ANTICANCER RESEARCH 27: 1369-1386 (2007)

ABSTRACTS OF THE INTERNATIONAL CONFERENCE ON GLOBAL mRNA AND ANALYSIS

7 - 8 September 2006 Dublin, Ireland

Edited by L. O’Driscoll, R. O’Connor, M. Clynes

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Abstracts of the International Conference on Global mRNA and Protein Analysis

Abstracts of Oral Presentations cancer. Through the application of these technologies, improved methods for cancer diagnosis and monitoring 1 involving minimally-invasive, sensitive, specific tests could CLINICAL APPLICATIONS OF contribute significantly to cancer detection, on-going EXPRESSION PROFILING AND monitoring, and treatment. We recently completed a whole PROTEOMICS IN PROSTATE CANCER genome microarray analysis of 104 archived breast tumour biopsies (with normal breast tissue as control). Additionally, John R. Masters using cancer cell line models, we have optimised methods The Prostate Cancer Research Centre, enabling us to establish that cancer cells apparently selectively Institute of Urology, University College London, secrete mRNAs. Aim: The aim of our recent pilot study was to 67 Riding House Street, London W1W 7EJ, England establish if it is possible to apply a modification of these technologies to investigating global expression of extracellular Prostate cancer is the most frequently diagnosed cancer in transcripts in human sera, to enable identification of novel men and the second leading cause of death due to cancer. panels of possible diagnostic/prognostic or predictive mRNA Treatment options for clinically localized disease are broad, biomarkers. Materials and Methods: RNA was isolated from including radical prostatectomy and radiotherapy and active pre-surgery serum specimens from four recently diagnosed surveillance, reflecting the wide spectrum of outcomes for breast cancer patients, from their breast tumour and matched early disease. Serum levels of prostate specific antigen (PSA) normal biopsies, and from serum specimens procured 2-4 are used for diagnosis and monitoring of prostate cancer. ~ months post-surgery. Serum specimens from 6 volunteers with PSA is of limited value diagnostically, as approximately 75% no history of cancer were included as controls. Up to 100 ng of men with levels in the abnormal range (4-10 ng/ml) have of each specimen was amplified and labelled using Affymetrix benign disease and about 15% of cancer present with PSA GeneChip Eukaryotic 2 Cycle Labelling Assays for Expression levels in the normal range (<4 ng/ml). More accurate Analysis and was subsequently examined using Affymetrix markers are needed for diagnosis and to identify the men whole genome microarrays. Results: transcripts were who will benefit from and need radical treatment. detected in all specimens analysed. Overall, approximately 8% Expression profiling has identified many that are (8.34±1.06) of the 54675 probesets representing transcripts on differentially expressed in prostate cancer. While some are of the microarray were denoted as "Present" in serum specimens prognostic significance (e.g. hepsin and pim-1: Dhanasekaran and 45% (44.99±3.25) were detected in breast tissue et al. Nature 412: 822, 2001), they have not yet been used in ~ biopsies. The quality of transcripts detected extracellularly was clinical practice. Expression profiling has made important of an acceptable standard i.e. based on cell line QC contributions to our understanding of the disease. For parameters. Conclusion: In the first reported analysis of its example, the polycomb group protein EZH2 is overexpressed kind, this study indicates the potential of microarray analysis in hormone-relapsed metastatic prostate cancer (Varambally of extracellular mRNAs to be applied to larger cohorts of et al. Nature 419: 624, 2002) and fusions of TMPRSS2 with patient specimens and to other cancer types. This presentation ETS transcription factor genes were found in most prostate will summarise some novel findings from our microarray cancers (Tomlins et al. Science 310: 644, 2005). analysis of a large bank of breast tumour biopsies and will also Proteomic analyses of serum and urine also may identify highlight the successes and the challenges/limitations of our more accurate markers for diagnosis and monitoring. Many pilot serum mRNA study. studies have used SELDI-TOF-MS. It is likely that the global This work was supported by funding from HEA's PRTLI tools for assessing gene and protein expression will eventually Cycle 3, Dublin City University's Albert College Fellowship yield a limited number of markers that can be used to aid the and Dublin City University's Faculty of Science and Health management of patients using relatively inexpensive assays Targeted Research Initiative. for a limited number of molecular markers.

2 3 EXPRESSION MICROARRAY MICROARRAY ANALYSIS OF INVASION ANALYSIS IN HUMAN CANCER IN HUMAN CANCER CELLS IN VITRO Lorraine O'Driscoll Susan McDonnell, Linda Hughes and Catherine Malone National Institute for Cellular Biotechnology, UCD School of Chemical and Bioprocess Engineering, Dublin City University, Dublin 9, Ireland University College Dublin, Belfield, Dublin 4, Ireland

Background: profiling using microarrays has The process of tumour invasion and subsequent metastasis created new possibilities for the molecular characterisation of represents the most lethal aspect of cancer and is responsible

0250-7005/2007 $2.00+.40 1371 ANTICANCER RESEARCH 27: 1233-1386 (2007) for the majority of deaths among cancer patients. Whilst early candidate tissue markers important in the molecular detection and improved treatment regimes are reducing characterisation of prostate cancer and which have been mortality rates, it is imperative that better methods for validated using laser capture tissue samples. However tissue anticipating which cancers will metastasise are developed. The biopsies only randomly sample prostate cancer and may not be objective of this project was to identify invasion-specific genes an appropriate detection medium. (ii) Human serum represents in breast cancer cell lines using DNA microarray technology. a more global sampling site but this complex sample type An isogenic cell line model of increasingly invasive breast represents protein concentrations typically spanning a wide cancer, comprising two cell lines, Hs578T and Hs578Ts(i)8, was dynamic range. In order to overcome the technical limitations generated using the BD Biocoatì Matrigelì invasion assay associated with this fact, the serum samples were subjected to system. The new variant, Hs578Ts(i)8, was extensively affinity depletion using the Multiple Affinity Removal System characterised and shown to be 3-fold more invasive than the removing six abundant found in human serum, parental cell line. Cell lines were subjected to Affymetrix enriching lower abundance proteins. Differential In-Gel microarray analysis (HG-U133A chip) and data analysis using Electrophoresis (DIGE) was used to analyse the difference in GeneSpring 7.0 generated a list of 508 differentially expressed expression between Gleason 5 and 7. The fluorescent 2-D gel genes. Array data for 17 genes was validated using real-time images were subjected to analysis using Progenesis PG240 PCR and showed a correlation value of 0.9249. Expression of (Nonlinear Dynamics). This analysis identified 63 proteins these candidate invasion specific genes was investigated in a which showed differential protein expression between the two panel of human tumour cell lines. Of the up-regulated genes, groups. Forty two of these proteins have been identified using overexpression of several genes including PLAC8, Cathepsin Z, mass spectrometry. These candidates which can statistically CTAG1B and Pirin was evident in the majority of invasive cell differentiate between Gleason 5 and 7 could potentially hold a lines. Of the down-regulated genes, expression of Lumican, number of clinically relevant biomarkers distinguishing between Decorin and S100A4 was absent or at low levels in most of the different grades of cancer, thus identifying patients with a invasive cell lines. Based on these results it is hoped that higher risk of more aggressive cancer allowing for informed potential new biomarkers for the diagnosis, prognosis and patient management and appropriate treatment strategy from treatment of metastatic breast disease will be identified. “watchful waiting” to aggressive therapy.

