Monoacylglycerol Lipase: A Novel Potential Therapeutic Target and Prognostic Indicator for Hepatocellular Carcinoma

Monoacylglycerol lipase (MAGL) is a key enzyme in lipid metabolism that is demonstrated to be involved in tumor progression through both energy supply of fatty acid (FA) oxidation and enhancing cancer cell malignance. The aim of this study was to investigate whether MAGL could be a potential therapeutic target and prognostic indicator for hepatocellular carcinoma (HCC). To evaluate the relationship between MAGL levels and clinical characteristics, a tissue microarray (TMA) of 353 human HCC samples was performed. MAGL levels in HCC samples were closely linked to the degree of malignancy and patient prognosis. RNA interference, specific pharmacological inhibitor JZL-184 and gene knock-in of MAGL were utilized to investigate the effects of MAGL on HCC cell proliferation, apoptosis, and invasion. MAGL played important roles in both proliferation and invasion of HCC cells through mechanisms that involved prostaglandin E2 (PGE2) and lysophosphatidic acid (LPA). JZL-184 administration significantly inhibited tumor growth in mice. Furthermore, we confirmed that promoter methylation of large tumor suppressor kinase 1 (LATS1) resulted in dysfunction of the Hippo signal pathway, which induced overexpression of MAGL in HCC. These results indicate that MAGL could be a potentially novel therapeutic target and prognostic indicator for HCC.

and utilize FA from stored neutral fats [8][9][10] . Some of the released FFAs, including lysophosphatidic acid (LPA) 11 and prostaglandin E2 (PGE2) 12 , were found to be involved in signal cascades which induce carcinogenesis and tumor progression. Considering the abundance and importance of FFAs and lipid metabolism in the liver, we predict that MAGL will be essential to the initiation and progression of HCC, perhaps more so than the other aforementioned cancers. Nevertheless, the role and mechanism of MAGL in HCC carcinogenesis and progression remain unclear.
In this study, we investigated the relationship between MAGL levels and clinical features of HCC patients. We also explored the mechanism of MAGL in HCC cell proliferation, invasion and apoptosis. Additionally, the reason for MAGL overexpression in HCC was investigated and the efficacy of targeted inhibition of MAGL in vivo was investigated to evaluate its potential value for HCC Therapy.

Results
MAGL levels are significantly higher in HCC and poorly differentiated in clinical samples. In clinical patient samples ( Fig. 1A-C), RT-PCR, Western Blots and IHC were used to evaluate MAGL mRNA and protein levels, respectively, along with TMAs (Fig. 1D). Significantly higher MAGL mRNA and protein levels were found in HCC samples when compared with non-HCC tissues (para-carcinoma tissue and normal liver tissue, *p < 0.05). This result was repeated in poorly differentiated HCC tissue compared with well differentiated HCC tissue (*p < 0.05). However, no significant difference of MAGL mRNA or protein levels was found between para-carcinoma and normal liver tissues.
MAGL deteriorates the prognosis of HCC patients. TMA and IHC results indicated that MAGL levels were increased significantly with decreased degree of tumor differentiation in HCC [MAGL IOD values: I = 67758 ± 27697, II = 171019 ± 49765, III = 443878 ± 132285, IV = 848382 ± 91689 and NLT = 28024 ± 18200 (not showed), Fig. 1E]. This indicates that MAGL expression may be used to predict the degree of malignancy in HCC, confirming our previous findings 15 .
To further verify our results using TMA and IHC, logistic regression between MAGL protein levels and degrees of tumor differentiation, follow-up data of TMA, and Kaplan Meier method for survival analysis were completed. MAGL protein levels (MAGL IOD value), A-fetoprotein (AFP), tumor, node and metastasis (TNM) stage were significantly correlated to the degrees of tumor differentiation (p < 0.05, Table 1). Survival analysis also demonstrated MAGL low-expression group (MAGL IOD value < 200000) showed significantly better survival than MAGL high-expression group (MAGL IOD value > 200000, p < 0.05, Fig. 1F). Thus, MAGL protein levels could be considered as a biomarker for predicting tumor differentiation degree and prognosis.
Multiple linear regressions between MAGL protein levels, AFP, tumor sizes, tumor numbers, tumor embolus and survival times of HCC patients were performed using the following equation: COX regression of MAGL protein levels and mortality risk factor of HCC were applied to evaluate the mortality risk of MAGL protein level to HCC patients (Table 2). Statistical results indicated that MAGL protein levels were associated with shorter survival time of HCC patients (p = 0.004).
MAGL protein level is positively correlated with degree of malignancy in HCC cells. Western blot analysis of MAGL protein levels in various liver cell lines indicated that MAGL protein levels were significantly lower in L02 cells than HCC cell lines (Fig. 2B). MHCC-97H/L and HCC-M3/M6 represented high/low invasive HCC cell lines, respectively 16 . MAGL protein levels were significantly higher in HCC cell lines with high invasive scores, including MHCC-97H and HCC-M3 (Fig. 2B), compared to HCC cell lines with low invasive scores, including MHCC-97L and M6.

