Disclosing FATP1 as a therapeutic target in breast cancer

Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2018A neoplasia maligna da mama e a neoplasia mais frequente nas mulheres a nível mundial com cerca de 1.7 milhões de casos e 521,900 mortes em 2012, representando 25% de todos os casos de cancro e 15% de todas as mortes por cancro entre mulheres. Os países mais desenvolvidos são os que apresentam as maiores taxas de incidência enquanto que a mortalidade relativa e maior nos países menos desenvolvidos. A redução do uso de terapia de reposição hormonal na pós-menopausa e a participação em programas de rastreio através de mamografias baixaram as taxas de incidência nos países ocidentais, no entanto, em Portugal, estas taxas tem vindo a aumentar nos últimos anos. Entre as mulheres portuguesas o carcinoma da mama e a neoplasia maligna mais comum e a principal causa de morte por cancro, com 6088 novos casos estimados e 1570 mortes em 2012. Este aumento pode estar também relacionado com alterações nos padrões reprodutivos, obesidade e inactividade física. O cancro da mama e dividido em vários subtipos com características moleculares intrínsecas: luminal A, luminal B, sobre expressão de HER2 e basal. Os tipos luminal A e B são caracterizados pela expressão de receptores hormonais, enquanto que os restantes subtipos são menos propensos a expressar o receptor de estrogénio (ER) ou de progesterona (PgR), sendo que o basal não expressa ER, PgR e HER2 (tumores triplo negativos). A heterogeneidade biológica bem como a plasticidade dinâmica do microambiente tumoral são importantes desafios no estudo e o tratamento do carcinoma da mama. Exemplo disso, e a importância de conhecer o status do RE e HER2, uma vez que constituem indicadores de prognostico e alvos terapêuticos relevantes. A acção do estrogénio e mediada pela ligação a receptores de estrogénio (ER-α e/ou ER-β) que são expressos em vários tecidos. Actualmente, apenas o ER-α tem sido usado na clinica como marcador de prognóstico e alvo terapêutico devido a sua elevada expressão proteica em lesões mamárias malignas em comparação com o tecido normal. A administração de fármacos antagonistas de estrogénios, como o tamoxifen, e eficaz no tratamento de cancro da mama, no entanto, estes fármacos tem diversos efeitos secundários e apos tratamentos prolongados os tumores podem tornar-se resistentes. A doença oncológica e considerada mundialmente como um problema de saúde pública e a segunda maior causa de morte em termos globais. A acumulação de alterações genéticas e epigenéticas permite as células escaparem a rede de controlo que regula o equilíbrio homeostático entre a proliferação celular e morte celular, levando a uma proliferação anormal de células. Uma neoplasia e considerada maligna se as suas células tiverem adquirido independência mitogénica e a capacidade de invadir os tecidos adjacentes. Durante a carcinogénese, 6 propriedades celulares fundamentais são alteradas: aumento da sinalização proliferativa, elevado potencial replicativo, insensibilidade aos sinais de antimitogénese, capacidade de invasão tecidular e metastização, angiogénese sustentada e resistência a apoptose. Estas capacidades biológicas adquiridas durante esta transformação gradual e complexa são as chamadas hallmarks do cancro que foram recentemente revisitadas, emergindo 2 novas hallmarks, a reprogramação do metabolismo energético e evasão ao controlo imunitário. Para além das células tumorais, as células não malignas do microambiente tumoral, incluindo fibroblastos, adipócitos, células imunes e endoteliais são determinantes na biologia do cancro visto que actuam como uma rede funcional na qual factores solúveis e moléculas orgânicas são continuamente partilhados. Para cumprir os requisitos biossintéticos associados a proliferação, uma célula deve aumentar a importação de nutrientes que suportam a sobrevivência e alimentam o crescimento celular ocorrendo uma remodelação metabólica. Num nicho tumoral proliferativo, os ácidos gordos são essenciais visto que podem ser usados simultaneamente como combustível e como blocos de construção de forma a manter a renovação e divisão celular. A desmoplasia é relativamente frequente em carcinoma da mama, apesar de não ser característica exclusiva, e consiste numa alteração estromal caracterizada pela presença de estroma fibrótico que tem como principal componente celular os fibroblastos. Para além das células tumorais, os fibroblastos associados a cancro (CAFS) são a componente principal do microambiente tumoral e pensa-se que eles desempenhem um papel determinante no início e na progressão da doença. Conceptualmente, os adipócitos são os principais fornecedores de ácidos gordos, no entanto, o nosso grupo de investigação demonstrou, em modelos in vitro e in vivo, que os CAFs cooperam com células de cancro da mama ao produzirem e fornecerem ácidos gordos a estas. As células de carcinoma da mama expostas a meio condicionado por CAFs aumentavam a absorção de lípidos bem como a expressão de FATP1/SLC27A1 (FA transport protein 1), promovendo a transferência de ácidos gordos. FATP1 e uma proteína membranar responsável pela absorção de ácidos gordos de cadeia longa e muito longa. A regulação da síntese, absorção e degradação de ácidos gordos e essencial para sustentar a fisiologia celular. Assim, limitar a disponibilidade de ácidos gordos as células tumorais de forma a perturbar o crescimento tumoral e uma possível abordagem terapêutica que poderá usar o FATP1 como alvo. Considerando a transferência de ácidos gordos dos CAFs as células de carcinoma da mama, o FATP1 parece ser um candidato adequado para tratar o cancro da mama e um possível marcador de prognóstico. Em 2013, foram desenvolvidos uns fármacos que foram caracterizados como inibidores eficazes do FATP1, as arilpiperazidinas. Este projecto tem como objectivo demonstrar o papel do FATP1 na sobrevivência e comportamento das células de cancro da mama, de forma a validar o seu uso como factor de prognóstico e alvo terapêutico. Para