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Background: CDC4/FBXW7, encoding a ubiquitin ligase, maps to 4q32 and has been implicated as a tumor suppressor gene and therapeutic target in many tumor types. Mutations in colonic adenomas, and the frequent losses on 4q described in gastric cancer prompt speculation about the role of CDC4/FBXW7 in gastric carcinogenesis. Methods: We assessed the role of CDC4/FBXW7 in gastric cancer, through loss of heterozygosity (LOH) and multiplex ligation-dependent probe amplification (MLPA) on 47 flow-sorted gastric carcinomas including early-onset gastric cancers (EOGC) and xenografted conventional gastric carcinomas. Ploidy analysis was carried out on 39 EOGCs and immunohistochemistry of CDC4/FBXW7 and its substrates c-myc, c-jun, Notch and cyclin E was performed on 204 gastric carcinomas using tissue microarrays (TMAs). Sequence analysis of CDC4/FBXW7 was carried out on gastric carcinoma cell lines and xenografts. Results: Loss of heterozygosity of CDC4/FBXW7 occurred in 32% of EOGCs, and correlated with loss of expression in 26%. Loss of expression was frequent in both EOGC and conventional gastric cancers. No CDC4/FBXW7 mutations were found and loss of CDC4/FBXW7 did not correlate with ploidy status. There was a significant correlation between loss of CDC4/FBXW7 expression and upregulation of c-myc. Conclusions: Loss of CDC4/FBXW7 appears to play a role in both EOGC and conventional gastric carcinogenesis, and c-myc overexpression is likely to be an important oncogenic consequence of CDC4/FBXW7 loss.

ÖZET HEPATOSELÜLER KARSINOMDA ROL ALAN GENLERİN TANIMLANMASI: hCdc4 VE B-Raf GENLERİNİN DEĞERLENDİRİLMESİ Banu SÜRÜCÜ Yüksek Lisans Tezi, Moleküler Biyoloji ve Genetik Bölümü Tez Yöneticisi: Yard. Doç. Dr. Cengiz YAKICffiR Eylül 2003, 56 sayfa Karaciğer kanserinin başlıca türü olan hepatoselüler karsinom (HCC) dünya çapında yaygın kanserlerden biridir. HCC'nin oluşması ve gelişimi çok basamaklı bir süreç olup onkogenlerin aktivasyonunu ve/veya tümör baskılayıcı genlerin inaktivasyonunu gerektirir. Her ne kadar p53, aksin gibi tümör baskılayıcı genlerin inhibisyonu ve 0- katenin gibi onkogenlerin aktivasyonu gösterildiyse de hepatoselüler karsinomun moleküler mekanizması belirgin değildir. Bu nedenle, hepatokarsinogenezde rolü olan genlerin tanımlanması sadece bu sürecin anlaşılmasında değil aynı zamanda koruma veya tedavi için yeni stratejiler geliştirilmesinde de önemli bir basamaktır. Bu çalışmanın amacı hCdc4 ve B-Raf genlerinin HCC'deki olası rollerini değerlendirmektir. Kromozom 4q üzerinde sık görülen delesyonlar nedeniyle bu bölgede bulunan gen ya da genlerin HCC 'de rol alabileceği düşünülmektedir. Yakın zamanda bu bölgede bulunan bir gen olan hCdc4 klonlanmış ve olası bir tümör baskılayıcı gen olarak gösterilmiştir. hCdc4 bir F-box proteini olup siklin E'nin ubikütinlenmesinde dolayısıyla da yıkımında rol aldığı gösterilmiştir. Yıkımındaki bir bozukluk dolayısıyla olabilecek siklin E ekspresyon seviyesindeki artış, HCC'nin de içinde bulunduğu çeşitli kanserlerde sık görülen bir olaydır. 15 HCC hücre hattında hCdc4 geninin ekspresyon ve mutasyon çalışmaları RT-PCR ve bunu takiben PCR ürünlerinin direkt DNA dizi analizi yöntemleri kullanılarak incelenmiştir. Test edilen HCC hücre hatlarında mutasyon veya anormal transkript gözlemlenmemiştir. Bu bulgular hCdc4 genindeki değişimlerin hepatokarsinogenezde sık rastlanan bir olay olmadığına işaret etmektedir.RAF proteininin insan isoformlanndan biri olan B-RAF onkogenik RAS tarafından aktive edilerek hücrelerin büyüme faktörlerine yanıt vermesinde işlev görür.. B-Raf mutasyonlan birçok kanser türünde gösterilmiştir. RAS mutasyonları insan hepatokarsinogenezinde sık görülen bir olay olmasa da HCC örneklerinde az sayıda rapor edilmiştir. Bu nedenle onkogenik MAP kinaz yolağının aktivasy onunun B-RAF gibi başka bir bileşeni tarafından aktive edilmesi olasıdır. B-Raf mutasyonları HCC hücre hatları ve tümörlerinde araştırılmıştır ve aktive edici B-Raf mutasyonlan HCC'lerin %3'ünde (2/72) tanımlanmıştır. Bu bulgular B-Raf geninin, dolayısıyla bu yolağın hepatokarsinogenezde rol alabileceğini ancak ne B-Raf ne de ras geninin hepatokarsinogenezde