PAK5-mediated AIF Phosphorylation Inhibits Its Nuclear Translocation And Promotes Breast Cancer Cell Proliferation

Objective: Breast cancer is the most commonly diagnosed cancer in women,ranking second among cancer-related death causes in women.More and more evidence showed that with the continuous progress of modern surgical technology and the continuous innovation of chemotherapy drugs,the prevention and treatment of breast cancer had made significant progress,significantly reducing the mortality of breast cancer in developed regions of the world.But for advanced breast cancer,the prognosis of patients is still very poor,and the treatment is also limited.The development of early diagnostic indicators and new therapies will require a deeper understanding of cancer progression.Therefore,it is particularly important to explore the molecular mechanism and therapeutic targets of breast cancer occurrence and development for the treatment of breast cancer patients.P21 activated kinases(PAKs)are a family of serine / threonine protein kinases,which are composed of six members.According to different biochemical and structural characteristics,they are divided into two categories,each with three members.The first group included PAK1,PAK2 and PAK3,and the second group included PAK4,PAK5 and PAK6.Among the three members of the second group,PAK5(also known as PAK7)is a newly discovered PAK family member,which plays an indispensable role in the regulation of cytoskeletal microtubule stability and cell survival.More and more evidences showed that PAK5 was a carcinogenic protein,which was related to a variety of cancers,including pancreatic cancer,colon cancer,gastric cancer,breast cancer,lung cancer and liver cancer.Although these studies indicated that PAK5 was a key factor in tumor progression,its potential molecular mechanism had not been fully elucidated.Mitochondria is the key factors for initiation and execution of apoptosis.When stimulated by a series of external signals,mitochondria releases apoptosis inducing factors such as AIF(apoptosis inducing factor),endonuclease G and cytochrome c into the cytoplasm,and activated caspase-dependent or caspase-independent pathways to induce apoptosis.Under normal physiological conditions,AIF is located in the intercellular space of mitochondrial membrane and plays the role of oxidoreductase and electron transfer.A series of evidences showed that when cells were stimulated by apoptotic signals,calpain could cleave AIF together with other cysteine proteases(lysosomal cathepsin),making it a 57 k Da soluble Pro apoptotic protein AIF(mature AIF),and could increase the permeability of mitochondrial membrane,released AIF from mitochondria to cytoplasm and enter into nucleus,which could interact with DNA Binding leads to chromatin condensation and DNA fragmentation,which induced caspase-independent apoptosis.The process of AIF from mitochondria to nucleus was regulated by many factors.However,the effect of PAK5 on AIF function had not been reported,and the clinical effect of AIF on breast cancer progression was still unclear.In this study,we found that PAK5 could prevent the release of AIF from mitochondria and inhibit caspase-independent apoptosis by phosphorylating AIF and reducing its nuclear translocation.Importantly,our study showed that with the development of breast cancer,the nuclear translocation of AIF was significantly reduced,which was closely related to the high expression of PAK5 in breast cancer.These results suggested that PAK5-AIF signaling pathway might be a new potential target for the treatment of breast cancer.Methods: In order to study the expression of PAK5 and AIF in clinical samples of breast cancer and their correlation with the prognosis of patients,the expression of AIF in breast cancer tissue was predicted by TCGA database,and the overall survival rate of AIF was predicted by TCGA and GSE31519 database.The expressions of PAK5 and AIF in breast cancer were detected by Western blot and immunohistochemistry,and their correlation with tumor grade was analyzed.According to the different expression levels of PAK5 and AIF,the survival of breast cancer patients was analyzed.The interaction between PAK5 and AIF was identified by protein immunoprecipitation assay and GST pull-down assay.The co localization of PAK5 and AIF was detected by immunofluorescence assay.To investigate the effect of PAK5 on the release of AIF from mitochondria.Firstly,the localization of AIF in mitochondria was detected by immunofluorescence assay and mitochondrial protein extraction.The ratio of BCL2 / Bax in mitochondria was detected to evaluate the effect of PAK5 on mitochondrial membrane permeability.JC-1 staining and flow cytometry were used to detect the effect of PAK5 on mitochondrial membrane potential.In order to prove that PAK5 can affect caspase-independent apoptosis,flow cytometry was used to detect the effect of PAK5 on apoptosis.After caspase inhibitor Z-VAD-FMK and apoptosis inducer were added,the expression of cleaved-PARP was detected by Western blot.To study the specific mechanism of PAK5 regulating nuclear translocation of AIF.Firstly,the specific domain of PAK5 binding to AIF was detected by GST-pulldown assay.The effect of PAK5 on nucleocytoplasmic localization of AIF was detected by nucleocytoplasmic separation and Western blot.In order to explore the specific mechanism of PAK5 affecting the nuclear