Conditional cell reprogramming involves non-canonical β-catenin activation and mTOR-mediated inactivation of Akt

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-267362, converts primary epithelial cells growing in vitro into an undifferentiated adult stem cell-like state that is characterized by long-term proliferation. This cell culture method also maintains the proliferation of adult epithelial stem cells from various tissues. Both primary and adult stem cells retain their tissue-specific differentiation potential upon removal of the culture conditions. Due to the ability to modulate the proliferation and differentiation of the cells, this method is referred to as conditional reprogramming and it is increasingly being used in studies of tumor heterogeneity, personalized medicine and regenerative medicine. However, little is known about the biology of these conditionally reprogrammed (CR) cells. Previously we showed that β-catenin activation, a hallmark of stem cells in vivo, occurs in CR human ectocervical cells (HECs). Here we show that β-catenin-dependent transcription is necessary for the induction of epithelial stem cell markers, and that β-catenin is activated via a non-canonical pathway that is independent of Wnt and Akt/GSK-3. Active Akt actually decreases due to increased mTOR signaling, with a consequent increase in dephosphorylated, active GSK-3. Despite the increase in active GSK-3, β-catenin associates with protein phosphatase 2A (PP2A) and is activated. Inhibition of PP2A catalytic activity reduces both the level of active β-catenin and the acute induction of stem cell markers, suggesting an important role for PP2A in the activation of β-catenin. Moreover, we demonstrate similar results using human prostate and breast cells, indicating that these changes are not restricted to ectocervical epithelial cells and may represent a more fundamental property of conditional reprogramming.

辐照成纤维细胞饲养层（irradiated fibroblast feeder cells）与Rho激酶抑制剂Y-267362的组合，可将体外培养的原代上皮细胞转化为具备长期增殖能力的未分化成体干细胞样状态。该细胞培养方法同样可维持多种组织来源的成体上皮干细胞的增殖活性。原代细胞与成体干细胞在移除培养条件后，均能保留其组织特异性分化潜能。鉴于该方法能够调控细胞的增殖与分化过程，因此被称为条件重编程（conditional reprogramming, CR）技术，目前正越来越多地应用于肿瘤异质性、个性化医疗与再生医学的相关研究中。然而，目前学界对这类条件重编程细胞的生物学特性仍知之甚少。此前我们的研究证实，β-连环蛋白（β-catenin）的激活——这是体内干细胞的标志性特征——会出现在条件重编程的人宫颈外上皮细胞（human ectocervical cells, HECs）中。本研究表明，依赖β-连环蛋白的转录过程对于上皮干细胞标志物的诱导是必需的，且β-连环蛋白的激活通过不依赖Wnt与Akt/GSK-3的非经典通路实现。活化的Akt实际会因mTOR信号通路增强而下调，进而导致去磷酸化的活性GSK-3水平升高。尽管活性GSK-3水平上升，β-连环蛋白仍可与蛋白磷酸酶2A（PP2A）结合并被激活。抑制PP2A的催化活性可同时降低活性β-连环蛋白的水平与干细胞标志物的快速诱导效果，这表明PP2A在β-连环蛋白的激活过程中发挥着关键作用。此外，我们在人前列腺细胞与乳腺细胞中得到了一致的实验结果，说明上述变化并非仅局限于宫颈外上皮细胞，可能代表了条件重编程过程中更为普遍的核心机制。