Research ArticleExperimental Studies

ABHD12 Knockdown Suppresses Breast Cancer Cell Proliferation, Migration and Invasion

SEMO JUN, SEOK WON KIM, JI-YEON LIM and SEON-JOO PARK
Anticancer Research May 2020, 40 (5) 2601-2611; DOI: https://doi.org/10.21873/anticanres.14231

Abstract

Background/Aim: Alpha/beta-hydrolase domain containing 12 (ABHD12) is a serine hydrolase that regulates immunological and neurological mechanisms. This study aimed to elucidate the oncogenic effect of ABHD12 on human breast cancer. Materials and Methods: ABHD12 expression was confirmed in breast cancer tissues and breast cancer cell lines by immunohistochemistry and quantitative RT-PCR. To determine the role of ABHD12, ABHD12 siRNA-suppressed breast cancer cells (MCF7 and MDA-MB-231 cells) were investigated for cell proliferation, migration, and invasion capabilities using MTT assays, EdU assays, colony formation assays, and Boyden chamber assays. Results: Immunohistochemical staining showed a higher ABHD12 expression in breast cancer tissues than in normal tissues. Additionally, ABHD12 knockdown was found to inhibit cell growth, proliferation, migration, and invasion in breast cancer cells. Conclusion: ABHD12 plays a crucial role in cell proliferation, migration, and invasion of breast cancer cells.

Lipids are essential components of the cell membrane, that are used for energy storage, and also act as secondary messengers for cellular signalling pathways or as steroid hormones. When energy supplies are adequate, cells have been observed to store excess lipids as lipid droplets, which are broken down via lipolysis and autophagy when cells require energy (1, 2). Changes in lipid metabolism have been shown to cause metabolic disorders, immune disorders and central nervous system disorders, as well as various diseases, such as cancer. Cancer cells have been shown to undergo lipid metabolic reprograming to obtain a steady source of energy and nutrients for regulating cancer cell proliferation and progression (3).

Human serine hydrolases are known to be large clusters of enzymes that include serine proteases and metabolic serine hydrolases (4). Metabolic serine hydrolases contain serine nucleophile at the active site, which is used to hydrolyse amide, ester, and thioester bonds of small molecules such as neutral fatty acyl esters, acyl thioesters, phospholipids, lipid amides, and other ester metabolites. Metabolic serine hydrolases have been reported to play roles in various physiological and pathophysiological processes including neurotransmission, metabolism, immune response, oxidative stress, and cancer (5-7).

Alpha/beta-hydrolase domain containing 12 (ABHD12) has been identified as a serine hydrolase that can hydrolyse endocannabinoid 2-arachidonoylglycerol (2-AG) (8). 2-AG has been reported as a neural messenger that can regulate neurotransmission and neuroinflammation by binding and activating cannabinoid receptors (CB1R and CB2R) (9). In the endocannabinoid system (ECS), 2-AG has been shown as an endogenous cannabinoid (CB) ligand that is expressed on response to specific stimuli and degraded by intracellular enzymatic hydrolysis (10). The equilibrium between the production and degradation of 2-AG is strictly regulated by the metabolic serine hydrolase family, which includes monoacylglycerol lipases (MAGL), ABHD6, and ABHD12 (11).

In addition, 2-AG has been reported to be an important mediator for lipid metabolism (12, 13). ABHD family members when accompanied by hydrolytic activity of 2-AG, are shown to play role in lipid synthesis and degradation, while mutations in the respective genes have been associated with inherited alterations in lipid metabolism (14, 15). ABHD12 is an integrated membrane glycoprotein of approximately 45 kDa with an active site near the extracellular space. ABHD12 transcripts have been observed to be highly expressed in the brain and other cell types, such as microglia and macrophages (16).

