Browsing by Subject "Carcinogenesis."

Now showing 1 - 10 of 10

Open Access
Characterization of FAM134B in the context of hepatocellular carcinoma and endoplasmic reticulum protein stress
(2011) Yılmaz, Mustafa
Family with sequence similarity 134, member B (FAM134B) is a replicative senescence associated gene, previously identified in studies of our group as a result of microarray analysis in spontaneously senescent clones of Huh7 hepatocellular carcinoma cell line and their immortal counterparts. Originating from this finding, this study primarily focused on characterization of FAM134B in the context of hepatocellular carcinoma and endoplasmic reticulum stress. At the beginning, the relationship between senescence and FAM134B was experimented by inducing premature senescence in Huh7 cells. Adriamycin or TGF-β induced premature senescence did not result in amplification of FAM134B gene expression, suggesting that upregulation of FAM134B expression in spontaneous replicative senescence is not directly associated with a senescence phenotype. Then, FAM134B mRNA and protein levels were analyzed in both well- and poorly-differentiated HCC cell lines. Results showed that FAM134B expression is greater in poorly-differentiated cell lines, which represent advanced and metastatic HCC in vitro. On the other hand, our studies on the relationship between FAM134B and endoplasmic reticulum (ER) stress showed that FAM134B is an ER stress response gene, whose expression is upregulated by induction of ER stress with chemicals, such as thapsigargin, tunicamycin or DTT. Therefore, high protein and mRNA levels of FAM134B in poorly-differentiated cell lines are linked to the presence of a basal level ER stress response in this group of cell lines. Furthermore, overexpression studies in Huh7 cells indicated that FAM134B cannot trigger an ER stress response or autophagic response in these cells. However, FAM134B was detected as an effector in cellular response, when ER stress is artificially induced by thapsigargin or tunicamycin treatments. FAM13B4 overexpression in Huh7 resulted in increased sensitivity to thapsigargin or tunicamycin induced apoptosis. Moreover, increased FAM134B expression was also associated with decreased proliferative capacity in response to ER stress induction with the same chemicals. Consequently, FAM134B was suggested to affect the severity of stress in the ER when ER stress is started with an inducer. In addition, our tissue based experiments revealed that FAM134B is expressed in the brain and liver. Taken together, FAM134B might be an important protein contributing to the liver tissue damage and pathogenesis of HCC.
Open Access
Characterization of novel monoclonal antibodies that target proteins differentially expressed in hepatocellular carcinoma : a proteomics approach
(2011) Öztaş, Emin
Hepatocellular carcinoma (HCC) is the sixth common cancer in the world. Because of the late diagnosis of the disease, survival rates are still poor in the HCC patients. Surveillance strategies have to be developed in populations with high risk groups having premalignant diseases for HCC, such as liver cirrhosis. The usage of serum and histology-based biomarkers assists health professionals to evaluate the patients. Despite of the advances in diagnostic methods, there is still a need to develop novel biomarkers for early detection of HCC. Therefore, we aimed to develop new biomarkers with higher sensitivity and specificity for HCC to improve the surveillance of the patients. Using an apoptotic HCC cell line, HUH7, and SIP1 proteins, we generated novel monoclonal antibodies (mAbs). 6D5, 1C6 and 6E5 hybridoma clones were chosen for characterization studies because of their strong reactivity in cell-ELISA assays. We found differential reactivity pattern for those novel mAbs in a panel of human sections consisting of tumors, benign liver diseases, normal tissues and a variety of cell lines. Using proteomics methods, we identified candidate target proteins for the 6D5 mAb. Better characterization of these target proteins will provide a better understanding of the molecular pathways in the HCC and aid in the research for developing newer therapeutic agents. In conclusion, our candidate biomarker mAbs can be used in the early diagnosis of HCC as well as in drug development studies.
