Expression Of Phosphorylated AKT In Breast Carcinoma Tissue And Its Effect On Lymphatic Metastasis

ObjectiveIn female malignant tumor, breast carcinoma occupies an important firstgeneratal position, lymphatic metastasis in breast carcinoma, is the mostcommon route of metastasis, that is the most critical factors to determine thestaging of breast carcinoma and to choice therapeutic regimen. Accordingly,how to early to examine and control lymphatic metastasis becames the key linkof breast carcinoma. prevention and cure in the process of breast carcinomadiagnosis and treatment. Studies have found that phosphatidylinositol3-kinase/Sisu threonine protein kinase (PI3K/AKT) signal transduction pathway plays akey role in the development of tumor, AKT, also called protein kinase B (proteinkinase B, PKB), is the key intracellular effector proteins, AKT phosphorylationin the incidence of breast carcinoma development, invasion and metastasis playsa very important role. Tumor suppressor PTEN is a retro-regulatory factor,investigated most in PI3K/AKT signal transduction pathway presently, who hasdual phosphatase activity by PIP3to adjust many downstream signaling proteinsincluding AKTand to inhibit tumorous development. Vascular endothelialgrowth factor C (vascular en-dothelial growth factor-C, VEGF-C) is the mostwidely studied as one of lymphangiogenesis factors, that plays a vital role in theprocess of lymphatic metastasis by inducing tumor lymphangiogenesis andlymphatic metastasis. There may exist PI3K/AKT/VEGF-C signal transductionpathway in lymphatic metastasis of breast carcinoma. Accordingly, it maybecome a new target in inhibition of breast carcinoma lymphatic metastasis byinvestigating the relationship of PI3K/AKT signal transduction pathway andlymphatic metastasis in breast carcinoma and further elucidating the signaltransduction mechanisms of breast carcinoma lymphatic metastasis. Materials and MethodsThe surgical specimens of60cases were collected from July2011toDecember2012in Taishan Medical College affiliated Hospital′s GeneralSurgery. Immunohistochemical method, Western-blot were used to detect thelevel of expression of lymphangiogenesis and metastasis regulatory factorsVEGF-C, as well as P-AKT (Thr308), P-AKT (Ser473), and the tumorsuppressor PTEN protein by studing Paraffin specimens and fresh specimens ofhuman breast carcinoma. RT-PCR experiment was used to detect gene levelexpression of VEGF-C, P-AKT, PTEN in the fresh specimens of human breastcarcinoma. These three methods were used to analys the relationship betweenAKT phosphorylation and lymphatic growth factor, lymphatic metastasis.1Immunohistochemical method was used to detect the level ofexpression of VEGF-C, and P-AKT (Thr308), P-AKT (Ser473) and the tumorsuppressor PTEN protein of60cases paraffin sections in human breastcarcinoma fresh specimens.2Western-Blot was used to detect the expression of VEGF-C P-AKT(Thr308), P-AKT (Ser473) and the tumor suppressor PTEN protein levels in20cases fresh specimens of human breast invasive ductal carcinoma.3RT-PCR experimental technique was used to detect the expression ofVEGF-C, AKT and PTEN gene levels in20cases fresh specimens of humanbreast invasive ductal carcinoma.4Experimental all datas were analyzed by using SPSS13.0statisticalsoftware. Fourfold table χ~2test line×list χ~2test, Spearman correlation analysis,two independent sample test were used to analyze according to the types of dataand statistical purposes. Difference has statistical significance at P <0.05, thatwas used to analys the effect of PTEN in PI3K/AKT signal transduction pathway,the effect of phosphorylated AKT with lymphatic metastasis of breast carcinoma,correlated effect of phosphorylated AKT, PTEN and lymphangiogenesis relevantfactor VEGF-C, and to explore the role of PI3K/AKT signal transductionpathway in breast carcinoma lymphatic metastasis.Result1expression and its correlation of P-AKT, PTEN and VEGF-C in breastcarcinomaThe positive expression rate of P-AKT308was70%, the positive expression rate of P-AKT473was65%in the60cases of breast carcinomaspecimens. The expression rate of P-AKT308, P-AKT473in the group withlymphatic metastasis was significantly higher than that in the group withoutlymphatic metastasis(χ~2=15.660, P=0.000; χ~2=10.280, P=0.001).Expression of P-AKT308and P-AKT473had nothing to do with the tumor