The Mechanism Of ZNF8 On The Promotion Of Breast Cancer Metastasis By TGFβ Signaling Pathway

Background:Breast cancer is one of the most common malignancies among women worldwide,and the distant metastasis is the main reason for the decreased survival rate of breast cancer patients.It can metastasize to specific organs through different mechanisms,and the main metastases sits are bone,lung and liver.However,the mechanisms of distant metastasis and organ-preferred metastasis of breast cancer are still unclear.Therefore,screening the regulatory molecules related to breast cancer metastasis and exploring its specific metastasis mechanism play an important role in determining the predictive indicators and therapeutic targets of breast cancer-specific organ metastasis.Previous studies in our laboratory found that ZNF8 could regulate the metastatic ability of breast cancer cells in vitro and in vivo through TGFβ signaling pathway.ZNF8 is a member of the KRAB zinc finger protein family,and now,its specific function and mechanism of action have not been reported.KRAB zinc finger protein family is the largest family of transcription regulators in mammals which plays an important role in the normal development of organs and tissues,as well as in the occurrence and development of tumors.However,the function of most members in this family is unknown.The TGFβ signaling pathway is one of the most important signaling pathways in the human.It can regulate various life progresses such as cell growth,proliferation,differentiation,senescence,cell migration and adhesion,and extracellular matrix synthesis and remodeling.During the occurrence and development of tumors,the selective regulation of the TGFβ pathway can affect tumor progression and metastasis in a variety of tumor cells.However,the regulation mechanism of the TGFβ pathway is still unclear.Objective:1.To confirm that ZNF8 promotes the metastasis of breast cancer by regulating the activity of TGFβ signaling pathway through interacting with Smads.2.To explore the mechanism of ZNF8 regulating the activity of TGFβ signaling pathway to promote breast cancer metastasis.Methods:Identification of high-confidence interacting proteins of Samd3 in breast cancer MDA-MB-231 cells with high metastatic potential using immunoprecipitation and mass spectrometry.The GST-pulldown assay was used to verify the direct interaction of ZNF8 with TGFβ signaling pathway molecules Smad2 and Smad3 and the methylase SMYD3 in vitro.The struncats of ZNF8,Samd2,Smad3,and SMYD3 were constructed respectively,and used to identify the domains mediated the interactions between each other by co-immunoprecipitation experiment.A C-terminal deletion truncate of ZNF8 was constructed,and used to confirm the domain of ZNF8 that is important for the promotion effect of ZNF8 on the migration of breast cancer cells by transwell assay.Co-immunoprecipitation experiments were used to examine the effect of ZNF8 on the interaction of Smad4 with Smad2 and Smad3,and SMYD3 and Smad3;The change in the interaction of ZNF8 with Smad2 and Smad3 under TGFβ treatment;and the interaction between ZNF8 and the methylase SMYD3.A nude mouse tail vein metastasis model was used to confirm the effect of ZNF8 on breast cancer MDA-MB-231 cell metastasis in vivo,and explore whether the effect of ZNF8 on breast cancer cell metastasis was dependent on TGFβ.Phosphorylated Smad2和 Smad3 antibody and western blot were used to detect the effect of ZNF8 on the phosphorylation level of Smad2 and Smad3,and the effect of ZNF8 on the nucleocytoplasmic localization of Smad2 and Smad3 in MDA-MB-231 cells was detected by nucleocytoplasmic separation assay technology.Results:1.ZNF8 was identified as a highly reliable interacting protein of Smad3 by immunoprecipitation and mass spectrometry in breast cancer MDA-MB-231 cells.2.ZNF8 directly binded to Smad2,Smad3 and the methylase SMYD3 in vitro.3.The C-terminal MH2 domains of Smad2 and Smad3 mediated their interactions with ZNF8,and the CTD domains of SMYD3 mediated the interactions with ZNF8,while the C-terminal domain of ZNF8 mediates the interactions with Smad2,Smad3 and SMYD3.4.The deletion of the C-terminal domain of ZNF8 attenuated its promotion function on the invasion and metastasis of breast cancer cells.5.TGFβ treatment enhanced the interactions of ZNF8 with and Smad2 and Smad3.6.The interactions between Smad2、Smad3 and Smad4 was reduced in the ZNF8 knockout of MDA-MB-231 cells.7.ZNF8 knockout inhibited lung tumor metastasis in mouse tail vein injection models.TGFβ promoted lung tumor metastasis of ZNF8 wild-type cells,but had no effect on the lung metastasis of ZNF8 knockout MDA-MB-cells.8.ZNF8 did not affect the phosphorylation levels of Smad2 and Smad3,and their nucleoplasmic localization.9.In HEK293T and MDA-MB-231 cells,ZNF8 interacted with SMYD3 exogenous and exogenous in vivo.10.The interaction of SMYD3 and Smad3 was reduced in the ZNF8 knockout MDA-MB-231 cells.Conclusions and significance:Taken together,ZNF8 participates in the regulation of TGFβ signaling pathway through interaction with Smad2 and Smad3,and promotes lung metastasis of breast cancer cells in a TGFβ-dependent manner.In addition,the mechanism by which ZNF8 affects TGFβ signaling pathway is not achieved by affecting the phosphorylation level of Smad2 and Smad3 and their nuclear localization.ZNF8 can enhance TGFβ signaling pathway activity by recruiting SMYD3 and increasing Smad3-SMYD3 interaction.In this study,we studied a novel regulatory factor of the TGFβ signaling pathway,which would enrich and supplement the regulatory mechanism of the TGFβ signaling pathway,and at the same time revealed a new molecular and related mechanism of breast tumor invasion and metastasis.This work would provide specific therapeutic targets and predictive indicators for the distant metastasis of breast cancer cells.