Role of oncogenic BRAFV600E on cell death/survival decision in human skin melanoma

Cutaneous melanoma, the most aggressive form of skin cancer, remains one of the most difficult to treat human malignancies. 40-50% of melanomas are characterized by the expression of oncogenic BRAF, with BRAFV600E being the most represented mutation. The well-known BRAFV600E-induced resistance to therapy remains an obstacle to the succesfull treatment and highlights the urgent need of in-depht studies to increase our knowledge on biochemical and molecular characteristics of this cancer in order to develop novel effective and longlasting therapeutic strategies. Autophagy and ER stress are pro-survival mechanisms, strongly linked to each other, involved in melanoma development, progression and chemoresistance. In the first part of this Ph.D project we show that BRAFV600E induces a chronic ER stress status increasing basal cell autophagy. BRAFV600E-mediated p38 activation stimulates both IRE1/ASK1/JNK and PERK/ATF4/TRB3 pathways. Bcl-XL/Bcl-2 phosphorylation by active JNK releases Beclin1 whereas TRB3 inhibits mTor, together resulting in an increase in cell basal autophagy. Furthermore, we demonstrate that chemical chaperones relieve the BRAFV600E-mediated chronic ER stress status, consequently reducing basal autophagic activity resulting in re-sensitization of melanoma cells to apoptosis. Although the immunosurveillance constantly reduces the risk of cancer development, melanoma and other tumours evolved effective strategies to efficiently evade this control. On the other hand, it is now known that chemotherapeutic agents, such as mitoxantrone (MTX) and doxorubicine (DOXO), are able to induce cancer cell death pathways involving the immune system. The immunogenic cell death (ICD) process involves the emission of so-called DAMPs (damage-associated molecular patterns) from dying cancer cells, able to activate the immune system against the tumour. The combined activity of pro-death drugs and the immune system results in a most effective eradication of the tumour. In the second part of this Ph. D project we show that MTX and DOXO are able to induce cell death and stimulate, at the same time, the release/exposure of the four strictly required DAMPs, in melanoma cell lines: i) exposure of the endoplasmic reticulum (ER) resident protein calreticulin (CRT) on cell membrane; ii) ATP secretion; iii) type I IFNs production and iv) HMGB1 secretion. Importantly, the presence of oncogenic BRAF does not interfere with ICD induction. We further investigated the role of the ER stress in the exposure of CRT, concluding that this mechanism is not involved, at least in our model. Altogether, on one hand these results suggest that enhanced basal autophagy, typically observed in BRAFV600E melanomas, is a consequence of a chronic ER stress status, which ultimately results in the chemoresistance of such tumours, therefore targeted therapies that attenuate ER stress may represent a novel and more effective therapeutic strategy for BRAF mutant melanoma. On the other hand, our result suggest that exploiting the ability of chemotherapeutical compounds such as MTX and DOXO to stimulate the release/exposure of ICD-related DAMPs might represents a feasible route in the developing of a valid and alternative strategy for the treatment of cutaneous melanoma. Therefore it is necessary to deepen the understanding and the potentiality of this therapeutic strategy by using in vivo models.