Mutação 249Ser no TP53 no Carcinoma Hepatocelular (CHC), frequente em países da África e Ásia, é uma evidência molecular de exposição à aflatoxina. O objetivo deste estudo é analisar a freqüência de 249Ser em 74 amostras de CHC no Brasil. A mutação foi analisada por RFLP e sequenciamento. A presença de vírus da hepatite B (VHB) foi analisada por PCR em tempo real. 249Ser foi encontrada em 13/74 (28%) e VHB em 13/74 (16%). A mutação foi encontrada em maior freqüência em tumores indiferenciados (OR = 2,415, IC = 1,001 - 5,824). O tamanho médio de tumores com 249Ser foi de 9,4 cm contra 5,5 cm de amostras sem a mutação (p=0,012). Não foi encontrada relação entre VHB e 249Ser. Os resultados indicam que 249Ser é um fator importante na carcinogênese do CHC no Brasil sendo associada à uma forma maior e menos diferenciada de tumor. / TP53 249Ser mutation has been proved a molecular evidence for aflatoxin-related Hepatocellular Carcinoma (HCC) and is frequent in Africa and Asia. The aim of our study was to analyze the frequency of 249Ser mutation in HCC from Brazil. We studied 74 samples of paraffin embedded HCC. 249Ser mutation was analyzed by RFLP and sequencing. Presence of HBV DNA was determined by Real-Time PCR. 249Ser was found in 21/74 (28%) samples while HBV DNA was found in 13/74 (16%). Poorly differentiated HCC was more likely to have 249Ser mutation (OR = 2.415, IC = 1.001 - 5.824). The mean tumor size of 249Ser HCC was 9.4 cm versus 5.5cm on wild type (p=0.012). HBV DNA was not related with 249Ser. Results indicate that 249Ser is an important factor of HCC carcinogenesis in Brazil and is associated with large and poorly differentiated tumors.

Staphylococcal nuclease and tudor-domain containing 1 (SND1) is an oncogene for a wide variety of cancers, including hepatocellular carcinoma (HCC). SND1 is a multifunctional protein regulating gene expression of proto-oncogenes and tumor suppressor genes, making SND1 a prime target for developing cancer therapeutics. This notion is especially attributed to HCC as most patients are diagnosed in advanced stages and the therapeutic options available for these patients are severely limited. In this study, we evaluated the therapeutic potential of a replication-defective adenovirus vector delivering SND1 shRNA (Ad.SND1sh) to human HCC cell lines, HepG3, HuH-7, and Hep3B. Adenovirus infection in HCC cells was confirmed by Western blotting and immunofluorescence. The efficacy of Ad.SND1sh to knockdown SND1 expression was confirmed via Western blot, qRT-PCR, and immunofluorescence. Ad.SND1sh did not significantly affect proliferation of the three human HCC cells but significantly inhibited their invasive and migratory capacities, as determined by wound healing and Matrigel invasion assays, respectively. As a corollary, Ad.SND1sh treatment resulted in a decrease in mesenchymal markers, such as N-cadherin, Twist, Snail, and Slug, without affecting levels of epithelial marker E-Cadherin, indicating that SND1 knockdown induces mesenchymal conversion in HCC cells. Additionally, reductions in liver cancer stem cell marker CD133 and HCC marker α-fetoprotein (AFP) were observed with SND1 knockdown. HCC cells with aberrant expression of these markers are associated with tumor initiation, recurrence, and multi-drug resistance. Our findings indicate that Ad.SND1sh may potentially be an effective therapy for advanced HCC and needs to be studied further for its clinical application.

Liver cancer is the fourth leading cause of cancer-associated deaths globally, and among primary liver cancers, hepatocellular carcinoma (HCC) encompasses 75-85% of all cases. HCC is a highly lethal disease due to limited treatment options – only a small subset of patients qualify for surgical resection or transplantation; the remaining patients often display resistance to radiation therapy or chemotherapy. Overexpression of the oncogene astrocyte elevated gene-1 (AEG-1) is associated with poorer survival and increased tumor recurrence in HCC, and numerous studies show its role in initiation of hepatocarcinogenesis. A prior study also demonstrated AEG-1 expression inhibits senescence by diminishing the ATM/Chk1/Chk2/p53/p21 DNA damage response (DDR) pathway. The aim of this study is to understand if AEG-1 expression promotes radioresistance in HCC. A CRISPR/Cas9 plasmid system was used to delete AEG-1 in the QGY-7703, HuH7 and DihXY cell lines, which model HCC. The cell lines were then treated with ionizing radiation (IR). We find that knockout of AEG-1 in these cell lines induces sensitivity to IR at 2.5 Gy. In response to radiation, AEG-1 wildtype cells more profoundly upregulate ATR, Chk1, and Chk2 signaling; and also more rapidly induce γH2AX, ATM, and BRCA1 signaling, which sense dsDNA breaks to initiate homologous recombination repair. We conclude that AEG-1 expression protects HCC cells from IR through two mechanisms: 1) rapidly initiating the DNA damage response; and 2) increasing replication fork stabilization. These findings indicate AEG-1 can be a therapeutic target in combination with radiation treatment to improve outcomes for HCC patients who demonstrate radioresistance.

