Browsing by Author "Domingues, Rita"
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- Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid CancerPublication . Faria, Márcia; Domingues, Rita; Bugalho, Maria João; Silva, Ana Luísa; Matos, PauloThe functional expression of the sodium-iodide symporter (NIS) at the membrane of differentiated thyroid cancer (DTC) cells is the cornerstone for the use of radioiodine (RAI) therapy in these malignancies. However, NIS gene expression is frequently downregulated in malignant thyroid tissue, and 30% to 50% of metastatic DTCs become refractory to RAI treatment, which dramatically decreases patient survival. Several strategies have been attempted to increase the NIS mRNA levels in refractory DTC cells, so as to re-sensitize refractory tumors to RAI. However, there are many RAI-refractory DTCs in which the NIS mRNA and protein levels are relatively abundant but only reduced levels of iodide uptake are detected, suggesting a posttranslational failure in the delivery of NIS to the plasma membrane (PM), or an impaired residency at the PM. Because little is known about the molecules and pathways regulating NIS delivery to, and residency at, the PM of thyroid cells, we here employed an intact-cell labeling/immunoprecipitation methodology to selectively purify NIS-containing macromolecular complexes from the PM. Using mass spectrometry, we characterized and compared the composition of NIS PM complexes to that of NIS complexes isolated from whole cell (WC) lysates. Applying gene ontology analysis to the obtained MS data, we found that while both the PM-NIS and WC-NIS datasets had in common a considerable number of proteins involved in vesicle transport and protein trafficking, the NIS PM complexes were particularly enriched in proteins associated with the regulation of the actin cytoskeleton. Through a systematic validation of the detected interactions by co-immunoprecipitation and Western blot, followed by the biochemical and functional characterization of the contribution of each interactor to NIS PM residency and iodide uptake, we were able to identify a pathway by which the PM localization and function of NIS depends on its binding to SRC kinase, which leads to the recruitment and activation of the small GTPase RAC1. RAC1 signals through PAK1 and PIP5K to promote ARP2/3-mediated actin polymerization, and the recruitment and binding of the actin anchoring protein EZRIN to NIS, promoting its residency and function at the PM of normal and TC cells. Besides providing novel insights into the regulation of NIS localization and function at the PM of TC cells, our results open new venues for therapeutic intervention in TC, namely the possibility of modulating abnormal SRC signaling in refractory TC from a proliferative/invasive effect to the re-sensitization of these tumors to RAI therapy by inducing NIS retention at the PM.
- Antagonistic effects of RAC1 and tumor-related RAC1b on NIS expression in thyroidPublication . Faria, Márcia; Félix, Daniela; Domingues, Rita; Bugalho, Maria João; Matos, Paulo; Silva, Ana LuísaThyroid cancer (TC) is the most common endocrine malignancy. The Sodium Iodide Symporter (NIS), responsible for active transport of iodide into thyroid cells, allows the use of radioactive iodine (RAI) as the systemic treatment of choice for TC metastatic disease. Still, patients with advanced forms of TC often lose the ability to respond to RAI therapy, which results in worse survival rates. We have shown that the overexpression of RAC1b, a tumor-related RAC1 splice variant, is associated with less favorable clinical outcomes in differentiated TCs derived from the follicular epithelial (DTCs). RAC1b overexpression is also significantly associated with the presence of MAPK-activating BRAFV600E mutation, which has been previously implicated in the loss of NIS expression. Here, we show that increased RAC1b levels are associated with NIS downregulation in DTCs and demonstrate that ectopic overexpression of RAC1b in non-transformed thyroid cells is sufficient to decrease TSH-induced NIS expression, antagonizing the positive effect of the canonically spliced RAC1 GTPase. Moreover, we clearly document for the first time in thyroid cells that both NIS expression and iodide uptake are hampered by RAC1 inhibition, highlighting the role of RAC1 in promoting TSH-induced NIS expression. Our findings support a role for RAC1 and RAC1b signaling in the regulation of NIS expression in thyroid cells and suggest that RAC1b in cooperation with other cancer-associated signaling cues may be implicated in the response of DTCs to RAI therapy.
