Skin cancers are very common in humans. The two most frequent epithelial skin cancers are the basal cell carcinoma (BCC) and the squamous cell carcinoma (SCC). For the vast majority of cancers, the cell at the origin of tumour initiation is still unknown and assumptions concerning their origin rely mainly on morphological and immunohistochemical studies. Recently, adult stem cells (SCs) have been suggested to be at the origin of tumour initiation based on their long term self-renewing capacities. According to these, two important questions arise; do epithelial skin cancers arise from mutations in a specific cell lineage of the epidermis? And are the stem cells more competent to initiate tumors than committed cells?<p>BCCs result from aberrant activation of HH signaling and several mouse models carrying mutations in HH signaling genes are capable to form tumors resembling to human BCCs. <p>To identify the cell lineage at the origin of BCC and to investigate the role of stem cells in tumor initiation, we followed a genetic approach where we conditionally expressed SmoM2 oncogene (a constitutively active Smoothened mutant) in distinct skin epidermal compartments including SCs. Targeting basal epidermis cells, showed that only SmoM2-clones in the inter follicular epidermis (IFE) and the infundibulum can progress into BCC, whereas SmoM2 expression in Bulge SCs or in matrix transit amplifying progenitor cells never leads to BCC formation. Progressively after SmoM2 expression, tumor-initiating cells lose their normal differentiation to adopt a hair placode-like shape and markers, demonstrating that biochemical and morphological tumour features can be misleading in extrapolating their cellular origin.<p><p>The molecular changes occurring in tumor initiating cells and the mechanisms regulating the early steps of cancer development are poorly characterized for the majority of tumors. To address these questions in BCC, we took advantage of our ability to isolate SmoM2 expressing cells at different stages of tumor initiation and progression. Transcriptional profiling of SmoM2-basal IFE cells isolated one week (normal histology) and 4 weeks (dysplastic lesion), suggests that adult IFE cells undergo a reprogramming into embryonic hair follicle (EHFP) like fate. In addition, we showed that Wnt/β-catenin signaling is essential for BCC initiating cell reprogramming into EHFP like fate and for tumor initiation in a cell autonomous manner. Finally, we show that EHFP reprogramming occurs also in human BCCs in addition to the presence of a similar canonical Wnt activation signature to the one revealed in the SmoM2-BCC mouse model.<p><p>SCC is the second most frequent skin cancers after BCC and mutations in p53 and Ras genes has been suggested to be potentially the primary events in this tumour. SCCs present signs of squamous differentiation, suggesting that SCCs may originate from the inter follicular epidermis (IFE). To identify the cell lineage at the origin of SCC and the role of the hair follicle SCs in tumor initiation, we use a genetic tools driving oncogenic KRas (KRasG12D) expression at physiological levels in different epidermal compartments. <p>Targeting KRasG12D expression in bulge SCs and their progeny or in IFE results in benign tumor development with no sign of malignant transformation. In contrast, KRasG12D expression in HF Transit amplifying (TA) matrix cells do not promotes any macroscopic tumors or microscopic defects in the epidermis. Interestingly, papillomas arising from the IFE express follicular markers such as CD34 and K17, indicating that the expression of HF markers by tumor cells does not necessarily reflect their cellular origin. Using a combination of deletion of both p53 alleles together with KRasG12D expression, we showed that bulge SCs and/or their progeny but not HF matrix TA cells, promote SCC formation, suggesting that additional genetic hits such as p53 are required to promote full-blown invasive skin SCC. <p><p>In summary, our work demonstrated the non-follicular origin of BCC resulting from Smo mutation, as well as the implication of the IFE progenitors in tumor initiation. We also revealed the progressive reprogramming of BCC initiating cells towards an EHFP-like fate and the key role of Wnt/β-catenin pathway in this process. In contrast, we showed the competence of several epidermal lineages to initiate benign tumors upon expression of KRasG12D oncogene at physiological levels. We also demonstrated that lineage -specific markers expression within tumor cells does not necessarily reflect their cellular origin. Finally, we demonstrated the requirement of additional hits, such as P53 loss, to promote malignant progression in the context of oncogenic Ras.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

