Hypermethylierung von tumor-assoziierten Genen in Tumoren des Kopf-Hals-Bereiches mit besonderer Berücksichtigung der epigenetischen Regulation und Funktion von Erythropoietin

Jährlich erkranken über 500000 Menschen an Plattenepithelkarzinomen im Kopf-Hals-Bereich (head and neck squamous cell carcinoma = HNSCC). Ein Ziel dieser Arbeit war es, den Methylierungsstatus von 15 Tumor-assoziierten Genen in 54 HNSCC- und 23 Normalgewebsproben zu untersuchen und mit den klinisch-pathologischen Daten zu korrelieren. Es zeigte sich, dass die Gesamtmethylierungsfrequenz dieser Gene in den Tumorproben signifikant höher war als in den Normalgewebsproben. Von den untersuchten Genen zeigten besonders p16, MGMT, DAPK, RARbeta, MLH1, CDH1, RASSF5 und MST1 eine häufige Methylierung in den HNSCC-Proben. Methylierungen von RASSF1A, GSTP1, RASSF4, MST2, LATS1 und LATS2 waren eher seltener in den Tumorproben zu finden. Bei der Korrelation der Methylierungsdaten mit den klinisch-pathologischen Daten war eine erhöhte Methylierungsfrequenz in Zusammenhang mit fortgeschrittenerem Tumorstadium und schlechterer Differenzierung der HNSCC-Proben zu beobachten. Zum Beispiel fand sich eine signifikant höhere Methylierung von p16 in schlecht differenzierten HNSCC und RASSF5 war wesentlich häufiger methyliert in Tumoren eines fortgeschritteneren Stadiums. Eine Methylierung von RASSF4 war nur in Patienten zu finden, die an einem Rezidiv erkrankten. Die Methylierung der Tumor-assoziierten Gene war in den untersuchten HNSCC-Zelllinien ähnlich häufig wie in den primären Tumorproben. Da die Gene, die eine hohe Methylierungsrate in den HNSCC-Proben aufwiesen, auch in einer Vielzahl der Normalgewebeproben methyliert waren, sind sie als Biomarker für HNSCC eher ungeeignet. Jedoch zeigt diese Methylierung ihre Relevanz in der Entstehung von HNSCC und könnte zur Abschätzung des Risikos einer Erkrankung an HNSCC dienen. Nur RASSF1A und RASSF4 waren in den Normalgewebsproben unmethyliert. Häufig werden HNSCC-Patienten mit rekombinantem Erythropoietin behandelt um das Allgemeinbefinden zu verbessern. Ein weiteres Ziel dieser Arbeit war deshalb, zu untersuchen, welchen Einfluss epigenetische Regulationsmechanismen auf die Genaktivität von Erythropoietin haben und wie sich die Reexpression von Erythropoietin auf das Proliferationsverhalten von HNSCC auswirkt. In dieser Arbeit zeigte sich, dass Erythropoietin sowohl in der Niere als auch in der Leber stark exprimiert wurde. Auch in der Plazenta ließ sich eine geringe Expression nachweisen. In den analysierten Krebszelllinien war die Expression im Vergleich zu der Zelllinie HepB3, die bekannt dafür war, Erythropoietin zu exprimieren, nur sehr gering und Methylierungsanalysen zeigten, dass sowohl Promotor als auch Enhancer dieser Zelllinien aber auch vieler primärer Tumore, insbesondere HNSCC-Tumore, Methylierungen aufwiesen. Aber auch die Normalgewebsproben der HNSCC-Patienten zeigten Methylierungen. In weitergehende Untersuchungen konnte nachgewiesen werden, dass die Hypermethylierung des Erythropoietin-Gens sowohl im Promotor als auch im Enhancer eine wichtige Rolle in der Regulation der Genaktivität spielt. Des Weiteren zeigte sich in dieser Arbeit, dass es bei der Induktion der Expression von Erythropoietin durch Hypoxie zur Acetylierung des Chromatins im Promotor- und auch im Enhancer-Bereich kommt. Wahrscheinlich ist für diese Modifikation des Chromatins eine Interaktion des HIF1-Komplexes mit dem Enhancer notwendig, die jedoch durch die Methylierung der HIF1alpha-Interaktionssequenz im Erythropoietin-Enhancer unterbunden werden kann. Reexpressionsanalysen in der HNSCC-Zelllinie Hep2 zeigten einen Proliferations-steigernden Effekt von Erythropoietin auf die Zellen sowohl unter Normoxie als auch unter Hypoxie. Eine Proliferationsteigerung in Krebszellen kann zu einem schnelleren Tumorwachstum führen, was den Tumor aggressiver machen und in einer erhöhten Invasivität und Metastasierung des Tumors resultieren kann. Aus diesem Grund sollte die Behandlung von HNSCC-Patienten mit rekombinantem Erythropoietin unter Abwägung der Vor- und Nachteile für den Patienten nochmals überdacht werden

