scholarly journals 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

Cancers ◽  
2021 ◽  
Vol 13 (21) ◽  
pp. 5460
Author(s):  
Márcia Faria ◽  
Rita Domingues ◽  
Maria João Bugalho ◽  
Ana Luísa Silva ◽  
Paulo Matos
Keyword(s):  
Plasma Membrane ◽  
Thyroid Cancer ◽  
Actin Polymerization ◽  
Functional Characterization ◽  
Thyroid Tissue ◽  
Small Gtpase ◽  
Mrna Levels ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
And Function

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.

scholarly journals Expression and function of the novel proto-oncogene PBF in thyroid cancer: a new target for augmenting radioiodine uptake

2011 ◽  
Vol 210 (2) ◽  
pp. 157-163 ◽  
Author(s):  
Vicki E Smith ◽  
Jayne A Franklyn ◽  
Christopher J McCabe
Keyword(s):  
Thyroid Cancer ◽  
Current Data ◽  
Subcellular Localisation ◽  
Sodium Iodide Symporter ◽  
Iodide Uptake ◽  
Radioiodine Uptake ◽  
Novel Target ◽  
And Function

Pituitary tumor-transforming gene (PTTG)-binding factor (PBF; PTTG1IP) was initially identified through its interaction with the human securin, PTTG. Like PTTG, PBF is upregulated in multiple endocrine tumours including thyroid cancer. PBF is believed to induce the translocation of PTTG into the cell nucleus where it can drive tumourigenesis via a number of different mechanisms. However, an independent transforming ability has been demonstrated both in vitro and in vivo, suggesting that PBF is itself a proto-oncogene. Studied in only a limited number of publications to date, PBF is emerging as a protein with a growing repertoire of roles. Recent data suggest that PBF possesses a complex multifunctionality in an increasing number of tumour settings. For example, PBF is upregulated by oestrogen and mediates oestrogen-stimulated cell invasion in breast cancer cells. In addition to a possible role in the induction of thyroid tumourigenesis, PBF overexpression in thyroid cancers inhibits iodide uptake. PBF has been shown to repress sodium iodide symporter (NIS) activity by transcriptional regulation of NIS expression through the human NIS upstream enhancer and further inhibits iodide uptake via a post-translational mechanism of NIS governing subcellular localisation. This review discusses the current data describing PBF expression and function in thyroid cancer and highlights PBF as a novel target for improving radioiodine uptake and thus prognosis in thyroid cancer.

scholarly journals Crosstalk Between Abnormal TSHR Signaling Activation and PTEN/PI3K in the Dedifferentiation of Thyroid Cancer Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Fang Feng ◽  
Huiqin Han ◽  
Shuqi Wu ◽  
Hui Wang
Keyword(s):  
Thyroid Cancer ◽  
Cancer Cells ◽  
Mtor Signaling ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Cell Mobility ◽  
Signaling Activation ◽  
Thyroid Cancer Cells ◽  
Serum Tsh

Iodide uptake and the metabolism of thyroid cells are regulated by thyrotropin (TSH)-TSH receptor (TSHR) signaling. Thus, it is necessary to elevate serum TSH levels by T4 withdraw or rTSH administration to facilitate radioiodide (131I) therapy for differentiated thyroid cancer (DTC). However, non-iodide-avid metastases of DTC which is dedifferentiated do not respond to stimulation by high levels of TSH, suggesting abnormal TSH-TSHR signal transduction in cancer cells. In addition, PI3K/AKT/mTOR signaling activation has been shown to be associated with the dedifferentiated phenotype of thyroid cancer, but the mechanism remains elusive. Therefore, in this study, we aimed to explore the role of abnormal TSH-TSHR signaling activation in regulating iodide uptake and cell mobility in thyroid cancer and its relationship with PI3K/AKT/mTOR signaling. We found that in thyroid cancer cells, TSH binds TSHR coupled to the Gα12/13 protein and then activates RhoA through interacting with leukemia associated RhoA guanine exchange factor (LARG). This results in a promigration tumorigenic phenotype independent of canonical TSHR-GαS signaling that regulates the expression of molecules involved in iodine uptake and metabolism. We observed that signaling pathways downstream of Gα12/13 signaling were increased, while that of Gαs signaling was decreased in thyroid cancer cells undergoing dedifferentiation compared to control cells following stimulation with different levels of TSH. PI3K/AKT/mTOR signaling activation enhanced Gα12/13 signaling through increasing LARG levels but also inhibited the expression of molecules downstream of Gαs signaling, including thyroid-specific molecules, and iodide uptake. In summary, our results demonstrate the noncanonical activation of TSH-TSHR signaling and its role in increasing the cell mobility and dedifferentiation of thyroid cancer through crosstalk with PI3K/AKT/mTOR signaling.