acase@tulane.edu

MiR-17-92 is an oncogenic miRNA cluster implicated in the development of several human cancers; however, it remains unknown whether miR-17-92 cluster is able to regulate hepatobiliary carcinogenesis. This study was designed to investigate the biological functions and molecular mechanisms of miR-17-92 cluster in primary liver cancer.<br>In-situ hybridization and qRT-PCR analysis showed that miR-17-92 cluster is highly expressed in human cholangiocarcinoma cells compared to the non-neoplastic biliary epithelial cells. Forced overexpression of the miR-17-92 cluster or its members, miR-92a and miR-19a, in cultured human cholangiocarcinoma cells enhanced tumor cell proliferation, colony formation and invasiveness, in vitro. Overexpression of miR-17-92 cluster or miR-92a also enhanced cholangiocarcinoma growth in vivo in SCID hairless outbred mice. The tumor suppressor PTEN was identified as a bona fide target of both miR-92a and miR-19a in cholangiocarcinoma cells. Accordingly, overexpression of PTEN open reading frame protein (devoid of 3’UTR) prevented miR-92a- or miR-19a-induced cholangiocarcinoma cell growth. Microarray analysis revealed additional targets of miR-17-92 cluster in human cholangiocarcinoma cells, including APAF-1 and PRDM2. Moreover, we observed that the expression of miR-17-92 cluster is regulated by IL-6/Stat3, a key oncogenic signaling pathway pivotal in cholangiocarcinogenesis. Taken together, our findings in this study disclose a novel IL-6/Stat3 miR-17-92 cluster PTEN signaling axis that is crucial for cholangiocarcinogenesis and tumor progression.<br>We also found the miR-17-92 is highly expressed in tumor tissue compared to non-tumor adjacent tissue in hepatocellular carcinoma patient tissue. Forced overexpression of the miR-17-92 cluster in cultured human hepatocellular carcinoma cells enhanced tumor growth in vitro; on contrast, inhibition of miR-17-92 cluster inhibited cell growth. MiR-17-92 cluster promote diethylnitrosamine-induced hepatocarcinogenesis in liver-specific miR-17-92 cluster transgenic mice. Binding sequence and mice whole genome microarray analysis revealed about 300 possible targets. RNA-sequencing data analysis showed both individual miRNAs and the host gene of miR-17-92 cluster was highly expressed in hepatocellular carcinoma patients and had negative correlation with several genes (CREBL2, PRRG1, and NTN4), among which, CREBL2 may play an important role in the hepatocarcinogenesis. / acase@tulane.edu

Woodchucks are the preferred animal model to study chronic viral hepatitis and the development of hepatocellular carcinoma (HCC), which occurs as a result of infection with woodchuck hepatitis virus. Significant elevations in the phosphomonoester peak in 31P-MRS spectrum correlated to the presence of HCC. Ex vivo 31P-NMR determined that HCC tissue had significantly elevated concentrations of PC compared to uninfected control tissues, confirming that PME is specific to the tumour’s growth. Finally, a recombinant vaccinia virus was constructed to stimulate the immune systems of infected woodchucks against cells expressing core antigens. Despite reductions in surface antigen expression and viral load, elevations in serum GGT and the PME in 31P-MRS indicated that there was tumour growth in treated woodchucks. In conclusion, the PME peak represents a potential biomarker of cancerous growth when used in conjunction with serological tests to detect HCC in the liver due to chronic hepatitis virus infection. / May 2009

