FRMD3 Inhibits Breast Cancer By Mediating Vimentin Ubiquitination And Degradation

Breast cancer is the most prevalent cancer in the world,and its mortality rate has been the highest among female malignancies.Despite the breakthrough progress in the diagnosis and treatment of BRCA and the achievement of a high cure rate,there is still a risk of recurrence and distant metastasis,which are the main cause of breast cancer-related death.At present,it is still a great challenge to further improve the early diagnosis rate of breast cancer and prevent its recurrence and metastasis.Just like any other cancers,the development and progression of breast cancer involves the abnormal expression and dysfunction of multiple oncogenes and tumor suppressor genes.Therefore,it is of great significance to identify the key genes involved in the development and progression of breast cancer and deeply explore their mechanisms for finding more accurate diagnostic and prognostic markers and effective therapeutic targets.In our previous study,we compared the gene expression between a variety of malignant tumor tissues and their corresponding normal tissues through second-generation sequencing technology,FERM domain containing protein 3(FRMD3)was one of the most differentially expressed genes.FRMD3 is one member of the protein 4.1 superfamily,which is characterized by the presence of a conserved FERM(4.1-ezrin-radixin-moesin)domain.FERM domain is involved in binding membrane-related proteins and lipids,acting as the junction between cell membrane and cytoskeleton.FRMD3 is expressed in a variety of human tissues,but its function is unknown.The TCGA-RNAseq database shows that the expression of FRMD3 in a variety of tumor tissues is significantly lower than that in normal tissues.But so far,there is only one article reporting that it can suppress tumor probably by inducing cell apoptosis,which suggests that FRMD3 may be a tumor suppressor gene.However,there still lacks deep research on the roles of FRMD3 in the occurrence and development of malignant tumors,and its molecular mechanism remains unclear.This thesis reveals the biological roles and the underlying mechanisms of FRMD3 in the development of breast cancer,based on the fact of the significantly lower expression of FRMD3 in breast cancer cell lines and clinical tissue specimens.The specific results are shown as follows:Firstly,in combination of analyzing breast cancer RNAseq data in the TCGA database and detecting FRMD3 expression levels in breast cancer cell lines and breast cancer clinical tissue microarrays using RT-q PCR,Western blot and immunohistochemistry(IHC)staining,we confirmed that FRMD3 expression levels were significantly lower in breast cancer cell lines and tissues compared to the normal counterparts,and the positive expression rates in in situ and metastatic breast cancer tissues were significantly lower than those in paracancerous tissues.Kaplan-meier survival curve based on TCGA database showed that BRCA patients with low-expression FRMD3 had shorter survival time.These results suggest that the low expression of FRMD3 may be related to the development and progression of breast cancer,and predict the poor prognosis of breast cancer patients.Subsequently,we used lentivirus to stably overexpress FRMD3 in two breast cancer cell lines and stably knockdown FRMD3 in normal breast epithelial cells MCF10 A,to explore the effect of FRMD3 on the malignant phenotype of breast cancer from both positive and negative aspects.We conducted a series of in vitro assays to detect cell proliferation(MTT,Brd U incorporation,colony formation and soft agar cloning),cell migration(cell scratch wound-healing,Transwell migration and real-time cell tracing)and cell invasion(Transwell invasion and 3D sphere invasion)and demonstrated that FRMD3 significantly inhibited the proliferation,migration and invasion of breast cancer cells.Meanwhile,UALCAN database analysis reminded that cell adhesion was one of the main enrichment pathways of FRMD3-related genes,and then we also confirmed that FRMD3 significantly inhibited the adhesion ability of breast cancer cells by cell adhesion assay.Finally,in situ tumor-bearing of nude mice and tail vein injection experiments also proved that FRMD3 significantly inhibited the growth and metastasis of breast cancer xenograft in vivo.In addition,we demonstrated that FRMD3 significantly reduced the expression levels of PI3K/AKT/m TOR and ERK signaling proteins,adhesion complex proteins,and vimentin,but did not affect the expression of other EMT markers in breast cancer cells.At the same time,we observed that the protruding pseudopodia and focal adhesions of FRMD3 overexpressed breast cancer cells were significantly reduced by immunofluorescence staining.The above in vivo and in vitro results indicate that FRMD3 plays a suppressor role in breast cancer.At last,we reveal the molecular mechanism by which FRMD3 exerts its suppressive effect on breast cancer.Co-immunoprecipitation assay demonstrated that FRMD3 interacted with the non-helical amino head of vimentin through its N-terminal ubiquitinlike domain and mediated vimentin ubiquitination and subsequent proteasome degradation,resulting in the decrease of vimentin in breast cancer cells.Thereafter,by replenishing vimentin in FRMD3 overexpressed breast cancer cells and knockdown vimentin in FRMD3 knockdown breast epithelial cells,we finally verified that FRMD3-triggered vimentin degradation indeed mediated the inhibition of FRMD3 on the proliferation,migration,invasion and adhesion in breast cancer cells and the expression of related proteins.These results are consistent with previous studies revealing that vimentin,as a cytoskeletal intermediate filament protein,is involved in the regulation of cancer invasion,metastasis,adhesion formation and activation of PI3 K and ERK signaling pathways.We further demonstrated that FRMD3 lacking ubiquitin-related domain completely lost its anti-cancer effect,which further confirmes that FRMD3 inhibits breast cancer through the degradation of vimentin mediated by ubiquitin-proteasome pathway.In conclusion,this thesis reveals the anti-cancer effects of FRMD3,which is significantly down-regulated in breast cancer,and the underlying molecular mechanism involving the ubiquitin-proteasome degradation of vimentin.Our research suggests that FRMD3 may act as a novel prognosis biomarker and a potential therapeutic target of BRCA,especially for providing new clues for the prevention and treatment of invasion and metastasis of BRCA and it also provides a new mechanism and evidence for the posttranslational regulation of vimentin and its involvement in breast cancer progression.