atingir este objectivo foram delineados 3 objectivos específicos: o primeiro e confirmar que o estrogénio e os ácidos gordos regulam a expressão de FATP1/SLC27A1 e que esta proteína e crucial para as células de carcinoma da mama; o segundo objectivo surgiu aquando da análise dos resultados obtidos no primeiro objectivo e consiste em esclarecer o papel do ERb na regulação de FATP1/SLC27A1, e o terceiro objectivo e testar a inibição de FATP1 recorrendo a exposição a arilpiperazidinas 5k e 12a na tentativa de delinear uma possível estratégia terapêutica. Os resultados de RQ-PCR e western blotting mostraram que os ácidos gordos (acido linoleico (C18:2)) e estradiol estimulam a expressão de FATP1/SLC27A1, especialmente nas MDA-MB-231 que possui níveis basais mais elevados de mRNA FATP1/SLC27A1 em comparação com MCF7. Curiosamente, observamos que o estradiol estimula a ligação do ER-b ao promotor do FATP1/SLC27A1. Isto levou-nos a investigar o papel do ER-b na regulação do FATP1/SLC27A1 utilizando um agonista do ER-b, o ERB-041 e um antagonista do ER-b, o PHTPP. Os resultados revelaram que o PHTPP afecta a viabilidade celular, a absorção de ácidos gordos e aumenta os níveis de FATP1 na membrana celular em ambas as linhas celular indicando que para alem do ER-a, o ER-b actua como um factor de pro-sobrevivência em células de carcinoma da mama e deveria ser investigado como eventual marcador de prognostico. Observamos igualmente que os ácidos gordos e o estradiol induzem a migração e a proliferação celular. Os resultados in vitro estão de acordo com dados de doentes que mostram uma maior expressão de FATP1/SLC27A1 em carcinomas da mama mais agressivos e invasivos. Por conseguinte, a inibição do FATP1 com as arilpiperazidinas interferiu com a absorção de ácidos gordos e com a proliferação celular validando a importância do FATP1 como um potencial alvo terapêutico em cancro da mama. O nosso estudo inicia um percurso de investigação que acreditamos que permitira eleger o FATP1 como um marcador de mau prognostico do cancro da mama e um potencial alvo terapêutico.Breast cancer is the most frequent malignant neoplasia in women worldwide, with an estimated 1.7 million cases and 521,900 deaths in 2012, accounting for 25% of all cancer cases and 15% of all cancer deaths among females. In western countries, incidence rates have been declining, however, in Portugal breast cancer age-adjusted incidence rates have been increasing for years. Indeed, breast carcinoma is the leading cause of cancer mortality among Portuguese women, with an estimated 6088 new cases and 1570 deaths in 2012. Cancer is a complex group of diseases in which several cellular and molecular components of the tumor microenvironment contribute to the survival of cancer cells. The non-neoplastic cells of the tumor microenvironment including fibroblasts, adipocytes, immune and endothelial cells are determinant in cancer biology since they act as a functional network in which soluble factors and organic molecules are transiently shared. To fulfill the biosynthetic demands associated with proliferation, a cell must increase the import of nutrients that sustain survival and fuel cell growth, supported by a metabolic remodeling. In a proliferative tumor niche, fatty acids (FA) are crucial since they are both fuel and construction blocks, maintaining cell renewal and division. Conceptually, adipocytes are the main suppliers of FA, however our team published that cancer associated fibroblasts (CAFs) also function as hubs of FA to supply the needs of cancer cells. Breast cancer cells exposed to CAFs-conditioned media increased their lipid uptake and the expression of FATP1/SLC27A1 (FA transport protein 1), promoting FA transfer. FATP1 is an integral membrane protein known to enhance the uptake of long-chain and very long-chain FA into cells. Considering FA transfer from CAFs to breast cancer cells, FATP1 appears to be a suitable candidate to treat breast cancer and a possible marker of disease outcome. The present thesis aims to prove the role of FATP1 in breast cancer cells survival and behavior in order to validate its use as a therapeutic target. The main objective was divided in 3 specific aims: The first aim is to confirm that estrogen and FA regulate the expression of FATP1/SLC27A1, which is crucial for breast cancer cells. The second aim emerged from the evaluation of the results obtained in the first aim, and it is to evaluate the role of ER-b in FATP1/SLC27A1 regulation. The third aim is to target FATP1 with arylpiperazines, which were characterized as effective inhibitors of FATP1, testing a putative therapeutic approach. RQ-PCR and western blotting analysis showed that FA (linoleic acid (C18:2)) and estradiol stimulated FATP1/SLC27A1 expression, especially in MDA-MB-231, which has higher basal levels of FATP1/SLC27A1 mRNA in comparison with MCF7. Interestingly, we found that estradiol stimulated the binding of ER-b to the FATP1/SLC27A1 promoter. These findings prompted us to investigate the role of ER-b in FATP1/SLC27A1 regulation using an ER-b agonist, ERB-041 and an ER-b antagonist, PHTPP. Results revealed that PHTPP affects cell viability, the uptake of FA and increases the levels of FATP1 in the cell membrane in both cell lines. Also indicating that, besides ER-a, ER-b is a pro-survival effector in breast cancer cells and it should be tested as a prognostic marker. Wound healing and cell cycle analysis showed that FA and estradiol induced cell migration and proliferation. Our in vitro findings were supported by patients’ data showing a higher expression of FATP1/SLC27A1 in more aggressive and invasive breast carcinomas. Furthermore, the inhibition of FATP1 with arylpiperazines interfered with the uptake of FA and cell proliferation, validating the importance of FATP1 as a putative therapeutic target in breast cancer. Our study paves the path in order to elect FATP1 a suitable marker for breast cancer prognosis and a putative therapeutic target to better treat breast cancer patients

