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Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer that has been associated with the Merkel cell polyomavirus (MCPyV). Indeed, MCC is one of the cancers with the best-established viral carcinogenesis. Despite persistence of the virus in MCC cells and the subsequent expression of viral antigens, the majority of MCC tumors are able to escape the surveillance of the immune system. Therefore the aim of the here presented thesis was to scrutinize immune escape mechanisms operative in MCC. A better understanding of their underlying molecular processes should allow to improve immunotherapeutic treatment strategies for MCC patients. The manuscripts included in this thesis characterize three novel immune evasion strategies of MCC. I) the epigenetic silencing of the NKG2D ligands MICA and MICB via histone H3 hypoacetylation II) reduced HLA class I surface expression via epigenetic silencing of the antigen processing machinery (APM) III) the activation of the PI3K-AKT pathway in a mutation independent manner as potential immune escape strategy MCC tumors and MCC cell lines were analyzed for their expression of MICA/B, HLA and components of the antigen processing machinery as well as for the activation of the PI3K-AKT pathway in situ and in vitro. These analysis reviled MICA and MICB, as well as HLA class I were not expressed or at least markedly reduced in ~80% of MCCs in situ. The PI3K-AKT pathway, that had only recently been demonstrated to play a significant role in tumor immune escape, was activated in almost 90% of MCCs in situ. To determine the underlying molecular mechanisms of these aberrations well characterized MCC cell lines were further analyzed in vitro. The fact that the PI3K-AKT pathway activation was due to oncogenic mutations in the PIK3CA or AKT1 gene in only 10% of MCCs, suggested an epigenetic regulation of this pathway in MCC. In line with this MICA/B as well as components of the APM were indeed silenced epigenetically via histone hypoacetylation in their respective promoter region. Notably MICA/B and HLA class I expression on the cell surface of MCC cells could be restored after treatment with HDAC inhibitors in combination with the Sp1 inhibitor Mithramycin A in all analyzed MCC cell lines in vitro and in a xenotransplantation mouse model in vivo. Moreover inhibition of HDACs increased immune recognition of MCC cell lines in a MICA/B and HLA class I dependent manner. Several studies have accumulated evidence that immunotherapy is a promising treatment option for MCC patients due to the exquisite immunogenicity of this malignancy. However, current immunotherapeutic interventions towards solid tumors like MCC have to account for the plentitude of tumor immune escape strategies, in order to increase response rates. The immune escape mechanisms of MCC described in this thesis can be reverted by HDAC inhibition, thus providing the rationale to combine ‘epigenetic priming’ with currently tested immunotherapeutic regimens. / Das Merkelzellkarzinom (MCC) ist ein aggressiver neuroendokriner Hautkrebs, der mit dem Merkelzell-Polyomavirus (MCPyV) assoziiert ist. Das MCC ist eine der Krebserkrankungen mit der am besten etablierten viralen Karzinogenese. Trotz der Anwesenheit des MCPyV in MCC-Zellen und der daraus einhergehenden Expression viraler Antigene sind die meisten MCC-Tumoren in der Lage der Überwachung durch das Immunsystem zu entgehen. Aus diesem Grund war das Ziel der hier vorliegenden Arbeit, neue im MCC operative „immune escape“ Mechanismen zu ermitteln. Ein besseres Verständnis der hierbei zugrunde liegenden Mechanismen, sollte es ermöglichen, immuntherapeutische Behandlungsstrategien für MCC-Patienten zu verbessern. Die vorgestellten Manuskripte beschreiben drei neuartige „immune evasion“ Strategien des MCC: I) die epigenetische Inaktivierung der NKG2D-Liganden MICA und MICB mittels Histone-H3-Hypoacetylierung II) eine reduzierte HLA Klasse I-Oberflächenexpression aufgrund epigenetischer Inaktivierung der Antigenprozessierungsmaschinerie (APM) III) die mutationsunabhängige Aktivierung des PI3K-AKT-Signalweges, als potentieller „immune escape“ Mechanismus MCC-Tumoren und MCC-Zelllinien wurden sowohl bezüglich der Expression von MICA/B, HLA Klasse I und Komponenten der APM als auch auf die Aktivierung des PI3K Signalweges in situ und in vitro untersucht. Diese Analysen zeigten, dass sowohl MICA und MICB als auch HLA Klasse I in ca. 80% der MCC-Tumoren in situ nicht, oder nur sehr reduziert, exprimiert wurden. Der PI3K-AKT-Signalweg, welcher erst kürzlich mit Tumor „immune escape“ in Verbindung gebracht wurde, war in fast 90% aller MCC-Tumoren in situ aktiviert. Um die zugrunde