4 5 THE PROSTATE CANCER RESEARCH GENE EXPRESSION PROFILING OF CONSORTIUM: A BIO-RESOURCE FOR THE MALIGNANT MELANOMA: APPLICATION DISCOVERY OF TISSUE AND SERUM OF TAQMAN TECHNOLOGY FOR MARKERS FOR PROSTATE CANCER HIGH-THROUGHPUT DOWNSTREAM VALIDATION OF TRANSCRIPTOMIC STUDIES William Watson, Jennifer Byrne, Niaobh O'Donogue, Michael Dunn, Caitriona Scaife, John Fitzpatrick and Mairin Rafferty1, Stephen Donoghue2, William J. Faller1, Stephen Pennington Ilse-Maria Nolan1, Aedin C. Culhane2, Catherine Moss3, Janet McCormick3, David J. Easty2, Peter A. Dervan2,4 UCD School of Medicine and Medical Science, and William M. Gallagher1 Conway Institute, University College Dublin, Ireland 1UCD School of Biomolecular and Biomedical Science, Prostate cancer is the most common solid malignancy affecting 2UCD School of Medicine and Medical Science and men. Current methods for its detection and diagnosis rely 3Transcriptomics Core, UCD Conway Institute, heavily upon PSA. However, PSA has low specificity and is not University College Dublin, Belfield, Dublin 4; suitable as a general screening tool. As with many types of 4Mater Misercordiae Hospital, 44 Eccles St., Dublin 7, Ireland cancer, early indicators would have the potential to highlight the presence of disease and allow for appropriate intervention The incidence of melanoma is increasing rapidly, with with improved outcome. The Prostate Cancer Research advanced lesions generally failing to respond to conventional Consortium Bio-resource has allowed for the collection of chemotherapy. Previously, we utilised DNA microarray biological material including tissue, serum and urine from men technology to identify 66 melanoma progression-associated undergoing radical prostectomy for localised disease. This genes, which exhibited altered expression in a unique isogenic allows for the full pathological diagnosis of the disease for set of melanoma human cell lines, termed the WM793 series complete correlation with genomic and proteomic identified (1). Here, we further examined these melanoma progression- changes in tissue, serum and urine. This talk will focus on two associated genes via a high-throughput real-time quantitative aspects of the consortium’s work. (i) Bioinformatic analysis for reverse transcriptase-PCR assay. In particular, we designed a previous genechip publications has identified a number of tailored TaqMan Low Density Array (LDA), representing the

1372 Abstracts of the International Conference on Global mRNA and Protein Analysis majority of genes within our cohort of interest, using an online quantification of changes in mRNA abundance on the whole- catalogue of available probe and primer sets from Applied genome scale. However, the extremely diverse physicochemical Biosystems. Each LDA contained two independent sectors (for properties of proteins render them difficult to quantify, separate biological samples), each with 96 individual probe and particularly since pharmacological actions are often mediated primer sets in duplicate. Probe and primer sets were available by a handful of low-abundance proteins that reside within a for 64 of the 87 melanoma progression-associated gene cohort, milieu of higher-abundance 'housekeeping' proteins. with the remaining 32 sets being used to analyse 7 other As a testbed system, we investigated the effects of the melanoma-linked genes, 13 putative non-changing genes corticosteroid methylprednisolone (MPL) in rat liver. MPL (observed as such from the prior DNA microarray studies), 12 binds to a cytoplasmic receptor that translocates to the nucleus, phosphatase genes and 18S rRNA. Using this customised interacts with specific DNA motifs, and alters gene TaqMan LDA, the expression values for each gene were transcription. Of over 8000 genes probed in rat liver, more determined in WM793 cells, along with three isogenic than 1200 were MPL-responsive. Temporal patterns of derivative cell lines, WM793-P1, WM793-P2 and 1205-Lu, response clustered into at least 6-8 distinct patterns, which which exhibit more aggressive phenotypic characteristics than have been analyzed using mechanistic pharmacodynamic the parental cells. A high level of correlation was observed models. For the quantification of downstream protein-level between replicates across all LDAs analysed, with an average r responses, we developed a modification of ICAT (isotope- value >0.95. Considerable concordance was seen between the coded affinity tagging), which retrieves only Cys-bearing transcriptomic profiles determined using DNA microarray and peptides from whole-tissue homogenates. This reduces sample TaqMan LDA approaches. Studies using this TaqMan LDA complexity and improves the quantification of low-abundance for the analysis of 20 metastatic melanoma biopsies are proteins. Retrieved ICAT peptides were identified by capillary ongoing. Overall, TaqMan LDA technology provides an ideal LC/ion-trap MS, and tryptic ICAT-peptides derived from opportunity for high-throughput downstream validation of target proteins were sought. Putative detected target peptides DNA microarray studies, as well as offering a flexible medium were then synthesized and used both to optimize quantification to carry out sensitive gene expression studies on a standalone on a triple-quad MS and to confirm LC retention time and basis. Funding is acknowledged from the Health Research fragmentation. Using this approach, we elucidated the time Board and IRCSET. course of tyrosine aminotransferase (TAT) and ornithine decarboxylase (ODC) induction by MPL in rat liver at fmol 1 Gallagher WM et al: Multiple markers for melanoma sensitivity in small (10-50 Ìg) samples. progression regulated by DNA methylation: insights from transcriptomic studies. Carcinogenesis 26(11): 1856-1867, 7 2005. SERUM PROTEOMICS Paul Dowling 6 PROTEOMIC APPROACHES TO THE National Institute for Cellular Biotechnology, QUANTIFICATION OF PHARMACODYNAMIC Dublin City University, Dublin 9, Ireland EFFECT MARKERS: GENERAL APPLICATIONS IN DRUG TARGET AND BIOMARKER IDENTIFICATION Identifying proteins involved in human disease and to understand how their expression, structure and function Robert M. Straubinger and Jun Qu cause illness are major goals in proteomics. Human serum Department of Pharmaceutical Sciences, contains many proteins and small molecules which have the University at Buffalo, State University of New York, potential to be used as biomarkers for diagnosis. Cancer Amherst, NY, 14260-1200, U.S.A. biomarkers in serum can be distinguished into different types: serum proteins that are differentially expressed in Numerous pharmacological agents exert cellular effects at the patients with cancer, serum proteins that are cleaved or transcriptional, translational, or post-translational levels. modified in cancer patients, proteins that are secreted by Understanding these effects can provide a more integrated tumour cells into the circulation and intracellular tumour description of drug mechanisms of action on diverse tissues. If proteins that are released when tumour cells die. quantified with sufficient accuracy, sensitivity, and temporal Techniques used in biomarker discovery include 2D-PAGE, resolution, pharmacophore responses at the mRNA/protein multidimensional chromatography, novel MS based level can enable the application of computational, mechanistic approaches and the SELDI protein chip system used for pharmacodynamic (PD) models that describe the link between protein profiling. Many challenges remain in the world of the magnitude of drug concentration at the effect site and the serum proteomics. Frequently candidate biomarkers are magnitude of response. Numerous technologies permit facile found only at nanomolar to picomolar concentrations in

