Adipose triglyceride lipase promotes the proliferation of colorectal cancer cells via enhancing the lipolytic pathway

Abstract Abnormal lipid metabolism is the sign of tumour cells. Previous researches have revealed that the lipolytic pathway may contribute to the progression of colorectal cancer (CRC). However, adipose triglyceride lipase (ATGL) role in CRC cells remains unclear. Here, we find that elevated ATGL positively correlates with CRC clinical stages and negatively associates with overall survival. Overexpression of ATGL significantly promotes CRC cell proliferation, while knockdown of ATGL inhibits the proliferation and promotes the apoptosis of CRC cells in vitro. Moreover, in vivo experiments, ATGL promotes the growth of CRC cells. Mechanistically, ATGL enhances the carcinogenic function of CRC cells via promoting sphingolipid metabolism and CoA biosynthesis pathway‐related gene levels by degrading triglycerides, which provides adequate nutrition for the progression of CRC. Our researches clarify for the first time that ATGL is a novel oncogene in CRC and may provide an important prognostic factor and therapeutic target for CRC.


| INTRODUC TI ON
Colorectal cancer (CRC) is the fourth most common cancer worldwide and the fifth leading cause of cancer death in humans. 1 At present, people's eating habits are mainly high-fat diet and lack of high dietary fibre intake, which makes the incidence rate of CRC showing a rising trend. 1 The characteristics of the CRC progression are rapid infiltrating growth, early metastasis and unfavourable prognosis. 2 Unlike most normal cells, cancer cells exhibit uncontrolled cell proliferation. To cope with the unlimited growth, expansion and diffusion, cancer cells must efficiently generate energy, even in the microenvironment of hypoxia and lack of nutrition. 3 Warburg effect shows that cancer cells have increased glucose uptake and glycolysis dependent metabolism. 4 Remarkably, the lipolytic pathway is also reprogrammed in many types of cancer cells, which are depend on mitochondrial βoxidation. 5 CRC cells have been in the intestinal high-fat environment for a long time, which leads to the active function of β-oxidation.
Therefore, it is worthy to explore the lipolytic enzyme function in CRC.
At present, the main reported enzymes involved in lipolysis are adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoacylglycerol lipase (MAGL). 6 Among them, ATGL is the most critical rate-limiting enzyme, which is a member of the patatin-like phospholipase domain (PNPLA) family. 7 Also, the distribution of ATGL in normal human was mainly in adipose tissue, while medium to low expression was detected in other tissues. 7,8 Triglyceride (TAG) metabolism is initiated by ATGL through hydrolysing TAG into free fat acid (FFA) and diacylglycerol (DAG). 9 The next step is to decompose DAG into monoacylglycerol (MAG), which requires HSL. Lastly, MAG is further broken down by MAGL into FFA and glycerol. 10 In addition to energetic purposes, FFA is essential for membranes biosynthesis and also serves as a signalling molecule. 11 ATGL-mediated lipolysis releases a large amount of FFA, which is important to adapt to the high proliferation rates of tumour cells. Previous studies have shown that the lipolytic enzyme, MAGL, enhances tumour growth and metastasis via the FFA pathway. 12,13 But recent researches have also suggested that MAGL has an antitumour effect. 14 Lipolytic enzymes roles need to be further explores in tumour development. ATGL have been shown to promote the proliferation of hepatocellular carcinoma cells. 15,16 However, it is not clear whether ATGL promotes tumour growth or other functions in CRC.
Our study investigated the role of ATGL, a key new oncogene in the development of CRC. And we determined the hypothesis that ATGL promoted CRC proliferation by enhancing lipolysis. These collective findings of our research might provide a novel target for the treatment of CRC.

| Tumour xenograft
We bought 4-week-old male BALB/c nude mice from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were randomly divided into 2 groups based on a previously described standard protocol.
1 × 10 6 SW480-Vector or SW480-ATGL cells were injected into the inguinal folds of mice, respectively (n = 6 in each group). And a nude mouse injected with SW480-Vector cells did not form tumour. We measured the tumour volume with an external calliper and calculated its results by the following formula: Volume = length × width 2 ⁄2. The mice were killed at 27 days after injection. Then, tumours were dissected, weighed, photographed and stored at −80°C for further researches.

| Human samples
The CRC tissue microarrays (HColA180Su21-1-250, 94 cases) were purchased from Shanghai Outdo Biotech. All procedures were performed under consensus agreements and in accordance with the Chinese Ethical Review Committee. The clinical and biological characteristics of the patients were described in Table 1.