Recently, mutations and deletions in Abhd12 have been found to be associated with neurodegenerative diseases such as polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) (17). The ABHD12 dysfunction-related mechanism that causes PHARC may be due to regulation of 2-AG in specific cell types such as macrophages and microglia with high ABHD12 expression, or ABHD12 regulation in other signalling or structural lipids. Correspondingly, it has been reported that ABHD12 also acts as a lysophospholipase and metabolizes lysophosphatidylserine (lysoPS) pathway in the murine model of PHARC (18-20). In addition to its role in the central nervous system, the endocannabinoid system, which consists of endocannabinods (eCBs), cannabinoid receptors, and other enzymes that can synthesize and degrade eCBs, has been found to be closely associated with cancer (21). eCBs and their signalling pathway have been reported to inhibit cell proliferation, arrest cell cycle, induce cell death, and prevent tumour migration and invasion in different types of cancers (22-24). Serine hydrolase family, which is involved in the eCB signalling pathway by hydrolysing 2-AG, has also been reported to be associated with cancer. Specifically, MAGL has been reported to play an important role in diverse types of cancers such as melanoma, ovarian, colon, and breast cancer (25, 26). However, the correlation between ABHD12 expression and cancer has never been established.

In this study, we aimed to investigate the potential role of ABHD12 in breast cancer. At first, we analysed The Cancer Genome Atlas (TCGA) data and found that ABHD12 mRNA expression is elevated in tissues of patients with breast cancer compared to its expression in healthy tissues. This finding suggested that ABHD12 expression is strongly associated with breast cancer progression. Immunohistochemistry (IHC) analysis revealed an up-regulated ABHD12 expression in breast cancer tissues compared to normal tissues. Additionally, ABHD12 knockdown was found to significantly inhibit cell proliferation, and suppress migration and invasion in breast cancer cell lines, which was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2´-deoxyuridine (EdU) proliferation assay, anchorage-dependent and anchorage-independent cell growth assay. These results suggested that ABHD12 can act as a therapeutic target in the treatment of human breast cancer.

Materials and Methods

Data analysis. ABHD12 expression levels were analysed in breast cancer using a web server, Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cancer-pku.cn) (27) that provided mRNA expression analysis of 1,085 breast cancer tissues and 291 healthy samples from TCGA and Genotype-Tissue Expression (GTEx). Overall survival and disease-free survival in ABHD12-expressing breast cancer patients was also obtained from GEPIA.

Tissue microarray (TMA) analysis. IHC staining for ABHD12 expression was performed using human TMA for breast cancer-metastasis-normal (CBA; SuperBioChips Laboratories, Seoul, Republic of Korea) according to the manufacturer's instructions. Briefly, tissue slides were deparaffinized in 100% xylene at 60°C for 15 min, and rehydrated with serial incubations in 100%, 90%, and 80% ethanol. For antibody retrieval, tissue slides were heated in a microwave for 3 min in 10 mM citrate buffer containing 0.05% Tween-20. Further, endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 15 min. Slides were blocked with PBS containing 5% bovine serum albumin (BSA) for 1 h, and then incubated with an anti-ABHD12 antibody (Abcam, Cambridge, UK). After washing with PBS for 15 min, slides were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature, and developed using diaminobenzidine (DAB) substrate, and counterstained with haematoxylin. Following dehydration in graded ethanol and xylene, slides were mounted with coverslips. Slides were evaluated for ABHD12 expression by two pathologists based on staining intensity of positive immunoreactive cells. Staining intensity was graded as: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining).