Open Access
Genetic analysis of Smad4 gene in TGF-Beta signalling pathway in human liver cancer
(1998) Irmak, Meliha Burcu
HCC is a multistep genetic disease in which many genomic changes occur as a result of uncontrolled proliferation of hepatocytes. Molecular events leading to HCC is still unclear. Until now, neither an oncogene nor a tumor suppressor gene has been shown to be prefentially altered in HCC. Genetic alterations other than p53, pl6, BRCA2 (Breast Carcinoma Associated Protein), M6P/IGFIIR (lyiannose 6 Phosphate/ Insulin Like Growth Factor II Receptor), Rb (Retinoblastoma), PRLTS (Platelet Derived Growth Factor Receptor-|3-Like Tumor Suppressor Gene), and Tg737 (Candidate polycyctic kidney disease gene) genes remain unknown. TGF-P is a strong inhibitor of hepatocyte proliferation. In HCC and cirrhosis increased levels of TGF-P is observed, so this shows that the presence of high levels of TGF-3 does not avoid hepatocyte proliferation. Thus, there may be a disruption in the signalling pathway of TGF-p. The common mediator Smad4 gene, which is among the genes located in TGF-P signalling pathway, is found to be mutated in many cancer types. We decided to do the mutational analysis of Smad4 gene, which is located in the signalling pathway of the hepatocyte antiproliferative factor, TGF-p. Exons 8, 9, 10, and 11 which are in MH2 region, and exon 2 which is in MHl region of Smad4 is mutationally analysed by SSCP for 35 HCC cases. In the 35 HCC tumors, 5 alterations were observed (14%), 3 of them being in exon 8, one of them being in exon 9a, and the last one being in exon 10 of Smad4 gene. In the samples we tested, no big deletions were observed, but the alterations observed are probably single base changes. Also HCC cell lines namely, HepG2, Hep3B, Huh-7, FOCUS, Mahlavu, and PLC/PRF/5 were checked for their mutations and cell lines other then PLC/PRF/5 were analysed for their mRNA transcription. There were no big deletions or alterations in Nand C- terminals of the cell lines and we have shown mRNA transcription for all cell lines except Hep3B in which PCR has revealed very weak amplification. Our results suggest that Smad4 might be involved in at least a part of primary HCC tumor development.
Open Access
Genetic and epigenetic evaluation of the candidate genes in human hepatocellular carcinomas
(2010) Acun, Tolga
Hepatocellular carcinoma (HCC) is the fifth most-common cancer and the third most common cause of cancer related mortality worldwide. HCC is also the most common type of liver cancer. Hepatocarcinogenesis is a multistep process that is not completely understood until today. In this study, we genetically and epigenetically evaluated candidate genes and molecular pathways which may act in hepatocarcinogenesis. The RAS/RAF/MAPK pathway was genetically investigated and no mutation was described in HCC cell lines for the genes MEK1 (MAP2K1), MEK2 (MAP2K2), ERK1 (MAPK3), ERK2 (MAPK1) and PTP 11 (SHP2). TP53 pathway is also a common target for inactivation during liver carcinogenesis. Our analysis indicated that the presence of the MDM2-SNP309 G allele is inversely associated with the presence of somatic TP53 mutations. This finding suggests that the MDM2-SNP309 G allele may functionally replace TP53 mutations, and in addition to known etiological factors, may be partly responsible for differential HCC prevalence. Epigenetic silencing of SIP1 gene in HCC together with its reduced mRNA and protein level in tumors relative to normal liver tissue indicated SIP1 as a potential tumor suppressor role. Inconsistent with previously published findings in other types of cancers, our results showed for the first time that PTPRD gene is epigenetically downregulated and mutated in liver cancers. Among other candidates our results suggest; FBXL11, TUBA3C, TPTE2, IQSEC1, MIPOL1, CHUK, MCL1, MAGI-2 and PTPRCAP genes are involved in hepatocarcinogenesis.
Open Access
Genetic and epigenetic targets in liver cancer
(2010) Yüzügüllü, Haluk
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 welldifferentiated, contrary to 14% of poorly differentiated cell lines. TCF activity generated by ectopic mutant β-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 β-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.