liegenden molekularen Mechanismen dieser Aberrationen zu entschlüsseln, wurden gut charakterisierte MCC-Zelllinien in vitro untersucht. Die Tatsache, dass der PI3K-AKT-Signalweg in nur 10% der MCCs auf Mutationen im PI3KA- oder AKT1-Gen zurückzuführen war, suggeriert eine epigenetische Regulation dieses Signalwegs. In Übereinstimmung hiermit waren sowohl MICA/B als auch Gene der APM epigenetisch mittels Histon-Hypoacetylierung in ihren jeweiligen Promoterregionen inaktiviert. Bemerkenswerterweise konnten in vitro und in einem Xenotransplantations-Mausmodell in vivo sowohl die MICA/B als auch die HLA Klasse I-Oberflächenexpression aller untersuchter MCC-Zelllinien durch die Behandlung mit HDAC-Inhibitoren in Kombination mit dem Sp1-Inhibitor Mithramycin A wieder hergestellt werden. Des Weiteren erhöhte die Inhibition von HDACs die MCC-Immunerkennung auf eine MICA/B und HLA Klasse I-abhängige Weise. Zahlreiche aktuelle Studien bestärken die Annahme, dass aufgrund der besonderen Immunogenität des MCC, die Immuntherapie eine aussichtsreiche Behandlungsoption für MCC-Patienten darstellt. Nichtsdestotrotz müssen die derzeitigen immuntherapeutischen Methoden zur Behandlung solider Tumore die Vielzahl von Tumor „immune escape“ Mechanismen mitberücksichtigen, um die Ansprechrate zu erhöhen. Die Tatsache, dass die hier beschriebenen „immune escape“ Mechanismen durch HDAC-Inhibition aufgehoben werden können, spricht für die Hypothese, dass eine Kombination von „epigenetischem Priming“ mit derzeitig untersuchten immuntherapeutischen Ansätzen sinnvoll ist.

Diss.--Münster, 1993. / Contient le catalogue des oeuvres de G.A. Merkel p. 298-430. Bibliogr. p. 440-455. Index.

En 2008, le génome d’un nouveau virus a été caractérisé au sein d’un cancer cutané rare survenant préférentiellement chez l’immunodéprimé, le carcinome de Merkel. Ce nouveau virus appartenait à la famille des Polyomaviridae qui comprend des virus dont le caractère cancérigène chez l’animal est avéré depuis plus de 50 ans. Dénommé Polyomavirus de Merkel puisqu’il semblait lié à la survenue du cancer du même nom, il constituait le premier Polyomavirus impliqué de manière consistante dans un cancer humain. Cette implication reposant sur une étude unique limitée à 10 cas, l’objectif de notre travail de thèse était de confirmer le rôle étiologique du Polyomavirus de Merkel dans le carcinome de Merkel.Nous avons montré que le génome du Polyomavirus de Merkel était présent dans les trois quarts des cas de carcinome de Merkel, mais également que le virus infecte la population générale de manière quasi-ubiquitaire et de nombreux tissus en dehors de la peau. Les faits que chez les sujets atteints de carcinome de Merkel, l’ADN viral soit présent à des taux décelables de manière chronique dans différents tissus et que les titres d’anticorps sériques spécifiques du virus soient élevés suggèrent que ces sujets développent une infection chronique active. Celle-ci pourrait faciliter la survenue de mutations et d’intégrations de l’ADN viral qui sont spécifiquement associées aux carcinomes de Merkel. Ces modifications secondaires du génome viral aboutissent à la production d’oncoprotéines virales par les cellules tumorales, mais à l’abolition des capacités réplicatives donc lytiques du virus et constitueraient ainsi le support de la transformation tumorale. / Nucleotidic sequences defining the genome of a new virus, the Merkel Cell Polyomavirus, has been discovered in 2008 in Merkel cell carcinoma, a rare form of cutaneous cancer developing mostly in immunosupressed individuals. Whereas this new virus belongs to the Polyomaviridae family, which includes known oncogenic viruses in animals, it was the first study consistently implicating a Polyomavirus in human cancer. Because scientific arguments were only based on a ten-case-single report, the primary goal of our work was to confirm the role of the Merkel Cell Polyomavirus in Merkel Cell Carcinoma.Our work demonstrated that Merkel Cell Polyomavirus DNA was indeed present in three quarters of Merkel Cell Carcinoma cases, but also that Merkel Cell Polyomavirus was a near ubiquitous virus infecting various tissues among healthy individuals. Nonetheless, viral DNA is chronically detected in various tissues from Merkel Cell Carcinoma cases, which harbour elevated seric titters of specific antibodies. Those facts suggest that Merkel Cell Polyomavirus develop an active and chronic infection that could favour genomic mutation and integration events specifically associated to Merkel Cell Carcinoma. Those modifications, inducing both expression of truncated viral oncoproteins and abolishment of cell lysis mediated by viral replication, may support cell transformation.

