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

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with rising global incidence. Merkel cell polyomavirus (MCV) was discovered in 2008 in 80% of MCC samples and since then a causal link between MCV and the majority of MCC cases has been established. microRNAs (miRNA, miR) are a family of small non-coding RNAs which play a key role in post-transcriptional regulation of gene expression and are considered significant players in disease and development in many species. Whilst the focus of MCV research has thus far been on the oncogenic MCV early proteins, large tumour (LT) and small tumour (sT) antigens, there is a knowledge gap regarding MCV miRNA and its functional significance in MCV pathogenesis. Given the emerging importance of viral miRNAs in virus-host interaction and pathogenesis, the aim of this doctoral research project was to investigate alterations in host cell transcripts induced by MCV miRNA and determine any functional significance these might have on virus-host cell interaction. RNA sequencing (RNA-Seq) in the presence and absence of MCV miRNA uncovered a multitude of downregulated cellular transcripts. Gene ontology analysis revealed that MCV miRNA targets transcripts associated with multiple cellular processes, however, regulation of immune response was overrepresented in our datasets. Validation of RNA-Seq data using MCV miRNA mimics and a synthetic, fully replicative MCV genome (MCVSyn) confirmed RNA-Seq data at mRNA and protein expression level for several targets, including the cytokine stimulating gene, SP100, and the neutrophil stimulator chemokine, CXCL8. Moreover, dual luciferase assays revealed that SP100 and MAPK10 (a member of mitogen-activated protein kinases (MAPK) family which is involved in regulation of CXCL8 expression) are directly and specifically targeted and downregulated by MCV miRNA. The MCV miRNA-dependent dysregulation of CXCL8 secretion is associated with impaired neutrophil migration, suggesting that the virus miRNA may be implicated in evasion of the host immune response.

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.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with rising global incidence. Merkel cell polyomavirus (MCV) was discovered in 2008 in 80% of MCC samples and since then a causal link between MCV and the majority of MCC cases has been established. microRNAs (miRNA, miR) are a family of small non-coding RNAs which play a key role in post-transcriptional regulation of gene expression and are considered significant players in disease and development in many species. Whilst the focus of MCV research has thus far been on the oncogenic MCV early proteins, large tumour (LT) and small tumour (sT) antigens, there is a knowledge gap regarding MCV miRNA and its functional significance in MCV pathogenesis. Given the emerging importance of viral miRNAs in virus-host interaction and pathogenesis, the aim of this doctoral research project was to investigate alterations in host cell transcripts induced by MCV miRNA and determine any functional significance these might have on virus-host cell interaction. RNA sequencing (RNA-Seq) in the presence and absence of MCV miRNA uncovered a multitude of downregulated cellular transcripts. Gene ontology analysis revealed that MCV miRNA targets transcripts associated with multiple cellular processes, however, regulation of immune response was overrepresented in our datasets. Validation of RNA-Seq data using MCV miRNA mimics and a synthetic, fully replicative MCV genome (MCVSyn) confirmed RNA-Seq data at mRNA and protein expression level for several targets, including the cytokine stimulating gene, SP100, and the neutrophil stimulator chemokine, CXCL8. Moreover, dual luciferase assays revealed that SP100 and MAPK10 (a member of mitogen-activated protein kinases (MAPK) family which is involved in regulation of CXCL8 expression) are directly and specifically targeted and downregulated by MCV miRNA. The MCV miRNA-dependent dysregulation of CXCL8 secretion is associated with impaired neutrophil migration, suggesting that the virus miRNA may be implicated in evasion of the host immune response.

No description available.

Yes / Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is recognised as the causative factor in the majority of MCC cases. The MCPyV small tumour antigen (ST) is considered to be the main viral transforming factor, however potential mechanisms linking ST expression to the highly metastatic nature of MCC are yet to be fully elucidated. Metastasis is a complex process, with several discrete steps required for the formation of secondary tumour sites. One essential trait that underpins the ability of cancer cells to metastasise is how they interact with adjoining tumour cells and the surrounding extracellular matrix. Here we demonstrate that MCPyV ST expression disrupts the integrity of cell-cell junctions, thereby enhancing cell dissociation and implicate the cellular sheddases, A disintegrin and metalloproteinase (ADAM) 10 and 17 proteins in this process. Inhibition of ADAM 10 and 17 activity reduced MCPyV ST-induced cell dissociation and motility, attributing their function as critical to the MCPyV-induced metastatic processes. Consistent with these data, we confirm that ADAM 10 and 17 are upregulated in MCPyV-positive primary MCC tumours. These novel findings implicate cellular sheddases as key host cell factors contributing to virus-mediated cellular transformation and metastasis. Notably, ADAM protein expression may be a novel biomarker of MCC prognosis and given the current interest in cellular sheddase inhibitors for cancer therapeutics, it highlights ADAM 10 and 17 activity as a novel opportunity for targeted interventions for disseminated MCC. / In parts by the Medical Research Council (95505126) to AW, Royal Society (UF100419) to JM and Biotechnology and Biological Sciences Research Council (BB/R000352/1) to GEB and AW.

