[Show abstract][Hide abstract] ABSTRACT: Glioblastoma (GBM) tumour invasion is facilitated by cell migration and degradation of the extracellular matrix (ECM). Invadopodia are actin-rich structures that protrude from the plasma membrane in direct contact with the ECM and are proposed to participate in epithelial-mesenchymal-transition. We have characterised the invasiveness of nine established GBM cell lines using an invadopodia assay and performed quantitative MS-based proteomic analyses on enriched membrane fractions. All GBM cells produced invadopodia, with a 65% difference between the most (U87MG) and least invasive (LN229) cells (p=0.0001). Overall, 1,141 proteins were identified in the GBM membrane proteome, the levels of 49 proteins correlated with cell invasiveness. Ingenuity pathway analysis predicted the activation ‘cell movement’ (z-score=2.608, p=3.94E-04) in more invasive cells and a network of invasion-associated proteins with direct links to key regulators of invadopodia formation was generated. Gene expression data relating to invasion-associated proteins, ITGA5, CD97 and ANXA1 show prognostic significance in independent GBM cohorts. Fluorescence microscopy demonstrated ITGA5, CD97 and ANXA1 localisations in invadopodia assays and siRNA-knockdown of ITGA5 reduced invadopodia formation in U87MG cells. Invasion-associated proteins, including ITGA5, may prove to be useful anti-invasive targets. The use of volociximab, a therapeutic antibody against integrin α5β1 should be assessed in a GBM setting.
Journal of Neuropathology and Experimental Neurology 04/2015; 74(5). DOI:10.1097/NEN.0000000000000187 · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) reinfection of the liver allograft after transplantation is universal, with some individuals suffering severe disease recurrence. Predictive markers of recurrent disease severity are urgently needed. In this study, we used a cluster of differentiation (CD) microarray to predict the severity of HCV recurrence after transplantation.
The CD antibody microarray assays of live leukocytes were performed on peripheral blood taken in the first year after transplantation. The results were grouped into phases defined as; Pre-transplant (day 0), Early (day 3 to week 2), Mid (week 4 to week 10), and Late (week 12 to week 26). Hepatitis C virus severity was based on fibrosis stages in the first 2 years (F0-1 mild and F2-4 severe).
Serial blood samples from 16 patients were taken before and after liver transplantation. A total of 98 assays were performed. Follow-up was 3 years or longer. Comparing recurrence severity, significantly greater numbers of CD antigens were differentially expressed on the pretransplant samples compared to any posttransplant timepoints. Five differentially expressed CD antigens before transplantation (CD27 PH, CD182, CD260, CD41, and CD34) were significantly expressed comparing severe to mild recurrence, whereas expression of only CD152 was significant in the late phase after transplantation. No relationship was observed between the donor or recipient interleukin-28B genotypes and HCV recurrence severity.
This study shows that circulating leukocyte CD antigen expression has utility in assessing recurrent HCV disease severity after liver transplantation and serves as a proof of principle. Importantly, pretransplant CD antigen expression is most predictive of disease outcome.
[Show abstract][Hide abstract] ABSTRACT: Clinical trials of heat shock protein 90 (Hsp90) inhibitors have been limited by high toxicity. We previously showed that the Hsp90 inhibitor, SNX-7081, synergizes with and restores sensitivity to fludarabine nucleoside (2-FaraA) in human chronic lymphocytic leukemia (CLL) cells with lesions in the p53 pathway (Best OG, et al., Leukemia Lymphoma 53:1367-75, 2012). Here, we used label-free quantitative shotgun proteomics and comprehensive bioinformatic analysis to determine the mechanism of this synergy. We propose that 2-FaraA-induced DNA damage is compounded by SNX-7081-mediated inhibition of DNA repair, resulting in enhanced induction of apoptosis. DNA damage responses are impaired in part due to reductions in checkpoint regulators BRCA1 and cyclin D1, and cell death is triggered following reductions of MYC and nucleolin and an accumulation of apoptosis-inducing NFkB2 p100 subunit. Loss of nucleolin can activate Fas-mediated apoptosis, leading to the increase of pro-apoptotic proteins (BID, fas-associated factor-2) and subsequent apoptosis of p53-negative, 2-FaraA refractory CLL cells. A significant induction of DNA damage, indicated by increases in DNA damage marker γH2AX, was observed following the dual drug treatment of additional cell lines, indicating that a similar mechanism may operate in other p53-mutated human B-lymphoid cancers. These results provide valuable insight into the synergistic mechanism between SNX-7081 and 2-FaraA that may provide an alternative treatment for CLL patients with p53 mutations, for whom therapeutic options are currently limited. Moreover, this drug combination reduces the effective dose of the Hsp90 inhibitor and may therefore alleviate any toxicity encountered.
