l-779-450 and Melanoma

Mutated BRAF represents a critical oncogene in melanoma, and selective inhibitors have been approved for melanoma therapy. However, the molecular consequences of RAF inhibition in melanoma cells remained largely elusive. Here, we investigated the effects of the pan-RAF inhibitor L-779,450, which inhibited cell proliferation both in BRAF-mutated and wild-type melanoma cell lines. It furthermore enhanced apoptosis in combination with the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and overcame TRAIL resistance in melanoma cells. Enhanced apoptosis coincided with activation of mitochondrial pathways, seen by loss of mitochondrial membrane potential and release of cytochrome c, Smac (second mitochondria-derived activator of caspases), and apoptosis-inducing factor (AIF). Subsequently, caspase-9 and -3 were activated. Apoptosis induction by L-779,450/TRAIL was prevented by Bcl-2 overexpression and was dependent on Bax. Thus, activation of Bax by L-779,450 alone was demonstrated by Bax conformational changes, whereas Bak was not activated. Furthermore, the BH3-only protein Bim was upregulated in response to L-779,450. The significant roles of Smac, Bax, and Bim in this setting were proven by small interfering RNA (siRNA)-mediated knockdown experiments. L-779,450 also resulted in morphological changes indicating autophagy confirmed by the autophagy marker light chain 3-II (LC3-II). The pro-apoptotic effects of L-779,450 may explain the antitumor effects of RAF inhibition and may be considered when evaluating RAF inhibitors for melanoma therapy.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Autophagy; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Cell Proliferation; Drug Resistance, Neoplasm; HCT116 Cells; Humans; Melanoma; Membrane Proteins; Mitochondria; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-bcl-2; Pyridines; Skin Neoplasms; TNF-Related Apoptosis-Inducing Ligand

BRAF inhibition has been an instant, although short-lasting, success in BRAF-mutated melanoma treatment. Novel data by Berger et al. now suggest that BRAF-inhibitor-mediated "priming to death" facilitates tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis. We give an overview about the importance of the crosstalk of extrinsic and mitochondrial apoptotic signaling and propose other combination therapies that may prevent or overcome secondary resistance in melanoma.

Topics: Apoptosis; Humans; Melanoma; Proto-Oncogene Proteins B-raf; Pyridines; Skin Neoplasms; TNF-Related Apoptosis-Inducing Ligand

RAF kinase inhibitors can induce ERK cascade signaling by promoting dimerization of RAF family members in the presence of oncogenic or normally activated RAS. This interaction is mediated by a dimer interface region in the RAF kinase domain that is conserved in members of the ERK cascade scaffold family, kinase suppressor of RAS (KSR). In this study, we find that most RAF inhibitors also induce the binding of KSR1 to wild-type and oncogenic B-RAF proteins, including V600E B-RAF, but promote little complex formation between KSR1 and C-RAF. The inhibitor-induced KSR1/B-RAF interaction requires direct binding of the drug to B-RAF and is dependent on conserved dimer interface residues in each protein, but, unexpectedly, is not dependent on binding of B-RAF to activated RAS. Inhibitor-induced KSR/B-RAF complex formation can occur in the cytosol and is observed in normal mouse fibroblasts, as well as a variety of human cancer cell lines. Strikingly, we find that KSR1 competes with C-RAF for inhibitor-induced binding to B-RAF and, as a result, alters the effect of the inhibitors on ERK cascade signaling.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Dimerization; Extracellular Signal-Regulated MAP Kinases; Humans; MAP Kinase Signaling System; Melanoma; Mice; Protein Binding; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins B-raf; Pyridines; raf Kinases