CANCER GENOMICS & PROTEOMICS
Volume 5, Number
3-4, May-August 2008
Molecular Mechanisms of Action of Imatinib Mesylate in Human Ovarian Cancer: A Proteomic Analysis. B.B. PATEL, Υ.Α. HE, X.-M. LI, A. FROLOV, L. VANDERVEER, C. SLATER, R.J. SCHILDER, M. VON MEHREN, A.K. GODWIN, A.T. YEUNG (Philadelphia, PA; Birmingham, AL, USA)
Identification of F-box/LLR-repeated Protein 17 as Potential Useful Biomarker for Breast Cancer Therapy. G.G. XIAO, B.-S. ZHOU, G. SOMLO, J. PORTNOW, A. JUHASZ, F. UN, H. CHEW, D. GANDARA, Y.YEN(Duarte, Torrance, Sacramento, CA; Omaha, NE, USA)
Phosphoproteome and Transcriptome Analyses of ErbB Ligand-stimulated MCF-7 Cells. T. NAGASHIMA, M. OYAMA, H. KOZUKA-HATA, N. YUMOTO, Y. SAKAKI, M. HATAKEYAMA (Kanagawa;Minato-ku, Tokyo,Japan)
The Small Variant of the Apoptosis-associated X-Chromosome RBM10 Gene is Co-expressed with Caspase-3 in Breast Cancer.
E. MARTIN-GARABATO, F. MARTINEZ-ARRIBAS, M. POLLAN, A.R. LUCAS, J. SANCHEZ, J. SCHNEIDER (Madrid, Spain)
The Proteome Profile of Two Cell Lines and their Xenografts Isolated from a Patient with Clear Cell Sarcoma (Soft Tissue Melanoma). K. DIMAS, C. TSIMPLOULI, A.K. ANAGNOSTOPOULOS, L. MAHAIRA, E. ILIOPOULOU, S. PEREZ, K. VOUGAS, G.Th. TSANGARIS (Athens, Greece)
CANCER GENOMICS & PROTEOMICS 5: 137-150 (2008)
Molecular Mechanisms of Action of Imatinib Mesylate in Human Ovarian Cancer: A Proteomic Analysis
BHAVINKUMAR B. PATEL1, YIN A.HE1, XIN- MING LI1, ANDREY FROLOV3, LISA VANDERVEER2, CAROLYN SLATER2, RUSSELL J. SCHILDER2, MARGARET VON MEHREN2, ANDREW K. GODWIN2 ANTHONY T. YEUNG1
Division of 1Basic Science, and 2Medical Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania; 3Department of Surgery, University of Alabama at Birmingham, 1824 6th Ave South, Birmingham, Alabama, U.S.A.
Abstract: Background: Imatinib mesylate (Gleevec®, Novartis, Basel, Switzerland) is a small-molecule tyrosine kinase inhibitor with activity against ABL, BCR-ABL, c-KIT, and PDGFRα. Several clinical trials have evaluated the efficacy and safety of imatinib in patients with ovarian carcinoma who have persistent or recurrent disease following front-line platinum/taxane based chemotherapy. However, there is limited pre-clinical and clinical data on the molecular targets and action of imatinib in ovarian cancer. Materials and Methods: Human ovarian cancer cells (A2780) were treated with imatinib mesylate for either 6 or 24 h. We employed a 2D (two-dimensional) gel electrophoresis and mass spectrometry-based proteomics approach to identify protein expression patterns and signaling pathways that were altered in response to imatinib. Cells were analyzed for PDGFRα and AKT expression, which were then correlated with imatinib sensitivity. Results: Using 2D gel electrophoresis of overlapping pH ranges from pH 4 to 11, about 4,000 protein spots could be analyzed reproducibly. Proteins whose levels changed between two fold to 30 fold were grouped according to whether changes were in the same direction at both time points of treatment with respect to the control, or changed their levels only at one of the time points. Conclusion: Differentially regulated proteins following imatinib treatment of A2780 cells involved the regulation of actin cytoskeleton, metabolic pathways, cell cycle, cell proliferation, apoptosis, cell junctions, and signal transduction. Thus, exposure of cells to imatinib produces complex changes in the cell that require further investigation.
