Eunju Choi

High-throughput lectin magnetic bead array-coupled tandem mass spectrometry for glycoprotein biomarker discovery

Alterations in protein glycosylation occur during development and progression of many diseases, hence glycomics and glycoproteomics have emerged as important tools in glycobiomarker discovery. High‐throughput glycan profiling can now be achieved with the recent developments in MS‐based techniques. To enable identification and rapid monitoring of glycosylation changes in serum proteins, we developed a semi‐automated high‐throughput glycoprotein biomarker discovery platform termed lectin magnetic bead array‐coupled tandem mass spectrometry (LeMBA‐MS) which includes (i) effective single‐step serum glycoprotein isolation using a panel of 20 individual lectin‐coated magnetic beads in microplate format, (ii) on‐bead trypsin digestion, and (iii) nanoLC‐MS/MS with lectin exclusion list. With use of appropriate sequence databases, LeMBA‐MS can detect glycosylation changes regardless of the species. By spiking known amounts of titrated ovalbumin to a serum sample, we report nanomolar sensitivity, and linearity of response of LeMBA‐MS using concanavalin A‐coupled beads. Neuraminidase treatment led to reduction of binding to sialic acid‐binding lectins. Interestingly, we found that desialylation caused increased binding of haptoglobin and hemopexin to mannose‐specific lectins, pointing to the importance of identifying a signature of lectin‐binding. High‐throughput LeMBA‐MS to generate glycosylation signatures will facilitate glycobiomarker discovery. LeMBA can be coupled to down‐stream detection platforms for validation, making it a truly versatile platform.

PTRF/cavin-1 neutralizes non-caveolar caveolin-1 microdomains in prostate cancer

Caveolin-1 has a complex role in prostate cancer and has been suggested to be a potential biomarker and therapeutic target. As mature caveolin-1 resides in caveolae, invaginated lipid raft domains at the plasma membrane, caveolae have been suggested as a tumor-promoting signaling platform in prostate cancer. However, caveola formation requires both caveolin-1 and cavin-1 (also known as PTRF; polymerase I and transcript release factor). Here, we examined the expression of cavin-1 in prostate epithelia and stroma using tissue microarray including normal, non-malignant and malignant prostate tissues. We found that caveolin-1 was induced without the presence of cavin-1 in advanced prostate carcinoma, an expression pattern mirrored in the PC-3 cell line. In contrast, normal prostate epithelia expressed neither caveolin-1 nor cavin-1, while prostate stroma highly expressed both caveolin-1 and cavin-1. Utilizing PC-3 cells as a suitable model for caveolin-1-positive advanced prostate cancer, we found that cavin-1 expression in PC-3 cells inhibits anchorage-independent growth, and reduces in vivo tumor growth and metastasis in an orthotopic prostate cancer xenograft mouse model. The expression of α-smooth muscle actin in stroma along with interleukin-6 (IL-6) in cancer cells was also decreased in tumors of mice bearing PC-3-cavin-1 tumor cells. To determine whether cavin-1 acts by neutralizing caveolin-1, we expressed cavin-1 in caveolin-1-negative prostate cancer LNCaP and 22Rv1 cells. Caveolin-1 but not cavin-1 expression increased anchorage-independent growth in LNCaP and 22Rv1 cells. Cavin-1 co-expression reversed caveolin-1 effects in caveolin-1-positive LNCaP cells. Taken together, these results suggest that caveolin-1 in advanced prostate cancer is present outside of caveolae, because of the lack of cavin-1 expression. Cavin-1 expression attenuates the effects of non-caveolar caveolin-1 microdomains partly via reduced IL-6 microenvironmental function. With circulating caveolin-1 as a potential biomarker for advanced prostate cancer, identification of the molecular pathways affected by cavin-1 could provide novel therapeutic targets.