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Objetivo: Identificar las características sociodemográficas y epidemiológicas del paciente con cáncer de piel diagnosticados en el servicio de anatomía patológica del Hospital Regional Lambayeque durante periodo enero del 2016 - diciembre del 2019. Métodos: La metodología empleada durante esta investigación estuvo basada en el diseño no experimental, descriptivo, de carácter retrospectivo, trasversal y observacional. Se incluyeron un total de 429 pacientes mayores de 18 años, diagnosticados con carcinoma cutáneo de tipo no melanoma (NPNM) y melanoma, mediante estudios anatomopatológicos de la lesión atendidos en Hospital Regional Lambayeque. La elección de la muestra fue mediante un muestreo no probabilístico de tipo censal, por la adaptabilidad al estudio. Resultados: De un total de 429 pacientes, 256(59,1%) tenían carcinoma basocelular (CBC), 146 (33,7%) carcinoma epidermoide (CsCC) y 31(7,2%) melanoma maligno cutáneo (MM). Siendo la edad promedio de aparición de 71 años en los NPNM y 62 años en el Melanoma Maligno Cutáneo, con predominio por el sexo femenino en el CBC y masculino en CsCC y MM. La ubicación anatómica más comprometida fue de la cabeza en los NPNM y miembros inferiores en MM, los cuales fueron identificadas mayormente por el servicio de Dermatología, seguido por Cirugía de Cabeza y Cuello del hospital. Los años con mayor número de carcinomas cutáneos fueron el 2019 para CBC y 2018 para los dos restantes. Conclusiones: La población general presenta más riesgo de presentar carcinomas no melanómico y en menor número el melanoma maligno, el cual predomina en áreas fotoexpuestas del cuerpo. / Objective: To identify the sociodemographic and epidemiological characteristics of the patient with skin cancer diagnosed in the pathological anatomy service of the Lambayeque Regional Hospital during the period January 2016 - December 2019. Methods: The methodology used during this investigation was based on the non-experimental design, descriptive, retrospective, cross-sectional and observational. A total of 429 patients over 18 years of age were included, diagnosed with non-melanoma skin carcinoma (NPNM) and melanoma, through anatomopathological studies of the lesion treated at Hospital Regional Lambayeque. The selection of the sample was by means of a non-probabilistic sampling of the census type, due to the adaptability to the study. Results: Of a total of 429 patients, 256 (59.1%) had basal cell carcinoma (BCC), 146 (33.7%) squamous cell carcinoma (SCC) and 31 (7.2%) cutaneous malignant melanoma (MM). Being the average age of appearance of 71 years in NPNM and 62 years in Cutaneous Malignant Melanoma, with a predominance of females in CBC and male in CsCC and MM. The most compromised anatomical location was the head in NPNM and lower limbs in MM, which were mostly identified by the Dermatology service, followed by Head and Neck Surgery at the hospital. The years with the highest number of skin carcinomas were 2019 for CBC and 2018 for the remaining two. Conclusions: The general population presents a higher risk of presenting non-melanoma carcinomas and a smaller number of malignant melanoma, which predominates in photo-exposed areas of the body.

In der vorliegenden Arbeit wurde die Expression von Angiotensinogen, Renin, Angiotensin-Converting-Enzym (ACE) und von den Agiotensin-Rezeptoren AT1 und AT2 in humaner Haut untersucht, um zu sehen, ob humane Haut ein lokales Gewebe Renin-Angiotensin-System (RAS) besitzt und fähig ist, Angiotensin II (Ang II) zu synthetisieren sowie welche physiologische Rolle Ang II in humaner Haut haben könnte. Außerdem wurde das Expressionsmuster von Angiotensinogen, Renin und ACE in gesunder humaner Haut mit dem in Psoriasis, Basaliom und Spinaliom (SCC) verglichen, um einen Einblick in pathophysiologische Funktionen des RAS zu gewinnen. Mit Hilfe von RT-PCR konnten alle Komponenten des RAS in vitro auf mRNA Ebene in kultivierten primären Keratinozyten, Melanozyten, dermalen Fibroblasten und dermalen mikrovaskulären Endothelzellen (MVEC´s) nachgewiesen werden, mit einer Ausnahme: Melanozyten scheinen keine AT2-Rezeptoren zu exprimieren. Immunhistochemische Untersuchungen zeigten die Expression aller Komponenten auf Proteinebene in Epidermis und dermalen Gefäßwänden in Gewebeschnitten humaner Haut. Zusätzlich erfolgte der Nachweis von Ang II in kultivierten Keratinozyten mittels enzymatischen immunometrischen Assays. Während Angiotensinogen, Renin und ACE bei