Cell culture. Human breast cancer cell lines (BT-549, Hs578T, MCF7, MDA-MB-231, and T47D cells) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in RPMI1640 media (Welgene Biotech Co., Taipei, Taiwan, ROC) containing 10% foetal bovine serum (FBS) and 1% penicillin streptomycin (Gibco, Life Technologies, Grand Island, NY, USA) in 5% CO2 at 37°C. Human mammary epithelial cells (MCF10A cells) were cultured in Mammary Epithelial Cell Growth Medium (MEGM; Lonza, Basel, Switzerland) without GA-1000 (gentamycin-amphotericin B mix).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR). Total RNA was extracted using the TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol, and cDNA (1 μg) was synthesized using Primescript™ Reverse Transcriptase reagent kit (TaKaRa Bio Inc., Japan) at 42°C for 60 min. qRT-PCR was performed using specific primers and TB Green™ Premix Ex Taq™ (TakaRa Bio Inc.). Primer sequences that were used for PCR, are as follows: ABHD12 forward, 5’-TCCCAGCACATGCAGAATGA-3’; ABHD12 reverse, 5’-AGCTCAGTCT AAG GCCAGGT-3’; CB2R forward, 5’-TTTCCCACTGATCCC CAATG-3’; CB2R reverse, 5’-AGTTGATGAGGCACAGCATG-3’, and GAPDH forward, 5’-TCAAGAAG GTGGTGAAGCAG-3’; GAPDH reverse, 5’-TCCACCACCCTGTTGCTGTA-3’. ABHD12 mRNA expression levels were analysed using the CFX Maestro software (Bio-Rad Laboratories Inc., Hercules, CA, USA), and the relative mRNA expression was evaluated by 2−ΔΔCt method. GAPDH was used to normalize gene expression levels.

Western blotting. For protein extraction, cells were cultivated with small interfering RNAs (siRNAs) for 48 h. Furthermore, cells were lysed using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1 mM dithiothreitol, 1 mM phenylmethanesulfonylfluoride, 10 μg/ml leupeptin, and 10 μg/mL aprotinin) for 30 min on ice, and then centrifuged at 15,000 × g for 15 min. Protein concentrations were determined by the Bio-Rad Protein Assay Kit. Equal quantities of proteins were electrophoresed on SDS-PAGE and then transferred onto a PVDF membrane (Pall Life Sciences, NY, USA). After blocking with 5% non-fat milk in TBS-T (0.1% Tween-20 in 1× TBS) to prevent non-specific binding, membranes were incubated overnight with primary antibodies for ABHD12 (Abcam) and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C. Furthermore, membranes were washed three times for 15 min with TBS-T buffer, and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. HRP-labelled proteins were detected by the EZ-Western Detection Kit (DoGEN Bio, Seoul, Republic of Korea).

Figure 1.
Figure 1.

ABHD12 expression in breast cancer tissues. (A) Alpha/beta-hydrolase domain containing 12 (ABHD12) mRNA expression levels in breast cancer tissues (n=1,085) and healthy tissues (n=291). Overall survival and disease-free survival rates were analysed from Gene Expression Profiling Interactive Analysis (GEPIA). (B) Tissue microarray (TMA) was performed to detect ABHD12 expression in breast cancer. Images were captured using microscope (magnification ×200 and ×400). (C) The graph shows the average staining score of ABHD12 expression in healthy and cancer tissues. (D) The graph shows staining score of ABHD12 expression based on cancer progression. ***p<0.001 compared to the control group.

siRNA transfection. For transfection, two siRNAs targeting ABHD12 transcripts were designed and synthesized, while AccuTarget Control siRNA was purchased from Bioneer (Daejeon, Republic of Korea). Following ABHD12 siRNA sequences were used in the experiment: ABHD12 siRNA #1; 5’-CCAGGUUCUUUCUGCACCU-3’ and ABHD12 siRNA #2; 5’-CUAUUACAAGUAGUGGAAUUA-3’. Cells were transfected with 50 nM of siRNAs using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions.

MTT assay. Cell viability was determined by the MTT assay. After siRNA transfection for 48 h, cells were seeded in 96-well plates at a density of 1×103 cells/well. MTT was added to each well and incubated at 37°C for 4 h. Media were discarded and cells were lysed with isopropanol containing 0.04 N HCl. Absorbance of each well was measured at 570 nm by a spectrophotometer. Percentage of cell viability was calculated as relative absorbance of knockdown cells to control cells.

