PI3K/Akt/mTOR信号通路调控FoxO3a诱导胰腺癌细胞EMT转化并获得干细胞干性特征的研究

Epithelial-mesenchymal transition is the initial stage of metastasis, which provides a molecular mechanism of invasion and metastasis in pancreatic carcinoma. Moreover, ethelial-mesenchymal transition not only confers pancreatic carcinoma cells with a distinct advantage for metastatic dissemination, but also provides those cells with self-renewal capacity and cancer stem cell-like characters, which finallly transforms tumor cells into cancer stem cells. Recently studies demonstrated that the PI3K/Akt/mTOR signaling pathway and FoxO3a may be present as the common signaling pathway in EMT and CSCs. However, FOXO3a can be translocated from the cytoplasm to the nucleus, which was regulated synergistically by phosphorylation and deacetylation mechanisms. Previous studies have shown that mTOR signal was over-activation in pancreatic carcioma. The invasive ability of pancreatic carcinoma cells was inhibited significantly by blocking mTOR signaling pathway. So we hypothesis that modulation of PI3K/Akt/mTOR signaling pathway on the transcriptional activitiy of FoxO3a through phosphorylation and deacetylation could induce epithelial-mesenchymal transition and acquire stemness characteristics in pancreatic cancinoma cells, which thus promotes the invasion and metastasis of pancreatic cancinoma. Finally, the results from this research may bring some novel clues for studying the synergistic molecular mechanism of epithelial-mesenchymal transition and cancer stem cells in the invasion and metastasis of pancreatic cancer.

上皮间质转化（EMT ）是胰腺癌转移早期的必经阶段，为胰腺癌细胞侵袭和转移提供了分子基础。EMT在促使胰腺癌侵袭转移的同时，也使转移的肿瘤细胞获得自我更新能力而具有干细胞特性，生成肿瘤干细胞（CSCs）。研究显示，PI3K/Akt/mTOR信号通路和FoxO3a可能是存在于EMT和CSCs中的共同信号通路。FoxO3a具有核浆转位的功能，同时受到磷酸化和去乙酰化机制的双重调控。前期研究发现，胰腺癌中mTOR呈过度激活状态，阻断mTOR信号可明显降低胰腺癌细胞的侵袭能力。预实验显示，阻断mTOR信号可显著提高FoxO3a转录因子的活性，降低胰腺癌迁移和侵袭能力。故我们设想：PI3K/Akt/mTOR信号通路可通过磷酸化和去乙酰化双重机制调控FoxO3a核浆转位，诱导胰腺癌细胞发生EMT转化并获得干细胞干性特征。其研究结果将进一步阐明EMT和CSCs在促进胰腺癌侵袭和转移中的协同作用机制。

胰腺癌是一种预后极差、极具侵袭性的消化系统肿瘤。上皮间质转化（EMT）是胰腺癌转移早期的必经阶段，EMT在促进胰腺癌侵袭转移的同时，也使转移的肿瘤细胞获得自我更新能力而转变为肿瘤干细胞。我们通过免疫组化染色检测胰腺癌组织中FoxO3a、p-AKT、mTOR、Ki67的表达，结果显示FoxO3a、p-AKT、mTOR在胰腺癌中的阳性表达率与肿瘤的分化程度、淋巴结转移及TNM分期密切相关，且FoxO3a的转录活性与p-AKT和mTOR的表达密切相关。此外在胰腺癌细胞系PANC-1和SW1990中研究调控FoxO3a诱导胰腺癌细胞EMT转化并获得干细胞干性特征的分子机制。结果显示，在胰腺癌细胞中转染FoxO3a siRNA下调FoxO3a基因表达后，胰腺癌细胞间黏附性下降，逐渐转变成长梭形。免疫荧光、qRT-PCR及Western blot显示转染FoxO3a siRNA后，胰腺癌细胞中上皮标记物E-cadherin表达明显下调，间叶标记VIM表达明显上调。侵袭及迁移实验显示转染FoxO3a siRNA后，胰腺癌细胞的侵袭和迁移能力明显增强，且肿瘤细胞体外裸鼠成瘤能力增强。转染FoxO3a siRNA后，Western blot显示转染后OCT4和SOX2显著上调，流式细胞鉴定表明CD24⁺CD44⁺ESA⁺胰腺癌肿瘤干细胞增多，成球实验及软琼脂克隆实验显示肿瘤细胞成球能力增强。反之，在胰腺癌细胞中转染FoxO3a TM上调FoxO3a基因表达后，胰腺癌细胞上皮标记物E-cadherin的表达明显上调，间叶标记VIM的表达明显下调，肿瘤细胞的侵袭和迁移能力明显减弱，CD24⁺CD44⁺ESA⁺胰腺癌肿瘤干细胞减少，OCT4和SOX2下调，肿瘤细胞成球能力减弱。这一研究提示下调胰腺癌细胞中FoxO3a基因的表达，可诱导胰腺癌细胞发生EMT转化，促进胰腺癌细胞侵袭和迁移，并获得干细胞干性特征，向肿瘤干细胞转化。进一步的研究显示，转染FoxO3a siRNA下调胰腺癌的FoxO3a基因后，下游基因β-catenin和TCF4的表达上调；而转染FoxO3a TM上调胰腺癌的FoxO3a基因后，β-catenin和TCF4的表达下调。以上研究结果揭示FoxO3a可能通过β-catenin/TCF4 complex信号通路诱导胰腺癌细胞发生EMT转化并获得干细胞干性特征。