Oncogenic BRAF induces chronic ER stress condition resulting in increased basal autophagy and apoptotic resistance of cutaneous melanoma

The notorious unresponsiveness of metastatic cutaneous melanoma to current treatment strategies coupled with its increasing incidence constitutes a serious worldwide clinical problem. Moreover, despite recent advances in targeted therapies for patients with BRAF^V600E mutant melanomas, acquired resistance remains a limiting factor and hence emphasises the acute need for comprehensive pre-clinical studies to increase the biological understanding of such tumours in order to develop novel effective and longlasting therapeutic strategies. Autophagy and ER stress both have a role in melanoma development/progression and chemoresistance although their real impact is still unclear. Here, we show that BRAF^V600E induces a chronic ER stress status directly increasing basal cell autophagy. BRAF^V600E-mediated p38 activation stimulates both the IRE1/ASK1/JNK and TRB3 pathways. Bcl-XL/Bcl-2 phosphorylation by active JNK releases Beclin1 whereas TRB3 inhibits the Akt/mTor axes, together resulting in an increase in basal autophagy. Furthermore, we demonstrate chemical chaperones relieve the BRAF^V600E-mediated chronic ER stress status, consequently reducing basal autophagic activity and increasing the sensitivity of melanoma cells to apoptosis. Taken together, these results suggest enhanced basal autophagy, typically observed in BRAF^V600E melanomas, is a consequence of a chronic ER stress status, which ultimately results in the chemoresistance of such tumours. Targeted therapies that attenuate ER stress may therefore represent a novel and more effective therapeutic strategy for BRAF mutant melanoma.Cutaneous melanoma represents one of the most aggressive and difficult to treat forms of human cancer, with a worldwide incidence that has steadily increased over the past 40 years.^{1, 2}Notoriously unresponsive to conventional chemotherapy, metastatic disease is highly invasive and evolves with an extensive repertoire of molecular defences against immunological and cytotoxic attack.³Although linked to exposure to ultraviolet light, it is widely accepted that both genotypic and phenotypic changes in melanocytes predispose to melanocyte transformation and the onset of melanoma.^{4, 5}Surprisingly, p53 mutations are very rare in melanoma, but activity is, however, impaired through direct or indirect inactivation of key elements of this pathway, including through the suppression of APAF-1 expression,⁶ loss of PTEN function,⁷ dysregulation of Bcl-2 expression,⁸ upregulation of the anti-apoptotic protein Mcl-1 together with its altered slice variant expression ^{9, 10} and the ER chaperone GRP78.^{11, 12, 13} Oncogenic mutations, however, in the Ras/Raf pathway are the most well-described genetic mutations associated with melanoma development and progression.¹⁴ Indeed, up to 90% of all melanomas harbour activating NRAS or BRAF mutations, with BRAF^V600E representing more than 90% of BRAF mutations,^{15, 16} the consequence of which is the constitutive activation of RAF-extracellular signal-regulated kinase/ERK signalling promoting melanoma proliferation and resistance to apoptosis.¹⁷ Nevertheless, mutation of NRAS/BRAF alone is not sufficient to initiate melanomagenesis, because these common mutations are also present in benign nevi, thereby highlighting the requirement of other factors to drive melanocyte transformation and melanoma development.^{15, 16} Dysregulation of autophagy has accordingly been postulated as a secondary event contributing to melanoma progression and, importantly, also has a key role in chemoresistance.^{18, 19, 20}Autophagy is the principal catabolic process for the bulk degradation and recycling of aged/damaged cellular components, organelles and proteins through the formation of a double-membraned cytosolic vesicle able to wrap targeted material. The subsequent fusion with lysosomes and degradation of cargo provide nutrients in times of environmental stress, such as nutrient deprivation or hypoxia.²¹ Though essential for the maintenance of cellular homeostasis under conditions of nutrient deprivation, paradoxically, autophagy promotes both tumour suppression and tumour development.²² Although the accumulation of damaged organelles/cytosolic proteins may lead to cellular transformation, autophagy may also sustain tumour growth in a microenvironment which is commonly poor of oxygen and nutrients.²² Thus, not surprisingly, autophagy activation is frequently observed in late-stage malignancy although the molecular mechanisms mediating its activation/regain of function remain unclear.ER stress may also constitute a key secondary event in melanoma development.²³ Primarily a cytoprotective pro-survival process, ER stress is activated as a result of accumulated unfolded proteins, protein overload or depletion of ER calcium stores and mediated through the activity of the master ER chaperone Grp78 and three signalling pathways; PERK/eIF2α/ATF4, IRE-1/Xbp-1 and ATF6 which collectively maintain ER homeostasis through the instigation of an unfolded protein response (UPR)²⁴ or sustained ER stress may lead to the induction of apoptosis.^{25, 26} Increasing evidence indicates that nutrient deprivation and hypoxia lead to activation of the UPR in various solid tumours, frequently correlating with resistance to chemotherapy.²⁷ The accepted hypothesis is thus that activation of the UPR in cancer cells enables their adaption to such ER stress resulting in the resistance to apoptosis through the persistent expression of pro-survival instead of pro-apoptotic proteins.²⁸Although under stress conditions, autophagy and ER stress seem to act in parallel, indeed they are closely related, because one can regulate the other and vice versa. In fact, ER stress is able to promptly stimulate autophagy,²⁶ whereas autophagy selectively removes the membranes of the endoplasmic reticulum at the end of the UPR, although the molecular mechanisms are still largely unclear.²⁹In the present study, we investigated the link between oncogenic BRAF^V600E and increased basal autophagy in melanoma cells, highlighting the pivotal role played by ER stress, possibly responsible for tumour growth and chemoresistance.