FAAH inhibition enhances anandamide mediated anti-tumorigenic effects in non-small cell lung cancer by downregulating the EGF/EGFR pathway

Abstract

The endocannabinoid anandamide (AEA), a neurotransmitter was shown to have anti-cancer effects. Fatty acid amide hydrolase (FAAH) metabolizes AEA and decreases its anti-tumorigenic activity. In this study, we have analyzed the role of FAAH inhibition in non-small cell lung cancer (NSCLC). We have shown that FAAH and CB1 receptor which is activated by AEA are expressed in lung adenocarcinoma patient samples and NSCLC cell lines A549 and H460. Since the synthetic analogue of anandamide (Met-F-AEA) did not possess significant anti-tumorigenic effects, we used Met-F-AEA in combination with FAAH inhibitor URB597 which significantly reduced EGF (epidermal growth factor)-induced proliferative and chemotactic activities in vitro when compared to anti-tumorigenic activity of Met-F-AEA alone. Further analysis of signaling mechanisms revealed that Met-F-AEA in combination with URB597 inhibits activation of EGFR and its downstream signaling ERK, AKT and NF-kB. In addition, it inhibited MMP2 secretion and stress fiber formation. We have also shown that the Met-F-AEA in combination with URB597 induces G0/G1 cell cycle arrest by downregulating cyclin D1 and CDK4 expressions, ultimately leading to apoptosis via activation of caspase-9 and PARP. Furthermore, the combination treatment inhibited tumor growth in a xenograft nude mouse model system. Tumors derived from Met-F-AEA and URB597 combination treated mice showed reduced EGFR, AKT and ERK activation and MMP2/MMP9 expressions when compared to Met-F-AEA or URB597 alone. Taken together, these data suggest in EGFR overexpressing NSCLC that the combination of Met-F-AEA with FAAH inhibitor resulted in superior therapeutic response compared to individual compound activity alone.

Figure 1. NSCLC cell lines and primary lung cancer tissues express CB1 and FAAH

(A) NSCLC cell lines were subjected to immunoblot analysis to determine the expression of CB1 and FAAH. GAPDH served as loading control. Representative photomicrographs of IHC staining of FAAH expression in lung adenocarcinoma (B) and respiratory epithelia (C). Representative photomicrographs of IHC staining of CB1 expression in lung adenocarcinoma (D) and respiratory epithelia (E).

Figure 2. FAAH inhibition enhances the anti-proliferative activity of Met-F-AEA in NSCLC cell lines

A549 (A) and H460 (B) cells were serum starved for 24h and treated with control, Met-F-AEA (MetF, 10μM), FAAH inhibitor URB597(Inh, 0.2μM) or MetF+Inh and analyzed for viability by MTT assay. 1000 individual A549 (C) and H460 (D) cells were plated in six well plates and subjected to colony formation assay by treating with control, Met-F-AEA (MetF, 10μM), FAAH inhibitor URB597 (Inh, 0.2μM) or MetF+Inh as shown for six days. The colonies were then fixed, stained and counted. (E) H460 cells were transfected with 100pmol of either the non targeting control (vector) or FAAH-siRNA (Dharmacon) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions for 36h and the expression of FAAH in siRNA and non targeted cells were evaluated by Western blot. (F) H460 cells which were transfected with either FAAH siRNA or non targeted control (vector) for 36h were treated with Met-F-AEA for 24h and subjected to MTT assay. P<0.05 (*) and P<0.005 (**) as calculated by Student's t test. Data represent the mean ± SD per experimental group.

Figure 3. FAAH inhibition enhances the anti-migratory and anti-invasive activities of Met-F-AEA in NSCLC cell lines

A549 (A) and H460 (B) cells were treated with control, Met-F-AEA (MetF, 10μM), FAAH inhibitor URB597 (Inh, 0.2μM) or MetF+Inh for 24h and subjected to EGF (100ng/ml)-induced migration using transwell plates. The number of cells migrated were stained with Hema stain and counted in five different fields. A549 (C) and H460 (D) cells were treated with control, Met-F-AEA (MetF, 10μM), FAAH inhibitor URB597 (Inh, 0.2μM) or MetF+Inh for 24h and subjected to EGF (100ng/ml)-induced invasion assay using transwell plates coated with matrigel. Invaded cells were stained with Hema stain and counted. (E) Confocal microscopy visualization of A549 cells treated with control, Met-F-AEA (MetF, 10μM), FAAH inhibitor URB597 (Inh, 0.2μM) or MetF+Inh for 24h and stimulated with EGF (100ng/ml) and stained for phalloidin (red), vinculin (green) expression and DAPI (blue). (F) A549 (upper panel) and H460 (lower panel) cells were treated with control, Met-F-AEA (MetF, 10μM), FAAH inhibitor URB597(Inh, 0.2μM) or MetF+Inh for 48h and the supernatants were collected, concentrated and run on zymogram gels to detect the active form of MMP2. P<0.05 (*) as calculated by Student's t test. Graphs represent the mean ± SD for each experiment repeated three times with similar results.

