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Karaciğer kanseri en sık rastlanan birincil kanserlerden olup, yılda 600.000 civarında ölüme sebep olmaktadır. Şu andaki en etkili tedavi yöntemi karaciğer transplantasyonudur ve tümör ablasyon yöntemi sadece tümör yeterince küçük ise uygulanabilmektedir. Kanserli hücreler kemoterapi ve radyoterapi gibi yöntemlere oldukça dayanıklıdırlar. Dahası, karaciğer kanseri sürekli siroz üzerine geliştiği için karaciğerin diğer hepatik fonksiyonlarını yerine getirmesini de engellediği için, sitotoksik ajanlar kullanmak oldukça tehlikelidir. Bu nedenle karaciğer kanserini yenmek için daha yeni ve geçerli terapatik yöntemlere ihtiyac duyulmaktadır.Bizim amacımiz karaciğer kanserinde yeni hedeflenebilir genleri keşfetmektir. Başka bir değişle, biz bu çalışma ile karaciğer kanserinde yeni hedefler ve yeni mekanizmalar keşfederek bunları potansiyel terapötik hedefler olarak kullanmayı amaçladık.Tez çalışmalarım sırasında, yeni hedefler ve yeni mekanizmalar keşfetmek için değişik yöntemler kullanmaya çalıştım:1-Karaciğer kanserinde kanonik Wnt sinyallenme yolağının rolü: karaciğer kanserı hücrelerini hepatosit spesifik, epitel ve mezenkimal markörler kullanarak ?iyi diferansiye? ve ?kötü diferansiye? hücre hatları olarak iki gruba ayırdık. Kötü diferansiye hücre hatlarında epitel ve hepatosit spesifik markörlerin negatif hale geldiğini, mezenkimal markörleri anlatmaya başladığını gösterdik. Wnt sinyal yolağında görev alan 45 genin, gen anlatım seviyelerini iki grupta inceledik. 6 Frizzled almaç geninin ve wnt3 ligandının her iki grupta da anlatıldığını gösterdik. Kanonik Wnt ligandı wnt8b`nin sadece iyi diferansiye grupta anlatıldığını ve Wnt4, Wnt5a, Wnt5b and Wnt7b gibi kanonik olmayan ligandların kötü diferansiye gruba spesifik olduğunu gösterdik. Kanonik Wnt sinyallenmesinin sadece iyi diferansiye hücrelerde seçici olarak aktif olduğunu değil, aynı zamanda kötü diferansiye tümörlerde de baskılandığını gösterdik. Kanonik olmayan hücre hattı, Snu449 da mutant beta-kateninin ektopik anlatımının TCF aktivitesini arttıramaması bu gruptaki hücrelerde baskılayıcı bir mekanizma olduğuna işaret etti. Kanonik olmayan wnt5a genini aday olarak test ettik. Wnt5a geninin mutant beta-kateninin aktive ettiği TCF aktivitesini baskıladığını gösterdik. Bazı kanser tiplerinde, APC, Axin ve beta-katenin gibi genlerde oluşan mutasyonlarla Wnt-beta-katenin sinyallenmesinin, Wnt ligandlarının olmadığı ortamda anormal olarak aktifleşip, beta-kateninin sitoplazma ve çekirdekte birikmesine sebep olup hedef genlerin aktivasyonuna yol açtığı biliniyor. Bu nedenle beta-kateninin hücre zarı, sitoplazma ve çekirdekteki fraksiyonlarını tanıyabilen monoklonal antikorlar üretip, bu antikorları kanserli dokularda test ederek klinik değerlerini karakterize ettik.2-Kinaz ve fosfataz genlerini potansiyel terapatik hedef olarak sistematik olarak taranması: Karaciğer kanserinde değişik sinyallenme yolaklarının aktif hale geldiği ve bu hastalarda multikinaz inhibitorü olan sorafenib ilacının, ileri derece hastaların ömürlerini uzattığı biliniyor. Bu amaçla yeni hedefler bulmak ve hücre yaşlanması indüklenmesi yönünde terapatik hedef gösterebilmek için çok fazla sayıda kinaz ve fosfatazları hedef alan RNA müdehalesi yöntemi ile tarama gerçekleştirdik. Toplamda 7 kinaz ve 5 fosfataz genini güçlü adaylar olarak gösterildi.3-Seçilen epigenetik hedeflerin potansiyel terapötik hedef olarak taranması: Yakın dönemde yapılan çalışmalar, hücre yaşlanması ve bundan kaçışın kromatin üzerindeki epigenetik değişiklikler ile düzenlendiğini gösterdi. Bu amaçla hücre yaşlanması ve bundan kaçış ile uyumlu histon asetillenmesi ve metillenmesi değişiklikleri tanımladık, bundan sorumlu histon enzimlerini bulup, bunları ?hücre yaşlanması öncüleyici? hedefler (kanserli hücreleri hücre yaşlanmasına götüren) olarak göstermeyi hedefledik. ATAD2 genini epigenetik hedef olarak tanımlayıp, karaciğer kanserinde normal hepatositlere göre daha fazla gen ifadesi olduğunu gösterdik. ATAD2 proteinin öncü-neoplastik ve ilk aşama karaciğer kanserlerine göre ileri aşama kanserlerde kademeli