| BackgroundBreast cancer is the most popular malignancy in female,while ERα positive type breast cancer accounts for about 70%.The over-activation of ERα signaling is regarded as the major driver for Luminal type breast cancer,which could be effective controlled via selective estrogen receptor modulators（SERM）,such as tamoxifen.The endocrine resistance is still a challenge for breast cancer treatment,while recently studies implicate the post-translational modification on ERα play important roles in endocrine resistance.The ubiquitination of ERαprotein is subject to a balance between ubiquitin ligases and deubiquitinases.Through deubiquitinases siRNA library screening,we discover PSMD14 as a critical deubiquitinase for ERα signaling and breast cancer progression.PSMD14 was firstly discovered as the component of 26 S proteasome non-ATP subunit,which also belongs to JAMM metalloprotease family.PSMD14 is elevated in breast cancer and relates to poor survival in Luminal type breast cancer.PSMD14 depletion inhibits breast cancer cell progression and estrogen signaling activity in vitro and in vivo.PSMD14 could associate with ERα at the Cterminus and enhance ERα stability possibly via inhibition K48-linked poly-ubiquitination on ERα.In conclusion,our results suggest a novel post-translational regulation of PSMD14 on estrogen signaling and breast cancer progression,which might uncover a promising target for Luminal type breast cancer therapeutics.ObjectiveThis project aims to clarify the role and molecular mechanism of PSMD14 in the progression of ER positive breast cancer,and to provide new drug targets and therapeutic strategies for the diagnosis and treatment of modern ER positive breast cancer.Methods1、 The siRNA screening using the DUBs siRNA library the study was to identifying novel DUBs in regulation estrogen signaling.2、 We use cell scratch assay,clone formation assay and CCK8 assay to investigate the phenotypic regulation of PSMD14 protein in estrogen receptor-positive breast cancer cells.Construction of a mouse transplantation tumor model assay is uesd to investigate the effect of PSMD14 on the proliferation of ER-positive breast cancer in vivo.sh PSMD14 stable transitional cell line was established in ER-positive breast cancer cells and the detection of tumor volume and weight in different groups of mice.3、The RNA-Seq to detect Analysis of novel regulatory mechanisms of PSMD14 protein in breast cancer4、Using q RT-PCR and Western blot to explore the molecular mechanism of PSMD14 protein regulating estrogen receptor signaling pathway.5、Observe the expression of PSMD14 in the tissues of ER-positive breast cancer patients by querying TCGA and Kmplot database.6、Using immunofluorescence staining and protein immunoprecipitation techniques to observe whether PSMD14 and ERα protein co-localize and interact with each other respectively.7、The MG132 assay,CHX assay and ubiquitination assay were used to verify through which modification pathway PSMD14 stabilizes ERα protein.Results1、 PSMD14 was identified as a candidate DUB of ERα,which regulate the expression of TFF1,a classical target gene of ERα,by screening in a deubiquitinating enzyme siRNA library.2、 Through endogenous and exogenous assays,we found that silencing PSMD14 could inhibit ER-positive breast cancer cells proliferation.3、 Using the TCGA database,we found that PSMD14 was increased in all subtypes of breast cancer samples and PSMD14 was positively correlated with the expression level of ERα.By RNA seq,we found that PSMD14 positively correlated with several ERα target genes,such as GREB1,IL20 and TFF1.Through the Kmplot database showed that PSMD14 correlated with poor survival in all breast cancer patients.4、 Using q RT-PCR and Western blot assay,we demonstrated that silencing PSMD14 could reduce ERα protein level,but had no significant effect on ERα m RNA level.Further studies revealed transcript levels of estrogen signaling pathway target genes have been reduced after silencing PSMD14.5、 By immunofluorescence assay and Co-IP assay indicated that PSMD14 and ERαco-localize spatially and have direct interaction.Further studies revealed that the 1-172 fragment of PSMD14 could interact with the AF1 fragment of ERα.6、 By CHX assay,we found that PSMD14 could affect the half-life of ERα.The MG132 and ubiquitin assays further demonstrated that PSMD14 ultimately affects the stability of ERα protein through the ubiquitin proteasome pathway.7、Through ubiquitination assay,we found that PSMD4 overexpression could inhibit ERα protein ubiquitination,and further study found that PSMD14 modified ERα protein polyubiquitination site is K48 site,and then through domain IP experiment we concluded that PSMD14 inhibited ERα protein K48 site through 1-172 fragment,and stabilizing the expression of ERα protein.ConclusionsThis article reveals the mechanism of PSMD14,a member of the JAMM family of deubiquitinating enzymes,regulates the estrogen signaling pathway in ER-positive breast cancer.PSMD14 directly interacts with ERα,the core factor of the estrogen signaling pathway,and promotes the ubiquitinated degradation of ERα protein,inhibits the expression of ERα downstream target genes,and suppresses the invasion and migration of ER-positive breast cancer cells by regulating the estrogen signaling pathway. |