MAGL promotes proliferation, invasion and apoptosis inhibition of HCC cell lines.
ShRNA-MAGL transfected cell lines displayed significantly reduced IOD values after 48 hours, indicating that at the peak of shRNA transcription, there was a significant inhibition of HCC cell proliferation. This effect was also observed in the JZL184 group at 12 hours after treatment. Due to its quick and transient inhibition of MAGL, proliferation recovered gradually. Accordingly, MAGL knock-in plasmids induced MAGL-overexpression which significantly increased the proliferation of HCC cell lines but not L02 cells (Fig. 2C). MAGL mRNA levels (A) and protein levels (B,C) were examined by RT-PCR and western blot. Both mRNA levels and protein levels increased as the malignancy increased [from normal liver tissue (NLT) and para-carcinoma tissue (Para-CT) to HCC tissue well-differentiated (HCC WD) and HCC tissue poor differentiated (HCC PD)] in tissue specimens from HCC patients (*p < 0.05). These results were confirmed with IHC of TMA (D, 100× magnification; 400× magnification). MAGL protein was stained brown by IHC in the tissue slices, and stain was deeper when malignance increased in liver and tumor tissues. Patients were distributed according to MAGL protein level measured by IOD value by Image-Pro Plus 6.0 software (E). MAGL protein levels increased significantly with decreasing of tumor differentiation degrees 15  Flow cytometric analysis of apoptosis indicated MAGL inhibition, though both shRNA and JZL184 treatment, significantly increased apoptosis of HCC cell lines and normal liver cell line. Overexpression of MAGL inhibited cell apoptosis of HCC cell lines but not normal liver cell line (Fig. 3A,B). Taken together these results indicate that MAGL expression promotes proliferation and inhibits apoptosis in HCC cells.
Due to the weaker invasive abilities of HepG2 and Huh 7.0 cell lines, SMMC-7721 was used for matrigel invasion assay. When MAGL was downregulated by shRNA or JZL184 in SMMC-7721 cells, a significant decrease in cell invasion was observed, indicating that downregulation of MAGL impaired invasive capacity of SMMC-7721 cells. In agreement with these results, overexpression of MAGL promoted invasion of SMMC-7721 through the matrigel (Fig. 3C). These results demonstrated that MAGL enhances the invasive capacity of HCC cells. Interestingly, although overexpression of MAGL did not promote proliferation of normal liver cell line L02, significant invasion ability was found in L02 cells that overexpressed MAGL (Fig. 3D). The result implied that MAGL may be involved in initiation and progression of HCC.
Nude mouse tumorigenicity assay. SMMC-7721 cells which stably expressed MAGL-shRNA (shMAGL group) or overexpressed MAGL protein (pMAGL group) were used in tumorigenicity assays in nude mice. After subcutaneous injection of cell suspension into nude mice, tumor volumes increased over time. Inhibition of MAGL slowed tumor growth, but overexpression of MAGL accelerated the growth of tumors. After 21 days, differences of tumor volumes between shMAGL group, pMAGL group, and blank group were significantly different (p < 0.05, Fig. 4A,B).
In the trial of MAGL-targeted pharmacological inhibition, JZL-184 oral administration significantly inhibited tumor growth of nude mice, while high fatty diet (HFD) promoted tumor growth (p < 0.05, Fig. 4D). After 42 days, gross samples of nude mice were collected and analyzed. The largest and smallest tumor sizes were recorded in HFD group and JZL-184 P.O. group, respectively (

