Role Of The Shh Pathway In Thyroid Cancer Self-renewal And Drug Resistance

The thyroid gland secretes thyroxine and calcitonin and plays an important role in maintaining energy equilibrium and body development.Due to the lifestyle change and ageing,thyroid diseases are increasing in both pets and humans.Among them,thyroid cancer is one of the most common tumors of the endocrine system,with the incidence rate steadily increasing annually.Conventional treatments such as surgery,radioactive iodine and thyroid hormone therapy are effective in most patients but not effective for undifferentiated or anaplastic thyroid cancer.Papillary thyroid cancer accounts for almost 80%of all thyroid cases.The BRAF gene is mutated approximately 40-60%of papillary and undifferentiated thyroid cancer.Although B-Raf kinase inhibitor such as PLX4032 is effective BRAF-mutated melanomas,it has little effect on thyroid cancer.The molecular mechanism underlying this remains to be further studied.Recent studies have shown that undifferentiated thyroid cancer contains a significantly higher number of cancer stem cells than those in well-differentiated thyroid cancer.Although it is well known that cancer stem cells play a key role in tumor metastasis,recurrence,drug resistance and anti-radiation therapy,whether B-Raf kinase inhibitor promotes the self-renewal of thyroid cancer stem cells to develop drug resistance has not been reported yet.The Shh signaling pathway plays an important role in cell proliferation and organogenesis.The cell surface receptor Patched(PTCH)binds one of its ligands(Sonic,India,deserted hedgehog),resulting in the activation of another transmembrane receptor Smoothened(SMO)and the downstream Glil transcription factor.Shh signaling pathway is highly activated in a variety of tumors,including thyroid cancer,and promotes tumor cell growth,invasion,and tumor metastasis.The previous work of our laboratory showed that the Shh signaling pathway can regulate the self-renewal of thyroid cancer stem cells and reduce the sensitivity of tumor cells to radiation killing,but the molecular mechanism is still unclear.This thesis employs in vitro cell culture and mouse models to study the molecular mechanism by which Shh signaling pathway regulates the self-renewal of thyroid cancer stem cells;to study how tumor cells develop drug resistance to B-Raf kinase inhibitors in thyroid tumors;to study whether novel combination therapy has synergistical effect on thyroid cancer.1.Mechanisms by which Shh signaling pathway regulates thyroid cancer stem cellsWe first investigated whether Shh signaling pathway inhibitors affect the expression of two stem cell-related genes(BMI1 and SOX2).We found that:1)two inhibitors of the Shh pathway.GANT61 and Cyclopamine(CP),inhibited the expression of BMI1 and SOX2 in undifferentiated thyroid cancer cell lines KAT-18 and SW1736;2)silencing Shh and Glil by siRNA led to the down-regulation of BMI1 and SOX2;3)Glil and Snail overexpression increased the expression of BMI1 and SOX2;4)GANT61 and CP down-regulated the promoter activity and mRNA level of Glil and SOX2,but had no effect on the promoter activity and mRNA level of BMI1;5)The expression levels of Glil,BMI1 and SOX2 were increased in ALDH-positive cells;6)Immunohistochemical analysis of clinical thyroid tumor samples revealed that BMI1 and SOX2 were highly expressed in papillary thyroid carcinoma and undifferentiated carcinoma,and correlated with the expression of Gli1.These results indicate that the Shh signaling pathway promotes the self-renewal of cancer stem cells by up-regulating BMI1 and SOX2.In vivo experiments using severely immunodeficient NCG mice show that:1)ALDH-positive cells have a significantly higher tumorigenic potential than unsorted tumor cells;2)GANT61 inhibits the occurrence and development of tumors in a mouse model of thyroid tumor stem cells;3)GANT61 treatment reduced the expression of Glil,ALDH,BMI1 and SOX2 in tumor tissue;4)GANT61 treatment of mice reduced the proportion of stem cells in tumor tissue.These results indicate that the Shh signaling pathway promotes self-renewal and tumorigenesis of thyroid cancer stem cells.2.B-RAF kinase inhibitor-mediated drug resistance in thyroid cancerWe further investigated whether the Shh signaling pathway is involved in the drug resistance of B-Raf kinase inhibitors in thyroid tumors.In vitro experiments were carried out with BRAF gene mutated thyroid cancer cells SW1736 and 8505C,papillary thyroid tumor cells WR082,and melanoma cells A375 cells.The cells were treated with B-Raf kinase inhibitor PLX4032.The results showed that PLX4032 could feedback activation of MAPK and MAPK in thyroid tumor cells.PI-3K signaling pathway,and increase the expression of genes related to tumor metastasis;PLX4032 increases the expression of genes related to stem cells;PLX4032 can increase the rate of ALDH-positive cells in thyroid tumor cells and the number of tumor microspheres;Add Her3 and AKT inhibitor Lapatinib And MK-2206 down-regulated the expression of genes related to cancer stem cells,reduced the rate of ALDH-positive cells,and reduced the number of tumor microspheres;at the same time,Lapatinib and MK-2206 reduced the promoter activity and mRNA levels of Glil and SOX2,but not The promoter activity of BMI1 has no effect on the mRNA level;the co-action with PLX4032 can reduce the increase caused by it;the co-action with PLX4032 and GANT61 can significantly reduce the expression of tumor stem cell-related genes.In vivo experiments used immunodeficient NOD/SCID mice and BALB/c nude mice as models.The results showed that:PLX4032 activates PI-3K signaling pathway in vivo and increases the expression of stem cell-related proteins;PLX4032 increases ALDH-positive cells in tumors Rate:The combination of PLX4032 and GANT61 significantly inhibited tumor growth.These results indicate that B-Raf kinase inhibitors exert their drug resistance by promoting the self-renewal of thyroid cancer stem cells,and their combined use with Shh signaling pathway inhibitors can overcome their negative effects in the treatment of thyroid tumors.In summary,we found that Shh signaling pathway promotes the self-renewal of tumor stem cells by inducing the expression of SOX2 and BMI1 in thyroid cancer;Shh signaling pathway inhibitors can inhibit tumor growth driven by thyroid CSCs.B-Raf kinase inhibitor PLX4032 activates the MAPK or PI-3K pathway through Her3,cross-activates the expression of Gli1 transcription factor,promotes self-renewal of thyroid tumor cells,and exerts drug resistance.The combination of PLX4032 and Gli1 inhibitor GANT61 has a synergistic anti-tumor effect.This study provides novel insights into the molecular mechanism of Shh signaling pathway-mediated regulation of thyroid cancer stem cell self-renewal and shed lights on the role of this pathway in targeted therapy of thyroid cancer.