Camille Buffet
email: camille.buffet@aphp.fr

Johanna Wassermann

Fabio Hecht

Laurence Leenhardt

Corinne Dupuy

Lionel Groussin

Charlotte Lussey-Lepoutre
email: charlotte.lepoutre@aphp.fr

Redifferentiation of Radioiodine-Refractory Thyroid Cancers

Keywords: NIS, radioiodine refractory thyroid cancers, redifferentiation, MAPK inhibition, NIS trafficking NIS, radioiodine refractory thyroid cancers, redifferentiation, MAPK inhibition, NIS trafficking, epigenetic regulation

teaching and research institutions in France or abroad, or from public or private research centers.

Redifferentiation of radioiodine-refractory thyroid cancers

Abstract

The management of radioiodine refractory thyroid cancers (RAIR TC) is challenging for the clinician. Tyrosine kinase inhibitors classically prescribed in this setting can fail due to primary or acquired resistance or the necessity of drug withdrawal because of serious or moderate but chronic and deleterious adverse effects. Thus, the concept of redifferentiation strategy, which involves treating patients with one or more drugs capable of restoring radioiodine sensitivity for RAIR TC, has emerged. The area of redifferentiation strategy leads to the creation of new definitions of RAIR TC including persistent non radioiodine-avid patients and "true" RAIR TC patients. The latter group presents a restored or increased radioiodine uptake in metastatic lesions but with no radiological response on conventional imaging i.e. progression of a metastatic disease thus proving that they are "truly" resistant to the radiation delivered by radioiodine. Unlike these patients, metastatic TC patients with restored radioiodine uptake offer the hope of prolonged remission or even cure of the disease as for radioiodine-avid metastatic TC.

We review here the different redifferentiation strategies based on the underlying molecular mechanism leading to the sodium iodide symporter (NIS) and radioiodine uptake reinduction i.e. by modulating signaling pathways, NIS transcription, NIS trafficking to the plasma membrane, NIS post-transcriptional regulation, by gene therapy and other potential strategies.

We discuss clinical trials and promising preclinical data of potential future targets.

Introduction

Although the majority of patients with differentiated thyroid cancers (DTC) could be successfully managed by surgery and radioiodine administration for which the indication is decided according to published guidelines [START_REF] Haugen | American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer[END_REF][START_REF] Luster | European Perspective on 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: Proceedings of an Interactive International Symposium[END_REF][START_REF] Ahuja | The Martinique Principles[END_REF], the evolution of a minority of these DTC will be less favourable and will require personalized management. Distant metastases encountered in less than 10% of DTC (https://seer.cancer.gov/statfacts/html/thyro.html) can lead to serious complications and even decrease the survival of patients [START_REF] Wassermann | Outcomes and Prognostic Factors in Radioiodine Refractory Differentiated Thyroid Carcinomas[END_REF]. Radioiodine therapy is the cornerstone of the treatment of distant metastases from DTC. This therapy is based on the expression at the plasma membrane of normal and tumoral thyroid epithelial cells of a symporter, the sodium iodide symporter (NIS), that transports two sodium ions and one iodide ion into the cytosol. Iodide is then handled by an iodine-metabolizing machinery that concentrates iodine into the thyroid cells which has probably a major impact on radioiodine efficacy. One-third of metastatic DTC patients will be in remission after one or more radioiodine sessions [START_REF] Durante | Long-term outcome of 444 patients with distant metastases from papillary and follicular thyroid carcinoma: benefits and limits of radioiodine therapy[END_REF]. The others are immediately or will later become radioiodine refractory (RAIR) which is correlated with high tumor burden (multiple and macrometastases) along with a less differentiated state of the tumor and worse prognosis [START_REF] Wassermann | Outcomes and Prognostic Factors in Radioiodine Refractory Differentiated Thyroid Carcinomas[END_REF][START_REF] Deandreis | Comparison of Empiric Versus Whole-Body/-Blood Clearance Dosimetry-Based Approach to Radioactive Iodine Treatment in Patients with Metastases from Differentiated Thyroid Cancer[END_REF]. Dedifferentiation is related to a decrease in or even a loss of the NIS expression and/or targeting to the plasma membrane where NIS is fully effective which results in the loss of iodine uptake in thyroid cells (Figure 1). Other histological types are less or not at all sensitive to radioiodine such as poorly DTC (PDTC) or anaplastic thyroid cancer (ATC) as these tumours have also lost the expression of thyroid specific genes, particularly those encoding for the iodine-metabolizing machinery during the dedifferentiation process. Therapeutic strategies for RAIR thyroid cancers include the implementation of local therapy (surgery, external radiotherapy, interventional radiology approach) whenever possible and in case of diffuse significant progression of distant metastatic disease systemic therapy, mainly tyrosine kinase inhibitors (TKI). However, TKI raise various issues such as primary and acquired resistance with tumor escape on the one hand and on the other hand serious and/or moderate but chronic and deleterious adverse effects, especially for antiangiogenic TKI. Thus, the concept of redifferentiation strategy has emerged with a view to finding one or more drugs capable of restoring radioiodine sensitivity for RAIR thyroid cancers (Figure 1). One of the challenges of this redifferentiation strategy is to find a treatment protocol involving the short-term use of systemic therapy so that even if side effects occur, they only have to be tolerated temporarily and frequently vanish with the discontinuation of re-differentiation treatment. Numerous compounds have been tested for several years with inconsistent results. A renewed interest for this strategy came from the publication of a pilot trial with a series of 24 patients showing promising results obtained with a pharmacological MEK inhibitor prescribed for 4 weeks [START_REF] Ho | Selumetinib-enhanced radioiodine uptake in advanced thyroid cancer[END_REF] which was in line with the pre-clinical mouse model validating this strategy [START_REF] Chakravarty | Small-molecule MAPK inhibitors restore radioiodine incorporation in mouse thyroid cancers with conditional BRAF activation[END_REF].

Here, we discuss the different redifferentiation strategies based on the underlying molecular mechanism leading to NIS and radioiodine uptake reinduction i.e. by modulating signaling pathways, NIS transcription, NIS trafficking to the plasma membrane, NIS posttranscriptional regulation, by gene therapy and other potential strategies. From a pre-clinical point of view, our review focuses on studies with data obtained from at least one cell line of authenticated thyroid cancer origin, as some cell lines tested in several studies were subsequently found to be of non-thyroid origin [START_REF] Landa | Comprehensive Genetic Characterization of Human Thyroid Cancer Cell Lines: A Validated Panel for Preclinical Studies[END_REF][START_REF] Schweppe | Deoxyribonucleic acid profiling analysis of 40 human thyroid cancer cell lines reveals cross-contamination resulting in cell line redundancy and misidentification[END_REF].

Finally, promising results of clinical trials on redifferentiation strategies are outlined. low expression of some thyroid differentiation genes such as SCLC5A5 gene encoding for the NIS, Thyroglobulin (Tg) or Thyroperoxydase (TPO) (Cancer Genome Atlas Research, 2014).

Modulation of signaling pathways

Several in vitro studies have demonstrated an increase in NIS expression and/or radioiodine uptake in human thyroid cancer-derived cell lines with various genetic backgrounds or patient-derived tumor tissue of ATC and PDTC with different MAPK pathway inhibitors namely BRAF V600E inhibitors such as vemurafenib (Cheng et al., 2016, Zhang and[START_REF] Zhang | Synergistic inhibition of MEK/ERK and BRAF V600E with PD98059 and PLX4032 induces sodium/iodide symporter (NIS) expression and radioiodine uptake in BRAF mutated papillary thyroid cancer cells[END_REF] or dabrafenib [START_REF] Fu | MAPK Inhibitors Enhance HDAC Inhibitor-Induced Redifferentiation in Papillary Thyroid Cancer Cells Harboring BRAF (V600E): An In Vitro Study[END_REF], multityrosine kinase inhibitor such as sorafenib [START_REF] Ruan | Iodide-and glucose-handling gene expression regulated by sorafenib or cabozantinib in papillary thyroid cancer[END_REF], Wachter et al., 2018a), or cabozantinib [START_REF] Ruan | Iodide-and glucose-handling gene expression regulated by sorafenib or cabozantinib in papillary thyroid cancer[END_REF], MEK inhibitors such as selumetinib [START_REF] Fu | MAPK Inhibitors Enhance HDAC Inhibitor-Induced Redifferentiation in Papillary Thyroid Cancer Cells Harboring BRAF (V600E): An In Vitro Study[END_REF], Wachter et al., 2018a, Wachter et al., 2018b), PD98059 [START_REF] Hou | Induction of thyroid gene expression and radioiodine uptake in thyroid cancer cells by targeting major signaling pathways[END_REF][START_REF] Vadysirisack | MEK signaling modulates sodium iodide symporter at multiple levels and in a paradoxical manner[END_REF][START_REF] Zhang | Synergistic inhibition of MEK/ERK and BRAF V600E with PD98059 and PLX4032 induces sodium/iodide symporter (NIS) expression and radioiodine uptake in BRAF mutated papillary thyroid cancer cells[END_REF] or refametinib [START_REF] Hou | Induction of thyroid gene expression and radioiodine uptake in thyroid cancer cells by targeting major signaling pathways[END_REF].

The first clinical phase II study testing a pharmacological inhibitor of the MAPK pathway reported the effect of the multityrosine kinase inhibitor sorafenib in 31 patients [START_REF] Hoftijzer | Beneficial effects of sorafenib on tumor progression, but not on radioiodine uptake, in patients with differentiated thyroid carcinoma[END_REF]. This study was based on in vitro data demonstrating that another multityrosine kinase inhibitor namely sunitinib was able to induce NIS expression in PTC cell lines harbouring RET/PTC1 rearrangement through inhibition of the MEK/ERK and SAPK/JNK pathway [START_REF] Fenton | Sunitinib inhibits MEK/ERK and SAPK/JNK pathways and increases sodium/iodide symporter expression in papillary thyroid cancer[END_REF] and that sunitinib increased iodine uptake in normal rat thyroid cells [START_REF] Salem | Effect of sunitinib on growth and function of FRTL-5 thyroid cells[END_REF]. Of the 20 patients evaluable for redifferentiation, after 26 weeks of treatment with sorafenib, only one restored a faint uptake of radioiodine in an occipital