| Cell lines and culture
The human CRC cell lines (HCI-H508, SW480, CaCO2, LoVo, HCT116, SW620, HT29) were obtained from the American Type Culture Collection. The normal intestinal epithelial cell lines (CCD841) were provided by Professor Peng Huang, from Sun Yat-sen University Cancer Center. Cell lines were authenticated by Cellcook Biotech Co., Ltd.

| RNA isolation and RT-qPCR
Total RNA was extracted using TRIzol (Thermo Scientific, 15596026) according to the manufacturer's protocol. First-strand cDNA synthesis was performed using 500 nanograms of total RNA, and the RT-qPCR analysis system was performed using iQ SYBR Green Supermix and the iCycler Real-time PCR Detection System (Bio-Rad).

| Immunofluorescence staining
After fixed in 4% paraformaldehyde, cells were blocked with goat serum at 37°C for 1hour. They were incubated with rabbit Ki67(Millipore, AB9260) antibodies at 4°C overnight, then were incubated with FITC conjugated goat anti-rabbit IgG (Dako, K500711) at 37°C for 1 hours after three times washing. Finally, the cell nucleus was stained with DAPI (Sigma-Aldrich, D9542).

| Cell Counting Kit-8 (CCK8)
Cell proliferation was measured via cell viability with a Cell Counting Kit-8 (Dojindo). CRC cells were seeded into 96-well plates and cultured for 24, 48 and 72 hours. Then, 10 μL CCK8 reagent was added to 96well plates and incubated for 2 hours. The absorbance (OD450 nm) was measured using a microplate reader (TECAN) and calculated.

| Colony formation assay
CRC cells were plated in 6-well dishes (500 cell/dish) and then incubated for 2 weeks for colony formation. After 14 days, cell colonies were then fixed in 4% polyformaldehyde and stained with 0.1% crystal violet. All colonies were counted separately for each sample, and the relative colony numbers were calculated.

| Terminal deoxynucleotidyl transferasemediated dUTP nick end labelling (TUNEL) staining
Tissue sections were deparaffinized and hydrated in xylene and gradient concentrations of ethanol, then incubated in proteinase K at room temperature for 30 minutes and stained with TUNEL kit (Sigma-Aldrich). Label solution was used instead of TUNEL reagent in the negative control group. All the images were captured by a fluorescence microscope (DFC700T, Leica). Cells that were positive for TUNEL staining and aligned with DAPI staining were considered apoptotic cells and counted.

| siRNA and lentivirus
ATGL siRNA and control siRNA were purchased from RiboBio.
According to the manufacturer's instructions, transfections were performed at approximately 60% confluency using RNAiMAX (Invitrogen). After 48 hours, confirmation of interference was carried out using real-time quantitative PCR (RT-qPCR) and Western blotting. Plasmids encoding ATGL were packaged as lentivirus by GeneChem Co., Ltd.

| Annexin V/propidium iodide flow cytometric analysis
CRC cell staining with Annexin V and PI was carried out using an Annexin V-FITC/PI Apoptosis Detection kit (Merck). A total of 1 × 10 6 cells were incubated at 37°C for 30 minutes before centrifugation to collect the cell pellet, then resuspended in a Ca2+-enriched binding buffer and analysed using a Beckman Coulter flow cytometry. Data were calculated using Cell Quest software. manufacturer's instructions. FFA was calculated from a standard curve for each assay, and the data were normalized to total protein.

| Oil Red O staining
For lipid droplet staining, different groups of SW480 and HCT116 cells incubated with 400 μM OA for 6 hours, or 10 μm cryostat sections from indicated CRC xenografts, were washed, fixed in 4% paraformaldehyde for 10 minutes and rinsed with 60% isopropanol. The slides were then placed in the freshly prepared working Oil Red O solution for 10 minutes at room temperature and rinsed again with 60% isopropanol. After lightly stained nuclei with haematoxylin and washed with distilled water, the slides were covered with glycerine jelly that will harden after a few hours. Relative lipid content was quantified by using Image Pro Plus 6.0.

| TCGA data analysis
The RNASeq data and clinical data for CRC were obtained from The Cancer Genome Atlas (TCGA) databases (https://genom e-cancer.ucsc. edu). For the association of ATGL expression with survival was used as a surrogate end-point and patients dichotomized by ATGL expression.
Gene set enrichment analysis (GSEA) was used based on CRC TCGA databases.

