Pemetrexed indirectly activates the metabolic kinase AMPK in human carcinomas

Abstract

The chemotherapeutic drug pemetrexed, an inhibitor of thymidylate synthase, has an important secondary target in human leukemic cells, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of purine biosynthesis. The purine intermediate aminoimidazolecarboxamide ribonucleotide (ZMP), which accumulates behind this block, transmits an inhibitory signal to the mTORC1 complex via activation of the cellular energy sensor AMP-activated kinase (AMPK). Given that the PI3K-AKT-mTOR pathway is frequently deregulated during carcinogenesis, we asked whether the indirect activation of AMPK by pemetrexed offers an effective therapeutic strategy for carcinomas with defects in this pathway. Activation of AMPK by ZMP in pemetrexed-treated colon and lung carcinoma cells and the downstream consequences of this activation were strikingly more robust than previously seen in leukemic cells. Genetic experiments demonstrated the intermediacy of AICART inhibition and the centrality of AMPK activation in these effects. Whereas AMPK activation resulted in marked inhibition of mTORC1, other targets of AMPK were phosphorylated that were not mTORC1-dependent. Whereas AMPK activation is thought to require AMPKα T172 phosphorylation, pemetrexed also activated AMPK in carcinoma cells null for LKB1, the predominant AMPKα T172 kinase whose deficiency is common in lung adenocarcinomas. Like rapamycin analogs, pemetrexed relieved feedback suppression of PI3K and AKT, but the prolonged accumulation of unphosphorylated 4E-BP1, a tight-binding inhibitor of cap-dependent translation, was seen following AMPK activation. Our findings indicate that AMPK activation by pemetrexed inhibits mTORC1-dependent and -independent processes that control translation and lipid metabolism, identifying pemetrexed as a targeted therapeutic agent for this pathway that differs significantly from rapamycin analogs.

Figure 1. AMPK activation by ZMP accumulating in pemetrexed-treated HCT116 cells

(A) Pemetrexed induces AMPK phosphorylation and activity. HCT116 cells were treated with 1 μM pemetrexed in the presence of 5.6 μM thymidine to offset the effects of TS inhibition. The phosphorylation of T172 on AMPKα was detected by western blot (upper panel), and the kinase activity of immunoprecipitated AMPKα1 was measured using SAMS peptide as a substrate (lower panel). (B) The secondary growth-inhibitory mechanism of pemetrexed correlates with ZMP accumulation. HCT116 cells were treated with pemetrexed for 72 hours in the absence (circles) or presence (open triangles) of thymidine. ZMP (closed triangles) was measured by HPLC following 15 hr exposure to pemetrexed and thymidine, and measured values in nmol/10⁶ cells were converted to mM concentrations in cell water. (C) siRNA knockdown (KD) of AICART mimics the effects of pemetrexed on AMPKα phosphorylation in HCT116 cells. (D) Accumulation of ZMP in HCT116 cells transfected with a pool of siRNAs against AICART. HCT116 cells were transfected with Dharmacon smart pools of AICART or scrambled siRNAs for 24 hours. Cells were lysed after 72 hours and ZMP assayed by HPLC. Where indicated, cells were treated with 100 μM AICAR for 4 hr immediately before harvest. The asterisks indicate values that were below the limits of the assay (< 0.001 mM). For details, see Materials and Methods.

Figure 2. mTORC1 inhibition by pemetrexed is mediated through AMPK activation

(A) Pemetrexed-induced activation of AMPK inhibits mTORC1 signaling. Immunoblots on HCT116 cells treated with pemetrexed for 15 hr were probed for Raptor p-S792 and for p-T389 of S6K1. (B) The AMPKα inhibitor, compound C (1 μM), blocks the effects of pemetrexed on mTORC1 signaling following 15 hr exposure to both agents in HCT116 cells. All treatments contained 0.1% DMSO and thymidine. (C) Depletion of AMPKα1 by siRNA knockdown (KD) in HCT116 cells blocks the effects of pemetrexed on mTORC1 signaling. siRNA pools were added to HCT116 cells for 24 hr and protein was harvested after 72 hr. Pemetrexed and thymidine were present during the last 15 hr.

Figure 3. Signaling events dependent on AMPK activity are maintained in LKB1-null carcinoma cells exposed to pemetrexed

(A) Sensitivity of carcinoma cells to pemetrexed-induced AMPK activation and to rapamycin. Cells were exposed to pemetrexed and thymidine or to rapamycin (25 nM) for 72 hr. The genotypes (Sanger Institute Cancer Cell Line Project http://www.sanger.ac.uk/genetics/CGP/CellLines/) is listed as mutant (M) for LKB1, PI3K, and KRAS. (B) Sensitivity of LKB1-null cell lines to growth inhibition after 72 hr exposure to pemetrexed in the presence of thymidine. LKB1 expression was assessed by immunoblot. (C) Signaling downstream of mTORC1 is inhibited in LKB1-null carcinoma cells following pemetrexed. (D) AMPK activation mediates mTORC1 inhibition in LKB1-null cells. Inhibition of S6K1 phosphorylation by pemetrexed is prevented by 1 μM compound C in H460 cells. (E-F) AMPK activation and ACC and eEF2 phosphorylation in cells of wild-type (HCT116) and -null (H460) LKB1 status following 1 μM pemetrexed plus thymidine or 250 μM AICAR for 15 hr.

Figure 4. Reactivation of mTORC1-dependent processes in response to relief of feedback inhibition of AKT activity following pemetrexed

(A) Time course of ZMP accumulation and AMPKα T172 phosphorylation in response to pemetrexed and thymidine in HCT116 cells. (B) mTORC1-dependent phosphorylation of 4E-BP1 is inhibited in pemetrexed-treated HCT116 cells, partially recovering by 48 hr (top panel). This is accompanied by substantial accumulation of hypophosphorylated 4E-BP1, which abundantly binds to a m⁷GTP sepharose bead (bottom panel). In the bottom panel, TdR and DMSO controls were for 24 hr. (C) Repression of S6K1 phosphorylation partially recovers following pemetrexed and thymidine treatment in HCT116 and LKB1-null H460 cells. (D) Recovery of mTORC1 signaling is concurrent with release of feedback inhibition of PI3K and AKT in HCT116 cells, as judged by phosphorylation of S6K1 and 4E-BP1. (E) AKT reactivation mediates recovery of mTORC1 signaling after pemetrexed. HCT116 cells were treated with pemetrexed and GSK690693 (100 nM) in the presence of thymidine for the denoted times. (F) The cytotoxicity of pemetrexed and GSK690693 in combination for 72 hr. Representative clonogenic survival plates are shown in the top panel and enumeration of colonies in the bottom panel.