Cell proliferation assay. Cell proliferation was determined by EdU incorporation assay using Click-iT™ EdU Alexa Fluor 488™ Imaging Kit (Invitrogen) according to the manufacturer's protocol. Images were analysed by IN Cell Analyzer 2500HS high content analysis (HCA) imaging system (GE Healthcare Life Science, Chicago, IL, USA).

Colony formation assay. Cells (500 cells/dish) were seeded on 6-cm culture dishes in triplicates and maintained in a humidified incubator. After 2 weeks, colonies were fixed using cold 95% methanol and stained with 0.1% crystal violet for 15 min at room temperature. Furthermore, culture dishes were washed with tap water and counted for viable colonies.

Soft agar assay. To coat the bottom layer of culture plates with agar, 0.6% agar (Duchefa Biochemie, Haarlem, the Netherlands) was diluted in complete media, poured in 60 mm plates, and allowed to solidity for 30 min at room temperature. Cells were suspended in complete media and added to 0.3% agar, and then poured on the solidified bottom layer of agar. After 30 min at room temperature, complete media were added on the top to prevent drying of agar. Cells were maintained at 37°C in 5% CO2 incubator for 3 weeks. Colonies were counted under microscope and images were analysed using the Image-Pro plus 4.5 software (Media Cybernetics, Rockvile, MD, USA).

Cell migration and invasion assays. Cell migration and invasion assays were performed using Boyden chamber system. For migration assay, equal amounts of siRNA-transfected cells were suspended in 100 μl of serum-fee medium and added to the upper chamber of 24-well transwell chamber with 8.0 μm Pore Polycarbonate Membrane Insert (Corning Incorporated, Corning, NY, USA). Correspondingly, the insert of chamber was pre-warmed with 100 μl of serum-free medium at 37°C. Further, 600 μl of complete medium was added to the lower chamber. After incubation for 24 h, cells remaining in the upper chamber were removed using cotton swabs, and the transwell inserts were washed twice with PBS. Cells that migrated to the lower surface of the membrane were fixed using 4% paraformaldehyde and stained with 0.1% crystal violet solution. Migrated cells were visualized and counted under microscope at a magnification of ×200. For invasion assay, matrigel-coated transwell chambers (Corning BioCoat™ Matrigel Invasion Chamber, Corning Costar) were used. Cells were suspended in FBS-free RPMI medium and seeded into the upper chamber. Further, 600 μl of RPMI medium containing 10% serum was added to the lower chamber. Cells were allowed to invade matrigel for 24-36 h at 37°C. Subsequent procedures were performed in the same manner as for the migration assay.

Figure 2.
Figure 2.

ABHD12 expression in breast cancer cell lines. ABHD12 mRNA expression was analysed by qRT-PCR in breast cancer cell lines and normal fibroblast (MCF10A) cells. Graphs represent mean±SD from three independent experiments. ***p<0.001 indicates a significant difference compared to the control group.

Statistical analysis. Data are presented as mean±standard deviation (SD). Comparisons between ABHD12-expressing cells and ABHD12-silenced cells were analysed by the two-tailed Student's t-test using Excel (Microsoft, Redmond, WA, USA) and the GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). *p<0.05 was considered statistically significant. All experiments were independently repeated thrice.

Results

ABHD12 expression is up-regulated in breast cancer tissues. To determine the correlation between ABHD12 expression and breast cancer, we analysed ABHD12 mRNA expression in breast cancer tissues and healthy tissues using the TCGA database and the publicly available, GEPIA database. mRNA expression profile revealed that the ABHD12 transcript is highly expressed in tissues of patients with breast cancer compared to that expressed in healthy tissues adjacent to tumour (Figure 1A). In addition, overall survival and disease-free survival of patients with breast cancer and high ABHD12 expression were found to be lower than in patients with breast cancer and low ABHD12 expression. Furthermore, we determined ABHD12 expression in breast cancer tissues by immunohistochemistry. TMA showed that ABHD12 expression was significantly up-regulated in breast cancer tissues compared to normal tissues (Figure 1B and C). Specifically, ABHD12 was found to be highly expressed depending on tumor grade (Figure 1D).