Title: Merkel with NSA on the telephone The aim of this essay is to study how Angela Merkel is being portrayed in context of NSAs surveillance in the Swedish newspapers Dagens Nyheter and Aftonbladet. The time period for the study is from the 24th of October to the 7th of November in the autumn of 2013. To concretize the aim three questions was formulated, how Angela Merkel was being portrayed in the material, how NSAs surveillance of Angela Merkel was being portrayed in the material and how different statements for various individuals in the material could affect the truth about the surveillance. The method chosen to study these questions was a discourse analysis, which also would contain parts of a linguistic analysis. The theories to support the method were Laclau & Mouffes discourse theory and Michel Foucault’s genealogical discourse analysis. The results of the analysis points to that Angela Merkel are being portrayed with titles, which represent her position in a democratic society. Words such as “head of government” “Bundeskansler” and “Kansler” are all frequently used to describe Angela Merkel’s position. Other findings points to that Angela Merkel’s position in the material is defined trough relationships with other political actors and leaders. The surveillance itself had in the material mostly negative effects on the relationships between the USA/Obama and EU/Germany/Merkel and was also pointed out as a problem for the on going talks of the FTA. More findings include the battle of the truth surrounding the surveillance, where at one side the USA with representative leaders from agencies like NSA and resides and on the other side journalists and leaders from EU resides. Both parties produce new knowledge with which they attempt to make a claim at the truth. Many conclusions that can be drawn from this essay the first is the way journalists use elites to verify and strengthen its articles. The second is the synoptic power that the media posses with which the members of societies can watch its leaders. And third, the way, in these case, the politicians gets there power and position verified due to different relationships with other political leaders of powerful individuals. / Titel: Merkel med NSA på andra sidan luren Syftet med den undersökning var att studera hur Angela Merkel blir framställd i en kontext av NSAs övervakning i de svenska tidningarna Dagens Nyheter och Aftonbladet. Tidsperioden som studeras är mellan den 24 oktober till och med den 7:e november 2013. Syftet konkretiserades genom tre frågor, hur Angela Merkel framställdes i materialet, hur NSAs övervakning av Angela Merkel framställdes samt hur olika uttalanden från olika personer i materialet kunde påverka sanningen om övervakningen. Metoden som användes för undersökningen var en diskursanalys, vilken även innehöll delar av en lingvistiskanalys. De teorier som valdes för att stötta metoden var Laclau & Mouffes diskursteori samt Michel Foucaults genealogiska diskursanalys. Undersökningens resultat pekar på att Angela Merkel blev framställd genom titlar, vilka representerar hennes position i ett demokratiskt samhälle. Ord som ”regeringschef, ”Bundeskansler” och Kansler var frekvent använda för att beskriva Angela Merkels position. Andra analysresultat pekar på att Angela Merkels position ofta blir definierad genom relationer med andra politiska aktörer och ledare. Själva övervakningen hade för det mesta negativa effekter på relationerna mellan USA/Obama och Eu/Tyskland/Merkel. Övervakningen pekades också ut som ett problem i de pågående förhandlingarna om frihandelsavtalet mellan EU och USA. Vidare framkom det en kamp om sanningen mellan aktörerna i materialet. På ena sidan fanns USA med representativa ledare från institutioner som NSA. På andra sidan fanns journalister och ledare från EU. Båda sidorna producerade ny kunskap om vad som hade skett, med vilken kunskap de försökte göra anspråk på sanningen. Många slutsatser går att dra från denna undersökning, den första är hur journalister använder ”eliter” för att verifiera och stärka sina artiklar. Den handlar om den synoptiska makten som medier innehar, genom vilken medlemmar av samhället kan övervaka sina ledare. Den tredje är på vilket sätt politiker kan få sina positioner och makt bekräftade genom olika relationer de har till andra politiskaledare eller mäktiga individer.