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skeletal metastasis on the diagnostic whole body scan (WBS) performed, which was not confirmed on the therapeutic WBS performed after the administration of 200 mCi [START_REF] Hoftijzer | Beneficial effects of sorafenib on tumor progression, but not on radioiodine uptake, in patients with differentiated thyroid carcinoma[END_REF]. A renewed interest for redifferentiation strategy with MAPK pathway pharmacological inhibitors came from the publication by the James Fagin laboratory (Memorial Sloan-Kettering Cancer Center, MSKCC) [START_REF] Chakravarty | Small-molecule MAPK inhibitors restore radioiodine incorporation in mouse thyroid cancers with conditional BRAF activation[END_REF]. His team developed an ingenious mouse model expressing the BRAF V600E oncogene specifically in the thyrocytes and with the ability to switch the expression of BRAF V600E on and off with the administration or withdrawal of doxycycline [START_REF] Chakravarty | Small-molecule MAPK inhibitors restore radioiodine incorporation in mouse thyroid cancers with conditional BRAF activation[END_REF]. Switching on BRAF V600E expression induced thyroid cancers in mice that recapitulate most human BRAF V600E -tumor characteristics and was associated with a virtual abolition of thyroidspecific gene expression along with radioiodine uptake. Switching off BRAF V600E not only restored thyroid follicular architecture but also thyroid-specific gene expression and radioiodine incorporation. Pharmacological inhibitors of MEK i.e. selumetinib or BRAF V600E inhibitor i.e. dabrafenib for 2 weeks partially restored thyroid-specific gene expression and radioiodine uptake. Following this preclinical in vivo model, Ho et al., from the MSKCC, performed a clinical study in 24 patients with PTC or follicular thyroid cancer (FTC) or poorly DTC confirming the ability of selumetinib to restore radioiodine in tumors previously shown to be radioiodine resistant [START_REF] Ho | Selumetinib-enhanced radioiodine uptake in advanced thyroid cancer[END_REF]. In this study, patients were treated with selumetinib for 4 weeks and then submitted to an iodine-124 PET-CT under thyrotropin stimulation to estimate the activity of iodine-131 required to deliver an arbitrary dose of 2000 cGy and above to the metastatic lesions. If it appeared that at least one lesion could be treated by an activity of less than 300 mCi the patient was then treated with a personalized therapeutic dose of 131-iodine. Out of the 20 patients evaluable, selumetinib increased radioiodine uptake in 12. Of these 12 patients, 8 reached the dosimetry threshold for radioiodine therapy, including all 5 patients with NRAS mutation but only one out of 9

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patients with BRAF V600E mutation. Of the 8 patients treated with radioiodine, 7 had a confirmed radiological partial response according to the RECIST 1.1 criteria, 6 months after selumetinib withdrawal and radioiodine therapy. The authors attributed their good results in large part to the dosimetric approach enabled by the iodine-124 PET-CT evaluation of the patients. This publication has opened the field of successful redifferentiation strategy with MAPK inhibitors (Table 1).

To confirm the data of the pilot study conducted by Ho et al., a multicenter UK single arm phase II trial (SEL-I-METRY) has opened [START_REF] Brown | Investigating the potential clinical benefit of Selumetinib in resensitising advanced iodine refractory differentiated thyroid cancer to radioiodine therapy (SEL-I-METRY): protocol for a multicentre UK single arm phase II trial[END_REF]. Patients with locally advanced or metastatic RAIR DTC or PDTC treated for 4 weeks with selumetinib and are evaluated by iodine-123 SPECT/CT under recombinant human TSH to select patients showing significant increase in iodine-123 uptake and who will subsequently be treated with a fixed activity of 150 mCi of 131-radioiodine.

Despite these encouraging preliminary results on RAIR DTC patients, the results of the ASTRA phase III study (NCT01843062) evaluating MEK inhibitors as an adjuvant therapy in non-proved RAIR DTC were disappointing. This trial included patients with DTC at high risk of recurrence after total thyroidectomy (i.e. pT> 4 cm, pT4, N1 with ≥ 5 lymph nodes or with at least 1 lymph node ≥ 1cm). Patients were randomized to receive placebo or selumetinib for 4 weeks prior radioiodine ablation to assess the effect of selumetinib on the complete remission rate at 18 months. The addition of selumetinib to radioiodine did not improve the complete remission rate (40% vs 38.5% in the placebo group) in this patient population, in any subgroup of patients, even when genotype was taken into account. However, the placebo group established a 38.5% complete remission rate with standard radioiodine alone in highrisk patients, suggesting the need for improved therapeutic approaches. Subgroup analyses of this trial suggest that treatment compliance and tailoring the targeted therapy approach to the F o r R e v i e w O n l y 10 oncogenic driver mutation, especially the absence of a BRAF mutation, may be critical design elements to consider for future trials (unpublished data from J. Fagin's team).

Owing to the relatively poor response of BRAF-mutated thyroid cancers in the study published by Ho et al., others have evaluated the capacity of the selective BRAF V600E inhibitor dabrafenib to specifically redifferentiate BRAF V600E [START_REF] Rothenberg | Redifferentiation of Iodine-Refractory BRAF V600E-Mutant Metastatic Papillary Thyroid Cancer with Dabrafenib-Response[END_REF]. Out of the 10 patients included with a RAIR BRAF-mutated PTC, defined as the absence of radioiodine uptake on a first WBS (diagnostic or therapeutic dose), 6 weeks of dabrafenib restored radioiodine uptake on a diagnostic WBS in 6 of them. Those 6 patients were then treated with 150 mCi iodine-131 with a therapeutic WBS confirming the data obtained with the diagnostic scan. Two patients showed partial response and 4 stable disease on standard radiological imaging obtained 3 months after radioiodine therapy and discontinuation of dabrafenib, while out of the 4 patients without restoration of radioiodine uptake, one had progressive disease and 3 stable disease. These results should be put into perspectives considering that at study entry 5 patients out of 10 had progression per RECIST v1.1 within the14 months prior to enrolment. Ho and his team published another pilot trial [START_REF] Dunn | Vemurafenib Redifferentiation of BRAF Mutant, RAI-Refractory Thyroid Cancers[END_REF] evaluating vemurafenib in 12 patients with a BRAF mutated-PTC or PDTC, with a methodology similar to their first study [START_REF] Ho | Selumetinib-enhanced radioiodine uptake in advanced thyroid cancer[END_REF]. Out of the 10 patients evaluable, 4 reached the dosimetry threshold on the iodine-124 PET scan and were retreated with a therapeutic dose of iodine 131. Of those 4 patients, radiological assessment revealed partial response (n = 2) and stable disease (n = 2) 6 months after radioiodine therapy. Interestingly the serum Tg value among iodine-124 responders was significantly higher than in non-responders. This suggests that cancers with better differentiation at baseline (high serum Tg) have a better chance to respond favourably to the redifferentiation strategy. Molecular tumor biopsy analysis performed before and under vemurafenib revealed that to some extent the degree of iodine avidity

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restoration is linked to the degree of MAPK pathway output inhibition and the induction of thyroid-specific gene expression.

The retrospective analysis of patients with RAIR DTC (PTC, FTC or PDTC) treated with MAPK inhibitors (BRAF V600E and/or MEK inhibitors), for a mean of 14 months, also confirmed the data described above [START_REF] Jaber | Targeted Therapy in Advanced Thyroid Cancer to Resensitize Tumors to Radioactive Iodine[END_REF][START_REF] Iravani | cancer: an evolving protocol[END_REF]. In the first study [START_REF] Jaber | Targeted Therapy in Advanced Thyroid Cancer to Resensitize Tumors to Radioactive Iodine[END_REF] 9 out of 13 patients were treated with a therapeutic dose of 150-250 mCi of radioiodine based on meaningful uptake of radioiodine on a diagnostic WBS except for one. All 9 patients had durable disease control. As previously reported, RAS-mutated tumors were the best responders compared to BRAF-mutated ones. In the second study [START_REF] Iravani | cancer: an evolving protocol[END_REF] out of 6 patients treated with MEK inhibitor (NRAS mutated) or BRAF and MEK inhibitors (BRAF mutated) for 4 weeks, 4 were considered suitable for radioiodine therapy based on the result of I124 PET/CT, with I131 post-therapeutic WBS confirming the restoration of radioiodine uptake. In this study all BRAF-mutated patients responded to the redifferentiation strategy while only one NRAS-mutated patient out of 3 did. Of these 4 patients 3 achieved a partial imaging response (while progressing or stable at study entry) and one stable disease (while progressing at study entry) after a median follow-up of 16.6 months.

Additional case reports demonstrated the potential redifferentiating effect of pharmacological MAPK inhibition in thyroid cancers [START_REF] Huillard | Restoring Radioiodine Uptake in BRAF V600E-Mutated Papillary Thyroid Cancer[END_REF][START_REF] Leboulleux | Redifferentiation of a BRAF(K601E)-Mutated Poorly Differentiated Thyroid Cancer Patient with Dabrafenib and Trametinib Treatment[END_REF]. In one of these case reports clinical hyperthyroidism developed, with free triiodothyronine and free thyroxine levels increasing to 6.6 and 4.4 times their upper reference limit, as ultimate evidence of efficient redifferentiation [START_REF] Leboulleux | Redifferentiation of a BRAF(K601E)-Mutated Poorly Differentiated Thyroid Cancer Patient with Dabrafenib and Trametinib Treatment[END_REF]). In the study of Iravani et al. 4 weeks of thyroid hormone withdrawal increased TSH to only 25.9 mUI/L in one patient submitted to a redifferentiation strategy with MEK and BRAF inhibitors suggesting the redifferentiation of functional thyroid cancer cells.

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As already observed in some patients in the study by Dunn, Huillard et al. (Dunn et al., 2019[START_REF] Huillard | Restoring Radioiodine Uptake in BRAF V600E-Mutated Papillary Thyroid Cancer[END_REF] showed that in the context of a redifferentiation strategy, a rise in thyroglobulin may indicate the success of redifferentiation rather than disease progression.

Interestingly, Huillard et al. demonstrated that a BRAF V600E inhibitor administered in a RAIR-PTC patient revealed unsuspected pulmonary miliary metastases on the therapeutic WBS performed. Finally, this case report also illustrated that the redifferentiation effect is transitory and disappears after a short period of discontinuation of the redifferentiation drug highlighting the importance of radioiodine administration while the patient is under treatment.

However, in all these studies, it is difficult to distinguish between tumor response resulting from a cytotoxic effect of the pharmacological inhibitor or restoration of radioiodine uptake.

Selecting patients with mixed response that is to say patients with lesions showing new or enhanced radioiodine uptake and lesions without any uptake could create the opportunity to distinguish between both effects [START_REF] Huillard | Redifferentiation of Iodine-Refractory BRAF V600E-Mutant Metastatic Papillary Thyroid Cancer with Dabrafenib-Letter[END_REF].

