| Objective:Breast cancer is the most common malignant cancer in women.In recent years,the incidence of breast cancer is increasing year by year in our country and even in the world.Although the diagnosis and treatment for breast cancer has made substantial progress,but the mortality of breast cancer is still in the forefront of female cancer.Metastasis is the leading cause of death in patients with breast cancer,and the death rate of more than 90% of patients with breast cancer is caused by metastasis,and the occurrence of distant metastasis severely limits the prognosis of patients with breast cancer.The 5 year survival rate of patients with breast cancer without metastasis is as high as 98%,while the survival rate of patients with breast cancer metastasis has dropped sharply to 23% year on year.At present,the molecular mechanism of breast cancer metastasis is not completely clear,so it will provide a theoretical basis for the diagnosis and treatment of breast cancer.The p21-activated kinases(PAKs)comprise a family of conserved serine/threonine protein kinases that were firstly identified as effectors for Rho GTPase.PAKs play crucial roles in a wide range of cell polarity,cell movement,cytoskeletal reorganization,cell cycle,gene transcription,especially cell transformation and tumor progression.Till now,mammalian PAKs family has six members,and is categorized into two subgroups based on their sequence similarities and regulatory mechanism.Group Ⅰ PAKs includes PAK1-3,and group Ⅱ PAKs includes 4-6.PAK5 is the last found member of the group Ⅱ PAKs.PAK5 is highly expressed in brain tissues and various tumor tissues.It is located in mitochondria and plays a critical role in promoting cytoskeletal reorganization,nerve growth,cell survival and other signaling pathways.PAK5 blocks the cell cycle,inhibits apoptosis,promotes tumor growth.However,the function of PAK5 in breast cancer metastasis remains unclear.Previous studies in our laboratory have found that PAK5 is highly expressed in breast cancer,which can change morphology of breast cancer cells.PAK5 can reduce the expression of E-cadherin,increase the expression of Fibronectin,and then promote the invasion and metastasis of breast cancer cells.Therefore,we hypothesized that PAK5 may play an important role in the development of breast cancer.In order to study the role of PAK5 in the process of breast cancer,we used co-immunoprecipitation assay enriching the binding proteins of PAK5 in MCF-7 cells.Then the binding proteins were analyzed by LC-ECI-MS/MS.The corresponding peptide sequence tags and identified new interacting proteins of PAK5 were obtained in the MCF-7 cells using database.DNPEP(aspartyl aminopeptidase)is a new interacting protein of PAK5,which is identified by protein immunoprecipitation and protein mass spectrometry.DNPEP is a kind of aminopeptidase,it is the unique mammalian member of the M18 family.Aminopeptidase is a key enzyme regulating signal peptide activity,it exists in all forms of life,and plays important roles in many physiological processes associated with signal peptides,such as angiogenesis,tumor growth,memory and maintenance of blood pressure,etc.It is highly conserved in evolution and is highly expressed in mammals.However,the physiological functions of DNPEP and its regulatory mechanism,especially its role in tumor progression are not fully clear.This article is based on the interaction proteins,first confirming PAK5 and DNPEP interaction in vitro and in vivo,then study the crosstalk between PAK5 and DNPEP and their functions in the growth and metastasis of breast cancer.That may provide a new clue to know the molecular mechanisms of breast cancer.Methods:1.The identification of phosphorylated sites of DNPEP by PAK5 and the confirmation of its interaction.1)To examine the interaction of PAK5 and DNPEP in vitro by GST-pull down.2)To examine the interaction of PAK5 and DNPEP in cells by Co-IP.3)In vitro kinase assay was used to detect the phosphorylation of DNPEP by PAK5.4)In vitro kinase assay was used to detect the specific phosphorylation sites of PAK5 to DNPEP.5)To generate the eukaryotic expression plasmids pcDNA3,tagged with triple Flag DNPEP SA.6)To detect the phosphorylation of DNPEP by PAK5 in breast cancer cells with Western Blot.2.Effect of PAK5-mediated phosphorylation of DNPEP on proliferation and metastasis of breast cancer cells.1)To observe the effect of CON,DNPEP WT,DNPEP SA on growth of breast cancer cells by clone formation.2)To observe the effect of CON,DNPEP WT,DNPEP SA on migration of breast cancer cells by wound healing assay.3)To observe the effect of CON,DNPEP WT,DNPEP SA on migration and invasion of breast cancer cells by transwell assay.4)To explore DNPEP whether have the ability to inhibit breast cancer cells growth and metastasis by nude mice xenograft model.3.USP4 is downstream of the PAK5-DNPEP axis.1)Protein chip technique was used to screen the interacting proteins of DNPEP.2)Overexpression of DNPEP,Western Blot was used to detect the protein expression of USP4.3)Determination of the interaction