[Show abstract][Hide abstract] ABSTRACT: An antibody microarray (DotScanTM) has been developed for profiling clinical melanoma specimens. Immobilized antibodies capture live cells expressing corresponding antigens to produce a dot pattern that represents the surface profile or immunophenotype. The unique signatures obtained may correlate with disease subtype, tumour progression and clinical outcome. Here we describe the rapid analysis of surgically resected metastatic melanoma. Leukocytes are separated from tumour cells using CD45 antibody-conjugated magnetic beads and separated cell populations are profiled on the microarray. This antibody microarray may be extended to include additional antibodies for cell surface biomarkers and therapeutic antibodies.
Methods in Molecular Biology, Melanoma: Methods and Protocols edited by Alexander Boiko, 01/2015: chapter Surface antigen profiling of surgical melanoma specimens; Springer protocols.
[Show abstract][Hide abstract] ABSTRACT: Extensive surface profiles of colorectal cancer (CRC) cells and tumour infiltrating lymphocytes (TIL) have been obtained from 45 surgical resection samples. Live cells were captured on an antibody microarray and stained with fluorescently-labeled antibodies. Minimal panels of 11 CRC antigens (CD13, CD24, CD26, CD49d, CD138, CD166, CA-125, CA19-9, EGFR, Galectin-4 and HLA-DR) and 11 T-cell antigens (CD10, CD11b, CD11c, CD25, CD31, CD95, CD151, CD181, Galectin-4, CA19-9, TSP-1) provide signatures for relapse and survival. Hierarchical clustering of profiles from CRC cells and TIL identified groups of patients for survival, systemic relapse and death. The groups from CRC and TIL profiles for systemic relapse showed 79.2% concordance, enabling prediction of relapse after surgery. The results demonstrate communication between CRC cells and TIL.
[Show abstract][Hide abstract] ABSTRACT: s P17.54. Glioblastoma multiforme (GBM) tumours are diffusely infiltrative making surgical resection virtually impossible. Invasion of brain parenchyma is facil-itated by cell migration and degradation of the extracellular matrix (ECM). Invadopodia are actin-rich organelles that protrude from the ventral side of the plasma membrane in direct contact with the ECM and play an important role in mesenchymal cell invasion. We have characterized the 'invasive poten-tial' of a panel of established GBM cell lines (n ¼ 9) using QCM gelatin inva-dopodia assay (Millipore) and performed comparative, quantitative membrane mass spectrometry-based proteomic analyses of highly invasive vs. less-invasive cell lines. All GBM cells produced invadopodia, and there was a significant difference between the most invasive (U87MG) and least in-vasive (LN229) cells (65%, percentage of total cell area; p ¼ 0.0001). Overall, 1667 quantifiable proteins were identified from duplicate analyses, of which 76% mapped to membrane structures using the David bioinformatics data-base (http://david.abcc.ncifcrf.gov). The differential abundance of 38 pro-teins significantly correlated with the degree of invasion (r 2 . 0.45 or r 2 , -0.45; n ≥ 5; p , 0.05) and are predominantly involved in cellular movement and cell-cell and interactions. Fluorescence microscopy demonstrates co-localisation of novel proteins to invadopodia structures and siRNA knock-down of a target protein confirmed its role in invadopodia-formation. Invadopodia-associated membrane proteins could be novel targets for anti-invasive GBM therapies. Neuro-Oncology 16:ii1 – ii112, 2014. doi:10.1093/neuonc/nou174
European Association of NeuroOncology, Turin, Italy; 10/2014
[Show abstract][Hide abstract] ABSTRACT: Outcomes for melanoma patients with stage III disease, differ widely even within the same sub-category. Molecular signatures that more accurately predict prognosis are needed to stratify patients according to risk. Proteomic analyses were used to identify differentially abundant proteins in extracts of surgically excised samples from patients with stage IIIc melanoma lymph node metastases. Analysis of samples from patients with poor (n = 14, < 1 year) and good (n = 19, > 4 years) survival outcomes identified 84 proteins that were differentially abundant between prognostic groups. Subsequent selected reaction monitoring analysis verified 21 proteins as potential biomarkers for survival. Poor prognosis patients are characterized by increased levels of proteins involved in protein metabolism, nucleic acid metabolism, angiogenesis, deregulation of cellular energetics and methylation processes, and decreased levels of proteins involved in apoptosis and immune response. These proteins are able to classify stage IIIc patients into prognostic sub-groups (p < 0.02). This is the first report of potential prognostic markers from stage III melanoma using proteomic analyses. Validation of these protein markers in larger patient cohorts should define protein signatures that enable better stratification of stage III melanoma patients.