Fig 1: Expression of fatty acid amide hydrolase (FAAH) in ReN cells under conditions of oxidative stress. Exposure of ReN cells to sublethal concentrations of tert-butylhydroperoxide (tBHP) led to an increase in FAAH expression (A,B). Green fluorescence is FAAH and blue fluorescence is DAPI. (C,D) Western blot analysis reveals that the expression of a 63 kDa band corresponding to FAAH is unaffected by exposure to 2 µM tBHP but upregulated 1.5-fold by 10 µM tBHP. The expected molecular weight of FAAH is 63 kDa. (E,F) Colocalization analysis between FAAH immunoreactivity with DAPI fluorescence (DNA) reveals low colocalization at resting untreated conditions (R = 0.18), but 2 µM tBHP exposure led to a 68% increase in Pearson’s R (from 0.17 to 0.28). Higher tBHP concentrations (10 µM) led to a large (3.6-fold) increase in FAAH-DAPI colocalization (from R of 0.17 to 0.62), suggesting significant FAAH localization in the nucleus near DNA. *p = 0.05, **p = 0.01.
Fig 2: Low FAAH expression in breast tumors is associated with poor patient prognosis.Kaplan-Meier curves for metastasis-free survival (a) and overall survival (b, c) in BC samples with high and low FAAH expression obtained from TMA #1 (a, b) and the METABRIC database15 (c). Kaplan-Meier curves were statistically compared by the log-rank test. d Matched FAAH expression in samples of primary tumor and lymph node (LN) metastasis obtained from TMA #2. Data were analyzed by 2-tailed Student’s t-test. e Representative IHC images (n > 10 biological replicates) showing FAAH expression decrease in LN metastasis compared to primary tumor. Scale bar = 250 µm. f Pie chart representing variations in FAAH expression from primary tumor to LN metastasis in TMA #2. g Kaplan-Meier curves for overall survival from patients included in TMA #2 according to the variations of FAAH expression from the primary tumor to the LN. Kaplan-Meier curves were statistically compared by the log-rank test. Source data are provided as a Source Data file.
Fig 3: FAAH is highly expressed in differentiated breast tumors.Relative FAAH mRNA expression along the four molecular subtypes of BC according to the TCGA16 (a) and METABRIC15 (b) datasets. Data were analyzed by 1-way ANOVA with post Tukey’s multiple comparison test. c Representative images (n > 10 biological replicates) showing FAAH expression scoring according to intensity staining in samples from TMA #1. Scores 0, 1, 2, and 3 correspond to no, weak, moderate, and high staining, respectively. Scale bar = 500 µm. d Association between FAAH expression and the molecular features of breast tumor samples included in TMA #1. Pearson’s chi-squared test was used for statistical analysis. Source data are provided as a Source Data file.
Fig 4: FAAH genetic silencing promotes tumor progression and lung metastasis in a mouse model of spontaneous breast tumor formation.a, b Experimental setup. c Tumor growth 50 days after first tumor arousal. Data were obtained from T2 and T3 mice. Data are presented as mean values ± SEM and were analyzed by two-tailed Student’s t-test. d Immunofluorescence analysis of MMTV-neu-derived tumors for cytokeratin 8 (CK8, red) and cytokeratin 14 (CK14, green). Data obtained from T3 mice (n = 3 biological replicates). Cell nuclei are stained in blue. Scale bar = 50 µm. e Percentage of animals with lung metastases at the time points established in panel b. f Number of metastasis per animal at T3. Data were analyzed by 2-tailed Student’s t-test. g Representative H&E images of lung metastases at T3 (n > 10 biological replicates). Scale bar = 250 µm. h Volcano plot showing data of the RT2 Profiler PCR array of mouse BC. Upregulated and downregulated genes in MMTV-neu:FAAH -/- derived tumors with a log2 fold change =0.58 (i.e., fold change =1.5) are depicted in yellow and blue, respectively, and statistically significant genes are over the horizontal line at -log10 p value = 1.3 (i.e., p = 0.05). Each point is the result of the mean of n = 3 biological replicates mice at T3. Source data are provided as a Source Data file.
Fig 5: FAAH modulation in BC cells alters the levels of invasion and metastasis-related genes.a Heatmap showing fold-change expression of the differentially expressed genes (DEGs = 491) in the RNA-seq of FAAH-modulated T-47D cells. DEGs considered in this analysis were only those significantly deregulated in T-47D FAAH KO cells vs. parental cells that also recovered their original levels in the rescue cells. b Volcano plot showing some of the top DEGs (downregulated in red, upregulated in green) in T-47D FAAH KO cells compared to parental/rescued cells. Dashed blue lines establish inclusion cut-off values at -log10 false discovery rate (FDR) =1.3 (i.e., FDR =0.05) and log2 fold change =0.58 (i.e., fold change =1.5). Some relevant upregulated and downregulated DEGs are shown in green and red, respectively. c Bar graph representing Gene Ontology (GO) Cellular Component (CC) analysis of DEGs in T-47D FAAH KO cells compared to parental/rescue cells. d Gene Set Enrichment Analysis (GSEA) of DEGs in T-47D FAAH KO cells compared to parental/rescue cells. e Bar graph showing results of the RT2 Profiler PCR array of human tumor metastasis in FAAH-modulated MDA-MB-231 cells. The expression of BC metastasis-related downregulated genes in FAAH-overexpressing MDA-MB-231 cells is depicted as log2 fold change vs. parental cells. Data are presented as mean values ± SEM of n = 3 biological replicates and were analyzed by two-tailed Student’s t-test. Source data are provided as a Source Data file.
Supplier Page from Abcam for Anti-FAAH1 antibody [EPR7549]