The Mary & John Knight Translational Ovarian Cancer Research Unit, Lawson Health Research Institute, London, ON, Canada.
Departments of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.
J Ovarian Res. 2020 May 11;13(1):58. doi: 10.1186/s13048-020-00660-5.
A hallmark of epithelial ovarian cancer (EOC) metastasis is the process of spheroid formation, whereby tumour cells aggregate into 3D structures while in suspension in the peritoneal cavity. EOC spheroids are subjected to bioenergetic stress, thereby activating AMP-activated protein kinase (AMPK) signaling to enter a metabolically quiescent state, which can facilitate cell survival under nutrient-limiting conditions. Independently, we have also demonstrated that EOC spheroids induce autophagy, a process that degrades and recycles intracellular components to restore energy and metabolites. Herein, we sought to examine whether AMPK controls autophagy induction as a cell survival mechanism in EOC spheroids.
We observed a co-ordinate increase in phosphorylated AMPK and the autophagy marker LC3-II during EOC spheroid formation. Reduced AMPK expression by siRNA-mediated knockdown of PRKAA1 and PRKAA2 blocked autophagic flux in EOC spheroids as visualized by fluorescence microscopy using the mCherry-eGFP-LC3B reporter. A complementary approach using pharmacologic agents Compound C and CAMKKβ inhibitor STO-609 to inhibit AMPK activity both yielded a potent blockade of autophagic flux as well. However, direct activation of AMPK in EOC cells using oligomycin and metformin was insufficient to induce autophagy. STO-609 treatment of EOC spheroids resulted in reduced viability in 7 out of 9 cell lines, but with no observed effect in non-malignant FT190 cell spheroids.
Our results support the premise that CAMKKβ-mediated AMPK activity is required, at least in part, to regulate autophagy induction in EOC spheroids and support cell viability in this in vitro model of EOC metastasis.
上皮性卵巢癌（EOC）转移的一个标志是球体形成过程，在此过程中，肿瘤细胞在腹腔悬浮时聚集形成 3D 结构。EOC 球体受到生物能量应激，从而激活 AMP 激活的蛋白激酶（AMPK）信号转导进入代谢静止状态，这可以促进细胞在营养有限的条件下存活。独立地，我们还证明 EOC 球体诱导自噬，这是一种降解和回收细胞内成分以恢复能量和代谢物的过程。在此，我们试图研究 AMPK 是否控制 EOC 球体中的自噬诱导作为细胞存活机制。
我们观察到在 EOC 球体形成过程中磷酸化 AMPK 和自噬标记物 LC3-II 的协调增加。siRNA 介导的 PRKAA1 和 PRKAA2 敲低降低了 AMPK 表达，使用 mCherry-eGFP-LC3B 报告荧光显微镜观察到 EOC 球体中的自噬流被阻断。使用药理学抑制剂 Compound C 和 CAMKKβ 抑制剂 STO-609 抑制 AMPK 活性的互补方法也产生了自噬流的有效阻断。然而，使用寡霉素和二甲双胍直接激活 EOC 细胞不足以诱导自噬。STO-609 处理 EOC 球体导致 9 个细胞系中的 7 个细胞活力降低，但在非恶性 FT190 细胞球体中没有观察到这种作用。
我们的结果支持这样一个前提，即 CAMKKβ 介导的 AMPK 活性至少部分需要调节 EOC 球体中的自噬诱导，并支持这种 EOC 转移体外模型中的细胞活力。
