Monoacylglycerol lipase exerts dual control over endocannabinoid and fatty acid pathways to support prostate cancer

Abstract

Cancer cells couple heightened lipogenesis with lipolysis to produce fatty acid networks that support malignancy. Monoacylglycerol lipase (MAGL) plays a principal role in this process by converting monoglycerides, including the endocannabinoid 2-arachidonoylglycerol (2-AG), to free fatty acids. Here, we show that MAGL is elevated in androgen-independent versus androgen-dependent human prostate cancer cell lines, and that pharmacological or RNA-interference disruption of this enzyme impairs prostate cancer aggressiveness. These effects were partially reversed by treatment with fatty acids or a cannabinoid receptor-1 (CB1) antagonist, and fully reversed by cotreatment with both agents. We further show that MAGL is part of a gene signature correlated with epithelial-to-mesenchymal transition and the stem-like properties of cancer cells, supporting a role for this enzyme in protumorigenic metabolism that, for prostate cancer, involves the dual control of endocannabinoid and fatty acid pathways.

Figure 1. MAGL is elevated in androgen-independent prostate cancer cells where it regulates monoacylglycerol and free fatty acid metabolism

(A) ABPP of serine hydrolase activities in the androgen-dependent LNCaP and androgen-independent PC3 and DU145 cells lines. Serine hydrolase activities were labeled in whole cell protoemes with the activity-based probe FP-rhodamine and detected by SDS-PAGE and in-gel fluorescence scanning (fluorescent gel shown in greyscale). MAGL and KIAA1363 are elevated in PC3 and DU145 cells compared to LNCaP cells. Proteomes were also prepared from cancer cells pretreated with DMSO or the selective MAGL inhibitor JZL184 (1 µM, 4 h in situ) to confirm the identities of the 33 and 35 kDa bands as MAGL. (B) The left panel shows C20:4 MAG hydrolytic activity of cancer cells in the presence or absence of JZL184 (1 µM, 4 h in situ). The middle panel shows MAGL-specific activity derived from subtracting the JZL184-insensitive portion from total MAG hydrolytic activity. The right panel shows spectral counts for MAGL in proteomes treated with FP-biotin and subjected to ABPP-MudPIT. (C,D) Inhibition of MAGL by JZL184 (1 µM, 4 h, in situ) raises MAG (C) and lowers FFA (D) levels in PC3 cells. (E–H) JZL184-treated PC3 cells also show elevations in lysophosphatidyl cholines (LPCs) (E), and reductions in phosphatidic acids (PAs) (F), lysophosphatidic acid (LPA) (G), and lysophosphatidyl ethanolamines (LPEs) (H). *p<0.05, **p<0.01 PC3 or DU145 versus LNCaP cells for (B) and JZL184-treated versus DMSO-treated control groups for (C, D). Data are presented as means ± standard error of the mean (SEM); n=4–5/group. See also Table S1, Table S2, and Figure S1.

Figure 2. Stable shRNA-mediated knockdown of MAGL lowers FFA levels in PC3 cells

(A) MAGL was stably knocked down using two independent short-hairpin RNA (shRNA) oligonucleotides (shMAGL1 and shMAGL2), resulting in >75 % reduction in MAGL activity in PC3 cells, as assessed by ABPP analysis of PC3 soluble proteomes, compared to shControl cells expressing an shRNA that targets a distinct serine hydrolase (DPP4). (B) Total C20:4 MAG hydrolytic activity of parental, shControl, and shMAGL1 and 2 PC3 whole cell lysate proteomes show significantly reduced MAGL activity in shMAGL cancer cells. (C, D) shMAGL cells show elevations in MAGs (C) and reductions in FFAs (D). The MAGL activity and MAG and FFA levels of shControl cells did not differ significantly from those of parental cancer cell lines. (E) Lipidomic analysis of PC3 shMAGL versus shControl cells shows not only elevations in MAGs and reductions in FFAs, but also lower levels of lysophosphatidyl ethanolamines (LPEs), phosphatidic acids (PAs), and lysophosphatidic acid (LPA). *p<0.05, **p<0.01 for shMAGL versus shControl groups. Data are presented as means ± SEM; n=4–5/group. See also Figure S1.

Figure 3. Pharmacological and shRNA blockade of MAGL impairs PC3 aggressiveness

(A–C, D–F) Both shMAGL and JZL184 (1 µM) PC3 cells show impaired migration (A, D), invasion (B, E), and serum-free survival (C, F). Cancer cells were pretreated with JZL184 in serum-free media for 4 h before migration (5 h migration time) and invasion (24 h invasion time) assays and 24 h before cell survival (20 h in serum and 4 h in serum-free media with JZL184). For PC3 migration, representative fields of migrated cells are shown at 200 × magnification. (G) Pharmacological (40 mg/kg JZL184, daily oral gavage) inhibition of MAGL causes impairments in PC3 tumor xenograft growth in immune-deficient SCID mice. Representative tumors are shown on the right. **p<0.01 for shMAGL versus shControl or JZL184 versus vehicle treatment groups. Data are presented as means ± SEM. For (A,B,D,E), n=4–5/group and for (C,F,G), n=6–8/group. See Figure S2 and Figure S3.

Figure 4. A combination of fatty acid and CB1 antagonist rescues the impaired migration and tumor growth of MAGL-disrupted PC3 cells

(A, B) The migratory defects observed with JZL184 (A) and in shMAGL (B) PC3 cells are partially rescued upon co-treatment with the CB1 receptor antagonist rimonabant (1 µM) or palmitic acid (10 µM), denoted as FFA in the figure), and fully rescued upon addition of both. (C) Tumor growth defects observed with shMAGL PC3 cells are also partially recovered upon daily administration of rimonabant (3 mg/kg, oral gavage) or high-fat diet (60 kcal % diet) and fully rescued with both. **p<0.01 for shMAGL or JZL184 groups versus control or shControl groups; ## p<0.01 for JZL184 or shMAGL groups treated with RIM, FFA, and/or HFD versus shMAGL or JZL184 groups. Data are presented as means ± SEM. For (A, B), n=4–5/group and for (C), n=5–8 mice/group. See Figure S4.

Figure 5. Gene signatures of aggressive human cancer cell lines

(A) Transcriptional profiling of aggressive (noted in red, PC3, DU145, C8161, SKOV, and 231MFP) versus non-aggressive (noted in blue, LNCaP, MUM2C, OVCAR3, and MCF7) cancer cell lines yields commonly dysregulated genes (left) (>3-fold changes in 4 out of 5 pairs of cell lines). (B) Several EMT and cancer stem cell markers are found among genes consistently elevated in aggressive cancer lines. (C) Metabolic enzymes consistently elevated (upper panel) or reduced (lower panel) in aggressive cancer lines. Heat maps were generated using Gene Tree View obtained from http://rana.lbl.gov/EisenSoftware.htm. Blue versus white denotes high versus low relative mRNA expression of each gene, respectively. Relative mRNA expression, as expressed by blue color, was normalized to the highest gene chip signal for each gene across the 9 cancer cell lines profiled. See also Table S3.