Frequent epigenetic inactivation of RASSF2 in thyroid cancer and functional consequences

<p>Abstract</p> <p>Background</p> <p>The Ras association domain family (RASSF) encodes for distinct tumor suppressors and several members are frequently silenced in human cancer. In our study, we analyzed the role of RASSF2, RASSF3, RASSF4, RASSF5A, RASSF5C and RASSF6 and the effectors MST1, MST2 and WW45 in thyroid carcinogenesis.</p> <p>Results</p> <p>Frequent methylation of the <it>RASSF2 </it>and <it>RASSF5A </it>CpG island promoters in thyroid tumors was observed. <it>RASSF2 </it>was methylated in 88% of thyroid cancer cell lines and in 63% of primary thyroid carcinomas. <it>RASSF2 </it>methylation was significantly increased in primary thyroid carcinoma compared to normal thyroid, goiter and follicular adenoma (0%, 17% and 0%, respectively; p < 0.05). Patients which were older than 60 years were significantly hypermethylated for <it>RASSF2 </it>in their primary thyroid tumors compared to those younger than 40 years (90% vs. 38%; p < 0.05). <it>RASSF2 </it>promoter hypermethylation correlated with its reduced expression and treatment with a DNA methylation inhibitor reactivated <it>RASSF2 </it>transcription. Over-expression of RASSF2 reduced colony formation of thyroid cancer cells. Functionally our data show that RASSF2 interacts with the proapoptotic kinases MST1 and MST2 and induces apoptosis in thyroid cancer cell lines. Deletion of the MST interaction domain of RASSF2 reduced apoptosis significantly (p < 0.05).</p> <p>Conclusion</p> <p>These results suggest that <it>RASSF2 </it>encodes a novel epigenetically inactivated candidate tumor suppressor gene in thyroid carcinogenesis.</p

The SARAH Domain of RASSF1A and Its Tumor Suppressor Function

The Ras association domain family 1A (RASSF1A) tumor suppressor encodes a Sav-RASSF-Hpo domain (SARAH), which is an interaction domain characterized by hWW45 (dSAV) and MST1/2 (dHpo). In our study, the interaction between RASSF1A and RASSF1C with MST1 and MST2 was demonstrated and it was shown that this interaction depends on the SARAH domain. SARAH domain-deleted RASSF1A had a similar growth-reducing effect as full-length RASSF1A and inhibited anchorage independent growth of the lung cancer cell lines A549 significantly. In cancer cells expressing the SARAH deleted form of RASSF1A, reduced mitotic rates (P = 0.001) with abnormal metaphases (P < 0.001) were observed and a significantly increased rate of apoptosis was found (P = 0.006) compared to full-length RASSF1A. Although the association with microtubules and their stabilization was unaffected, mitotic spindle formation was altered by deletion of the SARAH domain of RASSF1A. In summary, our results suggest that the SARAH domain plays an important role in regulating the function of RASSF1A

Peptide Receptor Radionuclide Therapy for a Phosphaturic Mesenchymal Tumor.

Tumor-induced osteomalacia is a very rare paraneoplastic syndrome. It can be caused by phosphaturic mesenchymal tumor (PMT), a generally benign tumor that produces fibroblast growth factor 23 (FGF-23), which can cause a severe renal phosphate wasting syndrome. Upon complete surgical removal of the tumor, FGF-23 normalizes and the osteomalacia is cured. In cases in which surgery is not feasible, radiofrequency ablation (RFA) is the treatment of choice. We describe a case with a PMT situated in the sacrum, in close proximity to the sacral plexus. Both surgery and RFA were considered potentially nerve damaging. Since the tumor showed expression of somatostatin receptors, we opted for a peptide receptor radionuclide therapy (PRRT) with 177Lu-DOTATOC. However, the therapy did not show the expected success, since the FGF-23 level had even temporarily increased. The patient was then successfully treated with RFA. A partial remission of the tumor was achieved and FGF-23 levels nearly normalized. Despite some severe neurological side effects, the patient showed a remarkable clinical improvement, with no symptoms of osteomalacia within a few weeks

Hepatic ILC2 activity is regulated by liver inflammation-induced cytokines and effector CD4+ T cells

In immune-mediated hepatitis, type 2 innate lymphoid cells (ILC2) as well as effector CD4+ T cells have been shown to drive disease pathology. However, less is known about mechanisms involved in the regulation of ILC2 function during liver inflammation. We showed that in homeostasis, hepatic ILC2 constituted a very small population with a naive, inactive phenotype. During immune-mediated hepatitis, the cytokines IL-33 and IFNγ were expressed in liver tissue. IL-33 induced strong activation and expression of type 2 cytokines as well as IL-6 by hepatic ILC2 while IFNγ suppressed cytokine production. Interestingly, this inhibitory effect was overcome by IL-33. The phenotype of activated hepatic ILC2 were stable since they did not show functional plasticity in response to liver inflammation-induced cytokines. Moreover, hepatic ILC2 induced a Th2 phenotype in activated CD4+ T cells, which increased ILC2-derived cytokine expression via IL-2. In contrast, Th1 cells inhibited survival of ILC2 by production of IFNγ. Thus, hepatic ILC2 function is regulated by IL-33, IL-2, and IFNγ. While IL-33 and IL-2 support hepatic ILC2 activation, their inflammatory activity in immune-mediated hepatitis might be limited by infiltrating IFNγ-expressing Th1 cells

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24-33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance

Orally active antischistosomal early leads identified from the open access malaria box.