Future prospects to improve tumor redifferentiation, especially BRAF V600E -mutated thyroid cancers, through MAPK inhibition, might come from compounds able to profoundly inhibit the MAPK pathway, such as CKI a MEK inhibitor that functions as a dominant-negative inhibitor of RAF which reduces the feedback reactivation of ERK signaling. James Fagin's laboratory published data, in vitro, in rat thyroid cells expressing BRAF V600E and in vivo in a mouse model with BRAF V600E -induced thyroid cancer, demonstrating that a small increase in ERK inhibition translates into a markedly increased expression of thyroid differentiation genes and increased iodide accumulation in cancer [START_REF] Nagarajah | Sustained ERK inhibition maximizes responses of BrafV600E thyroid cancers to radioiodine[END_REF]. A Highthroughput NIS enhancer screening platform also enabled the identification of a new tyrosine kinase inhibitor which increased NIS promoter activity along with the expression of NIS and other thyroid specific proteins (Tg, TPO, TSH-R, Pax 8 and TTF-1) in a BRAF V600E -mutated thyroid cancer cell line. Moreover this compound, which decreased the level of

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phosphorylated ERK i.e. the activation of the MAPK pathway, increased 125 I uptake in the same cell line and in vivo in xenograft mice models [START_REF] Oh | Reverting iodine avidity of radioactiveiodine refractory thyroid cancer with a new tyrosine kinase inhibitor (K905-0266) excavated by high-throughput NIS (sodium iodide symporter) enhancer screening platform using dual reporter gene system[END_REF]. The combination of BRAF V600E and MEK inhibition may be a promising strategy as the combination synergistically increased radioiodine uptake in human BRAF-mutated thyroid cancer cell lines possibly through inhibition of the rebound of ERK1/2 activation observed with only one drug [START_REF] Zhang | Synergistic inhibition of MEK/ERK and BRAF V600E with PD98059 and PLX4032 induces sodium/iodide symporter (NIS) expression and radioiodine uptake in BRAF mutated papillary thyroid cancer cells[END_REF]). An ongoing clinical trial performed by the French RAIR thyroid cancer TUTHYREF network is testing the association of MEK (Trametinib) alone (for RAS mutated tumors) or in combination with BRAF V600E (Dabrafenib) inhibitors (for BRAF V600E mutated tumors) followed by radioiodine therapy for the treatment of RAIR metastatic DTC (NCT 03244956).

Another strategy for BRAF V600E -mutated thyroid cancers would consists in be to combine inhibitors of the human epidermal growth factor receptor (Her) belonging to the epidermal growth factor receptor family with MAPK inhibitors. In thyroid cancer cells harbouring BRAF V600E mutation, inhibition of the MAPK pathway by pharmacological inhibitors were transient due to the release, by a RAF or MEK inhibitor, of a transcriptional repressor from the HER3 promoter and consequently induced HER3 gene overexpression [START_REF] Montero-Conde | Relief of feedback inhibition of HER3 transcription by RAF and MEK inhibitors attenuates their antitumor effects in BRAFmutant thyroid carcinomas[END_REF]). An autocrine secretion by thyroid cancer cells of a ligand able to bind to and activate by dimerization the tyrosine kinase receptors HER2/HER3 resulted in the reactivation of the MAPK and PI3K pathway. The Her kinase inhibitor lapatinib prevented MAPK rebound and overcame BRAF-mutated thyroid cancer cell resistance to MAPK inhibitors [START_REF] Montero-Conde | Relief of feedback inhibition of HER3 transcription by RAF and MEK inhibitors attenuates their antitumor effects in BRAFmutant thyroid carcinomas[END_REF]. In line with these data, Cheng et al. [START_REF] Cheng | HER inhibitor promotes BRAF/MEK inhibitorinduced redifferentiation in papillary thyroid cancer harboring BRAFV600[END_REF] demonstrated in vitro in BRAF V600E -mutated human thyroid cancer derived cell lines that the combination of lapatinib with dabrafenib or selumetinib increased radioiodine uptake. A clinical trial (NCT 02456701) performed at the MSKCC and which tests the ability of

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vemurafenib combined with an anti HER3 monoclonal antibody to restore iodine incorporation in BRAF mutant RAIR thyroid cancer patients is ongoing.

Iodide oxidation and tyrosine organification are not restored by MAPK blockade. As PI3K inhibition seems to prolong radioiodine retention in thyroid cells [START_REF] Lakshmanan | Modulation of thyroidal radioiodide uptake by oncological pipeline inhibitors and Apigenin[END_REF], the combination of MAPK and PI3K inhibitors may be an interesting strategy, but with the disadvantage of potential synergistic side effects.

The potential of combining MAPK inhibitors with HDAC inhibitors is developed in the 2.2 section of this review.

Concerning undifferentiated thyroid cancers namely ATC, hope for successful redifferentiation comes from a mouse model of lethally aggressive thyroid cancer, harboring BRAF V600E and PIK3CA H1047R mutations demonstrating that MEK inhibition for only 10 days could increase radioiodine uptake, which could then translate into a stable and profound reduction in tumor burden [START_REF] Elmokh | MEK Inhibition Induces Therapeutic Iodine Uptake in a Murine Model of Anaplastic Thyroid Cancer[END_REF].

Modulation of other pathways: preclinical evidence

The PI3K pathway

In a non-tumoral context, a central role for PI3K in the repression of NIS gene transcription by IGF1 has been demonstrated in the FRTL-5 rat thyroid cell line [START_REF] Garcia | PI3K is involved in the IGF-I inhibition of TSH-induced sodium/iodide symporter gene expression[END_REF]. Paradoxically, Quercetin, a compound with PI3K inhibitory properties, has been shown to reduce NIS mRNA levels in FRTL-5 cells [START_REF] Giuliani | The flavonoid quercetin regulates growth and gene expression in rat FRTL-5 thyroid cells[END_REF].

However, other publications have shown that PI3K pathway inhibition has the potential to restore radioiodine sensitivity of RAIR DTC (Figure 2). Song et al. [START_REF] Song | A somatic mutation of RasGRP3 decreases Na(+)/I(-) symporter expression in metastases of radioactive iodine-refractory thyroid cancer by stimulating the Akt signaling pathway[END_REF] demonstrated in vitro in thyroid cancer cell lines that a mutant RasGRP3 (Ras guanine nucleotide-releasing protein 3), which was frequently found in metastases of RAIR DTC, decreased iodine uptake ability besides promoting cell proliferation, migration and invasiveness. This was associated with PI3K pathway activation and the addition of the PI3K pathway pharmacological inhibitor LY294002 restored radioiodine uptake of the cells at their basal level i.e. without overexpression of RasGRP3 mutant. LY294002 also increased radioiodine uptake in human thyroid cancer cells expressing RET/PTC1 and engineered to constitutively express NIS [START_REF] Kogai | Phosphoinositide-3kinase inhibition induces sodium/iodide symporter expression in rat thyroid cells and human papillary thyroid cancer cells[END_REF] and in the non-cancerous PCCL3 rat thyroid cells (de Souza et al., 2010) mainly through NIS upregulation [START_REF] Liu | Modulation of sodium iodide symporter expression and function by LY294002, Akti-1/2 and Rapamycin in thyroid cells[END_REF].

Interestingly pharmacological Akt inhibition increased radioiodine uptake despite a decrease in NIS protein levels, in PCCL3 rat thyroid cells with doxycycline induced expression of constitutively active MEK. Indeed, this Akt1/2 inhibitor acted on iodine uptake through a decreased iodide efflux and a higher iodide affinity enhancing the NIS-mediated iodide transport rate [START_REF] Liu | Modulation of sodium iodide symporter expression and function by LY294002, Akti-1/2 and Rapamycin in thyroid cells[END_REF]. The same research team further demonstrated that among different pharmacological inhibitors targeting Akt, MEK, PI3K, Hsp90 or BRAF V600E tested in PCCL3 rat thyroid cells conditionally expressing RET/PTC3 or BRAF V600E , the PI3K inhibitor GDC-0941 outperformed other inhibitors in radioiodine increase [START_REF] Lakshmanan | Modulation of thyroidal radioiodide uptake by oncological pipeline inhibitors and Apigenin[END_REF]. Surprisingly the greatest effect was observed in BRAF V600E expressing thyroid cells, where MEK is the canonical downstream of BRAF V600E . Again, a decrease in iodide efflux was the main mechanism of action of this PI3K inhibitor which had limited effect on NIS protein levels. Of note Lakshmanan et al. [START_REF] Lakshmanan | Modulation of thyroidal radioiodide uptake by oncological pipeline inhibitors and Apigenin[END_REF] demonstrated that TGF present in the thyroid tumor microenvironment, reduces the extent of increase in radioiodine uptake induced by the pharmacological inhibitors tested. They also showed that Apigenin, a plant-derived flavonoid, associated with GDC-0941 provided the highest

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radioiodine uptake level in both BRAF V600E and RET/PTC3 expressing PCCL3 cells, corroborating previous data obtained with an Akt pharmacological inhibitor in the same cells and in a mouse thyroid tumor model [START_REF] Lakshmanan | Apigenin in combination with Akt inhibition significantly enhances thyrotropinstimulated radioiodide accumulation in thyroid cells[END_REF].

Another PI3K inhibitor, rapamycin was found to induce NIS protein levels along with radioiodine uptake in BRAF V600E and RET/PTC1 PTC derived cell lines (but not in a FTC derived cell line), possibly through a transcriptional effect dependent on the transcription factor TTF1 [START_REF] Plantinga | mTOR Inhibition promotes TTF1-dependent redifferentiation and restores iodine uptake in thyroid carcinoma cell lines[END_REF]. Rapamycin and its synthetic analog everolimus, led to controversial results in a non-tumoral thyroid in vitro model [START_REF] De Souza | MTOR downregulates iodide uptake in thyrocytes[END_REF][START_REF] Liu | Modulation of sodium iodide symporter expression and function by LY294002, Akti-1/2 and Rapamycin in thyroid cells[END_REF].

Other pathways

The NOTCH signaling pathway

Notch genes encode receptors for a signaling pathway that controls cell fate by regulating cell proliferation and differentiation, especially in oncogenic contexts. Notch 1 and Notch3 have been described as tumor suppressor genes in PTC, FTC and ATC [START_REF] Somnay | Notch3 expression correlates with thyroid cancer differentiation, induces apoptosis, and predicts disease prognosis[END_REF][START_REF] Xiao | Notch1 mediates growth suppression of papillary and follicular thyroid cancer cells by histone deacetylase inhibitors[END_REF][START_REF] Yu | Resveratrol induces differentiation markers expression in anaplastic thyroid carcinoma via activation of Notch1 signaling and suppresses cell growth[END_REF]. Moreover, Notch1 and 3 expression are inversely correlated to the degree of differentiation in thyroid cancers with ATC having the strongest decreased expression of both Notch [START_REF] Ferretti | Notch signaling is involved in expression of thyrocyte differentiation markers and is down-regulated in thyroid tumors[END_REF][START_REF] Somnay | Notch3 expression correlates with thyroid cancer differentiation, induces apoptosis, and predicts disease prognosis[END_REF]. From a clinical point of view, low Notch3 expression levels correlated with overall survival independently from the classical other prognostic factors [START_REF] Somnay | Notch3 expression correlates with thyroid cancer differentiation, induces apoptosis, and predicts disease prognosis[END_REF]. In vitro Notch3 constitutive activation in a FTC cell line increased the mRNA levels of thyroid specific genes i.e. NIS, TSH-R, TTF-1, TTF-2. In a non-tumoral context, in the normal FRTL-5 rat thyroid cell line, overexpression of Notch1 increased the expression of NIS and TPO [START_REF] Ferretti | Notch signaling is involved in expression of thyrocyte differentiation markers and is down-regulated in thyroid tumors[END_REF]. Pharmacological Notch1 activating compounds (Figure 2) such as resveratrol [START_REF] Yu | Resveratrol induces differentiation markers expression in anaplastic thyroid carcinoma via activation of Notch1 signaling and suppresses cell growth[END_REF] [START_REF] Patel | Hesperetin activates the Notch1 signaling cascade, causes apoptosis, and induces cellular differentiation in anaplastic thyroid cancer[END_REF] have been shown to induce NIS mRNA levels or other differentiation markers such as TSH-R, TTF-1, TTF-2 and PAX8, in vitro in ATC cell lines. The silencing of Notch1 by siRNA abrogated this effect, demonstrating the dependence upon Notch1 signaling [START_REF] Yu | Resveratrol induces differentiation markers expression in anaplastic thyroid carcinoma via activation of Notch1 signaling and suppresses cell growth[END_REF]. Interestingly other compounds with redifferentiation capacity, such as the HDAC inhibitors valproic acid and SAHA or retinoic acid outlined elsewhere in these reviews have also been reported as Notch activating compounds [START_REF] Lin | All-trans retinoid acid increases Notch1 transcript expression in acute promyelocytic leukemia[END_REF][START_REF] Xiao | Notch1 mediates growth suppression of papillary and follicular thyroid cancer cells by histone deacetylase inhibitors[END_REF].