Cavin-1/PTRF alters prostate cancer cell-derived extracellular vesicle content and internalization to attenuate extracellular vesicle-mediated osteoclastogenesis and osteoblast proliferation

Background Tumour-derived extracellular vesicles (EVs) play a role in tumour progression; however, the spectrum of molecular mechanisms regulating EV secretion and cargo selection remain to be fully elucidated. We have reported that cavin-1 expression in prostate cancer PC3 cells reduced the abundance of a subset of EV proteins, concomitant with reduced xenograft tumour growth and metastasis. Methods We examined the functional outcomes and mechanisms of cavin-1 expression on PC3-derived EVs (PC3-EVs). Results PC3-EVs were internalized by osteoclast precursor RAW264.7 cells and primary human osteoblasts (hOBs) in vitro, stimulating osteoclastogenesis 37-fold and hOB proliferation 1.5-fold, respectively. Strikingly, EVs derived from cavin-1-expressing PC3 cells (cavin-1-PC3-EVs) failed to induce multinucleate osteoblasts or hOB proliferation. Cavin-1 was not detected in EVs, indicating an indirect mechanism of action. EV morphology, size and quantity were also not affected by cavin-1 expression, suggesting that cavin-1 modulated EV cargo recruitment rather than release. While cavin-1-EVs had no osteoclastogenic function, they were internalized by RAW264.7 cells but at a reduced efficiency compared to control EVs. EV surface proteins are required for internalization of PC3-EVs by RAW264.7 cells, as proteinase K treatment abolished uptake of both control and cavin-1-PC3-EVs. Removal of sialic acid modifications by neuraminidase treatment increased the amount of control PC3-EVs internalized by RAW264.7 cells, without affecting cavin-1-PC3-EVs. This suggests that cavin-1 expression altered the glycosylation modifications on PC3-EV surface. Finally, cavin-1 expression did not affect EV in vivo tissue targeting as both control and cavin-1-PC3-EVs were predominantly retained in the lung and bone 24 hours after injection into mice. Discussion Taken together, our results reveal a novel pathway for EV cargo sorting, and highlight the potential of utilizing cavin-1-mediated pathways to attenuate metastatic prostate cancer.

Glyco-centric lectin magnetic bead array (LeMBA) − proteomics dataset of human serum samples from healthy, Barrett׳s esophagus and esophageal adenocarcinoma individuals

This data article describes serum glycoprotein biomarker discovery and qualification datasets generated using lectin magnetic bead array (LeMBA) – mass spectrometry techniques, “Serum glycoprotein biomarker discovery and qualification pipeline reveals novel diagnostic biomarker candidates for esophageal adenocarcinoma” [1]. Serum samples collected from healthy, metaplastic Barrett׳s esophagus (BE) and esophageal adenocarcinoma (EAC) individuals were profiled for glycoprotein subsets via differential lectin binding. The biomarker discovery proteomics dataset consisting of 20 individual lectin pull-downs for 29 serum samples with a spiked-in internal standard chicken ovalbumin protein has been deposited in the PRIDE partner repository of the ProteomeXchange Consortium with the data set identifier PRIDE: PXD002442. Annotated MS/MS spectra for the peptide identifications can be viewed using MS-Viewer (〈http://prospector2.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer〉) using search key “jn7qafftux”. The qualification dataset contained 6-lectin pulldown-coupled multiple reaction monitoring-mass spectrometry (MRM-MS) data for 41 protein candidates, from 60 serum samples. This dataset is available as a supplemental files with the original publication [1].