Identification of F-box/LLR-repeated Protein 17 as Potential Useful Biomarker for Breast Cancer Therapy
GARY GUISHAN XIAO1,2,5, BING-SEN ZHOU1, GEORGE SOMLO3, JANA PORTNOW3, AGNES JUHASZ1, FRANK UN1, HELEN CHEW4, DAVID GANDARA4,YUN YEN1
1Department of Clinical & Molecular Pharmacology, City of Hope National Cancer Center, Duarte, CA; 2Department of Pediatrics/Mass Spectrometry Lab Facility, Harbor-University of California Los Angeles, Torrance, CA; 3Department of Medical Oncology, 4University of California Davis Cancer Center, Sacramento, CA; 5Genomics & Functional Proteomics Laboratory, Osteoporosis Research Center, Creighton University, Omaha, NE, U.S.A.
Abstract: Background: The expression and activity of ribonucleotide reductase (RR) has been associated with resistance to multiple drugs in human cancer. The use of antisense oligonucleotide drug, GTI-2040, a 20-mer phosphorothioate oligonucleotide complemented to the human RR M2 subunit mRNA, represents an effective strategy for inhibiting RR. The increased specificity due to the anti-resistance effect of GTI-2040 may also lead to a more favorable therapeutic outcome. Materials and Methods: To understand the molecular mechanism underlying RR inhibition, patients' blood samples were analyzed using multiple dimensional proteomics technology via matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry. Results: A major difference occurred at 5k m/z in the MALDI profile, which appeared only in the non-responsive group and diminished after GTI-2040 treatment. This specific peptide peak remained at the basal level in responsive patients. The peak was identified to represent the F-box/LLR-repeat protein 17 (FBXL17) through nanoelectrospray ionization liquid chromatography-tandem mass spectrometry (nanoESI LC-MS/MS). Further characterization revealed that FBXL17/SKP2 directly interacts with the human RR M2 (RRM2) subunit to promote hRRM2 overexpression in the breast cancer cell line MCF-7. Conclusion: Validation of this protein using real-time RT-PCR indicates the F-box protein 17 (FBXL17) can serve as a therapeutic target and surrogate marker for breast cancer therapy.
1Computational and Experimental Systems Biology Group, RIKEN Genomic Sciences Center, Yokohama, Kanagawa; 2Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan
Abstract: Cellular signal transduction pathways and gene expression are tightly regulated to accommodate changes in response to physiological environments. In the current study, molecules were identified that are activated as a result of intracellular signaling and immediately expressed as mRNA in MCF-7 breast cancer cells shortly after stimulation of ErbB receptor ligands, epidermal growth factor (EGF) or heregulin (HRG). For the identification of tyrosine-phosphorylated proteins and expressed genes, a SILAC (stable isotopic labeling using amino acids in cell culture) method and Affymetrix gene expression array system, respectively, were used. Unexpectedly, the overlapping of genes appeared in two experimental datasets was very low for HRG (43 hits in the proteome data, 1,655 in the transcriptome data, and 5 hits common to both datasets), while no overlapping gene was detected for EGF (15 hits in the proteome data, 211 hits in the transcriptome data, and no hits common to both datasets). The HRG overlapping genes included ERBB2, NEDD9, MAPK3, JUP and EPHA2. Biological pathway analysis indicated that HRG-stimulated molecular activation is significantly related to cancer pathways including bladder cancer, chronic myeloid leukemia and pancreatic cancer (p<0.05). The proteome datasets of EGF and HRG contain molecules that are related to Axon guidance, ErbB signaling and VEGF signaling at a high rate.
