Dual inhibition of V600EBRAF and the PI3K/AKT/mTOR pathway cooperates to induce apoptosis in melanoma cells through a MEK-independent mechanism

BRAF is a main oncogene in human melanomas. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with RAF inhibitor Sorafenib induces apoptosis in NPA melanoma cells expressing oncogenic (V600E)BRAF. This effect is mediated through a MEK/ERK-independent mechanism, since treatment with the MEK inhibitor U0126 does not exert any effect. Moreover, we demonstrate that inhibition of the PI3K/AKT/mTOR cascade alone does not increase apoptosis in these cells. However, the blockage of this pathway in cells lacking either BRAF expression or activity cooperates to induce higher levels of apoptosis than those achieved by inhibition of BRAF alone. Consistently, we demonstrate that abrogation of BRAF expression increases AKT and mTOR phosphorylation, suggesting the existence of a compensatory pro-survival mechanism after BRAF depletion. Together, our data provide a rationale for dual targeting of BRAF and PI3K/AKT/mTOR signalling to effectively control melanoma disease

Oncogenic Ras, but not V600EB-RAF, protects from cholesterol depletion-induced apoptosis through the PI3K/AKT pathway in colorectal cancer cells

Cholesterol is necessary for proliferation and survival of transformed cells. Here we analyse the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in colorectal cancer cells carrying oncogenic Ras or B-V600E-RAF mutations. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment results in a significant increase in apoptosis in HT-29 and Colo-205 cells containing the B-V600E-RAF mutation, but not in HCT-116 and LoVo cells harbouring the (G13D)Ras mutation, or BE cells, which possess two mutations, (G13D)Ras and B-G463V-RAF. We also demonstrate that oncogenic Ras protects from apoptosis induced by cholesterol depletion through constitutive activation of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. The specific activation of the PI3K/AKT pathway by overexpression of the (V12)RasC40 mutant or a constitutively active AKT decreases the LPDS plus 25-HC-induced apoptosis in HT-29 cells, whereas PI3K inhibition or abrogation of AKT expression renders HCT-116 sensitive to cholesterol depletion-induced apoptosis. Moreover, our data show that LPDS plus 25-HC increases the activity of c-Jun N-terminal kinase proteins only in HT-29 cells and that the inhibition of this kinase blocks the apoptosis induced by LPDS plus 25-HC. Finally, we demonstrate that AKT hyperactivation by oncogenic Ras protects from apoptosis, preventing the activation of c-Jun N-terminal kinase by cholesterol depletion. Thus, our data demonstrate that low levels of cholesterol induce apoptosis in colorectal cancer cells without oncogenic Ras mutations. These results reveal a novel molecular characteristic of colon tumours containing Ras or B-RAF mutations and should help in defining new targets for cancer therapy

Targeting mitochondrial oxidative phosphorylation eradicates therapy-resistant chronic myeloid leukemia stem cells

Treatment of chronic myeloid leukemia (CML) with imatinib mesylate and other second-and/or third-generation c-Abl-specific tyrosine kinase inhibitors (TKIs) has substantially extended patient survival(1). However, TKIs primarily target differentiated cells and do not eliminate leukemic stem cells (LSCs)(2-4). Therefore, targeting minimal residual disease to prevent acquired resistance and/or disease relapse requires identification of new LSC-selective target(s) that can be exploited therapeutically(5,6). Considering that malignant transformation involves cellular metabolic changes, which may in turn render the transformed cells susceptible to specific assaults in a selective manner(7), we searched for such vulnerabilities in CML LSCs. We performed metabolic analyses on both stem cell-enriched (CD34(+) and CD34(+)CD38(-)) and differentiated (CD34(-)) cells derived from individuals with CML, and we compared the signature of these cells with that of their normal counterparts. Through combination of stable isotope-assisted metabolomics with functional assays, we demonstrate that primitive CML cells rely on upregulated oxidative metabolism for their survival. We also show that combination treatment with imatinib and tigecycline, an antibiotic that inhibits mitochondrial protein translation, selectively eradicates CML LSCs both in vitro and in a xenotransplantation model of human CML. Our findings provide a strong rationale for investigation of the use of TKIs in combination with tigecycline to treat patients with CML with minimal residual disease

Papel de B-RAF en la progresión del cáncer de tiroides: cooperación con TGFβ en la transición epitelio-mesénquima

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

TGFß induces epithelial-mesenchymal transition of thyroid cancer cells by both the BRAF/MEK/ERK and Src/FAK pathways

The epithelial-mesenchymal transition (EMT) is a crucial process in tumour progression, by which epithelial cells acquire a mesenchymal phenotype, increasing its motility and the ability to invade distant sites. Here, we describe the molecular mechanisms by which (V600E)BRAF, TGF beta and the Src/FAK complex cooperatively regulate EMT induction and cell motility of anaplastic thyroid cancer cells. Analysis of EMT marker levels reveals a positive correlation between TGF beta and Snail expression, with a concomitant downregulation of E-cadherin, accompanied by an increase of cell migration and invasion. Furthermore, we show that (V600E)BRAF depletion by siRNA or inhibition of its activity by treatment with its inhibitor PLX4720 reverses the TGF beta-mediated effects on Snail, E-cadherin, migration and invasion. Moreover, (V600E)BRAF induces TGF beta secretion through a MEK/ERK-dependent mechanism. In addition, TGF beta activates the Src/FAK complex, which in turn regulates the expression of Snail and E-cadherin as well as cell migration. The inhibition of Src with the inhibitor SU6656 or abrogation of FAK expression with a specific siRNA reverses the TGF beta-induced effects. Interestingly, we demonstrate that activation of the Src/FAK complex by TGF beta is independent of (V600E)BRAF signalling, since inhibition of this oncogene does not affect its phosphorylation. Our data strongly suggest that TGF beta induces EMT and aggressiveness of thyroid cancer cells by parallel mechanisms involving both the (V600E)BRAF/MEK/ERK and Src/FAK pathways independently. Thus, we describe novel functions for Src/FAK in mediating the EMT program and aggressiveness regulated by TGF beta, establishing the inhibition of these proteins as a possible effective approach in preventing tumour progression of (V600E)BRAF-expressing thyroid tumours