Proteomics

Proteomics

Gynecologic Oncology 88, S22–S24 (2003) doi:10.1006/gyno.2002.6678 Discussion: Somatic Genetics and Signaling/Genomics/Proteomics Sir Walter Bodmer,*...

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Gynecologic Oncology 88, S22–S24 (2003) doi:10.1006/gyno.2002.6678

Discussion: Somatic Genetics and Signaling/Genomics/Proteomics Sir Walter Bodmer,* Edison Liu, M.D.,† Joseph Gray, Ph.D.,‡ Karen Rodland, Ph.D.,§ David Smith, Ph.D., ¶ Benjamin Tsang, Ph.D.,㛳 and Georgia C. Trench, Ph.D.** *University of Oxford; †Genome Institute of Singapore; ‡University of California at Los Angeles; §Oregon Health Science University; ¶ Mayo Foundation; 㛳University of Ottawa; and **Queensland Institute of Medical Research

Progress has been made in defining the primary genetic events that lead to the development of cancers at several sites. These include gene amplification, deletion, mutation, and methylation. The phenotype of cancers must be determined by these genetic changes and by the basic properties of the tissue from which a cancer is derived. Use of expression arrays and proteomics should facilitate the identification of genes responsible for malignant transformation. Ultimately, if these fundamental genetic changes are understood within individual cancers, it should be possible to predict a patient’s clinical course and their response to treatment. In contrast to several other forms of cancer, relatively few specific mutations have been defined in ovarian cancers and there is not yet a well-defined sequence of genetic events in ovarian oncogenesis. p53 and PTEN are frequently mutated. Components of the PI3 kinase/AKT signaling pathway are frequently amplified. HER-2 can be amplified and overexpressed in a small fraction of cases. RAS can be mutated, particularly in mucinous ovarian cancers. Expression of the functional allele of imprinted genes such as ARHI and LOT1 can be lost by multiple mechanisms including loss of heterozygosity (LOH). A number of genes, such as the cadherins, are excellent candidates for regulating growth and determining progression of ovarian cancer, but have not received adequate attention. LOH AND TUMOR SUPPRESSOR GENES IN OVARIAN CANCER Dr. Georgia Trench addressed the discrepancy between sites of LOH and the detection of mutations in putative suppressor genes for ovarian cancer. In colon cancer and pancreatic cancer a good correlation has been found between LOH and mutations in tumor suppressor genes. Both p53 and APC had initially been located through studies of LOH. It is unlikely that ovarian cancer will, in the long run, be an exception to this general rule. However, few, if any, tumors suppressor genes have been identified in ovarian cancer using sites of LOH without assistance from hereditary syndromes. We do not yet understand whether the many sites of LOH are critical events in carcinogenesis or simply a background indication of genomic instability. With chromosome transfer, several relatively small 0090-8258/03 $35.00 © 2003 Elsevier Science (USA) All rights reserved.

functional regions on chromosones 6, 11, and 22 have been defined that seem to suppress tumor growth. We are still dominated by the Knudson “2 hit” model where the second hit occurs through an inactivating mutation. Instead of inactivating mutations, methylation may be important for silencing tumor suppressor genes, providing a second hit. Alternatively, haploinsufficiency may permit tumor progression so that a second hit is not required. Edison Liu’s presentation suggested that down-regulated genes clustered about certain chromosomal locations rather than within particular pathways. The critical factor in the genesis of ovarian cancer may be slight downregulation or haploinsufficiency of several linked genes, rather than complete inactivation of a single gene. Indeed, DNA copy number abnormalities and transcript levels are relatively well correlated. Subtle differences in expression may prove to be important. Transfection studies are not an effective way to evaluate such subtle changes in gene expression. Sir Walter Bodmer raised the interesting question of how much of the observed LOH is related to the severe aneuploidy very commonly observed in advanced ovarian cancers associated with tumor progression. In colorectal cancer, LOH may have been more informative due to the importance of mismatch repair defects in 15–20% of colorectal cancers, providing a near diploid karyotype in these cancers. Due to the “noise” produced by random genetic change, studies of LOH have proven difficult in ovarian cancer. Many tumors may have undergone telomeric crisis resulting in a markedly aberrant genome. In the absence of selection of specific aberrations, LOH may not identify critically important regions. Dr. Gray felt that despite the loss of chromosomal segments due to dramatic genetic instability in ovarian cancer, the observation of LOH at the same site in different tumors may still mark sites for likely candidates. Given a limited number of useful polymorphisms, mapping of LOH often depends on studies with relatively few tumors. Dozens of events will be required to define appropriate sites. Moreover, these sites must be linked to mutation or silencing of the contralateral allele to permit positional cloning. Alternative technologies such as array comparative genomic hybridization (CGH) may provide a global method for mapping areas of both heterozygous and homozygous loss. Multiple candidate genes may be found at a site of

