Biological Function And Molecular Mechanism Of TROP2 In Thyroid Cancer

Background:The incidence of thyroid cancer has sharply increased globally and has become the most common malignant tumor of the endocrine system.Clinically,papillary thyroid cancer（PTC）is the most common histological subtype,accounting for up to 85%-90%of all cases.The majority of PTC exhibit indolent biology with slow progression and favorable prognosis.Nevertheless,certain PTC patients exhibit extremely aggressive characteristics at the time of diagnosis,including extrathyroidal invasion,cervical lymph node metastasis,and even distant metastases,which has a negative impact on the prognosis of PTC patients.Currently,the mechanism of the rapid progression and evolution of PTC has not been fully clarified,and there are few ways to recognize thyroid cancer’s highly aggressive characteristics prior to surgery.In order to individualize and standardize the diagnosis and treatment of thyroid cancer patients,it is crucial to identify the key molecular events of high-risk thyroid cancer and to screen out highly invasive molecular indicators.An oncogene known as trophoblast cell-surface antigen 2（TROP2）is linked to the growth,invasion,and metastasis of malignant cells.The expression of TROP2 is typically low or absent in healthy human tissues,but it is markedly elevated in a variety of solid malignant tumors.The goal of this study is to examine TROP2’s biological role and molecular mechanism in thyroid cancer and to generate novel approaches and concepts for thyroid cancer molecular detection,development,and targeted therapy.Methods:1.Bioinformatics analysis was performed to investigate the differential expression of TROP2 in patients with thyroid cancer and its relationship to clinicopathological variables.The biological mechanism of TROP2 in the development of thyroid cancer was examined by functional enrichment analysis,and a nomogram based on TROP2 expression was built to predict cervical lymph node metastasis.Also,the Timer database and the Cell Miner database examined the connections between TROP2,immune cells,and medication sensitivity.2.To confirm the differential expression of TROP2 in thyroid carcinoma and its diagnostic function in cervical lymph node metastases.Immunohistochemistry（IHC）,western blotting（WB）,and RT-qPCR were utilized.The biological role of TROP2 was discovered by using the CCK-8,EDU,colony formation,transwell proliferation and invasion assays,tumor formation in nude mice,and tail vein pulmonary metastasis tests.The downstream targeted proteins and tumor signaling pathways were identified using the Gene MANIA database,string databases,and immunoprecipitation assays.3.CCK-8 was used to examine the effects of cisplatin（DDP）therapy at various concentrations and treatment time points,as well as to determine the IC50 value of DDP in the control group and sh TROP2group.Also,the combined anti-cancer effects of TROP2 knockdown were examined.Results:1.Bioinformatics analysis indicated that TROP2 was highly expressed in thyroid cancer,which was closely related to BRAF^V600Emutation,extrathyroidal invasion,and lymph node metastasis.A nomogram for predicting cervical lymph node metastases was successfully created based on TROP2 expression levels,which showed good predictive efficacy（AUC=0.751）.The gene enrichment analysis revealed TROP2 to be involved in the PI3K-AKT and TNF signaling networks.Immune cell infiltration analysis indicated a positive correlation between TROP2 expression and immune cell expression levels,as well as immune scores.The high expression of TROP2 was closely related to the sensitivity of Nutlin-3a（r=0.393,P<0.001）,Temozolomide（r=0.355,P<0.001）and nilaparib（r=0.350,P<0.001）.2.RT-qPCR and WB results demonstrated that thyroid cancer tissues had considerably greater levels of TROP2 expression than para-cancer tissues（P<0.01）.TROP2 expression was greatest in PTC and then found in thyroid follicular adenoma,medullary thyroid cancer,follicular thyroid cancer,and anaplastic thyroid cancer.In the meantime,Immunohistochemistry TROP2 scores in PTC with cervical lymph node metastasis and metastatic lymph node tissues were significantly greater than those in PTC without lymph node metastasis and normal lymph node tissues.（P<0.05）.Functional studies showed that TROP2 knockdown greatly reduced the proliferation and invasion of thyroid cancer cells in vitro while TROP2 overexpression markedly increased these processes.TROP2 reduction led to lower tumor volume,weight,and nodule number in in vivo tumor development and tail vein pulmonary metastasis experiments（P<0.05）.The expression of CLDN4 is favorably connected with TROP2,and mechanically,TROP2 physically interacted with CLDN4.CLDN4 expression is decreased when TROP2 is downregulated,and vice versa.Additionally,thyroid cancer cell invasion and proliferation were decreased by CLDN4 knockdown.Si-CLDN4 was able to counteract the biological effects of TROP2 overexpression,which encouraged thyroid cancer cell invasion and proliferation.TROP2knockdown decreased the expression levels of p-AKT and PDK1,while the expression levels of PI3K and AKT proteins remained largely unchanged.Overexpression of TROP2 could significantly increase the expression levels of p-AKT and PDK1,which subsequently declined upon co-transfection with oe-TROP2 and si-CLDN4.3.Chemotherapy sensitivity analysis revealed that DDP considerably reduced the IC50 of DDP in thyroid cancer cells,and this impact was time-and concentration-dependent.TROP2 knockdown in conjunction with DDP significantly slowed tumor growth in vivo tests,with a concomitant downregulation of Ki67 expression and upregulation of P53expression.Conclusions:1.Bioinformatics analysis suggested that TROP2 was highly expressed in thyroid cancer and closely associated with lymph node metastasis.The nomogram based on TROP2 expression levels could effectively predict cervical lymph node metastasis.TROP2 regulated the progression of thyroid cancer through PI3K-AKT and TNF signaling pathways,as well as by altering the tumor immune microenvironment.2.TROP2 could be used as a potential biomarker for distinguishing malignant from benign thyroid nodules and identifying metastatic lymph nodes.Furthermore,TROP2 increased thyroid cancer growth and metastasis by targeting CLDN4 via the PI3K/AKT signaling pathway.3.Knockdown of TROP2 enhanced the chemo-sensitivity of thyroid cancer cells to DDP.TROP2 knockdown in conjunction with DDP could significantly inhibited the proliferation of thyroid cancer cells in vivo.