Activated CAMKKβ-AMPK signaling promotes autophagy in a spheroid model of ovarian tumour metastasis

Abstract

Background: 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. Results: We observed a coordinate 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. Conclusions: 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.

Possible Role of Metformin as an Immune Modulator in the Tumor Microenvironment of Ovarian Cancer

“…Lastly, it is important to consider that glycolysis mediated by GLUT1 expression and AMPK activation has been observed to markedly enhance OvCa tumor cell proliferation [ 91 ]. More recently, AMPK was shown to be indispensable for autophagy-mediated OvCa spheroid survival and proliferation [ 92 ], although whether and how it was influencing this phenotype has yet to be clearly defined. Although the majority of in vitro data using metformin in HGSOC and other OvCa cell lines demonstrate inhibited proliferation and colony formation [ 18 , 85 ], both of these pro-tumorigenic mechanisms could potentially be promoted by metformin through its well-defined upregulation of GLUT1 expression and AMPK activation.…”

Insights into high-grade serous carcinoma pathobiology using three-dimensional culture model systems

“…However, it is regarded as a key mechanism that can promote cancer cell survival under hypoxia in avascular tumours, nutrient depletion in rapidly growing tumours, or in the face of chemotherapy [ 30 ]. Indeed, our group observed that autophagy is rapidly induced in EOC spheroids, and this is driven by two parallel signaling pathways working in opposite orientations: downregulation of Akt-mTOR and upregulation of AMPK [ 10 , 17 ]. Interestingly, we also determined that the canonical Beclin-1 mechanism for autophagy activation is not required in EOC spheroids, however ULK1 within the autophagy initiation complex (AIC) is activated and required in EOC spheroids [ 17 , 28 , 30 ].…”

Principles of dormancy evident in high-grade serous ovarian cancer

“…A major downstream effect mediated by AMPK activity in EOC spheroids is the induction of macroautophagy. AMPK expression and activity, particularly that controlled by CAMKKβ, are required for induction of autophagy in HGSOC spheroids [58]. AMPK activity also acts to control cell proliferation by inducing cytostasis under nutrient-limiting conditions [57].…”

“…Autophagy is rapidly induced in HGSOC spheroids (Fig. 2A), and this is driven by two anti-parallel signaling pathways that were introduced in the signaling section above, namely the downregulation of AKT and the upregulation of AMPK [53,58]. More recently, a primary target of AMPK, the unc51-like protein kinase 1 (ULK1) within the autophagy-initiation complex (AIC), was shown to be activated and required for survival in HGSOC spheroids [65].…”