immunhistochemischen Untersuchungen an Gewebeschnitten gesunder menschlicher Haut in allen Epidermalschichten gleichmäßig verteilt waren, zeigte sich bei der Psoriasis eine deutliche Betonung der unteren Epidermalschichten. Immunhistochemische Untersuchungen von Basaliomen erbrachten eine verminderte Expression von Angiotensinogen und Renin innerhalb der Tumornester. ACE wurde in den Tumorzellen noch weniger exprimiert. In immunhistochemischen Untersuchungen von Spinaliomen färbten sich die Tumorzellen deutlich homogen an. Die Experimente haben gezeigt, dass alle Komponenten des RAS in enger Lokalisation in menschlicher Haut vorkommen und dass folglich ein lokales Gewebe RAS in humaner Haut existiert sowie dass humane Haut fähig ist, Ang II ohne Zufuhr weiterer Komponenten und ohne regulatorische Einflüsse aus der Zirkulation zu synthetisieren. Eine mögliche physiologische Rolle von Ang II könnte die Regulation von Keratinozyten-Proliferation und –Differenzierung über seine Rezeptoren sein. Bezüglich der pathophysiologischen Rolle haben die Untersuchungen eine Fehlregulation des kutanen RAS in Epidermis psoriatisch veränderter Haut gezeigt, welches ein Hinweis auf eine pathogenetische Rolle des RAS bei der gestörten Keratinozyten-Proliferation und –Differenzierung sein könnte. Das Expressionsmuster in den untersuchten Tumoren war uneinheitlich, weshalb eine Interpretation der Rolle des RAS in kutanen Tumoren ohne weitere Untersuchungen kaum möglich erscheint. 1 / The present study was designed to elucidate whether a local tissue renin-angiotensin system (RAS) is expressed in human skin, whether cutaneous cells are able to autonomously synthesise angiotensin II (Ang II), and to get a first insight into a putative physiological role of Ang II in this location. For this purpose, the expression of angiotensinogen, renin, angiotensin-converting enzyme (ACE) and of the angiotensin receptors AT1 and AT2 was examined in human skin samples and in diverse cutaneous cells in primary culture on mRNA- and protein-level. Furthermore, the study compared the expression pattern of angiotensinogen, renin and ACE in healthy human skin with that in psoriasis, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) to look for possible differences between healthy and diseased skin. Using mRNA derived from cultured primary keratinocytes, melanocytes, dermal fibroblasts and dermal microvascular endothelial cells (MVECs), all components of the RAS could be demonstrated by RT-PCR except for AT2 receptors in melanocytes. Immunohistochemical stainings of cryostat sections of human skin revealed the expression of all components at protein level within the epidermis and in dermal vessel walls. In addition, the presence of Ang II in cultured keratinocytes and their supernatants could be proven by enzyme immunometric assay giving strong evidence for the ability of keratinocytes to autonomously synthesise Ang II. Regarding the comparison of RAS expression in healthy versus diseased skin, expression of angiotensinogen, renin and ACE was altered in all dermatoses examined. While in normal skin, RAS components were distributed equally and homogenously throughout all layers of the epidermis, in psoriatic skin their expression was more intense in the basal epidermal layers and less intense in the upper layers. In BCC sections, expression of angiotensinogen and renin was down-regulated, and tumour cells stained negatively for ACE. In SCC cryostat sections, tumour cells stained positively for all RAS components with an intensity comparable to normal skin. Taken together, the experiments revealed that a local tissue RAS exists in human skin, and that human skin is able to autonomously synthesise Ang II without any supply of components from the circulation. The physiological role of Ang II in normal skin may comprise the regulation of keratinocyte proliferation and differentiation. Concerning a putative pathophysiological role of Ang II in skin, this study provides evidence for a deregulation of the RAS in psoriatic skin and in BCC pointing to an involvement of the RAS in the pathomechanisms of these dermatoses. 1