1373 ANTICANCER RESEARCH 27: 1233-1386 (2007) cancer patient sera, 106- to 109-fold lower than albumin, farnesyltransferase inhibitors, heat shock protein inhibitors, making sensitive biochemical measurements and initial p38 inhibitors, statins, VEGF inhibitors. Future curative discovery difficult. Many other diseases lead to the therapies are likely to consist of a rational combination of appearance of various markers in the blood which agents that target the malignant plasma cells and the bone complicates the development of cancer-specific markers. marrow micro-environment which are selected on the basis However proteomics is constantly advancing and the of a detailed molecular and genomic analysis of each patient promise of sensitive and selective biomarkers is certainly on an individual basis. achievable. 9 8 EGFR AND HER-2 ANTAGONISTS UPDATE ON MYELOMA THERAPY IN BREAST CANCER Peter O'Gorman Norma O'Donovan1, Brigid Browne1, John Crown1,2, Michael J. Duffy2, Dennis Slamon3 and Martin Clynes1 Mater University Hospital, Dublin, Ireland 1National Institute for Cellular Biotechnology, Multiple myeloma (MM) remains an incurable disease; Dublin City University, Dublin 9; therefore new treatment strategies are needed to improve 2St. Vincent's University Hospital, Dublin, Ireland; the outcome of patients with this disease. The increased 3University of California, Los Angeles, U.S.A. knowledge about MM biology is already contributing to more specific drug design based on the understanding that HER-2 is overexpressed in 25-30% of breast cancers. the interaction of plasma cells with the bone marrow micro- Trastuzumab, a monoclonal antibody to HER-2, has shown environment is central to the pathogenesis of MM. promising results in HER-2-positive breast cancers. Thalidomide was initially used in MM because of its anti- However, not all HER-2-positive patients respond to angiogenic activity. Increased angiogenesis is evident in the trastuzumab and those that initially respond often develop bone marrow of MM patients. Thalidomide has additional resistance. Recent studies have shown that overexpression mechanisms of action; it inhibits the production of TNF·, of HER-2 is frequently associated with expression of EGFR, stimulates T cell proliferation, induces the secretion of IFNÁ which influences response to HER-2 inhibition. We have and IL-2, augments NK cytotoxicity, induces and examined dual targeting of HER-2 and EGFR as a means regulates the expression of adhesion molecules. of improving response to HER-2 targeting therapies. We Thalidomide can be used in combination with other agents also investigated the role of IGF-IR in acquired resistance including dexamethasone, adriamycin, melphalan and to trastuzumab. bortezomib. Synergistic effects of these combinations have Combinations of trastuzumab (Herceptin), gefitinib been demonstrated. Thalidomide was initially used in (Iressa) and lapatinib (Tykerb) were tested in HER-2- and relapsed and refractory patients only. More recently, we and EGFR-positive breast cancer cell lines. IGF-IR expression, others have demonstrated efficacy of thalidomide in activation and inhibition were examined in trastuzumab- combination with dexamethasone in newly diagnosed resistant cell lines. patients with MM. Earlier studies have shown a high rate of Dual targeting with trastuzumab and either gefitinib or deep vein thrombosis (DVT) and pulmonary embolism (PE) lapatinib showed additivity or synergy in inhibiting in patients receiving thalidomide and dexamethasone (up to proliferation of SKBR3 and BT474 cells. Trastuzumab or 15%). We have shown a low rate of thrombosis in this gefitinib alone did not induce significant apoptosis in setting using a simple prophylactic anti-coagulation SKBR3 cells, whereas lapatinib alone induced significant approach. Immunomodulatory drugs (IMID) developed as apoptosis. Trastuzumab combined with lapatinib further thalidomide analogues so far include two drugs: enhanced apoptosis induction. Pre-treatment with EGFR lenalidomide and actimid. Lenalidomide has just received plus HER-2 inhibitors also enhanced the cytotoxicity of FDA approval in the US. The results of phase II and phase chemotherapy drugs. III will be summarised. Bortezomib inhibits cellular IGF-IR protein levels were not altered in the trastuzumab- proteasomes which represent a large complex of proteolytic resistant cell lines. However, activation of IGF-IR was enzymes responsible for the intra-cellular degradation of increased in the resistant cells. IGF-IR siRNA transfection ubiquinated proteins. A combination of bortezomib with caused a significant decrease in proliferation and improved dexamethasone has resulted in overall response rates of 80- response to trastuzumab in the resistant cells. Our results 90% in patients with relapse\refractory myeloma. The suggest that dual targeting of HER-2 combined with current data will be summarised. Other novel anti-MM inhibitors of EGFR and/or IGF-IR may improve response to drugs under development include arsenic trioxide, treatment in patients with HER-2 overexpressing tumours.

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10 Institute of Anatomy, Medical Faculty, THE PHARMACOLOGY OF CANCER RESISTANCE Technical University of Dresden, Fetscherstr. 74, 01307 Dresden, Germany Robert O'Connor National Institute for Cellular Biotechnology, We demonstrate that bleomycin induced senescence and, to a Dublin City University, Dublin 9, Ireland lower extent, apoptosis in A549 cells and upregulated endogenous caveolin-1 expression. Inhibition of bleomycin- The administration of chemotherapy drugs is a standard induced apoptosis by the broad-spectrum caspase inhibitor Z- form of treatment for most advanced or metastatic cancers. VAD-fmk inhibited the bleomycin-induced active form of While such agents are normally extremely toxic, many caspase-3 but significantly failed to prevent phosphatidylserine cancer cells become resistant to the cytotoxic actions of externalization as shown in FACS analysis for annexin V binding these drugs through one or more resistance mechanisms and and did not reduce the amount of caveolin-1. Our results clearly this is a major cause of treatment failure. Active drug efflux indicate that treatment with bleomycin preferably induced pump proteins (ATP-binding cassette transporters) have cellular senescence which is characterized by arrest been well characterised for a number of years and function through activation of the p53/p21 pathway, as well as by an primarily by reducing the concentration of drug which can increase in senescence-associated ‚-galactosidase. Pre-senescent reach its cellular target. Two such proteins, P-gp (MDR-1, A549 cells did not phosphorylate Erk-1/2 after EGF stimulation, ABCB1) and MRP-1 (ABCC1), have been demonstrated to whereas young cells did. First experiments regarding the role of pump a wide selection of the most commonly used cancer caveolins in terms of senescence will be presented. drugs and correlate broadly with negative treatment response characteristics in many different forms of cancer. 12 Many clinical trials of P-gp inhibitors in combination with NEW APPROACHES TO TREATMENT substrate cancer drugs have been conducted, but these have OF BRAIN CANCER largely failed to show improved cancer treatment efficacy and Rolf Bjerkvig increased adverse toxicity is a common finding. Although MRP-1 has a significant overlap in substrate specificity with NorLux Neuro-Oncology, Department of Biomedicine, P-gp, it has a distinct and separate mechanism of action. University of Bergen Norway, and Centre Recherche Through in vitro screening we identified a number of Public Santé, Luxembourg competitive inhibitors of MRP-1 activity. One of these agents, sulindac, a non-steroidal anti-inflammatory drug (NSAID), Malignant brain tumours derived from glial cells (malignant was further tested and we have now concluded a Phase I gliomas) are characterized by their local infiltrative growth of clinical trial of sulindac in combination with epirubicin. This the normal brain. Despite intensive clinical and laboratory trial demonstrated that sulindac did not interfere with the research during the last decades, the survival benefit after pharmacology or pharmacokinetics of epirubicin and the therapy has been highly limited. Interestingly, many doses used produced concentrations anticipated to generate researchers have been successful treating brain tumour models therapeutic MRP-1 inhibition. Positive treatment responses in animals, but the success has been limited when the were also recorded. treatment principles have been translated into the clinic. One Targeted small molecular therapeutics, e.g. tyrosine reason for this failure is the lack of appropriate animal models kinase inhibitors, have recently revolutionised our that reflect the behavior of human tumours in situ. There are expectation of cancer treatment. However, it has been therefore obvious limitations in the preclinical animal models demonstrated that many such agents are also substrates for used to study brain tumour growth and progression and to drug efflux mechanisms, especially P-gp. Classical drug evaluate novel therapeutic strategies. Therapeutic progress resistance pumps may therefore be responsible at least in has been hampered by a limited delivery of effective part for the emerging evidence of resistance to such agents. therapeutic compounds to an extremely heterogeneic tumor Our research indicates that some of these agents such as cell population. At present, therapies are emerging which gefitinib (Iressa), by virtue of being substrates for P-gp, can include cell-based therapies, aspects of , stem cell effectively competitively inhibit the actions of this pump. therapy and immunotherapy. Several of these treatment modalities utilize the principle of transplanting cells or compounds that either directly or indirectly show therapeutic 11 efficacy. Many of these principles depend on an increased THE ROLE OF CAVEOLIN-1 IN BLEOMYCIN biological knowledge of brain tumours. The development of EXPOSED HUMAN LUNG CANCER A549 CELLS new therapeutic principles based on such knowledge, may Annett Linge, Michael Kasper and Kathrin Barth finally provide glioma patients with improved survival.

