common, making these patients especially vulnerable to catheter-associated complications. Chronic catheterization is associated with bacterial colonization and frequent catheter-associated urinary tract infections (CAUTI). One determinant of infection success and treatment resistance is production of catheter-associated biofilms, composed of microorganisms and host- and microbial-derived components. To better understand the biofilm microenvironment, we performed proteomics analysis of catheter-associated biofilms and paired urine samples from four people with SCI with chronic indwelling urinary catheters. We developed a novel method for the removal of adhered cellular components on catheters that contained both human and microbial homologous proteins. Proteins from seven microbial species were identified including: Escherichia coli, Klebsiella species (spp), Enterococcus spp, Proteus mirabilis, Pseudomonas spp, Staphylococcus spp, and Candida spp. Peptides identified from catheter biofilms were assigned to 4,820 unique proteins, with 61% of proteins assigned to the biofilm-associated microorganisms, while the remainder were human-derived. Contrastingly, in urine, only 51% were assigned to biofilm-associated microorganisms and 4,554 proteins were identified as a human-derived. Of the proteins assigned to microorganisms in the biofilm and paired urine, Enterococcus, Candida spp, and P. mirabilis had greater associations with the biofilm phase, whereas E. coli and Klebsiella had greater associations with the urine phase, thus demonstrating a significant difference between the urine and adhered microbial communities. The microbial proteins that differed significantly between the biofilm and paired urine samples mapped to pathways associated with amino acid synthesis, likely related to adaptation to high urea concentrations in the urine, and growth and protein synthesis in bacteria in the biofilm. Human proteins demonstrated enrichment for immune response in the catheter-associated biofilm. Proteomic analysis of catheter-associated biofilms and paired urine samples has the potential to provide detailed information on host and bacterial responses to chronic indwelling urinary catheters and could be useful for understanding complications of chronic indwelling catheters including CAUTIs, urinary stones, and catheter blockages.

Lineage Plasticity in SCLC Generates Non-Neuroendocrine Cells Primed for Vasculogenic Mimicry.

INTRODUCTION: Vasculogenic mimicry (VM), the process of tumor cell transdifferentiation to endow endothelial-like characteristics supporting de novo vessel formation, is associated with poor prognosis in several tumor types, including SCLC. In genetically engineered mouse models (GEMMs) of SCLC, NOTCH, and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch, and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs. METHODS: We analyzed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX, GEMMs, and patient biopsies. We evaluated their three-dimensional structure and defined collagen-integrin interactions. RESULTS: We found that VM vessels are present in 23/25 CDX models, 2 GEMMs, and in 20 patient biopsies of SCLC. Perfused VM vessels support tumor growth and only NOTCH-active non-NE cells are VM-competent in vivo and ex vivo, expressing pseudohypoxia, blood vessel development, and extracellular matrix organization signatures. On Matrigel, VM-primed non-NE cells remodel extracellular matrix into hollow tubules in an integrin ß1-dependent process. CONCLUSIONS: We identified VM as an exemplar of functional heterogeneity and plasticity in SCLC and these findings take considerable steps toward understanding the molecular events that enable VM. These results support therapeutic co-targeting of both NE and non-NE cells to curtail SCLC progression and to improve the outcomes of patients with SCLC in the future.

Measuring the multifaceted roles of mucin-domain glycoproteins in cancer.

Mucin-domain glycoproteins are highly O-glycosylated cell surface and secreted proteins that serve as both biochemical and biophysical modulators. Aberrant expression and glycosylation of mucins are known hallmarks in numerous malignancies, yet mucin-domain glycoproteins remain enigmatic in the broad landscape of cancer glycobiology. Here we review the multifaceted roles of mucins in cancer through the lens of the analytical and biochemical methods used to study them. We also describe a collection of emerging tools that are specifically equipped to characterize mucin-domain glycoproteins in complex biological backgrounds. These approaches are poised to further elucidate how mucin biology can be understood and subsequently targeted for the next generation of cancer therapeutics.

Integrated transcriptome-proteome analyses of human stem cells reveal source-dependent differences in their regenerative signature.

