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Role of arl13b in breast cancer cell migration and invasion
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RESUMO: O cancro da mama é a principal causa de morte, associada a cancro, na população
mundial feminina, maioritariamente devido à formação de metástases. A
disseminação das células cancerígenas resulta da sua capacidade em migrar a
partir do tumor primário e invadir o tecido circundante até chegar à corrente
sanguínea ou linfática, onde podem chegar a outros órgãos. Desta forma, a
capacidade de migração e invasão celular é essencial para a formação de
metástases e é controlada pela regulação das adesões célula-célula e célulamatriz extracelular, pela capacidade de degradar a matriz extracelular e pelo
rearranjo do citoesqueleto de actina. Assim, a identificação das proteínas
envolvidas na regulação da migração e invasão de células do cancro da mama
poderá contribuir para o estabelecimento de novas estratégias de prognóstico,
diagnóstico e de tratamento.
A família de fatores de ribosilação do ADP (Arf) é principalmente conhecida pela
sua função na regulação do tráfego vesicular. Curiosamente, vários estudos
mostram que células cancerígenas possuem uma expressão alterada e recrutam
membros desta família para migrar e invadir eficazmente. A proteína Arl13b
pertence a uma subfamília da família de proteínas Arf e foi primeiramente
identificada como sendo uma proteína ciliar. No entanto, vários estudos têm
demonstrado que a Arl13b pode ser encontrada e exercer funções fora do cílio. O
nosso grupo descobriu que a Arl13b regula o tráfego endocítico de reciclagem e a
migração celular em condições não-patológicas através da actina e da miosina
não muscular IIA (NMIIA), identificada como efector da Arl13b. Para além disso,
dados recentes do nosso laboratório sugerem uma correlação positiva entre a
expressão de Arl13b e a capacidade invasiva de células do cancro da mama.
Consequentemente, o principal objetivo desta tese foi avaliar o papel da Arl13b na
migração e invasão de células do cancro da mama e determinar os mecanismos
envolvidos.
Os resultados obtidos durante este trabalho mostram que a Arl13b regula
positivamente a migração e invasão de células de cancro da mama, in vitro. Para
além disso, estes resultados são corroborados por dados do nosso grupo que
mostram que a Arl13b regula o tamanho do tumor primário e o número de metástases pulmonares, in vivo. Curiosamente, o cancro da mama é o terceiro
tipo de cancro em que foi relatada uma função da Arl13b como regulador positivo
da formação e progressão tumoral. Nos outros dois modelos tumorais,
nomeadamente meduloblastoma e cancro gástrico, foi proposto um mecanismo
mediado pela via de sinalização “Sonic Hedgehog” canónica, que é dependente
do cílio. Em cancro da mama, nós propomos um mecanismo alternativo para a
regulação da migração e invasão celular por Arl13b, independente do cílio. Em
células de cancro da mama, observámos que a Arl13b está localizada e regula
negativamente as interações célula-célula. Adicionalmente, detetámos que a
Arl13b interage com a β3-integrina e colocaliza com outros dois componentes das
adesões focais (AF) com a matriz extracelular, nomeadamente Vinculina e
Paxilina. Quando a Arl13b é silenciada em células de cancro da mama, verificase um aumento do tamanho das AFs. Nós sugerimos que este aumento de
tamanho das AFs resulta de um aumento do crescimento destas estruturas,
mediado pela tensão gerada pela NMIIA, uma vez que nós observámos um
aumento dos níveis de NMIIA em células onde foi silenciada a Arl13b. Para além
disso, as AFs maiores podem resultar de uma redução da taxa de desmontagem,
que pode ser explicada pelos menores níveis de ativação da via de sinalização
mediada pelas integrinas, que se sabe estar envolvida na desmontagem de AFs.
Por fim, detetámos a presença de Arl13b em estruturas envolvidas na degradação
da matriz extracelular, designadas por invadopodia. No futuro, deverá ser avaliado
se a Arl13b regula a formação de invadopodia, já que isso poderia explicar a
função de Arl13b na regulação da invasão de células de cancro da mama.