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13 problematic. Our laboratory has systematically analyzed and DETECTION AND MOLECULAR PROFILING optimized the RNA sample preparation stages for OF CIRCULATING TUMOR CELLS microarray analysis for several commercial platforms. We will present the history and results of our RNA amplification W.H. Albert, V. Zieglschmid, S. Hauch, S. Zimmermann, technology development. This will include several case S. Lankiewicz and O. Böcher studies where we attempted to measure the amplification AdnaGen AG, Ostpassage 7, D-30853 Langenhagen, characteristics of low total RNA input amounts. RNA Germany amplification was performed on purified total RNA from many different sources (mouse brain samples, cell cultures, Circulating tumor cells (CTC) in tumor patients indicate tumour biopsies, whole blood and high quality control disease progression. Their presence reflects an active growth samples). The results of this survey and other preliminary (relapse or metastases), since shedded tumor cells survive studies will be discussed. Microarray analysis of some of only a short time in the circulation. AdnaGen has developed these samples gives some indication that expression analysis the “Combination-of-Combinations-Principle”, a unique of ng amounts of total RNA provides valid information. and proprietary technology for the sensitive and specific A second focus of our laboratory is improving the detection and molecular analysis of circulating tumor cells. technologies for detecting and measuring microRNA A specificity of >97% was reached and the analytical (miRNA). We will present an overview of our current miRNA sensitivity (limit of detection) allows the detection of one tools and provide a summary of our upcoming technology tumor cell in 5 mL blood. The prognostic value as well as development for using qRT-PCR and microarrays for the usefulness in therapy monitoring of testing for the extending the analysis of transcriptome to include miRNA. presence of CTC has been demonstrated. Furthermore, the molecular profiling of CTC may be 15 used to identify therapeutic targets such as Her2 or EGFR CLINICAL ADJUSTED IN VITRO SIMULATION for personalized treatment, because the molecular targets OF ANTICANCER DRUG EFFICIENCY on CTC reflect the nature of the metastases, and, therefore, Timo Schinothe obliterates the sole reliance on the histopathology of the primary tumor. The expression levels of these therapeutic TumorTec GmbH, Kölner Weg 4, 50858 Köln, Germany targets for drugs like Herceptin and Erbitux may differ significantly between the primary tumor and the CTC in Small molecules with an anticancer activity are designed to metastatic patients, which is likely to have an important act in a targeted manner. An increase of target-specific impact on the therapeutic efficacy of these drugs. treatments will also increase the problem of individualized In conclusion, a sensitive and specific method to detect treatment efficiency. The preclinical efficiency of targeted circulating tumor cells in peripheral blood of breast treatments with a pathway specific activity must therefore be carcinoma patients is provided. This innovative method is simulated on a wide range of representative samples. In this an option for clinicians as a predictive tool with respect to case, cell lines mostly demonstrate only an artificial test tool. metastases formation and may result in an appropriate New methods for preclinical testing of anticancer drugs are selection of patients for adjuvant therapy. necessary. These methods should handle the problem of 14 individual treatment response and must also be predictable AMPLIFICATION AND DETECTION OF mRNA in clinical use. Beside this, it would be helpful to combine these methods with further analysis (e.g. gene expression). Robert A. Setterquist, Sharmili Moturi and John P. Whitley To solve these problems we designed a new method Ambion Inc., 2130 Woodward St., Austin, TX, U.S.A. based on a diagnostic tool which is able to predict the clinical outcome of an anticancer treatment with an RNA amplification using T7 RNA polymerase transcription accuracy of more than 98%. The method can also be of cDNA populations is a very useful method for preparing combined with further analysis like gene expression or microarray samples and has become a routine procedure for protein quantification. This tool acts under GMP/GLP gene expression research. In many cases, the method is conditions and enables the measurement of the anticancer necessary for producing sufficient concentrations of labelled efficiency on wide scale of independent samples of cancer target nucleic acid for detection in array hybridization. While patients. It is designed as a short-term cell culture assay the amplification of Ìg amounts of RNA (millions of cells) is using primary patient material by quantifying tumour cell fairly robust and has been successfully used for many survival in dependence on drug concentration. expression profiling experiments, amplification with ng This method is also an additional step to large-scale drug (hundred to thousands of cells) to pg (few cells) still appears screening. Pharmaceutical companies usually have the

1376 Abstracts of the International Conference on Global mRNA and Protein Analysis opportunity to analyse the efficiency of hundreds of shared by cancer stem cells. Malignant teratomas or thousands of drugs against only a few different cell types. teratocarcinomas are malignant tumours that occur in the testes This new method supports the opposite. The efficiency of a and are largely composed of undifferentiated cells which are few interesting candidates can be determined on a large- capable of differentiating into a vast array of different tissues. scale number of patient samples. Beside the opportunity of The aim of our study was to establish gene expression profiles combining this method with further analysis, this test is able for a normal stem cell and a cancer stem cell using teratoma to transfer parts of clinical phase I and phase II studies into tumourigenesis as our model system, thus allowing us to study the preclinical situation. genes that are required to drive cancer cell proliferation and progression. To do this, we performed comparative whole- 16 genome cDNA array analysis on murine embryonic stem cells PROTEOMIC ANALYSIS ANTIPROLIFERATIVE (ES) in their undifferentiated state and compared it to murine EFFECT OF 5-FLUOROURACIL ON NORMAL AND pluripotent and nullipotent teratocarcinoma cells, which CANCER EPITHELIAL CELLS OF THE LUNG represented our malignant/cancer stem cell. All cDNA arrays were performed in triplicate using Applied Biosystems William Bryan, Paula Meleady, Andrew Dowd, technology. Data was analysed using a combination of R and Paul Dowling and Martin Clynes Spotfireì programmes. ES array data was compared to the National Institute for Cellular Biotechnology, malignant cell lines to obtain a list of differentially expressed Dublin City University, Dublin 9, Ireland genes in the non-malignant and malignant states. Only genes with a fold change of >2 and false discovery rate (FDR) of 0.05 The antimetabolite drug 5-fluorouracil (5-FU) is the most were considered. The gene ontology site Panther was used to commonly used chemotherapeutic in the treatment of cancer. identify the pathways, biological processes and molecular It exerts its effect through incorporation into DNA and RNA. functions associated with significant genes. This analysis However, use of this drug is not always successful and generated a list of 1655 genes differentially expressed in understanding the underlying mechanisms that allow cancer malignant and non-malignant stem cells. We propose that this cells to survive exposure to 5-FU is important. Here we take list results in a putative profile for these malignant/cancer stem a proteomics approach to characterize the effect of 5-FU on cells and a putative profile for non-malignant/normal stem cells. the proteome of two lung carcinomas of epithelial origin and bronchial epithelial cells in order to determine how cancer 18 cells and normal cells respond to exposure to 5-FU. Using a DIFFERENTIAL PROTEIN PROFILING OF combination of 2-dimensional electrophoresis and Cy Dye SECRETED PROTEINS FROM HUMAN protein labelling chemistry it is possible to do this. Proteins CANCER CELL LINES BY SURFACE-ENHANCED that were found to show statistically significant fold changes LASER DESORPTION IONIZATION TIME-OF- were investigated and identified using MALDI-ToF mass FLIGHT MASS SPECTROMETRY (SELDI) spectrometry. Here we show a common and cell specific Priyanka Maurya, Paula Meleady, Paul Dowling and response to 5-FU exposure. Martin Clynes 17 GLOBAL mRNA ANALYSIS TO DETERMINE A National Institute for Cellular Biotechnology, TRANSCIPTOME PROFILE OF CANCER STEMNESS Dublin City University, Dublin 9, Ireland