rol oynayan başlıca genler olmadığını göstermektedir. Bu nedenle MAP kinaz yolağının diğer üyelerinin HCC 'deki mutasyonları değerlendirilmelidir. vı ABSTRACT IDENTIFICATION OF GENES INVOLVED IN HEPATOCELLULAR CARCINOMA; EVALUATION OF hCdc4 and B-Raf GENES Banu SÜRÜCÜ M.S. in Molecular Biology and Genetics Supervisor: Assist. Prof. Dr. Cengiz YAKICIER September 2003, 56 pages Hepatocellular carcinoma (HCC), the major type of primary liver cancer, is one of the most common cancers worldwide. Development and progression of HCC occur as a multistep process, requiring the activation of oncogenes and the inactivation of several tumor suppressor genes. Although, inactivation of tumor suppressor genes, including p53, Axin and activation of oncogene (3-catenin, have been shown to be involved in HCC development, the molecular mechanism of hepatocarcinogenesis is still unclear. The identification of additional genes that are involved in hepatocarcinogenesis is, not only, an important task to understand this process, but also for development of novel strategies for prevention or therapy. The aim of this study was to elucidate a possible function of hCdc4 and B-Raf genes in HCCs. A gene or genes on chromosome 4q have been implicated in hepatocarcinogenesis by the observation of frequent deletions of this region in HCCs. More recently, hCdc4, a gene in this region has been proposed as a candidate tumor suppressor gene. hCdc4 is an F-box protein which is shown to be involved in ubiquitination of cyclin E, thus targeting it for destruction. Cyclin E overexpression, is reported to be a frequent event in different cancers including HCC suggesting a problem in its destruction. 15 HCC cell lines were analysed for expression and mutation of hCdc4 gene by RT-PCR and direct sequencing of PCR products. No abnormal transcript and mutation observed in HCC cell lines tested. Our findings suggest that alteration of this gene is not a frequent event in hepatocarcinogenesis. inB-Raf, which is one of the human isoforms of RAF, is activated by oncogenic Ras (leading to cooperative effects in cells responding to growth factor signals). B-Raf mutations are found in a wide range of cancers. Eventhough, mutational activation of Ras is not a frequent event in human hepatocarcinogenesis, few Ras mutations were reported in HCC cases. Thus activation of oncogenic MAP kinase pathway by another component of this pathway such as BRAF is worth to analyze. HCC cell lines and tumours were searched for B-Raf mutations. Activating BRAF missense mutations were identified in 2/72 HCCs (3%). Our results suggest that B-Raf may be occasionally involved in hepatocarcinogenesis. Thus MAP kinase pathway might be involved in hepatocarcinogenesis but neither B-Raf nor Ras are the major players of this pathway in this event. For this reason, other members of this pathway should be evaluated for mutations in HCC. Keywords: HCC, hCdc4, B-Raf, oncogene, tumor suppressor gene, mutation. IV 56

hCDC4 (FBW7, FBXW7) is a new potential tumor suppressor gene which provides substrate specificity for SCF (Skp–Cullin–F-box) ubiquitin ligases and thereby regulates the degradation of potent oncogenes such as cyclin E, Myc, c-Jun and Notch. Mutations in the hCDC4 gene have been found in several solid tumors such as pancreas, colorectal or endometrial cancer. We carried out a mutation analysis of the hCDC4 gene in 35 samples of patients with Acute Myeloid Leukemia (AML) to elucidate a possible role of hCDC4 mutations in this disease. By direct DNA sequencing and digestion with Surveyor nuclease one heterozygous mutation in the 5' untranslated region of exon 1, transcript variant 3 was detected. Additionally, we could identify a new intronic SNP downstream of exon 10. The new variation was present in 20% of AML samples and was furthermore confirmed in a panel of 51 healthy individuals where it displayed a frequency of 14%. In conclusion we provide first data that in contrast to several solid tumors, mutations in the hCDC4 gene may not play a pivotal role in the pathogenesis of AML. Furthermore, we describe a new intronic polymorphism with high frequency in the intron sequence of the hCDC4 gene.