MAPK Inhibition Requires Active RAC1 Signaling to Effectively Improve Iodide Uptake by Thyroid Follicular Cells

Abstract

The 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.

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

“…These observations were consistent with AJ integrity being required to sustain NIS permanence at the PM of thyroid cells. To further validate these findings, we repeated these experiments in TPC1-HA-NIS cells co-expressing the halide-sensitive mutant of yellow fluorescent protein (YFP) F46L/H148Q/I152L (HS-YFP), which is quenched by iodide, and that we had previously used successfully to study NIS-mediated iodide transport in these and other thyroid cells [ 10 , 12 ]. As shown in Figure 1 E,F, disruption of AJ integrity through EGTA treatment led to a decrease in the rate of NIS-mediated iodide influx (confirmed by its blocking in the presence of NIS inhibitor ClO 4 − ) comparable to the observed decrease in NIS PM levels ( Figure 1 C,D).…”

“…In view of our findings, one could speculate that by compromising cell–cell adhesion, this loss of E-cadherin in advanced TCs could contribute to precluding NIS PM residency. Coupled with a decreased transcriptional expression of the symporter, namely via the hyperactivation of the MAP kinase pathway through NRAS and BRAF mutations [ 12 , 22 ], a reduced PM residency could be sufficient to promote RAI refractoriness in advanced TCs. Initial studies towards RAI resensitization therapy in refractory TC have highlighted that approaches promoting TC epithelial redifferentiation often led to improvements, albeit partial, in RAI uptake by TC cells in vitro and in vivo [ 23 ].…”

“…Cell surface protein biotinylation assay in TPC1 and PCCL3 cells was performed as described [ 10 , 12 ]. Briefly, cells were incubated with ice-cold PBS++ (PBS pH 8.0 containing 0.1 mM CaCl 2 and 1 mM MgCl 2 ) to arrest the endocytic traffic and cell surface proteins were labeled with sulfosuccinimidyl 3-[[2-(Biotinamido)ethyl] di-thio] pro-pionate sodium salt (0.5 mg/mL, sc-212981, Santa Cruz Biotechnology, Santa Cruz, CA, USA).…”

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 ].…”