Open Access
Histone methylation and methyltransferase enzyme Set8 in hepatocellular carcinoma
(2010) Harputlugil, Eylül
Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers worldwide. The epigenetic modifications, which are involved in virtually all cellular processes are also involved in the carcinogenic process, and this is a growing new field of investigation. HCC has also been associated with several epigenetic aberrations which include the ones in histone modifications, histone methyltransferase enzymes, and the epigenetic machinery. The transition from cirrhosis to HCC is related to senescence bypass, and the distinctions between senescence and immortality in HCC cell lines. Global levels of H3K4me3, H3K9me3, H3K27me3, H3K36me3, H3R2me2, H3R17me2 and H4K20me3 histone marks were evaluated in well-differentiated and poorly differentiated HCC cell lines in the presence and absence of TGF-β induced senescence. No prominent changes in the levels of these histone modifications were indentified in response to TGF-β induced senescence. However, H4K20me3 levels appeared to correlate with the differentiation status of the cell lines, where a loss of methylation was observed in poorly differentiated cell lines. In order to address the mechanism of this loss, H4K20 specific methyltransferases were analyzed in terms of their transcript levels, and only the expression pattern of monomethyl transferase Set8 was found to correlate with the H4K20me3 methylation patterns. A potential role played by Set8 in HCC development was investigated via overexpression and knockdown studies. But no significant role could be attributed to this enzyme in this study.
Open Access
Molecular mechanisms of senescence response to transforming growth factor-beta in liver cancer
(2010) Şentürk, Şerif
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. HCC is associated with several etiological factors including infections with hepatitis B and C viruses, heavy alcohol consumption and chronic aflatoxin B1 exposure. Due to its multi-step disease hallmark characterized with genetic heterogeneity, liver cancer has very limited therapeutic options. In light of many previous findings, cellular senescence acts as a barrier against immortalization and prohibits the proliferation of premalignant cells in various tumors including HCCs. However, implications of this anti-tumor mechanism in hepatic tissues are not wellknown. TGF-β is a multifunctional cytokine implicated in diverse cellular processes including senescence arrest as well as liver physiology and pathophysiology. Although TGF-β-induced senescence has been described in different cell types, this issue has never been addressed for hepatic cells. According to our recent data, TGF- β1 expression pattern in various HCC malignancies closely correlated with reported frequencies of SABG activities in these corresponding disease stages. Therefore, we hypothesized that TGF-β signaling might play key role in hepatocellular senescence. Well-differentiated (WD) five cell lines characterized with epithelial-like morphology displayed TGF-β-induced growth inhibition associated with SABG activity, with lack of evidence of apoptosis induction. Even a brief exposure to TGF- β was sufficient to trigger a massive senescence response. Senescence arrest in WD cell lines was linked to c-myc down-regulation and a reciprocal increase in p21Cip1 and p15Ink4b protein levels. In addition, TGF-β-induced senescence was correlated with Nox4 induction, intracellular accumulation of reactive oxygen species (ROS) and sustained 53BP1 foci formation as a mark of DNA-damage response. Moreover, intratumoral injection of TGF-β in human HCC tumors, generated subcutaneously in immunodeficient mice, induced expanded SABG that was associated with a strong anti-tumor response activity. On the other hand, poorly differentiated (PD) HCC cell lines with mesenchymal-like characteristics appeared to be resistant to TGF−β-induced senescence. However, PD cell lines had intact TGF-β signaling from cell membrane to nucleus. Resistance of PD cell lines was partially due to zeb2 overexpression, homozygous p15Ink4b deletion and lack of pRb expression. Besides, PD cells did not display Nox4 upregulation and also lacked ROS accumulation upon TGF-β stimulation. In addition, we demonstrated that sustained exposure to TGF-β established resistant Huh7 subclone. The resistance was partially attributed to deregulated Smad signaling, permanent epithelial-mesenchymal transition-like transformation. Surprisingly enough, removal of TGF-β from culture medium of continuously treated Huh7 subclone did not resolve the resistance phenotype in the rescued subclone. Epigenetic regulations mainly histone modifications are considered as candidate mechanisms responsible for irreversible TGF-β-resistance and maintenance of mesenchymal-like phenotype. Taken together, our results establish a close link between senescence arrest and anti-tumor activity of TGF-β signaling pathway in WD cell lines by delineating the mechanisms underlying TGF-β-induced growth arrest. Moreover, we propose partial explanation for the resistance to TGF-β- mediated growth arrest in PD cell lines and thoroughly signify the potential mechanisms of acquired resistance to TGF-β in continuously treated cultures. Further studies to enlighten our knowledge about implications of TGF-β signaling in less differentiated HCCs are necessary. As a conclusion, we identify TGF-β signaling as a potent therapeutic option for well-differentiated early HCCs.
