Proteomics: Advances in Biomarker Discovery

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Nov 23, 2007 - Ashley Martin (Cancer Research UK Institute for Cancer ... Martin presented data from a study of patients .... Ward DG, Suggett N, Cheng Y et al.
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Proteomics: Advances in Biomarker Discovery Expert Rev. Proteomics 5(1), 21–23 (2008)

Matthew L Cowan† and Jaime Vera

Proteomics: Advances in Biomarker Discovery 23 November, 2007, BioPark, Herefordshire, UK



Author for correspondence St George’s, University of London, Section for Infection, Division of Cellular & Molecular Medicine, London SW17 0RE, UK Tel.: +44 208 725 3474 Fax: +44 208 725 3486 [email protected]

The challenges encountered by proteomic researchers seeking diagnostic, prognostic and mechanistic markers were the subject of the 1-day meeting, Proteomics: Advances in Biomarker Discovery hosted by EuroSciCon. The speakers had a broad range of clinical and basic science interests, and presented data using a number of proteomic platforms to search for discriminant biomarkers of disease in easily accessible bodily fluids including serum and urine. Several potential pitfalls for proteomic researchers were mentioned and the potential of collaborative networks between research institutions to increase the size and power of clinical studies was discussed. Overall, the meeting highlighted the exciting opportunities that proteomic techniques offer for discovering not only diagnostic but also prognostic and mechanistic markers of a number of clinically important diseases.

The applications of proteomics technologies to discover diagnostic, prognostic and mechanistic markers in diverse medical and scientific fields was the subject of the 1-day meeting, Proteomics: Advances in Biomarker Discovery, hosted by EuroSciCon. The meeting was chaired by Ayesha De Souza (St George’s, University of London, UK). The meeting began with a presentation by Ashley Martin (Cancer Research UK Institute for Cancer Studies, University of Birmingham, UK), who gave an introduction to the challenges of discovering which of the many changes in the proteome are clinically useful. Only 5–10% of patients referred to specialist centers with the potential diagnosis of cancer have malignant disease. Early-stage tumors amenable to curative therapies are the most difficult to detect. Ashley Martin presented data from a study of patients referred for investigation of possible bowel cancer [1]. A total of 13 protein peaks were found to be differentially expressed in those with cancer using SELDI-TOF mass spectrometry (MS). An artificial neural network identified the combination of protein peaks with the greatest discriminant power of differentiating cancer from control. Identification of some of these discriminant proteins was performed with liquid chromatography (LC)-MS/MS of a trypsin digest of proteins purified by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) and www.future-drugs.com

10.1586/14789450.5.1.23

confirmed by depletion with monoclonal antibodies. A model containing a traditional serum marker and features discovered using proteomic techniques was able to classify subjects with an accuracy of 80%. Upregulation of proteolytic activity is a feature of malignancy. Serum protein fragments are excreted unchanged in urine. A model using MALDI-TOF-MS of urine was able to distinguish between colon cancer and controls and also between early- and late-stage tumors. Radiolabeled peptides were used to validate this quantitative technique. The identification of discriminant urinary biomarkers has been hindered by the lack of availability of protein fragment antibodies. Robin Wait (Kennedy Institute of Rheumatology, Imperial College, London, UK) spoke about the search for biomarkers of arthritis. Degradation of collagen leads to irreversible destruction of the joint surface in osteoarthritis. Elucidating the mechanisms of collagenolysis may identify novel therapeutic targets. Proteins in articular cartilage were investigated with 2D-SDS-PAGE and quadrupole-TOF-MS. The cartilage proteome of patients with osteoarthritis was more complex than that of patients undergoing amputation or joint replacement for reasons other than osteoarthritis. The level of type II collagen, a biomarker of osteoarthritis, was increased in subjects, as were a number of growth factors and pro-inflammatory cytokines.