translocation of AIF,the location of the interaction between AIF and PAK5,the interaction between AIF and importins,and the effect of PAK5 on the interaction between AIF and importins were detected by immunoprecipitation assay.In order to prove that the effect of PAK5 on the nuclear translocation of AIF depends on the phosphorylation level,we first predicted the possible phosphorylation sites of PAK5 on AIF by bioinformatics,and detected the changes of Flag,AIF WT,AIF S202 A and AIF T281 A nuclear translocation by nuclear cytoplasmic separation and Western blot.The T281 site of PAK5 phosphorylated AIF was verified by phosphorylated protein mass spectrometry and immunoprecipitation assay.The effects of Flag,AIF WT and AIF T281 A on apoptosis were detected by flow cytometry.The binding of Flag,AIF WT and AIF T281 A to PAK5 and importins was detected by immunoprecipitation assay.The effects of Flag,AIF WT and AIF T281 A on the proliferation of breast cancer cells were detected by colony formation and CCK-8 assay.The effect of phosphorylated AIF on the proliferation of breast cancer cells was observed and analyzed in nude mice.To study the clinical correlation between PAK5 and AIF nuclear translocation.The expression and localization of AIF were detected in breast cancer adjacent tissues,breast benign tumor and breast cancer tissues,and the correlation between different expression levels and localization of AIF and breast cancer grade was analyzed.Immunofluorescence was used to detect the co localization of PAK5 and AIF in para-cancerous tissue,breast benign tumor and breast cancer tissues.Results: 1.PAK5 and AIF were highly expressed and positively correlated in breast cancer.TCGA database predicted high expression of AIF in breast cancer.TCGA and GSE31519 databases predicted that the overall survival rate of patients with high AIF expression is low.Western blot and immunohistochemistry showed that PAK5 and AIF were highly expressed in clinical cases of breast cancer,and there was a positive correlation between them.It was also positively correlated with tumor grade.Survival analysis showed that patients with high expression of PAK5 and AIF had poor prognosis.The interaction between PAK5 and AIF was confirmed by immunoprecipitation assay.GST pull-down assay confirmed that PAK5 and AIF directly combined in vitro.Immunofluorescence assay confirmed that PAK5 and AIF co-located in the cytoplasm of breast cancer cells.2.PAK5 inhibited the release of AIF by regulating mitochondrial membrane permeability and membrane potential.Immunofluorescence assay confirmed that PAK5 and AIF could co-locate in mitochondria,and PAK5 could increase the expression of AIF in mitochondria.Western blot showed that PAK5 could increase the ratio of BCL2 / Bax and decrease the permeability of mitochondrial membrane.Flow cytometry showed that PAK5 could increase mitochondrial membrane potential.3.PAK5 mediated AIF phosphorylation inhibited nuclear translocation and promoted breast cancer proliferation.Western blot analysis confirmed that PAK5 could inhibit the apoptosis of caspase independent cells.GST-pulldown assay confirmed that PAK5 directly binds to the 263-480 a.a.A domain of AIF.It was confirmed that PAK5 could prevent AIF from entering the nucleus.Immunoprecipitation assay confirmed that PAK5 could attenuate the interaction between AIF and importin ?3.The Thr-281 site of PAK5 phosphorylated AIF was verified by phosphorylated protein mass spectrometry and immunoprecipitation assay.Nuclear cytoplasmic separation and immunoprecipitation experiments confirmed that PAK5 inhibited nuclear translocation of AIF by phosphorylating the Thr-281 site of AIF and weakening its binding to importin ?3.CCK-8,clone formation and tumor formation in nude mice confirmed that phosphorylated AIF could promote the proliferation of breast cancer cells.Immunohistochemical results showed that the expression of AIF in breast cancer tissues was higher than that in adjacent tissues and benign breast diseases,and the nuclear translocation of AIF was less.The expression of AIF was positively correlated with the grade of breast cancer,while the nuclear translocation of AIF was negatively correlated with the grade of breast cancer.Immunofluorescence assay showed that PAK5 and AIF were more co-localized in the cytoplasm of breast cancer tissues than in the adjacent tissues and benign breast diseases.Conclusion: 1.The expression of PAK5 and AIF in clinical samples of breast cancer was positively correlated with tumor grade.Patients with high expression of PAK5 and AIF had poor prognosis.2.There was interaction between PAK5 and AIF,and PAK5 was colocated in the cytoplasm.3.PAK5 inhibited the release of AIF from mitochondria by decreasing mitochondrial membrane permeability and increasing membrane potential.4.PAK5 could inhibit the apoptosis of caspase-independent cells.5.PAK5 could reduce the interaction between AIF and importin ?3 to prevent AIF from entering the nucleus.6.PAK5 inhibited nuclear translocation of AIF by phosphorylating Thr-281 of AIF.7.PAK5 mediated AIF phosphorylation could promote the proliferation of breast cancer cells.8.In clinical cases of breast cancer,AIF nuclear translocation was negatively correlated with the grade of breast cancer.9.The co-localization of PAK5 and AIF in the cytoplasm of breast cancer tissues was stronger than that of adjacent tissues and breast benign tumor.