Loss of aquaporin-4 expression and putative function in non-small cell lung cancer

<p>Abstract</p> <p>Background</p> <p>Aquaporins (AQPs) have been recognized to promote tumor progression, invasion, and metastasis and are therefore recognized as promising targets for novel anti-cancer therapies. Potentially relevant AQPs in distinct cancer entities can be determined by a comprehensive expression analysis of the 13 human AQPs.</p> <p>Methods</p> <p>We analyzed the presence of all AQP transcripts in 576 different normal lung and non-small cell lung cancer (NSCLC) samples using microarray data and validated our findings by qRT-PCR and immunohistochemistry.</p> <p>Results</p> <p>Variable expression of several AQPs (AQP1, -3, -4, and -5) was found in NSCLC and normal lung tissues. Furthermore, we identified remarkable differences between NSCLC subtypes in regard to AQP1, -3 and -4 expression. Higher transcript and protein levels of AQP4 in well-differentiated lung adenocarcinomas suggested an association with a more favourable prognosis. Beyond water transport, data mining of co-expressed genes indicated an involvement of AQP4 in cell-cell signalling, cellular movement and lipid metabolism, and underlined the association of AQP4 to important physiological functions in benign lung tissue.</p> <p>Conclusions</p> <p>Our findings accentuate the need to identify functional differences and redundancies of active AQPs in normal and tumor cells in order to assess their value as promising drug targets.</p

Epigenetic modulation by histone deacetylase inhibition as new therapeutic option against hepatocellular carcinoma

Acetylierung und Deacetylierung DNA-assoziierter Histonproteine durch das gegensätzlich arbeitende Enzympaar Histondeacetylase (HDAC) und Histonacetyltransferase (HAT) erlauben feinste Modulierungen der zellulären Genexpression auf epigenetischer Ebene. In malignen Zellen können HDAC-Inhibitoren (HDAC-I) eine Veränderung der Nukleosomenstruktur und damit eine Reaktivierung von Genen bewirken, welche zentrale Kontrollfunktionen bei Proliferation, Differenzierung und dem zellulären Selbstmordprogramm (Apoptose) ausüben. In der Konsequenz dieser epigenetisch vermittelten „Reprogrammierung“ können die entarteten Zellen im Wachstum gehemmt und in die Apoptose getrieben werden. In dieser Dissertation wurden zwei neuartige HDAC-I, das Antikonvulsivum Valproat (VPA) und der Wirkstoff ITF2357 unter Verwendung der humanen Hepatomzelllinien HepG2, PLC/PRF/5 und HuH7 und zusätzlich auch von primären humanen Hepatozyten (PHH) charakterisiert. Die HDAC-I Behandlung führte in den Hepatomzellen zu einer konzentrationsabhängigen Hemmung der Proliferation und Induktion von Apoptose. Im Gegensatz hierzu führte eine identische Behandlung bei PHH zu keinen meßbaren Zeichen einer Zytotoxizität. VPA und ITF2357 führten weiterhin zur einer erhöhten Expression des regulatorischen Zellzyklusproteins p21 und von pro-apoptotischen Mitgliedern der Bcl-2 Familie, wie Bid und Bax, jedoch auch zu einer Verminderung des anti-apoptotischen Bcl-XL Proteins. Eine artifizielle Überexpression von Bcl-XL durch Transfektion eines Bcl-XL kodierenden Plasmids konnte die durch HDAC-I hervorgerufene Zelltodrate deutlich reduzieren, so daß ein Übergewicht pro-apoptotischer Faktoren in den Hepatomzellen entscheidend zu der HDAC-I vermittelten Induktion von Apoptosephänomenen beizutragen scheint. Der zweite Teil der Dissertation befaßte sich mit einer Kombinationsbehandlung aus HDAC-I und dem Zytokin TNF-related apoptosis inducing ligand (TRAIL). Interessanterweise konnte gezeigt werden, daß eine durch HDAC-I hervorgerufene „Reprogrammierung“ der Tumorzellen in der Lage ist die TRAIL-Resistenz von Hepatomzellen zu überwinden und eine Sensibilisierung gegenüber TRAIL zu erreichen. Während TRAIL alleine keine relevante Apoptose auslösen konnte, führte eine Präinkubation mit einem HDAC-I zu einer deutlichen Zunahme der Zelltodrate nach TRAIL-Exposition in allen Hepatomzellen. Auf der Suche nach dem hierfür verantwortlichen Mechanismus zeigten sich zwar keine Veränderungen pro-apoptotischer TRAIL-Rezeptoren an der Zelloberfläche, jedoch konnte eine Herunterregulation des intrazellulären anti-apoptotischen Caspase-8 Inhibitors FLIP identifiziert werden. Die Kombination von HDAC-I und TRAIL zeigte in gesunden Leberzellen fünf verschiedener Spender ein ausgesprochen gutes Toleranzprofil ohne Zeichen der Toxizität, während gleichartig behandelte Hepatomzellen deutliche Zeichen eines massiven Zelluntergangs boten. Die Selektivität der HDAC-I gesteuerten TRAIL-Sensibilisierung korrelierte mit einer differentiellen Regulation von FLIP, welches in den PHH im Gegensatz zu den malignen Hepatomzellen hochreguliert war und somit die Apoptoseschwelle nicht maligner Zellen als Schutzfaktor zusätzlich erhöhte. Zusammenfassend eröffnet die untersuchte Kombinationsbehandlung von TRAIL mit einer HDAC-inhibitorisch wirkenden Substanz die Perspektive für eine innovative Therapiestrategie in der Behandlung des Hepatozellulären Karzinoms, die weiter klinisch evaluiert werden sollte.Due to a particular resistance against conventional chemotherapeutics, palliative treatment of hepatocellular carcinomas (HCC) is highly ineffective. Recent demonstration of both proliferation-inhibition and apoptosis of hepatoma cells by a histone deacetylase inhibitor (HDAC-I) treatment opens up a promising new approach. HDAC-I substances with favourable in vivo profiles, valproate (VPA) and ITF2357, were investigated on HCC cell lines and primary human hepatocytes (PHH). Histone acetylation and apoptosis-modulating proteins were investigated by western-blotting, proliferation by sulforhodamin B binding, toxicity by enzyme release, apoptosis by FACS analysis. VPA and ITF2357 inhibited proliferation in HCC cell lines. Both substances induced considerable cellular damage in HCC-derived cells, but PHH tolerated these substances well. A downregulation of anti- and upregulation of proapoptotic factors was found. Moreover, Bcl-X(L) transfection into HCC cells abrogated apoptosis induced by both substances, indicating that modulation of intracellular pro- and anti-apoptotic proteins is a key event in VPA or ITF2357 induced tumor-cell death.Preferential induction of cell death in HCC-derived cell lines, without toxicity in PHH, demonstrates the potential of VPA and ITF2357 to become promising new tools in the fight against HCC. Hepatocellular carcinoma displays further a striking resistance to innovative tumor cell apoptosis-inducing agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In TRAIL-sensitive cancer cells, the mechanism of HDAC-I-induced cell death has been identified to be TRAIL-dependent by inducing apoptosis in an autocrine fashion. In contrast, in HCC-derived cells, a prototype of TRAIL-resistant tumor cells, we found a HDAC-I-mediated apoptosis that works independently of TRAIL and upregulation of death receptors or their cognate ligands. Interestingly, TRAIL resistance could be overcome by a combinatorial application of HDAC-I and TRAIL, increasing the fraction of apoptotic cells two- to threefold compared with HDAC-I treatment alone, whereas any premature HDAC-I withdrawal rapidly restored TRAIL resistance. Furthermore, a tumor cell-specific downregulation of the FLICE inhibitory protein (FLIP) was observed, constituting a new mechanism of TRAIL sensitivity restoration by HDAC-I. In contrast, FLIP levels in primary human hepatocytes (PHH) from different donors were upregulated by HDAC-I. Importantly, combination HDAC-I/TRAIL treatment did not induce any cytotoxicity in nonmalignant PHH. In conclusion, HDAC-I compounds, exhibiting a favorable in vivo profile and inherent activity against HCC cells, are able to selectively overcome the resistance of HCC cells toward TRAIL. Specific upregulation of intracellular FLIP protein levels in nonmalignant hepatocytes could enhance the therapeutic window for clinical applications of TRAIL, opening up a highly specific new treatment option for advanced HCC