Cynthia C.B.B. Heffron, Michael F. Gallagher and Selection of the human drug-sensitive and invasive cell line John J. O'Leary (MDA-MB-435-S-F) with the chemotherapeutic agent, paclitaxel, resulted in the development of drug-resistant variant Molecular Pathology Research Laboratory, Department of (MDA-MB-435-S-F/Taxol10p4pSI) displaying enhanced Pathology, Coombe Women's Hospital, Dublin 8; invasion-related characteristics. Since the secretome of cells and Department of Histopathology, University of Dublin, tissues may reflect a broad variety of pathological conditions Trinity College Dublin, Dublin 2, Ireland and represents a rich source of information, comparative protein profiling of differentially expressed secreted proteins in Similarities between stem cells and cancer cells led to the conditioned medium of drug sensitive and invasive versus concept of the existence of a cancer stem cell. This concept was resistant and superinvasive cells might explain associated first documented in haematological malignancies and has since mechanisms involved in drug resistance and invasion/metastasis, been recognised in breast, brain and prostate tumours. The or for establishing biomarkers associated with this phenotype. defining features of a normal stem cell are its ability to self In order to investigate changes in the secretome, serum- renew and to generate differentiated progeny, properties also free conditioned media from MDA-MB-435-S-F and MDA-

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MB-435-S-F/Taxol10p4pSI were analyzed with surface- underlying the differing invasive and adhesive capacities of the enhanced laser desorption ionization time-of-flight (SELDI) sub-clones which might possibly play an important role in mass spectrometry using IMAC-Cu2+, CM10, Q10 and H50 adhesion and inhibition of invasion in pancreatic cancer. ProteinChip Arrays. Five differentially expressed peptides/proteins were observed at 7.6 kDa on IMAC-Cu2+, 20 10.5, 10.7 and 10.9 kDa on H50 and 8.5 kDa on CM10 ANTIPROLIFERATIVE ACTIVITY OF THIOPHENE chromatographic surfaces. The peak intensity of the 7.6 kDa SUGAR CONJUGATES IN ENDOTHELIAL CELLS protein was 4-fold greater in the superinvasive phenotype Violeta Zaric2, Sarah L. Rawe1, Dearbhla Doyle1, with a statistically significant p-value of <0.04. The 7.6 kDa Paul V. Murphy1 and Kathy O'Boyle2 species was also found to be present in MDA-MB-435S- F/Adr-10p10pSI, melanoma cell line M14, SKMEL-28 cells 1Centre for Synthesis and Chemical Biology and but absent in melanoma cell line HT144, breast cell lines 2UCD School of Biomolecular and Biomedical Science, MCF-7, BT20 and pancreatic cell line MiapaCa-2 and its Conway Institute, University College Dublin, Belfield, clones. Work is ongoing to identify these differentially Dublin 4, Ireland expressed proteins using MALDI-ToF mass spectrometry. Background: The inhibition of angiogenesis, defined as the 19 process of new blood vessel formation, represents a promising INVESTIGATION INTO THE MOLECULAR therapeutic strategy for treating cancer and angioproliferative MECHANISMS OF ALTERED INVASION AND diseases. Aim: We are interested in developing novel non ADHESION IN SUB-CLONES OF THE anticoagulant sugar derivatives, which have the potential to PANCREATIC CANCER CELL LINE, MiaPaCa-2 alter endothelial cell growth. Materials and Methods: The inhibitory properties of deacetylated and acetylated Naomi Walsh, Norma O'Donovan and Martin Clynes monosaccharide analogs of N-glycosylthiophene-2-carboxamide National Institute for Cellular Biotechnology, (GTC) on the growth of bovine aortic endothelial cells Dublin City University, Dublin 9, Ireland (BAECs), stimulated by either fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF), were assessed Pancreatic cancer is a devastating disease and exhibits a highly using cell cycle analysis, cell counting and [3H]thymidine malignant phenotype characterised by invasion and early incorporation. Results: Deacetylated GTC conjugates (500 ÌM) metastasis to distant organs. The aim of this study is to completely prevented the stimulation of BAEC growth by FGF. investigate the mechanisms of invasion and the relationship Significantly, acetylated analogues were active at a between invasion and adhesion in pancreatic cancer, using cell concentration of 80 ÌM whereas their respective deacetylated line models. Materials and Methods: Single cell populations derivatives were inactive at this concentration (1). The were isolated from the pancreatic cell line, MiaPaCa-2. acetylated compounds also reduced [3H]thymidine Adhesion and invasion assays using matrigel, fibronectin, incorporation into DNA induced by VEGF. Complementary laminin, collagen type IV and I were performed comparing analysis of the cell cycle showed that the acetylated compounds parental cell line to sub-clones, clone #3 and #8. Results: Two prevented the increase of the S-phase induced by FGF for up to MiaPaCa-2 sub-clones #3 and #8 showed altered invasion 16 h. The increase in cell number induced by FGF (4.2±0.2x105 (higher and lower than the parent, respectively) through cells/mL) was reduced by 14% (3.6±0.2x105 cells/mL) by matrigel and were selected for further analysis. Clone #3 acetylated compounds (p<0.05, t-test). Mass spectrometry showed significantly higher invasion through matrigel and results suggest that the acetylated compounds freely diffused slightly increased invasion through fibronectin and laminin, through the cell membrane and were then rapidly deacetylated while clone #8 showed significantly decreased invasion by the action of non-specific intracellular esterases. In contrast, through the three matrices, compared to the parent cell line. deacetylated derivatives showed limited ability to enter cells. Clone #8 cells displayed preferential adhesion to laminin and Conclusion: Using a number of approaches, acetylated GTC matrigel and also displayed increased adhesion to fibronectin. conjugates were found to inhibit BAEC proliferation. The Clone #3 displayed significantly lower adhesion to matrigel, increased potency of acetylated derivatives compared to their fibronectin, laminin and collagen IV and I. Conclusion: We deacetylated counterparts is probably correlated to improved have established a model of pancreatic cancer cell invasion, transportation across the cell membrane. The promising results which displays increased invasion through laminin and obtained with acetylated monosacharride derivatives as fibronectin correlating with decreased adhesion to extracellular inhibitors of BAEC growth could lead to the development of matrices. Our results suggest that further studies on the novel angiogenesis inhibitors. involvement of integrins (‚1, ‚4, ·5, ·6) and MMPs (MMP- 14, MMP-15) may be performed to elucidate the mechanisms 1 Bioorg Med Chem Lett 16(5): 1316-1319, 2006. Epub 2005.

