CCL2 is a negative regulator of AMP-activated protein kinase to sustain mTOR complex-1 activation, survivin expression, and cell survival in human prostate cancer PC3 cells

Abstract

CCL2 is a cytokine prevalent in the prostate cancer tumor microenvironment. Recently, we reported that CCL2 induces the mammalian target of rapamycin (mTOR) pathway to promote prostate cancer PC3 cell survival; however, the mechanism used by CCL2 to maintain mTOR complex-1 (mTORC1) activation requires clarification. This study demonstrates that upon serum starvation, CCL2 functions as a negative regulator of AMP-activated protein kinase (AMPK) by decreasing phosphorylation at its major regulatory site (Thr(172)) in PC3, DU145, and C4-2B prostate cancer cells. The CCL2-mediated AMPK regulation decreased raptor phosphorylation (Ser(792)) resulting in hyperactivation of mTORC1. D942, a pharmacological activator of AMPK, stunted CCL2-induced mTORC1 activity, survivin expression, and cell survival without significantly affecting Akt activity. CCL2, however, conferred some resistance to the lethal effect of D942 compared with untreated cells. By using Akt-specific inhibitor X, it was shown that Akt inactivation did not cause an increase in AMPK phosphorylation in CCL2-stimulated cells, suggesting that CCL2-mediated negative regulation of AMPK is independent of Akt. Furthermore, bisindolylmaleimide-V, a specific inhibitor of p70(S6K), stunted survivin expression and induced cell death in CCL2-treated PC3. Altogether, these findings suggest that CCL2 hyperactivates mTORC1 through simultaneous regulation of both AMPK and Akt pathways and reveals a new network that promotes prostate cancer: CCL2-AMPK-mTORC1-survivin.

Figure 1

CCL2 induces mTORC1 activation and downregulates AMPK/raptor phosphorylation in serum-starved PC3 cells. (A and B) Immunoblot analysis depicting the time-dependent effect of CCL2 (100 ng/ml) on (A) phosphorylation of the p70^S6 kinase (Thr³⁸⁹ residue), a direct target of mTORC1, and (B) AMPK/raptor phosphorylation and its correlation with survivin up-regulation. Reduction of AMPK and raptor phosphorylation, in response to CCL2 treatment, was quantified by densitometric analysis; the relative change in spot density was compared with total respective proteins and was normalized to actin (shown in respective bar graphs to the right). (C) Quantification of PC3 cellular ATP 48 and 72 hours for control and CCL2-treated cells. Bar graph depicts ATP concentration (µM per 1 x 10⁵ cells), and is representative of n = 3 bioluminescent ATP determination assays.

Figure 2

AMPK activator D942 induces raptor phosphorylation and downregulates mTORC1 signaling promoting cell death in PC3, but CCL2 opposes the lethal effect of D942. Cell viability was evaluated by WST-1 dye conversion at 24-hour increments up to 96 hours. (A) a and b: Time course for PC3 cell survival in response to D942: 10 µM (a) or 20 µM (b), ±CCL2. Bar graphs corresponding to control and CCL2-treated cells, without D942, are also included. Improved PC3 survival in response to CCL2 treatment was determined to be significant compared to control at 72 and 96 hours after stimulation, as evaluated by the Student's t-test; P < .0001 were observed. c: Relative effect of increasing D942 from 10 to 20 µM for control and CCL2-stimulated cells. Data for all graphs are representative of n = 5 assays, and SDs are depicted by ±Y error bars for each data point. (B) Western blot analysis using anti-P-Thr¹⁷² AMPK and anti-P-Ser⁷⁹² raptor phospho-specific antibodies depicts increased AMPK/raptor signaling, in response to D942 treatment (20 µM), 48 hours after treatment. Reduction in raptor phosphorylation compared with total protein was verified by densitometric analysis (shown in respective bar graph). Akt activation was also evaluated in response to D942, and the relative change in phospho and total Akt was quantified and analyzed by densitometric analysis with n = 3 samples (shown in bar graph). Phospho-PRAS40 (Thr246) immunoblots were included as a control for Akt-dependent signaling. (C) Western blot analysis representing the effect of D942 on: p70^S6K phosphorylation (Thr³⁸⁹), survivin expression, and the autophagic marker LC3-II. Phospho-specific expression was verified by comparison with total respective proteins.

Figure 3

AMPK inhibitor, compound C, stunts Akt-dependent signaling, and PC3 cell survival. (A) Cell viability was evaluated by WST-1 dye conversion at 24 and 48 hours. PC3 cell viability graph bars in response to compound C (20 µM) are shown for control and CCL2-stimulated cells. The data are representative of n = 5 assays for each CCL2-stimulated and control cells. SD bars are depicted for each data point. (B) Western blot depicting reduced AMPK activity in response to compound C (20 µM) in cells treated or not with CCL2 as evaluated by raptor phosphorylation (Ser⁷⁹²). The blots also show how compound C hinders Akt activation by inhibiting phosphorylation of key activation sites (Ser⁴⁷³ and Thr³⁰⁸) and thus preventing phosphorylation of a downstream target PRAS40 (Thr²⁴⁶). (C) Immunoblot analysis illustrating the effect of compound C at 24 hours on p70^S6K phosphorylation (Thr³⁸⁹), survivin expression, and the autophagic marker LC3-II. (D) CCL2 regulates AMPK signaling through an Akt-independent mechanism. Serum-starved, CCL2-stimulated PC3 cells were treated with increasing concentrations (1.25, 2.5, and 5 µM) of Akt inhibitor Akti-X (Calbiochem). Western blot analysis reveals how Akti-X effectively inhibits Akt activation/phosphorylation (Ser⁴⁷³ and Thr³⁰⁸) resulting in a reduced PRAS40 phosphorylation (Thr²⁴⁶). AMPK phosphorylation (Thr¹⁷²) and the induction of autophagy (LC3-II) were evaluated in response to Akti-X. Phospho-specific expression was verified by comparison with total respective proteins, and β-actin is included as a loading control.

Figure 4

CCL2-mediated survivin up-regulation and prolonged PC3 cell survival is dependent on p70^S6K phosphorylation. (A) Cell viability was evaluated at increasing time points (24, 48, and 72 hours) in response to specific p70^S6K inhibitor, Bis-V (8 µM). Data are representative of n = 5 assays. SD bars are depicted for each data point. P value was calculated by the Student's t-test (B) Western blot analysis of cells treated with 8 µM Bis-V in the presence and absence of CCL2. The inhibitor effect was evaluated by the analysis of p70^S6K phosphorylation (Thr³⁸⁹), LC3 conversion, and survivin expression. Phospho-specific expression of p70^S6K was verified by comparison with the total protein, and β-actin was included as a loading control.

Figure 5

CCL2 regulates AMPK signaling in C4-2B and DU145 prostate cancer cells. (A and B) Western blot analysis revealing the inhibition of AMPK/raptor phosphorylation by CCL2 in C4-2B (A) and DU145 (B) prostate cancer cell lines. Reduction in AMPK phosphorylation for both C4-2B and DU145, with respect to total protein, was quantified by densitometric analysis (bar graphs, right). Phosphospecific expression was verified by comparison with total respective proteins, and β-actin was evaluated as a loading control.

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^S6K phosphorylation over time, leading to survivin up-regulation and prolonged cell survival.