Analysis of MAGL-PGE2/LPA pathway in HCC cells.
ELISAs were performed to detect the levels of two potential downstream effector molecules of MAGL, PGE2 and LPA, in both secreted and intracellular levels. A similar trend in the secreted and intracellular levels of PGE2 and LPA as MAGL was observed in HCC cell lines when MAGL levels were modulated. However, only PGE2 levels correlated with MAGL in the normal L02 liver cell line (Fig. 5). Upon knockdown or pharmacologic inhibition of MAGL, secretory and intracellular levels of PGE2 decreased in both HepG2 and SMMC-7721 cell lines. However, only intracellular levels of LPA were reduced in both HCC cell lines. Secretory level of LPA was only significantly decreased in SMMC-7721 but not HepG2 cells. Conversely, MAGL upregulation in all cell lines led to significant increases in both PGE2 and LPA levels in all HCC and normal liver cells, except for LPA levels in L02 cells. These results demonstrate that tissue-specific differences existed in the levels of both PGE2 and LPA and these differences were controlled by MAGL expression. These results support the notion that both PGE2 and LPA are downstream products of MAGL.  www.nature.com/scientificreports/ CCK8 assay was used to examine the proliferation ability changes when MAGL was regulated by iRNA, gene knock-in and JZL-184. MAGL was quickly inhibited by JZL-184 (blue lines; compared with blank group, black lines, *p < 0.05) which suppressed cell proliferation; gradually increasing, following drug metabolism. However, MAGL iRNA showed long-term and stable proliferation inhibition by MAGL knock-down (green lines; compared with blank group, *p < 0.05). In contrast, MAGL gene knock-in by MAGL gene-loading plasmid (pMAGL) transfection promoted proliferation of cell lines (red lines, compared with blank group, *p < 0.05).

Promoter of LATS1 methylation induces MAGL overexpression by blocking Hippo signal pathway.
MSP was utilized to evaluate the methylation of the LATS1 promoter in HCC cell lines HepG2 and SMMC-7721 and normal liver cell line L02 (Fig. 6A). Methylation specific primers were detected in all HCC cell lines but in L02 liver cells. Non-methylated specific primers, however, were only detected in the L02 normal liver cell line. To further confirm the methylation patterns of the LATS1 promoter in HCC cell lines, DAC was used to demethylate the LATS1 promoter in HCC cell lines. DAC treatment of HCC cells led to a decrease in methylation specific primers while non-methylation specific primers increased. In summary, methylation of LATS1 promoter was observed in HCC cells but not in normal liver cells and DAC led to demethylation of LATS1 in HCC cells.
In HCC cell lines HepG2 and SMMC-7721 ( Fig. 6B-D), expression of LATS1 protein was suppressed. Demethylation of LATS1 promoter by DAC and Knock-in LATS1 gene by plasmid transfection elevated LATS1 protein levels, which confirmed our findings that methylation of the LATS1 promoter in HCC cells induced decreased expression of the LATS1 protein. Deregulation of LATS1 in HepG2 and SMMC-7721 cell lines using the methods described above led to significant changes in the YAP and pYAP protein levels. LATS1 and YAP displayed a negative correlation, whereas, LATS and pYAP demonstrated a positive correlation. These results demonstrated LATS1 is involved in the phosphorylation of YAP which induces nucleus retention of YAP and advancement of the Hippo-YAP signaling pathway 17 . These results agree with the report by Tang et al. 18 which demonstrated that YAP induced MAGL mRNA transcription. In conclusion, in HCC cell lines, methylation of the LATS1 promoter induced downregulation of LATS1 and a subsequent decrease in the phosphorylation and nuclear entry of YAP which may induce excessive transcription of MAGL (Fig. 7).