PDGFR blockade

Lopez-Campistrous et al. [START_REF] Lopez-Campistrous | PDGFRalpha Regulates Follicular Cell Differentiation Driving Treatment Resistance and Disease Recurrence in Papillary Thyroid Cancer[END_REF] have demonstrated in thyroid cancer cell lines and mouse xenograft models that activation of PDGFR decreases Tg and NIS expression which promotes a decrease in radioiodine uptake (Figure 2). Blocking PDGFR in these same models improved radioiodine uptake and also reduced migration and invasion potentials of the cells as well as tumor volume in animal models. The relocation of the transcription factor TTF1 from the nucleus to the cytoplasm is the main mechanism of action of PDGFR activation. In human PTC, PDGFR expression was strongly associated with metastatic disease and radioiodine resistance.

Modulation of NIS transcription

Epigenetic modification is responsible for the regulation of gene expression without involving modification of the genomic DNA sequence. Epigenetic dysregulation is emerging as a contributor to carcinogenesis and tumor progression and as a therapeutic target. The restoration of iodine uptake in thyroid tumors that were considered refractory to radioiodine, suggesting reversible repression of the SLC5A5 gene encoding for the NIS, is an indirect demonstration of an epigenetic regulation of the NIS gene.

Retinoic acid

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Retinoids are chemical compounds related to vitamin A. They act on the nuclear receptors retinoic acid receptor (RAR) and retinoid X receptor (RXR). All-trans-retinoic acid (RA)-RAR or RA-RXR complexes bind to the responsive elements in gene promoter sites and activate the transcription of their target genes. The retinoid pathway is involved in cellular differentiation, proliferation, and apoptosis. Retinoic acid is a well-known drug for certain dermatological diseases.

In thyroid carcinoma cell lines in vitro experiments showed that retinoids affect thyroid specific functions (induction of type I 5'-deiodinase activity, NIS gene expression), cell-cell or cell-matrix interaction, differentiation markers, growth, and tumorigenicity [START_REF] Jeong | Effect of all-trans retinoic acid on sodium/iodide symporter expression, radioiodine uptake and gene expression profiles in a human anaplastic thyroid carcinoma cell line[END_REF][START_REF] Kurebayashi | Alltrans-retinoic acid modulates expression levels of thyroglobulin and cytokines in a new human poorly differentiated papillary thyroid carcinoma cell line, KTC-1[END_REF][START_REF] Schmutzler | Retinoic acid redifferentiation therapy for thyroid cancer[END_REF], Schmutzler et al., 2002[START_REF] Schreck | Retinoids stimulate type I iodothyronine 5'-deiodinase activity in human follicular thyroid carcinoma cell lines[END_REF][START_REF] Van Herle | Effects of 13 cis-retinoic acid on growth and differentiation of human follicular carcinoma cells (UCLA R0 82 W-1) in vitro[END_REF]. Moreover, retinoids partly re-differentiate follicular thyroid carcinoma cell lines [START_REF] Schmutzler | Retinoic acid redifferentiation therapy for thyroid cancer[END_REF] (Figure 2).

The first clinical series of 10 RAIR-DTC patients treated with isotretinoin (13-cis-retinoid acid, a first-generation retinoid) was published by Simon et al. Radioiodine uptake was restored in 4 patients [START_REF] Simon | Redifferentiation therapy of differentiated thyroid carcinoma with retinoic acid: basics and first clinical results[END_REF]. Other pilot studies reported an increase in radioiodine uptake in about 20 to 40% of patients [START_REF] Courbon | Defective efficacy of retinoic acid treatment in patients with metastatic thyroid carcinoma[END_REF][START_REF] Fernandez | Effectiveness of retinoic acid treatment for redifferentiation of thyroid cancer in relation to recovery of radioiodine uptake[END_REF][START_REF] Gruning | Retinoic acid for redifferentiation of thyroid cancer--does it hold its promise?[END_REF][START_REF] Grunwald | Changes in the blood picture after radioiodine therapy of thyroid cancer[END_REF][START_REF] Kim | Efficacy on anaplastic thyroid carcinoma of valproic acid alone or in combination with doxorubicin, a synthetic chenodeoxycholic acid derivative, or lactacystin[END_REF][START_REF] Oh | Combined therapy with 131I and retinoic acid in Korean patients with radioiodine-refractory papillary thyroid cancer[END_REF][START_REF] Simon | Clinical impact of retinoids in redifferentiation therapy of advanced thyroid cancer: final results of a pilot study[END_REF]. However, tumor response did not always correlate with increased radioiodine uptake and other direct antiproliferative effects of isotretinoin might have been involved [START_REF] Simon | Clinical impact of retinoids in redifferentiation therapy of advanced thyroid cancer: final results of a pilot study[END_REF]. Prospective clinical trials failed to demonstrate a clinical utility of isotretinoin. In an open-label phase II trial, isotretinoin did not significantly increase radioiodine uptake in 16 RAIR-DTC [START_REF] Short | A phase II study using retinoids as redifferentiation agents to increase iodine uptake in metastatic thyroid cancer[END_REF]. In another published phase II trial, increased radioiodine uptake was observed in 17% (n=9/53) of patients. However, this did not translate into tumor response or clinical benefit [START_REF] Handkiewicz-Junak | 13-cis-retinoic acid redifferentiation therapy and recombinant human thyrotropin-aided radioiodine treatment of non-Functional metastatic thyroid cancer: a single-center, 53-patient phase 2 study[END_REF].

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Few clinical data exist with other retinoids. In a series of 11 patients treated with tretinoin (all-trans-retinoic acid [RA]), also a first-generation retinoid, authors reported increased radioiodine uptake in 4 patients and partial response in 5 patients [START_REF] Vadysirisack | MEK signaling modulates sodium iodide symporter at multiple levels and in a paradoxical manner[END_REF].

Another series of 13 patients treated with tretinoin for RAIR-DTC showed a weak radioiodine uptake in 6 patients with no tumor response among those patients [START_REF] Damle | Retinoic acid therapy in patients with radioiodine negative differentiated thyroid cancer and clinical or biochemical evidence of disease: An initial experience[END_REF]. In a pilot study with bexarotene (RXR activator), increased radioiodine uptake was observed in 8/11 patients, but no significant clinical benefit was demonstrated (Liu et al., 2006a).

A recent meta-analysis on retinoids in RAIR-DTC reported a pooled effect of 27.6% for increased radioiodine uptake and 17% for tumor response by RECIST criteria [START_REF] Pak | Response of Retinoic Acid in Patients with Radioactive Iodine-Refractory Thyroid Cancer: A Meta-Analysis[END_REF].

