Isolation, Cultivation And Identification Of Human Lung Adenocarcinoma Stem Cells And Identification The Role Of PI3K/AKT Signal Transduction Pathway In Regulating Its Self-renewal And Proliferation

Part I Expression and clinical significance of the PI3K/AKT signaltransduction pathway in non-small cell lung carcinomaObjective:To investigate the expression of phosphatidylinositol3-kinase(PI3K) andphosphorylated AKT B (p-AKT) protein and its clinical significance in non-small-cell lungcarcinoma (NSCLC).Methods: The clinical records of157patients with NSCLC（70cases inⅠ-ⅢA stageand87cases in ⅢB-Ⅳ stage）, consisting of75cases of squamous cell carcinoma (SCC)and82cases of adenocarcinoma (AdC), together with30cases of resection of lung cancertumor-adjacent tissues, were retrospectively evaluated. PI3K and p-AKT expression inNSCLC cancer tissues and tumor-adjacent tissues were measured using animmunohistochemical method and its association with clinicopathological data andprognosis in advanced NSCLC were also evaluated.Results: PI3K and p-AKT expression were significantly higher in cancer tissues thanthose in tumor-adjacent tissues （χ2=14.8455， P=0.000； χ2=14.2615， P=0.000;respectively). The overexpression of p-AKT in NSCLC withⅠ-ⅢA stage wasrelated to lymph node metastasis and TNM stage (χ2=6.1189,P=0.013，χ2=8.9752,P=0.011;respectively) and no correlation was observed with gender, age,histological categories or histological grade. The overexpression of p-AKT in NSCLC withⅢB-Ⅳ stage was only related to TNM stage（χ2=5.7501,P=0.016） and no correlationwas observed with gender, age, histological categories,histological grade or ECOGperformance status. The overexpression of PI3K was not related to above clinicopathological variables in all patients. Survival rates are significantly better inadvanced NSCLC with PI3K and p-AKT negative expression than positive expression[17.699months (95%CI15.114-20.283)/13.426months (95%CI11.832-15.021),P=0.004and17.134months (95%CI14.927-19.341)/13.067months (95%CI11.316-14.817),P=0.007]. Multivariate analysis showed that PI3K [HR=2.143(95%CI1.211-3.790)，P=0.009]， p-AKT [HR=1.991(95%CI1.009-3.927)，P=0.047]，TNM stage[HR=4.788(95%CI2.591-8.848)，P=0.000]， ECOG performance status [HR=3.272(95%CI1.701-6.296)，P=0.000] were independent predictors for survival in NSCLC with ⅢB-Ⅳ stage.Conclusion: p-AKT overexpression is closely correlated with unfavorable prognosticfactors in NSCLC. PI3K and p-AKT overexpression are independent poor prognosticmarkers in advanced NSCLC. PartⅡ Isolation, cultivation and identification of human lungadenocarcinoma stem cellsObjective：Isolation, cultivation and identification the highly tumorigenic cancerstem-like cells with stem cell features from the tissues of lung adenocarcinoma．Methods：The CD133(+) cells were isolated from the single cell suspension of the lungadenocarcinoma tissue by using magnetic activated cell sorting(MACS) technique, andenriched by serum free cultures．The markers of CD133on the isolated and enriched cellswere detected by using flow cytometry instrument and immunofluorescence． Thetumorigenic potent of the isolated cells was evaluated via the transplantation intoNOD-SCID mice．Results：The CD133(+) cells were successfully isolated by using MACS technique,and enriched by serum free cultures,and had capacity for self-renewal,multi-potentdifferentiation, and induction of xenograft tumors in vivo. Tumor could be induced inNOD-SCID mice by transplantation of100stem-like cells per mouse．Conclusion： MACS combined with serum-free culture are effective methods to isolate and enrich the human lung cancer stem cells. Part Ⅲ RNAi targeting AKTl and PI3K P85suppresses proliferation andself-renewal of lung cancer stem cellsObjective: To investigate the effect of RNA interference(RNAi) targeting AKTl andPI3K P85on the proliferation and self-renewal of lung cancer stem cells.Methods: Real-time PCR was carried out to detect the expression pattern of AKTland PI3K P85in lung cancer stem cells.The recombinant adenovirus expressionvector,which contained short hairpin RNA(shRNA) targeting open reading frames of AKTland PI3K P85(rAd5-siAKTl-siPI3K P85)，was transfected into lung cancer stem cells.AKTl and P13K P85protein expressions were detected by Western blotting analysis.Theexpressions of PCNA,cyclin Dl,and P53were also detected by Western blottinganalysis．The effects of AKTl and PI3K P85on self-renewal and proliferation of lungcancer stem cells were investigated using MTT assay,sphere forming assay and xenograftformation. In addition, cell cycle assay was also detected using flow cytometry after AKTland PI3K P85silence in lung cancer stem cells.Results: AKTl and PI3K P85expressions are up-regulated in lung cancer stem cellscompared with the lung cancer primary cells in mRNA level. Recombinant adenovirusvector rAd5-siAKTl-siPI3K P85significantly down-regulated AKTl and PI3K P85mRNAand protein expressions in lung cancer stem cells；The downstream factors PCNA andcyclin D1were also down-regulated，while P53was up-regulated. Following silence ofAKTl and PI3K P85, cell proliferation, tumor sphere self-renewal and tumor formation inNOD/SCID mice was reduced.Conclusion: AKTl and PI3K P85expressions are up-regulated in lung cancer stemcells. shRNA targeting AKTl and P13K P85can significantly down-regulate theexpression of AKT1and PI3K P85in lung cancer stem cells,and inhibit the proliferation,self-renewal of lung cancer stem cells in vitro．...