14-3-3ζ与二甲双胍在AMPK信号通路及克服化疗耐药中的相互作用研究

Metformin has been noted for its anticancer potential, however, it was reported that use of metformin alone seemed not to have the activity, it is important to find the starting point that metformin is involved in anticancer therapy. Our published data, as well as others, showed chemotherapy with metformin might improve survival of cancer patients, the exact mechanisms have been unclear and some important issue remain to be clarified. In our project, we got 12 colon cancer samples from surgical resection in diabetic patients, and 12 those from nondiabetic patients with matched clinicopathological features. iTRAQ-based quantitative proteomic analysis reveals the expression of 14-3-3ζ was obviously upregulated in colon cancer with diabetes, compared with those from nondiabetes. 14-3-3ζ is involved in some important life activities and is associated with tumorigenesis, invasion, epithelial–mesenchymal transition, and drug resistance. The finding of elevated 14-3-3ζ under the background of diabetic condition suggests it may be involved in cancer metabolism. It was also found to inhibit LKB1/AMPK signaling in recent research, which suggest it may interact with metformin whose anticancer effect is through activating the signaling pathway. In this study, we aim to study the influence of 14-3-3ζ on antitumor effects of metformin by upregulating or interfering the expression of 14-3-3ζ, as well as the interaction of metformin and 14-3-3ζ in drug resistant cancer models, which may be helpful for understanding the mechanism and its further application in anticancer therapy with chemotherapy.

二甲双胍的潜在抗癌作用引起了广泛的关注，但单独使用似乎疗效不佳，寻找合适的切入点或联合应用可能是成功的关键。我们及他人的研究初步提示二甲双胍与化疗联用可增强抗肿瘤作用并改善癌症患者的预后，值得进一步深入研究。我们对12对特征匹配的糖尿病或非糖尿病患者的结肠癌手术标本进行了iTRAQ定量蛋白质组学分析，发现14-3-3ζ蛋白的表达在糖尿病的结肠癌组织中明显增高。该蛋白广泛参与了细胞内重要的生命活动，并与肿瘤发生，侵袭增强，上皮间质转换，药物抵抗等密切相关。糖尿病背景下发现的14-3-3ζ增高提示它可能参与了肿瘤代谢，新近的研究发现它能抑制LKB1/AMPK通路，提示它可能影响二甲双胍的抗肿瘤代谢作用。我们拟通过体内外抑制或过表达14-3-3ζ来观察它对二甲双胍抗肿瘤作用及相关信号通路的影响，并在耐化疗药肿瘤模型中观察二者的相互作用，为阐明其机制及二甲双胍与化疗的合理联合提供帮助。

二甲双胍为广泛用于2型糖尿病的有效降糖药，其特点为安全性高，且价格低廉。二甲双胍被报道在肿瘤的预防和治疗方面有较好前景。然而，在不同临床特征的人群或不同类型的肿瘤患者中，二甲双胍的使用与肿瘤发生风险或预后的关系并不一样。在目前的报道中，二甲双胍的抗肿瘤作用在糖尿病背景下的癌症似乎更加明确。虽然目前二甲双胍的抗癌机理还没得到确切的阐明，但是AMPK信号通路的激活被认为是最重要的机制。我们前期通过对这两组病理标本的iTRAQ同位素标记色谱联合质谱分析显示，不管在II期还是III期结肠癌，14-3-3ζ蛋白的表达在糖尿病的结肠癌组织中均明显高于非糖尿病的结肠癌组织。14-3-3蛋白家族中14-3-3ζ通过调控多条关键的信号通路而参与了多种癌症的形成和进展，其表达在多种肿瘤中增高。而有报道发现14-3-3ζ能够抑制LKB1，从而抑制其底物AMPK的磷酸化，从而减少了G1期停滞和凋亡的发生。因此，我们不禁思考14-3-3ζ是否能影响二甲双胍的抗肿瘤作用。我们首先通过干扰14-3-3ζ的表达探索其在糖尿病背景下结直肠癌细胞中的表达及角色，并通过体内外模型探索14-3-3ζ与二甲双胍抗肿瘤作用的关系及潜在机制。我们发现14-3-3ζ在合并糖尿病的结直肠癌组织表达增加，而14-3-3ζ与结直肠癌的形成，增殖和凋亡有关；发现二甲双胍在14-3-3ζ高水平表达的结肠癌中能诱导凋亡，减缓肿瘤生长；且在高表达14-3-3ζ条件下，二甲双胍能取消14-3-3ζ对AMPK活性及线粒体相关凋亡信号的抑制。我们实验发现合并糖尿病的结直肠癌中 14-3-3ζ 升高，二甲双胍对 14-3-3ζ高表达的结肠癌的疗效增加，因此本课题有助于我们理解糖尿病患者肿瘤发病风险和致死性增加的原因以及二甲双胍在糖尿病背景下的抗肿瘤作用，也提示二甲双胍在14-3-3ζ高表达的结肠癌患者中的潜在临床应用。该研究结果对于未来研究二甲双胍的抗肿瘤作用提供了新的思路，对二甲双胍在肿瘤人群中的临床应用提供了一定的指导。