CANCER GENOMICS & PROTEOMICS
Volume 1, Number
2, March-April 2004
CONTENTS
PAGE
Interferon-A Sensitises Human Osteosarcoma Cells to Trail-mediated Apoptosis. I. M. MIKKELSEN, C. LOKKE, T. FLAGSTAD, O. S. BRULAND (Tromso; Oslo, Norway)
95
Genomic Alterations in Gastrointestinal Stromal Tumors as Revealed by Conventional and Array-based Comparative Genomic Hybridization. S. R. GRANITTO, P. KOLACHANA, C. R. ANTONESCU, R. C. JOHNSON, R. P. DEMATTEO, S. C. JHANWAR (New York, NY; Houston, TX, U.S.A.)
105
*Real-time Quantitative PCR Arrays for Virally-associated Cancers. D. P. DITTMER, W. VAHRSON, M. R. STAUDT, J. F. PAPIN, R. HINES-BOYKIN, F. D. FAKHARI (Oklahoma City, OK, U.S.A.)
117
Protein Profiling of the Supratentorial Primitive Neuroectodermal Tumor (PNET) Cell Line PFSK-1. A. PEYRL, K. KRAPFENBAUER, L. AFJEHI-SADAT, T. STROBEL, IRENE SLAVC, G. LUBEC (Vienna, Austria; Basel, Switzerland)
125
Alterations in Gene Expression Associated with Head and Neck Squamous Cell Carcinoma Development. M. M. SEREWKO-AURET, A. L. DAHLER, L. SMITH, C. FAI WONG, C. POPA, L. M. BARNES, W. COMAN, N. A. SAUNDERS (St Lucia, Queensland, Australia)
137
Human Papillomavirus High-risk Genotypes: Relationship to Apoptosis and p53 Expression in Egyptian Patients with Laryngeal Carcinoma. S. K. KASSIM, L. SEADA, S. IBRAHIM (Cairo; Benha, Egypt)
149
*Prediction of Drug Sensitivity and Resistance of Cancer by Protein Expression Profiling. M. VOLM, R. KOOMAGI, T. EFFERTH (Heidelberg, Germany; London, U.K)
157
New Target Genes for Tumor-derived Soluble Factors in Primary Monocytes.T. HOFMANN, B. SCHMITT, B. MACK, S. LANG1, OLIVIER GIRES, R. ZEIDLER (Munich, Germany)
167
LKW, a Putative Dual-specificity Kinasewhich is Down-regulated in Several Invasive Systems.
S. LOSCH, M. BUCHHOLZ, T. M. GRESS, U. H. WEIDLE (Penzberg; Ulm, Germany)
177
CYP1A1 and CYP1B1 Polymorphism and Lung Cancer Risk in Relation to Tobacco Smoking. J. SCHNEIDER, U. BERNGES, M. PHILIPP, H-J. WOITOWITZ (Giessen, Germany)
189
*Reviews
(pages 117,157)
CANCER GENOMICS & PROTEOMICS 1: 189-198 (2004)
CYP1A1 and CYP1B1 Polymorphism and Lung Cancer Risk in Relation to Tobacco Smoking
JOACHIM SCHNEIDER, ULRIKE BERNGES, MONIKA PHILIPP, HANS-JOACHIM WOITOWITZ
Institut und Poliklinik fur Arbeits - und Sozialmedizin der Justus-Liebig Universitat, Giessen, Germany
Abstract: Background: The impact of genetic polymorphisms in CYP1A1 or CYP1B1 on susceptibility to lung cancer has received particular attention since these enzymes play a central role in the activation of major classes of tobacco carcinogens. Several polymorphisms in the CYP1A1 locus have been identified and their genotypes appear to exhibit population frequencies that depend on ethnicity. In the current German study, we investigated the role of CYP1A1 and CYP1B1 polymorphisms as a genetic modifier of risk for individuals with lung cancers as susceptible genotypes, especially in relation to tobacco smoking. Materials and Methods: Three polymorphisms, the CYP1A1 T6235C (CYP1A1 MspI), the CYP1A1 A4889G-position (CYP1A1 iva) as well as the CYP1B1 codon 432 polymorphism were determined by real-time PCR analysis in 446 lung cancer patients and in 622 controls. Results: The observed allele frequencies in the population were within the range described for Caucasians. Multivariate analyses of lung cancer patients, who carried at least one mutant variant allele of CYP1A1 T6235C (OR=1.06; 95%-CI: 0.7-1.6), CYP1A1 A4889G (OR=1.09; 95%-CI: 0.63-1.88) or CYP1B1 Val432Leu (OR=1.01 CI: 0.73-1.39) did not show any elevated risks. When analysed by histology, no individual subtype of lung cancer was significantly associated with the polymorphisms. Lung cancer risk rose significantly with higher cumulative cigarette consumption. Stratified analysis between tobacco smoking and variant genotypes revealed, for heavy smokers (>60 pack-years), increasing risks with the presence of at least one copy of the CYP1A1 T6235C variant allele OR=27.74 (95%-CI: 4.34-177.25), the CYP1A1 A4889G position OR=33.23 (95%-CI: 3.11-354.99) and the CYP1B1 OR=418.70 (95%-CI: 45.45-3856.89). By analysing the interaction between tobacco smoking and the genotypes, the combination of smoking and having the susceptible genotypes did not show a joint effect. Conclusion: In this study polymorphism of the CYP1A1 T6235C- or A4889G-position as well as CYP1B1-codon 432-polymorphisms had no relevant modifying effect on lung cancer risk and cumulative smoking dose.
