The cellular response to a shortage of environmental nutrients and resultant loss in energy is a critical pathologic event. Autophagy is an adaptive response to nutrient deprivation and cellular stress to recycle and remove damaged macromolecules and organelles [40
]. By mediating catabolism of intracellular contents, autophagy maintains cellular bioenergetics during metabolic stress. Starvation, protein aggregation, and anticancer treatments increase autophagic activity above the basal levels. In solid tumors, cancer cells at the center of a tumor are poorly vascularized, and the induction of autophagy may allow these cells to survive the nutrient depletion stress. However, if the induction of autophagy surpasses the physiological range, it could contribute to cell death, and therefore, strict regulation is crucial in these nutrient-poor settings [15,41–43
]. Furthermore, most chemotherapy-resistant cancers exhibit defective apoptosis, and cell death involves the activation of a secondary mechanism [44
mTOR serves as a major negative regulator of autophagy [14,45
]. mTOR functions as a sensor for cellular energy and amino acid levels and acts as a metabolic rheostat-controlling protein synthesis during cellular stress. mTOR links this information with external signals originating from cell surface receptors, and the sensory input is biochemically integrated and coupled to a coordinated response that controls cellular functions. In addition, AMPK is a master regulator of energy balance in the cell, and once activated, it shuts down energy-consuming processes and stimulates the catabolic pathways that generate ATP [8
]. The AMPK pathway is linked to mTOR signaling through the ability of AMPK to inhibit mTOR. Thus, the energy demands of protein synthesis can be reduced when cellular energy levels are diminished. We previously reported that CCL2, a chemokine highly prevalent in the tumor microenvironment, induces the mTOR pathway and promotes prostate cancer PC3 cell survival by inhibiting autophagic death through activation of PI3K/Akt signaling and survivin up-regulation [25,26
]. In this work, we have examined the possible role of CCL2 in regulating the AMPK activity to control the activation of mTORC1, survivin expression, and cell survival.
First, it was demonstrated that CCL2 hyperactivates mTORC1 in prostate cancer PC3 cells and sustains its activation over time as monitored by phosphorylation of p70S6K
(). mTORC1 stimulation was accompanied by a negative regulation of AMPK phosphorylation on the crucial residue (Thr172
] (). Furthermore, raptor, an essential component of mTORC1 and a direct target of AMPK [10
], was also negatively regulated by CCL2. The AMPK regulation by CCL2 also correlates with the up-regulation of survivin expression that was shown to enhance survival through inhibition of autophagic cell death () [25
The significance of AMPK regulation by CCL2 to sustain the mTORC1 activation and PC3 cell survival was demonstrated by using the specific AMPK activator D942. Indeed, D942 induced the AMPK and raptor phosphorylation (), resulting in a significant decrease of p70S6K
activation by mTORC1 (). Consequently, the CCL2-induced survivin up-regulation was stunted, correlating with an increase in autophagosome formation (higher LC3-II reflects higher autophagy [19,25
]) and rapid induction of cell death (, A, a
, and C
). These findings also corroborate our previous data showing that CCL2 modulates the amount and localization of LC3 and that survivin short hairpin RNA induces changes in LC3 punctate pattern that could not be reversed by CCL2 [25
]. Furthermore, CCL2-treated cells exhibited a higher resistance to AMPK activator when compared with control cells (, a, b
, and c
), indicating a crucial role of AMPK signaling in the survival mechanism induced by this cytokine. Concurrently, D942 showed insignificant alteration of Akt activation (, bar graph
), suggesting that the changes inmTORC1 and cell survival are a direct consequence of AMPK activation.
Conversely, the ATP competitive inhibitor of AMPK, compound C, induced a strong inhibition of Akt/PRAS40 phosphorylation, and consequently, mTORC1 was also inhibited (, B
). Although the inhibitor resulted in lower raptor phosphorylation (), the mTORC1 complex remained inactive, which could be explained by the inhibition of Akt signaling. Inhibition of autophagy by using compound C has been reported in other mammalian cells such as HT-29 and HeLa cells [5
]. However, in PC3 cells, compound C induced autophagic death, as evidenced by the increase in autophagosome formation (higher LC3-II) and lower survivin expression (, A
). In addition, reports in other cells suggest that Akt negatively regulates AMPK phosphorylation [36,37
]. However, it was found that CCL2-mediated AMPK regulation is independent of Akt signaling. In fact, the Akt-specific inhibitor X (Akti-X) effectively blocked Akt/PRAS40 phosphorylation and stimulated autophagy (increase in LC3-II); conversely, AMPK was not activated by Akti-X (). In summary, these results suggest that AMPK functions in parallel to Akt signaling; nevertheless, both pathways are indispensable for the induction of mTORC1 signaling and promotion of cell survival. Independent inactivation of any of these two CCL2-induced signals will blunt mTORC1 activity and induce cell death. Moreover, the presented findings also suggest an important physiological role of raptor phosphorylation by AMPK in the control of autophagy. Importantly, this study also shows that under serum deprivation, CCL2 induces a negative regulation of AMPK in other prostate cancer cells: C4-2B and DU145 (, A
). In these cells, survivin is also upregulated by CCL2 [25
], suggesting that CCL2 induces an analogous survival mechanism.
Further significance of p70S6K phosphorylation at Thr389 came from the findings that the p70S6K-specific inhibitor, Bis-V, also stunted survivin expression and induced autophagy and cell death (, A and B). Because CCL2 is a negative regulator of AMPK, and p70S6K phosphorylation is inhibited by AMPK, these results imply that this is a major mechanism used by CCL2 to control survivin expression and induce prostate cancer cell survival. summarizes this mechanism: CCL2 induces a negative regulation of AMPK and a positive regulation of Akt, and both signaling pathways act in parallel and are necessary to sustain mTORC1 activation and p70S6K phosphorylation to induce survivin up-regulation and promote cell survival.
Figure 6 Proposed mechanism of mTORC1 regulation by CCL2 in human prostate cancer PC3 cells. CCL2 induces negative regulation of AMPK and positive regulation of Akt; both signaling pathways act in parallel and are necessary to sustain mTORC1 activation and p70 (more ...)
Growing evidence suggests that signaling abnormalities within nutrient signaling pathways can lead to cancer. For example, the AMPK kinase, LKB1, is a tumor suppressor protein that activates AMPK when the AMP/ATP ratio increases in starved cells [47,48
]. Mutations in the AMPK kinase, LKB1, are associated with several types of related cancers [47,49,50
]. Although it is not clear how CCL2 exerts AMPK regulation, the findings presented here suggest that CCL2 does not induce metabolic changes that affect the ATP concentration in the cell (). Nevertheless, the fact that CCL2 regulates AMPK signaling pathway to sustain mTORC1 activation, survivin expression, and survival in prostate cancer cells suggests that CCL2 and AMPK may serve as therapeutic targets for the treatment of prostate cancer.