干预SGLT1/AMPK通路诱导自噬增强Cetuximab抗结肠癌疗效的研究

Autophagy could serve as a newt anti-cancer mechanism of Cetuximab since downregulating decsodium/glucose cotransporter 1 (SGLT1) in kinase-independent EGFR pathway could trigger autophagic cell death. We have found that cetuximab induced autophagy by down regulating the expression of SGLT1 and that the expression of LC3, a marker of autophagy, showed a correlation with the efficacy of cetuximab, so more than ten papers were published in professional publications, including 5 SCI papers. The mTOR complex as well as Beclin1 complex is the major factor serving as regulation signals and transduction pathways in the process of autophagy, while our pilot experiments showed that Beclin1 had no role in the autophagy induced by Cetuximab. We plan to explore whether that autophagy by Cetuximab is dependent on the pathway of mTOR through Cetuximab regulating SGLT1 and AMPK, and to clarify if SGLT1 is another new way to active AMPK, and show if interposing AMPK, mTOR and p70S6k could sensitize Cetuximab, while analysis the correlations between their expression in colorectal cancer with the effect of cetuximab, therefore find the newest anti-cancer mechanism of Cetuximab by studying autophagy, and explore the new molecular indicator for the efficacy of Cetuximab, and provide theoretical basis for the next research program of improving the effect of cetuximab combined with the chemotherapy through interposing the autophagy.

EGFR非酪氨酸激酶途径的信号分子SGLT1下调可致细胞自噬性死亡，为研究Cetuximab抗瘤机制的新方向！我们已发现Cetuximab下调结肠癌细胞SGLT1而诱导自噬；肠癌组织中自噬标志物LC3表达与Cetuximab疗效相关；相关内容发表论文10余篇(5篇SCI)。自噬的主要信号通路是mTOR与Beclin1，预实验证实Beclin1不参与Cetuximab诱导自噬。本课题研究Cetuximab是否通过SGLT1/AMPK介导的mTOR路径诱发肠癌细胞自噬；SGLT1是否为激活AMPK的新途径；干预AMPK、mTOR及p70S6k是否增敏Cetuximab抑制细胞增殖和这些分子在癌组织的表达对Cetuximab疗效的预测价值；旨在从自噬角度阐明Cetuximab新的抗瘤机制，探索Cetuximab疗效新的预测指标，为下一步干预自噬增敏Cetuximab联合化疗疗效研究提供实验基础。

背景：结直肠癌是最常见的恶性肿瘤，一半的病人初诊时即伴有转移，另外一半的病人大多数最终也会发生转移，化疗及靶向药物治疗是晚期结直肠主要的治疗方法。晚期结直肠的有效药物不多，一线治疗中主要是3种化疗药物及两个靶向药物，如何提高现有药物的疗效是一个重要的研究方向。Cetuximab是作用于EGFR的人鼠嵌合型单抗，主要通过EGFR的MAPK途径发挥抗肿瘤作用。在晚期结直肠的临床实践中发现Cetuximab抗癌效应并不理想，与常用化疗方案联合平均生存期仅两年余。探究Cetuximab抗癌新机制从而发现增强Cetuximab抗癌效应新靶点为精准医学的研究方向。. 研究内容：Cetuximab调控肠癌细胞SGLT1/AMPK诱发依赖mTOR激酶途径的自噬；Cetuximab诱发的自噬与Beclin1信号通路无关；应用免疫组化方法检测临床患者的标本，从依赖mTOR激酶的自噬途径寻找影响Cetuximab疗效的分子指标。.结果：1.Cetuximab可诱发肠癌细胞株HCT-8及CACO-2的自噬，应用western-blot检测到LC3-II表达的增加，GFP-LC3免疫荧光发现自噬小体的增加；2. Cetuximab诱导自噬的作用独立于EGFR下游的MAPK途径，是通过EGFR共沉淀并下调细胞膜上SGLT1，激活AMPK后抑制mTOR路径诱发，而Beclin1水平没有明显变化；3.建立了SGLT1稳定表达的细胞株后，再使用Cetuximab处理结肠癌细胞，细胞发生自噬的水平下降，进一步证明Cetuximab可通过下调SGLT1而诱导自噬；4.使用自噬阻断剂氯喹后Cetuximab的抗肿瘤效应并没有增加，故推测诱导自噬水平上升是Cetuximab抗肿瘤机制，而不是耐药机制。以上基础研究的论文正在投稿中。5.建立了Cetuximab的病例数据库：搜集了中山大学肿瘤防治中心近十年应用Cetuximab的病例并完善了其临床、血常规及生化检验、病理及分子表达的详细数据，已完成部分病例自噬方面的研究，前期发表2篇SCI，全部病例的研究结果论文正在投稿中。.科学意义：此课题已经基本按计划完成，为今后进一步的研究奠定了坚实的基础。