BACKGROUND: Worldwide hundreds of millions of schistosomiasis patients rely on treatment with a single drug, praziquantel. Therapeutic limitations and the threat of praziquantel resistance underline the need to discover and develop next generation drugs. METHODOLOGY: We studied the antischistosomal properties of the Medicines for Malaria Venture (MMV) malaria box containing 200 diverse drug-like and 200 probe-like compounds with confirmed in vitro activity against Plasmodium falciparum. Compounds were tested against schistosomula and adult Schistosoma mansoni in vitro. Based on in vitro performance, available pharmacokinetic profiles and toxicity data, selected compounds were investigated in vivo. PRINCIPAL FINDINGS: Promising antischistosomal activity (IC50: 1.4-9.5 µM) was observed for 34 compounds against schistosomula. Three compounds presented IC50 values between 0.8 and 1.3 µM against adult S. mansoni. Two promising early leads were identified, namely a N,N'-diarylurea and a 2,3-dianilinoquinoxaline. Treatment of S. mansoni infected mice with a single oral 400 mg/kg dose of these drugs resulted in significant worm burden reductions of 52.5% and 40.8%, respectively. CONCLUSIONS/SIGNIFICANCE: The two candidates identified by investigating the MMV malaria box are characterized by good pharmacokinetic profiles, low cytotoxic potential and easy chemistry and therefore offer an excellent starting point for antischistosomal drug discovery and development

The Novel Immunosuppressive Protein Kinase C Inhibitor Sotrastaurin Has No Pro-Viral Effects on the Replication Cycle of Hepatitis B or C Virus

The pan-protein kinase C (PKC) inhibitor sotrastaurin (AEB071) is a novel immunosuppressant currently in phase II trials for immunosuppression after solid organ transplantation. Besides T-cell activation, PKC affects numerous cellular processes that are potentially important for the replication of hepatitis B virus (HBV) and hepatitis C virus (HCV), major blood-borne pathogens prevalent in solid organ transplant recipients. This study uses state of the art virological assays to assess the direct, non-immune mediated effects of sotrastaurin on HBV and HCV. Most importantly, sotrastaurin had no pro-viral effect on either HBV or HCV. In the presence of high concentrations of sotrastaurin, well above those used clinically and close to levels where cytotoxic effects become detectable, there was a reduction of HCV and HBV replication. This reduction is very likely due to cytotoxic and/or anti-proliferative effects rather than direct anti-viral activity of the drug. Replication cycle stages other than genome replication such as viral cell entry and spread of HCV infection directly between adjacent cells was clearly unaffected by sotrastaurin. These data support the evaluation of sotrastaurin in HBV and/or HCV infected transplant recipients

Epigenetic Silencing of Erythropoietin in Human Cancers

The glycoprotein hormone erythropoietin (EPO) is a key regulator in the production of red blood cells. EPO is produced mainly in the embryonic liver and kidney of adults. Other organs are also known to express varying amounts of EPO. In our study, we have analyzed the epigenetic regulation of EPO in human cancer cell lines by DNA methylation assays, chromatin immunoprecipitation, RT-PCR, and promoter analysis under different growth conditions. Moreover, the growth-related effects of ectopic EPO expression were analyzed in a head and neck cancer cell line. We found frequent DNA hypermethylation of the CpG island promoter and enhancer of EPO in different cancer cell lines. Aberrant methylation of EPO promoter was observed in primary lung, head and neck, breast, and liver cancers. Hypermethylation of EPO was associated with a decreased expression of EPO in cancer cells. Treatment of cancer cell lines with 5-aza-2′-deoxycytidine (Aza), an inhibitor of DNA methylation, reactivated EPO expression under hypoxia. In contrast, in the liver cancer cell line HepB3, the EPO promoter was unmethylated, and a high EPO expression was observed independently of Aza treatment. Moreover, in vitro hypermethylation of the EPO promoter and enhancer reduced expression of a reporter gene under normoxia and hypoxia. Induction of EPO under hypoxia was accompanied by increased histone H3 acetylation and reduced histone H3 lysine 9 trimethylation. In a head and neck cancer cell line, which exhibited low EPO levels, ectopic expression of EPO significantly enhanced proliferation under normoxia and hypoxia. In summary, we show that hypermethylation of regulatory sequences of EPO is frequently observed in tumors and that this aberrant methylation induces epigenetic silencing of EPO in cancer cells