FoxO3a在PC9细胞中对吉非替尼耐药性的调控机制研究

Acquired resistance is a major clinical problem in almost all non-small-cell lung cancer (NSCLC) patients using gefitinib. A hyperactive PI3K/AKT pathway has been also found in tumour samples from advanced cancer patients failing EGFR-targeted therapy. Forkhead/winged helix box class O protein FOXO3a, the critical downstream targets of Akt pathway, are implicated in the regulation of several important biological processes and involved in the resistance mechanisms to several anti-tumor drugs. However, little information is available about FoxO3a in the acquired resistance to gefitinib in NSCLC. We have found the acquired resistance to gefitinib could be reversed through FoxO3a over expression in PC9 cells. Thus we propose a hypothesis that FoxO3a may be involved in the resistance mechanism to gefitinibin NSCLC. In this project, we will study the role of FoxO3a in the acquired resistance of gefitinib in NSCLC and its underlying mechanisms. We will establish gefitinib-resistant cell line PC9/GR from gefitinib-sensitive NSCLC cell line PC9 and compare the expression and phosphorylation of FoxO3a and other signaling molecules between PC9/GR and PC9 cell lines. Then, we will verify the role of FoxO3a in drug-resistance by protein overexpression and knockdown; explore the downstream target gene and their effects. Based on those findings we will clarify the regulatory roles and mechanisms of FoxO3a in resistance to gefitinib in NSCLC.

表皮生长因子受体(EGFR)酪氨酸激酶抑制剂——吉非替尼的获得性耐药是导致其用于非小细胞肺癌(NSCLC)治疗失败的主要原因。PI3K/Akt通路是EGFR信号的重要传导途径。FoxO3a作为Akt的直接下游靶标参与了多种肿瘤耐药性机制，但其在NSCLC耐药中的作用至今未有报道。我们发现，高表达FoxO3a可以逆转吉非替尼耐药细胞株PC9细胞的耐药性。那么FoxO3a通过何种机制逆转了吉非替尼的耐药性？本课题通过信号阻断剂、过表达、基因敲除和导入定点突变的质粒等多种手段，在PC9耐药细胞株中检测相关信号分子的表达和磷酸化的改变，研究FoxO3a的核转位动力学变化和对EGFR信号通路的负反馈作用，寻找参与耐药机制的下游靶基因，以期明确FoxO3a在调控NSCLC对吉非替尼耐药性中的作用和机制。

表皮生长因子受体(EGFR)酪氨酸激酶抑制剂——吉非替尼的获得性耐药是导致其用于非小细胞肺癌(NSCLC)治疗失败的主要原因。FoxO3a作为Akt 的直接下游靶标参与了多种肿瘤耐药性机制。我们前期发现，高表达FoxO3a可以逆转吉非替尼耐药细胞株PC9细胞的耐药性。本课题通过信号阻断剂、过表达、基因敲除和导入质粒等多种手段，在PC9细胞株中检测相关信号分子的表达水平的改变。结果发现，PC9细胞和PC9/GR细胞正常培养状态时，FoxO3a-GFP均分布于细胞质中；剥夺血清可使FoxO3a-GFP浓集于细胞核中；EGF可使FoxO3a-GFP逐渐重新回到胞质中。通过对基因表达程度的调节，FoxO3a过表达和EPS8沉默后，PC9细胞的增殖指数PI从原来的47.31%分别下降到24.76%和37.56%； FoxO3a沉默和EPS8过表达时，PC9细胞的增殖指数PI较原来的47.31%分别上升至56.4%和57.71%。据此我们进证实FoxO3a能够抑制肿瘤细胞的增殖而EPS8能促进细胞增殖。同时在荷瘤小鼠的在体实验中，我们发现FoxO3a siRNA组及高表达EPS8组荷瘤小鼠肿瘤体积及肿瘤重量显著增加；高表达FoxO3a组及EPS8 siRNA组的肿瘤体积和重量却显著减小。说明EPS8具有显著的促肿瘤生长的作用，而FoxO3a可以显著抑制肿瘤的生长。进而，我们研究了FoxO3a对EPS8的调控作用。EPS8在FoxO3a过表达时反而其mRNA含量较NC组降低1倍。说明FoxO3a很可能通过直接或间接的作用于EPS8而抑制其基因的表达。.通过上述研究我们基本可以确定在PC9细胞中FoxO3a通过直接或间接的抑制EPS8表达对PC9细胞的耐药起到抵抗作用。FoxO3a-EPS8通路可能在调控NSCLC对吉非替尼耐药性中的起到关键作用，并具有成为治疗靶点的潜力。