Department of Surgery, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA.
Mol Cancer Res. 2010 Sep;8(9):1217-26. doi: 10.1158/1541-7786.MCR-10-0162. Epub 2010 Aug 24.
Although autophagy is generally considered a prosurvival mechanism that preserves viability, there is evidence that it could drive an alternative programmed cell death pathway in cells with defects in apoptosis. Because the inhibition of autophagic activity promotes resistance to both chemotherapy and external beam radiation in papillary thyroid cancer (PTC), we determined if RAD001, a potent activator of autophagy, improves the efficacy of either therapy. We found that RAD001 increased the expression level of light chain 3-II, a marker for autophagy, as well as autophagosome formation in cell lines and in human PTC ex vivo. RAD001 sensitized PTC to doxorubicin and external beam radiation in a synergistic fashion, suggesting that combination therapy could improve therapeutic response at less toxic concentrations. The effects of RAD001 were abrogated by RNAi knockdown of the autophagy-related gene 5, suggesting that RAD001 acts, in part, by enhancing autophagy. Because the synergistic activity of RAD001 with doxorubicin and external radiation suggests distinct and complementary mechanisms of action, we characterized how autophagy modulates signaling pathways in PTC. To do so, we performed kinome profiling and discovered that autophagic activation resulted in Src phosphorylation and Met dephosphorylation. Src inhibition did not reverse the effects of RAD001, whereas Met inhibition reversed the effects of autophagy blockade on chemosensitivity. These results suggest that the anticancer effects of autophagic activation are mediated largely through Met. We conclude that RAD001 induces autophagy, which enhances the therapeutic response to cytotoxic chemotherapy and external beam radiation in PTC.
尽管自噬通常被认为是一种维持细胞存活的生存机制，但有证据表明，在凋亡缺陷的细胞中，它可能会驱动另一种程序性细胞死亡途径。由于自噬活性的抑制会促进甲状腺乳头状癌 (PTC) 对化疗和外照射的抵抗，我们确定了 RAD001（一种有效的自噬激活剂）是否能提高这两种治疗方法的疗效。我们发现，RAD001 增加了细胞系和人 PTC 体外的轻链 3-II 的表达水平，这是自噬的标志物，以及自噬体的形成。RAD001 以协同的方式使 PTC 对多柔比星和外照射敏感，这表明联合治疗可以在毒性较小的浓度下提高治疗反应。RNAi 敲低自噬相关基因 5 可消除 RAD001 的作用，这表明 RAD001 的部分作用是增强自噬。由于 RAD001 与多柔比星和外照射协同作用的效果，提示其具有独特而互补的作用机制，我们研究了自噬如何调节 PTC 中的信号通路。为此，我们进行了激酶组谱分析，发现自噬的激活导致了Src 的磷酸化和 Met 的去磷酸化。Src 抑制不能逆转 RAD001 的作用，而 Met 抑制则逆转了自噬阻断对化疗敏感性的影响。这些结果表明，自噬激活的抗癌作用主要通过 Met 介导。我们得出结论，RAD001 诱导自噬，从而增强了 PTC 对细胞毒性化疗和外照射的治疗反应。