© 2008 Future Drugs Ltd

ISSN 1478-9450

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MeetingReview Report

Cowan & Vera

Activin A, a homodimer of two inhibin β-A chains, was also upregulated. Inhibin β-A mRNA and activin A secretion were stimulated in human chondrocytes by pro-inflammatory cytokines and inhibited by fibroblast growth factor receptor inhibitor. Activin induces tissue inhibitor of matrix metalloprotein-1, and thus may be protective against joint destruction. Expression of fibroblast activation protein α, a serine protease responsible for activating collagenolytic enzymes, was also increased. Rheumatoid arthritis is a chronic debilitating autoimmune disease with no curative treatment. Citrullinated autoantibodies have a better sensitivity for diagnosis than rheumatoid factor. Anticitrullinated α-enolase autoantibodies were identified as a serum biomarker of high specificity in four cohorts with rheumatoid arthritis. The degree of immunogenicity was dependent upon the site of citrullination [2]. A currently unpublished longitudinal treatment study demonstrated a correlation between antibody level and disease activity. Pancreatic adenocarcinoma is another malignancy where early diagnosis and treatment can significantly impact upon survival, explained Mark Weeks (Institute of Cancer, Barts and the London School of Medicine and Dentistry, London, UK). His group has optimized quantitative methods for examining the urine proteome to distinguish between healthy controls, and benign and malignant pancreatic disease. Using pooled urine samples collected in two sites and a 2D-DIGE platform, 42 differentially expressed proteins were discovered in patients with pancreatic cancer correlating to 19 unique gene products. By saturating the high-abundance proteins on the gels, a further 76 differentially expressed proteins were noted, of which 59 were identified. One of these proteins was identified by MALDI-TOF-MS as S100P. S100P is a calcium-binding protein that increases with increasing stage in pancreatic carcinoma in situ. The expression of S100P is increased in the nucleus of cancer cell lines. The proteome of these cells demonstrates an increase in the expression of a number of cytokeratins with abnormal phosphorylation patterns of MALDI-TOF-MS/MS fragmentation spectra. Functional wound healing assays show reduced motility of these tumor cells. Clones of cells overexpressing S100P have high levels of cathepsin D, which is associated with degradation of extracellular matrix and tissue invasion [3] . Robert Rees (Department of Life Sciences, Nottingham Trent University, UK) discussed the need for biomarkers that can diagnose cancers, detect metastatic cancer and predict therapeutic outcome. The MALDI-TOF-MS serum proteome was used to train neural network algorithms to differentiate not only patients with melanoma from controls, but also individual cancer stages. The performance of the neural networks improved as the number of markers included increased. Professor Rees stressed the importance of collaborative networks in providing large numbers of well-characterized samples. Pooled sera from patients with prostate cancer were screened for autoantibodies using a cDNA library. Of the sera from patients with prostate cancer, 50% contained antibodies