Open Access
Role of dietary ethiological factors in the molecular pathogenesis of liver cancer
(2011) Gürsoy Yüzügüllü, Şehriban Özge
Hepatocellular carcinoma is ranked third foremost cause of cancer deaths. Dietary factors play a crucial role in the molecular pathogenesis of liver cancer. Oxidative stress is usually coupled with the malignancy and progression of HCC since it is considered as a common factor during inflammation after chronic viral infection. Chemical stress caused by aflatoxin exposure, metabolic stress produced by alcohol abuse and selenium deficiency as a risk factor for HCC are associated with oxidative stress. It should be eliminated with an intact antioxidant defense mechanism. It is a major cause of genotoxicity endogenously through metabolic stress and exogenously produced by chemical and physical carcinogens. Even though the contribution of dietary factors in HCC progression has been established, the underlying molecular mechanism has not been fully understood. Cancer cells may respond to genotoxic stress with a cryptic development of survival advantage mechanisms. Therefore we wanted to investigate this idea with dietary factors involved in liver cancer. In this work, we studied the implication of Se-deficiency in tumorigenesis of hepatocytes and the mechanism underlying the selective selection of aflatoxins for p53-249 mutation in HCC. Aflatoxins are the most potent naturally occurring carcinogens and may play a causative role in 5-28% of hepatocellular carcinomas, worldwide. Aflatoxins are activated in liver cells and induce principally G- >T mutations, including a codon 249 (G->T) hotspot mutation of TP53 gene that is specifically associated with aflatoxin-related hepatocellular carcinoma. However, our comparative analysis showed that R249S does not provide survival advantage at heterozygous state. Thus, the selection could be at the mutation induction stage. The lack of p53 activation in Aflatoksin B1 exposed HCC cells led us to test DNA damage response after aflatoxin exposure. Unexpectedly, DNA damage checkpoint response to aflatoxins has not been studied thoroughly before. Although, DNA damage checkpoint response acts as an anti-tumor mechanism by protecting genome integrity against genotoxic agents, this highly critical aspect of aflatoxin carcinogenicity is poorly known. Our findings provide evidence for the contribution of ERK, p38MAPK and PI3K/Akt survival pathways under selenium supplementation in some HCC cell lines. Apart from the effect of selenium deficiency, our results enlighten the aflatoxin carcinogenicity in vitro. Our study pointed out for a negligent G1 and G2/M checkpoint response to aflatoxin B1-induced DNA damage. This defective response may account mostly for mutagenic and carcinogenic influences of aflatoxins. It may also associate with the frequent induction of TP53 hotspot mutation in aflatoxin-related human HCC.
Open Access
The role of FLT3 in hepatocellular carcinogenesis
(2010) Bayın, N. Sumru
Hepatocellular carcinoma (HCC) is one of the most prevalent cancer types and it has a high mortality rate. Its high incidence is a consequence of lack of biomarkers that could track the progression of the disease. Identification of a marker, which involves in different stages of cancer progression, through fibrosis to HCC, would be a good candidate for diagnosis, prediction of prognosis and targeted therapies. Therefore we decided to identify a novel marker for HCCs, to overcome these consequences. Previously our group has shown that oval cell marker FLT3, a known hematopoetic stem cell marker and which is known to be constitutively active in many of the leukemias, has a role in liver regeneration. Also our immunohistochemical analysis of cirrhotic liver tissues have shown that FLT3 is expressed in liver injury. Therefore, we decided to analyze the role of FLT3 in hepatocellular carcinogenesis. Expression analysis of FLT3 on mRNA and protein level and the expression analysis of adult stem cell, cancer stem cell, and epithelial and mesenchymal lineage markers on mRNA level in 14 HCC cell lines (HepG2, Hep3B, Hep40, Huh7, PLC/PRF/5, Mahlavu, Focus, Sk-Hep-1, Snu182, Snu387, Snu398, Snu423, Snu449, Snu475) was performed. Four of these cell lines (Snu182, Snu398, Huh7 and Hep40) were chosen due to their different expression levels of FLT3 and the functional role of FLT3 in HCCs was assessed by blocking its activity by a small molecule inhibitor K-252a Nocardiopsis sp.. Functional studies had shown that upon inhibitor treatment, subcellular localization of the protein was changed and its invasion ability in vitro was impaired. Also nude mice xenografts had shown that upon inhibitor treatment tumor forming ability of FLT3 expressing cells were highly diminished. Therefore we suggest that FLT3 has a role in hepatocellular carcinogenesis and it might be another link between liver regeneration and hepatocellular carcinogenesis.