artış olduğu gösterildi. Kanserli hücrelerde siRNA tekniği kullanarak, ATAD2 proteinini azaltıp ve buna bağlı global histon asetillenmesinin arttığını, hücre çoğalmasının azaldığını, hücreleri apoptoza götürdüğünü ve MRC5 akciğer fibroblastlarında ise hücre yaşlanmasını indüklediğini gösterdik. ATAD2 geninin potansiyel artırıcı (östrojen, androjen ve myc hedefleri üzerinde) etkisinden dolayı karaciğer kanserinde aday terapötik hedef olduğunu işaret etmektedir. Hepatocellular carcinoma (HCC) kills nearly 600.000 people each year and the only effective therapy for this cancer is liver transplantation or tumor ablation only when the tumor is small enough. These tumors are surprisingly resistant to conventional therapies such as chemotherapy and radiotherapy. Moreover, as HCC is almost always associated with cirrhosis, the treatment with cytotoxic agents is dangerous as they will also affect hepatic functions of the diseased liver. Therefore, there is urgent need to find novel therapeutic approaches against HCC in order to diminish death toll. Our overall goal is to discover ?druggable target genes? in HCC. In other words, we wish to identify novel genes and novel mechanisms involved in these cancers in order to use them as potential therapeutic targets.During my thesis work, I developed different approaches to find new mechanisms and novel targets:1- Deciphering the role of canonical Wnt signaling in HCC: We classified human HCC cell lines into `well-differentiated` and `poorly differentiated` subtypes, based on the expression of hepatocyte lineage, epithelial and mesenchymal markers. Poorly differentiated cell lines lost epithelial and hepatocyte lineage markers, and overexpressed mesenchymal markers. Also, they were highly motile and invasive. We compared the expression of 45 Wnt pathway genes between two subtypes. Likewise, six Frizzled receptors, and canonical Wnt3 ligand were expressed in both subtypes. In contrast, canonical ligand Wnt8b and noncanonical ligands Wnt4, Wnt5a, Wnt5b and Wnt7b were expressed selectively in well- and poorly differentiated cell lines, respectively. Canonical Wnt signaling activity, as tested by a TCF reporter assay was detected in 80% of well-differentiated, contrary to 14% of poorly differentiated cell lines. TCF activity generated by ectopic mutant b-catenin was weak in poorly differentiated SNU449 cell line, suggesting a repressive mechanism. We tested Wnt5a as a candidate antagonist. It strongly inhibited canonical Wnt signaling that is activated by mutant b-catenin in HCC cell lines.2. Systematic screening of protein kinases and phosphatases as potential therapeutic targets: There is evidence of aberrant activation of several signaling cascades in HCC, and a multikinase inhibitor, sorafenib, has shown survival benefits in patients with advanced HCC. We used siRNAs to screen a large number of kinases and phosphatases to identify related genes involved in HCC cell survival. A total of 7 kinases and 5 phosphatases were identified as strong candidate targets.3-Screening of a set of selected epigenetic regulators as potential therapeutic targets: Recent studies have indicated that senescence arrest or senescence escape could be regulated by epigenetic changes on chromatin. We wanted to identify key histone methylation and acetylation changes associated with senescence or senescence escape, and select key histone modifying enzymes, as potential targets for ?pro-senescence? interventions (therapeutic interventions that allow senescence induction in cancer cells). We identified ATAD2 as an epigenetic target, and found ATAD2 gene overexpressed in HCC compared to normal liver. We also found a stepwise increase of ATAD2 protein expression in late stages with respect to pre-neoplastic and early stage during hepatocellular carcinogenesis. ATAD2 knockdown using siRNAs in cancer cells leads to increase in global histone acetylation and inhibit cell proliferation and induce caspase-3 dependent apoptosis in HCC and induce senescence in MRC5 cells. Its potentiator role (coactivator role with estrogen, androgen and myc targets) indicates ATAD2 as a potential therepeutic target for HCC. 218

