Atransgenic mousestrain that expresses the hepatitis Bvirus (HBV)large envelope protein intheliver was usedtodetermine theextent ofoxidative DNA damagethat occurs during chronic HBV infection. Thismousestrain de- velops achronic necroinflammatory liver disease that mimics theinflammation, cellular hyperplasia, andincreased risk for cancer that isevident inhumanchronic active hepatitis. When perfused insitu withnitroblue tetrazolium, anindicator for superoxide formation, theliver oftransgenic micedisplayed intense formazan deposition inKupffer cells, indicating oxygen radical production, andS-phase hepatocytes werecommonly seenadjacent tothestained Kupffer cells. Similar changes were notobserved innontransgenic control livers. Todetermine whether these events wereassociated withoxidative DNA damage, genomic DNAfromthelivers oftransgenic miceand nontransgenic controls wasisolated andexamined for8-oxo- 2'-deoxyguanosine, anoxidatively modified adduct ofdeoxy- guanosine. Results showed asignificant, sustained accumula- tion insteady-state 8-oxo-2'-deoxyguanosine that started early inlife exdusively inthetransgenic miceandincreased pro- gressively withadvancing disease. Themostpronounced in- crease occurred inlivers exhibiting microscopic nodular hy- perplasia, adenomas, andhepatocellular carcinoma. Thus, HBV transgenic micewithchronic active hepatitis display greatly increased hepatic oxidative DNAdamage. Moreover, theDNA damageoccurs inthepresence ofheightened hepa- tocellular proliferation, increasing theprobability offixation of theattendant genetic andchromosomal abnormalities andthe development ofhepatocellular carcinoma. Chronic inflammatory diseases affect millions ofpeople world-wide andmaybeaprimary factor inthedevelopment ofuptoone-third ofallcancers (1). A major association between a persistent infection andcancerisevident in chronic active hepatitis Bvirus (HBV)infection (2). Epide- miological studies showthatincidence ofhepatocellular carcinoma (HCC)correlates strongly togeographical areas of endemic HBV infection (3). Furthermore, patients with chronic hepatitis havea muchhigher incidence ofliver cirrhosis andHCCthanindividuals notinfected withHBV (4). Also, HBV antigens inseraofpatients areassociated withHCC(5). Because thevirus infects >300million people throughout theworld, HBV hasbeendescribed assecond only totobacco asaknownhumancarcinogen (6). Despite theapparent epidemiological association of chronic active hepatitis infection andcancer, themechanisms involved arestill notentirely understood. Research hasbeen hampered duetothelonglatency period between theonset ofinfection andcancer development, andthelack ofsuitable animal models that mimicchronic active humanhepatitis. HBV doesnotcontain anyknownacutely transforming oncogenes, andonlyrarely dointegrated HBV sequences activate cellular protooncogenes (7-10). Incontrast, persis- tentinflammation associated withchronic active hepatitis canbeassociated withmutations intumorsuppressor genes (11)andextensive chromosomal abnormalities. Persistent HBVinfection often leads tocirrhosis that often precedes the appearance ofHCC(12). Thenecroinflammatory liver dis- easeseenduring HBVinfection isthought tobemediated by thehostimmuneresponse toviral antigens. Activated mac- rophages andother recruited leukocytes release powerful reactive oxygen species suchasHOONO(from NOandO-), HOCI,andH202atsites ofinfection, causing areas offocal necrosis andcompensatory cell division (13,14). These oxidants notonlykill target cells butmayalso overwhelm cellular antioxidant defenses ofneighboring cells, leading to damage ofimportant biomolecules, including DNA (15). We hypothesize that oxidative DNA damage maybean important underlying eventthatleads tocancerduring chronic active hepatitis andother persistent inflammatory diseases. Wetested this hypothesis using atransgenic mouse modelinwhichoverexpression oftheHBV large envelope protein causes anecroinflammatory liver disease that inex-

Extensive oxidative DNA damageinhepatocytes oftransgenic mice withchronic active hepatitis destined todevelop hepatocellular carcinoma

