Nuevos mecanismos moleculares implicados en la carcinogénesis producida por B-RAF
- Autores: Irene Sánchez Hernández
- Directores de la Tesis: Antonio Chiloeches Gálvez (dir. tes.)
- Lectura: En la Universidad de Alcalá ( España ) en 2010
- Idioma: español
- Tribunal Calificador de la Tesis: M. Josefa Toro Nozal (presid.), Santiago Ropero Salinas (secret.), Marina Lasa Benito (voc.), Marina I. Garín Ferreira (voc.), Andrés Castellanos Martín (voc.)
- Materias:
- Enlaces
- Resumen
- español
La proteína B-RAF forma parte de la vía de señalización de Ras-RAF-MEK-ERK y regula, entre otros, la proliferación y la supervivencia celulares, dos procesos clave para la transformación celular. Se han encontrando mutaciones activadoras de B-RAF en un 10-20% de tumores humanos y, en concreto, V600EB-RAF se considera un oncogén. Por ello, es importante y necesario un mejor entendimiento de la regulación de los procesos biológicos modulados por V600EB-RAF en cáncer. En este trabajo nosotros demostramos que V600EB-RAF es el principal activador de la vía MEK-ERK y el responsable de la proliferación de las células tumorales que lo contienen. Por otro lado nuestros resultados demuestran que V600EB-RAF protege de la apoptosis por regulación de proteínas de la maquinaria apoptótica celular como BIM y caspasa 3. Sorprendentemente, su papel en apoptosis es independiente de la activación de MEK-ERK. Además, nosotros observamos que la vía PI3K-AKT-mTOR coopera con B-RAF en la supervivencia de las células tumorales estudiadas. Por último, nosotros demostramos que V600EB-RAF regula la transcripción dependiente de NF-?B por dos mecanismos distintos, uno a través de la vía canónica y otro aumentando la transactivación de p65/RelA a través de la quinasa MSK1. Además, la activación de NF-?B por V600EB-RAF es dependiente de MEK-ERK y, por lo tanto, no está implicada en la supervivencia celular inducida por este oncogén.
- English
B‐RAF protein is included in the Ras‐RAF‐MEK‐ERK pathway that regulates several signalling processes. In particular, it has been implicated in cellular proliferation and apoptosis, two key steps in transformation. Due to point mutations in B‐RAF, this protein is hyperactivated in approximately 10 % of human cancers. The most common mutation is V600EB‐RAF, which account for 90% of the cases. This substitution transforms the wild type protein in a constitutively activated kinase. Previous studies have shown that most of the processes regulated by B‐RAF occur through the activation of MEK‐ERK pathway, but it also exist data suggesting that oncogenic B‐RAF can produce its effects through other pathways. Therefore, it is important and necessary a better understanding of the biological processes regulated by V600EB‐RAF in cancer. In the present work, the role of V600EB‐RAF in tumour cells proliferation and apoptosis, was studied, as well as other signalling pathways that may be related to V600EB‐RAF in these processes. We used two recognized strategies in the study of RAF proteins functions with the aim to achieve these goals: treatment with the RAF inhibitor BAY 43‐9006 and silencing of B‐RAF and C‐RAF expression by using small interference RNA. Moreover, the MEK specific inhibitor U0126 was used for determining the contribution of the MEK‐ERK pathway in those processes. The model used includes four cell lines derived from different human tumours: two of them carrying the wild type B‐RAF protein and the other two containing the oncogenic protein V600EB‐RAF. Thus, we could compare the role of either wild‐type B‐RAF or the constitutively activated protein in such processes. Our results demonstrate that V600EB‐RAF is the main MEK‐ERK activator when this mutant is present in the cells. Thus, V600EB‐RAF‐MEK‐ERK pathway promotes proliferation of tumour cells and its inhibition diminishes cell proliferation. We also show that the inhibition of either B‐RAF activity or its expression increases cellular apoptosis, so V600EB‐RAF is a survival signal for the cells harbouring it and its silencing sensitize cells to death by treatment with the chemotherapeutic drug etoposide. Furthermore, this oncoprotein protects cells from apoptosis by regulation of expression levels of some proteins involved in the apoptotic machinery like BIM, Bcl‐ 2, Bcl‐XL, and the activation of caspase 3. Surprisingly, the MEK‐ERK pathway does not act downstream V600EB‐RAF in the prevention of apoptosis, since the inhibition of this pathway does not have any effect in the apoptotic levels. In addition, our data establish that PI3K also participates in proliferation and survival in cells carrying the V600EB‐RAF mutation. Firstly, inhibition of PI3K with LY294002 reduces proliferation, and this effect is bigger when cells are treated simultaneously with LY294002 and the inhibitors of the RAF‐MEK‐ERK pathway. Secondly, PI3K‐AKT‐mTOR pathway cooperates with V600EB‐RAF in the survival of the studied tumour cell lines. The inhibition of the PI3K pathway alone does not exert any effect on apoptosis; however, together with V600EB‐RAF inhibition, it has a bigger effect on the levels of apoptosis than the inhibition of B‐RAF alone. Remarkably, the presence of V600EB‐RAF downregulates the activation of the pathway since its silencing induces the phosphorylation of AKT and p70S6K proteins. Finally, we demonstrate that V600EB‐RAF regulates NF‐κB‐dependent transcription by two different mechanisms: one through the canonical pathway, inducing IκBα degradation and increasing the p65/RelA nuclear translocation, and the other one by increasing p65/RelA transactivation potential by a MSK1‐dependent mechanism. V600EB‐RAF depends on MEK‐ERK pathway for activating NF‐κB, so this transcription factor does not participate in the survival induced by oncogenic V600EB‐RAF. However, the role of NF‐κB is not yet elucidated. All together, these data demonstrate that oncogenic V600EB‐RAF plays a crucial role in carcinogenesis, regulating both tumour cell proliferation and survival, two key processes in tumoral progression. Therefore, B‐RAF must be considered as a therapeutical target in cancer treatment, specially, in combination with drugs which act on other signalling pathways such as PI3K and NF‐κB pathways.
- español