Diss.--Historisch-Philosophische Fakultät--Universität Göttingen, Wintersemester 1987/88. / Bibliogr. p. 257-287.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer. In approximately 80% of cases, genomic integration of the Merkel cell polyomavirus (MCPyV) is observed and overexpression of the two MCPyV T antigens (TAgs) is regarded as the main oncogenic determinant of MCPyV-positive MCC cases. However, the nature of the cells from which MCC arises is unknown. Therefore, the goal of the present work was to determine the cell of origin of MCC. First, we characterized MCC patients’ tumors and demonstrated a high similarity of MCPyV- negative MCC with extracutaneous neuroendocrine carcinoma while MCPyV-positive MCC differs from these two groups with respect to morphology, immunohistochemical profile, genetics, origin and behavior. Based on the analysis of a trichoblastoma/MCC combined tumor, we demonstrated that a MCPyV-positive MCC can arise following MCPyV integration in an epithelial cell. In addition, the high similarity between trichoblastoma cells and Merkel cell (MC) progenitors of the hair follicle suggests that these hair follicle cells may represent a general start point for the development of MCPyV-positive MCC. A contribution of the viral TAgs to the development of the characteristic Merkel cell-like MCC phenotype is suggested by experiments demonstrating induction of Merkel cell markers upon TAg expression in human primary keratinocytes or hair follicle cells. As potential mechanisms mediating these phenotypic changes, we identified the capability of MCPyV LT to repress degradation of master regulator of MC development, i.e. the transcription factor ATOH1. To conclude, our work suggests that MCPyV integration in epithelial cells of the hair follicle may represent an important path for MCC development. / Das Merkelzellkarzinom (MCC) ist ein seltener und aggressiver Hautkrebs. In etwa 80% der Fälle wird die genomische Integration des Merkelzell-Polyomavirus (MCPyV) beobachtet und die Überexpression der beiden MCPyV-T-Antigene (TAgs) gilt als die wichtigste onkogene Determinante der MCPyV-positiven MCC-Fälle. Die Ursprungszelle des MCC ist jedoch bisher unbekannt. Daher war das Ziel der vorliegenden Arbeit, die Hinweise auf die Herkunftszelle zu generieren. ...