HDAC inhibitors

Post-translational modification of histones bound to DNA such as acetylation is a well-known mechanism of tumorigenesis initiation and progression, especially for thyroid cancers [START_REF] Russo | Targeting histone deacetylase in thyroid cancer[END_REF]. By altering the structural state of chromatin, histone acetylation by histone acetyl transferases favors transcriptional activation. On the other hand, histone deacetylation by histone deacetylase (HDACs) is associated with transcriptional repression. Several publications have demonstrated that the acetylation of histones of the SLC5A5 promotor regulates its transcription and consequently NIS expression [START_REF] Fu | MAPK Inhibitors Enhance HDAC Inhibitor-Induced Redifferentiation in Papillary Thyroid Cancer Cells Harboring BRAF (V600E): An In Vitro Study[END_REF][START_REF] Puppin | Cooperative effects of SAHA and VPA on NIS gene expression and proliferation of thyroid cancer cells[END_REF][START_REF] Lakshmanan | Apigenin in combination with Akt inhibition significantly enhances thyrotropinstimulated radioiodide accumulation in thyroid cells[END_REF]. Various HDAC inhibitors (HDACi), such as panobinostat [START_REF] Fu | MAPK Inhibitors Enhance HDAC Inhibitor-Induced Redifferentiation in Papillary Thyroid Cancer Cells Harboring BRAF (V600E): An In Vitro Study[END_REF][START_REF] Pugliese | Histone deacetylase inhibition affects sodium iodide symporter expression and induces 131I cytotoxicity in anaplastic thyroid cancer cells[END_REF], Wachter et al., 2018c, Wachter et al., 2018a), valproic acid [START_REF] Haghpanah | The Beneficial Effects of Valproic Acid in Thyroid Cancer Are Mediated through Promoting Redifferentiation and Reducing Stemness Level: An In Vitro Study[END_REF][START_REF] Massimino | Effect of Combined Epigenetic Treatments and Ectopic NIS Expression on Undifferentiated Thyroid Cancer Cells[END_REF][START_REF] Shen | Valproic acid inhibits growth, induces apoptosis, and modulates apoptosis-regulatory and differentiation gene expression in human thyroid cancer cells[END_REF][START_REF] Frohlich | Induction of iodide uptake in transformed thyrocytes: a compound screening in cell lines[END_REF], vorinostat [START_REF] Cheng | Robust Thyroid Gene Expression and Radioiodine Uptake Induced by Simultaneous Suppression of BRAF V600E and Histone Deacetylase in Thyroid Cancer Cells[END_REF][START_REF] Clinckspoor | 1,25-dihydroxyvitamin D3 and a superagonistic analog in combination with paclitaxel or suberoylanilide hydroxamic acid have potent antiproliferative effects on anaplastic thyroid cancer[END_REF][START_REF] Hou | Induction of thyroid gene expression and radioiodine uptake in thyroid cancer cells by targeting major signaling pathways[END_REF][START_REF] Puppin | Effects of histone acetylation on sodium iodide symporter promoter and expression of thyroid-specific transcription factors[END_REF][START_REF] Puppin | Cooperative effects of SAHA and VPA on NIS gene expression and proliferation of thyroid cancer cells[END_REF], Wachter et al., 2018a), romidepsin [START_REF] Furuya | Histone deacetylase inhibitors restore radioiodide uptake and retention in poorly differentiated and anaplastic thyroid cancer cells by expression of the sodium/iodide symporter thyroperoxidase and thyroglobulin[END_REF][START_REF] Kitazono | Low concentrations of the histone deacetylase inhibitor, depsipeptide (FR901228), increase expression of the Na(+)/I(-) symporter and iodine accumulation in poorly differentiated thyroid carcinoma cells[END_REF][START_REF] Xu | Histone deacetylase inhibitor depsipeptide represses nicotinamide N-methyltransferase and hepatocyte nuclear factor-1beta gene expression in human papillary thyroid cancer cells[END_REF], sodium butyrate [START_REF] Puppin | Effects of histone acetylation on sodium iodide symporter promoter and expression of thyroid-specific transcription factors[END_REF] (Wachter et al., 2018a[START_REF] Zarnegar | Increasing the effectiveness of radioactive iodine therapy in the treatment of thyroid cancer using Trichostatin A, a histone deacetylase inhibitor[END_REF] or analogs targeting HDAC [START_REF] Jang | Novel analogs targeting histone deacetylase suppress aggressive thyroid cancer cell growth and induce redifferentiation[END_REF], have been shown in human thyroid cancer derived cell lines or patient-derived thyroid cancer tissue to reinduce NIS mRNA levels (Figure 2) or even other thyroid differentiation genes such as Tg, TPO or TSH receptor. More interestingly, some studies have demonstrated in cellular or in in vivo models an increase in iodine uptake following HDACi treatment and/or proper targeting of the NIS to the plasma membrane where this transporter can be effective [START_REF] Cheng | Robust Thyroid Gene Expression and Radioiodine Uptake Induced by Simultaneous Suppression of BRAF V600E and Histone Deacetylase in Thyroid Cancer Cells[END_REF][START_REF] Fu | MAPK Inhibitors Enhance HDAC Inhibitor-Induced Redifferentiation in Papillary Thyroid Cancer Cells Harboring BRAF (V600E): An In Vitro Study[END_REF][START_REF] Furuya | Histone deacetylase inhibitors restore radioiodide uptake and retention in poorly differentiated and anaplastic thyroid cancer cells by expression of the sodium/iodide symporter thyroperoxidase and thyroglobulin[END_REF][START_REF] Hou | Induction of thyroid gene expression and radioiodine uptake in thyroid cancer cells by targeting major signaling pathways[END_REF][START_REF] Kitazono | Low concentrations of the histone deacetylase inhibitor, depsipeptide (FR901228), increase expression of the Na(+)/I(-) symporter and iodine accumulation in poorly differentiated thyroid carcinoma cells[END_REF][START_REF] Massimino | Effect of Combined Epigenetic Treatments and Ectopic NIS Expression on Undifferentiated Thyroid Cancer Cells[END_REF][START_REF] Pugliese | Histone deacetylase inhibition affects sodium iodide symporter expression and induces 131I cytotoxicity in anaplastic thyroid cancer cells[END_REF][START_REF] Puppin | Cooperative effects of SAHA and VPA on NIS gene expression and proliferation of thyroid cancer cells[END_REF], Wachter et al., 2018a). However the results of four clinical trials [START_REF] Amiri-Kordestani | Phase I trial of a new schedule of romidepsin in patients with advanced cancers[END_REF][START_REF] Kelly | Phase I study of an oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced cancer[END_REF][START_REF] Nilubol | A phase II trial of valproic acid in patients with advanced, radioiodineresistant thyroid cancers of follicular cell origin[END_REF], Sherman et al., 2013) that tested HDACi in RAIR human thyroid cancers were in apparent contradiction with the promising results of preclinical studies.

Nilubol et al. [START_REF] Nilubol | A phase II trial of valproic acid in patients with advanced, radioiodineresistant thyroid cancers of follicular cell origin[END_REF] tested the effect of valproic acid in 13 patients with RAIR DTC without any improvement in radioiodine uptake in the 10 patients evaluable. Two trials have tested romidepsin [START_REF] Amiri-Kordestani | Phase I trial of a new schedule of romidepsin in patients with advanced cancers[END_REF], Sherman et al., 2013) in RAIR DTC.

In these phase 1 and 2 trials, the 131I diagnostic WBS showed restoration of faint radioiodine uptake after various time periods of treatment in 2 out of 6 evaluable patients for the phase 1 study and 2 out of 16 patients for the phase 2. In the phase 2 trial [START_REF] Sherman | Evaluation of romidepsin for clinical activity and radioactive iodine reuptake in radioactive iodine-refractory thyroid carcinoma[END_REF], the faint radioiodine uptake observed on the diagnostic WBS was confirmed on the therapeutic WBS but without long-term benefit from the treatment. In a phase 1 trial testing vorinostat also known as Suberoylanilide Hydroxamic Acid (SAHA) [START_REF] Kelly | Phase I study of an oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced cancer[END_REF] in patients with advanced thyroid cancers, Kelly et al. reported an improvement in the radioiodine uptake evaluated by post-therapeutic WBS after SAHA administration for an unknown duration in one out of the 3 PTC patients included. These trials have classically been considered disappointing. However, several points should be highlighted before definitively buring HDACi as an effective redifferentiation strategy. First, from a clinical point of view, besides

F o r R e v i e w O n l y
the small number of patients included that preclude any definite conclusion, the evaluation of a proper iodine uptake increase or restoration after HDACi treatment on a diagnostic WBS can be criticized as this imaging lacks sensitivity [START_REF] Haugen | American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer[END_REF]. Second, preclinical studies show the complexity of triggering NIS and other thyroid-differentiation gene reinduction. In several of these studies the redifferentiation effect i.e. the increase in NIS mRNA and/or protein levels and/or increase in iodine uptake was considerably variable among the human thyroid cancer-derived cell lines tested, suggesting variable effects according to the genetic background of the cell lines [START_REF] Clinckspoor | 1,25-dihydroxyvitamin D3 and a superagonistic analog in combination with paclitaxel or suberoylanilide hydroxamic acid have potent antiproliferative effects on anaplastic thyroid cancer[END_REF][START_REF] Fu | MAPK Inhibitors Enhance HDAC Inhibitor-Induced Redifferentiation in Papillary Thyroid Cancer Cells Harboring BRAF (V600E): An In Vitro Study[END_REF][START_REF] Massimino | Effect of Combined Epigenetic Treatments and Ectopic NIS Expression on Undifferentiated Thyroid Cancer Cells[END_REF][START_REF] Shen | Valproic acid inhibits growth, induces apoptosis, and modulates apoptosis-regulatory and differentiation gene expression in human thyroid cancer cells[END_REF]. Illustrating the fine and complex regulation of the system, Shen et al. [START_REF] Shen | Valproic acid inhibits growth, induces apoptosis, and modulates apoptosis-regulatory and differentiation gene expression in human thyroid cancer cells[END_REF] reported a paradoxical inefficacy to restore NIS mRNA levels of a higher dose of valproic acid (1.5 mM) in comparison with a lower dose (0.5 mM).

Moreover the timing of HDACi administration and the evaluation of the redifferentiation effect might also be critical, as in vitro the increase in radioiodine uptake may vary with the timing of the pharmacological inhibitor administration (Wachter et al., 2018c).

Several publications suggest that drug combination may be a powerful redifferentiation strategy. MAPK inhibition with MEK inhibitor or BRAF V600E inhibitor combined with HDACi such as, panobinostat or SAHA, have been shown to have a synergistic effect on NIS and other differentiation thyroid gene (TSH-R, TPO, Tg) reinduction in various human thyroid cancer cell lines, in comparison with either drug used alone [START_REF] Cheng | Robust Thyroid Gene Expression and Radioiodine Uptake Induced by Simultaneous Suppression of BRAF V600E and Histone Deacetylase in Thyroid Cancer Cells[END_REF][START_REF] Fu | MAPK Inhibitors Enhance HDAC Inhibitor-Induced Redifferentiation in Papillary Thyroid Cancer Cells Harboring BRAF (V600E): An In Vitro Study[END_REF][START_REF] Hou | Induction of thyroid gene expression and radioiodine uptake in thyroid cancer cells by targeting major signaling pathways[END_REF]. This effect was even further enhanced by treatment with TSH, which not only enhanced the expression of thyroid genes, but also promoted NIS targeting to the plasma membrane [START_REF] Cheng | Robust Thyroid Gene Expression and Radioiodine Uptake Induced by Simultaneous Suppression of BRAF V600E and Histone Deacetylase in Thyroid Cancer Cells[END_REF][START_REF] Hou | Induction of thyroid gene expression and radioiodine uptake in thyroid cancer cells by targeting major signaling pathways[END_REF]. PI3K pathway inhibitors, such as mTOR or Akt inhibitors in combination with HDACi also have the same synergistic effect [START_REF] Hou | Induction of thyroid gene expression and radioiodine uptake in thyroid cancer cells by targeting major signaling pathways[END_REF]. Finally, the triple combination of MEK inhibitor, Akt inhibitor and SAHA had a synergistic effect on radioidine uptake, further enhanced by TSH, in human ATC [START_REF] Hou | Induction of thyroid gene expression and radioiodine uptake in thyroid cancer cells by targeting major signaling pathways[END_REF]. These pre-clinical data provide evidence which supports the proposal to use a drug combination strategy targeting the MAPK pathway and/or PI3K pathway with HDACi.

Finally, beyond HDACi, a Poly ADP ribose polymerase-1 (PARP-1) pharmacological inhibitor has been demonstrated to increase radioiodine uptake in different thyroid cancer cell lines, through upregulation of histone modification activation marks especially the acetylation of Lysine 9 and 14 of Histone 3 (H3K9K14Ac) [START_REF] Lavarone | The PARP inhibitor PJ34 modifies proliferation, NIS expression and epigenetic marks in thyroid cancer cell lines[END_REF].

PPAR agonists

Peroxisome proliferator-activated receptors (PPAR)- belongs to the nuclear receptor family of transcription factors. PPAR- is the master regulator of adipogenesis and plays a role in tumorigenesis [START_REF] Raman | Pax-8-PPAR-gamma fusion protein in thyroid carcinoma[END_REF]. Of note the rearrangement of PPAR- /PAX8 occurs in 30-35 % of FTC and in some follicular variants of PTC [START_REF] Raman | Pax-8-PPAR-gamma fusion protein in thyroid carcinoma[END_REF].

