IGF2 is a potential factor in RAI-refractory differentiated thyroid cancer.

Differentiated thyroid cancer (DTC) is the most frequent endocrine tumor with a good prognosis after primary treatment in most cases. By contrast, 30-40% of patients with metastatic DTC are unresponsive to 131I radioactive iodide (RAI) treatment due to tumor dedifferentiation. Currently, underlying molecular mechanisms of dedifferentiation remain elusive and predictive biomarkers are lacking. Therefore, the present study aimed to identify molecular biomarkers in primary tumors associated with RAI refractoriness. A retrospective cohort was gathered consisting of RAI-sensitive patients with DTC and RAI-refractory patients with poorly DTC. In all patients, extensive intratumoral mutation profiling, gene fusions analysis, telomerase reverse transcriptase (TERT) promoter mutation analysis and formalin-fixed paraffin-embedded-compatible RNA sequencing were performed. Genetic analyses revealed an increased mutational load in RAI-refractory DTC, including mutations in AKT1, PTEN, TP53 and TERT promoter. Transcriptomic analyses revealed profound differential expression of insulin-like growth factor 2 (IGF2), with up to 100-fold higher expression in RAI-refractory DTC compared with in RAI-sensitive DTC cases. ELISA revealed significant lower IGF2 plasma concentrations after surgery and subsequent 131I RAI therapy in patients with DTC compared with pretreatment baseline. Overall, the current findings suggested that the tumor-promoting growth factor IGF2 may have a potential role in acquiring RAI refractoriness.

Digoxin treatment reactivates in vivo radioactive iodide uptake and correlates with favorable clinical outcome in non-medullary thyroid cancer.

PURPOSE: Non-medullary thyroid cancer (NMTC) treatment is based on the ability of thyroid follicular cells to accumulate radioactive iodide (RAI). However, in a subset of NMTC patients tumor dedifferentiation occurs, leading to RAI resistance. Digoxin has been demonstrated to restore iodide uptake capacity in vitro in poorly differentiated and anaplastic NMTC cells, termed redifferentiation. The aim of the present study was to investigate the in vivo effects of digoxin in TPO-Cre/LSL-BrafV600E mice and digoxin-treated NMTC patients. METHODS: Mice with thyroid cancer were subjected to 3D ultrasound for monitoring tumor growth and 124I PET/CT for measurement of intratumoral iodide uptake. Post-mortem analyses on tumor tissues comprised gene expression profiling and measurement of intratumoral autophagy activity. Through PALGA (Dutch Pathology Registry), archived tumor material was obtained from 11 non-anaplastic NMTC patients who were using digoxin. Clinical characteristics and tumor material of these patients were compared to 11 matched control NMTC patients never treated with digoxin. RESULTS: We found that in mice, tumor growth was inhibited and 124I accumulation was sustainably increased after short-course digoxin treatment. Post-mortem analyses revealed that digoxin treatment increased autophagy activity and enhanced expression of thyroid-specific genes in mouse tumors compared to vehicle-treated mice. Digoxin-treated NMTC patients exhibited significantly higher autophagy activity and a higher differentiation status as compared to matched control NMTC patients, and were associated with favourable clinical outcome. CONCLUSIONS: These in vivo data support the hypothesis that digoxin may represent a repositioned adjunctive treatment modality that suppresses tumor growth and improves RAI sensitivity in patients with RAI-refractory NMTC.

Digitalislike Compounds Restore hNIS Expression and Iodide Uptake Capacity in Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is a rare malignancy that accounts for 1%-2% of all thyroid cancers. ATC is one of the most aggressive human cancers, with rapid growth, tumor invasion, and development of distant metastases. The median survival is only 5 mo, and the 1-y survival is less than 20%. Moreover, as a result of severe dedifferentiation, including the loss of human sodium iodide symporter (hNIS) expression, radioactive iodide (RAI) therapy is ineffective. Recently, we have demonstrated beneficial effects of autophagy-activating digitalislike compounds (DLCs) on redifferentiation and concomitant restoration of iodide uptake in RAI-refractory papillary and follicular thyroid cancer cell lines. In the current study, the effects of DLCs on differentiation and proliferation of ATC cell lines were investigated. Methods: Autophagy activity was assessed in ATC patient tissues by immunofluorescent staining for the autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3). In addition, the effect of autophagy-activating DLCs on the proliferation, gene expression profile, and iodide uptake capacity of ATC cell lines was studied. Results: Diminished autophagy activity was observed in ATC tissues, and in vitro treatment of ATC cell lines with DLCs robustly restored hNIS and thyroglobulin expression and iodide uptake capacity. In addition, proliferation was strongly reduced by induction of cell cycle arrest and, to some extent, cell death. Mechanistically, reactivation of functional hNIS expression could be attributed to activation of the transcription factors activating transcription factor 3 and protooncogene c-fosConclusion: DLCs could represent a promising adjunctive therapy for restoring iodide avidity within the full spectrum from RAI-refractory dedifferentiated to ATC.