Purpose Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide with. lack of effective systemic chemotherapy. In this study, we aimed to evaluate the value of ATPase family AAA domain-containing protein 2 (ATAD2) as a biomarker and potential therapeutic target for HCC. Methods The expression of ATAD2 was tested in different HCC patient cohorts by immunohistochemistry and comparative transcriptional analysis. The co-expression of ATAD2 and proliferation markers was compared during liver regeneration and malignancy with different bioinformatics tools. The cellular effects of ATAD2 inactivation in liver malignancy was tested on cell cycle, apoptosis and colony formation ability as well as tumor formation using RNA interference. The genes affected by ATAD2 inactivation in three different HCC cell lines were identified by global gene expression profiling and bioinformatics tools. Results ATAD2 is overexpression is closely correlated with HCC tumor stage. There was gradual increase from dysplasia, well differentiated and poorly differentiated HCC, respectively. We also observed transient upregulation of ATAD2 expression during rat liver regeneration in parallel to changes in Ki-67 expression. ATAD2 knockdown resulted in apoptosis and decreased cell survival in vitro and decreased tumor formation in some HCC cell lines. However, three other HCC cell lines tested where not affected. Similarly, gene expression response to ATAD2 inactivation in different HCC cell lines was highly heterogeneous. Conclusions ATAD2 is a potential proliferation marker for liver regeneration and HCC. It may also serve as a therapeutic target despite heterogeneous response of malignant cells.

Cataloged from PDF version of article. Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths. Aflatoxins, which may play a causative role in 5-28% of HCCs worldwide, are activated in liver cells and induce principally G→T mutations, including the TP53 codon 249(G→T) hotspot mutation. The DNA damage checkpoint response acts as an antitumour mechanism against genotoxic agents, but its role in aflatoxin-induced DNA damage is unknown. Aim: We studied the DNA damage checkpoint response of human cells to aflatoxin B1 (AFB1). Methods and results: The treatment of HepG2 hepatoma cells with mutation-inducing doses (3-5μmol/l) of AFB1 induced DNA adducts, 8-hydroxyguanine lesions and DNA strand breaks that lasted several days. Persistent phospho-H2AX and 53BP1 foci were also detected, but cell growth was not affected. AFB1-exposed HepG2 cells formed phospho-H2AX and 53BP1 foci, but failed to phosphorylate both Chk1 and Chk2. Huh7 hepatoma and HCT116 colorectal cancer cell lines also exhibited a similarly incomplete checkpoint response. p53 phosphorylation also failed, and AFB1-exposed cells did not show p53-dependent G1 arrest or a sustained G2/M arrest. These observations contrasted sharply with the fully functional DNA damage response of cells to Adriamycin. Cotreatment of cells with AFB1 did not inhibit p53 and p21Cip1 accumulation induced by Adriamycin. Thus, the deficient checkpoint response to AFB1 was not due to an inhibitory effect, but could be explained by an inefficient activation. Conclusion: Genotoxic doses of AFB1 induce an incomplete and inefficient checkpoint response in human cells. This defective response may contribute to the mutagenic and carcinogenic potencies of aflatoxins. © 2011 John Wiley & Sons A/S.

BACKGROUND: Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, "normal-like", and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity. METHODOLOGY/PRINCIPAL FINDINGS: A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21(Cip1) correlated with senescence in these cell lines. p21(Cip1) knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some ICP cell lines generated only CD44+/CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and "normal-like" tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice. CONCLUSIONS/SIGNIFICANCE: Luminal A and "normal-like" breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential.