Digoxin treatment is associated with thyroid cancer differentiation and radioactive iodide treatment response

Non-medullary thyroid cancer (NMTC) is the most frequent endocrine tumor with in most cases a good prognosis. Unfortunately, 30-40% of patients with metastatic NMTC are unresponsive to 131-I radioactive iodide (RAI) treatment as a result of tumor dedifferentiation. Autophagy has emerged as an important mechanism involved in NMTC dedifferentiation. Furthermore, activation of autophagy by cardiac glycosides such as digoxin has been demonstrated to induce effective in vitro redifferentiation of poorly differentiated and anaplastic thyroid cancer cell lines, thereby restoring sensitivity to RAI treatment. However, the in vivo effects of digoxin treatment on tumor differentiation in NMTC patients remains unclear. In the present retrospective clinical study, archived tumor material obtained from NMTC patients that received digoxin as treatment of heart disease before and after NMTC diagnosis was investigated. By a national PALGA-PHARMO database search, 11 digoxin-treated NMTC patients were included encompassing all major histological NMTC subtypes. In addition, 11 control NMTC patients never treated with digoxin were included that were matched for age, gender, histological tumor type, TNM staging and genetic profile. From the collected tumor material, autophagy activity has been determined by LC3 immunofluorescent staining and RNA expression profiles have been generated by RNA sequencing to assess differential expression of thyroid-specific genes. Interestingly, the results indicate that tumor material from digoxin-treated NMTC patients exhibit significantly higher autophagy activity as compared to tumor material of matched control NMTC patients. Moreover, in all 11 tumor tissues obtained from digoxin-treated NTMC patients the differentiation status was profoundly higher as compared to the matched control NMTC patients, of which the effect size was however dependent on histological NMTC subtypes and genetic profile of the tumor. In conclusion, treatment of NMTC with digoxin before and after NMTC diagnosis is associated with a higher tumor differentiation grade as compared to tumor tissue from closely matched NMTC patients not treated with digoxin. These in vivo data confirm our previous in vitro findings and provide accumulating evidence that digoxin could represent a beneficial adjunctive treatment modality to improve RAI sensitivity in patients with RAI-refractory thyroid carcinoma. Archived, paraffin-embedded, formalin-fixed primary tumor material obtained from 25 patients with non-medullary thyroid cancer. RNA was isolated and prepared for RNA sequencing by the NuGEN SoLo kit and Illumina NextSeq500.

非髓样甲状腺癌（Non-medullary thyroid cancer, NMTC）是最常见的内分泌肿瘤，多数患者预后良好。然而，30%~40%的转移性NMTC患者因肿瘤去分化而对131-I放射性碘（radioactive iodide, RAI）治疗无响应。自噬已被证实是参与NMTC去分化的重要机制。此外，洋地黄毒苷（digoxin）等强心苷类药物可激活自噬，在体外有效诱导低分化及间变性甲状腺癌细胞系发生再分化，进而恢复其对RAI治疗的敏感性。然而，洋地黄毒苷治疗对NMTC患者体内肿瘤分化的影响仍不明确。本回顾性临床研究对NMTC诊断前后因心脏病接受洋地黄毒苷治疗的NMTC患者的存档肿瘤组织样本进行了分析。通过全国PALGA-PHARMO数据库检索，共纳入11例接受洋地黄毒苷治疗的NMTC患者，涵盖所有主要的NMTC组织学亚型。此外，纳入11例未接受洋地黄毒苷治疗的NMTC对照患者，其年龄、性别、肿瘤组织学类型、TNM分期及遗传特征均与实验组匹配。从收集的肿瘤组织样本中，通过LC3免疫荧光染色检测自噬活性，并通过RNA测序生成RNA表达谱，以评估甲状腺特异性基因的差异表达情况。有趣的是，结果显示，与匹配的对照患者肿瘤组织相比，接受洋地黄毒苷治疗的NMTC患者的肿瘤组织自噬活性显著更高。此外，11例接受洋地黄毒苷治疗的NMTC患者的肿瘤组织分化程度均显著高于匹配的对照患者，不过该效应的大小取决于肿瘤的NMTC组织学亚型及遗传特征。综上，与未接受洋地黄毒苷治疗的匹配NMTC患者的肿瘤组织相比，在NMTC诊断前后接受洋地黄毒苷治疗的患者肿瘤分化程度更高。这些体内数据验证了我们此前的体外研究结果，并提供了更多证据表明，洋地黄毒苷可作为一种有效的辅助治疗手段，以提高RAI难治性甲状腺癌患者对RAI治疗的敏感性。本研究纳入25例非髓样甲状腺癌患者的存档福尔马林固定石蜡包埋原发性肿瘤组织样本。采用NuGEN SoLo试剂盒及Illumina NextSeq500平台完成RNA提取与RNA测序文库构建。