[en] Mutations in the oncogenic protein kinase BRAF are involved in the onset and progression of several tumours, including more than 50% of melanoma cases and around 8% of all cancers worldwide. Currently, BRAF/MEK inhibitors (BRAF/MEKi) are the standard therapy for BRAFmutated melanoma and have radically changed the treatment landscape of advanced BRAF mutation-positive tumours. However, limited efficacy and emergence of drug resistance are major handicaps for successful treatments. The channel protein connexin43 (Cx43), allows the exchange of small molecules such as ions, metabolites or small RNAs (sRNAs) via hemichannels, gap junctions and thought small extracellular vesicles (EVs), affecting of cell growth, metabolism, and differentiation. Cx43 has also been detected at the mitochondrial associated membranes (MAMs) and it has been identified as a potential tumour suppressor in primary melanoma. However, the molecular basis of its role in disease progression remains controversial. In this project, we aim to unravel de role of Cx43 in the context of BRAF mutated cancer and resistance to therapy in BRAF-mutated tumours. Our findings show that Cx43 enhances the efficacy of BRAF/MEKi in various BRAF-driven tumours in vitro and in vivo models, by attracting DNA repair complexes to the nuclear membrane by interacting with lamin-A/C and different DDR proteins such as HMGB1, PRPF19, XCRR6 or CDCL5. This nuclear compartmentalisation restrict HR pathways and delay DNA repair leading to persistent DNA damage, contribuing to genome instability and synthetic lethality. The results of this thesis demonstrated that targeting Cx43 to interfere with DNA repair could emerge as a potential therapeutic strategy to increase efficacy and to overcome drug resistance in tumours with a BRAF mutant background. In light of our findings, we designed an innovative drug combination by using EVs as drug delivery systems to deliver the full-Cx43 (mRNA and protein) in combination with the BRAF/MEKi. Our results have shown that Cx43 not only increases the internalisation capacity of EVs into receptor cells but also modifies their cargo through the recruitment of new proteins and different molecules of RNAs. The modification of the cargo of these EVs could have an impact on their physiological functions, as we have observed that both sensitive and resistant tumour cells are affected by treatment with these particles, especially when they are in combination with BRAF/MEKi. Furthermore, it has been observed that Cx43 impacts the metabolism of tumour cells by enhancing mitochondrial content and increasing their capacity for respiration through oxidative phosphorylation in BRAFV600E melanoma cells. In addition, Cx43 is present in the endoplasmic reticulum (ER) of melanoma cells, suggesting its role as a novel regulator of ERrelated stress pathways, potentially leading to cell death. Overall, our study highlights novel functions of Cx43, demonstrating for the first time that Cx43 is a novel factor contributing to DNA repair response and BRAF/MEKi efficacy, as well as a key player in the regulation of ER stress, highlighting the potential of Cx43 as an antitumour agent and enhancer of therapy efficacy, as well as a bypass for drug resistance acquisition in BRAF-mutant tumours.

[gl] As mutacións na proteína quinasa oncoxénica BRAF están directamente relacionadas coa aparición e progresión de varios tipos de tumores, incluíndo máis do 50% dos casos de melanoma e aproximadamente o 8% de todos os cancros no mundo. Na actualidade, os inhibidores de BRAF/MEK (BRAF/MEKi) son considerados a terapia estándar para os melanomas que presentan mutacións en BRAF, e modificaron drasticamente o panorama do tratamento dos tumores avanzados con esta característica xenética. Con todo, a súa efectividade é limitada e a resistencia a estes fármacos constitúe un obstáculo significativo para o éxito do tratamento. A proteína transmembrana conexina43 (Cx43) facilita o intercambio de pequenas moléculas, como ións, metabolitos e ARN pequenos, a través de diferentes mecanismos celulares como unións comunicantes (GJs), hemichanles ou vesículas extracelulares (EVs), afectando aspectos clave como o crecemento, metabolismo e diferenciación celular. Ademais, identificouse á Cx43 nas membranas asociadas ás mitocondrias (MAMs) e suxeriuse como un potencial supresor tumoral no melanoma primario. Con todo, o seu papel molecular exacto na progresión da enfermidade aínda é motivo de controversia. Neste proxecto, o noso obxectivo é dilucidar o papel da Cx43 no contexto de cancros con mutación en BRAF e na resistencia á terapia asociada ás devanditas mutacións. Os nosos achados indican que a Cx43 mellora a eficacia dos inhibidores de BRAF/MEK en varios modelos de tumores BRAF-mutados, tanto in vitro como in vivo. Este efecto lógrase atraendo complexos de reparación do ADN cara á membrana nuclear a través da interacción con diversas proteínas, como lamin-A/C e diferentes proteínas de reparación do ADN. Esta compartimentación nuclear restrinxe as vías de reparación do ADN, o que conduce a danos persistentes no xenoma e contribúe á inestabilidade xenómica e á letalidade sintética. Os nosos resultados suxiren que a Cx43 podería ser usada como unha estratexia terapéutica prometedora para mellorar a eficacia e superar a resistencia aos fármacos en tumores con mutacións en BRAF. En base aos nosos resultados, desenvolvemos unha combinación innovadora de fármacos utilizando EVs como vehículos de administración para entregar a Cx43 completa (ARNm e proteína) xunto cos BRAF/MEKi. Descubrimos que a Cx43 non só aumenta a capacidade de internalización das EVs nas células receptoras, senón que tamén modifica o seu contido, o que afecta tanto as células tumorais sensibles como ás resistentes, especialmente cando se combinan cos BRAF/MEKi. Ademais, observamos que a Cx43 impacta no metabolismo celular das células de melanoma BRAF mutado, aumentando a tensión do retículo endoplásmico (RE), o que podería estar a provocar un aumento no contido mitocondrial e a capacidade respiratoria das células. En conxunto, o noso estudo destaca novas funcións da Cx43, demostrando a súa contribución á resposta de reparación do ADN, a eficacia de BRAF/MEKi e a regulación da tensión do RE en tumores con mutacións en BRAF. Estes achados resaltan o potencial da Cx43 como un axente antitumoral e potenciador da eficacia terapéutica, así como un obxectivo para combater a resistencia aos fármacos en tumores con mutacións en BRAF.