Effects Of PDK1 Nuclear Translocation On Breast Cancer Cell Growth,Migration And Invasion

ObjectiveTo investigate the effects of pyruvate dehydrogenase kinase 1(PDK1)nuclear translocation on growth,migration and invasion in human breast cancer MCF-7 and T47 D cells.Methods1.The sub-cellular localization of PDK1 in breast cancer cell lines containing two luminal A(MCF-7 and T47D),one luminal B (BT474),one HER2+(SKBR3)and two basal-like TNBC(BT549, MDA-MB-231)cell lines were performed with immunofluorescence(IF)and western blot analysis.The localization of PDK1 in breast normal tissue and breast duct carcinoma were detected by Immunohistochemistry.2.Firstly,human breast cancer MCF-7 and T47 D cells were treated with low glucose medium,serum-free medium,CoCl2 with a final concentration of 100 ?M,and human recombinant EGF with a final concentration of 100 ng / ml for 12 and 24 hours.Western blot was used to detect the sub-cellular localization of PDK1 with or without treatment.Immunohistochemistry and Western blot were used to detect the expression of EGFR in breast cancer tissue and MCF-7,T47 D cell.MCF-7 and T47 D cells were treated with 100 ng/ml EGF for 24 hr with or without prior treatment with Gefitinib (10 nM),the sub-cellular localization of PDK1 with or without treatment were detected by Western blotting.3.The protein-protein interaction between PDK1 and Importin ? was verified by co-immunoprecipitation(co-IP)assay.The presence of NLS in the amino acid sequence of PDK1 was predicted by NLS mapper.The recombinant plasmids carrying control(NC),wild-type PDK1 gene(WT-PDK1),and NLS mutant PDK1 gene(MUT-PDK1) PDK1 gene were transfected by liposome into MCF-7 and T47D cells,respectively.The sub-cellular localization of PDK1 in MCF-7 and T47 D cells were detected by Western blotting.4.The recombinant plasmids carrying control(NC),wild-type PDK1 gene(WT-PDK1),and NLS mutant PDK1 gene(MUT-PDK1)PDK1 gene were transfected by liposome into MCF-7 and T47 D cells,the growth of cells was measured by CCK-8 assay,the percentage of apoptotic cells was measured by flow cytometry,the migration of cells was measured by wound healing assay,The cell motility was evaluated by the transwell assay,the expression of proliferation- related protein(C-myc),apoptosis-related proteins(Bcl-2 and Bcl-xl) and epithelial markers(N-cadherin and Vimentin)were detected by Western blotting.Results 1.The results of immunofluorescence and western blot indicated that MCF-7 and T47 D cells exhibited cytoplasmic and nuclear PDK1 distribution.Western blot analysis showed that PDK1 emerged in the nucleus of basal-like TNBC cells,HER2+ and luminal cells.Immunohistochemistry analysis showed that PDK1 highly expressed in breast duct carcinoma and nuclear PDK1 expression in breast duct carcinoma,compared with breast normal tissue.2.Subcellular fractionation followed by western blot demonstrated hat hypoxia and EGF treatment promoted PDK1 to locate in the ucleus in a time-dependent manner,while glucose deficiency and utritional restriction have no effect.Western blot showed that GFR was expressed in MCF-7 and T47 D cells,immunohistochemistry showed that EGFR highly expressed in breast duct carcinoma tissue compared with normal breast tissue, and Western blot analysis showed that PDK1 nuclear accumulation was reduced by treatment with Gefitinib(an EGFR tyrosine kinase inhibitor)for 24 hr.3.Co-IP analysis demonstrated that PDK1 was bound to importin ? in CF-7 and T47 D cells,and EGF treatment promoted the ombination.NLS mapper prediction results showed that there was NLS(405-kaawkhyntnheaddwcvpsrepkdmttfr-434)at the C- erminus of the amino acid sequence of PDK1 protein,then we utated the basic amino acid containing Arg 425/434 and the Lys 05/409/428 to Ala.Western blot analyses demonstrated that UT-PDK1,unlike WT-PDK1,was unable to be translocated into he nuclear in MCF-7 and T47 D cells.4.The results of CCK8 revealed that the expression of WT-PDK1,in ontrast to the expression of MUT-PDK1,promoted the roliferation of MCF-7 and T47 D cells,and treatment with EGF on he basis of the expression of WT-PDK1 and MUT-PDK1 further romoted cell proliferation.Flow cytometry showed that compared ith the expression of WT-PDK1,when MUT-PDK1 was xpressed,the percentage of apoptotic cells was significantly ecreased,and EGF treatment enhanced this inhibition.The xpression of MUT-PDK1 induced cell apoptosis.Proliferation and poptosis were further evaluated using Western blot.Compared ith the expression of WT-PDK1,the level of C-myc,Bcl-2 and cl-xl in MCF-7 and T47 D cells were decreased significantly hen cell expressed MUT-PDK1.The wound healing assay indicated that compared to control cells,WT-PDK1 over-expressing cells exhibited stronger migration ability,and meanwhile,the migration of MUT-PDK1 over-expressing cells were severely impaired.Transwell analysis showed that the number of WT-PDK1 over-expressing cells through matrigel increased significantly,while MUT-PDK1 over-expressing cells barely penetrated Matrigel.Western blot was performed to further confirm that WT-PDK1 expression increased the expression of mesenchymal markers,such as N-cadherin and Vimentin,and MUT-PDK1 expression had completely opposite effect.Conclusions 1.PDK1 is present in the nucleus of human breast cancer cells 2.Hypoxia and EGF treatment promoted PDK1 to locate in the nucleus while glucose deficiency and nutritional restriction have no effect.3.The C-terminus amino acid sequence of PDK1 contains a functional NLS,PDK1 relies on this NLS to bind to the nuclear import protein Importins to enter the nucleus.4.Nuclear PDK1 facilitates the growth,migration and invasion of human breast cancer cells.