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

Abstract

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.

Targeting GLI1 Transcription Factor for Restoring Iodine Avidity with Redifferentiation in Radioactive-Iodine Refractory Thyroid Cancers

“…Besides, several factors, including blockade of phosphatidylinositol glycan anchor biosynthesis class U (PIGU) by activating an MAPK signaling pathway, increased leukemia-associated RhoA guanine exchange factor (LARG) owing to PTEN deficiency, induction of NIS proteolysis by valosin-containing protein as a component of endoplasmic reticulum-associated degradation, and overexpression of pituitary tumor-transforming gene 1 (PTTG1)-binding factor (PBF), have been shown to trigger impairment of NIS membrane targeting [ 35 , 36 , 37 , 38 , 39 , 40 , 41 ]. On the other hand, recognition of ADP-ribosylation factor 4 (ARF4) by VAPK motif in the NIS C-terminus, regulation of SRC/RAC1/PAK1/PIP5K/EZRIN pathway, activation of RAC1 via MAPK inhibition, application of transient receptor potential vanilloid type 1 (TRPV1) agonist and others were shown to promote NIS functionality and trafficking towards the cell membrane [ 41 , 42 , 43 , 44 , 45 ]. Recently, an article suggested the role of protospacer adjacent motif (PDZ)—PDZ interaction which means PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS to localize at proper basolateral plasma membrane [ 46 ].…”

Adherens Junction Integrity Is a Critical Determinant of Sodium Iodide Symporter Residency at the Plasma Membrane of Thyroid Cells

“…HA-NIS-TPC-1- cells were established from the human PTC-derived cell line TPC-1, modified to stably express the NIS construct containing an extracellular triple HA tag and co-express the halide-sensitive yellow fluorescent protein YFP-F46L/H148Q/I152L (HS-YFP), as previously described [ 10 ]. Cells were maintained in 10% v/v FBS-supplemented RPMI (Gibco, Grand Island, NY, USA).…”

“…Plasmid pCDNA3-MYC-RAC1-V12 [ 11 ] transfection into HA-NIS-TPC1 cells was performed as previously described [ 10 ].…”

“…Finding new targets to render a functional NIS protein at the membrane is emerging as a requirement for overcoming iodine refractoriness in TC. Since little is known about the mechanisms regulating NIS PM residency, in a previous work, we employed a new experimental strategy to purify and characterize proteins which are selectively associated with NIS at the PM, potentially modulating its abundance and stability on the surface of TC cells [ 10 ].…”

“…We identified a set of NIS interactors involved in the regulation of the actin cytoskeleton, from which it was possible to characterize an intracellular signaling pathway derived from SRC kinase, which, acting through RAC1, leads to the recruitment and binding of the actin-anchoring EZRIN to NIS, promoting its retention at the PM of thyroid cells [ 10 ].…”