Prediction of Drug Sensitivity and Resistance of Cancer by Protein Expression Profiling
MANFRED VOLM1, REET KOOMAGI2, THOMAS EFFERTH3
1German Cancer Research Center, Heidelberg, Germany; 2University Brunel, Uxbridge W-London, U.K.; 3Center for Molecular Biology of the University Heidelberg (ZMBH), Germany
Abstract: Although the statistical probability of therapeutic success is known for larger groups of cancer patients, the clinical response to chemotherapy of the individual patient remains uncertain. It would be of great value to know whether or not an individual tumor responds to the proposed therapy. The concept of sensitivity testing of tumors for individualized therapy traces back to the 1970s. Currently, an astonishing revival has taken place due to the thriving development of genomic and proteomic technologies. This review discusses our own results on protein expression profiles of non-small cell lung cancer, kidney carcinoma and acute lymphoblastic leukemia regarding the prediction of drug sensitivity or resistance. A great diversity of drug resistance mechanisms are operative in the clinical drug resistance of cancer e.g., resistance proteins, proliferative, apoptotic, angiogenic factors, proto-oncogenes and tumor suppressor-genes. Hierarchical cluster analyses and cluster image maps reveal different resistance profiles even within cancer types of homogeneous histology. Protein arrays may be appropriate to perform sensitivity or resistance tests for individual patients because thousands of proteins may be detected in a single experiment. On the other hand, results suggest that already a set of a limited number of factors may be sufficient to detect the sensitivity or resistance of a cancer.
Interferon-ã Sensitises Human Osteosarcoma Cells to Trail-mediated Apoptosis
IDUN M. MIKKELSEN1, CECILIE LOKKE1, TROND FLAGSTAD1,3, OYVIND S. BRULAND 2,4
1Department of Paediatrics and 2Department of Oncology, Institute of Clinical Medicine, University of Tromso, N-9037 Tromso;. 3Department of Paediatrics, University Hospital of North Norway, N-9037 Tromso; 4The Norwegian Radium Hospital, Montebello, Oslo, Norway
Abstract: Background: Nearly half of all patients with osteosarcoma are still not cured by the currently employed multimodal treatment. New strategies are therefore needed to further improve the outcome. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2) is able to induce programmed cell death in transformed cells, while normal cells remain unaffected. Materials and Methods: We have investigated the effect of TRAIL in combination with IFN-ã on four human osteosarcoma cell lines; Saos-2, U2OS, KPDXM and OHS, and on one normal human fibroblast cell line, MRC-5. Results: One of the four osteosarcoma cell lines was TRAIL-resistant, but was sensitised to TRAIL-mediated cell death upon pre-incubation with IFN-ã. In two of the three TRAIL-sensitive cell lines, the effect of TRAIL was enhanced by IFN-ã. The normal human fibroblast cell line MRC-5 was not affected by treatment with TRAIL and IFN-ã. A caspase cascade involving the activation of caspase-8, caspase-7 and PARP was associated with the onset of apoptosis in the osteosarcoma cell lines. Apoptosis was partly inhibited by the addition of caspase inhibitors zVADfmk (general inhibitor) and zIETDfmk (selective caspase-8 inhibitor). These findings further emphasize the important role of the caspases in cell death signalling. Conclusion: Our results show that treatment with both IFN-ã and TRAIL efficiently induces cell death in osteosarcoma cell lines and should be further investigated as a potential future therapy.