Different pre-clinical studies have demonstrated the re-differentiation effect of several thiazolidinediones, which bind to PPAR and are known to be PPAR agonists, such as rosiglitazone [START_REF] Frohlich | Induction of iodide uptake in transformed thyrocytes: a compound screening in cell lines[END_REF][START_REF] Frohlich | Action of thiazolidinediones on differentiation, proliferation and apoptosis of normal and transformed thyrocytes in culture[END_REF], troglitazone {Frohlich, 2005#265, Park et al., 2005) and pioglitazone [START_REF] Frohlich | Action of thiazolidinediones on differentiation, proliferation and apoptosis of normal and transformed thyrocytes in culture[END_REF], in addition to their anti-proliferative effects on DTC-derived cell lines (Figure 2). Interestingly Frolich et al. [START_REF] Frohlich | Action of thiazolidinediones on differentiation, proliferation and apoptosis of normal and transformed thyrocytes in culture[END_REF] demonstrated that in FTC cell lines thiazolidinediones increased radioiodine uptake and the amount of NIS in the membrane fraction, especially troglitazone which showed greater effects than other thiazolidinediones. However, these effects seem to be independent of PPAR activation as the addition of a PPAR antagonist did not reverse their effects. Several case reports [START_REF] Elias | Enhanced radioiodine uptake in a patient with poorly differentiated papillary thyroid cancer after treatment with rosiglitazone[END_REF]Lizotte, 2006, Elola et al., 2011), pilot study and phase II clinical trials [START_REF] Kebebew | Results of rosiglitazone therapy in patients with thyroglobulin-positive and radioiodinenegative advanced differentiated thyroid cancer[END_REF][START_REF] Kebebew | A phase II trial of rosiglitazone in patients with thyroglobulin-positive and radioiodine-negative differentiated thyroid cancer[END_REF][START_REF] Philips | Effect of peroxisome proliferator-activated receptor gamma agonist, rosiglitazone, on dedifferentiated thyroid cancers[END_REF][START_REF] Rosenbaum-Krumme | Effects of rosiglitazone on radioiodine negative and progressive differentiated thyroid carcinoma as assessed by (1)(2)(4)I PET/CT imaging[END_REF][START_REF] Tepmongkol | Rosiglitazone effect on radioiodine uptake in thyroid carcinoma patients with high thyroglobulin but negative total body scan: a correlation with the expression of peroxisome proliferatoractivated receptor-gamma[END_REF] suggest a more or less efficient redifferentiation effect of the PPAR agonist rosiglitazone. Kebebew [START_REF] Kebebew | Results of rosiglitazone therapy in patients with thyroglobulin-positive and radioiodinenegative advanced differentiated thyroid cancer[END_REF][START_REF] Kebebew | A phase II trial of rosiglitazone in patients with thyroglobulin-positive and radioiodine-negative differentiated thyroid cancer[END_REF] published the results of a phase II trial including 20 patients with RAIR DTC treated for 8 weeks with rosiglitazone of whom 4 showed a positive diagnostic WBS after treatment. Despite the administration of 50 to 213 mCi I131 in those 4 patients, none had complete or even partial response according to the RECIST 1.1 criteria at 3 months of followup. There was no relationship between the level of PPAR mRNA and protein expression in patients who had radioiodine uptake compared with those who did not. These last results were in contradiction with the study of [START_REF] Tepmongkol | Rosiglitazone effect on radioiodine uptake in thyroid carcinoma patients with high thyroglobulin but negative total body scan: a correlation with the expression of peroxisome proliferatoractivated receptor-gamma[END_REF]. Out of 23 patients with RAIR DTC treated for 6 weeks with rosiglitazone, 6 patients demonstrated restoration of radioiodine uptake on a therapeutic WBS after rosiglitazone. Of these, 5 had strong PPAR-positive staining in thyroid biopsies and one a weak staining. On the other hand, none of the seven patients with negative staining had positive therapeutic WBS after rosiglitazone therapy. In the study of, Rosenbaum-Krumme et al. [START_REF] Rosenbaum-Krumme | Effects of rosiglitazone on radioiodine negative and progressive differentiated thyroid carcinoma as assessed by (1)(2)(4)I PET/CT imaging[END_REF] out of 9 patients, 4 were retreated after 3 or 6 months of rosiglitazone, and of these, 3 had at best partial response using RECIST 1.1 criteria after rosiglitazone therapy. A small pilot study in 2004 [START_REF] Philips | Effect of peroxisome proliferator-activated receptor gamma agonist, rosiglitazone, on dedifferentiated thyroid cancers[END_REF] had shown less encouraging results than the trials described above, as out of 5 patients with RAIR DTC only one experienced restoration of a faint uptake of radioiodine in lung metastases after 3 months of rosiglitazone.

Pre-clinical data for potential future targets

Demethylating agents

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Hypermethylation of the promoter region of the SCLC5A5 gene encoding for the NIS have been demonstrated in several publications, both in benign and malignant thyroid tumors [START_REF] Galrao | Hypermethylation of a New Distal Sodium/Iodide Symporter (NIS) enhancer (NDE) is associated with reduced NIS expression in thyroid tumors[END_REF][START_REF] Galrao | Methylation levels of sodium-iodide symporter (NIS) promoter in benign and malignant thyroid tumors with reduced NIS expression[END_REF], Smith et al., 2007[START_REF] Stephen | DNA methylation in thyroid tumorigenesis[END_REF][START_REF] Venkataraman | Restoration of iodide uptake in dedifferentiated thyroid carcinoma: relationship to human Na+/I-symporter gene methylation status[END_REF][START_REF] Xing | Methylation of the thyroid-stimulating hormone receptor gene in epithelial thyroid tumors: a marker of malignancy and a cause of gene silencing[END_REF] (Figure 2). In PTC, specific CpG-island regions of the promoter are hypermethylated through DNA methyltransferase 1 (DNMT1) upregulation following NFB activation by BRAF V600E [START_REF] Choi | B-RafV600E inhibits sodium iodide symporter expression via regulation of DNA methyltransferase 1[END_REF]. Moreover an inverse correlation between NIS expression and the degree of methylation of some of this CpG island has been described [START_REF] Galrao | Hypermethylation of a New Distal Sodium/Iodide Symporter (NIS) enhancer (NDE) is associated with reduced NIS expression in thyroid tumors[END_REF]. However, preclinical studies analyzing the rededifferentiation effect of demethylating agents such as 5-azacytidine in human thyroid cancer cell lines had shown disappointing results without any significant effect on NIS mRNA reinduction and/or radioiodine uptake increase [START_REF] Massimino | Effect of Combined Epigenetic Treatments and Ectopic NIS Expression on Undifferentiated Thyroid Cancer Cells[END_REF][START_REF] Provenzano | Increased iodine uptake in thyroid carcinoma after treatment with sodium butyrate and decitabine (5-Aza-dC)[END_REF][START_REF] Tuncel | The comparative effects of gene modulators on thyroid-specific genes and radioiodine uptake[END_REF], unless 5-azacytidine was associated with the sodium butyrate HDACi [START_REF] Provenzano | Increased iodine uptake in thyroid carcinoma after treatment with sodium butyrate and decitabine (5-Aza-dC)[END_REF]. Only one study showed an increase in radioiodine uptake in vitro after 5-azacytidine treatment, correlated with a reduced methylation level of SCLC5A5 promoter [START_REF] Galrao | Hypermethylation of a New Distal Sodium/Iodide Symporter (NIS) enhancer (NDE) is associated with reduced NIS expression in thyroid tumors[END_REF]. To date clinical trials evaluating demethylating agent effects on RAIR DTC redifferentiation are still lacking.

NADPH oxidase 4 (NOX4)

The link between BRAF V600E mutation, reduction of NIS expression and dedifferentiation is well established. Even if a link between BRAF V600E -induced TGF production and repression of NIS, Tg and TPO has been demonstrated [START_REF] Costamagna | The functional interaction between the paired domain transcription factor Pax8 and Smad3 is involved in transforming growth factor-beta repression of the sodium/iodide symporter gene[END_REF][START_REF] Kawaguchi | Transforming growth factor-beta1 suppresses thyrotropin-induced Na+/I-symporter messenger RNA and protein levels in FRTL-5 rat thyroid cells[END_REF][START_REF] Nicolussi | TGF-beta control of rat thyroid follicular cells differentiation[END_REF][START_REF] Riesco-Eizaguirre | The oncogene BRAF V600E is associated with a high risk of recurrence and less differentiated papillary thyroid carcinoma due to the impairment of Na+/I-targeting to the membrane[END_REF], the whole underlying molecular mechanism remains largely unknown. [START_REF] Azouzi | NADPH Oxidase NOX4 Is a Critical Mediator of BRAF(V600E)-Induced Downregulation of the Sodium/Iodide Symporter in Papillary Thyroid Carcinomas[END_REF] shed light on the role of the NADPH oxidase NOX4, an enzyme specialized in reactive oxygen species production

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upregulated in thyroid cancers [START_REF] Weyemi | Intracellular expression of reactive oxygen species-generating NADPH oxidase NOX4 in normal and cancer thyroid tissues[END_REF]. Azouzi et al. demonstrated that NOX4 upregulation by the BRAF V600E -activated TGF-Smad3 pathway correlated with NIS repression and dedifferentiation in thyroid cancer cell lines, patient tissue samples and in mouse BRAF V600E -induced thyroid tumors. Remarkably NOX4 silencing by siRNA reversed BRAF V600E -induced NIS repression at the mRNA and protein levels making NOX4 a potential future therapeutic target (Figure 2).

Consistent with this data the antioxidant alpha-lipoic acid was able to increase radioiodine uptake in a human thyroid cancer derived cell line [START_REF] Choi | Alpha-lipoic acid induces sodium iodide symporter expression in TPC-1 thyroid cancer cell line[END_REF].

Bromodomain-containing protein 4 (BRD4)

BRD4 is a member of the BET (bromodomain and extra terminal) family which binds to acetylated histones promoting gene transcription and plays a role in carcinogenesis. BRD4 is up-regulated in thyroid cancer tissues and cell lines [START_REF] Gao | Inhibition of BRD4 suppresses tumor growth and enhances iodine uptake in thyroid cancer[END_REF] and its pharmacological inhibition, besides having an antiproliferative effect, restored radioiodine uptake in thyroid cancer cell lines and in a mouse model of human papillary xenograft [START_REF] Gao | Inhibition of BRD4 suppresses tumor growth and enhances iodine uptake in thyroid cancer[END_REF] (Figure 2).

Digitalis-like components

Autophagy, which facilitates the degradation of cytoplasmic components in response to stimuli such as DNA damage or hypoxia, has emerged as a key player in carcinogenesis. In thyroid cancers, autophagy has been shown to be associated with dedifferentiation and reduced clinical response to radioiodine [START_REF] Plantinga | Autophagy activity is associated with membranous sodium iodide symporter expression and clinical response to radioiodine therapy in non-medullary thyroid cancer[END_REF]. Based on this association, a systematic high-throughput screening has identified among autophagy-activating small molecules 5 digitalis-like compounds capable of restoring NIS expression and iodine uptake,

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at micromolecular concentrations, in PTC, FTC [START_REF] Tesselaar | Digitalislike Compounds Facilitate Non-Medullary Thyroid Cancer Redifferentiation through Intracellular Ca2+, FOS, and Autophagy-Dependent Pathways[END_REF] and ATC cell lines [START_REF] Tesselaar | Digitalislike Compounds Restore hNIS Expression and Iodide Uptake Capacity in Anaplastic Thyroid Cancer[END_REF] (Figure 2). The increase in transcriptional activation of NIS following up-regulation of transcription factors such as FOS or ATF3 (activating transcription factor 3) [START_REF] Tesselaar | Digitalislike Compounds Restore hNIS Expression and Iodide Uptake Capacity in Anaplastic Thyroid Cancer[END_REF][START_REF] Tesselaar | Digitalislike Compounds Facilitate Non-Medullary Thyroid Cancer Redifferentiation through Intracellular Ca2+, FOS, and Autophagy-Dependent Pathways[END_REF] after digitalis-like compound treatment may account for the redifferentiation effect. Future studies involving mouse models and addressing the issue of the narrow therapeutic index of digitalis-like compounds as well as the dosage required for thyroid cancer redifferentiation are mandatory before clinical implementation.