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LOH. Systematic evaluation of each gene for mutations can point to putative tumor suppressors. Dr. Gordon Mills suggested that there are many examples in the genetic literature where loss of one gene copied has had functional significance. Sir Walter pointed out that a majority of genes may require two active copies for full function. For those genes regulated by methylation, silencing of the initial copy must provide a selective advantage. In this setting, haploinsufficiency must provide a selective force. Dr. Gray pointed out that twofold changes in p53 and bcl-2 levels can be critically important in regulating apoptosis. Dr. Tom Hamilton added that uniparental disomy of normally imprinted genes can be associated with developmental defects and mental retardation, presumably related to a twofold difference in expression. Dr. Trench cited examples with murine systems where haploinsufficiency of certain genes predisposed animals to the development of cancers, but did not know of a comparable example in human cancers. Sir Walter pointed to the Li– Fraumeni and FAP syndromes where haploinsufficiency was observed in the germline. In the cancers that arise in these families, second hits are observed. GENE AMPLIFICATION IN OVARIAN CANCER Dr. Mills questioned why a modest increase in copy number should dramatically affect cell function, given multiple mechanisms of transcriptional, translational, and posttranslational regulation of gene expression and protein function. Moreover, why is only modest amplification of some genes observed, rather than the marked amplification of myc and HER-2neu? Dr. Gray suggested that cells may not tolerate marked amplification of growth regulatory genes. Growth of amplicons may be limited by the inclusion of linked genes whose overexpression is not compatible with survival. Future studies in murine systems should focus on modest changes in copy number, rather than on marked overexpression or total deletion of genes. Dr. David Smith suggested that amplicons might map to areas of genomic instability such as “fragile sites.” With regard to amplified genes, Dr. Agamemnon Epenetos had asked whether myc might be an appropriate target for prevention or therapy. The gene is amplified in as many as 70% of ovarian cancers, but overexpressed in 30%. Further, amplification of myc is a very strong prognostic indicator. Dr. Gray pointed out that to date only anti-sense strategies are available, but in the future new approaches might be developed. Dr. Nina Einhorn had studied CGH in borderline and invasive ovarian cancers. Regional amplification occurred 13 times more frequently in borderline tumors than in invasive cancers. Dr. Gray had asked how often changes in borderline cancers matched changes found in invasive neoplasms. Additional studies to compare the changes in benign lesions, borderline tumors, and invasive ovarian cancers may provide important insights into the etiology of these diseases.

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TRANSCRIPTIONAL PROFILING OF OVARIAN CANCER Dr. Smith discussed transcriptional profiling of ovarian cancers performed in collaboration with Dr. Viji Shridar at the Mayo Clinic and investigators at M. D. Anderson Cancer Center and Millennium Pharmaceuticals. Expression analysis was conducted with cDNA arrays on nylon membranes that included 25,000 genes and ESTs. In addition, 4000 clones were sequenced from subtraction suppression hybridization libraries. When ovarian cancers were compared to epithelial cells scraped from normal ovaries, some 12,000 genes demonstrated a twofold increase or decrease in expression. More downregulated genes were observed than upregulated genes. Similar patterns of abnormalities were observed in early- and late-stage ovarian cancers, although a smaller fraction of early-stage cancers had abnormalities. Even when fivefold differences in expression were analyzed, similar abnormalities were observed in early- and late-stage high-grade cancers. Thus, early-stage, high-grade cancers do not appear to have a different spectrum of genetic changes than late-stage, high-grade cancers. Of importance, when PCR or Northern blot analysis was performed, results with arrays could be confirmed with only 70% of the genes that were thought to be up- or down-regulated. Dr. Gray suggested that the stringency of hybridization may be important in determining the apparent degree of up- or downregulation. Dr. Andrew Berchuck asked Dr. Liu whether expression profiling might aid in predicting penetrance of mutant BRCA1 and BRCA2 genes. Dr. Liu pointed out that distinguishing sporadic cancers from cancers that arise in women with BRCA1 and BRCA2 abnormalities was difficult in that profiles overlapped in a significant fraction of cases. Similarly, there is overlap in the gene expression profiles in the ovarian cancers between patients with germline BRCA1 and BRCA2 mutations. This overlap precludes the definitive use of arrays as a diagnostic. PROTEOMIC ANALYSIS OF OVARIAN CANCERS Dr. Karen Rodland characterized the proteomic profile of ovarian cancers based on her studies at the Pacific Northwest National Laboratory in Oregon. Proteins were resolved with multidimensional liquid chromatography and reverse phase liquid chromatography rather than with 2D gels before analysis with mass spectroscopy. 2D gels are limited in detecting lowprevalence proteins and all proteins of high molecular weight. Highly basic and highly acidic proteins are also underrepresented on 2D gels. Plasma membrane proteins were studied in SKOv3 and CAOv3 ovarian cancer cells that exhibited different growth characteristics in heterograft models. Fourteen proteins were identified, including a number of integral membrane protein tyrosine kinase receptors. Direct quantitative information cannot be obtained by this technique. Methods for quan-

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titative mass spectroscopy are being developed using a deuterium-labeled linker to cysteine residues in native proteins. These methods may permit comparison of data obtained with CGH, expression arrays, and proteomics. Protein–protein interactions are also being studied by immunoprecipitation and matrix-assisted laser desorption and ionization time of flight mass spectroscopy. Sir Walter pointed out that molecules such as cadherins and cateinins would not have been detected among the membrane proteins but were seen to be up-regulated on the expression arrays. APOPTOTIC SIGNALING IN OVARIAN CANCER Dr. Ben Tsang reviewed his studies of cancer cell survival and chemoresistance that depend upon the XIAP (CIAP1) protein. XIAP inhibits a number of caspases including caspases

3 and 9, thus affecting both membrane- and mitochondrialmediated apoptosis. Cisplatin can decrease XIAP levels in cancer cells that are sensitive to this drug. Down-regulation of XIAP in cisplatin-resistant cells increases sensitivity to this agent in the presence of wild-type p53, but not in the presence of mutant p53. Transfection of wild-type p53 and down-regulation of XIAP restore cisplatin sensitivity. XIAP can also enhance phosphorylation of AKT by mechanisms that remain to be studied. Caspase-mediated cleavage and ubiquitin-mediated proteolysis of XIAP are likely to be important determinants of apoptosis. For molecular therapeutics, use of antisense or dominant negative mutants may provide proof of concept for XIAP modulation in potentiating chemotherapy. Sir Walter suggested that an examination of mutations in the caspase pathway might aid in the understanding of drug resistance.