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Altered glycosylation is commonly observed in colorectal cancer. In vitro models are frequently used to study this cancer but little is known about the differences that may exist between these model cell systems and tumour tissue. We have compared the membrane protein glycosylation of five colorectal cancer cell lines (SW1116, SW480, SW620, SW837, LS174T) with epithelial cells from colorectal tumours using liquid chromatography tandem mass spectrometry. Remarkably, there were five abundant O-glycans in the tumour cells that were undetected in the low-mucin producing cell lines, although two were found in the mucinous LS174T cells. The O-glycans included the well-known glycan cancer marker, sialyl-Tn, which has been associated with mucins. Using qRT-PCR, sialyl-Tn expression was found to be associated with an increase in α2,6-sialyltransferase gene (ST6GALNAC1) and a decrease in core 1 synthase gene (C1GALT1) in LS174T cells. The expression of a subset of mucins (MUC2, MUC6, MUC5B) was also correlated with sialyl-Tn expression in LS174T cells. Overall, the membrane protein glycosylation of the model cell lines was found to differ from each other and from the epithelial cells of tumour tissue. These findings should be noted in the design of biomarker discovery experiments particularly when cell surface targets are being investigated.
The extent of protein glycosylation differences between in vitro cell lines and ex vivo tumours in colorectal cancer research is unknown. Our study expands current knowledge by characterising the membrane protein glycosylation profiles of five different colorectal cancer cell lines and of epithelial cells derived from resected colorectal cancer tumour tissue, using liquid chromatography tandem mass spectrometry. The detailed structural differences found in both N- and O-linked glycan structures on the membrane glycoproteins were determined and correlated with the mRNA expression of the relevant proteins in the cell lines. The glycosylation differences found between cultured cancer cell lines and epithelial cells from tumour tissue have important implications for glycan biomarker discovery.
Journal of Proteomics 05/2014; 108. DOI:10.1016/j.jprot.2014.05.002 · 3.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fludarabine (2-FaraAMP) is a purine analog that is effective against chronic lymphocytic leukemia (CLL) and non-Hodgkins lymphoma (NHL). For some cases of CLL, 2-FaraAMP as a single agent can clear the blood of leukemia cells, but leukemia stem cells usually remain protected in sanctuary sites. It is clear that 2-FaraAMP has multiple mechanisms of action that may collectively result in strand breaks in DNA, accumulation of phosphorylated p53 and apoptosis. We have demonstrated using the human Burkitt's lymphoma B-cell line, Raji, that p53, p63 and p73 all accumulate in the nucleus, following treatment of cells with fludarabine nucleoside (2-FaraA). In addition, phosphorylated p53 accumulates in the cytosol and at mitochondria. Using sophisticated methods of proteomic analysis with mass spectrometry, proteins that become differentially abundant after treatment of cells with 2-FaraA have been identified, providing considerable additional information about the cellular responses of B-lymphoid cancers to this purine analog. The levels of proteins involved in the unfolded protein response increase, indicating that endoplasmic reticulum stress is likely to be one mechanism for induction of apoptosis. The levels of a number of proteins found on the outer plasma membrane change on cells treated with 2-FaraA, suggesting that signaling from the B-cell antigen receptor (BCR) is stimulated, resulting in induction of apoptosis through the intrinsic pathway. Increased levels of the cell surface proteins, CD50, CD100 and ECE-1, would promote survival of these cells; the balance between these survival and death responses would determine the fate of the cell.
