Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK

Abstract

The multifunctional AMPK-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth, and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because although on one hand active AMPK inhibits mammalian target of rapamycin (mTOR) and lipogenesis--two crucial arms of cancer growth--AMPK also ensures viability by metabolic reprogramming in cancer cells. AMPK activation by two indirect AMPK agonists AICAR and metformin (now in over 50 clinical trials on cancer) has been correlated with reduced cancer cell proliferation and viability. Surprisingly, we found that compared with normal tissue, AMPK is constitutively activated in both human and mouse gliomas. Therefore, we questioned whether the antiproliferative actions of AICAR and metformin are AMPK independent. Both AMPK agonists inhibited proliferation, but through unique AMPK-independent mechanisms and both reduced tumor growth in vivo independent of AMPK. Importantly, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation. Metformin directly inhibited mTOR by enhancing PRAS40's association with RAPTOR, whereas AICAR blocked the cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. Together, our results suggest that although AICAR and metformin are potent AMPK-independent antiproliferative agents, physiological AMPK activation in glioma may be a response mechanism to metabolic stress and anticancer agents.

Keywords: glioma; metabolism.

Fig. 1.

Phosphorylated (active) AMPK is abundantly expressed in gliomas and the direct AMPK activator A769662 does not inhibit glioma growth. (A) IHC of active AMPK (pAMPK) in six human GBMs (12 tumors were analyzed). (Inset) High magnification of A. (B) Immunoblot shows pAMPK in glioma cell lines and normal astrocytes. Histology (C), IHC (D), and immunoblot (E) of mouse high-grade gliomas (HGGs). Magnification: A, 20×; A, Inset, 60×; C, 10×; D, 40×. A total of 12 tumors were analyzed. N, contralateral normal brain; T, tumor tissue. (F) Immunoblot analysis of glioma cells treated with AMPK agonists and (G) the effect AICAR, metformin, and A769662 on the proliferation of glioma cells. *P ≤ 0.005. Data shown is representative of three to six independent experiments. Error bars represent mean ± SD.

Fig. 2.

Inhibition of lipogenesis is not a mechanism of AICAR and metformin’s antiproliferative action. Proliferation of FASN-silenced (A) and ACC-silenced (B) T98G and U87EGFRvIII glioma cells treated with AICAR and metformin. nt, nontarget. Immunoblot with FASN (A, Inset) and ACC (B, Inset) antibodies. nt, nontarget shRNA. *P ≤ 0.005. (C) Proliferation of glioma cells in the presence of lipogenesis inhibitors [C75 (10 µg/mL) and atorvastatin (1 µM)]. Data shown is representative of two to four independent experiments. Error bars in A–C represent mean ± SD.

Fig. 3.

AMPK-dependent mTOR inhibition is not required by AICAR and metformin to suppress glioma cell proliferation. (A) Immunoblots showing the effects of AICAR, metformin, and A769662 on phosphorylation of AMPK substrates (ACC and RAPTOR) and mTOR effectors (S6 and 4EBP1) in T98G glioma cells. In B, eIF4E was immunoprecipitated with m7GDP-Sepharose and bound 4EBP1 was detected with 4EBP1 antibody. (C, Upper) Proliferation of control (nt shRNA) or AMPKβ1 shRNA cells treated with AMPK agonists. nt, nontarget. *P ≤ 0.005. (C, Lower) Immunoblots show AMPK, ACC, and RAPTOR phosphorylation by AMPK agonists. Densitometry of pAMPK levels is also shown. Error bars represent mean ± SD. (D) Immunoblots demonstrate the effects of AMPK agonists on mTOR effectors (S6 and 4EBP1) in control (nt) and AMPKβ1 shRNA T98G glioma cells. (E) The CAP-binding assay was done as in B in control or AMPKβ1 shRNA T98G cells. Data shown is representative of two to five independent experiments.

Fig. 4.

AICAR and metformin do not suppress glioma proliferation by inducing chronic energy crisis. (A) HPLC analysis shows the energy content of T98G glioma cells treated with AMPK agonists. (B) O₂ consumption (OCR) and (C) glycolysis (ECAR) of T98G glioma cells treated with AICAR or metformin. OCR (D) and ECAR (E) were also measured in control (nt) and AMPKβ1 shRNA T98G cells. nt, nontarget. (F) Proliferation of control and Rho-0 T98G cells treated with AICAR or metformin. Inset shows RT-PCR analysis of mitochondrial transcripts [cytochrome oxidase II (CoxII) and NADH dehydrogenase 4 (ND4)] in control and Rho-0 cells. Data shown is representative of at least three independent experiments. *P ≤ 0.001. Error bars in A–F represent mean ± SD.

Fig. 5.

Metformin and AICAR inhibit glioma growth in vivo and AICAR inhibits glioma proliferation by degrading cdc25c independent of AMPK. (A) Metformin and AICAR’s effect on growth of control (NT shRNA) and AMPKβ1 shRNA expressing U87EGFRVIII glioma xenografts in Nu/Nu mice (n = 10 per condition). *P ≤ 0.03 shown for both metformin and AICAR in NT and shRNA tumors. (B) Photomicrographs of representative control and treated gliomas. Histogram of the cell cycle analysis of three glioma cells (C) and control (nt) and AMPKβ1-silenced T98G glioma cells (D), treated with AMPK agonists. *P ≤ 0.005. Immunoblots show the effects of AICAR on G2M regulators in two glioma cells (E), and in control (nt) and AMPKβ1shRNA T98G glioma cells (F). (G) Effect of AICAR on cdc25c RNA levels. (H) Effects of AICAR alone or in the presence of the proteasomal inhibitor MG132 on protein levels of cdc25c in control (nt) and β1 shRNA T98G glioma cells. (I) Proliferation and cell cycle analysis of control (nt) and cdc25c shRNA expressing T98G glioma cells. (I, Inset) Immunoblot shows knockdown of cdc25c protein by three independent shRNA. Data are representative of at least three independent experiments. nt, nontarget. *P ≤ 0.005. Error bars in A and I represent mean ± SD.

Fig. 6.

Metformin suppresses proliferation through PRAS40-mediated mTOR inhibition. (A and B) Immunoblot using mTOR and 4EBP1 antibodies showing shRNA-mediated knockdown of mTOR and 4EBP1 in T98G glioma cells. Actin was used as a loading control. (C) Proliferation of control (NT) and mTOR shRNA-expressing and 4EBP1 shRNA-expressing T98G cells treated with AICAR and metformin. (D) Immunoprecipitation of RAPTOR followed by immunoblot analysis showing the effect of metformin and other cell stressors on RAPTOR–PRAS40 association. Loading control lysates are shown in the bottom three panels. (E) Proliferation of metformin-treated glioma cells expressing NT or PRAS40 shRNA. (E″) Immunoblot showing PRAS40 knockdown. (F) The model shows active AMPK is highly expressed in glioblastoma. The direct AMPK activator A769662 or the indirect activator AICAR does not effectively suppress mTOR. Although A769662 has no effect on glioma proliferation, AICAR suppresses glioma proliferation by degrading a crucial G2M phosphatase cdc25c through the proteasome, independent of AMPK. The other AMPK agonist metformin represses glioma proliferation through mTOR inhibition by increasing PRAS40-RAPTOR interaction, independent of AMPK. Data are representative of two to four independent experiments. *P ≤ 0.001. Error bars in C and E represent mean ± SD.