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Dissertação de mestrado em Genética Molecular, O própolis é um produto natural resinoso produzido por abelhas melíferas. Diferentes grupos de compostos têm sido identificados no própolis e associados a diversas bioatividades. Algumas amostras de própolis Português exibem potencial antioxidante e antimicrobiano pelo que, neste trabalho, se avaliou esses atributos numa amostra de própolis obtida no Gerês em 2018. A atividade antitumoral do própolis Português só foi descrita no carcinoma renal, cancro da próstata, colorretal e mama. Até à data, nada se sabe sobre o efeito deste produto em melanoma com mutação BRAF, o cancro de pele mais agressivo e letal. Assim, para além da avaliação das propriedades antioxidantes e antimicrobianas do própolis do Gerês, este trabalho teve como objetivo avaliar também o seu potencial contra melanoma. Primeiramente, as propriedades antioxidantes e antimicrobianas do G18.EE e de micro-extratos etanólicos de própolis do Gerês recolhido em 2020 (G20.miEEs) foram avaliadas, demonstrando atividade antioxidante e antimicrobiana significativas, sendo G18.EE o mais eficaz. Adicionalmente, G18 revelou cumprir os requisitos de qualidade estabelecidos para própolis. Assim, G18.EE foi selecionado e fracionado – n-hexano, acetato de etilo (EtOAc) e n-butanol (n-BuOH) – para uso nos ensaios antitumorais in vitro. Quanto à atividade antitumoral, o G18.EE e as suas frações afetam a viabilidade de células de melanoma mutadas em BRAF, sendo n-BuOH/G18.EE e n BuOH/EtOAc as frações mais ativas para a linha A375 e WM9, respetivamente. O efeito de G18.EE e das frações selecionadas (n-BuOH e EtOAc) na proliferação, migração, crescimento, apoptose, metabolismo, produção de ROS e potencial mitocondrial foi avaliado. Conclui-se que os tratamentos diminuem a migração celular e o crescimento e a migração dos esferóides. Também se observou um aumento da produção de lactato, consistente com o aumento da expressão de LDHA e MCT-1, assim como uma acumulação de ROS em células de melanoma tratadas. Por fim, através da avaliação da expressão de proteínas pró e anti-apoptóticas, validou-se a indução da apoptose. Em suma, pela primeira vez, apresentam-se evidências de atividade antitumoral do própolis português em melanoma, sendo que este modula importantes marcadores tumorigénicos – crescimento tumoral, proliferação e migração– e promove a morte celular via apoptose mediada por ROS. Este produto natural é uma potencial fonte de compostos para o desenvolvimento de novas terapias anticancerígenas., Propolis is a natural resinous product produced by Apis mellifera L. (worker honeybees). Different groups of compounds have been identified in propolis and associated with different bioactivities. Portuguese propolis is a powerful antioxidant and antimicrobial agent. Thus, in this work, the last properties were evaluated in a propolis sample from Gerês collected in 2018. Regarding its antitumoral activity, it has only been verified in renal cell carcinoma, prostate, colorectal, and breast cancer. Nothing is known about the potential of this national natural product in BRAF-mutated melanoma, the most aggressive and lethal type of skin cancer. In light of this, besides the evaluation of the antioxidant and antimicrobial properties from Gerês propolis, this work aims to assess its potential against melanoma. To start, antioxidant and antimicrobial properties of G18.EE and of ethanol micro-extracts harvested in 2020 in Gerês (G20.miEEs) were evaluated, demonstrating significant antioxidant and antimicrobial activities, with G18.EE showing the highest efficacy. Additionally, it was established that G18 fulfills the quality requirements established for propolis. Considering this, G18.EE was selected and fractionated - n-hexane, ethyl acetate (EtOAc), and n-butanol (n-BuOH) fractions - to use in the in vitro antitumoral assays. About the antitumoral activity, we first observed that G18.EE and its fractions affect the BRAF-mutated melanoma cells viability, being n-BuOH/G18.EE and n BuOH/EtOAc the most active fractions for A375 and WM9 cells, respectively. The effect of G18.EE and selected fractions (n-BuOH and EtOAc) on proliferation, migration, growth, apoptosis, metabolism, ROS production, and mitochondrial potential was assessed. It was concluded that propolis treatments decrease cell migration and the growth and migration of tumor spheroids. Additionally, we observed an increase in lactate production, which is consistent with the increased expression of LDHA and MCT-1, as well as an accumulation of ROS in melanoma-treated cells. Lastly, the upregulation of pro-apoptotic and the downregulation of anti-apoptotic proteins show a stimulation of apoptosis. To summarize, for the first time, we provided evidence that Portuguese propolis has anticancer activity against melanoma, since it modulates important hallmarks that dictate tumorigeneses, namely cell proliferation, cell migration, and tumor growth; and promotes cancer cell death via ROS-mediated apoptosis. This natural product is a potential source of compounds for the development of new anticancer therapies.

