Antagonistic effects of RAC1 and tumor-related RAC1b on NIS expression in thyroid

Faria, Márcia; Félix, Daniela; Domingues, Rita; Bugalho, Maria João; Matos, Paulo; Silva, Ana Luísa

doi:10.1530/jme-19-0195

Cited by 11 publications

(19 citation statements)

References 47 publications

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“…In agreement with this hypothesis, we found that in the core of this regulatory network, alongside actin, was the small GTPase RAC1. We and others have reported that RAC1 signaling potentiates the TSH-induced transcription of the NIS (SLC5A5) gene in non-transformed thyroid cells [24], and also in MCF7 breast cancer cells, in this case through the activation of p38 MAPK signaling [59]. Here, we found that RAC1 signaling also has an important role in the posttranscriptional regulation of NIS PM residency.…”

Section: Discussionsupporting

confidence: 66%

“…The cells expressing the HS-YFP were selected with 250 µg/mL Hygromycin B (Santa Cruz Biotechnology [SCBT], Santa Cruz, CA, USA). PCCL3 cells expressing HS-YFP were previously described [24].…”

Section: Cell Line Generationmentioning

confidence: 99%

“…Second, the NIS function was assessed in the TPC1 cell line by the co-expression of HA-NIS or WT-NIS, together with the halide-sensitive mutant of yellow fluorescent protein (YFP) F46L/H148Q/I152L (HS-YFP), which is quenched by iodide and has been successfully employed to study iodide transport in various cellular systems [27,29]. Using a live cell imaging-based iodide influx assay [24] (Figure 1D), we observed a significant and, again, equivalent decrease in HS-YFP fluorescence over time (Figure 1E) in TPC1 cells expressing either HA-NIS or WT-NIS, which is indicative of an effective rate of iodide uptake into TPC1 cells by the tagged protein at the PM (Figure 1F). We further confirmed that this iodide uptake was dependent on HA-NIS activity, as the effect was abolished upon incubation with perchlorate (ClO 4 − ), a specific inhibitor of iodide uptake by NIS proteins [35,36].…”

Section: Production and Functional Validation Of The Stable Ha-nis-expressing Tpc1 Cell Linementioning

confidence: 99%

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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 Cancer

Faria

Domingues

Bugalho

et al. 2021

Cancers

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The 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.

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Section: Discussionsupporting

confidence: 66%

Section: Cell Line Generationmentioning

confidence: 99%

Section: Production and Functional Validation Of The Stable Ha-nis-expressing Tpc1 Cell Linementioning

confidence: 99%

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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 Cancer

Faria

Domingues

Bugalho

et al. 2021

Cancers

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“…We have previously reported NF-kB activation as a key mechanism through which the tumor-related RAC1b exerts its oncogenic action in the context of PTC [19]. Additionally, we have also recently shown an inverse correlation between RAC1b overexpression and NIS levels in DTCs [45]. This prompted us to explore, in the present study, the impact of the NF-κB signaling on NIS expression.…”

Section: Fig 3 Effect Of Tnf-α and Pma On Tsh-induced Iodide Uptake mentioning

confidence: 84%

“…HPRT1 and TBP were used as endogenous control genes for rat and human, respectively. RT-qPCR preformed as previously described [45]. The different cell lines were subjected to a 96h starvation period followed by stimulation with TSH (1 mU/mL for 48h).…”

Section: S1 Fig Effect Of Tsh On Nis Transcriptional Expression In Tmentioning

confidence: 99%

TNFα-mediated activation of NF-κB downregulates sodium-iodide symporter expression in thyroid cells

et al. 2020

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The 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.

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Targeting Cancer by Using Nanoparticles to Modulate RHO GTPase Signaling

Matos

Pereira

Jordan

2022

Advances in Experimental Medicine and Biology

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Antagonistic effects of RAC1 and tumor-related RAC1b on NIS expression in thyroid

Cited by 11 publications

References 47 publications

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 Cancer

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 Cancer

TNFα-mediated activation of NF-κB downregulates sodium-iodide symporter expression in thyroid cells

Targeting Cancer by Using Nanoparticles to Modulate RHO GTPase Signaling

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