Discussion
Since Nomura et al. first reported the tumor promoting effects of MAGL 7 , increasing studies investigating the relationship between MAGL, carcinogenesis, and tumor progression have been performed to reveal insights into the relevant mechanisms. Until now, the role of MAGL in tumorigenesis and progression remains controversial due to the fact that tumor suppressing effects of MAGL are observed in some colorectal cancers 8,19 . In prostate cancer, ovarian cancer and colorectal cancer MAGL has been shown to be a key enzyme in the lipid metabolism network, promotes tumor progression by supplying FFA for β -oxidation, provides components to build cell This outcome was also found in matrigel invasion assay. HCC cell line SMMC-7721 showed enhanced invasion ability when overexpressed MAGL by gene knock-in, and impaired invasion ability when MAGL was suppressed by iRNA or JZL-184 (C), *p < 0.05). Interestingly, a certain level of invasion ability was observed in normal liver cell line L02, which was unable to invade after overexpressed MAGL by gene knock-in (D, *p < 0.05). This result implied MAGL is involved in malignant change or tumorigenesis.
structures and effector molecules which are involved in cell proliferation, invasion, apoptosis resistance and stemness. Additionally, MAGL facilitates tumor growth by degrading 2-arachidonoylglycerol (2-AG) and inhibiting activation of cannabinoid receptor-1 (CB1) 20 . In contrast, the anti-tumor properties of MAGL have been shown to be mediated by the PI3K-ATK signal pathway which suppresses anchorage-independent growth (AIG) and metastasis in certain tumors 18,19 .
As a central organ in lipid metabolism, the liver is likely to be affected by aberrant lipid metabolism. Furthermore, disorders of lipid metabolism demonstrate a significant correlation with HCC 21-23 . Therefore, due to the pivotal role of MAGL in lipid metabolism, its likely involvement in carcinogenesis and progression of HCC were investigated. This study, for the first time, demonstrated that MAGL expression levels were positively correlated with aggressiveness of HCC cell lines and negatively correlated with HCC tissue differentiation degrees. Additionally, MAGL was shown to play an important role in proliferation, apoptosis inhibition, invasion, and tumorigenesis of HCC cells. The tumor-promoting functions of MAGL were shown to be caused by the downstream effector molecules, LPA and PGE2. Furthermore, we demonstrated that the Hippo signaling pathway was responsible for MAGL overexpression in HCC cells. Specifically, methylation of the LATS1 promoter induced LATS1 protein downregulation, and subsequently, decreased phosphorylation and increased nuclear entry of YAP, which induces transcription of MAGL.
Inhibition of MAGL expression and function with shRNA or pharmacological methods, respectively, suppressed HCC growth and progression in vivo. Thus, we propose that MAGL may be a novel HCC therapeutic target due to its numerous significant tumor promoting effects. Furthermore, we found that MAGL could be considered as a prognostic indicator for HCC since increased MAGL levels correlate with decreased HCC tissue differentiation degree.
To go a step further, an equation was designed with multiple linear regressions between MAGL protein levels, AFP, tumor sizes, tumor numbers, and tumor emboli to roughly calculate the survival time of HCC patients. Due to the use of data from follow-up times that spanned only 48 months, the R 2 value of this equation was small and the linear correlation is not strong, however, with larger samples and longer study times we do believe a stronger correlation will be reached. We demonstrated that an increase of MAGL protein levels was correlated with decreased short-term survival (one-year survival) of HCC patients in binary logistic regression. The shortened survival time of HCC patients associated with MAGL was confirmed with COX regression, and may be considered as an independent mortality risk of HCC patients. Taken together, these data support the use of MAGL as a prognostic indicator for HCC patients along with a potential therapeutic target.
Despite the recent research indicating the relationship between MAGL and tumor progression, the mechanisms of the protumor activity of MAGL are unknown. Here, we provide insights regarding the mechanism of action of MAGL in HCC, however, some key questions remain unanswered. Are there other mechanisms, aside from the MAGL-LPA/PGE2 and MAGL-2-AG-CB1 pathways, that are involved in the protumor effects of MAGL? Aside from tumor cell proliferation and invasion, what effect does MAGL have on tumor development and progress, for example, does it modulate tumor cell metabolism through the MAGL-FFA pathway? Finally, will MAGL be an effective therapeutic target and prognostic indicator for HCC? It remains unclear whether histological examination may be suitable for clinical application of HCC diagnosis and prognosis prediction but it may be worth investigating whether serum levels of MAGL may provide a more reliable and convenient clinical test for HCC diagnosis and prognosis.