The Small Variant of the Apoptosis-associated X-Chromosome RBM10 Gene is Co-expressed with Caspase-3 in Breast Cancers
E. MARTÍN-GARABATO1, F. MARTÍNEZ-ARRIBAS1,2, M. POLLÁN3, A.R. LUCAS1, J. SÁNCHEZ1,4 and J. SCHNEIDER1,2
1Fundación Tejerina-Centro de Patología de la Mama, Madrid; 2Departamento de Ciencias de la Salud-2, Universidad Rey Juan Carlos, Alcorcón (Madrid); 3Cancer Epidemiology Department, National Centre for Epidemiology, Madrid; 4Universidad de Alcala, Alcalá de Henares, Madrid, Spain
Abstract: Background: There are very few studies on the final triggers of apoptosis, the caspases, in breast cancer. Materials and Methods: Caspase-3 expression was studied by means of reverse transcript polymerase chain reaction (RT-PCR) in a series of 108 previously untreated patients with breast cancer. Expression levels were correlated with those obtained using the same technique of the apoptosis-associated X-chromosome genes RBMX, RBM3, RBM10 small and RBM10 large variant; Bcl-2 and Bax; the angiogenesis-associated genes VEGF and CD105 (endoglin); hMAM and Nup88. The correlation with the expression of hormone receptors, c-erb-B2, mutant p53 and Ki-67, all measured by means of immunohistochemistry, was also studied, as well as that with standard clinical parameters such as histological type, tumor size, axillary metastasis and DNA-ploidy. Results: The only statistically significant correlations observed between caspase-3 mRNA expression and the parameters tested were a direct one with both the Bax (p=0.007) and the small variant of the X-chromosome RBM10 gene (p=0.018), and an inverse one with the angiogenesis-associated CD105 (endoglin) gene (p=0.044). Conclusion: These results indicate that very few genes are involved in the last steps of the apoptotic cascade in breast cancer, among them one of the X-chromosome RBM family. They also support the relatively unexplored link between apoptosis and angiogenesis.
The Proteome Profile of Two Cell Lines and their Xenografts Isolated from a Patient with Clear Cell Sarcoma (Soft Tissue Melanoma)
KONSTANTINOS DIMAS1, CHRISIIDA TSIMPLOULI1, ATHANASSIOS K. ANAGNOSTOPOULOS2, LOUISA MAHAIRA1,3, ELENI ILIOPOULOU4, SONIA PEREZ4, KONSTANTINOS VOUGAS2 GEORGE T. TSANGARIS2
1Division of PharmacologyPharmacotechnology, Centre of Basic Research I, and 2Proteomics Research Unit, Centre of Basic Research II, Biomedical Research Foundation of the Academy of Athens, Athens; 3Anavex Life Sciences, Pallini; 4Cancer Immunology and Immunotherapy Centre, St. Savvas Hospital, Athens, Greece.
Abstract: We report the establishment of two novel clear cell sarcoma (CCS) cell lines (soft tissue melanoma) from a patient and the production of the corresponding xenografts after xenotransplantation of those cells to NOD/SCID mice. As no comprehensive study on the relevant proteomes of this type of cancer has been reported to date, proteomics technologies were applied in a first attempt to analyze the proteins of the two cell lines and their corresponding primary xenografts. Total protein extracts were separated by two dimensional gel electrophoresis (2-DE) and analysed by MALDI-MS and MALDI-MS-MS following in-gel digestion with trypsin. Protein identification was carried out by peptide mass fingerprint (PMF) and post source decay (PSD), respectively. Comparative analysis revealed that 124 proteins were common between the cell lines and the xenografts; 249 proteins were found to be expressed only in the proteome of the cell lines, while 178 proteins were expressed only in the proteome of xenografts. Our results demonstrated that both cell lines and xenografts were positive for vimentin and S100 reported as markers for CCS. After functional analysis, 27 different protein groups were identified in the analysed proteomes, including apoptosis-related proteins, oncogenes and several proteins closely related to TP-53 and NF-κB pathways. Furthermore, the proteins nestin, stem cell growth factor CLC11 and mdr-1, closely related to malignant-melanoma-initiating cells, were found to be expressed in both the cell lines and their corresponding xenografts. Since there are no data concerning protein expression in CCS, this study may contribute to the understanding of the molecular basis of the disease, while the cell lines as well as the developed xenografts may be used as tools for the development of new therapeutic strategies to tackle this rare but fatal malignancy.