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans—anteaters, sloths, and armadillos—have essential functions forecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosys-tem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts withdomestic dogs, these species have been threatened locally, regionally, or even across their fulldistribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths.Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae(3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data onDasypus pilo-sus(Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized,but new genetic studies have revealed that the group is represented by seven species. In thisdata paper, we compiled a total of 42,528 records of 31 species, represented by occurrence andquantitative data, totaling 24,847 unique georeferenced records. The geographic range is fromthe southern United States, Mexico, and Caribbean countries at the northern portion of theNeotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regardinganteaters,Myrmecophaga tridactylahas the most records (n=5,941), andCyclopessp. havethe fewest (n=240). The armadillo species with the most data isDasypus novemcinctus(n=11,588), and the fewest data are recorded forCalyptophractus retusus(n=33). Withregard to sloth species,Bradypus variegatushas the most records (n=962), andBradypus pyg-maeushas the fewest (n=12). Our main objective with Neotropical Xenarthrans is to makeoccurrence and quantitative data available to facilitate more ecological research, particularly ifwe integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, andNeotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure,habitat loss, fragmentation effects, species invasion, and climate change effects will be possiblewith the Neotropical Xenarthrans data set. Please cite this data paper when using its data inpublications. We also request that researchers and teachers inform us of how they are usingthese data

Neotropical xenarthrans: a dataset of occurrence of xenarthran species in the Neotropics.

International audienceXenarthrans—anteaters, sloths, and armadillos—have essential functions forecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosys-tem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts withdomestic dogs, these species have been threatened locally, regionally, or even across their fulldistribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths.Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae(3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data onDasypus pilo-sus(Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized,but new genetic studies have revealed that the group is represented by seven species. In thisdata paper, we compiled a total of 42,528 records of 31 species, represented by occurrence andquantitative data, totaling 24,847 unique georeferenced records. The geographic range is fromthe southern United States, Mexico, and Caribbean countries at the northern portion of theNeotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regardinganteaters,Myrmecophaga tridactylahas the most records (n=5,941), andCyclopessp. havethe fewest (n=240). The armadillo species with the most data isDasypus novemcinctus(n=11,588), and the fewest data are recorded forCalyptophractus retusus(n=33). Withregard to sloth species,Bradypus variegatushas the most records (n=962), andBradypus pyg-maeushas the fewest (n=12). Our main objective with Neotropical Xenarthrans is to makeoccurrence and quantitative data available to facilitate more ecological research, particularly ifwe integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, andNeotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure,habitat loss, fragmentation effects, species invasion, and climate change effects will be possiblewith the Neotropical Xenarthrans data set. Please cite this data paper when using its data inpublications. We also request that researchers and teachers inform us of how they are usingthese data