scholarly journals Expression of the Ring Ligase PRAJA2 in Thyroid Cancer

2012 ◽  
Vol 97 (11) ◽  
pp. 4253-4259 ◽  
Author(s):  
Silvia Cantara ◽  
Francesco D'Angeli ◽  
Paolo Toti ◽  
Luca Lignitto ◽  
Maria Grazia Castagna ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Differentiated Thyroid Cancer ◽  
Cultured Cells ◽  
Thyroid Tumor ◽  
Mrna Levels ◽  
Malignant Phenotype ◽  
Hormone Production ◽  
Thyroid Cells ◽  
Mutational Status ◽  
Benign Nodules

Introduction: In thyroid cells, binding of TSH to its receptor increases cAMP levels, sustaining thyrocytes growth and hormone production. The main cAMP effector enzyme is protein kinase A (PKA). Praja2 is a widely expressed RING (Really Interesting New Gene) ligase, which degrades the regulatory subunits of PKA, thus controlling the strength and duration of PKA signaling in response to cAMP. Differentiated thyroid cancer expresses a functional TSH receptor, and its growth and progression are positively regulated by TSH and cAMP signaling. Aim: We aimed to analyze the expression of praja2 in a group of 36 papillary thyroid cancer (PTC), 14 benign nodules, and six anaplastic thyroid cancers (ATC). Methods: We measured praja2 mRNA levels by quantitative RT-PCR and praja2 expression by Western blot and immunohistochemistry. Possible association between praja2 mRNA and the presence of known mutations was evaluated. Results: We found a statistical significant increase of mRNA levels in PTC tissue samples, compared with benign nodules and ATC. In particular, mRNA levels were maximal in differentiated thyroid cancer (PTC), progressively decreasing in more aggressive tumors, ATC having the lowest amount of praja2 mRNA. Accordingly, higher levels of praja2 protein were detected in lysates from PTC, compared with ATC. By immunohistochemistry, in PTC sections we observed a marked increase of cytoplasmic praja2 signal, which significantly decreased in less differentiated thyroid tumors, completely disappearing in ATC. Studies in cultured cells stably expressing RET/PTC1 oncogene or mutant BRAF revealed a direct correlation between praja2 mRNA levels and malignant phenotype of transformed cells. Similar results were obtained using thyroid cancer tissues carrying the same mutations. Conclusions: praja2 is markedly overexpressed in differentiated thyroid cancer, and its levels inversely correlate with the malignant phenotype of the tumor. Thus, praja2 is a novel cancer-related gene whose expression is linked to the histotype and mutational status of the thyroid tumor.

scholarly journals DIAGNOSIS OF ENDOCRINE DISEASE: Thyroglobulin measurement using highly sensitive assays in patients with differentiated thyroid cancer: a clinical position paper

2014 ◽  
Vol 171 (2) ◽  
pp. R33-R46 ◽  
Author(s):  
Luca Giovanella ◽  
Penelope M Clark ◽  
Luca Chiovato ◽  
Leonidas Duntas ◽  
Rossella Elisei ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Clinical Guidelines ◽  
Differentiated Thyroid Cancer ◽  
Clinical Care ◽  
Thyroid Tissue ◽  
Position Paper ◽  
Analytical Sensitivity ◽  
Thyroid Cells ◽  
Highly Sensitive

Differentiated thyroid cancer (DTC) is the most common endocrine cancer and its incidence has increased in recent decades. Initial treatment usually consists of total thyroidectomy followed by ablation of thyroid remnants by iodine-131. As thyroid cells are assumed to be the only source of thyroglobulin (Tg) in the human body, circulating Tg serves as a biochemical marker of persistent or recurrent disease in DTC follow-up. Currently, standard follow-up for DTC comprises Tg measurement and neck ultrasound combined, when indicated, with an additional radioiodine scan. Measurement of Tg after stimulation by endogenous or exogenous TSH is recommended by current clinical guidelines to detect occult disease with a maximum sensitivity due to the suboptimal sensitivity of older Tg assays. However, the development of new highly sensitive Tg assays with improved analytical sensitivity and precision at low concentrations now allows detection of very low Tg concentrations reflecting minimal amounts of thyroid tissue without the need for TSH stimulation. Use of these highly sensitive Tg assays has not yet been incorporated into clinical guidelines but they will, we believe, be used by physicians caring for patients with DTC. The aim of this clinical position paper is, therefore, to offer advice on the various aspects and implications of using these highly sensitive Tg assays in the clinical care of patients with DTC.