sizeand histological type(P>0.05).The positive expression rate of VEGF-C was68.3%, the positiveexpression rate of PTEN was56.7%in the60cases of breast carcinomaspecimens. The expression rate of VEGF-C in the group with lymphaticmetastasis was significantly higher than that in the group without lymphaticmetastasis(χ~2=20.863, P=0.000). The expression rate of PTEN in the groupWith lymphatic metastasis was significantly lower than that in the group withoutlymphatic metastasis(χ~2=12.996, P=0.000). Expression of VEGF-C andPTEN had nothing to do with the tumor size and histological type(P>0.05).VEGF-C positive expression rate of P-AKT308positive cases was81%,PTEN positive expression rate was45%; VEGF-C positive expression rate ofP-AKT308negative cases was38.9%, PTEN positive expression rate was83%.The relationship of P-AKT308and VEGF-C expression was positivecorrelation(r=0.511, p<0.05), and the relationship of P-AKT308and PTENexpression was negative correlation (r=-0.502, p<0.05), according to Spearmancorrelation analysis.VEGF-C positive expression rate of P-AKT473positive cases was82.1%,PTEN positive expression rate was48.7%; VEGF-C positive expression rate ofP-AKT473negative cases was42.9%, PTEN positive expression rate was71%.The relationship of P-AKT473and VEGF-C expression was positivecorrelation(r=0.505, p<0.05), and the relationship of P-AKT473and PTENexpression was negative correlation(r=-0.334, p<0.05), according to Spearmancorrelation analysis.2protein expression of P-AKT, PTEN and VEGF-C in breast carcinomaThere had12cases with lymph node metastasis,8cases with no lymphnode metastasis in20cases fresh specimens of human breast invasive ductalcarcinoma. In lymph node metastasis-positive cases, P-AKT308, P-AKT473andVEGF-C protein all expressed, but three cases of PTEN protein did not express.In lymph node metastasis-negative cases,2cases of P-AKT308did not express, 3cases of P-AKT473did not express, one case of VEGF-C protein did notexpress, but all PTEN cases expressed.Raw datas measured by ImageJ were derived by the two independentsamples test which were the absorbance value ratios of the protein of eachsample and the internal reference protein. The expression of P-AKT308,P-AKT473, VEGF-Cand PTEN protein was different whether with lymph nodemetastasis or not in20cases of human breastinfiltrating ductal carcinoma(U=-2.200, P=0.025; U=-2.512, P=0.010; U=-2.394, P=0.016; U=-2.474,P=0.012).3gene expression of AKT, PTEN and VEGF-C in breast carcinomaThere had12cases with lymph node metastasis,8cases with no lymphnode metastasis in20cases fresh specimens of human breast invasive ductalcarcinoma. In lymph node metastasis-positive cases, gene expression of AKTand VEGF-C expressed, but three cases of PTEN gene did not express. In lymphnode metastasis-negative cases, one cases of AKT did not express,3cases ofVEGF-C did not express, but all PTEN cases expressed.Raw datas measured by ImageJ were derived by the two independentsamples test which were the absorbance value ratios of the protein of eachsample and the internal reference protein.The expression of AKT, VEGF-C andPTEN gene was different whether with lymph node metastasis or not in20casesof human breast infiltrating ductal carcinoma (U=-3.703, P<0.05; U=-3.632,P<0.05; U=-3.709, P<0.05).Conclusion1P-AKT, VEGF-C expression of breast cancer tissue in the group withlymphatic metastasis significantly higher than it in the group without lymphaticmetastasis, PTEN expression of breast cancer tissue in the group with lymphaticmetastasis significantly lower than it in the group without lymphatic metastasis,it suggested that AKT phosphorylation promoted breast carcinoma lymphaticmetastasis, and PTEN played a negative regulatory role.2The positive correlation in the relationship of phosphorylated AKT andVEGF-C expression, negative correlation in the relationship of phosphorylatedAKT and PTEN expression in breast carcinoma suggested that AKTphosphorylation may promote the expression of VEGF-C, and further promotedbreast carcinoma lymphatic metastasis. 3Maybe AKT phosphorylation participate in starting intracellularPI3K/AKT signal transduction pathways to promote lymphangiogenesis andlymphatic metastasis, by the cross dialogue with VEGF-C/VEGFR-3signalingpathways in breast carcinoma. It may be the mechanism of action in breastcarcinoma lymphatic metastasis.