Modulation of NIS trafficking to the plasma membrane

The restoration of NIS expression in RAIR thyroid cancers can be insufficient as NIS targeting to the plasma membrane can be impaired (Figure 1). Interestingly, overexpression of BRAF V600E in normal rat PCCL3 thyroid cells has the same effect as TSH withdrawal from the culture medium i.e. besides decreasing NIS protein levels it also impairs NIS targeting to the plasma membrane [START_REF] Riesco-Eizaguirre | The oncogene BRAF V600E is associated with a high risk of recurrence and less differentiated papillary thyroid carcinoma due to the impairment of Na+/I-targeting to the membrane[END_REF]. Inhibition of the MAPK pathway by a MEK inhibitor partially restored NIS protein expression albeit without recovering proper localization to the plasma membrane [START_REF] Riesco-Eizaguirre | The oncogene BRAF V600E is associated with a high risk of recurrence and less differentiated papillary thyroid carcinoma due to the impairment of Na+/I-targeting to the membrane[END_REF].

Regarding the issue of the sub-cellular localization of NIS, several publications have shed light on the role of the proto-oncogene pituitary tumor transforming gene (PTTG)-binding factor (PBF) which may represent a novel therapeutic target for increasing radioiodine uptake [START_REF] Smith | Expression and function of the novel protooncogene PBF in thyroid cancer: a new target for augmenting radioiodine uptake[END_REF]. PBF, which specifically binds to PTTG, is over-expressed in thyroid cancers [START_REF] Smith | Expression and function of the novel protooncogene PBF in thyroid cancer: a new target for augmenting radioiodine uptake[END_REF]. After its phosphorylation on a tyrosine residue by the kinase Src, PBF represses iodide uptake in vitro and in murine models [START_REF] Read | Proto-oncogene PBF/PTTG1IP regulates thyroid cell growth and represses radioiodide treatment[END_REF], through 2 mechanisms: transcriptional inhibition of NIS expression [START_REF] Boelaert | PTTG and PBF repress the human sodium iodide symporter[END_REF] and the binding and redistribution of the NIS from the plasma membrane to the cytoplasm [START_REF] Smith | A novel mechanism of sodium iodide symporter repression in differentiated thyroid cancer[END_REF]. Interestingly, a pharmacological Src inhibitor was able to stimulate radioiodine

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uptake in human thyroid cancer cell lines and in human primary thyroid cells [START_REF] Smith | Manipulation of PBF/PTTG1IP phosphorylation status; a potential new therapeutic strategy for improving radioiodine uptake in thyroid and other tumors[END_REF]. Future clinical therapeutic strategies may combine drugs targeting NIS protein reinduction associated with drugs modulating NIS sub-cellular localization, more specifically to the plasma membrane where the NIS is fully active.

Moreover [START_REF] Thompson | Dimerization of the Sodium/Iodide Symporter[END_REF] recently demonstrated in vitro that NIS dimerization may be critical to its trafficking to the plasma membrane. Impaired dimerization of the NIS and its subsequent mis-localization in RAIR DTC remains a field for future investigation.

Lan et al. [START_REF] Lan | Inhibiting beta-catenin expression promotes efficiency of radioiodine treatment in aggressive follicular thyroid cancer cells probably through mediating NIS localization[END_REF] demonstrated that -catenin activation subsequent to HIF1 overexpression relocated the NIS into intracellular location near the nucleus in FTC cells. As a consequence, radioiodine uptake decreased, but -catenin knockdown restored the iodine uptake capacity and the NIS localization on the periphery of the cells. Experiments in xenograft mice tumors confirmed these data as -catenin silencing promotes the efficacy of radioiodine therapy in FTC cells overexpressing HIF-1 and -catenin.

Finally, two novel NIS interactors namely ARF4 (ADP-ribosylation) and VCP (valosincontaining protein) have been involved in NIS trafficking to the plasma membrane. ARF4 enhanced NIS trafficking to the plasma membrane while VCP governed NIS proteolysis.

ARF4 overexpression and VCP inactivation resulted in increased iodine uptake in human thyroid cancer cells. Selective VCP inhibitors promoted RAI uptake in these same models as well as in primary thyrocytes isolated from a mouse model. Interestingly VCP and ARF4 are associated with poorer survival characteristics in RAI-treated patients [START_REF] Fletcher | Targeting novel sodium iodide symporter interactors ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP) enhances radioiodine uptake[END_REF].

Modulation of NIS post-transcriptional regulation

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Few data are published on the role of microRNAs (miR), which are small interfering RNAs modulating the translation, on NIS and other thyroid differentiation genes expression.

Among the miR studied, the following ones appear to be good candidates to modulate NIS expression: miR-146b, miR let-7f-5p, miR-21 and miRNA-106a (Li et al., 2015, Wachter et al., 2018b, Riesco-Eizaguirre et al., 2015, Damanakis et al., 2016, Haghpanah et al., 2016[START_REF] Shen | Valproic acid inhibits growth, induces apoptosis, and modulates apoptosis-regulatory and differentiation gene expression in human thyroid cancer cells[END_REF]. Interestingly, in the TCGA miR-146b-5p, miR-146b-3p and miR-21 were inversely correlated with the thyroid differentiation score of the PTC studied (Cancer Genome Atlas [START_REF] Research | Integrated genomic characterization of papillary thyroid carcinoma[END_REF].

Gene therapy

Using various delivery techniques radioiodine uptake was induced in vitro in different thyroid cancer cell lines and sometimes in vivo in xenograft models by NIS gene delivery and reviewed in 2009 by Sptizweg [START_REF] Spitzweg | Gene therapy in thyroid cancer[END_REF]. All studies [START_REF] Haberkorn | 9-cis-Retinoic acid regulation of four UGT isoforms in hepatocytes from rats with various thyroid states[END_REF][START_REF] Lee | Kinetics of iodide uptake and efflux in various human thyroid cancer cells by expressing sodium iodide symporter gene via a recombinant adenovirus[END_REF][START_REF] Lin | Application of the Cre/loxP system to enhance thyroid-targeted expression of sodium/iodide symporter[END_REF][START_REF] Smit | Reestablishment of in vitro and in vivo iodide uptake by transfection of the human sodium iodide symporter (hNIS) in a hNIS defective human thyroid carcinoma cell line[END_REF] demonstrated that NIS gene therapy was able to increase radioiodine uptake but almost always along with rapid iodide efflux which, as a consequence, was not sufficient to allow the therapeutic effects of radioiodine in vivo in animal models, except in one study [START_REF] Smit | Iodide kinetics and experimental (131)I therapy in a xenotransplanted human sodium-iodide symporter-transfected human follicular thyroid carcinoma cell line[END_REF].

Other studies suggested that gene therapy with both the transcription factors TTF1 and PAX8 known to control the expression of the NIS gene as well as Tg and TPO, was able to induce radioiodine uptake in thyroid cancer cell lines and could limit radioiodine efflux, while gene therapy with the transcription factor TTF1 alone was associated with rapid iodine efflux [START_REF] Mu | Combining transfer of TTF-1 and Pax-8 gene: a potential strategy to promote radioiodine therapy of thyroid carcinoma[END_REF]. The adenovirus mediated transfer of TTF1 in thyroid cancer cell lines that also stably express the NIS rescued this rapid radioiodine efflux issue [START_REF] Furuya | Histone deacetylase inhibitors restore radioiodide uptake and retention in poorly differentiated and anaplastic thyroid cancer cells by expression of the sodium/iodide symporter thyroperoxidase and thyroglobulin[END_REF]. Pax-8 gene transfer alone in thyroid cancer cell lines seems to be sufficient to promote radioiodine [START_REF] Mu | Combining transfer of TTF-1 and Pax-8 gene: a potential strategy to promote radioiodine therapy of thyroid carcinoma[END_REF].

NIS gene transfer driven by telomerase promoters in a human thyroid cancer cell line markedly reduced cell viability after exposure to radioiodine [START_REF] Riesco-Eizaguirre | Telomerase-driven expression of the sodium iodide symporter (NIS) for in vivo radioiodide treatment of cancer: a new broad-spectrum NIS-mediated antitumor approach[END_REF].

Finally, based on EGFR overexpression in ATC, EGFR-targeted synthetic polymers was used to target NIS expression in ATC cell lines as well as in DTC cell lines [START_REF] Schmohl | Reintroducing the Sodium-Iodide Symporter to Anaplastic Thyroid Carcinoma[END_REF].

Radioiodine uptake was significantly increased in vitro and in vivo in a mouse xenograft model leading to significant reduction in tumor growth and prolonged survival in comparison with control animals.

Other potential strategies

A south Korean group recently published 3 papers describing the potential of the inverse agonist of estrogen-related receptor (ERR on the redifferentiation of thyroid tumors (Kim et al., 2019[START_REF] Singh | Inverse Agonist of Estrogen-Related Receptor gamma Enhances Sodium Iodide Symporter Function Through Mitogen-Activated Protein Kinase Signaling in Anaplastic Thyroid Cancer Cells[END_REF][START_REF] Singh | A Novel Orally Active Inverse Agonist of Estrogen-related Receptor Gamma (ERRgamma), DN200434, A Booster of NIS in Anaplastic Thyroid Cancer[END_REF]. The inverse agonist of ERR increased radioiodine uptake in ATC cell lines by inducing the expression of iodine handling genes and enhancing the membrane localization of the NIS [START_REF] Kim | Discovery of Potent, Selective, and Orally Bioavailable Estrogen-Related Receptorgamma Inverse Agonists To Restore the Sodium Iodide Symporter Function in Anaplastic Thyroid Cancer[END_REF][START_REF] Singh | Inverse Agonist of Estrogen-Related Receptor gamma Enhances Sodium Iodide Symporter Function Through Mitogen-Activated Protein Kinase Signaling in Anaplastic Thyroid Cancer Cells[END_REF][START_REF] Singh | A Novel Orally Active Inverse Agonist of Estrogen-related Receptor Gamma (ERRgamma), DN200434, A Booster of NIS in Anaplastic Thyroid Cancer[END_REF]. Surprisingly pharmacological MEK inhibitors abolished the effects of the inverse agonist of ERR on radioiodine uptake of the cells [START_REF] Singh | Inverse Agonist of Estrogen-Related Receptor gamma Enhances Sodium Iodide Symporter Function Through Mitogen-Activated Protein Kinase Signaling in Anaplastic Thyroid Cancer Cells[END_REF][START_REF] Singh | A Novel Orally Active Inverse Agonist of Estrogen-related Receptor Gamma (ERRgamma), DN200434, A Booster of NIS in Anaplastic Thyroid Cancer[END_REF].