Gap junctions and cell polarity: connexin32 and connexin43 expressed in polarized thyroid epithelial cells assemble into separate gap junctions, which are located in distinct regions of the lateral plasma membrane domain

1995 ◽  
Vol 108 (7) ◽  
pp. 2609-2617 ◽  
Author(s):  
A. Guerrier ◽  
P. Fonlupt ◽  
I. Morand ◽  
R. Rabilloud ◽  
C. Audebet ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Gap Junctions ◽  
Cell Polarity ◽  
Laser Scanning ◽  
Thyroid Tissue ◽  
Thyroid Cell ◽  
Membrane Domain ◽  
Thyroid Cells ◽  
Connexin43 Gap Junctions

Epithelial cells of the thyroid gland present an uncommon connexin expression pattern, they coexpress connexin32 and connexin43. In the present work, we have analyzed the membrane distribution of these two connexins to determine: (i) whether they co-assemble in the same gap junctions or form separate gap junctions; and (ii) whether their location is somehow related to the thyroid cell polarity. Immunofluorescence analyses of the localization of the two connexins in thyroid tissue sections revealed that connexin32 and connexin43 are located in different regions of the plasma membrane. We further analyzed the location of each of the two connexins with regard to that of the tight junction-associated protein, ZO1. Laser scanning confocal microscope observations of connexin32 or connexin43 and ZO1 double-immunolabelled thyroid cells, gave evidence for a separate localization of gap junctions made of each of these two connexins. Connexin32 gap junctions appeared as fluorescent spots scattered over the lateral membrane domain, while connexin43 gap junctions formed a meshed network superimposable with that of tight junctions in the subapical region of the cells. Western blot analyses of the distribution of connexins in thyroid plasma membrane subfractions obtained by ultracentrifugation on a sucrose gradient led to the identification of membrane sub-populations enriched in either connexin32 gap junctions or connexin43 gap junctions. Connexin32 gap junctions and connexin43 gap junctions were found to differ in their resistance to solubilization by N-lauroylsarcosine. Increasing concentrations of this detergent from 0.12% to 0.42% caused a progressive solubilization of connexin43 while connexin32 remained membrane-bound.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte Scaffolding Protein p55 Functions as An Essential Regulator of Neutrophil Polarity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 318-318
Author(s):  
Brendan J. Quinn ◽  
Athar H. Chishti
Keyword(s):  
Plasma Membrane ◽  
Actin Polymerization ◽  
Leading Edge ◽  
Small Gtpase ◽  
Scaffolding Protein ◽  
Knockout Mouse Model ◽  
Surface Receptors ◽  
Lipid Product ◽  
Chemotactic Gradient

Abstract Erythrocyte p55 is a prototypical member of a family of scaffolding proteins known as Membrane Associated Guanylate Kinase Homologues (MAGUKs). MAGUKs are multi-domain proteins that couple signals from specialized sites at the plasma membrane to intracellular signal transduction pathways and the cytoskeleton. P55 was originally identified in the erythrocytes as part of a ternary complex with protein 4.1R and glycophorin C, providing a critical linkage between the actin cytoskeleton and the plasma membrane. Although p55 is expressed in a variety of tissues, especially hematopoietic cells, its biological function is unclear. Here, using a p55 knockout mouse model, we show that p55 plays a prominent role in the regulation of neutrophil polarization. Neutrophils are the first respondents during infection and injury, adopting a highly polarized morphology when stimulated with chemotactic factors. G proteincoupled surface receptors recognize the external chemotactic gradient and translate it into an internal gradient of signaling molecules. At the front of the cell, accumulation of the lipid product phosphatidylinositol-3,4,5-trisphosphate (PIP3), activation of the small GTPase Rac, and polymerization of F-actin stimulates a positive feedback loop promoting pseudopod formation. Here, we show that neutrophils lacking p55 form multiple transient pseudopods at the sides and back of the cell upon stimulation. P55 is required for limiting the pseudopod in the direction of chemoattractant. As a result, these neutrophils do not migrate efficiently up a chemotactic gradient in vitro. Biochemical analysis indicates that total F-actin polymerization and total Rac activation is similar between wild type and p55 knockout neutrophils. However, we found that phosphorylation of AKT, the major kinase downstream of the phosphatidylinositol 3-kinase (PI3K)-PIP3 pathway, is almost completely blocked in p55 knockout neutrophils. This finding suggests that p55 exerts its functional effect by regulating PIP3 accumulation or its localization at the membrane, which is responsible for amplification of the frontness signal and stability of the leading edge pseudopod. Consistent with this finding, the p55 null mice are significantly more susceptible to spontaneous and induced infections. Taken together, we have identified p55 as a novel mediator of the frontness signal in neutrophils that promotes polarization and efficient chemotaxis.