Figure 4. FAAH inhibition enhances Met-F-AEA mediated inhibition of EGFR signaling in NSCLC cell lines

A549 (A) and H460 (B) cells were treated with control, Met-F-AEA (MetF, 10μM), FAAH inhibitor URB597(Inh, 0.2μM) or MetF+Inh for 24h and transfected with either wild-type or NF-kB plasmid for 24h, stimulated with EGF (100ng/ml) for additional 24h, lysed and analyzed for luciferase activity. Renilla luciferase vector served as internal control. A549 (C) and H460 (D) cells were treated with control, Met-F-AEA (MetF, 10μM), FAAH inhibitor URB597(Inh, 0.2μM) or MetF+Inh for 24h, stimulated with EGF (100ng/ml) for 0 and 15 min and subjected to Western blot analysis to determine expression of phopho-EGFR, ERK or AKT (P-EGFR, P-ERK, P-AKT) and total EGFR, ERK and AKT (T-EGFR, T-ERK, T-AKT). (E) H460 cells which were transfected with either FAAH siRNA or non targeted control (vector) for 36h were treated with either Met-F-AEA or control for 24h, stimulated with EGF (100ng/ml) for 0 and 15 min and analyzed by Western blotting to determine expression levels of phopho-ERK or AKT (P-ERK, P-AKT) and total ERK and AKT (T-ERK, T-AKT). P<0.05 (*) as calculated by Student's t test. Data represent the mean ± SD for each experiment repeated three times with similar results.

Figure 5. FAAH inhibition enhances Met-F-AEA induced cell cycle arrest and apoptosis at later stage

(A) A549 cells were pre-treated with control, Met-F-AEA (MetF, 10μM), FAAH inhibitor URB597 (Inh, 0.2μM) or MetF+Inh for 48h, stained with propidium iodide and analyzed for cell cycle arrest by flow cytometry. (B) Representative images of TUNEL positive cells in H460 cells. H460 (C) and A549 (D) cells were pre-treated with control, Met-F-AEA (MetF, 10μM), FAAH inhibitor URB597 (Inh, 0.2μM) or MetF+Inh for 48h and subjected to TUNEL assay. The percentage of apoptotic cells was calculated as the percentage of fluorescent positive cells. A549 (E) and H460 (F) cells were pre-treated with control (1), Met-F-AEA (2), FAAH inhibitor URB597(3) or MetF+Inh (4) for 48h and subjected to Immunoblot analysis to determine the expression of cell cycle markers cyclin D1, CDK4 and apoptotic markers pro-caspase-9 and pro-PARP. GAPDH served as loading control. P<0.05 (*) as calculated by Student's t test. Data represent the mean ± SD for each experiment repeated three times with similar results.

Figure 6. FAAH inhibition enhances Met-F-AEA mediated inhibition of NSCLC tumor growth in vivo by downregulating EGFR signaling

(A) H460 cells were subcutaneously injected in nude mice and palpable tumors were treated with control, Met-F-AEA (MetF, 5mg/kg), FAAH inhibitor URB597 (Inh, 1mg/kg) or MetF+Inh every third day for 3 weeks. Tumor volume was assessed periodically and calculated using the formula= length × (width) ² /2. (B) Tumor weight measured various experimental groups. (C) Representative tumors dissected from various experimental groups. (D) Representative photomicrographs of H&E and (E) Ki67 staining of tumors extracted from various experimental groups. (F) The number of Ki67 positive cells was counted in four different fields using bright field microscopy in each experimental group and the average was calculated. (G) Xenograft tumors isolated from (A) were subjected to Western blot analysis to determine expression of phospho-EGFR, ERK or AKT (P-EGFR, P-ERK, P-AKT) and total EGFR, ERK and AKT (T-EGFR, T-ERK, T-AKT). Control, Met-F-AEA (MetF), FAAH inhibitor URB597(Inh) or MetF+Inh. (H) Xenograft tumors were subjected to Real Time PCR analysis to determine the expression of MMP2 and MMP9. P<0.05 (*) and P<0.005 (**) as calculated by Student's t test. Data represent the mean ± SD for each experiment repeated three times with similar results.