Akt signaling leads to stem cell activation and promotes tumor development in epidermis.

Hair follicle stem cells (HF-SCs) alternate between periods of quiescence and proliferation, to finally differentiate into all the cell types that constitute the hair follicle. Also, they have been recently identified as cells of origin in skin cancer. HF-SCs localize in a precise region of the hair follicle, the bulge, and molecular markers for this population have been established. Thus, HF-SCs are good model to study the potential role of oncogenic activations on SC physiology. Expression of a permanently active form of Akt (myrAkt) in basal cells leads to Akt hyperactivation specifically in the CD34(+)Itga6(H) population. This activation causes bulge stem cells to exit from quiescence increasing their response to proliferative stimuli and affecting some functions such as cell migration. HF-SC identity upon Akt activation is preserved; in this sense, increased proliferation does not result in stem cell exhaustion with age suggesting that Akt activation does not affect self-renewal an important aspect for normal tissue maintenance and cancer development. Genome-wide transcriptome analysis of HF-SC isolated from myrAkt and wild-type epidermis underscores changes in metabolic pathways characteristic of cancer cells. These differences manifest during a two-step carcinogenesis protocol in which Akt activation in HF-SCs results in increased tumor development and malignant transformation.

Establishment of a murine epidermal cell line suitable for in vitro and in vivo skin modelling.

BACKGROUND: Skin diseases are a major health problem. Some of the most severe conditions involve genetic disorders, including cancer. Several of these human diseases have been modelled in genetically modified mice, thus becoming a highly valuable preclinical tool for the treatment of these pathologies. However, development of three-dimensional models of skin using keratinocytes from normal and/or genetically modified mice has been hindered by the difficulty to subculture murine epidermal keratinocytes. METHODS: We have generated a murine epidermal cell line by serially passaging keratinocytes isolated from the back skin of adult mice. We have termed this cell line COCA. Cell culture is done in fully defined media and does not require feeder cells or any other coating methods. RESULTS: COCA retained its capacity to differentiate and stratify in response to increased calcium concentration in the cell culture medium for more than 75 passages. These cells, including late passage, can form epidermis-like structures in three-dimensional in vitro models with a well-preserved pattern of proliferation and differentiation. Furthermore, these cells form epidermis in grafting assays in vivo, and do not develop tumorigenic ability. CONCLUSIONS: We propose that COCA constitutes a good experimental system for in vitro and in vivo skin modelling. Also, cell lines from genetically modified mice of interest in skin biology could be established using the method we have developed. COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations.

Transgenic mice expressing constitutively active Akt in oral epithelium validate KLFA as a potential biomarker of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a common human neoplasia, of poor prognosis and survival, which frequently displays Akt overactivation. Previously, we reported that mice expressing high levels of constitutively Akt activity (myrAkt) in oral epithelia develop lesions and tumors in the oral cavity. MATERIALS AND METHODS: Functional genomics of primary keratinocytes from different transgenic mouse lines and immunostaining of mouse and human samples were performed in order to identify and validate putative biomarkers of oral cancer progression. RESULTS: The expression of KLF4 was found to be increased only in tumor prone samples from mice bearing overactivation of Akt. Such increased expression was confirmed in oral dysplasias and tumors arising in those mice. Tissue microarray analysis of human samples confirmed the association between active Akt and increased KLF4 expression. CONCLUSION: These data support the notion that KLF4 is potentially a reliable marker of HNSCC, and that myrAkt transgenic mice are valuable tools for preclinical research of HNSCC.

Análisis de la situación de juego en el diseño de tareas en baloncesto formativo / Analysis of Game Situations in the Design of Tasks in Training Basketball / Análise da situação do jogo na concepção de tarefas no basquetebol formativo

Resumen: Introducción: el conocimiento de los entrenamientos es fundamental para optimizar la formación de los jugadores; por ello, una de las estrategias más empleadas por los entrenadores para optimizar el proceso de entrenamiento es modificar el número de jugadores (Situación de Juego). Objetivo: el objetivo del estudio fue analizar el proceso de enseñanza-aprendizaje de un entrenador de baloncesto en edad escolar, así como conocer la asociación de la Situación de Juego y las variables pedagógicas, de carga externa y organizativa empleadas que influyen en el diseño de las tareas. Método: para ello, se analizaron un total de 20 sesiones de entrenamiento formadas por 100 tareas, registradas a través del Sistema integral para el análisis de las tareas de entrenamiento. Se realizó un análisis descriptivo e inferencial entre las variables planteadas en la investigación con relación a la Situación de Juego. Resultados: los resultados demostraron la relación significativa de las variables analizadas con la Situación de Juego. La forma de agrupar los jugadores durante el desarrollo de las tareas de entrenamiento condiciona los resultados del resto de las variables que definen una tarea de entrenamiento, tanto en el nivel pedagógico, organizativo, como de carga externa. Conclusión: estas evidencias revindican la importancia de conocer los factores que influyen en las tareas de entrenamiento. Además, los resultados muestran que existen diferencias en el empleo de las Situaciones de Juego en función del resto de variables empleadas.