Pathological processes and therapeutic advances in radioiodide refractory thyroid cancer.

While in most patients with non-medullary thyroid cancer (TC), disease remission is achieved by thyroidectomy and ablation of tumor remnants by radioactive iodide (RAI), a substantial subgroup of patients with metastatic disease present tumor lesions that have acquired RAI resistance as a result of dedifferentiation. Although oncogenic mutations in BRAF, TERT promoter and TP53 are associated with an increased propensity for induction of dedifferentiation, the role of genetic and epigenetic aberrations and their effects on important intracellular signaling pathways is not yet fully elucidated. Also immune, metabolic, stemness and microRNA pathways have emerged as important determinants of TC dedifferentiation and RAI resistance. These signaling pathways have major clinical implications since their targeting could inhibit TC progression and could enable redifferentiation to restore RAI sensitivity. In this review, we discuss the current insights into the pathological processes conferring dedifferentiation and RAI resistance in TC and elaborate on novel advances in diagnostics and therapy to improve the clinical outcome of RAI-refractory TC patients.

Digitalis-like Compounds Facilitate Non-Medullary Thyroid Cancer Redifferentiation through Intracellular Ca2+, FOS, and Autophagy-Dependent Pathways.

Up to 20%-30% of patients with metastatic non-medullary thyroid cancer have persistent or recurrent disease resulting from tumor dedifferentiation. Tumor redifferentiation to restore sensitivity to radioactive iodide (RAI) therapy is considered a promising strategy to overcome RAI resistance. Autophagy has emerged as an important mechanism in cancer dedifferentiation. Here, we demonstrate the therapeutic potential of autophagy activators for redifferentiation of thyroid cancer cell lines. Five autophagy-activating compounds, all known as digitalis-like compounds, restored hNIS expression and iodide uptake in thyroid cancer cell lines. Upregulation of hNIS was mediated by intracellular Ca2+ and FOS activation. Cell proliferation was inhibited by downregulating AKT1 and by induction of autophagy and p21-dependent cell-cycle arrest. Digitalis-like compounds, also designated as cardiac glycosides for their well-characterized beneficial effects in the treatment of heart disease, could therefore represent a promising repositioned treatment modality for patients with RAI-refractory thyroid carcinoma. Mol Cancer Ther; 16(1); 169-81. ©2016 AACR.

Autophagy activity is associated with membranous sodium iodide symporter expression and clinical response to radioiodine therapy in non-medullary thyroid cancer.

Although non-medullary thyroid cancer (NMTC) generally has a good prognosis, 30-40% of patients with distant metastases develop resistance to radioactive iodine (RAI) therapy due to tumor dedifferentiation. For these patients, treatment options are limited and prognosis is poor. In the present study, expression and activity of autophagy was assessed in large sets of normal, benign and malignant tissues and was correlated with pathology, SLC5A5/hNIS (solute carrier family 5 member 5) protein expression, and with clinical response to RAI ablation therapy in NMTC patients. Fluorescent immunostaining for the autophagy marker LC3 was performed on 100 benign and 80 malignant thyroid tissues. Semiquantitative scoring was generated for both diffuse LC3-I intensity and number of LC3-II-positive puncta and was correlated with SLC5A5 protein expression and clinical parameters. Degree of diffuse LC3-I intensity and number of LC3-II-positive puncta scoring were not discriminative for benign vs. malignant thyroid lesions. Interestingly, however, in NMTC patients significant associations were observed between diffuse LC3-I intensity and LC3-II-positive puncta scoring on the one hand and clinical response to RAI therapy on the other hand (odds ratio [OR] = 3.13, 95% confidence interval [CI] =1.91-5.12, P = 0.01; OR = 5.68, 95%CI = 3.02-10.05, P = 0.002, respectively). Mechanistically, the number of LC3-II-positive puncta correlated with membranous SLC5A5 expression (OR = 7.71, 95%CI = 4.15-11.75, P<0.001), number of RAI treatments required to reach remission (P = 0.014), cumulative RAI dose (P = 0.026) and with overall remission and recurrence rates (P = 0.031). In conclusion, autophagy activity strongly correlates with clinical response of NMTC patients to RAI therapy, potentially by its capacity to maintain tumor cell differentiation and to preserve functional iodide uptake.