Les genodermatoses sont des maladies génétiques rares à expression cutanée. Parmi elles, les dysplasies ectodermiques (DE) caractérisées par des anomalies du développement d’au moins deux structures ectodermiques (dents, ongles, glandes sudorales et poils), constituent un groupe hétérogène de genodermatoses de plus de 200 syndromes rares. Si la plupart de ces syndromes associent des anomalies des seuls dérivés ectodermiques, d'autres plus complexes, tels que le syndrome de Bazex-Dupré-Christol et l’Incontinentia Pigmenti, rassemblent en plus des manifestations disparates. Le syndrome de Bazex-Dupré-Christol (SBDC) associe une DE à la prédisposition aux carcinomes basocellulaires (CBCs) de survenue précoce. L’étude de 6 familles nous a permis d’identifier, chez 2 d’entre elles, une mutation tronquante dans le gène ACTRT1, codant la protéine Arp-T1. Dans l’épiderme, la protéine Arp-T1 est diminuée chez tous les patients atteints de SBDC, porteurs ou non de mutations dans le gène ACTRT1. Le séquençage à haut débit de la région candidate a permis d’identifier des mutations dans des régions transcrites, régulatrices du gène ACTRT1 chez les patients des 4 autres familles. Notant que la voie Hedgehog est dérégulée dans 70 % des CBCs, nous avons démontré qu’ACTRT1 est un nouvel inhibiteur de cette voie, en particulier par sa liaison au promoteur de GLI1 dont il inhibe l’expression. Enfin, ACTRT1 est un nouveau gène suppresseur de tumeur capable de réduire in vivo la progression tumorale de certaines lignées cancéreuses par la régulation de gènes impliqués dans la prolifération, la mort et la survie cellulaire, ou encore la migration. L’Incontinentia Pigmenti (IP) est une affection multisystémique caractérisée par une atteinte de la peau, des dents, des yeux et parfois du système nerveux central. Elle résulte de mutation dans le gène NEMO et l’abolition de l’activation de la voie NF-KB. L’étude d’une famille concernée par l’IP à permis d’identifier une nouvelle mutation d’épissage du gène NEMO aboutissant à l’expression d’une protéine tronquée. Cette protéine conserve l’intégralité des domaines fonctionnels de NEMO connus à ce jour. Sa caractérisation a révélé une perte d’interaction avec SHARPIN, composants du complexe LUBAC permettant l’ubiquitination linéaire. Il s’agit de la première mutation humaine de NEMO montrant l’importance de son ubiquitination linéaire dans l’activation de la voie NF-KB. Mes travaux de thèse ont ainsi mis en évidence de nouveaux mécanismes physiopathlogiques responsables de deux formes de dysplasie ectodermique. Ces mécanismes reflètent la complexité des voies moléculaires impliquées dans le développement de la peau et le maintien de son homéostasie durant la vie adulte. / Genodermatoses are rare genetic diseases with cutaneous expression. Among them, ectodermal dysplasia (ED) characterized by abnormal development of at least two ectodermal structures (teeth, nails, sweat glands and hair) constitute a heterogeneous group of genodermatoses of more than 200 rare syndromes. While most of these syndromes associate only abnormalities of the ectodermal derivatives, others more complex, such as Bazex-Dupré-Christol syndrome and Incontinentia Pigmenti, bring together disparate manifestations. Bazex-Dupré-Christol syndrome (BDCS) associates ED with predisposition to early basal cell carcinoma (BCCs). The study of 6 families allowed us to identify, in 2 of them, a truncated mutation in the ACTRT1 gene, encoding the Arp-T1 protein. In the epidermis, Arp-T1 protein is decreased in all patients with BDCS, carrying or not of mutations in ACTRT1 gene. High-throughput sequencing of the candidate region allowed to identify mutations in transcribed enhancer regions, regulating the ACTRT1 gene in patients of the remaining 4 families. Noting that the Hedgehog pathway is deregulated in more than 70% of BCCs, we have demonstrated that ACTRT1 is a novel inhibitor of this pathway, via its binding to GLI1 promoter and inhibiting its expression. Finally, ACTRT1 is a new tumor suppressor gene capable of reducing in vivo the tumor progression of certain cancer lines by the regulation of genes involved in proliferation, death and cell survival, or migration. Incontinentia Pigmenti (IP) is a multisystemic disorder characterized by involvement of skin, teeth, eyes and sometimes the central nervous system. It results from mutation in the NEMO gene and the abolition of activation of NF-KB pathway. The study of a family concerned with IP allowed to identify a new splicing mutation of NEMO gene leading to a truncated protein expression. This protein retains all the functional domains of NEMO known. Its characterization revealed a loss of interaction with SHARPIN, components of LUBAC complex allowing linear ubiquitination. This is the first human mutation of NEMO showing the importance of its linear ubiquitination in the activation of the NF-KB pathway. Thus, my thesis work revealed novel physiopathological mechanisms responsible for two forms of ectodermal dysplasia. These mechanisms reflect the complexity of the molecular pathways involved in the development of the skin and the maintenance of its homeostasis during adult life.