Abstract 4950: Hypercholesterolemia promotes prostate cancer PC-3 metastases in orthotopic xenograft mice

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Hypercholesterolemia has been proposed as a potential risk factor for advanced prostate cancer, and use of cholesterol-lowering drugs, statins, inversely correlates with advanced prostate cancer risk. Hypercholesterolemia increases growth of androgen-sensitive LNCaP xenograft in vivo, potentially via androgen signaling. The aim of this study was to determine if hypercholesterolemia affects castration-resistant prostate tumor progression using an androgen receptor-negative prostate cancer cell line PC-3. Compared to control media, cholesterol-deficient media reduced PC-3 proliferation, migration and anchorage-independent growth in vitro. While adding cholesterol did not significantly increase proliferation or anchorage-independent growth, cholesterol replacement in cholesterol-deficient media significantly increased PC-3 transmigration. In order to determine the in vivo effect, mice were randomly assigned to normal or hypercholesterolemic, isocaloric diet groups (N=14 and 15, respectively). After two weeks, hypercholesterolemic diet significantly increased circulating cholesterol but did not increase body weight. PC-3 cells stably expressing luciferase were orthotopically injected into the dorsolateral prostate. Tumor progression and metastases were monitored by in vivo and ex vivo optical bioluminescence imaging for 6 weeks. Strikingly, the results show that diet-induced hypercholesterolemia accelerated tumor metastases to lymph nodes, lung, proximal and distant bones without significantly affecting primary tumor growth. The metastases were confirmed histopathologcally. Hypercholesterolemia was not associated with elevated weight or circulating testosterone. This is the first study to directly demonstrate a causal relationship between hypercholesterolemia and prostate tumor metastases mediated through androgen-independent mechanisms, highlighting the potential clinical benefit of cholesterol lowering therapy such as statins in advanced, castration-resistant, prostate cancer patients. Citation Format: Hyeongsun Moon, Laura Sharpe, Eunju Choi, Helle Bielefeldt-Ohmann, Zeyad Nassar, Marie-Odile Parat, Mathias Francois, C Soon Lee, Andrew Brown, Pamela Russell, Kerry Inder, Michelle Hill. Hypercholesterolemia promotes prostate cancer PC-3 metastases in orthotopic xenograft mice. [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 4950. doi:10.1158/1538-7445.AM2014-4950

Abstract 2492: Discovery and validation of novel serum glycoprotein biomarkers for Barrett's esophagus and esophageal adenocarcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND: Esophageal adenocarcinoma (EAC) is one of the most rapidly increasing cancers globally. The majority of EAC cases are diagnosed at very late stages during pathogenesis hence <15% of the patients survive 5 year post-diagnosis. There is an urgent need to improve diagnosis of EAC and its pre-cancer metaplastic condition, Barretts esophagus (BE). BE patients are monitored using upper gastro-esophageal endoscopy with biopsy for early neoplastic changes. However, being an asymptomatic condition, it is very difficult to identify BE patients for screening. Moreover, endoscopy is unsuitable for population screening due to high cost, requirement of technical expertise and patient non-compliance. The aim of this project is to identify serum biomarkers for diagnosis of BE and EAC, with the goal of translating to blood tests. APPROACH & METHODOLOGY: We focused on alterations in circulatory protein glycosylation, using a panel of 20 lectins to isolate different glycan structures on serum glycoproteins, as reported recently [1, 2]. Serum samples from healthy (n = 9), BE (n = 10) and EAC (n = 10) patient groups were analyzed by lectin magnetic bead array-coupled mass spectrometry (LeMBA-MS/MS) [1, 2]. Data analysis was performed using a customized database and analysis package “GlycoSelect” which incorporates outlier detection and sparse Partial Least Squares regression discriminant analysis (sPLS-DA) [3]. RESULTS & DISCUSSION: We identified a ranked list of candidate glycobiomarkers that distinguish a) EAC from BE b) BE from healthy and c) EAC from healthy group. In general, glycoproteins bound several lectins, reflecting heterogeneity and multiplicity of glycosylation. Specific glycan structure changes were observed as loss and gain of binding to a single lectin while maintaining binding to other lectins. Top two candidate biomarkers were validated using orthogonal validation technique LeMBA-immunoblotting in an independent patient cohort (n = 80). The biomarkers showed Area under Receiver Operating Characteristic curve (AUROC) of 0.74 to discriminate EAC from BE and 0.71 to discriminate BE from healthy patient group. Future work will validate all candidate protein-lectin pairs using lectin-affinity array coupled with triple quadrupole quantitative mass spectrometry measurements for the independent patient cohort. The specificity and sensitivity of panels of glycoprotein biomarkers will be determined for formulating a serum screening test for BE and EAC. [1] Choi et al., Electrophoresis 32, 3564-3575 (2011) [2] Loo et al., J Proteome Res 9, 5496-5500 (2010) [3] Le Cao et al., BMC Bioinformatics 12, 253-268 (2011) Citation Format: Alok K. Shah, David Chen, Kim-Anh Le Cao, Eunju Choi, Derek Nancarrow, David Whiteman, Nicholas A. Saunders, Andrew P. Barbour, Michelle M. Hill. Discovery and validation of novel serum glycoprotein biomarkers for Barretts esophagus and esophageal adenocarcinoma. [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 2492. doi:10.1158/1538-7445.AM2014-2492

