PKA Suppresses Papillary Thyroid Cancer Progression Through Hippo Pathway

Purpose:Thyroid cancer is the most common endocrine malignancy,and its incidence has increased year by year in the world over the past few decades,especially in women with thyroid cancer.PTC is the most common histopathological type of thyroid cancer,accounting for more than 90% of all thyroid malignancies.It is necessary to investigate the molecular mechanisms and genetic changes of PTC.Protein kinase A(PKA)is the most well-studied member of the serine-threonine protein kinase superfamily and is involved in the biological regulation of various cells.The main function of cAMP in eukaryotes is to activate cAMP-dependent protein kinase.PKA is involved in the development and progression of a variety of tumors,where normal thyroid cell proliferation is greatly affected by cAMP levels.cAMP and its major intracellular target protein,PKA,constitute pathways involved in a variety of cellular functions,of which regulation of cell proliferation and differentiation is of paramount importance.In addition,cAMP plays a mitogenic role in neurons and several endocrine-derived cells,and changes in cAMP downstream pathways are associated with tumorigenesis in several endocrine tumors.Therefore,whether PKA affects cell function in PTC is investigated,and it is important to study how PKA regulates the specific functions of cell biology.The Hippo signaling pathway plays an important role in tissue homeostasis,which ensures that organs develop at an appropriate size.The Yes-associated protein(YAP)transcriptional coactivator is the major effector of the Hippo pathway and is phosphorylated and inactivated by the Hippo pathway kinase LATS1/2(Large tumor suppressor 1/2).It has recently been shown that YAP activity is regulated by G protein coupled receptor signaling.Activation of Gas-coupled receptors typically leads to accumulation of cAMP,an important second messenger with multiple physiological functions,including cell proliferation and differentiation.Despite extensive research,the precise molecular mechanisms by which cAMP regulates cell proliferation and differentiation are not fully understood.In this study,we investigated the biological effects of PKA on PTC,and whether PKA affects theprogression of PTC through the Hippo signaling pathway.Method:1.A total of 132 patients with PTC who underwent surgery at the Tianjin Medical University Cancer Hospital were selected,including 40 paracancerous normal tissues.The tissue samples were stained by ICH,the expression level of p-PKA was detected,and the ICH results were scored.We analyzed the difference of p-PKA expression in PTC and paracancerous normal tissues,and discussed the relationship between the expression level of p-PKA and the clinicopathological characteristics of PTC and its clinical significance.2.The effect of PKA on proliferation,migration,apoptosis and cell cycle of PTC cells.CCK8 and clone formation experiments were performed to investigate the proliferation of PTC cells by PKA inhibitors KT5720,H89 and activator FI.Effects of scratch test on the migration of PTC cells by PKA inhibitors KT5720,H89 and activator FI;Transwell assay to investigate the effects of PKA inhibitors KT5720,H89 and activator FI on the invasion of PTC cells;Flow cytometry was conducted to investigate the effects of PKA inhibitors KT5720,H89 and activator FI on the cycle and apoptosis of PTC cells.3.PKA inhibits the progression of PTC through the Hippo signaling pathway.KTC-1was treated with PKA activator FI and DMSO for RNA-seq,and the bioinformatics analysis of RNA-seq was conducted.Realtime PCR was applied to detect the expressions of downstream target genes of YAP1,such as abcb1,ankrd,cat,ctgf,cyr6,gpatch4,imnb2 and txn.ICH staining of p-PKA and YAP1 in 132 paraffin-embedded specimens of PTC was performed to investigate the role of p-PKA in thyroid cancer localization.DMSO,PKA inhibitor and activator were added to process TPC-1 and KTC-1 cells,respectively.The changes in the levels of major proteins in the Hippo signaling pathway as well as proteins related to apoptosis and migration were detected by WB;Subsequently,DMSO,PKA inhibitor H89,KT5720 and activator FI were used to treat TPC-1 and KTC-1 of PTC cells,respectively,and immunofluorescence was used to