Figure 3.
Figure 3.

siRNA-mediated ABHD12 knockdown in MCF7 and MDA-MB-231 cells. (A) For knockdown of ABHD12 expression, MCF7 and MDA-MB-231 cells were transfected with two different siRNAs targeting ABHD12. mRNA expression levels of ABHD12 were evaluated by qRT-PCR. ***p<0.001 indicates a significant difference compared to the control group. (B) ABHD12 expression levels were evaluated by western blotting. β-actin was used as a loading control.

ABHD12 is highly expressed in breast cancer cell lines. To determine ABHD12 expression levels in breast cancer cell lines, qRT-PCR analysis was performed in various breast cancer cell lines (BT-549, Hs578T, MCF7, MDA-MB-231, and T47D cells) and human breast epithelial cell lines (MCF10A cells). As shown in Figure 2, ABHD12 mRNA expression was found up-regulated in breast cancer cell lines compared to its expression in MCF10A. These results suggested that ABHD12 expression may be associated with breast cancer. Furthermore, we studied two breast cancer cell lines (MCF7 and MDA-MB-231 cells) as they showed relatively high ABHD12 expression and are widely used in breast cancer research.

ABHD12 knockdown inhibits cell proliferation in MCF7 and MDA-MB-231 cells. To determine the role of ABHD12 in breast cancer cells, negative control siRNA and siRNAs targeting ABHD12 were transfected in breast cancer cell lines, MCF7 and MDA-MB-231. Knockdown efficacy of ABHD12 siRNA was confirmed by qRT-PCR and western blot. As shown in Figure 3A and B, mRNA and protein expression of ABHD12 were found to be effectively down-regulated in both ABHD12-silenced breast cancer cell lines; however, not in control siRNA-transfected cells. These results showed that ABHD12 expression was effectively down-regulated by ABHD12 siRNA.

Furthermore, we investigated whether ABHD12 plays role in proliferation of MCF7 and MDA-MB-231 cells. The effect of ABHD12 knockdown on cell proliferation was evaluated from cell growth curve of the MTT assay. We found that ABHD12 siRNA-transfected MCF7 cells showed a significant decrease in cell proliferation at 4 days after incubation (Figure 4A). In addition, ABHD12-silenced MDA-MB-231 cells showed similar results. Following MTT assays, we performed EdU cell proliferation assays to detect proliferating cells by analysing those that incorporate EdU into cellular DNA during DNA replication. EdU-stained cells were observed by fluorescence microscopy, and the percentage of EdU-positive cells relative to DAPI-positive cells was determined. As shown in Figure 4B, results confirmed that the number of proliferating cells was lower in ABDH12 siRNA-transfected MCF7 and MDA-MB-231 cells compared to that of proliferating cells in control siRNA-transfected MCF7 and MDA-MB-231 cells.

Figure 4.
Figure 4.

ABHD12 knockdown inhibits cell proliferation in MCF7 and MDA-MB-231 cells. (A) Cell proliferation in control and ABHD12-silenced cells was investigated by the MTT assay at days 1, 2, 3, and 4. (B) EdU proliferation assay was performed in ABHD12-silenced cells. Graph represents mean±SD from three independent experiments. *p<0.05 and ***p<0.001 indicate a significant difference compared to the control group.

Figure 5.
Figure 5.

ABHD12 knockdown inhibits the transforming ability in MCF7 and MDA-MB-231 cells. (A) A colony formation assay was performed in ABHD12 siRNA-transfected MCF7 and MDA-MB-231 cells. (B) Soft agar colony formation assay was performed in ABHD12 siRNA-transfected MCF7 and MDA-MB-231 cells. Graph represents mean±SD of colonies formed from three independent experiments. *p<0.05, **p<0.01, and ***p<0.001 indicate significant difference compared to the control group.