Cadherin-6 is a putative tumor suppressor and target of epigenetically dysregulated miR-429 in cholangiocarcinoma

<p>Cholangiocarcinoma (CC) is a rare malignancy of the extrahepatic or intrahepatic biliary tract with an outstanding poor prognosis. Non-surgical therapeutic regimens result in minimally improved survival of CC patients. Global genomic analyses identified a few recurrently mutated genes, some of them in genes involved in epigenetic patterning. In a previous study, we demonstrated global DNA methylation changes in CC, indicating major contribution of epigenetic alterations to cholangiocarcinogenesis. Here, we aimed at the identification and characterization of CC-related, differentially methylated regions (DMRs) in potential microRNA promoters and of genes targeted by identified microRNAs. Twenty-seven hypermethylated and 13 hypomethylated potential promoter regions of microRNAs, known to be associated with cancer-related pathways like Wnt, ErbB, and PI3K-Akt signaling, were identified. Selected DMRs were confirmed in 2 independent patient cohorts. Inverse correlation between promoter methylation and expression suggested miR-129-2 and members of the miR-200 family (miR-200a, miR-200b, and miR-429) as novel tumor suppressors and oncomiRs, respectively, in CC. Tumor suppressor genes <i>deleted in liver cancer 1</i> (<i>DLC1</i>), <i>F-box/WD-repeat-containing protein 7</i> (<i>FBXW7</i>), and <i>cadherin-6</i> (<i>CDH6</i>) were identified as presumed targets in CC. Tissue microarrays of a representative and well-characterized cohort of biliary tract cancers (n=212) displayed stepwise downregulation of CDH6 and association with poor patient outcome. Ectopic expression of <i>CDH6</i> on the other hand, delayed growth in the CC cell lines EGI-1 and TFK-1, together suggesting a tumor suppressive function of <i>CDH6</i>. Our work represents a valuable repository for the study of epigenetically altered miRNAs in cholangiocarcinogenesis and novel putative, CC-related tumor suppressive miRNAs and oncomiRs.</p