[Show abstract][Hide abstract] ABSTRACT: There is an urgent need to identify more accurate prognostic biomarkers in melanoma patients, particularly in those with metastatic disease. This study aimed to identify melanoma and leukocyte surface antigens predictive of survival in a prospective series of AJCC stage IIIb/c melanoma patients (n = 29). Live cell suspensions were prepared from melanoma metastases within lymph nodes (LN). The suspensions were immuno-magnetically separated into CD45+ (leukocyte) and CD45- (non-hematopoietic, enriched melanoma cell) fractions. Surface antigens on CD45- and CD45+ cell populations were profiled using DotScan™ microarrays (Medsaic Pty. Ltd.) and showed differential abundance levels for 52 and 78 antigens respectively. Associations of the surface profiles with clinicopathologic and outcome data (median follow-up 35.4 months post LN resection) were sought using univariate (log-rank test) and multivariate (Wald's test; modelled with patient's age, gender and AJCC staging at LN recurrence) survival models. CD9 (p = 0.036), CD39 (p = 0.004) and CD55 (p = 0.005) on CD45+ leukocytes were independently associated with distant metastasis-free survival using multivariate analysis. Leukocytes with high CD39 levels were also significantly associated with increased overall survival (OS) in multivariate analysis (p = 0.016). LNs containing leukocytes expressing CD11b (p = 0.025), CD49d (p = 0.043) and CD79b (p = 0.044) were associated with reduced OS on univariate analysis. For enriched melanoma cells (CD45- cell populations), 11 surface antigens were significantly correlated with the disease-free interval (DFI) between diagnosis of culprit primary melanoma and LN metastasis resection. Nine antigens on CD45+ leukocytes also correlated with DFI. Following validation in independent datasets, surface markers identified here should enable more accurate determination of prognosis in stage III melanoma patients and provide better risk stratification of patients entering clinical trials.
[Show abstract][Hide abstract] ABSTRACT: There is an urgent need to identify more accurate prognostic biomarkers in melanoma patients, particularly in those with metastatic disease. This study aimed to identify melanoma and leukocyte surface antigens predictive of survival in a prospective series of AJCC stage IIIb/c melanoma patients (n=29). Live cell suspensions were prepared from melanoma metastases within lymph nodes (LN). The suspensions were immuno-magnetically separated into CD45+ (leukocyte) and CD45- (non-hematopoietic, enriched melanoma cell) fractions. Surface antigens on CD45- and CD45+ cell populations were profiled using DotScanTM microarrays (Medsaic Pty. Ltd.) and showed differential abundance levels for 52 and 78 antigens respectively. Associations of the surface profiles with clinicopathologic and outcome data (median follow-up 35.4 months post LN resection) were sought using univariate (log-rank test) and multivariate (Wald’s test; modelled with patient’s age, gender and AJCC staging at LN recurrence) survival models. CD9 (p=0.036), CD39 (p=0.004) and CD55 (p=0.005) on CD45+ leukocytes were independently associated with distant metastasis-free survival (DMFS) using multivariate analysis. Leukocytes with high CD39 levels were also significantly associated with increased overall survival (OS) in multivariate analysis (p=0.016). LNs containing leukocytes expressing CD11b (p=0.025), CD49d (p=0.043) and CD79b (p=0.044) were associated with reduced OS on univariate analysis. For enriched melanoma cells (CD45- cell populations), 11 surface antigens were significantly correlated with the disease-free interval (DFI) between diagnosis of culprit primary melanoma and LN metastasis resection. Nine antigens on CD45+ leukocytes also correlated with DFI. Following validation in independent datasets, surface markers identified here should enable more accurate determination of prognosis in stage III melanoma patients and provide better risk stratification of patients entering clinical trials.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT Chronic lymphocytic leukaemia (CLL) is clinically heterogeneous. While some patients have indolent disease for many years, 20-30% will progress and ultimately die of their disease. CLL may be classified by the Rai or Binet staging systems, mutational status of the immunoglobulin variable heavy-chain gene (IGVH), ZAP-70 over-expression, cytogenetic abnormalities (13q-, +12, 11q(-), 17p(-)) and expression of several cell surface antigens (CD38, CD49d) that correlate with risk of disease progression. However, none of these markers identify all CLL cases at risk. In a recent review (Huang et al. Leukemia & Lymphoma 2012; 53:1046-56), we summarised those CD antigens known to correlate with the prognosis of CLL. The present study has identified surface profiles of CD antigens that distinguish clinically progressive CLL from slow-progressive and stable CLL. Using an extended DotScan CLL antibody microarray (Version 3, 182 CD antibodies), and with refined analysis of purified CD19+ B-cells, the following 27 CD antigens were differentially abundant for progressive CLL: CD11a, CD11b, CD11c, CD18, CD19, CD20 (2 epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD102, CD148, CD180, CD196, and CD270. The extensive surface profiles obtained provide disease signatures with an accuracy of 79.2%, a sensitivity of 83.9% and a specificity of 72.5% that could provide the basis for a rapid test to triage CLL patients by probability of clinical progression and potential earlier requirement for treatment.