Curiosamente, a desregulação das interações célula-matriz extracelular e do
rearranjo de actina são dois eventos observados nas fases iniciais da progressão
do cancro da mama. Dessa forma, seria interessante avaliar se um aumento de
expressão da Arl13b também é observado nas fases iniciais da progressão da
doença. De qualquer forma, como nós observámos que a Arl13b regula o rearranjo
de actina e a dinâmica das AFs e que a sua expressão está positivamente
correlacionada com a capacidade invasiva das células, nós propomos Arl13b
como um possível marcador prognóstico ou mesmo como um alvo terapêutico em
cancro da mama. Num cenário em que uma expressão aumentada de Arl13b é
detetada em lesões iniciais do cancro da mama e é preditiva de progressão tumoral, a inibição da Arl13b poderia bloquear a transição de lesões pré-malignas
para invasivas por afetar o rearranjo de actina e a desmontagem das AFs e desta
forma a migração e invasão celular.
ABSTRACT: Breast cancer is the leading cause of cancer-associated deaths in women worldwide, mainly due to metastasis. To disseminate, cancer cells migrate from the primary tumor and invade surrounding tissues, until they reach the blood and lymphatic vessels, whereby they travel to a new organ. Therefore, cell migration and invasion are two essential cellular processes required for cancer metastasis and involve coordinated regulation of cell-cell and cell-extracellular matrix (ECM) adhesion, ECM degradation, and actin cytoskeletal remodeling. Importantly, the identification of molecular regulators of breast cancer cell migration and invasion can contribute to the establishment of new prognosis, diagnosis and therapeutic strategies. ADP-ribosylation factor (Arf) family of proteins are mainly known as regulators of vesicular trafficking. Interestingly, various reports show that cancer cells abnormally express and hijack members of this family to migrate and invade efficiently. Arl13b belongs to the Arf-like (Arl) subfamily and was first described as a ciliary protein. Nevertheless, it has been shown that Arl13b is also found and exert functions outside cilia. Our group has shown that Arl13b regulates endocytic recycling traffic and cell migration in non-pathological conditions through its effector non-muscle myosin heavy chain IIA (NMIIA). Moreover, recent data from our lab suggests that Arl13b expression levels positively correlate with the invasive capacity of breast cancer cells. Consequently, the main aim of this PhD thesis was to determine the role of Arl13b in breast cancer cell migration and invasion and identify the underlying molecular mechanisms. The results obtained in this work show that Arl13b positively regulates breast cancer cell migration and invasion in vitro. Corroborating these results, additional data from our lab shows that Arl13b regulates primary tumor size and the formation of lung metastases, in vivo. Interestingly, breast cancer is the third type of cancer where a function for Arl13b as a positive regulator of tumor formation and/or progression has been reported. In the other two types, namely medulloblastoma and gastric cancer, a mechanism mediated by ciliary canonical Sonic hedgehog (Shh) signaling was proposed. In breast cancer, we have evidence for an alternative mechanism of cancer cell migration and invasion regulation by Arl13b,independent of cilia. Indeed, we observed that Arl13b localizes to and negatively regulates cell-cell contacts. Moreover, we found that Arl13b interacts with β3- integrin and colocalizes with Vinculin and Paxillin, two focal adhesion (FA) components. Furthermore, when Arl13b is silenced in breast cancer cells, we found an increase in the size of FAs. Thus, we propose that the larger FAs in Arl13bsilenced cells results from increased FA growth through NMIIA-mediated tension, since we observed elevated NMIIA levels, and decreased FA disassembly, due to defective integrin-mediated signaling involved in FA disassembly. Finally, we observed that Arl13b localizes to structures involved in ECM degradation named invadopodia. In the future, a function of Arl13b in the formation of invadopodia, which could explain the role of Arl13b in breast cancer cell invasion, should be assessed. Interestingly, it has been observed that dysregulated cell-ECM interactions and abnormal actin cytoskeleton remodeling are two early events in breast cancer progression. Therefore, it would be interesting to evaluate if the altered expression of Arl13b is also an early event in breast cancer. Even tough, as we have found that Arl13b regulates actin cytoskeleton remodeling and FA dynamics and its expression is positively correlated with breast cancer cell invasive capacity, we propose Arl13b as a potential prognostic marker or even a therapeutic target. In a scenario where abnormal Arl13b expression is detected in early breast cancer lesions and predictive of tumor progression, targeting of Arl13b could block the transition of pre-malignant lesions to invasive cancer by impairing actin cytoskeleton remodeling, FA turnover and subsequently cell migration and invasion.