Merkel cell carcinoma (MCC) is a rare but highly aggressive neuroendocrine cutaneous cancer whose molecular biology is poorly characterized. Our broad research objective is to identify microRNAs (miRNA) that are biologically and/or clinically important in MCC. While attempting to establish an MCC-specific miRNA signature, we observed that microRNA-375 was the most highly expressed miRNA in primary MCC tumours relative to normal skin – an observation that I propose reflects miR-375’s specific association with neuroendocrine (NE) and secretory subpopulations within normal tissues. Here, I report that miR-375 is strikingly elevated in a range of NE tumour types compared with tissue-matched cancers of non-neuroendocrine origin. Furthermore, I show that miR-375 is expressed abundantly in a subset of MCC cell lines that possess the biochemical and immunohistochemical characteristics of NE cells, but is silenced in cell lines that fail to retain these markers. I demonstrate that the enforced expression of miR-375 induces a NE gene expression signature – a phenomenon that is mechanistically driven by the post-transcriptional repression of multiple Notch pathway components by miR-375. This work identifies the Notch pathway as a novel mechanistic link between the association of miR-375 and a NE cell fate, provides new insights into the cellular ancestry of MCC, and suggests that miR-375 could facilitate clinicopathological diagnosis of MCC and other NE tumours as a novel biomarker. miR-375 is silenced in “variant” MCC cell lines, and inversely correlates with cell doubling time and overall aggressiveness. Therefore, despite its high expression in most MCC tumours, I propose that miR-375 is an endogenous tumour suppressor. I show that the enforced expression of miR-375 inhibits cell viability, impairs cell migration and invasion, can oppose survival under stress, and represses the AKT pro-survival signaling pathway. Only siRNA-mediated inhibition of Notch2 and Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) phenocopied the effects of miR-375 overexpression. Because variant (miR-375low) cell lines originate from more aggressive tumours in both MCC and small cell lung carcinoma, I postulate that miR-375 silencing occurs in a subset of MCC patients and might predispose them to a highly virulent clinical course through the disinhibition of Notch signaling. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-08-15 09:59:09.832

by Senok, Silas Solomon. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 194-217). / ACKNOWLEDGEMENT --- p.ii / ABSTRACT --- p.v / Chapter 1 --- INTRODUCTION AND OBJECTIVES --- p.1 / Chapter 2 --- LITERATURE REVIEW --- p.8 / Chapter 2.1 --- Classification of Cutaneous Mechanoreceptors --- p.8 / Chapter 2.2 --- Characteristics of Some Mammalian Cutaneous Mechanoreceptors --- p.21 / Chapter 2.3 --- Vibrissae --- p.30 / Chapter 2.4 --- Mechanosensory Transduction --- p.40 / Chapter 2.5 --- The Merkel Cell Controversy --- p.44 / Chapter 2.6 --- Intracellular Calcium Mobilisation --- p.53 / Chapter 3 --- METHODS --- p.59 / Chapter 3.1 --- The Isolated Vibrissal Preparation --- p.59 / Chapter 3.2 --- Experimental Protocol --- p.77 / Chapter 3.3 --- Data Analysis --- p.79 / Chapter 4 --- RESULTS --- p.82 / Chapter 4.1 --- Characterisation of The Receptors --- p.82 / Chapter 4.2 --- Stability And Viability of The Preparation --- p.94 / Chapter 4.3 --- Pharmacologic Studies --- p.103 / Chapter 5 --- DISCUSSION --- p.160 / Chapter 5.1 --- Outcome of Project --- p.160 / Chapter 5 2 --- The Isolated Vibrissal Preparation --- p.162 / Chapter 5.3 --- The Vibrissal Slowly Adapting Mechanoreceptors --- p.165 / Chapter 5 4 --- Mechanism of Chloroquine Inhibtion of Merkel Cell Receptors --- p.170 / Chapter 5.5 --- Evidence For CICR In Merkel Cell Endings --- p.175 / Chapter 5.6 --- Mechano-Electric Transduction In Merkel Cell Receptors --- p.182 / Chapter 5.7 --- Transduction in St II Nerve Terminals --- p.190 / Chapter 5.8 --- What Next? --- p.192 / Chapter 5.9 --- Conclusion --- p.193 / REFERENCES --- p.194

Touch sensation is initiated by diverse mechanosensory neurons that innervate distinct skin structures; however, little is known about how touch receptors are patterned during mammalian skin development. During the course of my PhD training, I analyzed embryonic and neonatal development of mouse touch domes, which contain Merkel cell-neurite complexes that encode pressure and object features. I found that developing touch domes share three key features with canonical sensory placodes: discrete patches of specialized epithelial, co-clustered mesenchymal cells capable of engaging in molecular crosstalk with the epithelium, and selective recruitment of sensory neurons. During embryogenesis, molecularly distinct patches of epithelial Merkel cells and keratinocytes clustered with a previously unsuspected population of BMP4-expressing dermal fibroblasts in nascent touch domes. Concurrently, two populations of sensory neurons preferentially targeted touch domes compared with other skin regions. Surprisingly, only one neuronal population persisted in mature touch domes. Overexpression of Noggin, a BMP antagonist, in epidermis at embryonic age 14.5 resulted in fewer touch domes, a loss of Merkel cells, and decreased innervation density in skin areas where touch domes are typically found. Thus, touch domes bear hallmarks of placode-derived sensory epithelia that require BMP signaling for proper specification.