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. An extensive array of growth factors and their receptors have been identified and may act as positive and negative modulators in different stages of liver carcinogenesis. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from conditioned medium of Huh-7 hepatoma cell line. HDGF has growth stimulating activity for various types of cells. Recent evidence indicates that HDGF upregulation is associated with poor survival outcome and tumor progression in HCC, non-small cell lung carcinoma and melanoma. However, the exact function and molecular mechanism of HDGF overexpression during HCC progression remain largely unknown. In the first project (Chapter 2) of this thesis study, we started with characterizing in HDGF release and response to exogenous HDGF between benign HepG2 and malignant SK-Hep-1 hepatoma cells. It was found that serum deprivation significantly stimulated the HDGF secretion in SK-Hep-1 cells but not HepG2 cells. Interestingly, SK-Hep-1 cells did not increase the secretion of vascular endothelial growth factor (VEGF), a potent angiogenic factor, during serum deprivation. Besides, SK-Hep-1 cells were more responsive to the growth- and migration-promoting effect of exogenous HDGF. We also validated the angiogenic functions of recombinant HDGF protein in vitro and in vivo. In the second project (Chapter 3), we investigated the influence of cellular HDGF level on the neoplastic potential of hepatoma cells. Adenovirus vectors encoding HDGF, Ad-HDGF, and antisense HDGF, Ad-HDGF (-), were generated to modulate the cellular HDGF levels in SK-Hep-1 cells. Adenovirus-mediated HDGF gene delivery increased the HDGF expression and release, and stimulated the proliferation, migration and anchorage-independent growth of SK-Hep-1 cells. In contrast, infection with Ad-HDGF (-) reduced the HDGF expression and secretion, and attenuated the oncogenic behaviors of SK-Hep-1 cells. Implanting HDGF-overexpressing SK-Hep-1 cells led to the accelerated growth of xenografted hepatoma in SCID mice while implantation of HDGF-downregulated SK-Hep-1 cells caused retarded tumor growth. Histological analysis revealed the increased proliferation and neovascularization in HDGF-overexpressing tumors. This could be attributed to elevated VEGF expression and activation of the nuclear factor kappa B (NF£eB) activities by HDGF upregulation in SK-Hep-1 cells. In the third project (Chapter 4), we delineated the mechanism underlying HDGF-induced VEGF secretion and activation of NFB pathway in SK-Hep-1 cells. Adding recombinant HDGF protein enhanced the VEGF release by SK-Hep-1 cells particularly during serum starvation. This was associated with a concomitant increment in VEGF protein and mRNA levels in SK-Hep-1 cells. Like many mitogens, HDGF increased the production of reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide in a dose-dependent manner. Pretreatment with antioxidants abolished the HDGF-induced VEGF secretion. NF£eB is a pivotal transcription factor for regulation of pro-inflammatory cytokines and genes such as VEGF and cycloxygenase¡V2 (COX-2). Application of HDGF stimulated NF£eB-driven luciferase activities. This was correlated with a dose- and time-depedent increment of NF£eB (p65) by HDGF. HDGF treatment also elevated the COX-2 protein levels and activities in SK-Hep-1 cells. In addition, blockade of COX-2 by NS-398 attenuated the HDGF-induced VEGF secretion, suggesting the involvement of COX-2. Finally, it was found that HDGF stimulated the phosphorylation of Akt, Erk1/2, and p38 MAPK. Inhibition of Akt by LY294002 also diminished the HDGF-induced VEGF secretion. These studies suggest that HDGF induces oxidative stress to activate NF£eB/COX-2/Akt pathway, thereby stimulating VEGF expression and release. In summary, this thesis study brings functional and mechanistic insights on how aberrant HDGF expression contributes to angiogenesis and tumorigenesis during liver carcinogenesis.

Hepatocellular carcinoma (HCC) is one of the most common and devastating malignant tumors in Taiwan. Due to an imbalanced between reactive oxygen species (ROS) production and detoxification, oxidative stress, has been implicated in liver carcinogenesis. Superoxide dismutases (SODS) play a key role in the detoxification of superoxide radicals and thus protect cells from damage induced by free radicals. Manganese superoxide dismutase (MnSOD or SOD2) is a member of the superoxide dismutase family located in mitochondria. SOD2 transforms toxic superoxide, a byproduct of the mitochondrial electron transport chain, into hydrogen peroxide and diatomic oxygen. Though reduced SOD2 protein level and activities have been reported in hepatoma tissues, it remains unclear how SOD2 expression affected the tumorigenic processes of hepatoma cells. Expression analysis of an array of human HCC cell lines revealed that SOD2 were down-regulated in poorly differentiated SK-Hep-1 hepatoma cells. Moreover, SOD2 is downregulated in 68.8% of resected HCC samples (97 out of 141 cases). Adenovirus-mediated SOD2 gene delivery increased the cellular SOD2 protein level and H2O2 production, but reduced the superoxide anion level in SK-Hep-1 cells. Furthermore, SOD2 restoration significantly reduced the proliferation, motility, and colony formation of SK-Hep-1 cells. In vivo animal model, the finding of SOD2 overexpression inhibited the proliferation of Sk-Hep-1 hepatoma cells while reduced the tumor growth in mice. Flow cytometry analysis showed that SOD2 gene transfer inhibited the growth of hepatoma cells through induction of cell cycle arrest at G2/M phase. This was associated with declined cdc2/cdk1 and cyclin B1 expression and upregulation p21Cip1 by SOD2 gene delivery. However, SOD2 overexpression had no effect on the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9.In conclusion, SOD2 overexpression suppresses the tumorigenicity of hepatoma cells and may hold promise for HCC treatment.

