Blockage Of Ciliary Assembly And Hedgehog Signaling Increased The Chemosensitivity To The BRAF Inhibitor In Papillary Thyroid Carcinoma

Thyroid cancer is the most common endocrine tumor,differentiated thyroid cancer include thyroid papillary carcinoma（Papillary thyroid carcinoma,PTC）and thyroid follicular carcinoma.Drug resistance（verofinil,a specific BRAF inhibitor）and refractory papillary thyroid carcinoma are important causes of thyroid cancer-related deaths,although the majority of patients with papillary thyroid carcinoma have a good prognosis after surgery plus radioactive iodine and regular review.Targeting Hedgehog signaling may enhance the sensitivity of thyroid cancer cells to BRAF inhibitors,which activate Hedgehog signaling pathway to produce therapeutic resistance.Meanwhile,the components of the Hedgehog signaling pathway are all located on the primary cilia,and it is not clear whether the activation of the Hedgehog signaling pathway by BRAF inhibitors depends on the completeness of primary cilia structure.Object:To clarify whether GLI1 inhibitor（HPI-4）can enhance the sensitivity of PTC cells to BRAF inhibitor（PLX7904）.Whether the activation of Hedgehog signaling pathway by PLX7904 depends on primary ciliary assembly.Whether the use of the ciliary assembly inhibitor（Nocodazole）increases the sensitivity of PTC to PLX7904.Methods:1.The viability of thyroid papillary cancer cells was detected by MTT,the IC₅₀ was calculated and the appropriate drug concentrations of PLX7904,HPI-4 and Nocodazole were selected.2.Western blotting was used to detect the expression of GLI1 protein in thyroid papillary carcinoma cell lines after adding PLX7904,the combination of PLX7904 and HPI-4 and the combination of PLX7904 and Nocodazole.3.The assembly of primary cilia was detected by immunofluorescence after the addition of PLX7904.4.The correlation between the expression of GLI1protein and clinicopathological features in thyroid papillary carcinoma was analyzed,and the survival analysis was carried out.5.The effects of PLX7904combined with HPI-4 on the proliferation,migration and invasion of thyroid papillary carcinoma cells were studied by MTT,plate cloning test,scratch test,Transwell migration and invasion test.6.The effect of PLX7904 combined with HPI-4 on apoptosis of PTC cells was detected by flow cytometry.7.Immunofluorescence was used to detect the effect of PLX7904 combined with HPI-4 or Nocodazole on primary ciliated assembly of PTC cells.Results:1.The IC₅₀values of PLX7904,HPI-4 and Nocodazole in KTC-1cells were 1.3μM,33.3μM and 0.07μM.The IC₅₀ values of TPC-1 cells were9.8μM,21.1μM and 0.07μM.2.BRAF inhibitor PLX7904 promotes GLI1expression and primary cilia assembly.3.GLI1 inhibitor HPI-4 increased the effect of BRAF inhibitor PLX7904 on promoting apoptosis,inhibiting proliferation and blocking cell cycle.4.HPI-4 enhanced the inhibitory effect of PLX7904 on the migration and invasion of PTC cells.5.HPI-4 enhanced the inhibitory effect of PLX7904 on ERK signaling pathway in PTC cells.6.HPI-4inhibited the promoting effect of PLX7904 on primary ciliary assembly of PTC cells.7.Partial reversal of the ciliary assembly inhibitor Nocodazole upregulation of GLI1 by BRAF inhibitor.Conclusion:Activation of Hedgehog signaling pathway is the underlying molecular mechanism leading to chemotherapy resistance of BRAF inhibitors.Activation of the Hedgehog signaling pathway depends on assembly of primary cilia.Targeting Hedgehog signaling and inhibiting assembly of primary cilia is a novel therapeutic strategy for increasing sensitivity to BRAF inhibitors in thyroid papillary carcinoma cells.