Open Access
Senescence and immortality genes as markers of hepatocellular carcinogenesis
(2009) Arslan Ergül, Ayça
Cellular senescence is a tumor-suppression mechanism, and immortalization facilitates neoplastic transformation. Both mechanisms may be highly relevant to hepatocellular carcinoma (HCC) development. We worked on two major aspects of cellular senescence and immortality in HCC. First, we analyzed the role of ZEB2 (Smad-interacting protein SIP1, ZFXH1B) gene for a senescence-related role in HCC. Then, we extended our work on the identification and analysis of a senescence and immortality gene network (SIGN) in relation to hepatocellular carcinogenesis. ZEB2 is a transcriptional repressor of E-cadherin, and induces epithelial-mesenchymal transition (EMT), a key process involved in tumor metastasis and progression. However, ZEB2 is also a repressor of telomerase reverse transcriptase (TERT) gene, which encodes a key enzyme required for telomere maintenance and tumor cell immortality. We performed in-vivo, in-silico and in-vitro studies to explore potential implications of ZEB2 in hepatocellular carcinoma (HCC). Tissue expression of ZEB2 transcripts displayed stepwise decreases in HCC lesions, as compared to liver cirrhosis. This inverse correlation suggested that sustained ZEB2 expression is not compatible with HCC progression. Next, vekt¨or¨u ile transfekte edilen Hep3B h¨ucrelerinin, ya¸slanma ili¸skili !-galaktozidaz aktivitesi ile, kalıcı h¨ucre d¨ong¨us¨u hapsine girdiklerini g¨ord¨uk. ZEB2 ile ind¨uklenen ya¸slanma hapsi, TERT ifadesinin baskılanması ve e¸slik eden siklin-ba˘gımlı kinaz engelleyici p21Cip1’in ifadesindeki artı¸s ile ba˘gıntılı idi. ZEB2’nin ge¸cici ifadesi, p21Cip1 ifadesindeki artı¸sı ind¨uklemedi. Son olarak, ZEB2’yi a¸sırı ifade eden Hep3B ve A431 klonlarının, in vitro k¨ult¨urde dereceli olarak azalması ile ZEB2 a¸sırı ifadesinin, kanser h¨ucrelerinin in vitro ya¸samı ile uyumlu olmadı˘gı sonucuna varıldı. Bu g¨ozlemler, ZEB2 geninin, EMT’deki rol¨un¨un dı¸sında, HCC h¨ucre b¨uy¨umesi ve ya¸samasında negatif rol oynadı˘gını d¨u¸s¨und¨urd¨u. Di˘ger ¸calı¸smamızda SIGN imzasını olu¸sturmak i¸cin, ya¸slanmaya programlanmı ¸s ve ¨ol¨ums¨uz HCC h¨ucrelerinden gelen gen ifade datasını, siroz ve HCC dokularından gelen data ile birle¸stirdik. SIGN imzası normal karaci˘ger, siroz, displazi ve HCC lezyonlarını do˘grulukla sınıflandırdı ve ya¸slanmadan ¨ol¨ums¨uzl¨u˘ge d¨on¨u¸s¨um¨un, ilk olarak displaziden erken HCC’ye ge¸ci¸ste ger¸cekle¸sti˘gini belirledi. Bu d¨on¨u¸s¨um, t¨um¨or ilerlemesine de katkıda bulunur. Ya¸slanmadan ¨ol¨ums¨uzl¨u˘ge d¨on¨u¸s¨ume, hepatik geri ba¸skala¸sım ile ve h¨ucre ¸co˘galması, kromozom de˘gi¸simi ve DNA hasar yanıtı genlerindeki ifade artı¸sı e¸slik etti. Bu nedenle, HCC ¨ol¨ums¨uzl¨u˘g¨u, k¨ok h¨ucre/¨onc¨u h¨ucre benzeri ¨ozellikler ile yakından ili¸skilidir. Son olarak, DNA hasar kontrol noktası ve DNA tamir genlerindeki artı¸s ile, t¨um¨or ba¸slangıcı ve ilerlemesi arasında ili¸ski bulduk. Bu genler, HCC’nin engellenmesinde ve terapisinde potansiyel hedefler olabilirler.