Targeting Therapy For Differentiated Thyroid Cancer

Part One:In vitro and in vivo anti-tumour effects of integrin antagonist in papillary thyroid cancerBackground:Patients with papillary thyroid cancer?PTC?generally have good prognosis,but the disease recurrence and metastasisisnot rare.Despite the effectiveness of radioactive iodine?RAI?therapy in most PTC patients,some patients showa poor response towards RAI therapy.Therefore,it is very necessary to explore other targeted therapies for these patients.Considering the crucial role of integrin in tumour activities,in this study we applied the integrin antagonist cilengitide in PTC and elucidated its antitumour effect in PTC cell lines and animal model.Methods:?v?3 and?v?5 integrin,the targets of cilengitide,were first determined with fluorescence-activated cell sorting?FACS?and immunofluorescence assay.Cell proliferation was examined by Cell Counting Kit-8 assay,and apoptosis was measured by FACS.Cell migration and invasion were determined with Transwell assays.Subcutaneous nude mouse models were used to evaluate the in vivo effect of clilengitide.Tumour volumes were recorded to monitor tumour growth while CD34staining and TUNEL staining were respectively applied to detect tumour angiogenesis and cell apoptosis at the histology level.Results:We observed that all PTC cell lines examined expressed both integrin?v?3and?v?5 and cilengitide was able to induce cell detachment and reduce proliferation?P?0.05?.Cell apoptosis was induced by cilengitide while TPC1 showed the most obvious cell apoptosis as compared to K1 and BCPAP cell line?37.8%vs 6.67%and9.43%,P?0.05?.The transwell assays revealed that a significant inhibition of migration and invasion was induced by cilengitide in three PTC cell lines?P?0.05?.In subcutaneous nude mouse models,a daily dosage of 50mg/kg cilengitide did not inhibit tumour growth in a period of two weeks.However,the immunostaining of tumour tissues showed a significant decrease of functional tumour vessels and increased numbers of apoptosis cells?P?0.05?.Conclusion:Cilengitide inhibited cell proliferation,migration and invasion,caused cellular detachment and apoptosis in PTC cell lines.Angiogenesis of subcutaneous tumour was decreased after the treatment of cilengitide,but such an anti-angiogenesis effect was not reflected in inhibition of tumour growth which was probably attributed to the relatively short monitoring time.The available data in this study provided us first-hand information of integrin inhibition therapy in PTC.Part Two:Comparison of ¹⁸F-AIF-NOTA-PRGD2 and ¹⁸F-FDG uptake in lymph node metastasis of differentiated thyroid cancerObject:A widespread application of integrin?_v?₃ imaging has been emerging in both pre-clinical and clinical studies.But few studies reported its value as compared with¹⁸F-FDG PET,especially for differentiated thyroid cancer?DTC?.In this study,we compared the tracer uptake of ¹⁸F-AIF-NOTA-PRGD2 and ¹⁸F-FDG in lymph node metastasis of DTC to evaluate ¹⁸F-AIF-NOTA-PRGD2 as compared with ¹⁸F-FDG.Methods:20 DTC patients with presumptive lymph node metastasis were examined with ¹⁸F-AIF-NOTA-PRGD2 and ¹⁸F-FDG PET/CT.16 patients undergone fine needle aspiration biopsy?FNAB?were evaluated by cytology results.For lesions without FNAB,the findings of clinical staging procedures served as the standard of reference?including neck ultrasound and serum thyroglobulin?.A maximum number of 4 lesions per patient was chosen for further analysis to avoid a bias by patients with an exceptionally high number of lesions?n>4?.The mean standardized uptake value?SUV?was calculated for further analysis.Results:A total of 39 presumptive lymph node metastases were visualized on PET/CT images.35 lesions were confirmed as malignant by FNAB and other clinical findings.The mean ¹⁸F-AIF-NOTA-PRGD2 in radioactive iodine-refractory?RAIR?lesions and benign lesions were 2.5±0.9 and 2.8±0.9 respectively.The mean SUV for¹⁸F-FDG in all malignant lesions was 4.5±1.6 while in benign lesions it was 3.3±1.2.For all malignant lesions,the mean SUV for ¹⁸F-FDG was significantly higher than that for ¹⁸F-AIF-NOTA-PRGD2?P<0.05?.No significant correlation was found between the SUVs of ¹⁸F-AIF-NOTA-PRGD2 and ¹⁸F-FDG for 35 lesions?r=0.114,P=0.515?.Moreover,there were 25 lymph nodes of which the diameter larger than1cm.No statistically difference of mean SUVs has been found between the lesions larger than 1cm and lesions smaller than 1cm.However,15 lesions of which the diameter larger than 1.5cm have higher ¹⁸F-AIF-NOTA-PRGD2 uptake as compared with the lesions smaller than 1.5cm.Conclusion:Although most lymph node metastases of DTC showed abnormal uptake of ¹⁸F-AIF-NOTA-PRGD2,its diagnosis value was inferior to ¹⁸F-FDG.No correlation was found between the uptake of ¹⁸F-AIF-NOTA-PRGD2 and ¹⁸F-FDG,which may suggest the two tracers provide complementary information in DTC lesions.Part Three:Robust Thyroid Gene Expression and Radioiodine Uptake Induced by Simultaneous Suppression of BRAF V600E and Histone Deacetylase in Thyroid Cancer CellsContext:Use of BRAFV600E inhibitors to restorethyroid iodide-handing gene expression and radioactive iodine?RAI?avidity is an attractive therapeutic strategyforRAI-refractory thyroid cancer,but recent initial clinical responseswere modest.Given histone deacetylation at the sodium/iodide symporter?NIS?promoter by histone deacetylase?HDAC?as a mechanism,simultaneously targetingBRAFV600E and HDAC could be a more effectivestrategy.Objectives:To test if suppressing both BRAFV600Eand HDAC could more effectively induce thyroid gene expression and RAI uptake in thyroid cancer cells.Research Design:We testedthe BRAFV600Einhibitor PLX4032?Vemurafenib?and the HDAC inhibitor SAHA?Vorinostat?,two major anticancer drugs currently approved for clinical use,in inducingthyroid gene expression and RAI uptake in thyroid cancer cells.Results:PLX4032 alone induced a modest expression of thyroid genes and RAI uptake preferentially in thyroid cancer cells harboringBRAFV600E.SAHA showed an effect in a genetic-independent manner in all the cells.A robust synergistic effect on thyroid gene expression and RAI uptake was observed in BRAFV600E-positive thyroid cancer cells when the two inhibitors were simultaneously used.This was dramatically enhanced further by thyroid-stimulating hormone?TSH?;triple combination of PLX4032,SAHA and TSH showed the most robust effect on thyroid gene expression and RAI uptake in cells harboring BRAF V600E.Abundant NIS protein expression in thyroid cancer cells under these conditions was confirmed by immunofluorescent microscopy.Conclusions:Simultaneously suppressing BRAFV600E and HDAC,particularly when co-treated with TSH,induced a far more robust expression of thyroid genes and RAI uptake in thyroid cancer cells than suppressing BRAF V600E alone.Triple combination of PLX4032,SAHA,and TSH is a specific robust regimen to restore RAI avidity in RAI-refractoryBRAFV600E-positive thyroid cancer,which warrants clinical trials to confirm.