scholarly journals Iodide symporter gene expression in normal and transformed rat thyroid cells

1999 ◽  
pp. 447-451 ◽  
Author(s):  
F Trapasso ◽  
R Iuliano ◽  
E Chiefari ◽  
F Arturi ◽  
A Stella ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Mrna Levels ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Transformed Cell ◽  
Nis Gene ◽  
Rat Thyroid ◽  
Transformed Cell Lines

OBJECTIVE: Decrease or loss of the Na+/I- symporter (NIS) activity profoundly affects the suitability of the use of radioiodine to detect or treat metastatic thyroid tissues. The aim of our study was to verify whether specific oncogene abnormalities were responsible for the alteration in NIS activity in thyroid cells. DESIGN AND METHODS: Expression of the NIS gene was investigated by Northern blot analysis in normal and in some oncogene-transformed cell lines with different degrees of malignancy which had lost the iodide uptake ability. RESULTS: NIS gene expression was up-regulated by TSH in a dose-dependent and time-dependent way in normal PC Cl 3 cells. The same effect was observed by activating the cAMP-dependent pathway by forskolin. Conversely, insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA) showed a partial inhibitory effect on NIS gene expression. The oncogene-transformed cell lines PC v-erbA, PC HaMSV, PC v-raf, and PC E1A cells showed reduced NIS mRNA levels compared with the normal PC Cl 3 cells. Conversely, an almost complete absence of NIS gene expression was found in PC RET/PTC, PC KiMSV, PC p53(143ala), and PC PyMLV cell lines. CONCLUSIONS: Our data show that oncogene activation could play a role in affecting the iodide uptake ability in thyroid tumoral cells; different mechanisms are involved in the oncogene-dependent loss of NIS activity in transformed thyroid cells.

IN-VITRO STUDIES OF NORMAL HUMAN THYROID CELLS: RESPONSES TO THYROTROPHIN AND DIBUTYRYL CYCLIC AMP

1977 ◽  
Vol 72 (1) ◽  
pp. 87-96 ◽  
Author(s):  
S. P. BIDEY ◽  
P. MARSDEN ◽  
J. ANDERSON ◽  
C. G. McKERRON ◽  
H. BERRY
Keyword(s):  
Cyclic Amp ◽  
Thyroid Tissue ◽  
Three Dimensional ◽  
Human Thyroid ◽  
Dibutyryl Cyclic Amp ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Normal Human

SUMMARY Follicular cells isolated from normal human thyroid tissue have been cultured for up to 140 h with bovine thyrotrophin (TSH) or dibutyryl cyclic AMP (DBcAMP). Both compounds induced marked reorganization of the cells into three-dimensional follicular structures, whilst non-supplemented cells assumed a monolayer form. Cultures treated initially with TSH or DBcAMP showed a greater iodide uptake capacity, in comparison with unsupplemented cultures, in which iodide uptake was markedly diminished after 24 h. The release of tri-iodothyronine (T3) and thyroxine (T4) into the medium was determined by radioimmunoassay. Both TSH- and DBcAMP-treated cells showed a significant increase in iodothyronine output compared with unsupplemented control cells. In contrast to the 'classical' TSH-induced depression of the T4:T3 ratio in vivo, an increase in the ratio was observed for both TSH- and DBcAMP-supplemented cells in vitro. The ratio was also significantly greater after TSH than after DBcAMP, and possible implications of this finding are discussed.

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