The fatty-acid amide hydrolase inhibitor URB597 inhibits MICA/B shedding

MICA/B proteins are expressed on the surface of various types of stressed cells, including cancer cells. Cytotoxic lymphocytes expressing natural killer group 2D (NKG2D) receptor recognize MICA/B and eliminate the cells. However, cancer cells evade such immune recognition by inducing proteolytic shedding of MICA/B proteins. Therefore, preventing the shedding of MICA/B proteins could enhance antitumor immunity. Here, by screening a protease inhibitor library, we found that the fatty-acid amide hydrolase (FAAH) inhibitor, URB597, suppresses the shedding of MICA/B. URB597 significantly reduced the soluble MICA level in culture medium and increased the MICA level on the surface of cancer cells. The effect was indirect, being mediated by increased expression of tissue inhibitor of metalloproteinases 3 (TIMP3). Knockdown of TIMP3 expression reversed the effect of URB597, confirming that TIMP3 is required for the MICA shedding inhibition by URB597. In contrast, FAAH overexpression reduced TIMP3 expression and the cell-surface MICA level and increased the soluble MICA level. These results suggest that inhibition of FAAH could prevent human cancer cell evasion of immune-mediated clearance.

www.nature.com/scientificreports/ production of smaller sized MICA proteins, suggesting cleavage by proteases (Fig. 2b), addition of URB597 did not affect the size (Fig. 2b). Therefore, the effect of URB597 on MICA/B shedding is indirect and likely mediated by changes in gene expression.
Tissue inhibitor of metalloproteinases 3 (TIMP3) expression is associated with the effect of URB597 on MICA/B shedding. Because URB597 did not appear to regulate MICA/B shedding directly, its effect on gene expression was determined using a genome-wide cDNA array. Among the genes upregulated by URB597, we selected SERPINA3, SERPINA7, SERPIND1, and TIMP3 as candidate genes (Fig. 3a), because they are related to proteolytic pathways. Among them, TIMP3 expression was increased by URB597 in a dosedependent manner (Fig. 3b).
To confirm the involvement of TIMP3 in MICA/B shedding, we established stably TIMP3-specific shorthairpin (sh) RNA-expressing cells (Fig. 3c). Although the MICA/B mRNA levels were not changed in these cells (Fig. 3d), surface MICA/B expression was significantly decreased (Fig. 3e). Moreover, the soluble MICA/B levels were significantly increased by knockdown of TIMP3 (Fig. 3f). Importantly, in TIMP3-knockdown cells, URB597 had no effect on MICA shedding (Fig. 3e,f). Therefore, the increased TIMP3 expression induced by URB597 inhibits MICA/B shedding.

URB597-induced inhibition of MICA/B shedding is dependent on FAAH.
Although URB597 is a known FAAH inhibitor and it efficiently suppressed FAAH activity in our experimental setting (Supplementary Figure S1), whether its inhibition of MICA/B shedding is dependent on FAAH is unclear. To examine this, we first established stably FAAH-specific shRNA-expressing HepG2 cells (Fig. 4a). In the same manner as with URB597, FAAH knockdown led to the upregulation of TIMP3 mRNA levels and surface MICA/B protein levels and the downregulation of soluble MICA/B levels without influencing MICA/B transcriptional activities ( Fig. 4b-e). The same results were obtained in Hep3B cells (Supplementary Figure S2). Consistent with these results, blocking of FAAH by another inhibitor, PF-3845, yielded similar results (Supplementary Figure S3). We then established stably FLAG-tagged FAAH-expressing cells (Fig. 5a). Interestingly, the TIMP3 mRNA level was significantly decreased in these cells (Fig. 5b). Moreover, although the MICA/B mRNA levels were unchanged (Fig. 5c), the surface MICA/B protein levels were significantly decreased in FLAG-tagged FAAH-overexpressing cells (Fig. 5d), and the soluble MICA/B levels were significantly increased (Fig. 5e). Therefore, FAAH promotes MICA/B shedding at least in part by modulating TIMP3 expression. TIMP3 expression and MICA/B shedding are regulated by the endocannabinoid system. Because FAAH is the primary enzyme responsible for hydrolyzing bioactive amides including anandamide, an agonist of cannabinoid receptor types I and II, TIMP3 