1378 Abstracts of the International Conference on Global mRNA and Protein Analysis

Abstracts of Poster Presentations counting. HER-2 and IGF-IR activity status were determined by immunoprecipitation with HER-2 and IGF-IR antibodies 21 followed by western blotting with a phospho-tyrosine antibody. MICROARRAY ANALYSIS OF TAXOL The effects of IGF-I/IGF-IR inhibition on proliferation and RESISTANT LUNG CANCER CELL LINES response to trastuzumab were investigated. No significant correlation was observed between levels of Laura Breen, Padraig Doolan, Eoin Ryan, HER-2 and response to trastuzumab in ten HER-2 positive Patrick Gammell, Jai Prakash Mehta and Martin Clynes breast cancer cell lines. However, IGF-IR levels correlated National Institute for Cellular Biotechnology, with resistance to growth-inhibition by trastuzumab in these Dublin City University, Dublin 9, Ireland ten cell lines. Phosphorylated IGF-IR was not detectable with western blot in any of these HER-2-positive cell lines. One of the major obstacles to successful treatment of lung However, high levels of phosphorylated IGF-IR were cancer is the development of resistance to chemotherapy, also detected in three trastuzumab-conditioned cell lines (BT474- known as multidrug resistance (MDR). In order to H, SKBR3-H and MDA-MB-361-H). The trastuzumab- investigate the development of drug resistance, a panel of conditioned cell lines were treated with IGF-binding protein four lung cancer cell lines were chosen for pulse-selection 3 (IGFBP3) or IGF-IR antibody (·IR3) to block IGF-IR, but with taxol. The four novel cell lines obtained were tested for neither significantly inhibited the growth of the cells or sensitivity to eight chemotherapeutic agents. The cell lines enhanced the anti-proliferative effects of trastuzumab. displayed resistance to taxol in the range of two- to eight-fold In conclusion, our results suggest that increased expression compared to their parent cell lines, and some of the cell lines and/or activation of IGF-IR may play a role in response to showed cross-resistance to other chemotherapeutic agents. trastuzumab. Therefore, a combination of anti-IGF-IR The results from toxicity testing indicate that no specific therapy and trastuzumab may be advantageous over trastuzu- pattern of MDR was present in the selected variants. The mab alone. Further in vitro investigation, however, is required low-level resistance observed in these selected cell lines may to determine if anti-IGF-IR inhibitors would be successful in be more clinically relevant to study than higher levels of overcoming IGF-IR-mediated trastuzumab resistance. resistance obtained by other methods of selection. Microarray analysis was used to identify genes associated with the 23 development of taxol resistance. The taxol-selected variants PROTEOMIC ANALYSIS OF DLKP AND ITS subjected to microarray analysis provide a unique opportunity SUBPOPULATIONS: DLKP-SQ, DLKP-I AND to study less well-characterised mechanisms of taxol DLKP-M AND INTERPRETATIONS OF ALTERED resistance, since these variants do not display classical MDR. INVASION RATES OBSERVED IN THESE CLONES William Bryan, Andrew Dowd, Helena Joyce, 22 Paula Meleady and Martin Clynes THE ROLE OF INSULIN-LIKE GROWTH FACTOR I RECEPTOR (IGF-IR) IN TRASTUZUMAB National Institute for Cellular Biotechnology, RESISTANCE IN BREAST CANCER CELLS Dublin City University, Dublin 9, Ireland Brigid Browne1, Norma O'Donovan1, Natarajan The lung carcinoma DLKP is described as a poorly Venkatesan3, Mark Pegram3, Martin Clynes1, Michael J. differentiated non-small cell lung carcinoma (NSCLC) with Duffy2, John Crown2 and Dennis J. Slamon3 neuroendocrine markers. It consists of three populations, 1National Institute for Cellular Biotechnology, DLKP-SQ, DLKP-I and DLKP-M which exist as Dublin City University, Dublin 9; approximately 70%, 25% and 5% of the DLKP population, 2St. Vincent's University Hospital, Dublin, Ireland; respectively. DLKP-SQ and DLKP-M interconvert with the 3University of California at Los Angeles, U.S.A. third population DLKP-I. These populations display distinct morphologies and invasive characteristics. A proteomics IGF-IR signaling interferes with the growth inhibitory action of approach was undertaken to understand the mechanisms trastuzumab (Herceptin) in breast cancer cell lines and may that govern the inter-conversion between these populations play a role in conferring resistance to trastuzumab treatment in and the mechanisms controlling invasion in these patients. The aim of this study was to define the role of IGF- subpopulations of DLKP. Using the combination of IR in trastuzumab resistance. HER-2 and IGF-IR protein levels 2-dimensional electrophoresis and Cy Dye protein labelling were determined using western blotting in twenty-four breast chemistry it is possible to do this. Proteins that were found to cancer cell lines. The anti-proliferative effect of trastuzumab, show statistically significant fold changes were investigated on HER-2-positive breast cancer cell lines was measured by cell and identified using MALDI-ToF mass spectrometry.

1379 ANTICANCER RESEARCH 27: 1233-1386 (2007)

24 All compounds were soluble in organic solvents but 4 of 6 INDUCTION OF APOPTOTIC CELL structures could not be tested due to their complete insolubility DEATH BY 1,10-PHENANTHROLINE (PHEN), in culture conditions. Two agents were soluble using solvent

[Ag2(phen)3(mal)]Ø2H2O, [Cu(phen)2(mal)]Ø2H2O vehicles and were found to be non-toxic. Using combination AND [Mn(phen)2(mal)]Ø2H2O (malH2ØMALONIC toxicity assays, we examined the resistance inhibitory potential ACID) USING HUMAN CANCER CELLS of these compounds, KG1, KG2, KG3, KG4, KG5 and KG6. Two non-small lung cell carcinomas, DLKP and DLKP- C. Deegan, M. McCann, M. Devereux, A, with differing multidrug resistance (MDR) characteristics B. Coyle and D.A. Egan were used. DLKP is resistant to several cytotoxic drugs due ITT Dublin, Tallaght, Dublin 24, Ireland to the expression of multidrug resistance protein 1 [MRP- 1, ABCC1 (ABC transporter)], whereas DLKP-A is highly Phenanthroline (phen) and substituted derivatives, both in the resistant due to the over expression of multidrug resistance metal-free state and as ligands coordinated to transition metals, gene 1 (MDR-1, ABC transporter). disturb the functioning of a variety of biological systems (1, 2). No synergy was evident in the DLKP cell line indicating that Phen and its metal complexes display a range of biological these compounds have no interaction with MRP-1, however, effects, including fungicidal activity, disruption of mitochondrial KG-4 demonstrated synergic potentiation of the MDR-1 function and induction of oxidative stress (3). Our research substrates, epirubicin and taxotere in DLKP-A (MDR-1+). No group has studied the chemotherapeutic potential of phen and synergy was evident with non-MDR-1 substrates. its three metal complexes, namely [Ag2(phen)3mal)]Ø2H2O, Similar toxicological analysis of related and precursor [Cu(phen)2(mal)]Ø2H2O and [Mn(phen)2(mal)]Ø2H2O (malH2 molecules suggests that drug resistance inhibition is = malonic acid) using two human cell lines (A-498 and Hep- associated with a specific core structure which may be G2). We showed that phen and these metal-phen complexes amenable to further chemical substitution to generate a induced a concentration-dependant cytotoxic response, with large new family of MDR-1 inhibitors. metal-phen complexes demonstrating a greater cytotoxic response than phen (4). In the current study, we sought to 26 probe the mechanism underlying the induction of this anti- WHOLE-GENOME MICROARRAY ASSESSMENT proliferative effect. Here we have shown that genomic DNA OF TAXOL- AND MELPHALAN-RESISTANT RPMI from drug-treated cells, once separated by electrophoresis, was CELL LINES AS A MODEL FOR DRUG-INDUCED cleaved to produce a ladder pattern characteristic of apoptotic INVASION IN CANCER AND IDENTIFICATION cell death. The induction of apoptosis was confirmed using OF POTENTIAL THERAPEUTIC TARGETS cellular morphology (phase-contrast and DAPI staining) and selected biochemical markers including activation of caspase-3 Padraig Doolan, Isabella Bray, Jai Prakash Mehta, and 9, cell cycle disruption and cleavage of PARP. Additional Patrick Gammell and Martin Clynes studies are currently underway to probe the identity of other Microarray and Bioinformatics Laboratory, cellular targets. In conclusion, these results suggest that phen National Institute for Cellular Biotechnology, and metal-phen complexes may have a therapeutic role to play Dublin City University, Dublin 9, Ireland in the successful treatment and management of cancer.

