Study On The Mechanism Of Hippo Pathway Inhibits Tumor Cell Migration Via Yki-Src42A-JNK Axis

With the deepening understanding of tumor migration,the control of tumor migration to inhibit the development of cancer has been paid more and more attention.At the same time,the development of cancer is closely related to the signaling pathway.By deepening the understanding of the signaling pathway,it may be possible to find the key therapeutic targets and apply them to the clinic.The Hippo signaling pathway controls organ development by regulating cell proliferation and apoptosis.The core of Hippo signaling pathway is a cascade of kinases.Upstream Hpo kinase interacts with Sav to phosphorylate and activate downstream kinase Wts,which phosphorylates transcriptional coactivator Yki and inhibits its accumulation in the nucleus.When Hippo pathway is inactivated,Yki translocates into the nucleus and drives target gene expression under the action of transcription factor Sd.The Hippo signaling pathway can regulate cell apoptosis and proliferation to control organ size,but the specific role of this pathway in the regulation of cell migration is still unclear and needs further investigation.The JNK signaling pathway belongs to an important branch of the MAPK pathway and is highly conserved from fruit flies to mammals.It is involved in many physiological processes such as cell cycle,proliferation,apoptosis and stress through a series of kinase cascades.Studies have shown that overactivation of the JNK pathway can induce cell migration.The activity of JNK signaling pathway is also considered as a marker of invasion and metastasis of tumor cells.In this paper,it is found that the inactivation of Hippo signaling pathway affects cell migration,and JNK signaling pathway is involved in the process of cell migration mediated by Hippo signaling pathway,co-regulating cell migration and participating in the occurrence and invasion of tumors.The specific experimental results obtained in this study are as follows:（1）Hippo signaling pathway regulates tumor cell migrationIn this study,a classical tumor migration model was used in Drosophila melanogaster.Knockout of tumor polar gene scrib induced cell migration at the A/P boundary using the ptc-gal4 system.In order to investigate whether Hippo pathway was involved in regulating cell migration,gene regulation of the activity of this pathway was conducted under this migration model.Silencing of Hippo signaling pathway elements hpo,wts and overexpression of Yki can promote cell migration,while knockdown of yki can save cell migration phenotypes caused by knockdown of hpo and wts.These results suggest that inhibition of Hippo pathway enhances scrib-RNAi induced cell migration in a Yki-dependent manner.（2）Yki activated JNK signaling pathway to promote tumor cell migrationPrevious studies have shown that the activation of JNK（c-Jun N-terminal kinase）signaling pathway leads to tumor cell invasion and migration,and Hippo pathway silencing promotes tumor cell migration.Does JNK signaling pathway participate in the cell migration process regulated by Hippo pathway?We used Bsk^DN mutant Drosophila melanogila to inhibit JNK pathway activity and found that JNK pathway inactivation could inhibit Hippo pathway regulated cell migration.In vivo immunofluorescence staining experiments also showed that Yki activation could upregulate the levels of JNK signaling pathway target genes MMP1 and puc-lac Z.Moreover,the level of JNK phosphorylation was also increased,indicating that Hippo pathway activated JNK signaling pathway through effect factor Yki to promote cell migration.（3）Transcription factor Sd is required for Yki to activate JNK signaling pathwayAs a transcriptional co-activator,Yki does not have the ability to bind to DNA and needs to act together with the transcription factor Sd to drive gene expression.In order to confirm that the binding of Yki to Sd is essential for the activation of JNK signaling pathway by Yki,this study created a Yki mutant with defective binding to Sd,which could not normally bind to Sd and play an activation role.The experimental results showed that after inhibiting Sd activity,Yki was unable to activate the levels of MMP1 and puc-lac Z,the target genes of JNK signaling pathway,indicating that the activation of JNK signaling by Yki also required the participation of transcription factor Sd.