Study On The Mechanism Of Ubiquitin Ligase PELI1 Cooperating With Epidermal Growth Factor Receptor To Promote Breast Cancer Metastasis

Breast cancer is a frequently occurred malignancy in female.Metastasis,a representative hallmark of the development of most cancers,is the leading cause of death from breast cancer patients.Triple-negative breast cancer(TNBC)having a significantly higher rate of metastasis and mortality than other types of breast cancer.Breast cancer metastasis is a cellular process in which breast cancer cells enter the vascular system and spread to distant organs as well as colonize new tumor lesions.One of the main mechanisms is epithelial-mesenchymal transition(EMT),which further leads to drug resistance in patients during clinical treatment.EMT is a complex cellular process in which cells lose epithelial characteristics and acquire mesenchymal characteristics.It is regulated by epigenetic modification,transcriptional regulation,and protein stability regulation in cells.The regulatory mechanism of the EMT process in cells is very complex and involves multiple signaling pathways,among which the epidermal growth factor receptor EGFR and ubiquitin ligase play an important role in the regulation of EMT.The activation of EGFR mediates a variety of signal transduction pathways,which is closely related to cell proliferation,migration and invasion.However,the studies on its regulatory mechanism are still limited.The ubiquitin ligase is an indispensable part of the ubiquitin-proteasome system,which specifically promotes the binding of ubiquitin to target proteins and regulates the levels of substrate proteins.PELI1,an ubiquitin ligase,is a member of Pellino family.When PELI1 is activated by phosphorylation,it not only participates in the regulation of multiple immune signaling pathways,but also plays an important role in the deterioration and metastasis of tumors.PELI1 regulates EMT by stabilizing EMT-related proteins SNAIL and SLUG through K63-linked ubiquitination.As the research on ubiquitin ligases is more and more in-depth,therapeutic intervention based on ubiquitin ligases has attracted more attention and more small molecule inhibitors targeting ubiquitin ligases have been developed.However,studies on the molecular mechanisms underlying the abnormal activation and oncogenic effects of PELI1 in tumors are still limited.This thesis mainly focuses on the active mechanism of ubiquitin ligase PELI1 and epidermal growth factor receptor EGFR in triple-negative breast cancer metastasis,and the study on screening and activities of small molecule inhibitors of PELI1 are carried out based on this mechanism.Firstly,we investigated the interaction and regulatory mechanism between PELI1 and EGFR in breast cancer cells.Immunohistochemical staining of tumor tissue microarrays revealed that PELI1 is a pan-oncoprotein and positively correlated with the levels of EGFR in breast cancer cells.The results of survival curve analysis showed that breast cancer patients with both high levels of PELI1 and EGFR had lower survival rates.Subsequently,EGFR was identified as a potential binding protein for PELI1 by IP assay and mass spectrometry.Interaction between PELI1 and EGFR was found by exogenous as well as endogenous Co-IP experiments.PELI1 was also found to co-localize with EGFR.The truncated mutant plasmids of PELI1 and EGFR were further constructed,and the key structural domains for the interaction were found to be the FHA domain of PELI1 and the TK domain of EGFR.Further study on the regulatory mechanism found that inhibition of PELI1 in TNBC cells could accelerate the degradation of EGFR protein.PELI1 enhanced the stability of EGFR protein and promoted the recycling of EGFR to the membrane by promoting the K63-linked ubiquitination of EGFR.The activation of EGFR promoted the phosphorylation of PELI1 at Tyr154 and Thr264,which was required for PELI1 to play ubiquitin ligase activity.It was also found that EGFR promoted auto-ubiquitination of PELI1 at Lys169.The role of PELI1/EGFR in breast cancer metastasis was then under investigation.We found that the co-inhibition of PELI1/EGFR by shRNA as well as kinase inhibitors effectively inhibited the migration,invasion,and tumor sphere formation ability of breast cancer cells.Further mechanism study showed that PELI1/EGFR induced EMT in TNBC cells.Coinhibition of PELI1/EGFR was found to significantly inhibit the growth of breast cancer through a subcutaneous xenograft tumor model in mice.Co-inhibition of PELI1/EGFR was also found to significantly inhibit lung metastasis of breast cancer cells in a mouse tail vein injection tumor metastasis model.We further found that inhibition of PELI1 enhanced the sensitivity of EGFR inhibitors.Therefore,we revealed a novel regulatory mechanism by which PELI1 and EGFR cooperated to promote breast cancer metastasis in this study.Finally,the aryl thioether-like compound S62,an inhibitor of PELI1,was screened based on the mechanism of PELI1/EGFR in triple negative breast cancer and its mechanism of inhibition of breast cancer metastasis was explored.We found that compound S62 was able to interrupt the PELI1/EGFR interaction and decreased the protein levels of PELI1 and EGFR.Further studies revealed that S62 inhibited the migration,invasion as well as the EMT process of TNBC cells.It also showed that compound S62 was able to inhibit the metastasis of breast cancer in vivo experiment.In addition,the combination of S62 with EGFR inhibitor Gefitinib exhibits significant synergistic effects.Taken together,our study not only reveals the role of PELI1/EGFR interaction in the breast cancer progression,which helps to gain insight into the pathogenesis of breast cancer metastasis as well as provides clues and evidence for further discovery and development of anti-breast cancer drug targets,but also provides an effective strategy for EGFR-targeted therapy patients to overcome drug resistance.In addition,our study has identified a small molecule inhibitor S62,which provides a new lead compound for the development of targeted drugs for breast cancer.