The Role Of S-phase Kinase-associated Proten2in Regulation Of Cell Proliferation And Metastasis In Triple Negative Breast Cancer

Objective Breast cancer is the most common female malignancy.The incidence andmortality of breast cancer increased in the past few years in our country. It has beenknown that several signaling pathways and various factors play critical roles in thedevelopment and progression of breast cancer. It is worth noting that exactmechanisms by which breast cancer arises remain largely unclear. Breast cancer hasbeen clinically characterized by the expression of hormone and growth factorreceptors such as estrogen receptor (ER), progesterone receptor(PR), and humanepidermal growth factor receptor2(Her2).Triple-negative breast cancers (TNBC) aredefined as tumors that lack expression of ER, PR,and Her2. TNBC account for about15%of all invasive breast cancers, and they usually have a high histologicgrade.Multiple studies have indicated that TNBC are associated with an adverseprognosis. No biologic therapy is available for TNBC.Women with triple-negativebreast cancer do not benefit from endocrine therapy or trastuzumab.Therefore,TNBCattracted more and more attention these years.PI3K/Akt signal pathway is animportant intracellular signal transduction pathway. It plays important roles in cellapoptosis and survival by affecting the activity of downstream effector molecules,and it is closely associated with the development and progression of most humanmalignant tumors.S-phase kinase associated protein2belongs to ubiquitin proteasomesystem (UPS) that plays vital roles in regulating many biological processes bycontrolling the timely turn-over of proteins It is known that as an F-box protein Skp2is one of the components of the Skp1–Cullin1–F-box (SCF) E3ligase complex. Skp2has important functions in the regulation of many cellular processes such as cell cycleregulation, proliferation, differentiation, apoptosis, and survival due to degradation ofits substrates, most of which are tumor suppressor proteins.Many studies have shown that overexpression of Skp2is observed in a variety of human cancers.Skp2expression has been considered as a biomarker for poor prognosis in many humancancers.The PI3K/Akt pathway has been reported to be involved in Skp2pathway.Arecent research suggested that Skp2is critically involved in ErbB family-induced Aktubiquitination.These evidence indicated strong interaction between PI3K/Akt pathwayand Skp2.Pagano’s group found that higher levels of Skp2are present more frequentlyin ER-negative tumors than in the ER-positive cases.And phosphorylation of Aktkinase was significantly higher in triple-negative breast cancers. These researchsuggested PI3K/Akt/Skp2pathway maybe significantly activated in TNBC,whichmight be connected with those biological characteristics and clinicopathologic featureof TNBC.Targeting at keypoint of this pathway may provide a novel newer strategyfor TNBC.Mechanism of this pathway in TNBC has not been revealed. In thisstudy,we investigated expression of key proteins in PI3K/Akt/Skp2pathway,analyzed the relationship between their expression and clinical pathologicalcharacteristics.Then we investigated the role of the pathway in TNBC cell growth,proliferation and metastasis in vitro, and finally, we constracted TNBC xenografts innude mice to explore the roles key proteins of the pathway played in TNBCtumorigenesis in vivo.Methods1.Immunohistochemestry was used to assess the expression of pAkt and Skp2in190cases of BC and30cases of benign breast lesions．The relationship between theexpression of key proteins related to P13K/Akt/Skp2signaling pathway and theclinicpathological parameters in BC was also analyzed．2.We constructed two pairs of specific recombinant plasmids targeting Skp2,andtransfected them into TNBC cells, and the expression of Skp2and other ralatedproteins were determined by Western blotting.The inhibition ration of TNBC cellsafter the Skp2shRNA trsanfected was examined by methyl-thiazolyl-tetrazolium(MTT) and cell colony formation assay. Flow cytometer was used to evaluated thedifference of cell cycle distribution and apoptosis rate in these different treatment groups.To detect invasion and migration abilities we used transwell migration assay.3.We established the xenograft model.The nude mice and subcutaneoustransplantation tumor growth were observed daily and the tumor volume werecalculated, then draw the tumor growth curve.Results1.Immunohistochemestry(1) Expression of pAkt and Skp2protein were remarkably higher than benign breastlesions(P=0.029,P=0.004).(2) High expression of pAkt and Skp2was not significantly correlated with patient’sage and tumor size(P=0.807,P=0.227; P=0.523,P=0.513), but correlated with lymphnode metastasis,degree of tumor differentiation and TNM staging (P=0.004, P=0.001,P=0.033; P=0.001,P=0.022, P=0.002).(3) Positive expression rates of pAkt protein in luminal A,luminal B, Her2positiveand TNBC substypes were18.8%,13.8%,42.8%,58.9%.And the expression rates ofSkp2protein were20.8%,20.7%,42.8%,53.8%. Positive expression of pAkt、Skp2inTNBC substype were remarkably higher than luminal A subtype and luminal Bsubtype(P=0.000,P=0.000; P=0.000,P=0.006),but not Her2positive subtype(P=0.233,P=0.417).(4) Expression of pAkt and Skp2protein were in negative correlation with theexpression of ER(r=-0.365，P<0.01；r=-0.296，P<0.01).(5) In TNBC, there is significantly positive correlation between pAkt andSkp2(r=0.692，P<0.01).2.The recombinant plasmids targeting Skp2was successfully constructed, and can besuccessfully transfected into TNBC cells, of which LV-Skp2-B was more efficient.3.Skp2shRNA inhibit Skp2mRNA expression.The expression of Skp2down-regulation could couse up-regulation of p27,caspase9,and down-regulation ofpAkt and cyclinD1.4.Cell cycle of TNBC cells arrested in the G0/G1phase,and TNBC cells were inducedapoptosis. 5.The growth,colony formation,invasion and migration abilities of K562cells wassignificantly inhibited after RNA interference.6.The TNBC xenograft model was successfully constructed, tumorigenecity of TNBCcells was suppressed after the Skp2shRNA trsanfection in vivo.Conclusion1.Expression of pAkt and Skp2in breast cancer was significantly higher than benignbreast lesions.High expression of pAkt and Skp2was correlated with lymph nodemetastasis,degree of tumor differentiation and TNM staging, which indicated thatpAkt and Skp2played important roles in the pathogenesis of breast cancer.2.Expression of pAkt and Skp2protein were in negative correlation with theexpression of ER.In TNBC, pAkt was significantly positive correlated with Skp2.Thisindicated that pAkt and Skp2may play important roles in TNBC.3.Knock-down of Skp2inhibited cell proliferation, colony formation as well as cellinvasion and migration abilities in breast cancer.5.Tumorigenecity of TNBC cells was suppressed in vivo by knock-down of Skp2suggested that Skp2could be a promising therapeutic target in combating TNBC.