Protein Profiling of the Supratentorial Primitive Neuroectodermal Tumor (PNET) Cell Line PFSK-1
ANDREAS PEYRL1, KURT KRAPFENBAUER2, LEILA AFJEHI SADAT1, THOMAS STROBEL3, IRENE SLAVC1, GERT LUBEC1
1Department of Pediatrics and 3Institute of Neurology, Medical University of Vienna, Vienna, Austria; 2Roche Center for Medical Genomics LtD, Basel, Switzerland
Abstract: Background: Supratentorial primitive neuroectodermal tumors (PNETs) are rare embryonal cerebral hemispheric tumors proposed to arise from primitive neuroepithelial cells. The permanent cell line PFSK-1 is widely used in studies of this tumor entity and it was the aim of this study to generate a proteome map to serve as a basis for further studies to search for tumor-related proteins. Materials and Methods: The PNET-related cell line PSFK-1 was cultivated and proteins from cell lysates were subject to two-dimensional gel electrophoresis with in gel-digestion of protein spots and subsequent MALDI-MS identification. Results: Among the 157 proteins identified by this method we observed structural, metabolic, chaperone, antioxidant, transcriptional / translational proteases as well as miscellaneous proteins. Hypothetical proteins similar to pyrroline-5-carboxylate reductase isoform, similar to 3-hydroxyisobutyryl-Coenzyme A hydrolase, thioredoxin domain containing protein 5 precursor, potential helicase with zinc-finger domain, an unnamed protein product and proteins P1.11659_4 and Pro1512 were detected. Conclusion: No neuronal, glial or other specific markers were found; the presence of vimentin may point to a mesenchymal rather than an epithelial origin; expression of developmentally expressed potential helicase P42694 indicates immaturity and FUSE binding protein 1 provides a link to myc, a major protooncogene, and to differentiation per se. We provide an analytical tool unambiguously identifying structures of several protein classes and show the existence of several hypothetical proteins, that had so far been predicted from nucleic acid sequences only and never detected in mammalian cell lines or tissues at the protein level.
Human Papillomavirus High-risk Genotypes: Relationship to Apoptosis and p53 Expression in Egyptian Patients with Laryngeal Carcinoma
SAMAR K. KASSIM1, LAILA SEADA2, SAMER IBRAHIM3
1Medical Biochemistry and 3Otolaryngology Departments, Ain Shams Faculty of Medicine, Cairo; 2Pathology Department, Zagazig University, Benha, Egypt
Abstract: Human papillomavirus (HPV) infection is suspected of causing laryngeal carcinoma. The relationship of HPV-16 and 18 genotypes to apoptosis and p53 protein expression in Egyptian laryngeal carcinoma patients was studied. Biopsy specimens from 82 patients with laryngeal carcinoma and 28 with minimal pathological lesions serving as a control group were examined. In all specimens, HPV-16 and-18 were examined using PCR, p53 expression was studied by immunohistochemistry and DNA fragmentation to assess apoptosis was assayed using a biochemical method and gel electrophoresis. HPV-16 was detected in 51.2% of laryngeal carcinoma patients versus 14.3% of the control group (p=0.001). The surrounding areas of positive tumors were negative in 52.4% of them. HPV-16 was significantly higher in tumors with higher expression of p53 (p=0.026). An inverse significant relationship was found between HPV-16 and DNA fragmentation in the laryngeal carcinoma group (p=0.022). HPV-18 was detected in only 2.4% of laryngeal carcinoma patients. p53 protein was expressed in 76.8% of the malignant group with significant increasing positivity with increasing stage of the disease (p=0.025). Non of the control group was p53-positive. Our results suggest that highly oncogenic types of HPV may play a role in the pathogenesis of laryngeal carcinoma through inactivation of wild-type p53 with subsequent decrease in apoptosis and by induction of p53 mutation, which itself can induce malignant transformation.