Le carcinome de Merkel est un cancer cutané de différenciation neuroendocrine rare, mais agressif, dont le facteur étiologique principal est le polyomavirus de Merkel (MCPyV). L’objectif de ce travail a été d’identifier des marqueurs virologiques et cellulaires pronostiques ou théranostiques à l’aide d’une cohorte historicoprospective de patients ayant un carcinome de Merkel. Les patients ayant des titres élevés d’anticorps dirigés contre la protéine de capside VP1 du MCPyV ont un pronostic favorable, tandis les anticorps dirigés contre les oncoprotéines virales reflètent l’évolution tumorale. Par ailleurs, il existe une hétérogénéité d’expression des récepteurs à la somatostatine dans les carcinomes de Merkel. Ce marqueur cellulaire peut constituer un outil théranostique lors de thérapies ciblées utilisant les analogues de la somatostatine. Enfin, nos travaux actuels portent sur l’évaluation de l’immunité cellulaire chez ces patients, avec une étude ayant montré la valeur pronostique du ratio neutrophiles/lymphocytes sanguin. / Merkel Cell Carcinoma is a rare and aggressive neuroendocrine skin cancer. The Merkel cell polymavirus has been identified as the main etiological agent of such cancer. We aimed to identify viral and cellular markers relevant as prognostic and theranostic tools in patients with Merkel Cell Carcinoma. Using serological immunoassays, we observed that patients with high levels of serum antibodies against the MCPyV major capsid protein at baseline had better outcomes, whereas antibodies directed against the MCPyV oncoproteins reflected the tumor burden and course of disease. We also demonstrated that Merkel Cell Carcinoma tumors displayed a heterogeneous expression of receptors to somatostatin, which could constitute a theranostic tool for the use of targeted therapies using somatostatin analogs. Finally, current studies focus on the assessment of cellular immunity in Merkel Cell Carcinoma patients. Our results indicate that the blood neutrophil-to-lymphocyte ratio is an independent marker of mortality in Merkel Cell Carcinoma patients.

Le rôle des cellules de Merkel dans la sensibilité cutanée reste imprécis. Elles assurent la décharge continue du récepteur de Merkel lors d'une pression sur la peau, mais on ne connait ni les autres stimuli capables de les activer, ni les médiateurs régulant leur activité. Cette ignorance est en partie liée à la difficulté d'isoler ces cellules qui ne représentent que 3 à 5% des cellules de la peau.Dans ce travail nous avons développé une technique de culture des cellules de Merkel à partir de peau humaine, en utilisant un tri cellulaire basé sur l'expression du récepteur CD56. Nous avons alors montré que les cellules de Merkel sont thermosensibles. Leur sensibilité aux températures fraiches est associée au fonctionnement du canal TRPM8. Cette sensibilité thermique ne module pas le fonctionnement du récepteur à une stimulation tactile. En revanche, les contacts entre les fibres cutanées C et Aδ, qui sont connues pour véhiculer les sensations thermiques, et les cellules de Merkel suggèrent que ces récepteurs pourraient intervenir aussi dans la thermosensation. Nous proposons donc pour la première fois que les récepteurs de Merkel soient aussi des récepteurs thermosensibles assurant une détection concordante de la pression et de la température cutanée. / The role of Merkel cells in cutaneous sensitivity remains imprecise. They provide continuous discharge of the receptor to a pressure. Nevertheless, other stimuli able to activate this complex, mediators regulating their activity are unknown. This ignorance is partly related to the difficulty to isolate these cells that represent only 3 to 5 % of skin cells.In this work, we have developed a Merkel cells cultured technique from human skin, using cell sorting based on the expression of the CD56 receptor. In this work, we show that Merkel cells are temperature sensitive. Their cool sensitivity is associated to TRPM8 channel. This thermal sensitivity does not modulate the discharge of the receptors during tactile stimulation. However, contacts between cutaneous Aδ and C fibres, which are known to carry the thermal sensations, and Merkel cells suggest that these receptors may also be involved in thermosensation. We propose for the first time that Merkel receptors are also temperature sensitive receptors providing a concurring detection of cutaneous pressure and temperature.

Merkel cell carcinoma (MCC) is a rare neuroendocrine malignancy of the skin that is highly aggressive and often metastasizes early. MCC is diagnosed based on histopathological findings and is most commonly treated with surgical resection, which may be accompanied by chemotherapy and/or radiation. This report describes a 55-year-old male with history of recurrent malignant melanoma of the right pinna and subsequent excision. Three years following the excision of melanoma, he presents with a lesion to the right forehead as well as a right-sided neck mass that were found to be metastatic Merkel cell carcinoma. Although there have been reports describing the development of second cancers following the treatment of MCC, the development of MCC after the treatment of other malignancies has not been well-described. Merkel cell carcinoma remains a highly aggressive and frequently metastatic malignancy that should not be overlooked, especially when developed after the diagnosis and treatment of other primary cutaneous malignancies such as melanoma.

Yes / Virus-encoded miRNAs are emerging as key regulators of persistent infection and host-cell immune evasion. Merkel cell polyomavirus (MCPyV), the predominant aetiological agent of Merkel cell carcinoma (MCC), encodes a single miRNA, MCV-miR-M1, which targets the oncogenic MCPyV large T antigen (LT). MCV-miR-M1 has previously been shown to play an important role in establishment of long-term infection, however, the underlying mechanism is not fully understood. A key unanswered question is whether, in addition to auto-regulating LT, MCV-miR-M1 also targets cellular transcripts to orchestrate an environment conducive for persistent infection. To address this, we adopted an RNA-Seq-based approach to identify cellular targets of MCV-miR-M1. Intriguingly, bioinformatics analysis of transcripts that are differentially expressed in cells expressing MCV-miR-M1 revealed several genes implicated in immune evasion. Subsequent target validation led to the identification of the innate immunity protein, SP100, as a direct target of MCV-miR-M1. Moreover, MCV-miR-M1-mediated modulation of SP100 was associated with a significant decrease in CXCL8 secretion, resulting in the attenuation of neutrophil chemotaxis towards Merkel cells harbouring synthetic MCPyV. Based on these observations we propose that MCV-miR-M1 targets key immune response regulators to help facilitate persistent infection, which is a pre-requisite for cellular transformation in MCC. / Funded in part by a University of Bradford studentship to PA and a Royal Society research award to JRB.