The overexpression of chondroitin sulfate proteoglycan 4 (CSPG4) is associated with several tumor types, including malignant melanoma, squamous cell carcinoma, triple‑negative breast carcinoma, oligodendrocytomas or gliomas. Due to its restricted distribution in normal tissues, CSPG4 has been considered a potential target for several antitumor approaches, including monoclonal antibody (mAb) therapies. The aim of the present study was to characterize the impact of the CSPG4‑specific mAb clone 9.2.27 on its own or in combination with the commonly used BRAF‑selective inhibitor, PLX4032, on different functions of melanoma cells to assess the potential synergistic effects. The BRAF V600‑mutant human melanoma cell lines, M14 (CSPG4‑negative) and WM164 (CSPG4‑positive), were exposed to the CSPG4‑specific 9.2.27 mAb and/or PLX4032. Cell viability and colony formation capacity were evaluated. A 3D‑cell culture spheroid model was used to assess the invasive properties of the treated cells. In addition, flow cytometric analysis of apoptosis and cell cycle analyses were performed. Incubation of the WM164 cell line with CSPG4‑specific 9.2.27 mAb decreased viability, colony formation ability and the invasive capacity of CSPG4‑positive tumor cells, which was not the case for the CSPG4‑negative M14 cell line. Combined treatment of the WM164 cells with 9.2.27 mAb plus PLX4032 did not exert any significant additional effect in comparison to treatment with PLX4032 alone in the clonogenic and invasion assays. M14 cell cycle distribution was not influenced by the CSPG4‑specific 9.2.27 mAb. By contrast, the exposure of WM164 cells to the mAb resulted in an arrest of the cells in the S phase. Moreover, combined treatment of the WM164 cells led to a significantly increased accumulation of cells in the subG1 phase, combined with a decrease of cells in the G2/M phase. On the whole, findings of the present study indicate that the CSPG4‑specific 9.2.27 mAb exerts an anti‑invasive effect on CSPG4‑positive melanoma spheroids, which is not enhanced by BRAF inhibition. These findings provide the basis for further investigations on the effects of anti‑CSPG4‑based treatments of CSPG4‑positive tumors.