Alterations in Gene Expression Associated with Head and Neck Squamous Cell Carcinoma Development
MAGDALENA M. SEREWKO-AURET1, ALISON L. DAHLER1, LOUISE SMITH1, CHUNG FAI WONG1, CLAUDIA POPA1, LIAM M. BARNES1, WILLIAM COMAN2, NICHOLAS A. SAUNDERS1,3
1Epithelial Pathobiology Group, Cancer Biology Program, Centre For Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Queensland, Australia, 4102; 2Department of Surgery, University of Queensland, Princess Alexandra Hospital, Queensland, Australia, 4102; 3Department of Physiology and Pharmacology, University of Queensland, St Lucia, Queensland, Australia, 4076
Abstract: Background: Normal keratinocytes (KC) and neoplastic cells derived from a head and neck lesion (SCC-25) were grown as organotypic raft cultures to mimic in vivo architecture in the absence of contaminating cell types. Alterations in gene expression between normal keratinocytes and a head and neck squamous cell carcinoma (HNSSC) cell line (SCC-25) were analysed using gene arrays. Materials and Methods: RNA from the organotypic raft cultures were used to probe four gene arrays. Gene expression alterations between the normal and neoplastic cells were identified and analysed using both fold differences and 2-tailed t-test. Four genes from different functional groups were used for immunohistochemical staining of patient tumours to confirm the gene array data. Results: Statistical analysis of the array data revealed 124 significantly altered genes between normal and neoplastic HNSCC cells. These gene expression alterations are associated with a variety of different functional groups and indicate the complexity of gene de-regulation associated with HNSCC. Conclusion: This study identified many novel gene alterations associated with HNSCC. The significantly altered gene alterations belong in a variety functional groups including: growth control, apoptosis and detoxication and present new targets for investigating the molecular basis of HNSCC formation.
Real-time Quantitative PCR Arrays for Virally-associated Cancers
DIRK P. DITTMER, WOLFGANG VAHRSON, MICHELLE R. STAUDT, JAMES F. PAPIN, REBECCA HINES-BOYKIN, FARNAZ D. FAKHARI
Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, U.S.A.
Abstract: Virally-associated cancers are unique in that their origin is typically well defined and suitable to genomic analysis on a smaller scale. We recently reported the transcription profile of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in Kaposi's sarcoma (KS) using a real-time quantitative PCR (QPCR) array. This review explores the advantages and limitations of such an approach as well as the possibilities of extending PCR-based profiling to human cancers. Since real-time QPCR records a truly quantitative transcription profile, this technology will improve statistical analysis and solidify clinical decision-making.
Genomic Alterations in Gastrointestinal Stromal Tumors as Revealed by Conventional and Array-based Comparative Genomic Hybridization
SELENA R. GRANITTO1, PREMA KOLACHANA1, CRISTINA R. ANTONESCU2, ROBERT C. JOHNSON4, RONALD P. DEMATTEO3, SURESH C. JHANWAR1
Departments of 1
Medicine, 2Pathology and 3Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, N.Y. 10021, and 4Spectral Genomics, 8080 N. Stadium Drive, Houston, TX 77054, U.S.A.
Abstract: Gastrointestinal stromal tumor (GIST) is the most commonly occurring mesenchymal neoplasm of the gastrointestinal tract, accounting for 80 percent of these tumors. GIST is highly unresponsive to standard chemotherapy, particularly in patients with advanced or metastatic disease. Recent molecular studies have shown that activating c-kit (KIT) mutations are detectable in a large proportion (>75%) of tumors, between 78% (1) and 89% (2). Approximately 30% of tumors without an identifiable KIT mutation exhibit PDGFRA mutations (3). Furthermore, the KIT mutations are heterogeneous, some being known to confer a relatively better prognosis than others (1). Gross cytogenetic abnormalities associated with GIST appear to be similar regardless of whether a KIT mutation is identified. The molecular genetic alterations associated with multistep GIST tumorigenesis, particularly those which confer intrinsic or acquired resistance to both standard as well as targeted therapeutic approaches, however, are not fully recognized. As an initial approach to identify chromosomal sites of candidate gene(s), which may predict overall clinical and biologic behavior of GISTs, as they relate to response to the specific therapeutic drug Gleevec, we analyzed six GIST samples using both conventional as well as array-based Comparative Genomic Hybridization (CGH). The common abnormalities detected by CGH in low and high grade tumors included loss of all or part of chromosome 14; an entire chromosome 14 was lost in four tumor samples, with the remaining two samples exhibiting loss of the 14q22-q32.3 region. Other recurrent abnormalities included loss of the 1p chromosomal region (four tumor samples), loss of part or entire chromosome 9 (only in metastatic tumors), loss of chromosomes 15 and 22, and a gain of the chromosome 3q region in three samples each. Array- based CGH performed using human BAC arrays (1400V11 Spectral Genomics Chip™) on the other hand, not only detected recurrent abnormalities of the chromosomes and chromosomal sites mentioned above, but also identified losses of additional chromosomal sites on chromosomes 6q and 13q. Array-based CGH further delineated the regions of loss or gain to specific chromosome bands or sub bands based on location of chromosomal site-specific BACs. The specific regions of losses are located at 1p36.2-36.3,6q12,9p13-ter,13q33.33-q34, and gain or amplification of chromosomal DNA at bands 3q26-27. It is interesting to note that some of the chromosomal sites of losses and gains also harbor gene(s) such as AFAR, RIZ1, p73(1p35-36), Akt-1(14q32), p14 and p16(9p21), NF-2(22q12), ZASCI, p63 and PIK3CA oncogenes (3q26-27), all of which are known to play a major role in solid tumor pathogenesis. The role of these genes in GIST, however, remains to be seen. Thus, the results of our current study demonstrate that such a combined approach to detect global genomic alterations in GIST is significant in providing information related to chromosomal sites of potential tumor suppressor genes associated with multistep tumorigenesis; some of which also may be predictive of prognosis and clinical outcome following specific targeted therapy, which is the focus of future studies.