Methods
Patients and tissue microarrays. Tissue microarray of 353 HCC patients was gifted from the Affiliated Zhongshan Hospital of Fudan University with complete patients 'authorization and full ethical approval of the Institutional Clinical Ethics Review Board of Fudan University (Table 3). Fifty pairs of HCC and para-carcinoma tissue (untreated, 36 males and 14 females, aged from 35-84), 11 normal liver tissue (traumatic hepatorrhexis) samples were obtained from the Second Affiliated Hospital of Chongqing Medical University and Fuling Center Hospital with histopathological confirmation, prior patient consent, and the approval of the Institutional Clinical Ethics Review Board in the Second Affiliated Hospital of Chongqing Medical University. All experiments with human tissue samples mentioned above were carried out in accordance with the consent and approval   Table 4. Plasmids carrying MAGL or LATS1 gene codes and blank plasmid vector (Genepharma, China) were used as controls. Transfection was performed on cells in 24-well plates. Briefly, cells were transfected with 2 μ l (1 μ g/μ l) shRNA per well using our self-developed transfection agent Large-scale mesoporous silica nanospheres (LPMSNs) 13 . Transfection was performed at least five times in each experiment and each experiment was repeated at least three times.
Western Blot analysis. Cells were seeded in 6-well plate and treated with JZL184 (0.5 μ g/μ l, Sigma, USA) for 12 h, DAC (5-Aza-2′ -deoxycytidine, 1 M, Sigma, USA) for 72 h or transfected with shRNA for 72 h, then harvested for total protein extraction and estimation. Polyacrylamide gel electrophoresis (PAGE) was performed and PAGE gels transferred to nitrocellulose membranes using previously described protocols (reference: Rego, S. L. Angiogenesis, 2014). After transfer membranes were blocked with 5% nonfat milk, incubated with polyclonal antibodies for MAGL, LATS1, YAP1 or pYAP1 (Abcam, USA) respectively, and a monoclonal antibody for β -actin (Boster, China) was used as a loading control. After incubation with horseradish peroxidase conjugated secondary antibody (Boster, China), membranes were developed using the enhanced chemiluminescence (ECL, Boster, China) detection system (Amersham, USA).

RT-PCR and methylation specific PCR (MSP).
The integrity of total RNAs was test by agarose gel electrophoresis (AGE). Primers used to amplify fragments using real time-polymerase chain reaction (RT-PCR) are shown in Table 5. RT-PCR was performed using StepOnePlus ™ RT-PCR System (Applied Biosystems, USA) with SYBR-Green PCR Master Mix (Applied Biosystems, USA) using routine methods 14 . MSP was performed utilizing standard methods. Data was analyzed with StepOne Software V2.1 (Applied Biosystems, USA).

Cell proliferation analysis.
Cell Counting Kit-8 (CCK8) method was used to analyze cell proliferation after modulating MAGL. Cells were plated in 96-well plates at a density of 10,000/well and cultured for 24 hours. Transfection or JZL-184 treatment was performed directly in 96-well plates. 10 μ l CCK8 solution was added to each well at corresponding time points and plates incubated for one hour. The optical density was measured at 450 nm using a Spectrophotometer (Beckman, USA).
Cell apoptosis analysis. Flow cytometry was utilized for cell apoptosis analysis. After treatments cells were digested and washed with phosphate buffered saline (PBS). 5 μ l FITC-Annexin and 5 μ l PI (250 μ g/ml) were added into the cell suspensions, and incubated on ice in dark for 10 minutes. Next, cells were washed with PBS two times, and fluorescence analyzed using a flow cytometry instrument (BD Bioscience, USA).

Enzyme-linked immunosorbent assay (ELISA).
In order to confirm the involvement of LPA and PGE2 in MAGL pathway, ELISAs were used to detect the levels of LPA and PGE2 in cell culture mediums (secreted levels) and cell lysate (intracellular levels). Cell culture media containing secreted proteins was collected 24 hour after treatments. Cell lysis buffers were prepared by repetitive freeze thaw method after transfection for 48 hours and JZL184 treatment for 12 hours. ELISA was operated according to manufacturer's recommendations (Cusabio Biotech, China).     . The mice were maintained in a pathogen-free facility with a 12-h light, 12-h dark cycle and were provided food (both normal diets and high fat diets) and water ad libitum. All efforts were made to minimize animal suffering, including euthanasia, and to reduce the number of animal used. In order to evaluate the tumorigenesis abilities of cells after MAGL manipulation, stably transfected SMMC-7721 (MAGL-knockdown or MAGL-overexpressed) were screened with G418 for nude mouse tumorigenicity assay. Cell suspension with 1 × 10 6 /ml was injected subcutaneously into subscapular region of nude mice. Tumor size was recorded every three days and calculated as ab 2 /2 (a stands for the long diameter, b stands for the short diameter). JZL-184 oral administration (50 mg/kg, qod) was used for of MAGL-targeted inhibition therapy in vivo.
Statistical analysis. The data were analyzed using the Statistical Package for the Social Sciences 18.0 (SPSS, USA) with a p < 0.05 taken as statistically significant. The measurement data were expressed as mean ± SD of at least three experiments. Rank sum test and chi-square test were used for categorical data, and the one-way analysis of variance (ANOVA) test was used for continuous data. The power analysis for the final sample size was calculated with Graph Pad Stat Mate (Graph Pad Software, CA).  Table 5. Amplified fragments of RT-PCR.