- MAPK Inhibition Requires Active RAC1 Signaling to Effectively Improve Iodide Uptake by Thyroid Follicular CellsPublication . Faria, Márcia; Domingues, Rita; Bugalho, Maria João; Matos, Paulo; Silva, Ana LuísaThe Sodium/Iodide Symporter (NIS) is responsible for the active transport of iodide into thyroid follicular cells. Differentiated thyroid carcinomas (DTCs) usually preserve the functional expression of NIS, allowing the use of radioactive iodine (RAI) as the treatment of choice for metastatic disease. However, a significant proportion of patients with advanced forms of TC become refractory to RAI therapy and no effective therapeutic alternatives are available. Impaired iodide uptake is mainly caused by the defective functional expression of NIS, and this has been associated with several pathways linked to malignant transformation. MAPK signaling has emerged as one of the main pathways implicated in thyroid tumorigenesis, and its overactivation has been associated with the downregulation of NIS expression. Thus, several strategies have been developed to target the MAPK pathway attempting to increase iodide uptake in refractory DTC. However, MAPK inhibitors have had only partial success in restoring NIS expression and, in most cases, it remained insufficient to allow effective treatment with RAI. In a previous work, we have shown that the activity of the small GTPase RAC1 has a positive impact on TSH-induced NIS expression and iodide uptake in thyroid cells. RAC1 is a downstream effector of NRAS, but not of BRAF. Therefore, we hypothesized that the positive regulation induced by RAC1 on NIS could be a relevant signaling cue in the mechanism underlying the differential response to MEK inhibitors, observed between NRAS- and BRAF-mutant tumors. In the present study, we found that the recovery of NIS expression induced through MAPK pathway inhibition can be enhanced by potentiating RAC1 activity in thyroid cell systems. The negative impact on NIS expression induced by the MAPK-activating alterations, NRAS Q61R and BRAF V600E, was partially reversed by the presence of the MEK 1/2 inhibitors AZD6244 and CH5126766. Notably, the inhibition of RAC1 signaling partially blocked the positive impact of MEK inhibition on NIS expression in NRAS Q61R cells. Conversely, the presence of active RAC1 considerably improved the rescue of NIS expression in BRAF V600E thyroid cells treated with MEK inhibitors. Overall, our data support an important role for RAC1 signaling in enhancing MAPK inhibition in the context of RAI therapy in DTC, opening new opportunities for therapeutic intervention.
- Modulação combinada das vias de sinalização RAC1 e MAPK para potenciar a captação de iodo radioactivoPublication . Faria, Márcia; Domingues, Rita; Félix, Diana; Bugalho, Maria João; Matos, Paulo; Silva, Ana LuísaO simportador de Sódio e Iodo (NIS) é responsável pela captação de iodo pelas células foliculares da tiroide. A retenção da expressão funcional de NIS na maioria dos tumores bem diferenciados da tiroide permite o uso de iodo radioativo (RAI) como terapêutica sistémica para doença metastática. No entanto, uma percentagem de doentes com formas avançadas de carcinoma da tiroide perde a capacidade de resposta a esta terapia, sendo as alternativas disponíveis terapêuticas crónicas, dispendiosas e associadas a efeitos adversos importantes. A principal razão para esta reduzida captação de iodo é a deficiente expressão funcional de NIS, tendo esta sido associada à atividade de várias vias de sinalização pró-tumorigénicas. A sobreativação da via MAPK está implicada na tumorigénese da tiroide, assim como na regulação negativa de NIS. Assim, várias estratégias têm sido desenvolvidas no sentido de inibir a via MAPK com o objetivo de aumentar a captação de iodo em tumores refratários. A relevância deste tipo de estratégia foi anteriormente documentada num ensaio clínico que demonstrou um ganho significativo de incorporação de iodo num subgrupo de pacientes com neoplasia tiroideia refratária ao iodo radioativo, após o tratamento com selumetinib (AZD6244), um inibidor de MEK 1/2. No entanto, a recuperação da expressão de NIS, utilizando inibidores desta via, nem sempre foi suficiente para viabilizar a terapia com RAI, tornando-se clinicamente relevante a identificação de alvos adicionais envolvidos na regulação positiva do NIS. Assim, este estudo pretende avaliar se a recuperação da expressão de NIS induzida pela inibição da via MAPK pode ser otimizada aumentando a atividade da GTPase RAC1, um potenciador da expressão de NIS em sistemas celulares da tiroide.