1Department of Otorhinolaryngology and 2Clinical Cooperation Group Molecular Oncology, Department of Head and Neck Research, Ludwig-Maximilians-University, D-81377 Munich, Germany
Abstract: Background: Tumor cells have developed several strategies to escape the immune system. One of these strategies consists of the secretion of immunosuppressive factors like interleukin-10 or prostaglandin E2 (PGE2), which impair the immune system. We have demonstrated recently that tumor-derived PGE2 down-regulates the expression of the integrin Mac-1 and the chemokine receptor CCR5 on primary monocytes, resulting in reduced adhesion and migration. Materials and Methods: In order to identify new target genes for tumor-derived factors in monocytes, we set up an in vitro system consisting of cDNA micro arrays and 2D gel electrophoresis. Results: We identified 25 genes that were differentially expressed upon incubation of cells in conditioned tumor cell supernatants as compared to cells incubated in cell culture medium. We describe in more detail that IL-1â secretion is induced by tumor supernatants and that IL-1â overexpression is also evident in monocytes from tumor patients in vivo, where expression correlates with the tumor stage. In addition, up-regulation of the plasminogen activator inhibitor-2, PAI-2, and down-regulation of the urokinase-type plasminogen activator receptor, uPAR, resulted in a reduced capability of monocytes to degrade and invade extracellular matrices. Conclusion: In summary, we describe interesting novel targets of soluble tumor-derived factors that are probably involved in the tumor-mediated immunosuppression commonly found in cancer patients.
LKW, a Putative Dual-specificity Kinase which is Down-regulated in Several Invasive Systems
STEPHANIE LOSCH1, MALTE BUCHHOLZ2, THOMAS M. GRESS2, ULRICH H. WEIDLE1
1Roche Diagnostics GmbH, Pharma Research, 82377 Penzberg; 2Department of Internal Medicine I, University Hospital, University of Ulm, 89081 Ulm, Germany
Abstract: Using Affymetrix GeneChip® arrays, we have established the transcriptional profiles of two sublines of the human pancreatic adenocarcinoma cell lines SUIT-2, S2-007 (metastatic) and S2-028 (non-metastatic). By comparison of ESTs corresponding to differentially regulated mRNAs, we have identified the putative dual-specificity kinase LKW as down-regulated in the metastatic cell line S2-007. LKW is composed of 358 amino acids and contains catalytic domains corresponding to those of Ser/Thr- and Tyr-kinases. The gene encoding LKW consists of four exons separated by three introns and is located on chromosome 20p12.2-p13. In parallel LKW has been identified in different experimental settings by other groups and is referred to as NIPK, SKIP3 and TRB3. We noticed that the metastatic propensity of two additional pancreatic cellular systems correlates with the down-regulation of mRNA levels corresponding to LKW. Evaluation of a panel of mammary carcinoma cell lines scored as non-invasive and invasive based on an in vitro invasion assay indicated down-regulation of LKW mRNA levels in the invasive cell lines. mRNA for LKW was shown to be overexpressed in pancreatic carcinomas compared to normal pancreas and tissues derived from patients with chronic pancreatitis as well as in colon, kidney, breast and ovarian carcinomas compared to their matching normal tissues. Analysis of colorectal carcinomas covering Duke's stages A, B, C and D revealed down-regulation of LKW mRNA in specimens corresponding to Duke's stages C and D, representing invasive and metastatic stages compared to the less invasive stages corresponding to Duke's stages A and B. Our results indicate that down-regulation of mRNA encoding LKW correlates with an invasive status of mammary carcinoma cell lines and the metastatic propensity of colorectal carcinoma.