Mesenchymal stem cells (MSCs) are gaining increasing prominence as an effective regenerative cellular therapy. However, ensuring consistent and reliable effects across clinical populations has proved to be challenging. In part, this can be attributed to heterogeneity in the intrinsic molecular and regenerative signature of MSCs, which is dependent on their source of origin. The present work uses integrated omics-based profiling, at different functional levels, to compare the anti-inflammatory, immunomodulatory, and angiogenic properties between MSCs from neonatal (umbilical cord MSC [UC-MSC]) and adult (adipose tissue MSC [AD-MSC], and bone marrow MSC [BM-MSC]) sources. Using multi-parametric analyses, we identified that UC-MSCs promote a more robust host innate immune response; in contrast, adult-MSCs appear to facilitate remodeling of the extracellular matrix (ECM) with stronger activation of angiogenic cascades. These data should help facilitate the standardization of source-specific MSCs, such that their regenerative signatures can be confidently used to target specific disease processes.

SU086, an inhibitor of HSP90, impairs glycolysis and represents a treatment strategy for advanced prostate cancer.

Among men, prostate cancer is the second leading cause of cancer-associated mortality, with advanced disease remaining a major clinical challenge. We describe a small molecule, SU086, as a therapeutic strategy for advanced prostate cancer. We demonstrate that SU086 inhibits the growth of prostate cancer cells in vitro, cell-line and patient-derived xenografts in vivo, and ex vivo prostate cancer patient specimens. Furthermore, SU086 in combination with standard of care second-generation anti-androgen therapies displays increased impairment of prostate cancer cell and tumor growth in vitro and in vivo. Cellular thermal shift assay reveals that SU086 binds to heat shock protein 90 (HSP90) and leads to a decrease in HSP90 levels. Proteomic profiling demonstrates that SU086 binds to and decreases HSP90. Metabolomic profiling reveals that SU086 leads to perturbation of glycolysis. Our study identifies SU086 as a treatment for advanced prostate cancer as a single agent or when combined with second-generation anti-androgens.

Protein signatures to distinguish aggressive from indolent prostate cancer.

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.

Multiomics Analysis of Spatially Distinct Stromal Cells Reveals Tumor-Induced O-Glycosylation of the CDK4-pRB Axis in Fibroblasts at the Invasive Tumor Edge.

The invasive leading edge represents a potential gateway for tumor metastasis. The role of fibroblasts from the tumor edge in promoting cancer invasion and metastasis has not been comprehensively elucidated. We hypothesize that cross-talk between tumor and stromal cells within the tumor microenvironment results in activation of key biological pathways depending on their position in the tumor (edge vs. core). Here we highlight phenotypic differences between tumor-adjacent-fibroblasts (TAF) from the invasive edge and tumor core fibroblasts from the tumor core, established from human lung adenocarcinomas. A multiomics approach that includes genomics, proteomics, and O-glycoproteomics was used to characterize cross-talk between TAFs and cancer cells. These analyses showed that O-glycosylation, an essential posttranslational modification resulting from sugar metabolism, alters key biological pathways including the cyclin-dependent kinase 4 (CDK4) and phosphorylated retinoblastoma protein axis in the stroma and indirectly modulates proinvasive features of cancer cells. In summary, the O-glycoproteome represents a new consideration for important biological processes involved in tumor-stroma cross-talk and a potential avenue to improve the anticancer efficacy of CDK4 inhibitors. SIGNIFICANCE: A multiomics analysis of spatially distinct fibroblasts establishes the importance of the stromal O-glycoproteome in tumor-stroma interactions at the leading edge and provides potential strategies to improve cancer treatment. See related commentary by De Wever, p. 537.

Oncogene-mediated metabolic gene signature predicts breast cancer outcome.

