Resumen de Papel de B-RAF en la progresión del cáncer de tiroides: cooperación con TGFβ en la transición epitelio-mesénquima
- español
El cáncer de tiroides es la segunda patología después del melanoma en la que el mutante V600EB-RAF se encuentra presente con mayor frecuencia. Numerosos estudios han demostrado que V600EB-RAF juega un papel importante en las primeras etapas del desarrollo del cáncer tiroideo y, recientemente se ha demostrado que este oncogén es, además, indispensable para el mantenimiento y la progresión del cáncer de tiroides a un carcinoma más agresivo y con peor pronóstico. A pesar de los esfuerzos, poco se conoce sobre los mecanismos moleculares mediados por V600EB-RAF implicados en este aumento de agresividad tumoral. Por ello, en este trabajo nos planteamos estudiar el papel de V600EB-RAF en los mecanismos moleculares implicados en la progresión del cáncer de tiroides, en concreto, en los mecanismos relacionados con los procesos de migración e invasión celular. En la primera parte del trabajo demostramos que el mutante V600EB-RAF causa un aumento en la migración y en la invasión celular y, a su vez, participa en la transición epitelio-mesénquima (EMT) a través del aumento de los niveles de Snail1 y de la represión de E-cadherina. Asimismo, confirmamos el efecto represor de Snail1 sobre E-cadherina y demostramos que la expresión del primero es necesaria para la represión de E-cadherina inducida por V600EB-RAF. Adicionalmente, mostramos que la sobreexpresión de Snail1 favorece los procesos de migración e invasión celular, indicando un posible mecanismo por el que V600EB-RAF participa en la progresión del cáncer de tiroides. Por otra parte, mostramos que la citoquina multifuncional TGFβ promueve la EMT y la migración celular en células tumorales tiroideas, demostrando así su papel estimulador del desarrollo tumoral en etapas avanzadas del cáncer de tiroides. Asimismo, demostramos que V600EB-RAF tiene un papel dual sobre los efectos de TGFβ. Por una parte, aumenta los niveles de TGFβ secretado al medio. Además, tanto su expresión como su actividad son necesarias para la EMT y la migración producidas por TGFβ, demostrando una cooperación entre V600EB-RAF y TGFβ en la inducción de estos procesos en cáncer de tiroides. Por tanto, este estudio, además de aportar nuevos datos para comprender los mecanismos tumorogénicos mediados por V600EB-RAF y TGFβ, muestra la importancia de considerar la cooperación entre estas dos vías de señalización y poder actuar de forma conveniente a la hora del desarrollo de nuevos protocolos terapéuticos para frenar la progresión del cáncer de tiroides.
- English
Cancer development is a multistep process in which a succession of genetic alterations leads to the progressive conversion of normal cells into highly malignant derivatives by conferring growth advantage. In this way, transformation allows cancer cells to survive and proliferate under conditions that would normally be deleterious. In tumor cells, there are certain types of genes that are often mutated or highly expressed, called oncogenes. Normally, the protein products of these oncogenes belong to signalling pathways involved in key regulatory processes, such as proliferation, survival and migration. One example is the serine/threonine kinase B-RAF included in the Ras-RAF-MEK-ERK, which is mutated in approximately 10% of human cancers. The most common genetic alteration in thyroid carcinoma is the oncogenic T1799A transversion of B-RAF. This mutation results in a V600E substitution which transforms the wild type protein in a constitutively activated kinase. Recent studies have shown that this mutant is important not only in early stages of the tumour, but also for the maintenance and progression of the disease. The aim of this work is to study the implication of V600EB-RAF in processes related to cancer progression, such as migration and invasion, and elucidate new molecular mechanisms mediated by this oncogene in the development of thyroid carcinoma. For this purpose, we used three thyroid cancer cell lines, two of them harbouring V600EB-RAF mutation and one containing WTB-RAF. This model, together with several molecular strategies including the treatment with the specific V600EB-RAF inhibitor PLX4720, silencing B-RAF by small interference RNA and treatment with the MEK inhibitor U0126, allowed us to describe de role of B-RAF in thyroid carcinoma progression. Furthermore, to confirm some of the results obtained in this study, we overexpressed the mutant V600EB-RAF, by lentiviral transduction, in thyroid cells containing wild type B-RAF Overall, in the first part of this work, we show that V600EB-RAF promotes cell migration and invasion. Moreover, this oncogen is involved in epithelial to mesenchymal transition (EMT) through the increase of Snail1 expression and E-cadherin repression. In addition, these effects are related to cell scattering observed when we overexpressed V600EB-RAF in WTB-RAF-cells. We also demonstrate that the expression of Snail1 is required for V600EB-RAF-induced E-cadherin repression. Furthermore, we show that overexpression of Snail1 promotes migration and invasion, indicating that one possible mechanism by which V600EB-RAF participates in the progression of thyroid cancer may be through regulating Snai1 and E-cadherin levels. On the other hand, we demonstrate that, in our model, the multifunctional cytokine TGFβ lacks its antiproliferative and proapoptotic effects characteristic of some types and cellular contexts. By contrast, TGFβ promotes both EMT and cell migration, demonstrating a tumor promoting role in advanced stages of thyroid cancer. Importantly, these TGFβ-mediated effects were blocked by inhibition of V600EB-RAF and MEK, suggesting a cooperation between the MEK/ERK pathway and TGFβ signalling in thyroid cancer progression. In addition, our data establish that V600EB-RAF induces secretion of functional TGFβ through a MEK-ERK dependent mechanism. Interestingly, the blocking of TGFβ signaling doesn´t rescue the V600EB-RAF-induced aggressive phenotype, showing that V600EB-RAF-induced cancer progression, is not mediated through TGFβ overexpression. Finally, we observed that TGFβ affects two major focal contacts associated proteins. TGFβ increases ILK expression through a V600EB-RAF dependent mechanism but interestingly, this is not related to the EMT or the induction of a more migratory phenotype. On the other hand, treatment with TGFβ also raises FAK activity and its interaction with Src. In contrast with ILK, we show that both Src and FAK are involved in TGFβ-mediated EMT, since its inhibition decrease Snail1 levels. In this context, the role of V600EB-RAF appears to be independent of Src activity, suggesting the wide range of mechanisms by which TGFβ can induce EMT and clarifying alternatives to inhibit tumor aggressiveness of thyroid carcinoma.
Although further studies are warranted to characterize the molecular details of V600EB-RAF-mediated signal transduction and TGFβ function in thyroid cancer, the results reported here contribute to a better understanding of the molecular mechanisms underlying the progression and tumor malignancy of these kinds of tumors. Indeed, all these data demonstrate that oncogenic V600EB-RAF plays an important role in the induction of an invasive phenotype, and provide more evidences that support the therapeutic intervention by using V600EB-RAF inhibitors in advanced stages of thyroid cancer.