activation by URB597 may be involved in activation of the endocannabinoid system. Supporting this hypothesis, anandamide elevated TIMP3 expression levels and cell surface MICA/B levels without influencing the transcriptional activity of MICA/B (Fig. 6). Although URB597 was reported not to increase 2-arachidonoyl glycerol (2-AG) levels 24 , 2-AG is a well-known endocannabinoid and administration of 2-AG resulted in enhanced TIMP3 expression and cell surface MICA/B levels (Supplementary Figure S4). Moreover, a CB1 receptor agonist, AM1241, activated TIMP3 expression and inhibited MICA/B shedding (Supplementary Figure S5). As expected, another CB1 receptor antagonist, rimonabant, partially but significantly attenuated the effect of URB597 on TIMP3 expression and MICA/B shedding (Fig. 7). These results suggest that the increased endocannabinoid is responsible for overexpressing TIMP3 and inhibiting MICA/B shedding. (a) A plot of the distribution of data from a cDNA microarray. The X-and Y-axes represent the average values of the log2-converted expression levels and the differences of log2-converted expression levels, respectively. SERPINA3, SERPINA7, SERPIND1, and TIMP3 (red) were identified as candidate inhibitors of MICA/B shedding. (b) qRT-PCR analysis of the TIMP3 mRNA level in HepG2 cells after treatment with URB597 or DMSO (control). Data are means ± SD from three independent experiments. A P-value of 0.44 (two-sided Welch's t-test) was not considered to indicate significance *P = 7.4 × 10 −5 ; *P = 2.0 × 10 −4 ; ***P = 3.3 × 10 −5 (twosided Welch's t-test). (c) Western blots of lysates from HepG2 cells without or with stable TIMP3-specific shRNA expression (HepG2 shTIMP3 ). Representative images of three independent experiments are shown. (d) qRT-PCR analysis of the MICA/B mRNA levels in HepG2 and HepG2 shTIMP3 cells. Data are means ± SD from three independent experiments. A P-value of 0.22 (two-sided Welch's t-test) was not considered to indicate significance. (e) Flow cytometry of surface MICA protein levels in HepG2 cells treated with DMSO (black line) and HepG2 shTIMP3 cells treated with DMSO (blue line) or URB597 (blue line). Gray-shaded histograms represent background staining with the isotype IgG. Representative results from three independent experiments are shown. Relative MFI data (n = 3) are shown in the right panel. Data are means ± SD. A P-value of 0.44 (twosided Welch's t-test) was not considered to indicate significance. *P = 0.027 (two-sided Welch's t-test). (f) Soluble MICA/B quantification by NanoLuc assay in the culture medium of HepG2 or HepG2 shTIMP3 cells treated with DMSO or URB597. Data are means ± SD from three independent experiments. A P-value of 0.40 (two-sided Welch's t-test) was not considered to indicate significance. *P = 0.0020 (two-sided Welch's t-test).

Discussion
We found that URB597, an inhibitor of FAAH, suppresses MICA/B shedding by increasing TIMP3 expression. As shown in our genome-wide association study, MICA gene polymorphisms and MICA protein levels are significantly correlated with the incidence of HCC in patients with chronic hepatitis 4 , an effect possibly mediated by modulating the elimination of pre-malignant cells by cytotoxic lymphocytes 1,2 . Therefore, MICA/B-mediated oncoimmunity is crucial in HCC oncogenesis. Tumors, including HCC, evade immune recognition by proteolytic shedding of MICA/B. Although a disulfide isomerase (ERp5), a matrix metalloproteinase (MMP), and a disintegrin and metalloproteinase (ADAM) family member have already been identified as promoters of MICA/B shedding 25-28 , we identified a novel candidate inhibitor of MICA/B shedding. This may be advantageous because a high serum MICA concentration is associated with the progression of several human cancers 29 . URB597, [3-(3-carbamoylphenyl)phenyl]N-cyclohexylcarbamate, is a selective inhibitor of FAAH. FAAH is the primary enzyme that degrades anandamide, an agonist of cannabinoid receptor types I and II. Therefore, inhibition of FAAH results in activation of cannabinoid receptor-mediated intracellular signaling pathways 30 . www.nature.com/scientificreports/ Indeed, URB597 elevates anandamide levels in vitro and