Targeted High-Throughput Glycoproteomics for Glyco-Biomarker Discovery

Biomarker discovery has become a major research area in proteomics as protein markers are more readily developed into clinical diagnostic tests than nucleic acid biomarkers. This is reflected by the fact that all United States Food and Drug Administration (US FDA)approved biomarkers currently available for clinical use are protein molecules (Srivastava, Verma, and Gopal-Srivastava 2005). Proteomic technologies for the global study of proteins have evolved in the past decade, in response to the growing demand for body fluid biomarker development (Anderson and Hunter 2006; Wang, Whiteaker, and Paulovich 2009). While mass spectrometry technology is improving in sensitivity and speed, several technical challenges in protein biomarker discovery still requires optimization. These include maximizing sample throughput to process adequate number of samples, reaching high sensitivity, specificity and reproducibility required for FDA approval, and managing the costs for biomarker discovery and assay development. This chapter will discuss the application of a targeted proteomics approach using lectins as affinity reagent throughout the biomarker discovery pipeline, and automation with magnetic beads to increase throughput.

Abstract 2499: Diagnostic serum biomarkers for canine hemangiosarcoma: a potential model of human angiosarcoma.

People suffering from uncommon diseases often do not benefit from the advances in medical research due to lack of research. One such disease is angiosarcoma, a highly aggressive cancer originating from blood vessels with risk factors including toxic exposure, radiotherapy and lymphedema. Angiosarcoma in dogs (commonly called hemangiosarcoma) is a very similar disease with much higher prevalence, comprising up to 7% of all canine malignancies. For both species, an accurate diagnostic method is needed for more favorable outcome. We developed a biomarker discovery pipeline consisting of a high-throughput biomarker discovery platform and a database for data mining and statistical analysis for candidate identification. The platform screens for differential glycosylation of serum glycoproteins as potential biomarkers utilizing 20 individual lectins followed by direct coupling to tandem mass spectrometry. The database facilitates the identification of differential glycosylation and produces a ranked list of potential biomarker candidates. Using this pipeline, sera from 10 dogs with hemangiosarcoma with 10 age and sex-matched control sera were processed and analyzed. Candidates from the database were ranked based on its ability to classify normal and cancer. The ranked list of candidates for this screen composed of glycoproteins involved in cell migration, tumor immune response, and glycoproteins reported to be altered in human and canine angiosarcoma and other cancer types. Some examples will be shown to illustrate the change in specific glycosylation structures associated with cancer as measured by binding to specific lectin(s) while binding to other lectins are not changed. These glycosylation-specific changes are potentially more specific diagnostic biomarkers than circulating proteins alone. We are currently validating the identified candidates and pursuing the development of clinical tests to improve diagnosis and clinical outcomes of angiosarcoma for both dogs and humans. Citation Format: Eunju Choi, David Chen, Kim-Anh Le Cao, Helle Bielefeldt-Ohmann, Caroline A. O9Leary, Michelle M. Hill. Diagnostic serum biomarkers for canine hemangiosarcoma: a potential model of human angiosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2499. doi:10.1158/1538-7445.AM2013-2499