Identification Of Nuclear Localization Signal On SIPA1 Protein And Mechanism Study On Its Regulation Of ABCB1 Activity In Breast Cancer Cells

Breast cancer is the most common cancer among women worldwide.With the rapid development of early diagnosis and treatment technology for breast cancer,most of breast cancer patients get effective treatment.Due to lack of specific receptors and target molecules,some malignant breast cancer patients,especially high metastatic triple negative breast cancer(TNBC)patients,failed to get effective treatment,leading to drug resistance and even treatment failure.Therefore,it is urgent to further clarify the mechanism of breast cancer metastasis and identify new drug targets to improve the clinical breast cancer treatment effect.SIPA1,Signal-induced proliferation associated Protein1,is a member of G-protein family which hydrolyses the Rap1 GTP into Rap1 GDP.Recently,SIPA1 was found be involved in cancer cell proliferation and metastatic melanoma in colorectal cancer,prostate cancer,breast cancer and so on.SIPA1 was located in either nucleus or cytoplasm in distinct cancer cells.In TNBC,nuclear localized SIPA1 is significantly correlated with the breast cancer progression.How SIPA1 translocates into nucleus remains unclear.In this thesis,the potential nuclear localization sequence(NLS)in SIPA1 protein was analyzed by bioinformatics tools and searched by comparing with the classical NLSs,but no typical NLSs or similar sequence was found in SIPA1 primary sequence.In order to identify the NLS in SIPA1 protein,a series of truncated mutants were constructed and overexpressed in SIPA1 low expressing HEK293 cells,and the subcellular distribution of mutant SIPA1 was detected by immunofluorescence imaging and Western Blot after the cytosol-nucleus extraction.Results showed that the nuclear localization region(NLR)of SIPA1 was located in the 140-179 aa region.The NLR of SIPA1 could guide the known cytoplasm localization GFP-GFP(2×GFP)protein into nucleus.Three dimension structure of NLR was analysed by Swiss-Model.SIPA1-NLR owns a specific structure with two?-sheets linked by a coiled-coil.Taken together,SIPA1-NLR is a new nuclear localization region(NLR)which may be crucial for translocate protein into the nucleus.In general,nuclear localization protein which contains the NLS was recognized and then transported into the nucleus by transportin protein.Whether SIPA1-NLR can be recognized and interacted by transportin protein remains to be clarified.By analyzing the results of Co-IP assay and Mass Spectrum assay,two transportin(importin?1 and importin7)were identified to potentially interact with SIPA1 protein in MDA-MB-231 cells.Then,the recombinant GST with NLR domain(GST-NLR)were expressed in E.coli and purified with GST beads.The interaction between importin7 and GST-NLR was confirmed by in-vitro pull-dowm assay,indicating that importin7 could interact with the NLR of SIPA1 protein and transport SIPA1 into the nucleus.In order to clarify the function of SIPA1 in different subcellular distribution status,MCF7 cell lines stable overexpression of the full-length of SIPA1 protein(MCF7/SIPA1)and the NLR deleted SIPA1(MCF7/SIAP1-?NLR)were established respectively.The results showed that migration ability of MCF7/SIAP1-?NLR was greatly decreased compared to that of MCF7/SIPA1 cells,indicating that NLR of SIPA1 may be indispensable domainfor SIPA1 to improve breast cancer cell migration.Furthermore,MCF7/SIAP1-?NLR cells showed more sensitive to epirubicin treatment compared to MCF7/SIPA1 cells.After epirubicin treated,the mRNA level of ABCB1 or ABCG2 gene were significantly decreased in MCF7/SIAP1-?NLR cells compared to that in MCF7/SIPA1 cells,indicating that the NLR-mediated nuclear localization may be necessary for SIPA1 to regulate ABCB1 expression.In summary,a novel NLR in SIPA1 protein was identified.Importin7 was found to interact with this NLR and transport SIPA1 protein into nucleus.The nuclear localizalization SIPA1 could probably regulate ABCB1 expression to decrease the epirubicin-sensitivity in breast cancer cells.These findings provide a new potential drug target site to facilitate the effectiveness of breast cancer chemotherapy.