1 Brandt WW et al: Chem Rev 54: 959, 1954. The progression of invasion and metastasis in cancer is a 2 MacLeod RA: J Biol Chem 197: 751, 1952. complex process involving interactions between cancer cells and 3 McCann M et al: Metal-Based Drugs 7: 4, 2000. their surroundings. The development in the NICB of taxol- and 4 Deegan C et al: Cancer Lett, 2006. melphalan-resistant sublines of the nasal epithelial cell line RPMI 2650 which differ in their invasive potential, provided a 25 unique opportunity to identify various genes and proteins that MACROCYCLES, A POTENTIAL ANTICANCER AGENT are altered in expression to exert the invasive effect. The non- invasive cell line RPMI 2650 was exposed to increasing Aoife Devery1, Brian Deegan2, Ewa Kowalska2, concentrations of the chemotherapeutic drugs, taxol and Nick Gathergood2, Robert O'Connor1 and Martin Clynes1 melphalan, in order to isolate two drug-resistant sublines. 1National Institute of Cellular Biotechnology and Invasion assays demonstrated that the melphalan-resistant 2School of Chemical Science, Dublin City University, subline displayed markedly increased invasion, whereas the Dublin 9, Ireland taxol-resistant subline did not display any change in invasion, when compared to the parental RPMI. RNA was isolated from Using in vitro cytotoxicity assays we examined the toxicity and three replicates of the control RPMI 2650, RPMI-Mel and resistance inhibitory potential of novel macrocycles. RPMI-Tax cell lines and examined using Affymetrix U133_Plus

1380 Abstracts of the International Conference on Global mRNA and Protein Analysis

2.0 microarray chips. Unsupervised 2-way clustering analysis melanocytes but at a lower level. ABCB5 mRNA was (Pearson correlation, Average linkage) and Principal detected in normal melanocytes and in 5 of the melanoma Components Analysis dispersed the samples as expected, cell lines. ABCG5, which is believed to be involved in sterol demonstrating clear differences in expression profiles between transport, was detected in the normal melanocytes and the replicates. To identify the genes that contribute to these melanoma cells. However, ABCG5 protein levels were lower altered profiles, rigorous statistics applied to the dataset in the melanoma cells than in the normal melanocytes. (Student's t-test, p<0.01, fold change >4, Benjamini and Our results suggest that MRP2 and ABCB5 play a role in Hochberg FDR multiple testing correction) identified 2769 drug resistance in melanoma. Further studies, including genes as differentially expressed (DE) in the RPMI-Tax cell siRNA inhibition of ABCB5 and MRP2, will be conducted line and 3456 DE genes in the RPMI-Mel cell line. Overlapping to elucidate the effects of these ABC transporters on these lists identified 1179 melphalan-specific genes up-regulated response to chemotherapy in melanoma cells. in expression and 934 genes down-regulated. Stringent (p<0.01) GOstat analysis of the overlapped up-regulated genelist 28 revealed over-representation of ontologies concerned with A CELLULAR MODEL SYSTEM response to stress, cell locomotion and motility and CHARACTERISING THE INVASIVENESS OF angiogenesis. GOstat analysis of the down-regulated gene list A HUMAN LUNG CARCINOMA CELL LINE revealed over-representation of ontologies concerned mainly Helena Joyce and Martin Clynes with transcription and transcriptional regulation. Pathway Assist pathway analysis of the invasion-specific up-regulated genelist National Institute for Cellular Biotechnology, identified a number of canonical pathways which were Dublin City University, Dublin 9, Ireland siginificantly affected, such as MAPK-ERK in growth and differentiation, EGF signalling, integrin signaling and IL-6 The human lung carcinoma cell line DLKP-SQ was exposed to signaling. In addition, direct-interaction mapping of the sequential pulses of the chemotherapeutic drugs mitoxantrone submitted genelist identified several proteins as important and 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). The cell interactors within the gene list, including the transcription populations derived from each pulse were tested for factors ETS1, STAT3, FOS, FOSL1, SMAD3, MITF and JUN invasiveness and for sensitivity to the original selecting agent. B, the kinases MAPK1 MAPK3 and JAK1, the receptors Invasion assays showed that BCNU exposure did not make the PLAUR, EGFR and TNFRSF1A and the SPP1 ligand protein. DLKP-SQ cells more invasive after five pulses. Preliminary toxicity assays showed that while the cells developed resistance 27 to mitoxantrone after the fourth pulse, a significant invasive EXPRESSION OF ABC TRANSPORTERS phenotype was not observed until the sixth pulse. Further IN MELANOMA CELL LINES characterisation of the cell populations from pulses four (not invasive) and six (invasive) is currently underway using DNA Alex Eustace, Robert O'Connor, Martin Clynes and microarray and proteomics technologies. Norma O'Donovan Microarray analysis was also carried out on the DLKP National Institute for Cellular Biotechnology, Dublin City parent, the adriamycin-selected DLKP-A10, DLKP-A and University, Dublin 9, Ireland its clones, DLKP-A2B and DLKP-A5F cell line. These cell Malignant melanoma is notoriously resistance to all forms of lines have varying levels of adriamycin resistance and treatment. Current knowledge of the mechanisms of drug invasiveness. This data combined with data from the cellular resistance in melanoma is limited, as is evidenced by the lack model system described above will be used to identify of significant improvements in outcome for this disease. The reliable gene targets for siRNA knockdown. aim of this study was to examine the role of ABC transporter proteins in drug resistance in melanoma cell lines. 29 Expression of p-glycoprotein (Pgp/ABCBl), multi-drug PROTEOMIC ANALYSIS TO DISSECT resistance protein 1 (MRP1/ABCC1), MRP2 (ABCC2), DRUG-RESISTANCE ASSOCIATED PROTEINS ABCB5, breast cancer resistance protein (BCRP1/ABCG2) IN A DLKP-MITOXANTRONE RESISTANT VARIANT and ABCG5 were measured in a panel of melanoma cell Joanne Keenan, Lisa Murphy and Martin Clynes lines and in normal melanocytes using RT-PCR and Western blotting. National Institute for Cellular Biotechnology, Dublin City Pgp protein was detected, but at very low levels, in 5 of University, Glasnevin, Dublin 9, Ireland the 6 melanoma cell lines and was not detected in the normal melanocytes. MRP2 mRNA was detected in 5 of the As cancer is normally a multi-faceted disease with alterations 6 melanoma cell lines. MRP2 was also expressed in normal in expression of many proteins, proteomic techniques provide

1381 ANTICANCER RESEARCH 27: 1233-1386 (2007) a unique opportunity to study the global changes in protein lines. The combination of imatinib at 13.52 ÌM and docetaxel at expression. The development of multidrug resistance, in 14.88 nM, however, synergistically inhibited cell growth and particular, presents poor outcome for patients. While invasion activity, which could not be reversed by drug removal. inhibitors to classical MDR mechanisms such as Pgp have This combination might be a promising step forward in the performed poorly in clinical trials, other mechanisms may direction to tackle glioma invasion and, in combination with a offer greater potential. In order to study the global effects of cytotoxic treatment, could present a new treatment regimen for the development of drug resistance, differential protein malignant astrocytomas. expression of a squamous lung carcinoma cell line, DLKP, and its mitoxantrone-resistant variant was investigated using 2D- 31 DIGE technology. Analysis using Decyderì in Difference In- GENERATION AND PRELIMINARY gel analysis detected 2221 protein spots of which 217 were CHARACTERISATION OF MAbs down-regulated (9.8%) and 174 were up-regulated (7.8%). RAISED TO THE INVASIVE BREAST Biological Variation Analysis revealed a total of 343 proteins CELL LINE, MDA-MB-435-SF to be differentially regulated with p<0.05 of which 147 were Annemarie Larkin, Dermot O'Sullivan, Helena Joyce, p<0.01. The fold differences ranged from 3.96-fold down- Sharon Glynn and Martin Clynes regulation to 3.85-fold up-regulation. Identification of proteins of interest by MALDI mass spectrometry revealed changes in National Institute for Cellular Biotechnology, proteins involved in many cellular processes including cellular Dublin City University, Dublin 9, Ireland reorganisation, detoxification, protein synthesis and folding. Monoclonal antibodies (MAbs) provide a powerful tool for the 30 identification of novel tumour-associated antigens in human IMATINIB AND DOCETAXEL IN COMBINATION cancer. In order to identify potentially interesting antigens CAN EFFECTIVELY INHIBIT GLIOMA associated with invasion and/or resistance mechanisms, we INVASION IN AN IN VITRO 3D INVASION ASSAY generated a panel of MAbs directed against a taxol-resistant variant of the MDA-MB-435-SF cell line, widely used as a Paula Kinsella1, Geoff Pilkington2, Martin Clynes1 and model of metastatic breast cancer. Following cell fusion, Verena Amberger-Murphy1 hybridoma supernatants were screened against the target 1National Institute for Cellular Biotechnology, immunogen using newly developed selective screening methods Dublin City University, Dublin 9, Ireland; involving high throughput invasion assays. To selectively identify 2School of Pharmacy and Biomedical Sciences, those hybridomas recognising cell surface targets, hybridomas University of Portsmouth, Portsmouth, Hampshire, U.K. were also screened using live cell immunofluorescence. Preliminary results indicate that antibodies 9E1, 4E5 and 102C The main problem in the treatment of malignant astrocytomas appeared to have an effect on the invasion process in the is their invasive behaviour. Even successful resection of the parental MDA-MB-435-S cell line, the highly invasive MDA- main tumour mass cannot prevent recurrence due to single MB-231 breast cell line and the invasive LOX melanoma cell cells, which had already invaded the surrounding brain line. Antibodies 104D and 4E5 appeared to recognise a cell parenchyma at the time of diagnosis. The classical combination surface epitope, whilst a more intracellular pattern of reactivity therapy PCV (procarbazine, CCNU and vincristine) has been was associated with antibody 9E1. Furthermore, an anti- used for 30 years; however, its clinical effectiveness in the proliferative effect in the LOX cell line was also observed with treatment of malignant astrocytomas and glioblastomas is still antibodies 4E5 and 9E1. Results from these functional assays doubtful. Using an in vitro 3D invasion model, we tested the and immunofluorescence studies will be discussed. effect of the tyrosine kinase inhibitor imatinib and the microtubule inhibitor docetaxel on the invasion activity of a 32 panel of astrocytic tumour cell lines, including two established CYP3A4 AND CYP3A5 ARE UP-REGULATED glioma cell lines, IPSB-18 and SNB-19, and two primary cell IN PULSE-SELECTED CELL LINES: lines originating from glioblastomas, CLOM002 and UPHHJA. ROLE IN CHEMOTHERAPY RESISTANCE The IC concentrations for each drug were determined and 50 V. Martinez, R. O'Connor and M. Clynes used to calculate the concentrations for the invasion assays. The