ABHD12 knockdown inhibits the transforming ability of MCF7 and MDA-MB-231 cells. We confirmed the inhibition of cell proliferation on ABHD12 knockdown by colony formation assay. As expected, ABHD12 knockdown was found to reduce colony formation in MCF7 as well as MDA-MB-231 cells, suggesting that cell proliferation was inhibited on ABHD12 knockdown. These results suggested that ABHD12 is involved in proliferation of MCF7 and MDA-MB-231 cells. To determine the transforming ability of ABHD12 in breast cancer cells, we examined soft agar colony formation in control siRNA- and ABHD12 siRNA-transfected MCF7 as well as MDA-MB-231 cells. As shown in Figure 5, the colony formation ability of ABHD12 siRNA-transfected MCF7 and MDA-MB-231 cells was found to significantly decrease compared to that of control siRNA-transfected cells.

ABHD12 knockdown suppresses cell migration and invasion. Furthermore, we investigated the role of ABHD12 in migration and invasion capabilities of breast cancer cells. Cell migration was assessed using transwell chamber with 8.0 μm Pore Polycarbonate Membrane Insert. As shown in Figure 6A, we found that cell migration capability of ABHD12-silenced MCF7 cells was reduced compared to migration capability of control cells. Similarly, MDA-MB-231 cells were observed with impeded cell migration ability on ABHD12 knockdown. These results suggested that ABHD12 may be involved in cell migration of breast cancer cells. To determine cell invasion, matrigel-coated transwell assay was performed using ABHD12 siRNA-transfected MCF7 and MDA-MB-231 cells. The number of invaded cells was found to be significantly less in both the ABHD12-silenced breast cancer cells lines, compared to control cells (Figure 6B). These results showed that ABHD12 may be involved in regulating invasion ability of breast cancer cells.

Figure 6.
Figure 6.

Effect of ABHD12 knockdown on cell migration and invasion in MCF7 and MDA-MB-231 cells. (A) A migration assay was performed in ABHD12 siRNA transfected MCF7 and MDA-MB-231 cells. (B) An invasion assay was performed in both cell lines. Data are shown as mean±SD of three independent experiments. *p<0.05, indicates a significant difference compared to the control group.

ABHD12 knockdown increases mRNA expression of CB receptors. The activation of CB receptors by eCB, including 2-AG, inhibits cell proliferation, and induces cell cycle arrest and cell death. Since ABHD12 is particularly involved in 2-AG-CB2R signaling in CNS, we examined whether ABDH12 knockdown regulates the expression of CB2R mRNA in MCF7 and MDA-MB-231 cells. As shown in Figure 7, CB2R mRNA expression levels were found to be up-regulated in ABHD12 siRNA-transfected MCF and MDA-MB-231 cells. These results suggested that ABHD12 knockdown was conferred to have anti-proliferative effect on breast cancer cells via activation of CB receptors.

Discussion

The endocannabinoid system (ECS) is reported as a biological organization composed of two G protein-coupled cannabinoid receptors (CB1R and CB2R); their endogenous activating ligands (eCB); and enzymes that regulate synthesis, release, and degradation of eCB (21). ECS plays important roles in regulating various physiological and neuronal mechanisms in the human central nervous system and peripheral tissues. In addition to its role in the central nervous system, increasing evidence revealed that ECS is involved in cancer progression. Recent studies have established that eCBs can act as potential target in breast cancer therapy (28). eCBs including anandamide and 2-AG activate their receptors and their signalling cascade has been shown to exhibit anti-proliferative activity by inhibiting cell growth, stimulating cell-cycle arrest, and promoting cell death in breast cancer cells (22-24). Furthermore, eCB-degrading enzymes have been studied for their role in cancer. Specifically, MAGL, an enzyme that hydrolyses 2-AG, is well-known for its anti-tumour effect in diverse cancers (25, 26). However, the association between other 2-AG hydrolases (ABHD6 and ABHD12) and cancer has not been studied. Nonetheless, the potential correlation of ABHD12 expression and colorectal cancer has been reported via analysis of copy number aberration and gene expression (29).