[Show abstract][Hide abstract] ABSTRACT: Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.
PLoS ONE 06/2013; 8(6):e66755. DOI:10.1371/journal.pone.0066755 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The proteomic effects of the Hsp90 inhibitor, SNX-7081, have been determined on the p53-mutated B-cell chronic lymphocytic leukemia (CLL) cell line, MEC1. Following SNX-7081 treatment (500 nM, 24 h), 51 proteins changed abundance by more than 2-fold (p<0.05); 7 proteins increased, while 44 proteins decreased. Proteins identified as differentially abundant by LC-MS/MS were validated by Western blotting (DDB1, PCNA, MCM2, Hsp90, Hsp70, GRP78, PDIA6, HLA-DR). RT-PCR showed that SNX-7081 unexpectedly modulates a number of these proteins in MEC1 cells at the mRNA level (PCNA, MCM2, Nup155, Hsp70, GRP78, PDIA6, and HLA-DR). Pathway analysis determined that 3 of the differentially abundant proteins (cyclin D1, c-Myc and pRb) were functionally related. p53 levels did not change upon SNX-7081 treatment of p53 wild-type Raji cells or p53-mutated MEC1 and U266 cells, indicating that SNX-7081 has a p53-independent mechanism. The decreases in DDB1, MCM2, c-Myc, and PCNA, and increases of pRb and cyclin D1 were confirmed in MEC1, U266, Raji, and p53 null HL60 cells by Western blotting. These data suggest that SNX-7081 arrests cell cycle and inhibits DNA replication and repair, and provide evidence for the mechanism of the observed synergy between Hsp90 inhibitors and drugs that induce DNA strand breaks.
Journal of Proteome Research 03/2013; 12(4). DOI:10.1021/pr301055y · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose. Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, some patients may survive for many years, while 20-30% of patients progress and may die within several years. Currently, there is not a single procedure that enables accurate prognosis and triaging of those patients who need immediate and aggressive treatment. All CLL cells are characterised by the expression of the B-cell antigens CD19, CD20, CD21, CD22 and CD23, with aberrant expression of the T-cell antigen CD5. Methods. We have developed a CD antibody microarray (DotScan) containing 182 immobilised CD antibodies that has been used to obtain extensive surface profiles of CLL cells obtained from 96 patients. Results. Of these 182 antigens, 27 were significantly differentially expressed between stable, stable-progressive and progressive CLL. Some of these antigens are not expressed on normal B-cells and may be targets for therapeutic antibodies against CLL. Unsupervised hierarchical clustering of the surface profiles from 96 patients showed that those with progressive CLL could be distinguished based solely upon this 'disease signature'. The sensitivity (proportion of actual positives correctly identified) was 67.9%, the specificity (proportion of negatives correctly identified) was 77.5%, and the accuracy was 71.9%. Conclusions. Considerable effort by a number of research groups has resulted in identification of individual markers for progressive CLL, but their collective use is yet to provide a test that identifies CLL patients at risk. Data presented here provide a basis for development of a simple test using an antibody microarray. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Journal of Pharmacy and Pharmaceutical Sciences 01/2013; 16(2):231-7. · 1.86 Impact Factor