in vivo 30,31 and exerts antinociceptive and anxiolytic effects in vivo 32 . In addition, cannabinoid-mediated signaling exerts an antitumor effect in several cancers [33][34][35][36] . However, the underlying mechanisms are unclear 37 . The inhibition of MICA/B shedding by URB597 may enhance the immune surveillance of cancer cells, which may explain the antitumor effect of cannabinoid-mediated signaling. If so, this would facilitate the development of novel immunotherapeutics for cancer. The inhibition of MICA/B shedding by URB597 was dependent on TIMP3. Because knockdown of TIMP3 led to increased MICA/B shedding, and URB597 significantly increased TIMP3 expression, URB597 inhibition of MICA/B shedding was likely mediated by increased TIMP3 expression. Because TIMPs (TIMP1, TIMP2, TIMP3, and TIMP4) are inhibitors of MMPs, which induce MICA/B shedding 26,27 , the suppression of MICA/B shedding caused by increased TIMP3 expression may be mediated by MMP inhibition 38 . Indeed, in acute myeloid leukemia cells, the shedding of MICA/B was significantly inhibited by increased expression of TIMP3 38 . Moreover, in patients with acute myeloid leukemia, low TIMP3 expression is significantly associated with an adverse cytogenetic prognosis, possibly due to decreased NK-cell-mediated immune recognition caused by increased The TIMP3 mRNA level was significantly upregulated by URB597. Gene expression is regulated by various mechanisms, such as promoter activity modulation, epigenetic modifications, chromatin regulation, and posttranscriptional mechanisms involving microRNAs. Because FAAH is involved in diverse intracellular signaling 42 , and URB597 may have effects other than FAAH inhibition, the mechanism by which URB597 increases TIMP3 mRNA expression is unclear. Nonetheless, the finding is intriguing, because regulation of MMP function is implicated in a variety of physiological and pathological conditions 43 .
In summary, URB597, an FAAH inhibitor, suppresses MICA/B shedding by increasing TIMP3 expression. Because MICA/B is a regulator of antitumor immunity 1-3 and the FAAH-mediated pathway is under evaluation www.nature.com/scientificreports/ as a target for neurological conditions, for example, post-traumatic stress disorder 44,45 , application of FAAH inhibitors in the cancer therapeutic field as a drug repositioning strategy may be a practical method to enhance anti-tumor immunotherapy.
The NanoLuc-conjugated MICA-expressing plasmid was constructed using a two-step cloning method described previously 23 . First, NanoLuc cDNA, amplified by polymerase chain reaction (PCR) using the pNL1.1 NanoLuc vector (Promega, Madison, WI) as the template, was cloned into the XbaI site of the pCDH vector (System Biosciences) using the In-fusion method (Clontech). Next, MICA cDNA, PCR amplified from a Halotagged MICA expression vector (Promega), was inserted immediately downstream of the NanoLuc cDNA at the EcoRI site of the pCDH NanoLuc vector. The following primers were used: 5′-CAT AGA AGA TTC TAG GCC ACC ATG GTC TTC ACA CTC -3′ and 5′-ATT CGC TAG CTC TAG CGC CAG AAT GCG TTC GCA CAG -3′ for NanoLuc cloning, and 5′-TAG AGC TAG CGA ATT CCA TGG GGC TGG GCC CGGT-3′ and 5′-ATT TAA ATT CGA ATT CTT AAA CGG CGC CCT CAG TGG A-3′ for MICA cloning.

Transfection and lentiviral transduction. Transient transfections were performed using Effectene
Transfection Reagent (Qiagen, Hilden, Germany). To generate polyclonal cells with stable Flag-FAAH expression, the Lentivirus Packaging System (System Biosciences) was used according to the manufacturer's instructions as described previously 47 . Briefly, 1.0 µg Flag-FAAH overexpression vector and 5.0 µg pPACKH1 packaging plasmid mix were transfected into HEK293T cells. After 24 h, the collected culture medium was mixed with one-fifth of its volume of PEG-it Reagent (System Biosciences) and incubated overnight at 4 °C to concentrate the virus. After centrifugation, the pellet was resuspended in 1 × phosphate-buffered saline (PBS). The viruses were transduced into HepG2 cells using polybrene (Santa Cruz Biotechnology, Dallas, TX), followed by selection with 6 mg/mL puromycin to obtain polyclonal cells stably expressing Flag-FAAH.