WW domain-containing oxidoreductase (WWOX) is a novel tumor suppressor gene discovered few years ago. Many researches indicate that expression of WWOX is reduced in a variety of cancers including heptocellular carcinoma (HCC). A recent report suggests that WWOX is implicated in Wnt/£]-catenin pathway which is frequently affected in HCC. In this study, we used immunohistochemical (IHC) staining to analyze the expression of WWOX and Wnt/£]-catenin pathway components in HCC and adjacent non-tumor tissues. Our result showed that WWOX was significantly downregulated in poor differentiated HCC. In addition, downregulation of WWOX was significantly correlated with cytoplasmic £]-catenin expression. We also found that TCF4 was strongly expressed in HCC tissues and the expression was associated with tumor grade and stage. Consequently, our result implied that downregulation of WWOX in HCC might lead to accumulation of £]-catenin in the cytoplasm and the subsequent activation of Wnt/£]-catenin signaling pathway.

<p>Liver cancer is a devastating disease that is the 5th most common cancer in men, 7th most common cancer in women, and the 3rd leading cause of cancer-related mortality. This disease arises from multiple etiological factors, including hepatitis viruses, environmental toxins, alcohol abuse, and metabolic syndrome, which induce a state of chronic inflammation. This diseased liver tissue background is a drastically different microenvironment from the healthy liver, especially with regards to immune cell prevalence and presence of mediators of immune function. It has been well-established that this altered tissue background contributes significantly to the tumorigenic process, yet its effects on the progression of the disease are more poorly understood. </p><p>To better understand the consequences of liver disease on tumor growth and the interplay with its microenvironment, we first utilized two standard methods of fibrosis induction and orthotopic implantation of tumors into the inflamed and fibrotic liver to mimic the liver condition in human HCC patients, and examined the immune infiltrate. Compared to non-diseased controls, tumor growth is significantly enhanced under fibrotic conditions. The immune cells that infiltrated the tumors are also drastically different, with decreased proportions of natural killer cells but greatly increased numbers of immune-suppressive CD11b+ Gr1hi myeloid cells in both models of fibrosis. In addition, there are model-specific differences: increased proportions of CD11b+ myeloid cells and CD4+ CD25+ T-cells are found in tumors in the bile duct ligation model but not in the carbon tetrachloride model. Importantly, the skewed immune infiltration into the tumor, while having some commonalities with the non-tumor tissue, had several distinct, tumor-specific populations. Induction of fibrosis also alters the cytokine production of implanted tumor cells, which could have far-reaching consequences on the immune infiltrate and its functionality. Taken together, this work demonstrates that the combination of fibrosis induction with orthotopic tumor implantation results in a markedly different tumor microenvironment and tumor growth kinetics. </p><p>Appreciating that the altered immune microenvironment dramatically shifts tumor progression, we sought to further explore the effects of individual inflammatory mediators on the development of the disease. Interleukin 18 (IL-18) is an inflammatory cytokine that is markedly increased in the circulation of patients with HCC correlated with poor prognosis. However, the precise role for IL-18 in HCC remains unclear, with reports presenting both pro- and anti-tumorigenic activities. To answer this question definitively, we interrogated in more detail the expression profiles of IL-18 in tissue specimens from HCC patients and conducted experimentation using multiple clinically relevant mouse models to explore the functional role of this cytokine in the context of HCC. Our results indicate that IL-18 exerts a tumor-suppressive effect mediated in large part by alterations in survival and functionality of T-lymphocytes which infiltrated the tumor microenvironment. This tumor-suppressive effect is however dependent upon the inflammatory milieu: In the absence of an inflammatory environment, whether from a chemical carcinogenesis model or a fibrosis induction model, loss of IL-18 signaling does not affect tumor growth. This effect is also stage-dependent. Taken together, our findings establish a tumor-suppressive role for IL-18 in established HCC and provide a mechanistic explanation for the complex relationship between its expression pattern and HCC prognosis. </p><p>In summary, this work demonstrates a dramatic shift in the microenvironment of developing HCC tumors in the presence of chronic inflammatory stimuli. This microenvironment, which more accurately models the situation in which tumors develop and progress in patients, alters the presence and functionality of many immune mediators. In particular, IL-18 signaling is a powerful mediator of tumor progression, however observation of its functionality is dependent on an inflammatory context. This work provides new insight into the complex processes underlying HCC tumor progression, and emphasizes the necessity for more accurate modeling of HCC progression in mice which takes into account the drastic changes in the tissue caused by chronic liver disease.</p> / Dissertation