Oncogenic B-RAFV600E Promotes Anchorage-Independent Survival of Human Melanocytes

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Amino terminal hydrophobic import signals target the p14ARF tumor suppressor to the mitochondria

The p14ARF tumor suppressor is frequently targeted for inactivation in many human cancers and in individuals predisposed to cutaneous melanoma. The functions of p14ARF are closely linked with its subcellular distribution. Nucleolar p14ARF dampens ribosome biosynthesis and nucleoplasmic forms of p14ARF activate the p53 pathway and induce cell cycle arrest. p14ARF can also be recruited to mitochondria where it interacts with many mitochondrial proteins, including Bcl-xL and p32 to induce cell death. It has been suggested that the movement of p14ARF to mitochondria requires its interaction with p32, but we now show that the ARF-p32 interaction is not necessary for the accumulation of p14ARF in mitochondria. Instead, highly hydrophobic domains within the amino-terminal half of p14ARF act as mitochondrial import sequences. We suggest that once this hydrophobic pocket is exposed, possibly in a stimulus-dependent manner, it accelerates the mitochondrial import of p14ARF. this allows the interaction of p14ARF with mitochondrial proteins, including p32 and enables p53-independent cell death.11 page(s

Oncogenic activation of MEK/ERK primes melanoma cells for adaptation to endoplasmic reticulum stress

Background: Endoplasmic reticulum (ER) stress is characterized by accumulation and aggregation of unfolded and/or misfolded proteins in the ER lumen. Cells respond by activating a range of signaling pathways to alter transcriptional and translational programs. Cancer cells are commonly subject to chronic ER stress, which must be adapted for survival and proliferation. We report here that in melanoma cells intrinsic activation of the ER stress response/unfolded protein response (UPR) is, at least in part, caused by increased outputs of protein synthesis driven by oncogenic activation of MEK/ERK. Aims: In this study, we examine the potential interaction between the constitutively activated MEK/ERK pathway and the UPR in melanoma cells. Methods: Melanoma cell lines were treated with PLX4720, U0126 or vemurafenib and subjected to analysis by western blot, QPCR, or flow cytometry. Melanocytes were lentivirally transduced with BRAFV600E before being analysed by western blot, QPCR or flow cytometry. Results: Inhibition of oncogenic BRAFV600E by PLX4720 or inhibition of MEK by U0126 attenuated activation of IRE1 and ATF6 signaling of the UPR in melanoma cells. This was associated with decreased phosphorylation of eIF4E and nascent protein synthesis, and was recapitulated by knockdown of eIF4E. In line with this, introduction of BRAFV600E into melanocytes led to increases in eIF4E phosphorylation and protein production, and triggered activation of the UPR. Paradoxically, treatment of melanoma cells with the clinically available BRAFV600E inhibitor vemurafenib resulted in induction of the UPR, an effect that may be independent of BRAFV600E inhibition. Conclusions: MEK/ERK signaling is necessary and sufficient for intrinsic activation of the UPR as a consequence of ER stress triggered by enhanced protein synthesis in melanoma cells. These results indicate that potentiation of adaptation to chronic ER stress is another mechanism by which activation of the MEK/ERK pathway promotes the pathogenesis of melanoma. The contrasting effect of PLX4720 and vemurafenib on the UPR in this study raises questions pertaining to the role of the UPR and ER stress in melanoma patients treated with BRAF inhibitors. Translational research aspect: T1: Basic Science This study illuminates the effect of PLX4720 and vemurafenib on activation of the UPR, which may be useful in developing combinational therapies or reducing side effects in melanoma patients being treated with mutant BRAF inhibitors.1 page(s