Flow cytometry. Flow cytometry was performed as described previously 21 . Briefly, cells were hybridized with anti-MICA (1:500; R&D Systems, Minneapolis, MN) or an isotype control IgG (1:500; R&D Systems) for 40 min at 4 °C. After washing, the cells were incubated with a goat anti-mouse antibody conjugated to Alexa Fluor 488 (1:1,000; Molecular Probes, Eugene, OR) for 20 min. Flow cytometry was performed, and the data were analyzed using Guava Easy Cyte Plus (GE Healthcare, Little Chalfont, UK). Mean fluorescence intensity (MFI) was used to estimate the statistical significance.
NanoLuc MICA shedding assay. A NanoLuc MICA shedding assay was performed as reported previously with slight modifications 23 . Briefly, cells seeded onto 10 cm dishes were transfected with 2.0 µg N-terminal NanoLuc-tagged MICA-expressing plasmid, incubated for 12 h, reseeded onto a 96-well plate, and incubated for another 12 h. After application of URB597 or DMSO as the control, the culture medium was collected and mixed with the luminescent substrate (Nano-Glo Luciferase Assay System; Promega), and luminescence values (Luc sup ) were measured using the GloMax Detection System (Promega). The cells were washed with PBS, and the luminescent substrate (Promega) was added to determine the luminescence values (Luc cell ) using the GloMax Detection System. Soluble MICA/B levels were determined as Luc sup /(Luc sup + Luc cell ) to adjust for transfection efficiency.
In vitro assay of MICA shedding. Recombinant  Genome-wide cDNA array. HepG2 cells were treated with URB597 (10 µM) or DMSO (control) for 24 h.
Next, total RNA was extracted, and reverse transcription was performed as described above. The resulting cDNA (3 µg) was subjected to a highly sensitive 3D-Gene microarray (Toray, Tokyo, Japan). The data and the detailed protocols were deposited in a public database (GEO accession #GSE143194).
Anandamide enzyme-linked immunosorbent assay (ELISA). Intracellular anandamide concentrations were determined using a Human Anandamide ELISA Kit (#MBS770230, MyBioSource, San Diego, CA) according to the manufacturer's instructions. Briefly, cells diluted in PBS were subjected to repeated freeze-thaw cycles to release the internal components, followed by centrifuging at 3000 rpm for 20 min. Then, the collected supernatants were added into wells pre-coated with anti-anandamide antibody and mixed with HRP-conjugated streptavidin to form an immune complex. After adding the color developing substrates, the optical density (OD) of each well was measured by a microplate reader (Multiskan FC, Thermo Fisher Scientific) set to 450 nm. The concentration of anandamide was calculated according to the concentration of the standard and the corresponding OD values. Three groups (control DMSO treatment followed by the addition of control DMSO; URB597 treatment followed by the addition of control DMSO; and URB597 treatment followed by the addition of rimonabant) were analyzed. (c) qRT-PCR analysis of TIMP3 mRNA levels in the three experimental groups. Data are means ± SD from three independent experiments. *P = 9.0 × 10 −4 ; **P = 0.016 (two-sided Welch's t-test). (d) qRT-PCR analysis of the MICA/B mRNA levels in the three experimental groups. Data are means ± SD from three independent experiments. P-values of 0.22 and 0.15 (two-sided Welch's t-test) were not considered to indicate significance. (e) Flow cytometry of surface MICA/B protein levels in the three experimental groups. Gray-shaded histograms represent background staining with the isotype IgG. Representative results from three independent experiments are shown. Relative MFI data (n = 3) are shown in the right panel. Data are means ± SD. *P = 0.0089; **P = 0.031 (two-sided Welch's t-test). (f) Soluble MICA/B quantification by NanoLuc assay in the culture medium of the three experimental groups. Data are means ± SD from three independent experiments. *P = 0.0054; **P = 0.029 (two-sided Welch's t-test).
◂ Scientific RepoRtS | (2020) 10:15556 | https://doi.org/10.1038/s41598-020-72688-y www.nature.com/scientificreports/ Statistical analysis. No statistical method was applied to determine the required sample size. Statistical analysis was conducted using R version 3.3.2 (R Foundation for Statistical Computing, Vienna, Austria). Continuous variables are reported as means ± standard deviation (SD) unless indicated otherwise. Welch's t-test was used for comparisons of continuous variables. P-values < 0.05 were considered indicative of statistical significance. The experiments were not randomized, and the investigators were not blinded to the group allocation during the experiments or the assessment of outcomes.