Moreover one orally active inverse agonist of ERR has also been demonstrated in vivo in xenograft tumor mice models of ATC to increase radioiodine activity and reduction of tumor growth after radioiodine therapy [START_REF] Singh | Inverse Agonist of Estrogen-Related Receptor gamma Enhances Sodium Iodide Symporter Function Through Mitogen-Activated Protein Kinase Signaling in Anaplastic Thyroid Cancer Cells[END_REF][START_REF] Singh | A Novel Orally Active Inverse Agonist of Estrogen-related Receptor Gamma (ERRgamma), DN200434, A Booster of NIS in Anaplastic Thyroid Cancer[END_REF].

TERT (telomerase reverse transcriptase) may be another potential candidate as the mutation of its promoter are more prevalent in the less differentiated form of thyroid cancers i.e. PDTC and ATC [START_REF] Landa | Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers[END_REF] and are associated with a decrease in NIS mRNA levels in an analysis of the TCGA database [START_REF] Tavares | NIS expression in thyroid tumors, relation with prognosis clinicopathological and molecular features[END_REF] TERT promoter mutation alone is associated with a loss of radioiodine avidity and even more so when associated with BRAF V600E mutation [START_REF] Liu | The Genetic Duet of BRAF V600E and TERT Promoter Mutations Robustly Predicts the Loss of Radioiodine Avidity in Recurrent Papillary Thyroid Cancer[END_REF], Yang et al., 2017).

Lithium salts known to increase the trapping of iodide by the thyroid glands showed inconsistent and disappointing results in term of potentiation of radioiodine uptake in benign thyroid diseases and DTC in clinical series (Liu et al., 2006b).

Other miscellaneous studies have highlighted the potential of other agents as candidates for redifferentiation strategy [START_REF] Bauriaud-Mallet | The cholesterol-derived metabolite dendrogenin A functionally reprograms breast adenocarcinoma and undifferentiated thyroid cancer cells[END_REF][START_REF] Chai | HMGB1-mediated autophagy regulates sodium/iodide symporter protein degradation in thyroid cancer cells[END_REF][START_REF] Goncalves | Flavonoid rutin increases thyroid iodide uptake in rats[END_REF][START_REF] Goncalves | Rutin Scavenges Reactive Oxygen Species, Inactivates 5'-Adenosine Monophosphate-Activated Protein Kinase, and Increases Sodium-Iodide Symporter Expression in Thyroid PCCL3 Cells[END_REF], Liu et al., 2017[START_REF] Marsee | Inhibition of heat shock protein 90, a novel RET/PTC1-associated protein, increases radioiodide accumulation in thyroid cells[END_REF][START_REF] Vella | DDR1 regulates thyroid cancer cell differentiation via IGF-2/IR-A autocrine signaling loop[END_REF].

Perspectives and concluding remarks

The differentiation of follicular cells and more particularly the expression of the NIS and the iodide-metabolizing machinery is subject to very complex regulation. The data discussed in this review are promising for the redifferentiation of DTC and there is even hope for the redifferentiation of ATC. The redifferentiation of thyroid cancers offers the hope of obtaining prolonged remission or even cure of the disease, similar to what is observed for radioiodine avid metastatic thyroid cancers. However, there is no long-term follow-up in any of the trials or case reports published nor data on repeated treatment and many issues remain to be addressed to optimize the redifferentiation approach.

The strategy to adopt and the objectives to achieve remain an open question. Three different scenarios are possible:

-The first would be a redifferentiation strategy implemented in the case of a RAIR DTC with a low metastatic tumoral volume (i.e. lesions under 1 cm) slowly progressing to achieve very prolonged stability of the disease or even remission -The second scenario would be a redifferentiation strategy implemented in the case of a RAIR DTC with a significant progressive metastatic tumoral volume (i.e. multiple lesions above 1 cm) to achieve stability of the disease or even a response to the strategy synergistically with the effect of antiproliferative systemic therapies -A third scenario of optimizing radioiodine uptake in thyroid cancer has emerged in the adjuvant situation after thyroid surgery and before the first radioiodine therapy for selected high risk DTC patients who present persistent/recurrent disease in 30 to 60 % of the cases.

In all scenarios, as already mentioned the timing of redifferentiating drug administration before new radioiodine treatment is a key element for the success of radioiodine uptake restoration. The future is certainly to move towards strategies combining two or more drugs with a complementary mechanism of action i.e. drugs inhibiting the MAPK pathway output combined with drugs acting on the epigenetic regulation of the NIS and drugs that target the NIS to the plasma membrane for example. However, many other combinations could be considered. To address the side effect counterpart of these multiple combinations the challenge is to find the minimal time period required to redifferentiate tumor cells and find the most efficient combination of drugs. In this context novel data on the redifferentiation potential of the latest generation well tolerated and highly selective RET or NTRK inhibitors may emerge. To this end, recent tools such as 124 I PET/CT make a dosimetric approach possible and, when performed sequentially, could also be powerful in determining the optimal timing of drug administration before radioiodine therapy. Other clinical studies propose qualitative evaluations with iodine-123 SPECT/CT or diagnostic 131-iodine WBS and a fixed dose of 150 mCi of 131-iodine in patients with increased uptake after MEK or BRAF inhibitors with comparable efficiency. In the future, these different strategies need to be correctly evaluated with direct comparisons in trials dedicated to this in order to prove the superiority of the dosimetric approach from a clinical point of view.

The question of whether to discontinue one or more redifferentiating drugs after radioiodine treatment remains to be answered, except for the third scenario (in the adjuvant setting) in which obviously redifferentiating treatments can be stopped after the administration of radioiodine. This raises the issue of the efficacy per se of radioiodine uptake increase or restoration vs the antiproliferative effect of systemic drugs used. However, in the first scenario of a low metastatic volume (i.e. lesions under 1 cm, based on RECIST measurability criteria usually taken into account for long term systemic treatment introduction), with slow progression, the benefit over the risk ratio may be in favor of discontinuing the redifferentiating drugs. In this scenario management would involve the administration of a shot of redifferentiating drugs for a minimum period followed by radioiodine therapy and discontinuation of the drugs. In the second scenario, systemic therapies may be continued in between radioiodine sessions. It is not described whether the continuation of systemic treatments could be associated with a loss of the redifferentiating effect and whether the temporary interruption of treatments followed by their recovery could be beneficial to this end.

Regarding the selection of the best candidates for redifferentiation strategy several issues arise. Is there a need for imaging to evaluate the potential of iodine uptake and, if there is, what is the degree of this uptake to select patients for treatment with a therapeutic dose of radioiodine? What are the predictive factors of redifferentiation? A simple and reliable biochemical marker of redifferentiation is probably the increase in the plasmatic Tg level without morphological tumoral progression or even with a decrease in tumoral metastatic volume. Besides Tg level, is there a molecular profile able to predict responders to the redifferentiation strategy? Could plasma drug level monitoring be useful in improving To conclude, at the time of redifferentiation strategy, the definition of RAIR thyroid cancers should be revisited. After the administration of redifferentiating drugs, 3 scenarios could be identified:

-restored radioiodine responsive patients: an ideal situation where radioiodine uptake is increased or restored along with an objective radiological prolonged stabilization or a decrease in a progressive metastatic tumoral mass; -true radioiodine resistance: a situation where there is a radioiodine uptake increase or restoration but with a radiological progression of the metastatic tumoral mass. In this case, the molecular mechanism underlying the resistance to radioiodine remains unclear.

-Persistent non avid patients: a situation where there is failure to restore radioiodine uptake. In this case, the molecular mechanism underlying the absence of a correct restoration of NIS activity remains to be elucidated.

In conclusion, redifferentiation strategy provides hope for the major issue of long term adverse events of systemic therapies currently used for RAIR-DTC thus creating new opportunities and a new choice for metastatic patients who should be back at the center of the decision making process.

Declaration of interest:

Figures/ Table legends

  of the MAPK pathway: preclinical and clinical evidenceSeveral publications demonstrate that MAPK pathway activation is associated with dedifferentiation and in particular NIS repression (Figure2). The comprehensive characterization of 496 papillary thyroid cancers (PTC) published in 2014 by the The Cancer Genome Atlas (TCGA) Research Network highlighted that BRAF V600E -mutated PTC which had the strongest activation of the MAPK pathway showed the most dedifferentiated state i.e.

  retention of radioiodine through up-regulation of NIS, TPO and Tg expression

  in identifying responders? The identification of predictive factors of redifferentiation is a main field on the agenda for future research, especially when considering this strategy in adjuvant setting where the benefit over the risk ratio should be carefully weighed up.

Figure 1

 1 Figure 1 Differentiation and redifferentiation of thyroid cancer schematic.The left panel illustrates a (re-)differentiated state where the Sodium iodide symporter (NIS) is normally expressed and localized at the plasma membrane, allowing radioactive iodine (RAI) to be uptaken by tumoral cells. The right panel illustrates a dedifferentiated state where the expression of the SLC5A5 gene, encoding for the NIS, is reduced and/or not correctly targeted to the plasma membrane and consequently with no RAI uptake. The bottom panels illustrate the case of a patient with metastatic RAI refractory papillary thyroid cancer treated for 8 weeks with dabrafenib. The post-therapeutic WBS after dabrafenib demonstrates a clear restoration of RAI uptake in the metastatic sites (lung and neck lymphadenopathy).

Figure--

  Figure 2 Molecular mechanism controlling NIS expression and current or future actionable targets

Figure 1

 1 Figure 1 Differentiation and redifferentiation of thyroid cancer schematic. The left panel illustrates a (re-)differentiated state where the Sodium iodide symporter (NIS) is normallyexpressed and localized at the plasma membrane, allowing radioactive iodine (RAI) to be uptaken by tumoral cells. The right panel illustrates a dedifferentiated state where the expression of the SLC5A5 gene, encoding for the NIS, is reduced and/or not correctly targeted to the plasma membrane and consequently with no RAI uptake. The bottom panels illustrate a personal case of a patient with metastatic RAI refractory papillary thyroid cancer treated for 8 weeks with dabrafenib. The post-therapeutic WBS after dabrafenib demonstrates a clear restoration of RAI uptake in the metastatic sites (lung and neck lymphadenopathy).

Figure 2

 2 Figure 2 Molecular mechanism controlling NIS expression and current or future actionable targets 306x185mm (300 x 300 DPI)

  . Moreover different studies suggest that

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  The authors declare that they have no conflict of interest for this 44 ZHANG, Y., JIA, S., LIU, Y., LI, B., WANG, Z., LU, H. & ZHU, C. 2007. A clinical study of all-trans-retinoidinduced differentiation therapy of advanced thyroid cancer. Nucl Med Commun, 28, 251-5. ZHANG, Z., LIU, D., MURUGAN, A. K., LIU, Z. & XING, M. 2014. Histone deacetylation of NIS promoter underlies BRAF V600E-promoted NIS silencing in thyroid cancer. Endocr Relat Cancer, 21, 161-73.

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erc@bioscientifica.com Manuscript submitted for review to Endocrine-Related Cancer

Funding: Not funding was received for this work