Breast cancer remains the second most lethal cancer among women in the United States and triple-negative breast cancer is the most aggressive subtype with limited treatment options. Trop2, a cell membrane glycoprotein, is overexpressed in almost all epithelial cancers. In this study, we demonstrate that Trop2 is overexpressed in triple-negative breast cancer (TNBC), and downregulation of Trop2 delays TNBC cell and tumor growth supporting the oncogenic role of Trop2 in breast cancer. Through proteomic profiling, we discovered a metabolic signature comprised of TALDO1, GPI, LDHA, SHMT2, and ADK proteins that were downregulated in Trop2-depleted breast cancer tumors. The identified oncogene-mediated metabolic gene signature is significantly upregulated in TNBC patients across multiple RNA-expression clinical datasets. Our study further reveals that the metabolic gene signature reliably predicts poor survival of breast cancer patients with early stages of the disease. Taken together, our study identified a new five-gene metabolic signature as an accurate predictor of breast cancer outcome.

MCM2-7 complex is a novel druggable target for neuroendocrine prostate cancer.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer that rarely develops de novo in primary tumors and is commonly acquired during the development of treatment resistance. NEPC is characterized by gain of neuroendocrine markers and loss of androgen receptor (AR), making it resistant to current therapeutic strategies targeting the AR signaling axis. Here, we report that MCM2, MCM3, MCM4, and MCM6 (MCM2/3/4/6) are elevated in human NEPC and high levels of MCM2/3/4/6 are associated with liver metastasis and poor survival in prostate cancer patients. MCM2/3/4/6 are four out of six proteins that form a core DNA helicase (MCM2-7) responsible for unwinding DNA forks during DNA replication. Inhibition of MCM2-7 by treatment with ciprofloxacin inhibits NEPC cell proliferation and migration in vitro, significantly delays NEPC tumor xenograft growth, and partially reverses the neuroendocrine phenotype in vivo. Our study reveals the clinical relevance of MCM2/3/4/6 proteins in NEPC and suggests that inhibition of MCM2-7 may represent a new therapeutic strategy for NEPC.

Enrichment of Intact Glycopeptides Using Strong Anion Exchange and Electrostatic Repulsion Hydrophilic Interaction Chromatography.

Glycosylation is a biologically important and complex protein posttranslational modification. The emergence of glycoproteomic technologies to identify and characterize glycans on proteins has the potential to enable a better understanding the role of glycosylation in biology, disease states, and other areas of interest. In particular, the analysis of intact glycopeptides by mass spectrometry allows information about glycan location and composition to be ascertained. However, such analysis is often complicated by extensive glycan diversity and the low abundance of glycopeptides in a complex mixture relative to nonglycosylated peptides. Enrichment of glycopeptides from a protein enzymatic digest is an effective approach to overcome such challenges. In this chapter, we described a glycopeptide enrichment method combining strong anion exchange, electrostatic repulsion, and hydrophilic interaction chromatography (SAX-ERLIC). Following enzymatic digestion of proteins into peptides, SAX-ERLIC is performed by solid phase extraction to enrich glycopeptides from biological samples with subsequent LC-MS/MS analysis. Glycopeptide data generated using the SAX-ERLIC enrichment yields a high number of total and unique glycopeptide identifications which can be mapped back to proteins. The enrichment strategy is robust, easy to perform, and does not require cleavage of glycans prior to LC-MS/MS analysis.

Discovery of CASP8 as a potential biomarker for high-risk prostate cancer through a high-multiplex immunoassay.

Prostate cancer remains the most common non-cutaneous malignancy among men in the United States. To discover potential serum-based biomarkers for high-risk prostate cancer, we performed a high-multiplex immunoassay utilizing patient-matched pre-operative and post-operative serum samples from ten men with high-grade and high-volume prostate cancer. Our study identified six (CASP8, MSLN, FGFBP1, ICOSLG, TIE2 and S100A4) out of 174 proteins that were significantly decreased after radical prostatectomy. High levels of CASP8 were detected in pre-operative serum samples when compared to post-operative serum samples and serum samples from patients with benign prostate hyperplasia (BPH). By immunohistochemistry, CASP8 protein was expressed at higher levels in prostate cancer tissues compared to non-cancerous and BPH tissues. Likewise, CASP8 mRNA expression was significantly upregulated in prostate cancer when compared to benign prostate tissues in four independent clinical datasets. In addition, mRNA levels of CASP8 were higher in patients with recurrent prostate cancer when compared to patients with non-recurrent prostate cancer and high expression of CASP8 was associated with worse disease-free survival and overall survival in renal cancer. Together, our results suggest that CASP8 may potentially serve as a biomarker for high-risk prostate cancer and possibly renal cancer.