observe the cytoplasmic localization of YAP1,so as to further verify the relationship between PKA and Hippo signaling pathway;next,use siRNA technology to knock down the key protein LATS1/2 of Hippo signalingpathway,and use FI to change PKA phosphorylation again.The level of YAP1 phosphorylation,localization and related functional changes were observed after phagocytosis;further,clone formation and Transwell experiments were used to perform cell function recovery experiments.Finally,immunodeficient mice were used as subjects to inject PTC cells into the small vein through the tail vein.Mice were housed for 2-3 weeks.The animal model was successfully used to verify whether the lung metastasis model was successfully established.DMSO and FI were administered by intraperitoneal injection for 0 days,2 days,4 days,6 days,8 days,10 days.The body weight of each group of mice in 12 days,and the in vivo size imaging technique was used to evaluate the size changes of the two groups of tumors by fluorescence brightness.The mice were sacrificed 12 days later,and the lung tissues of the mice were ICHly stained.Result:1.The results of ICH showed that p-PKA was significantly higher in paracancerous normal tissues than PTC(c2=20.803,P<0.001);the high expression rate of p-PKA in PTC was significantly lower than that in adjacent tissues;further analysis of the expression of p-PKA and the clinicopathological features of PTC.The results showed that in PTC,p-PKA was correlated with lymph node metastasis(2=8.707,p =0.003)and TNM stage(2=3.986,p =0.046),but not with age,gender,tumor size,multiple foci,and T stage(p >0.05).In the multivariate analysis,we found that the expression of p-PKA(OR 0.302;95% CI 0.136-0.675;P=0.004)and tumor size(OR 3.530;95%CI 1.555-8.015;P=0.003)is an independent prognostic factor for lymph node metastasis of PTC.2.CCK8 and colony formation experiments showed that PKA inhibitors KT5720 and H89 can promote the proliferation of PTC cells,PKA activator FI inhibits the proliferation of PTC cells;scratch assay and Transwell assay validate PKA inhibitors KT5720 and H89 can promote the migration and invasion of PTC cells.PKA activator FI inhibits the migration and invasion of PTC cells.Flow cytometry results showed that PKA activator FI induced the apoptosis of PTC cells,and could induce the G1 block of PTC cells.3.RNA-seq results show that PKA is associated with the Hippo signaling pathway,which may affect the biological function of PTC through the Hippo signaling pathway.Realtime PCR detection of downstream target genes of YAP1,a downstream target of Hippo signaling pathway,showed down-regulated expression of most downstream genes,which further confirmed the results of RNA-seq.ICH staining showed that YAP1 was located in the nucleus when p-PKA expression was low,and YAP1 was located in the cytoplasm when p-PKA expression was high.WB and immunofluorescence detection showed that PKA could affect the phosphorylation level of related proteins in the Hippo signaling pathway,and inhibit the expression of migration-related proteins and increase the expression of apoptosis-related proteins.Cloning formation and Transwell experiments for functional recovery experiments further validated the progression of PKA inhibition of PTC cells via the Hippo signaling pathway.Finally,we carried out animal experiments.Through the mouse lung metastasis model,it was found that after PKA activation,the growth of lung metastases was significantly slowed down,and the weight of the control group was significantly reduced.By ICH staining,p-PKA,p-LATS,p-YAP1,and p53 were positively expressed in the experimental group,while Vimentin was negative.The above experiments demonstrate that PKA inhibits the progression of PTC cells through the Hippo signaling pathway.Conclusion:1.The expression of p-PKA in adjacent tissues was significantly higher than that in PTC tissues;p-PKA is associated with lymph node metastasis and TNM staging in PTC;p-PKA expression and tumor size were independent prognostic factors for lymph node metastasis in PTC.2.PKA affects the proliferation,migration,invasion,apoptosis,cell cycle and other biological functions of PTC cells.3.PKA regulates the changes in biological functions of PTC through Hippo signaling pathway,thereby inhibiting the progression of PTC.