The Mechanisms And Functions Of Asporin In Thyroid Cancer

Thyroid cancer is the most common endocrine malignancy.Over the past two decades,rapidly rising incidence rates and comparatively stable mortality for thyroid cancer have been reported throughout much of the world.Papillary thyroid cancer(PTC)accounts for 84%of thyroid cancer.Although it is controversial that the prognostic significance of lymph node metastases(LNMs)in patients age 45 years or younger,their role in patients older than age 45 years has been well defined and was positively correlated with high mortality,recurrence,and poor prognosis.Furthermore,it was demonstrated that older patients with PTC and N1b disease at presentation have poorer disease-specific survival compared with patients with NO or N1a disease.However,the underlying molecular mechanisms are still obscure.Therefore,this research has two objectives.Firstly,we aim to explore the different protein profiles and functions among PTC-N0,PTC-N1a,and PTC-N1b tumorous tissues;Secondly,Compared with their paired normal tissues,we aim to explore the different protein profiles and functions among PTC-N0,PTC-N1a,and PTC-N1b tumorous tissues,investigate the underlying molecular mechanisms and find the new biomarkers.For the first aspect of our research,through volcano plot and non-supervised principal component analysis,the protein profiles among tumorous tissues with different degrees of LNMs are distinguished,while the protein profiles in normal tissues are remarkably similar.In an in-depth investigation of the molecular mechanisms involved in PTC LNM,Gene Ontology(GO)and biological pathway enrichment analyses were performed.The majority of differentially expressed proteins(DEPs)in tumorous tissues are mostly enriched in the categories associated with pathological hallmarks of cancer,including extracellular matrix,metabolism,and cell growth,which may account for the different tumorigenicity of PTC with different degrees of lymph node metastases.For the second aspect of our research,compared with paired normal tissues,the protein profile among PTC-N0,PTC-N1a,and PTC-N lb cancer tissues was different but somewhat similar to some extent.DEPs elevated in PTC-N0,PTC-Nla,and PTC-Nlb tumorous tissues were selected by bioinformatics analysis.Furthermore,Western Blotting has performed to verify the expression of five DEPs,including Versican,PL3,SERP1NA1,CD55,and Asporin,and we found that these five DEPs increased in PTC-NO,PTC-Nla and PTC-Nlb tumorous tissues,which was consistent with our proteomics data.Besides,we found that Asporin is mainly expressed in the extracellular matrix,cell membrane,and cytoplasm in PTC tumorous tissues via performing Immunohistochemistry.To investigate the clinical significance of Asporin in PTC patients,we used the non-parametric Mann-Whitney or Kruskal-Wallis tests to examine the relationship between the Z-scores of Asporin in tumorous tissues with the clinicopathological characteristics in the TCGA THCA cohort.The high-level Z-scores of Asporin were positively associated with PTC patients with larger tumor classification,lymph node ietastasis,bilateral location,high AJCC staging,and BRAFV600E mutation.Kaplan—Meier analysis indicated that high Z-scores of Asporin in tumorous tissues were associated with worse progression-free survival and overall survival than that of patients with normal/low Z-score.Therefore,the elevated Asporin expression was positively correlated with a poorer prognosis,thus implicating Asporin as a novel candidate prognostic biomarker.The viability of BCPAP and KTC-1 cells was significantly reduced by knockdown Asporin expression in CCK8 and colony-formation assays,suggesting that Asporin can promote the proliferation and survival of PTC cancer cells.Furthermore,the migration and invasion of BCPAP and KTC-1 cells were also significantly downregulated by knockdown Asporin expression in Transwell,suggesting that Asporin play vital roles in the metastasis of thyroid cancer cells.To further investigate the underlying mechanisms,we knock down Asporin expression in BCPAP,KTC-1,and BHT101 cell lines.This reduced BRAF and p-ERK1/2 expression but not the t-ERK1/2 level.Besides,knocking down Asporin further reduced EMT-related proteins,including β-catenin,SNAIL,SLUG,ZEB1 and ZEB2,which is downstream of the MAPK signaling pathway.Furthermore,Knocking down Asporin caused cytoskeleton remodeling and cytoskeletal network damage.Using FITC-phalloidin staining,we analyzed the distribution of F-actin and found that F-actin shows cross-linked architecture in the cytoplasm in scramble cells,whereas the three-dimensional reticular architecture of actin filaments is destroyed in BCPAP and KTC-1 cells knocking down Asporin expression.These results indicating that Asporin could regulate the cytoskeleton of thyroid cancer cells through promoting the activation of the MAPK signaling pathway and upregulating its downstream EMT-related proteins,which finally results in promoting the PTC invasiveness and tumorigenicity.The endogenous co-immunoprecipitation assay revealed that Asporin coprecipitated with HER2,but not with EGFR and SRC.Furthermore,we found that the co-localization signal of Asporin and HER2 on the BCPAP and KTC-1 cell membrane and cytoplasm in the immunofluorescence assay.These results indicate that Asporin and HER2 may associate together as a complex.We knock down Asporin expression in BCPAP and KTC-1 cell lines.This reduced HER2,p-HER2Y1248,p-SRCY418,p-EGFRY845,and p-EGFRY1173 expression,but not SRC and EGFR level.These results suggested that Asporin could directly bind HER2 to maintain its expression level,which could subsequently upregulate the expression of p-EGFR and p-SRC in BCPAP and KTC-1 cell lines.Furthermore,we found that recombinant human Asporin could not promote the MAPK signaling pathway via interacting with HER2,and the MAPK signaling pathway could not upregulate the Asporin expression to create a regulatory feedback loop.Collectively,Asporin/HER2/MAPK/EMT axis could promote the migration and invasion of thyroid cancer cells,and mouse models can be performed to explore whether Asporin could become a promising therapeutic target.To investigate the clinical application of Asporin,the serological expression of Asporin in PTC-N0,PTC-N1a,and PTC-N1b was higher than that of healthy controls by performing ELISA assay.Furthermore,the areas under the curve(AUC)of Asporin for discriminating PTC patients with healthy controls were 0.73.More importantly,the AUC of Asporin for discriminating PTC-N1a and PTC-N1b patients with PTC-N0 patients was 0.84.However,to achieve "Bench-to-bedside",large-scale multicenter research still needs to be performed to investigate whether Asporin may be regarded as a serological biomarker to identify healthy controls,PTC-N0,PTC-N1a,and PTC-N1b patients.