Y box binding protein 1 inhibition as a targeted therapy for ovarian cancer.

Y box binding protein 1 (YB-1) is a multifunctional protein associated with tumor progression and the emergence of treatment resistance (TR). Here, we report an azopodophyllotoxin small molecule, SU056, that potently inhibits tumor growth and progression via YB-1 inhibition. This YB-1 inhibitor inhibits cell proliferation, resistance to apoptosis in ovarian cancer (OC) cells, and arrests in the G1 phase. Inhibitor treatment leads to enrichment of proteins associated with apoptosis and RNA degradation pathways while downregulating spliceosome pathway. In vivo, SU056 independently restrains OC progression and exerts a synergistic effect with paclitaxel to further reduce disease progression with no observable liver toxicity. Moreover, in vitro mechanistic studies showed delayed disease progression via inhibition of drug efflux and multidrug resistance 1, and significantly lower neurotoxicity as compared with etoposide. These data suggest that YB-1 inhibition may be an effective strategy to reduce OC progression, antagonize TR, and decrease patient mortality.

A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry-Based Glycoproteomics.

Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography and its derivatives, porous graphitic carbon, reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as mass spectrometry instrumentation and software improve, so this review aims to help equip researchers with the necessary information to choose appropriate enrichment strategies that best complement these efforts.

Novel Aza-podophyllotoxin derivative induces oxidative phosphorylation and cell death via AMPK activation in triple-negative breast cancer.

BACKGROUND: To circumvent Warburg effect, several clinical trials for different cancers are utilising a combinatorial approach using metabolic reprogramming and chemotherapeutic agents including metformin. The majority of these metabolic interventions work via indirectly activating AMP-activated protein kinase (AMPK) to alter cellular metabolism in favour of oxidative phosphorylation over aerobic glycolysis. The effect of these drugs is dependent on glycaemic and insulin conditions.  Therefore, development of small molecules, which can activate AMPK, irrespective of the energy state, may be a better approach for triple-negative breast cancer (TNBC) treatment. METHODS: Therapeutic effect of SU212 on TNBC cells was examined using in vitro and in vivo models. RESULTS: We developed and characterised the efficacy of novel AMPK activator (SU212) that selectively induces oxidative phosphorylation and decreases glycolysis in TNBC cells, while not affecting these pathways in normal cells.   SU212 accomplished this metabolic reprogramming by activating AMPK independent of energy stress and irrespective of the glycaemic/insulin state. This leads to mitotic phase arrest and apoptosis in TNBC cells. In vivo, SU212 inhibits tumour growth, cancer progression and metastasis. CONCLUSIONS: SU212 directly activates AMPK in TNBC cells, but does not hamper glucose metabolism in normal cells. Our study provides compelling preclinical data for further development of SU212 for the treatment of TNBC.

Plectin is a regulator of prostate cancer growth and metastasis.

Prostate cancer is responsible for over 30,000 US deaths annually, attributed largely to incurable metastatic disease. Here, we demonstrate that high levels of plectin are associated with localized and metastatic human prostate cancer when compared to benign prostate tissues. Knock-down of plectin inhibits prostate cancer cell growth and colony formation in vitro, and growth of prostate cancer xenografts in vivo. Plectin knock-down further impairs aggressive and invasive cellular behavior assessed by migration, invasion, and wound healing in vitro. Consistently, plectin knock-down cells have impaired metastatic colonization to distant sites including liver, lung, kidney, bone, and genitourinary system. Plectin knock-down inhibited number of metastases per organ, as well as decreased overall metastatic burden. To gain insights into the role of plectin in prostate cancer growth and metastasis, we performed proteomic analysis of prostate cancer plectin knock-down xenograft tissues. Gene set enrichment analysis shows an increase in levels of proteins involved with extracellular matrix and laminin interactions, and a decrease in levels of proteins regulating amino acid metabolism, cytoskeletal proteins, and cellular response to stress. Collectively these findings demonstrate that plectin is an important regulator of prostate cancer cell growth and metastasis.