Simple Summary Leptomeningeal metastases are a devastating complication of solid tumors with poor survival, regardless of the type of treatments. The limited efficacy of targeted agents is due to the molecular divergence between leptomeningeal recurrences and primary site, as well as the presence of a heterogeneous blood-brain barrier and blood-tumor barrier that interfere with the penetration of drugs into the brain. The diagnosis of leptomeningeal metastases is achieved by neurological examination, and/or brain and spinal magnetic resonance, and/or a positive cerebrospinal fluid cytology. The presence of neoplastic cells in the cerebrospinal fluid examination is the gold-standard for the diagnosis of leptomeningeal metastases; however, novel techniques known as “liquid biopsy” aim to improve the sensitivity and specificity in detecting circulating neoplastic cells or DNA in the cerebrospinal fluid. Targeted therapies and immunotherapies have changed the natural history of metastatic solid tumors, including lung, breast cancer, and melanoma. Targeting actionable mutations, such as epidermal growth factor receptor-mutated and anaplastic lymphoma kinase-gene rearranged in lung cancer, human epidermal growth factor receptor 2-positive breast cancer, and BRAF-mutated melanoma, have led to encouraging results also in leptomeningeal metastases. On the other hand, immunotherapy or modified traditional chemotherapy are under investigation in LM from non-druggable tumors. Abstract Leptomeningeal metastases (LM) from solid tumors represent an unmet need of increasing importance due to an early use of MRI for diagnosis and improvement of outcome of some molecular subgroups following targeted agents and immunotherapy. In this review, we first discussed factors limiting the efficacy of targeted agents in LM, such as the molecular divergence between primary tumors and CNS lesions and CNS barriers at the level of the normal brain, brain tumors and CSF. Further, we reviewed pathogenesis and experimental models and modalities, such as MRI (with RANO and ESO/ESMO criteria), CSF cytology and liquid biopsy, to improve diagnosis and monitoring following therapy. Efficacy and limitations of targeted therapies for LM from EGFR-mutant and ALK-rearranged NSCLC, HER2-positive breast cancer and BRAF-mutated melanomas are reported, including the use of intrathecal administration or modification of traditional cytotoxic compounds. The efficacy of checkpoint inhibitors in LM from non-druggable tumors, in particular triple-negative breast cancer, is discussed. Last, we focused on some recent techniques to improve drug delivery.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a multifunctional transmembrane proteoglycan involved in spreading, migration and invasion of melanoma. In addition to the activating BRAF V600E mutation, CSPG4 was shown to promote MAPK signaling by mediating the growth-factor induced activation of receptor tyrosine kinases. However, it remains elusive which factors regulate CSPG4 expression. Therefore, the aim of the present study was to examine whether BRAF and MEK inhibitors have an effect on the expression of CSPG4. We exposed a panel of BRAF-mutant CSPG4-positive or -negative melanoma cell lines to BRAF and MEK inhibitors. Protein levels of CSPG4 were analyzed by flow cytometry (FACS), immunofluorescence microscopy (IF), and western blotting. CSPG4 mRNA levels were determined by quantitative PCR (qPCR). The prolonged exposure of cells to BRAF and MEK inhibitors resulted in markedly reduced levels of the CSPG4 protein in permanent resistant melanoma cells as well as decreased levels of its mRNA. We did not observe increasing levels of CSPG4 shedding into the culture supernatants. In addition, patient-derived matched tumor samples following therapy with kinase inhibitors showed decreased numbers of CSPG4-positive cells as compared to pre-therapy tumor samples. Our results indicate that BRAF and MEK inhibition downregulates CSPG4 expression until the cells have developed permanent resistance. Our findings provide the basis for further investigation of the role of CSPG4 in the development of drug-resistance in melanoma cells.