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against the T21 gene product [4]. T21, located on chromosome 12, encodes a transcription factor of unknown function. T21 mRNA was detected in prostate cancer tissue and prostate cancer cell lines, but not in normal tissues or in resected prostate tissue from patients with benign disease. Prostate cancer tissue is heterogeneous, consisting of areas of malignant and benign glands. Individual glands were sampled with laser-capture microdissection. The level of T21 in benign glands from malignant tissue was above baseline but less than that of malignant cells from the same sample. Knocking out T21 with silencing RNA reduced the rate of cell proliferation. The addition of siRNA downregulated 156 genes, the majority of which were involved in cellular proliferation or antiapoptosis, whilst a number of caspase and apoptosis markers were upregulated. Future priorities for research include assaying T21 in serum. The search for novel therapeutic targets in radiotherapyresistant breast cancer was the subject of the presentation given by Lynn Cawkwell (University of Hull, UK). Resistance to radiotherapy was induced in a number of breast cancer cell lines by use of a clinically equivalent radiation dose. Differentially expressed proteins in radioresistant cell lines relative to parent cells were analyzed by 2D-DIGE. MALDI-TOFMS was used to confirm the identity of candidate biomarkers. A western blot confirmed a twofold change in expression of a number of provisionally identified markers. Interestingly, examining the proteomes of the same cell lines with LC-MS and isobaric tags for relative and absolute quantitation (iTRAQ ®; a tagged LC-based approach) revealed a number of complementary biomarkers. Cawkwell presented some preliminary data from a protein microarray with antibodies to 725 proteins, which found a number of potential biomarkers of radioresistance in cell lines. A similar study identified biomarkers of doxorubicin resistance in breast cancer cells. Protein microarrays are reproducible and suitable for investigating low-abundance proteins and proteins that may have chemical characteristics which make it difficult to fractionate them using traditional methodologies. Unfortunately, protein microarrays are expensive, dependent upon the existence of antibodies and may not distinguish between post-translational modifications. In the final talk of the day, Judit Nagy (Imperial College, London, UK) presented some work on high-performance capillary electrophoresis (HPCE) with label-free intrinsic imaging (LFII™; Peregrine™ manufactured by deltaDOT), a new technique for the detection of potential biomarkers in a mouse model of insulin resistance. This technology uses a multiplex UV detector to quantitate biomolecules separated by capillary gel electrophoresis. The instrument can be used to investigate complex mixtures of proteins, DNA, RNA or glycoproteins. Previous 2D-gel work has detected a number of proteins differentially expressed in the liver of ob/ob mice with Type 2 diabetes mellitus. HPCE-LFII™ detected 14 proteins expressed at different levels between mice with and without diabetes. Nine

Expert Rev. Proteomics 5(1), (2008)

Proteomics: advances in biomarker discovery

proteins involved in processes including oxidative stress pathways and sugar metabolism were identified by molecular weight and validated by 2D-DIGE. The speakers throughout the day displayed a broad range of backgrounds, a variety of interests and presented data using a number of proteomic platforms. Several potential pitfalls for proteomic researchers were discussed. The development of collaborative networks between research institutions will increase the size and power of clinical studies, but requires standardization of sample collection and appropriate selection of control subjects to ensure success. It is likely that future models will involve both proteomic and nonproteomic data. Accurate identification of protein markers is crucial if the biological role of biomarkers is to be discovered and exploited as potential therapeutic targets. A number of the novel putative markers discussed represent fragments of known fragments or proteins that have undergone disease-specific post-translational modification. Protein identification techniques must be capable of detecting these subtle differences. In summary, proteomic techniques offer exciting opportunities to discover not only diagnostic, but also prognostic and mechanistic markers of a number of clinically important diseases. Financial & competing interests disclosure

Matthew L Cowan is the recipient of a Fellowship from the National Institute for Healthcare Research. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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Meeting Review Report

References 1

Ward DG, Suggett N, Cheng Y et al. Identification of serum biomarkers for colon cancer by proteomic analysis. Br. J. Cancer 94(12), 1898–1905 (2006).

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Kinloch A, Tatzer V, Wait R et al. Identification of citrullinated α-enolase as a candidate autoantigen in rheumatoid arthritis. Arthritis Res. Ther. 7(6), R1421–R1429 (2005).

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Whiteman HJ, Weeks ME, Dowen SE et al. The role of S100P in the invasion of pancreatic cancer cells is mediated through cytoskeletal changes and regulation of cathepsin D. Cancer Res. 67(18), 8633–8642 (2007).

4

Miles AK, Rogers A, Li G et al. Identification of a novel prostate cancer-associated tumor antigen. Prostate 67(3), 274–287 (2007).

Affiliations •

Matthew L Cowan, BSc, MRCP Section for Infection, Division of Cellular and Molecular Medicine, St George’s, University of London, London SW17 0RE, UK Tel.: +44 20 8725 3474 Fax: +44 20 8725 3486 [email protected]



Jaime Vera, MD, MRCP Section for Infection, Division of Cellular and Molecular Medicine, St George’s, University of London, London SW17 0RE, UK Tel.: +44 208 725 5097 Fax: +44 208 725 3486 [email protected]

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