IC50 concentration of imatinib was between 15.7 ÌM and 18.7 National Institute for Cellular Biotechnology, ÌM, at which it had no inhibitory effect on invasion of the Dublin City University, Dublin 9, Ireland tested cell lines. The IC50 concentration of docetaxel was between 0.7 nM and 19.8 nM, and at 14.88 nM docetaxel had a The development of drug resistance by tumour cells has slight transient inhibitory effect on invasion activity of all cell become a major challenge for cancer treatment. Different

1382 Abstracts of the International Conference on Global mRNA and Protein Analysis mechanisms are involved in this process and there is (http://www.ncbi.nlm.nih.gov/geo). These samples were pooled evidence that cancer cells can become resistant by reducing as a single experiment and normalized using the dCHIP drug activation or enhancing drug detoxification. algorithm. Since the tissue samples were on U133A and the Cytochromes P450 are able to metabolise a number of cell lines on U133_Plus 2.0, the genes not represented in chemotherapeutic drugs into less or non-toxic derivatives; U133A were removed from the cell line data, giving a total these enzymes therefore have the potential to influence the gene number of 22277. The two groups were compared for sensitivity of tumour cells to anticancer agents. To evaluate genes which were significantly up or down regulated (p<0.001, their relevance in the development of resistance, we pulse- fold change >2, difference >100). Following this, pathways selected two different tumour cell lines, Caco2 and HepG2, analysis was performed using Genmapp software to identify with the chemotherapy drugs cisplatin, taxol and taxotere: canonical pathways that differed in behaviour between the cell treated cells were found to be considerably more resistant lines and clinical samples. (2- to 100-fold) to the toxic effects of taxol, taxotere and A total of 2615 genes passed the above filtration criteria. vincristine than the parental ones, while cisplatin toxicity Some of the genes which were highly up-regulated in cell was unaffected. Expression of cytochrome P450 3A4 lines relative to tissue were TIP5, MGEA6, KIAA1, CLCP1, (CYP3A4) and 3A5 was found to be up-regulated in FMR1, MIR1, NUP205, A2RP, LFHL1 and HCGII. Genes selected cells, together with that of MDR1. Inhibition of highly up-regulated in tissue samples relative to cell line P450 activity by treatment with 17· ethynyl estradiol were IGCJ, LZM, IGLJ3, NACRA4, MIP-2, NTR2, HCAK1, (17AEE) did not revert resistance to parental levels. In KRTHA6, FAM30 and COLL6. A large number of genes up- contrast, treatment with the MDR1 inhibitor GF120918A regulated in cell lines relative to tissue were related to the greatly enhanced the cytotoxicity of the aforementioned cell cycle while many of the genes up-regulated in tissue anticancer agents, indicating that MDR1 is at least partly relative to cell lines were related to immunoglobin and responsible for the increase in resistance. Simultaneous immune response. The gene lists were further subjected to treatment of cells with taxol, GF120918A and 17AEE did Gene Ontology and Pathways analysis using Genmapp. This not result in greater toxicity than treatment with taxol or analysis revealed that some of the functions which are up- GF120918A alone, suggesting that P450 expression is not regulated in cell lines relative to tissue are mitosis, cell relevant for taxol-resistance in these cells, even when division and proliferation. Some pathways which were MDR1 is inhibited. Our results indicate that an increase in increased were those of the cell cycle, translation factors P450 expression does not result in enhanced resistance to and mRNA processing reactome. Conversely, functions like chemotherapy, suggesting that it might be the result of a immune response, defence response, response to biotic general cell response to toxic stimuli. stimulus, MHC class II receptor activity and extra-cellular matrix were significantly up-regulated in tissue relative to 33 cell lines. Some of the pathways which were down-regulated HOW REPRESENTATIVE ARE CELL LINE were Classical complement activation, matrix metallo- MODELS TO CLINICAL CONDITIONS? A proteinase, smooth muscle contraction, and inflammatory MICROARRAY APPROACH TO TRANSLATIONAL response pathway. MEDICINE IN BREAST CANCER 34 Jai Prakash Mehta, Padraig Doolan and Martin Clynes DETECTION OF POTENTIAL DRUG National Institute for Cellular Biotechnology, RESISTANCE MARKERS IN LUNG CANCER Dublin City University, Dublin 9, Ireland Lisa Murphy, Joanne Keenan and Martin Clynes Cell line models are widely used to represent particulars of National Institute for Cellular Biotechnology, a clinical condition. However, the degree to which these cell Dublin City University, Dublin 9, Ireland lines model their respective clinical conditions has never been fully assessed. The work presented here utilises Proteomics is the simultaneous analysis of complex protein microarray datasets to compare the gene expression profiles mixtures, like cell lysates and tissues, to look for qualitative of breast cancer cell lines and breast cancer tissue to and semi-quantitative changes of expression levels. Currently identify major transcriptional changes in the adaptation one of the most popular applications of proteomics is the process from a tissue to a cell line. The aim of this study is area of cancer research, where it has applications in drug to assess the suitability of cell lines as breast cancer models discovery, prognostics/diagnostics and prediction. The for clinical scenarios. objective is to study drug-resistance mechanisms in lung Gene expression profiles of 189 breast tissue and 19 cell cancer in vitro by proteomic technologies to increase our lines were obtained from Gene Expression Omnibus understanding of these mechanisms and to identify putative

1383 ANTICANCER RESEARCH 27: 1233-1386 (2007) markers of possible prognostic/diagnostic value. Differential phenotypic characteristics studied were minimal overall. protein expression between a lung cell line (DLKP) and its siRNA silencing at protein and mRNA levels will be assessed adriamycin-resistant variant (DLKP-A) was observed using in future work, as levels and duration of silencing can vary 2D-DIGE to focus in on proteins associated with adriamycin between cells depending on the efficiency of siRNA uptake. resistance. Difference In-gel Analysis using Decyder revealed a total of 3454±303 protein spots between DLKP and DKLP- 36 A. In Biological Variation Analysis,