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans—anteaters, sloths, and armadillos—have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths. Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the southern United States, Mexico, and Caribbean countries at the northern portion of the Neotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n = 5,941), and Cyclopes sp. have the fewest (n = 240). The armadillo species with the most data is Dasypus novemcinctus (n = 11,588), and the fewest data are recorded for Calyptophractus retusus (n = 33). With regard to sloth species, Bradypus variegatus has the most records (n = 962), and Bradypus pygmaeus has the fewest (n = 12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans data set. Please cite this data paper when using its data in publications. We also request that researchers and teachers inform us of how they are using these data

NEOTROPICAL CARNIVORES: a data set on carnivore distribution in the Neotropics

Mammalian carnivores are considered a key group in maintaining ecological health and can indicate potential ecological integrity in landscapes where they occur. Carnivores also hold high conservation value and their habitat requirements can guide management and conservation plans. The order Carnivora has 84 species from 8 families in the Neotropical region: Canidae; Felidae; Mephitidae; Mustelidae; Otariidae; Phocidae; Procyonidae; and Ursidae. Herein, we include published and unpublished data on native terrestrial Neotropical carnivores (Canidae; Felidae; Mephitidae; Mustelidae; Procyonidae; and Ursidae). NEOTROPICAL CARNIVORES is a publicly available data set that includes 99,605 data entries from 35,511 unique georeferenced coordinates. Detection/non-detection and quantitative data were obtained from 1818 to 2018 by researchers, governmental agencies, non-governmental organizations, and private consultants. Data were collected using several methods including camera trapping, museum collections, roadkill, line transect, and opportunistic records. Literature (peer-reviewed and grey literature) from Portuguese, Spanish and English were incorporated in this compilation. Most of the data set consists of detection data entries (n = 79,343; 79.7%) but also includes non-detection data (n = 20,262; 20.3%). Of those, 43.3% also include count data (n = 43,151). The information available in NEOTROPICAL CARNIVORES will contribute to macroecological, ecological, and conservation questions in multiple spatio-temporal perspectives. As carnivores play key roles in trophic interactions, a better understanding of their distribution and habitat requirements are essential to establish conservation management plans and safeguard the future ecological health of Neotropical ecosystems. Our data paper, combined with other large-scale data sets, has great potential to clarify species distribution and related ecological processes within the Neotropics. There are no copyright restrictions and no restriction for using data from this data paper, as long as the data paper is cited as the source of the information used. We also request that users inform us of how they intend to use the data

NEOTROPICAL ALIEN MAMMALS: a data set of occurrence and abundance of alien mammals in the Neotropics

Biological invasion is one of the main threats to native biodiversity. For a species to become invasive, it must be voluntarily or involuntarily introduced by humans into a nonnative habitat. Mammals were among first taxa to be introduced worldwide for game, meat, and labor, yet the number of species introduced in the Neotropics remains unknown. In this data set, we make available occurrence and abundance data on mammal species that (1) transposed a geographical barrier and (2) were voluntarily or involuntarily introduced by humans into the Neotropics. Our data set is composed of 73,738 historical and current georeferenced records on alien mammal species of which around 96% correspond to occurrence data on 77 species belonging to eight orders and 26 families. Data cover 26 continental countries in the Neotropics, ranging from Mexico and its frontier regions (southern Florida and coastal-central Florida in the southeast United States) to Argentina, Paraguay, Chile, and Uruguay, and the 13 countries of Caribbean islands. Our data set also includes neotropical species (e.g., Callithrix sp., Myocastor coypus, Nasua nasua) considered alien in particular areas of Neotropics. The most numerous species in terms of records are from Bos sp. (n = 37,782), Sus scrofa (n = 6,730), and Canis familiaris (n = 10,084); 17 species were represented by only one record (e.g., Syncerus caffer, Cervus timorensis, Cervus unicolor, Canis latrans). Primates have the highest number of species in the data set (n = 20 species), partly because of uncertainties regarding taxonomic identification of the genera Callithrix, which includes the species Callithrix aurita, Callithrix flaviceps, Callithrix geoffroyi, Callithrix jacchus, Callithrix kuhlii, Callithrix penicillata, and their hybrids. This unique data set will be a valuable source of information on invasion risk assessments, biodiversity redistribution and conservation-related research. There are no copyright restrictions. Please cite this data paper when using the data in publications. We also request that researchers and teachers inform us on how they are using the data