ABSTRACT: Breast cancer is the leading cause of cancer-associated deaths in women worldwide, mainly due to metastasis. To disseminate, cancer cells migrate from the primary tumor and invade surrounding tissues, until they reach the blood and lymphatic vessels, whereby they travel to a new organ. Therefore, cell migration and invasion are two essential cellular processes required for cancer metastasis and involve coordinated regulation of cell-cell and cell-extracellular matrix (ECM) adhesion, ECM degradation, and actin cytoskeletal remodeling. Importantly, the identification of molecular regulators of breast cancer cell migration and invasion can contribute to the establishment of new prognosis, diagnosis and therapeutic strategies. ADP-ribosylation factor (Arf) family of proteins are mainly known as regulators of vesicular trafficking. Interestingly, various reports show that cancer cells abnormally express and hijack members of this family to migrate and invade efficiently. Arl13b belongs to the Arf-like (Arl) subfamily and was first described as a ciliary protein. Nevertheless, it has been shown that Arl13b is also found and exert functions outside cilia. Our group has shown that Arl13b regulates endocytic recycling traffic and cell migration in non-pathological conditions through its effector non-muscle myosin heavy chain IIA (NMIIA). Moreover, recent data from our lab suggests that Arl13b expression levels positively correlate with the invasive capacity of breast cancer cells. Consequently, the main aim of this PhD thesis was to determine the role of Arl13b in breast cancer cell migration and invasion and identify the underlying molecular mechanisms. The results obtained in this work show that Arl13b positively regulates breast cancer cell migration and invasion in vitro. Corroborating these results, additional data from our lab shows that Arl13b regulates primary tumor size and the formation of lung metastases, in vivo. Interestingly, breast cancer is the third type of cancer where a function for Arl13b as a positive regulator of tumor formation and/or progression has been reported. In the other two types, namely medulloblastoma and gastric cancer, a mechanism mediated by ciliary canonical Sonic hedgehog (Shh) signaling was proposed. In breast cancer, we have evidence for an alternative mechanism of cancer cell migration and invasion regulation by Arl13b,independent of cilia. Indeed, we observed that Arl13b localizes to and negatively regulates cell-cell contacts. Moreover, we found that Arl13b interacts with β3- integrin and colocalizes with Vinculin and Paxillin, two focal adhesion (FA) components. Furthermore, when Arl13b is silenced in breast cancer cells, we found an increase in the size of FAs. Thus, we propose that the larger FAs in Arl13bsilenced cells results from increased FA growth through NMIIA-mediated tension, since we observed elevated NMIIA levels, and decreased FA disassembly, due to defective integrin-mediated signaling involved in FA disassembly. Finally, we observed that Arl13b localizes to structures involved in ECM degradation named invadopodia. In the future, a function of Arl13b in the formation of invadopodia, which could explain the role of Arl13b in breast cancer cell invasion, should be assessed. Interestingly, it has been observed that dysregulated cell-ECM interactions and abnormal actin cytoskeleton remodeling are two early events in breast cancer progression. Therefore, it would be interesting to evaluate if the altered expression of Arl13b is also an early event in breast cancer. Even tough, as we have found that Arl13b regulates actin cytoskeleton remodeling and FA dynamics and its expression is positively correlated with breast cancer cell invasive capacity, we propose Arl13b as a potential prognostic marker or even a therapeutic target. In a scenario where abnormal Arl13b expression is detected in early breast cancer lesions and predictive of tumor progression, targeting of Arl13b could block the transition of pre-malignant lesions to invasive cancer by impairing actin cytoskeleton remodeling, FA turnover and subsequently cell migration and invasion.
Descrição
Palavras-chave
Cancro da mama Breast cancer Cell migration ARL13B role