Purpose: To investigate SGI-110 as a “chemosensitizer” in ovarian cancer and to assess its effects on tumor suppressor genes (TSG) and chemoresponsiveness-associated genes silenced by DNA methylation in ovarian cancer. Experimental Design: Several ovarian cancer cell lines were used for in vitro and in vivo platinum resensitization studies. Changes in DNA methylation and expression levels of TSG and other cancer-related genes in response to SGI-110 were measured by pyrosequencing and RT-PCR. Results: We demonstrate in vitro that SGI-110 resensitized a range of platinum-resistant ovarian cancer cells to cisplatin (CDDP) and induced significant demethylation and reexpression of TSG, differentiation-associated genes, and putative drivers of ovarian cancer cisplatin resistance. In vivo , SGI-110 alone or in combination with CDDP was well tolerated and induced antitumor effects in ovarian cancer xenografts. Pyrosequencing analyses confirmed that SGI-110 caused both global ( LINE1 ) and gene-specific hypomethylation in vivo , including TSGs ( RASSF1A ), proposed drivers of ovarian cancer cisplatin resistance ( MLH1 and ZIC1 ), differentiation-associated genes ( HOXA10 and HOXA11 ), and transcription factors ( STAT5B ). Furthermore, DNA damage induced by CDDP in ovarian cancer cells was increased by SGI-110, as measured by inductively coupled plasma-mass spectrometry analysis of DNA adduct formation and repair of cisplatin-induced DNA damage. Conclusions: These results strongly support further investigation of hypomethylating strategies in platinum-resistant ovarian cancer. Specifically, SGI-110 in combination with conventional and/or targeted therapeutics warrants further development in this setting. Clin Cancer Res; 20(24); 6504–16. ©2014 AACR .

The novel, small-molecule DNA methylation inhibitor SGI-110 as an ovarian cancer chemosensitizer

Defects in DNA repair can result in oncogenic genomic instability. Cancers occurring from DNA repair defects were once thought to be limited to rare inherited mutations (such as BRCA1 or 2). It now appears that a clinically significant fraction of cancers have acquired DNA repair defects. DNA repair pathways operate in related networks, and cancers arising from loss of one DNA repair component typically become addicted to other repair pathways to survive and proliferate. Drug inhibition of the rescue repair pathway prevents the repair-deficient cancer cell from replicating, causing apoptosis (termed synthetic lethality). However, the selective pressure of inhibiting the rescue repair pathway can generate further mutations that confer resistance to the synthetic lethal drugs. Many such drugs currently in clinical use inhibit PARP1, a repair component to which cancers arising from inherited BRCA1 or 2 mutations become addicted. It is now clear that drugs inducing synthetic lethality may also be therapeutic in cancers with acquired DNA repair defects, which would markedly broaden their applicability beyond treatment of cancers with inherited DNA repair defects. Here we review how each DNA repair pathway can be attacked therapeutically and evaluate DNA repair components as potential drug targets to induce synthetic lethality. Clinical use of drugs targeting DNA repair will markedly increase when functional and genetic loss of repair components are consistently identified. In addition, future therapies will exploit artificial synthetic lethality, where complementary DNA repair pathways are targeted simultaneously in cancers without DNA repair defects.

Drugging the Cancers Addicted to DNA Repair

Chronic liver diseases pose a substantial health burden worldwide, with approximately two million deaths each year. Branched-chain amino acids (BCAAs)—valine, leucine, and isoleucine—are a group of essential amino acids that are essential for human health. Despite the necessity of a dietary intake of BCAA, emerging data indicate the undeniable correlation between elevated circulating BCAA levels and chronic liver diseases, including non-alcoholic fatty liver diseases (NAFLD), cirrhosis, and hepatocellular carcinoma (HCC). Moreover, circulatory BCAAs were positively associated with a higher cholesterol level, liver fat content, and insulin resistance (IR). However, BCAA supplementation was found to provide positive outcomes in cirrhosis and HCC patients. This review will attempt to address the contradictory claims found in the literature, with a special focus on BCAAs’ distribution, key signaling pathways, and the modulation of gut microbiota. This should provide a better understanding of BCAAs’ possible contribution to liver health.