between DNPEP and USP4 in cells by Co-IP.4)The DNPEP plasmid was overexpressed in different phosphorylation states,and the expression of USP4 protein was detected by Western Blot.4.PAK5 promotes DNPEP degradation.1)Western blot was used to detect the expression of PAK5,DNPEP and USP4 in breast cancer tissues and to analyze their expression in breast cancer tissues and paired adjacent nonneoplastic tissues.2)Overexpression of PAK5,to examine the expression of DNPEP and USP4 by Western blot.3)CHX treatment and overexpression of PAK5,to examine the effects of DNPEP expression by PAK5.4)MG132 treatment and overexpression of PAK5,to examine the effects of DNPEP expression byPAK5.5)MG132 treatment and overexpression of PAK5 with different phosphorylation states,to examine the ubiquitination levels of DNPEP by Co-IP.5.High PAK5 and USP4 expression contributes to breast cancer metastasis.1)Immunohistochemical assay was used to detect the expression of PAK5 and USP4 in breast cancer tissue samples,and to analyze the correlation between them and metastasis of breast cancer.2)Survival analysis was performed on breast cancer cases with different expression levels of PAK5 and USP4.6.DNPEP interacts with and modulates CD44 abundance.1)Western blot was used to detect the expression of DNPEP and CD44 in breast cancer tissue samples,and to analyze their expression in breast cancer tissues and paired adjacent nonneoplastic tissues,and their correlation.2)To examine the interaction ofDNPEP and CD44 in cells by Co-IP.3)To observe the localization and co-localization of DNPEP and CD44 by immunofluorescence.4)Overexpression of DNPEP,to examine the expression of CD44 by Western blot.5)Knockdown DNPEP,to examine the expression of CD44 by Western blot.7.DNPEP promotes CD44 hydrolysis and is involved in breast cancer cell stemness.1)CHX treatment and overexpression of DNPEP,to examine the effects of CD44 expression by DNPEP.2)MG132 treatment and overexpression of DNPEP,to examine the effects of CD44 expression byDNPEP.3)Overexpression of DNPEP with different hydrolase activity,to examine the expression of CD44 by western Blot.4)Cells treatment with L-DOPS and overexpression of DNPEP WT,to examine the expression of CD44 by western Blot.5)Overexpression of DNPEP,Western Blot was used to detect the protein expression of stem cell indexes.6)The effects of overexpression of DNPEP on the stem cell characteristics of breast cancer were examined by microsphere formation assay.Results:1.The interaction of PAK5 and DNPEP in vitro and in cells and PAK5 phosphorylates DNPEP at S119.1)The interaction of PAK5 and DNPEP in vitro is confirmed.2)The interaction of PAK5 and DNPEP in cells is confirmed.3)In vitro kinase assay confirmed that PAK5 could phosphorylate S119 site of DNPEP.2.Phosphorylation of DNPEP inhibits proliferation and metastasis of breast cancer cells.1)DNPEP inhibit proliferationof breast cancer cells while DNPEP Ser119 A cannot.2)DNPEP inhibit metastasis of breast cancer cells while DNPEP Ser119 A cannot.3.USP4 is downstream of the PAK5-DNPEP axis.1)The proteome microarray to screen new interacting protein USP4 of DNPEP.2)The interaction of DNPEP and USP4 in cells is confirmed.3)Overexpression of DNPEP,can down-regulate the expression of USP4.4.PAK5 promotes DNPEP degradation.1)PAK5 and USP4 were highly expressed in breast cancer tissues.2)DNPEP was low expressed in breast cancer tissues.3)Overexpression of PAK5,can down-regulate the expression of DNPEP,and up-regulate the expression of USP4.4)PAK5-mediated phosphorylation of DNPEP facilitates its ubiquitination.5.High PAK5 and USP4 expression contributes to breast cancer metastasis.1)Both PAK5 and USP4 were highly expressed in breast cancer tissues with distant metastasis.2)There was a positive correlation between the expression of PAK5 and USP4 in breast cancer.3)Patients with high expression of PAK5 and USP4 had shorter survival time.6.DNPEP promotes CD44 hydrolysis and inhibits breast cancer cell stemness.1)The high expression of CD44 was negatively correlated with DNPEP in breast cancer.2)The interaction of DNPEP and CD44 in vitro is confirmed.3)The co-localization of DNPEP and CD44 in cell membranes.4)DNPEP can down-regulate the expression of CD44 through hydrolysis.5)DNPEP has inhibitory effect on stemness of breast cancer cells.Conclusions:1)DNPEP is a PAK5 physiological substrate.2)Phosphorylation of DNPEP inhibit breast cancer cellular proliferation and metastasis.3)USP4 is downstream of the PAK5-DNPEP axis.4)PAK5-mediated DNPEP phosphorylation promotes its ubiquitin-mediated degradation.5)High PAK5 and USP4 expression contributes to breast cancer metastasis.6)There was a positive correlation between the expression of PAK5 and USP4 in breast cancer.7)Patients with high expression of PAK5 and USP4 had shorter survival times.8)There was a negative correlation between the expression of CD44 and DNPEP in breast cancer.9)CD44 is the hydrolytic substrate of DNPEP.10)DNPEP can inhibit the stemness of breast cancer cells. |
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