Figure 7.
Figure 7.

Effect of ABHD12 knockdown on activation of CB2R. CB2R expression in ABHD12 knockdown MCF7 (A) and MDA-MB-231 (B) cells was measured by qRT-PCR. Data are shown as mean±SD of three independent experiments. *p<0.05, **p<0.01 indicate a significant difference compared to the control group.

In this study, we analysed mRNA sequential data from TCGA, and found that ABHD12 expression is up-regulated in breast cancer. Moreover, ABHD12 expression was found elevated in tissues of patients with breast cancer compared to normal tissues adjacent to the tumor (Figure 1). These findings suggested that ABHD12 plays a vital role in breast cancer progression and development. Subsequently, we conducted experiments to confirm whether ABHD12 knockdown exerts anti-oncogenic effects on breast cancer cells. Following siRNA treatment, ABHD12-silenced MCF7 and MDA-MB-231 cells were observed to significantly inhibit cell proliferation, as determined by MTT and EdU cell proliferation assays (Figure 4). Furthermore, anchorage-dependent and anchorage-independent cell growth revealed that ABHD12 knockdown significantly inhibited cell growth and the transforming ability in breast cancer cells (Figure 7). These results suggested that ABHD12 is involved in regulating cancer cell proliferation along with potential metastasis. Furthermore, migration and invasion assays showed that ABHD12 knockdown suppresses oncogenic effect in MCF7 and MDA-MB-231 cells (Figure 5). Moreover, ABDH12 knockdown was found to trigger up-regulation of CB receptors in MCF7 and MDA-MB-231 cells (Figure 6). CB-induced activation of CB receptor has been shown to play an important role in a variety of signal transduction pathways. In breast cancer cells, CB has been reported to inhibit cell proliferation via CB receptor activation. While CB1R activation has been known to promote cell-cycle arrest at G1/S phase, CB2R activation has been reported to mediate G2/M phase arrest (28). Moreover, CB receptors have been found to inhibit cell migration and angiogenesis. Due to the role of CB receptors in carcinogenesis and cancer progression, the regulators of CB receptors act as key molecules in cancer treatment. These results suggest that ABHD12 plays a significant role in breast cancer cell proliferation and development via activation of CB receptors. Furthermore, we recommend to investigate additional comprehensive functions of ABHD12 and CB-related signalling pathway.

In conclusion, this study revealed that ABHD12 knockdown inhibits cell proliferation, and suppresses cell mobility and invasiveness in breast cancer. Furthermore, we showed that ABHD12 knockdown can up-regulate the expression of CB receptors, suggesting its anti-proliferative capability in MCF7 and MDA-MB-231 cells. Our results suggest that the up-regulation of ABHD12 expression is positively correlated with breast cancer progression, and thus ABHD12 is a promising therapeutic target for breast cancer. Accordingly, further studies are needed to identify ABHD12-specific inhibitor or microRNA, which may be a useful strategy to prevent or treat breast cancer expressing high level of ABHD12.

Acknowledgements

This work is supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning [NRF-2015R1A5A2009070].

Footnotes

  • ↵* These Authors contributed equally to this study.

  • Author's Contributions

    Seok Won Kim and Seon-Joo Park designed the study, analyzed the result and prepared the manuscripts. Semo Jun analyzed TCGA data, and performed experiment with Ji-Yeon Lim. Semo Jun and Ji-Yeon Lim were collected data and prepared the figures. All Authors participated in the writing and revision of the final manuscript.

  • Conflicts of Interest

    The Authors declare no conflicts of interest regarding this study.

  • Received March 10, 2020.
  • Revision received March 19, 2020.
  • Accepted March 20, 2020.

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ABHD12 Knockdown Suppresses Breast Cancer Cell Proliferation, Migration and Invasion
SEMO JUN, SEOK WON KIM, JI-YEON LIM, SEON-JOO PARK
Anticancer Research May 2020, 40 (5) 2601-2611; DOI: 10.21873/anticanres.14231
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