Compelling evidence indicates that the immediate heterogeneous adaptive response of BRAF-mutated melanoma cells plays a major role in the resistance to MAPK inhibition. A large number of compensatory mechanisms of adaptive response to BRAF inhibition rely on variations in gene expression level, many of which occur at the post-transcriptional and translational steps of gene expression and are being regulated by regulatory mRNA-binding proteins (mRBPs). HuR (ELAVL1), which is a ubiquitously expressed stress-response nuclear mRBP, has been shown to be involved in cell proliferation as well as cell differentiation. HuR's extensively studied repertoire includes many of the important nodes of the signaling network involved in the adaptive response to MAPK inhibition. We initiate our work in this thesis by experimentally demonstrating that the BRAF inhibitor-induced immediate paradoxical proliferation of a subpopulation of BRAF-mutated melanoma cells is prevented in HuR-overexpressing melanoma cells. We then conduct a series of observational studies ex vivo and in vivo in patients' metastatic tumors, and show that a larger proportion of HuRLow expressing cells (HuRLow cells) is associated with a higher propensity of the whole malignant cell population for adaptive response. In our subsequent experimental work, we demonstrate that a reversible increase in the proportion of HuRLow cells, which is equivalent to increasing HuR expression fluctuation, enhances the adaptive response to BRAF inhibition. Importantly, HuRLow cells display MAPK independency and a highly “plastic”, slow or fast proliferating expression profile. Conversely, lithium salts therapy suppresses the HuRLow cell subpopulation, restores partially the growth of adapted cells and attenuates the adaptive response of the whole malignant cell population both ex vivo and in vivo in a xenograft model of adaptive response. This effect is associated at the whole cell population level with a less resistant expression profile. Our work has immediate clinical consequences for the therapy of metastatic melanoma considering that we describe a therapeutically actionable mechanism to reduce the proportion of highly plastic and partly slowly dividing cells in which the adaptive proliferation emerges under BRAF inhibition. Indeed, we are already working in initiating a multicentric phase I/II trial to evaluate the combination of lithium salts with BRAF/MEK inhibitors in BRAF-mutated metastatic melanoma. In this trial, we will use the IHC-based automated quantification approach developed in this thesis to monitor the expression distribution of HuR. Our work has broad implications for the targeted therapies of solid malignancies and opens many research perspectives, which include the precise understanding of the mechanisms that differentiate malignant cells that tightly regulate the expression of RBPs, such as HuR, from those in which their expression is loosely regulated. Such studies will likely improve our therapeutic ability to reduce the plasticity of malignant cells and their adaptive response to targeted therapies.

In patients with BRAFV600 mutated unresectable stage IIIc or metastatic melanoma, molecular targeted therapy with combined BRAF/MEK-inhibitor vemurafenib plus cobimetinib has shown a significantly improved progression-free survival and overall survival compared to treatment with vemurafenib alone. Nevertheless, the majority of BRAFV600 mutation-positive melanoma patients will eventually develop resistance to treatment. Molecular imaging with 18F-Fluorodeoxyglucose (18F-FDG) PET has been used to monitor response to vemurafenib in some BRAFV600 mutated metastatic melanoma patients, showing a rapid decline of 18F-FDG uptake within 2 weeks following treatment. Furthermore, preliminary results suggest that metabolic alterations might predict the development of resistance to treatment. 18F-Fluoro-3′-deoxy-3’L-fluorothymidine (18F-FLT), a PET-tracer visualizing proliferation, might be more suitable to predict response or resistance to therapy than 18F-FDG. This phase II, open-label, multicenter study evaluates whether metabolic response to treatment with vemurafenib plus cobimetinib in the first 7 weeks as assessed by 18F-FDG/18F-FLT PET can predict progression-free survival and whether early changes in 18F-FDG/18F-FLT can be used for early detection of treatment response compared to standard response assessment with RECISTv1.1 ceCT at 7 weeks. Ninety patients with BRAFV600E/K mutated unresectable stage IIIc/IV melanoma will be included. Prior to and during treatment all patients will undergo 18F-FDG PET/CT and in 25 patients additional 18F-FLT PET/CT is performed. Histopathological tumor characterization is assessed in a subset of 40 patients to unravel mechanisms of resistance. Furthermore, in all patients, blood samples are taken for pharmacokinetic analysis of vemurafenib/cobimetinib. Outcomes are correlated with PET/CT-imaging and therapy response. The results of this study will help in linking PET measured metabolic alterations induced by targeted therapy of BRAFV600 mutated melanoma to molecular changes within the tumor. We will be able to correlate both 18F-FDG and 18F-FLT PET to outcome and decide on the best modality to predict long-term remissions to combined BRAF/MEK-inhibitors. Results coming from this study may help in identifying responders from non-responders early after the initiation of therapy and reveal early development of resistance to vemurafenib/cobimetinib. Furthermore, we believe that the results can be fundamental for further optimizing individual patient treatment. Clinicaltrials.gov identifier: NCT02414750. Registered 10 April 2015, retrospectively registered.