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

Abstract

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-Braf^V600E 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.

Keywords: Autophagy; Digoxin; Non‐medullary thyroid cancer; Radioactive iodide; Redifferentiation.

Fig. 1

Digoxin treatment inhibits growth of Braf^V600E mouse tumors. a Tumor volumes in time in TPO-Cre/LSL-Braf^V600E transgenic mice treated with either DMSO vehicle (N = 3), 20 µg digoxin (N = 6) or 60 µg digoxin daily (N = 6). Data are means ± SD, *P < 0.05 (repeated measures ANCOVA with baseline correction). b Percentages of Ki-67 positive cells in post-mortem tumor tissue of TPO-Cre/LSL-Braf^V600E transgenic mice treated with either DMSO vehicle (N = 3), 20 µg digoxin (N = 6) or 60 µg digoxin daily (N = 6). c Representative immunofluorescent images of Ki-67 expression in digoxin-treated (left) and vehicle control (right) murine tumors. Scoring results were generated in quintuple for each tissue section. Data are means ± SEM, *P < 0.05 (Mann-Whitney U test)

Fig. 2

Digoxin treatment enhances ¹²⁴I accumulation in Braf^V600E mouse tumors. Intratumoral ¹²⁴I accumulation in TPO-Cre/LSL-Braf^V600E transgenic mice treated with either DMSO vehicle (N = 3), 20 µg digoxin (N = 6) or 60 µg digoxin daily (N = 6) at baseline and at three time points after initiation of digoxin treatment. PET/CT scans were performed either 24 hours (a) or 72 hours (b) after ¹²⁴I injection at baseline and day 5, day 12, day 19 of digoxin treatment. Data are expressed as measured intratumoral ¹²⁴I activity (Bq/ml, upper panels) and as intratumoral percentage of injected ¹²⁴I activity (lower panels). Data are means ± SEM, *P < 0.05 (repeated measures ANCOVA with baseline correction). c Representative frontal PET/CT images of one vehicle and one digoxin treated mouse; dotted line represents the axial plane. Axial images are shown for ¹²⁴I uptake at baseline and day 5 after vehicle or digoxin treatment. Images were corrected for total injected ¹²⁴I activity. d Relative percentages (average and range) of ¹²⁴I accumulation 24 and 72 hours after ¹²⁴I injection in vehicle and digoxin-treated mice in relation to physiological ¹²⁴I uptake in thyroids of wild-type mice (100 % reference)

Fig. 3

Digoxin treatment enhances autophagy activity, thyroid-specific gene expression and nuclear FOS levels in Braf^V600E mouse tumors. a Quantification of the number of LC3-II positive puncta per 100 tumor cells in post-mortem tumor tissue of TPO-Cre/LSL-Braf^V600E transgenic mice treated with either DMSO vehicle (N = 3), 20 µg digoxin (N = 6) or 60 µg digoxin daily (N = 6). Scoring results were generated in quintuple for each tissue section. Data are means ± SEM, *P < 0.05 (Mann-Whitney U test). b Expression of 16 thyroid-specific genes, encompassing the Thyroid Differentiation Score (TDS), in post-mortem tumor tissue of TPO-Cre/LSL-Braf^V600E transgenic mice treated with either DMSO vehicle (N = 3), 20 µg digoxin (N = 6) or 60 µg digoxin daily (N = 6). Data are means ± SEM, *P < 0.05 (Mann-Whitney U test). c Percentage of FOS positive nuclei in post-mortem tumor tissue of TPO-Cre/LSL-Braf^V600E transgenic mice treated with either DMSO vehicle (N = 3), 20 µg digoxin (N = 6) or 60 µg digoxin daily (N = 6). d Representative immunofluorescent images of nuclear FOS expression in digoxin-treated (left) and vehicle control (right) murine tumors. Scoring results were generated in sextuple for each tissue section. Data are means ± SEM, *P < 0.05 (Mann-Whitney U test)

Fig. 4

RNA expression profiling, Ki-67 positivity, autophagy activity and nuclear FOS expression in NMTC tumor tissues. a Percentage of Ki-67 positive cells in tumor tissues of NMTC patients, stratified for histological tumor subtypes. Scoring results were generated in quintuple for each tissue section; all individual data points are plotted. *P < 0.05 (Mann-Whitney U test). b Quantification of the number of LC3-II positive puncta per 100 tumor cells. Scoring results were generated in quintuple for each tissue section; all individual data points are plotted. Results were stratified based on histological subtype. *P < 0.05 (Mann-Whitney U test). c RNA-seq-based expression analysis of thyroid-specific genes in tumor tissues of NMTC patients untreated (N = 11) or treated with digoxin at the time of NMTC diagnosis (N = 11) encompassing all 16 genes incorporated in the Thyroid Differentiation Score (TDS). d Percentage of FOS positive nuclei in tumor tissues of NMTC patients, stratified for histological tumor subtypes. Scoring results were generated in quintuple for each tissue section; all individual data points are plotted. *P < 0.05 (Mann-Whitney U test). FTC: follicular thyroid cancer; FVPTC: follicular-variant papillary thyroid cancer; HCC: Hürthle cell carcinoma; LC3-II: lipidated form of microtubule-associated protein 1A/1B light chain 3B; NMTC: Non-medullary thyroid cancer; PTC: papillary thyroid cancer;