Transcriptional reprogramming using a new dual NIS agonist enhances radioiodide uptake with implications for predicting thyroid cancer recurrence

Background: New approaches are urgently needed to enhance radioiodide (RAI) ablation of aggressive thyroid cancer. Previously, we reported that valosin-containing protein inhibitors (VCPi), such as disulfiram, markedly increase sodium iodide symporter (NIS) activity to promote RAI uptake. Disulfiram inhibits NPL4 activity – a critical VCP cofactor – via its copper bound diethyldithiocarbamate metabolite Cu(DDC)2. We hence hypothesised that disulfiram and its metabolites increase RAI uptake by interfering with ER-Associated Degradation (ERAD) via a VCP/NPL4 pathway, permitting more NIS protein to be trafficked to the plasma membrane.Aims: To determine the mechanistic impact and clinical relevance of Cu(DDC)2 in RAI therapy.Methods: We utilised RNA-Seq to identify Cu(DDC)2-regulated transcriptional pathways. NIS function was monitored in wild-type BALB/c mice via Technetium-99m pertechnetate (99mTc) uptake following intravenous administration. TCGA was appraised to investigate Cu(DDC)2-gene interactions in recurrent RAI-treated papillary thyroid cancer (PTC).Results: Cu(DDC)2 increased RAI uptake in multiple thyroid cancer cell lines (mean~3.4-fold). Subsequent RNA-Seq revealed potent transcriptional changes in Cu(DDC)2-treated 8505C cells (4661 genes; P<0.05). TaqMan RTPCR confirmed induction of transcription factors with key roles in regulating NIS expression, including PAX8, in thyroid cancer cell lines and human primary thyrocytes. In support, Cu(DDC)2 was unable to induce NIS mRNA or 125I uptake when PAX8 was depleted. Importantly, induction of thyroidal 99mTc-uptake (~35%;n=6-11;3mg/kg; P<0.05) in BALB/c mice treated intraperitoneally with Cu(DDC)2 was associated with increasing PAX8 mRNA (1.4-fold; P<0.01). Additionally, Cu(DDC)2 retained activity in the absence of NPL4 but not VCP in thyroid cancer cells and primary thyrocytes. LASSO regression analysis using TCGA further identified a 22-gene riskscore classifier based on Cu(DDC)2-associated transcription factors, which showed a significantly worse prognosis in RAI-treated PTC [Hazard Ratio=11.6;95%CI 5.8-23.31; P<0.001;n=256].Conclusions: Our work demonstrates that a new dual NIS agonist targets transcriptional and VCP pathways to enhance RAI uptake, with clinical relevance in impacting therapy and patient stratification for predicting recurrence

Transcriptional reprogramming using a new dual NIS agonist enhances radioiodide uptake with implications for predicting thyroid cancer recurrence

Background: New approaches are urgently needed to enhance radioiodide (RAI) ablation of aggressive thyroid cancer. Previously, we reported that valosin-containing protein inhibitors (VCPi), such as disulfiram, markedly increase sodium iodide symporter (NIS) activity to promote RAI uptake. Disulfiram inhibits NPL4 activity – a critical VCP cofactor – via its copper bound diethyldithiocarbamate metabolite Cu(DDC)2. We hence hypothesised that disulfiram and its metabolites increase RAI uptake by interfering with ER-Associated Degradation (ERAD) via a VCP/NPL4 pathway, permitting more NIS protein to be trafficked to the plasma membrane.Aims: To determine the mechanistic impact and clinical relevance of Cu(DDC)2 in RAI therapy.Methods: We utilised RNA-Seq to identify Cu(DDC)2-regulated transcriptional pathways. NIS function was monitored in wild-type BALB/c mice via Technetium-99m pertechnetate (99mTc) uptake following intravenous administration. TCGA was appraised to investigate Cu(DDC)2-gene interactions in recurrent RAI-treated papillary thyroid cancer (PTC).Results: Cu(DDC)2 increased RAI uptake in multiple thyroid cancer cell lines (mean~3.4-fold). Subsequent RNA-Seq revealed potent transcriptional changes in Cu(DDC)2-treated 8505C cells (4661 genes; P<0.05). TaqMan RTPCR confirmed induction of transcription factors with key roles in regulating NIS expression, including PAX8, in thyroid cancer cell lines and human primary thyrocytes. In support, Cu(DDC)2 was unable to induce NIS mRNA or 125I uptake when PAX8 was depleted. Importantly, induction of thyroidal 99mTc-uptake (~35%;n=6-11;3mg/kg; P<0.05) in BALB/c mice treated intraperitoneally with Cu(DDC)2 was associated with increasing PAX8 mRNA (1.4-fold; P<0.01). Additionally, Cu(DDC)2 retained activity in the absence of NPL4 but not VCP in thyroid cancer cells and primary thyrocytes. LASSO regression analysis using TCGA further identified a 22-gene riskscore classifier based on Cu(DDC)2-associated transcription factors, which showed a significantly worse prognosis in RAI-treated PTC [Hazard Ratio=11.6;95%CI 5.8-23.31; P<0.001;n=256].Conclusions: Our work demonstrates that a new dual NIS agonist targets transcriptional and VCP pathways to enhance RAI uptake, with clinical relevance in impacting therapy and patient stratification for predicting recurrence