- A role for the small GTPases RAC1 and RAC1b in the modulation of NIS expression: potentiation of therapy with radioactive iodine in differentiated thyroid carcinomaPublication . Faria, Márcia; Félix, Daniela; Domingues, Rita; Bugalho, Maria J.; Matos, Paulo; Silva, Ana L.Introduction or Background: The Sodium Iodide Symporter (NIS) is responsible for active transport of iodide into thyroid follicular cells. The retention of its functional expression in most of the well-differentiated thyroid carcinomas (DTCs) enables the use of radioactive iodine (RAI) for treatment of metastatic disease. Still, about 30% of patients with advanced forms of DTC became refractory to RAI which makes their management very challenging. The main reason for impaired iodide uptake in refractory-DTC is the defective functional expression of NIS. Several molecular players have been described as critical for TSH-induced NIS expression, an example being the p38 mitogenic kinase. In breast cancer cells, the small GTPase RAC1 was shown to mediate the positive impact of p38 kinase activity on NIS expression. We, on the other hand, have previously shown that overexpression of RAC1b, a tumor-related splicing variant of RAC1, is associated with worse outcomes in DTC and correlates with the MAPK-activating BRAFV600E mutation, which has been related to the loss of NIS. Since RAC1 and RAC1b may act in an antagonistic fashion to regulate specific cellular responses, we asked if RAC1b would be implicated in NIS downregulation observed in DTCs. Methods Section: NIS expression levels were analyzed by RT-qPCR in a RAC1/RAC1b expression model system developed in non-transformed thyroid cell lines. A non-radioactive iodide influx assay was used to confirm the impact of RAC1-signaling on the efficacy of iodide uptake. Results Section: We demonstrate that ectopic overexpression of RAC1b is sufficient to decrease TSH-induced NIS expression, antagonizing the positive effect of RAC1 GTPase. Moreover, we clearly document, for the first time in thyroid cells, that both NIS expression and iodide uptake are downregulated upon RAC1 inhibition, supporting the role of canonical RAC1 signaling in promoting TSH-induced NIS expression. Conclusion: Our findings provide evidence that RAC1 and RAC1b signaling are implicated in the regulation of NIS expression in thyroid cells and suggest that RAC1b overexpression may be one of the mechanisms contributing to the low levels of NIS observed in some subgroups of DTCs, antagonizing RAC1 stimulatory effect on the TSH/cAMP-mediated induction of NIS expression.
- TNFα-mediated activation of NF-κB downregulates sodium-iodide symporter expression in thyroid cellsPublication . Faria, Márcia; Domingues, Rita; Paixão, Francisca; Bugalho, Maria João; Matos, Paulo; Silva, Ana LuísaThe sodium-iodide symporter (NIS) mediates transport of iodide across the basolateral membrane of thyroid cells. NIS expression in thyroid cancer (TC) cells allows the use of radioactive iodine (RAI) as a diagnostic and therapeutic tool, being RAI therapy the systemic treatment of choice for metastatic disease. Still, a significant proportion of patients with advanced TC lose the ability to respond to RAI therapy and no effective alternative therapies are available. Defective NIS expression is the main reason for impaired iodide uptake in TC and NIS downregulation has been associated with several pathways linked to malignant transformation. NF-κB signaling is one of the pathways associated with TC. Interestingly, NIS expression can be negatively regulated by TNF-α, a bona fide activator of NF-κB with a central role in thyroid autoimmunity. This prompted us to clarify NF-kB's role in this process. We confirmed that TNF-α leads to downregulation of TSH-induced NIS expression in non-neoplastic thyroid follicular cell-derived models. Notably, a similar effect was observed when NF-κB activation was triggered independently of ligand-receptor specificity, using phorbol-myristate-acetate (PMA). TNF-α and PMA downregulation of NIS expression was reverted when NF-κB-dependent transcription was blocked, demonstrating the requirement for NF-kB activity. Additionally, TNF-α and PMA were shown to have a negative impact on TSH-induced iodide uptake, consistent with the observed transcriptional downregulation of NIS. Our data support the involvement of NF-κB-directed transcription in the modulation of NIS expression, where up- or down-regulation of NIS depends on the combined output to NF-κB of several converging pathways. A better understanding of the mechanisms underlying NIS expression in the context of normal thyroid physiology may guide the development of pharmacological strategies to increase the efficiency of iodide uptake. Such strategies would be extremely useful in improving the response to RAI therapy in refractory-TC.