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The Emerging Role of Branched-Chain Amino Acids in Liver Diseases

Simple Summary Cancer stem cells are a distinct tumor subpopulation associated with poor outcome. The epithelial cell adhesion molecule (EpCAM) and Thy-1 cell surface antigen (CD90) have been implicated as cancer stem cell (CSC) markers in hepatocellular carcinoma (HCC). Eradication of those CSCs is of high importance with respect to the outcome of treatment. We aimed to verify the impact of targeting HCC cells expressing the CSC marker CD90 and EpCAM with combined therapy of AKT and mTOR inhibitors. Our data demonstrated that combined targeting of AKT and mTOR is highly synergistic in vitro but leads to treatment resistance of xenograft tumors in an orthotopic mouse model in vivo. The development of resistance was observed in large numbers of MK2206/RAD001 treated cells. Restoration of phosphorylated AKT was observed in most tumors during AKT/mTOR therapy underlining the importance of restored AKT signaling as a resistance mechanism. Further work is required to verify the molecular mechanisms of HCC treatment resistance. Abstract The epithelial cell adhesion molecule (EpCAM) and Thy-1 cell surface antigen (CD90) have been implicated as cancer stem cell (CSC) markers in hepatocellular carcinoma (HCC). Expression of EpCAM and CD90 on HCC cells is associated with increased tumorigenicity, metastasis and poor prognosis. In this study, we demonstrate that combined treatment with AKT and mTOR inhibitors—i.e., MK2206 and RAD001—results in a synergistic reduction in proliferation of EpCAM+ and CD90+ HCC cells cultured either as adherent cells or as tumoroids in vitro. In addition, tumor growth was reduced by combined treatment with AKT and mTOR inhibitors in an orthotopic xenograft mouse model of an EpCAM+ HCC cell line (Huh7) and primary patient-derived EpCAM+ HCC cells (HCC1) as well as a CD90+ HCC-related cell line (SK-HEP1) in vivo. However, during AKT/mTOR treatment, outgrowth of therapy-resistant tumors was observed in all mice analyzed within a few weeks. Resistance was associated in most cases with restoration of AKT signaling in the tumors, intrahepatic metastases and distant metastases. In addition, an upregulation of the p38 MAPK pathway was identified in the AKT/mTOR inhibitor-resistant tumor cells by kinome profiling. The development of resistant cells during AKT/mTOR therapy was further analyzed by red-green-blue (RGB) marking of HCC cells, which revealed an outgrowth of a large number of Huh7 cells over a period of 6 months. In summary, our data demonstrate that combined treatment with AKT and mTOR inhibitors exhibits synergistic effects on proliferation of EpCAM+ as well as CD90+ HCC cells in vitro. However, the fast development of large numbers of resistant clones under AKT/mTOR therapy observed in vitro and in the orthotopic xenotransplantation mouse model in vivo strongly suggests that this therapy alone will not be sufficient to eliminate EpCAM+ or CD90+ cancer stem cells from HCC patients.

/pdf/combined-targeting-of-akt-and-mtor-inhibits-tumor-formation-dgp4skj9.pdf

Combined Targeting of AKT and mTOR Inhibits Tumor Formation of EpCAM+ and CD90+ Human Hepatocellular Carcinoma Cells in an Orthotopic Mouse Model

Despite advances in the treatment of cancers through surgical procedures and new pharmaceuticals, the treatment of hepatocellular carcinoma (HCC) remains challenging as reflected by low survival rates. The PI3K/Akt/mTOR pathway is an important signaling mechanism that regulates the cell cycle, proliferation, apoptosis, and metabolism. Importantly, deregulation of the PI3K/Akt/mTOR pathway leading to activation is common in HCC and is hence the subject of intense investigation and the focus of current therapeutics. In this review article, we consider the role of this pathway in the pathogenesis of HCC, focusing on its downstream effectors such as glycogen synthase kinase-3 (GSK-3), cAMP-response element-binding protein (CREB), forkhead box O protein (FOXO), murine double minute 2 (MDM2), p53, and nuclear factor-κB (NF-κB), and the cellular processes of lipogenesis and autophagy. In addition, we provide an update on the current ongoing clinical development of agents targeting this pathway for HCC treatments.

Miriam Wankell

Papers

Targeting the PI3K/Akt/mTOR Pathway in Hepatocellular Carcinoma.

The role of DNA damage and repair in liver cancer

Cell adhesion an important determinant of myogenesis and satellite cell activity.