Abstract: Introduction: Currently, Knowledge of training is essential to optimize player training. Therefore, one of the strategies most used by coaches to optimize the training process is to modify the number of players (Game Situation). Purpose: The objective of the study was to analyze the teaching-learning process of a school-age basketball coach, as well as to know the association of the Game Situation and the Pedagogical, External Load and Organizational variables used that influence the design of the tasks. Method: For this, a total of 20 training sessions consisting of 100 tasks, recorded through the Comprehensive System for the Analysis of Training Tasks, were analyzed. A descriptive and inferential analysis was carried out between the variables raised in the research in relation to the Game Situation. Results: The results demonstrated the significant relationship of the variables analyzed with the Game Situation. The way the players are grouped during the development of the training tasks conditions the results of the rest of the variables that define a training task, both at a pedagogical, organizational, and external load level. Conclusion: These evidences vindicate the importance of knowing the factors that influence training tasks. In addition, the results show that there are differences in the use of Game Situations depending on the rest of the variables used.

Resumo: Introdução: O conhecimento do treinamento é fundamental para otimizar o processo de formação dos jogadores. Portanto, uma das estratégias mais utilizadas pelos treinadores para otimizar o processo de treinamento é modificar o número de jogadores (Situação do jogo). Objetivo: O objetivo do estudo foi analisar o processo de ensino-aprendizagem de um treinador de basquetebol em idade escolar, e também descobrir a associação entre a Situação do Jogo e as variáveis Pedagógicas, de Carga Externa e Organizacionais utilizadas que influenciam o desenho das tarefas. Método: Para este fim, foram analisadas 20 sessões de treinamento compostas de 100 tarefas, registradas através do Sistema Integral de Análise de Tarefas de Treinamento. Foi realizada uma análise descritiva e inferencial entre as variáveis levantadas na pesquisa em relação à situação do jogo. Resultados: Os resultados mostraram uma relação significativa entre as variáveis analisadas e a situação do jogo. A forma de agrupar os jogadores durante o desenvolvimento das tarefas de treinamento condiciona os resultados das demais variáveis que definem uma tarefa de treinamento, tanto ao nível pedagógico, organizacional e de carga externa. Conclusão: Estas evidências reafirmam a importância de conhecer os fatores que influenciam as tarefas de treinamento. Além disso, os resultados mostram que existem diferenças no uso de Situações de Jogo dependendo do resto das variáveis utilizadas.

EMT and induction of miR-21 mediate metastasis development in Trp53-deficient tumours.

Missense mutations in TP53 gene promote metastasis in human tumours. However, little is known about the complete loss of function of p53 in tumour metastasis. Here we show that squamous cell carcinomas generated by the specific ablation of Trp53 gene in mouse epidermis are highly metastatic. Biochemical and genome-wide mRNA and miRNA analyses demonstrated that metastases are associated with the early induction of epithelial-mesenchymal transition (EMT) and deregulated miRNA expression in primary tumours. Increased expression of miR-21 was observed in undifferentiated, prometastatic mouse tumours and in human tumours characterized by p53 mutations and distant metastasis. The augmented expression of miR-21, mediated by active mTOR and Stat3 signalling, conferred increased invasive properties to mouse keratinocytes in vitro and in vivo, whereas blockade of miR-21 in a metastatic spindle cell line inhibits metastasis development. Collectively these data identify novel molecular mechanisms leading to metastasis in vivo originated by p53 loss in epithelia.

A functional role of RB-dependent pathway in the control of quiescence in adult epidermal stem cells revealed by genomic profiling.

Continuous cell renewal in mouse epidermis is at the expense of a pool of pluripotent cells that lie in a well defined niche in the hair follicle known as the bulge. To identify mechanisms controlling hair follicle stem cell homeostasis, we developed a strategy to isolate adult bulge stem cells in mice and to define their transcriptional profile. We observed that a large number of transcripts are underexpressed in hair follicle stem cells when compared to non-stem cells. Importantly, the majority of these downregulated genes are involved in cell cycle. Using bioinformatics tools, we identified the E2F transcription factor family as a potential element involved in the regulation of these transcripts. To determine their functional role, we used engineered mice lacking Rb gene in epidermis, which showed increased expression of most E2F family members and increased E2F transcriptional activity. Experiments designed to analyze epidermal stem cell functionality (i.e.: hair regrowth and wound healing) imply a role of the Rb-E2F axis in the control of stem cell quiescence in epidermis.