（4）src42A is a transcriptional target of Yki/SdCurrently,most transcription targets of Sd in Hippo signaling pathway are genes that promote cell proliferation and inhibit cell apoptosis.Does Yki activate JNK signaling pathway to mediate cell migration involve other potential transcription targets of Sd?In this study,we found that src42A is a potential transcriptional target of Yki/Sd by analyzing RNA-seq and Ch IP-seq.In order to verify whether Yki can activate src42A expression,we used src42A-lac Z reporter Drosophila to detect src42A transcription level,and made Src42A polyclonal antibody by injecting mice to detect endogenous Src42A protein level.In vivo immunofluorescence staining results showed that,Yki overexpression can activate src42A transcription and protein level expression.At the same time,it was found that src42A silencing blocked YKi-mediated cell migration phenotype and Yki activation of JNK signaling pathway.All these experimental results indicated that src42A was a transcriptional target of Yki/Sd.Src42A is necessary to regulate cell migration in the Hippo pathway and further activate the JNK signaling pathway.（5）transcription factor Sd binds to src42ATranscription factor Sd recognizes a conserved DNA sequence of HRE（Hippo Responsive Element）.We found three potential HRE sequences on src42A gene,which may be the binding site of Sd to src42A.These sequences were verified by dual luciferase reporting system,Ch IP-q PCR assay,EMSA assay and in vivo lac Z reporting factor.It was found that Yki-Sd activated src42A transcription by directly binding to the third HRE sequence of src42A gene.At the same time,CRISPR-Cas9 gene editing technology was used to delete the third HRE sequence,and the results showed that the deleted src42A was unable to respond to the activation of Yki/Sd.These experimental results indicated that src42A directly binds to the transcription factor Sd and mediates Yki to activate JNK signaling pathway to regulate cell migration.（6）Src42A inhibited Hippo pathway activityIn addition,overexpression of Src42A was found to increase the transcriptional activity of Yki.Overexpression of Src42A activates the expression of Yki target genes,while silencing of src42A reduces the level of Yki target proteins,suggesting that Src42A is a positive regulator of Yki activity,forming a feedback loop in Drosophila melanogaster.In order to reveal the mechanism of Src42A regulating Hippo pathway,genetic epistatic analysis showed that Src42A played a role in the upstream or parallel position of Wts,and Co-IP results also showed that Src42A interacted with Wts.Considering that non-receptor tyrosine kinase Src42A always acts on phosphorylation by binding substrates,this study examined whether Src42A phosphorylates Wts.The experiment showed that the addition of Src42A phosphorylates Wts,and the affinity between Wts and Mats is weakened,which alleviates the inhibition of Wts on Yki,activated Yki.Therefore,in the process that Yki promotes cell migration by regulating JNK signaling pathway through Src42A,there may also be a positive feedback loop of Src42A activating Yki,enhancing cell migration.（7）The conserved of the mechanism was verified in mammalsThese results suggest that the key effector of Hippo signaling pathway,Yki,activates the JNK signaling pathway through target factor Src42A to induce cell migration.Is this mechanism conserved in mammals?The results of more than 200 hepatocellular carcinoma（HCC）samples showed that SRC（Src42A homology）was positively correlated with YAP（Yki homology）expression.YAP can activate the expression of SRC.Tranwell experiments showed that the addition of YAP can induce the migration of hepatocellular carcinoma cell lines.The inactivation of SRC or the addition of SRC inhibitors inhibited YAP-induced hepatoma cell line migration.Therefore,YAP can also mediate tumor cell migration by regulating SRC in mammals.In conclusion,this study found that a conserved Yki/YAP-Src42A/SRC-JNK axis promotes tumor cell migration,and also offers hope for SRC inhibitors as a treatment for YAP-associated malignancies.This study not only revealed that Hippo signaling pathway regulates cell proliferation and apoptosis,but also regulates cell migration,improving the Hippo signal transduction network,and recognizing that Hippo signaling pathway and JNK signaling pathway also interact in regulating cell migration,providing potential drug targets and treatment plans for cancer metastasis therapy.