The self-renewal transcription factor Nanog and the phosphoinositide 3-kinase (PI3K)–Akt pathway are known to be essential for maintenance of mesenchymal stem cells. We evaluated their contribution to the maintenance of CD133(+) cancer stem-like cells (CSCs) and spheroid-forming cells in patient-derived cell lines from three human sarcoma subtypes: HT1080 fibrosarcoma, SK-LMS-1 leiomyosarcoma, and DDLS8817 dedifferentiated liposarcoma. Levels of Nanog and activated Akt were significantly higher in sarcoma cells grown as spheroids or sorted for CD133 expression to enrich for CSCs. shRNA knockdown of Nanog decreased spheroid formation 10- to 14-fold, and reversed resistance to both doxorubicin and radiation in vitro and in H1080 flank xenografts. In the HT1080 xenograft model, doxorubicin and Nanog knockdown reduced tumor growth by 34% and 45%, respectively, and the combination reduced tumor growth by 74%. Using a human phospho-kinase antibody array, Akt1/2 signaling, known to regulate Nanog, was found to be highly activated in sarcoma spheroid cells compared with monolayer cells. Pharmacologic inhibition of Akt using LY294002 and Akt1/2 knockdown using shRNA in sarcoma CSCs decreased Nanog expression and spheroid formation and reversed chemotherapy resistance. Akt1/2 inhibition combined with doxorubicin treatment of HT1080 flank xenografts reduced tumor growth by 73%. Finally, in a human sarcoma tumor microarray, expression of CD133, Nanog, and phospho-Akt were 1.8- to 6.8-fold higher in tumor tissue compared with normal tissue. Together, these results indicate that the Akt1/2–Nanog pathway is critical for maintenance of sarcoma CSCs and spheroid-forming cells, supporting further exploration of this pathway as a therapeutic target in sarcoma.

自我更新转录因子Nanog和磷酸肌醇3激酶（PI3K）–Akt途径是维持间充质干细胞必不可少的。我们评估了它们对三种人类肉瘤亚型：HT1080纤维肉瘤，SK-LMS-1平滑肌肉肉瘤和DDLS8817去分化的患者源性细胞系中CD133（+）癌症干样细胞（CSCs）和类球体形成细胞的维持的作用脂肪肉瘤。在成长为球形或按CD133表达分类以富集CSC的肉瘤细胞中，Nanog和活化的Akt的水平明显更高。Nanog的shRNA敲低将球体形成降低了10到14倍，并在体外和H1080侧翼异种移植物中逆转了对阿霉素和放射的抗性。在HT1080异种移植模型中，阿霉素和Nanog组合可降低肿瘤生长34％和45％，分别使该组合降低了74％的肿瘤生长。与单层细胞相比，使用人磷酸激酶抗体阵列，发现已知可调节Nanog的Akt1 / 2信号在肉瘤球形细胞中被高度激活。在肉瘤CSC中使用LY294002抑制Akt的药理作用，使用shRNA抑制Akt1 / 2的药理作用降低了Nanog表达和球体形成，并逆转了化疗耐药性。Akt1 / 2抑制结合阿霉素治疗HT1080侧翼异种移植物可使肿瘤生长降低73％。最后，在人肉瘤肿瘤微阵列中，与正常组织相比，肿瘤组织中CD133，Nanog和磷酸化Akt的表达高1.8至6.8倍。总之，这些结果表明Akt1 / 2–Nanog途径对于维持肉瘤CSC和球状细胞至关重要，