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Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-31T11:38:11Z No. of bitstreams: 1 mariliadepaduadornelascorrea.pdf: 1683962 bytes, checksum: 535fc7956722559af7dce14b15336986 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-12-15T12:56:51Z (GMT) No. of bitstreams: 1 mariliadepaduadornelascorrea.pdf: 1683962 bytes, checksum: 535fc7956722559af7dce14b15336986 (MD5) / Made available in DSpace on 2016-12-15T12:56:51Z (GMT). No. of bitstreams: 1 mariliadepaduadornelascorrea.pdf: 1683962 bytes, checksum: 535fc7956722559af7dce14b15336986 (MD5) Previous issue date: 2007-12-14 / O Carcinoma basocelular (CBC) é o câncer mais comum em humanos, tendo predileção pela cabeça e pescoço, e sua incidência vem aumentando nos últimos anos. Estudos utilizando recursos da biologia molecular e genética, associados à histomorfologia, permitem a identificação de fatores de risco no desenvolvimento de lesões mais recorrentes e agressivas e propõem métodos mais eficazes de prevenção. O objetivo foi correlacionar a expressão dos marcadores de apoptose (p53 e Bcl-2) e proliferação celular (Ki-67 e PCNA) com os indicadores histológicos de gravidade do tumor. Foram estudadas cinco amostras de cada uma das formas nodular, morfeiforme e superficial e um grupo controle com três pacientes livres de lesão. O teste de Mann Whitney foi utilizado na comparação da expressão destes marcadores com a forma de apresentação do CBC. A marcação do Bcl-2 foi expressiva nos CBCs ditos agressivos. A variante morfeiforme foi a que mais o expressou, seguida pela nodular. Dos tumores estudados, 66,7% (dez) expressaram fortemente o p-53 e, nos controles, um indivíduo o expressou fracamente na camada basal. Nossos resultados mostram maior expressão do Ki-67 no CBC nodular e superficial, sem expressão nos controles. O PCNA mostrou forte marcação em todos os tipos de tumores e nos controles. O Bcl-2 e a p-53 apresentam tendência para diagnosticar gravidade do Carcinoma Basocelular, e o Ki-67, por seu comportamento variável, não pode ser considerado como marcador de gravidade, assim como o PCNA que não foi um bom marcador de proliferação celular. / Basal Cell Carcinoma (BCC) is the most common form of human cancer, showing a predilection for the head and neck region. Its incidence has been increasing over the last years. Studies employing resources from molecular and genetic medicine associated with histomorphology allow the identification of risk factors in the development of more recurring and aggressive lesions, and propose more efficient prevention methods. The objective was to correlate the expression of markers of apoptosis (p53 and bcl-2) and cell proliferation (Ki-67 and PCNA) with histological indicators of the gravity of the tumor. In this study we analyzed five samples of the nodular, sclerosing and superficial forms, respectively, and one control group with three lesion-free patients. The Mann-Whitney test was used to compare the expression of these markers with the form of manifestation of BCC. Bcl-2 expression was significant in the BCCs said to be aggressive. The sclerosing variant expressed it the most, followed by the nodular variant. Of the studied growths, 66.7% (10) expressed p-53 strongly. Among the controls, one individual expressed it weakly in the basal layer. Our results show a larger expression of Ki-67 in nodular and superficial BCCs, with no expression whatsoever in the controls. PCNA was strongly expressed in all types of tumors and in the controls. Bcl-2 and p-53 have a tendency to indicate the gravity of Basal Cell Carcinoma. In contrast, Ki-67, due to its variable behaviour, can not be considered to be a marker of gravity. Furthermore, OCNA was not a good marker of cell proliferation.

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