| Statistical analysis
We presented the variability of the data as the SD (mean ± SD).
Between two groups, significant differences were determined using Student's t test. And we used one-way ANOVA to assessed multiple groups significant differences. χ 2 test was used to determine the relationships between clinicopathological characteristics and ATGL expression. Survival curves were plotted by the Kaplan-Meier method and further compared by the log-rank test. Statistical significance was defined at P < 0.05.

| Elevated ATGL is associated with tumour progression in CRC
To investigate the underlying role of ATGL in CRC, the expression of ATGL in a CRC Tissue Microarray was detected by immunohistochemistry (IHC) staining. Impressively, ATGL expression was significantly elevated in CRC specimens compared with adjacent specimens ( Figure 1A,B). In addition, ATGL expression increased along with the progression of CRC clinical stages ( Figure 1C,D).
Moreover, the chi-square test was used to analyse the association between clinicopathological characteristics and ATGL expression.
The result indicated that the expression of ATGL was related to the clinical stage (P = 0.018), T classification (P = 0.010) and CD8 expression (P = 0.007), but not with gender, age, N classification, M classification, intravascular tumour thrombus, nerve invasion, PDL1 expression and PD1 expression (Table 1). Meanwhile, Kaplan-Meier analysis showed that ATGL protein levels were negatively correlated with overall survival (P < 0.001; Figure 1E). Collectively, these results suggested that elevated ATGL contributes to the progression of CRC and may be a poor prognostic factor for CRC.

| ATGL promotes CRC cell proliferation
To reveal the effect of altered ATGL expression on CRC cell prolifera-  Figure S1B).
These data indicated that ATGL enhanced the proliferation of CRC cells.

| Knockdown of ATGL promotes apoptosis of CRC cells
In further experiments, we examined the apoptosis of CRC cells with ATGL knockdown by flow cytometric analysis and TUNEL staining.
CRC cells were incubated with PI and Annexin V staining for flow cytometric detection. Our results indicated that the apoptosis rate was apparently elevated after interfering with ATGL ( Figure 4A; Figure S2). Furthermore, the results of TUNEL staining suggested that the staining intensity elevated after ATGL knockdown, indicating that knockdown of ATGL promoted CRC cell apoptosis ( Figure 4B).

| ATGL promotes the lipolytic pathway of CRC cells
In To further demonstrate the molecular mechanisms of ATGL in lipid metabolic pathways in CRC, we analysed genes related to ATGL expression in the TCGA CRC dataset. Gene set enrichment analysis (GSEA) indicated that ATGL was positively correlated with sphingolipid metabolism ( Figure 5E) and CoA biosynthesis ( Figure 5F). The heat map demonstrated that ATGL was positively correlated with ACER2, SMPD1, SPHK2 and other genes in cholesterol metabolism, while ATGL was positively correlated with ENPP1 and other genes in CoA biosynthesis ( Figure S3A,B). Bioinformatic analysis results were further verified by qRT-PCR, and we found that the expression of ACER2 was most obviously changed in these cells ( Figure S3C,D).
Collectively, these results suggested that ATGL-mediated lipolysis releases FFA for cholesterol metabolism and CoA biosynthesis.

| ATGL promotes the growth of CRC cells in vivo
Animal research was performed further to confirm the role of ATGL carcinogenesis in vivo. The tumours generated by ATGL-transfected SW480 cells had a larger size than those generated by control cells ( Figure 6A-C). Noticeably, Oil Red O staining indicated that ATGL decreased lipid droplets accumulation in SW480 Xenograft Models, which was consistent with the results of in vitro experiments ( Figure 6D,E). Moreover, Ki67 staining of the SW480 Xenograft Models tumours showed that ATGL promoted CRC cell proliferation ( Figure 6F,G). Our findings revealed that ATGL accelerated CRC cells growth in vivo.

| D ISCUSS I ON
Abnormal lipid metabolism in CRC has been reported to be closely associated with the biological processes of tumorigenesis and development. Lipid mobilization or lipolysis means that one TG molecule is hydrolysed successively by ATGL, HSL and MAGL to release three free FFA molecules and one glycerol molecule. 9,10 Dysregulated lipid metabolism underlies CRC pathogenesis, facilitating substantial enhancement in FFA content and cancer cell survival. Remarkably, ATGL is the initial enzyme involved in the process of degrades fat into monoglycerides and starts fat mobilization. 9 However, the relationship between CRC and ATGL remains unclear.
To clarify the role of ATGL in CRC, the expression of ATGL in CRC tissue was examined by IHC. Our results showed that ATGL expression was remarkable elevated in CRC and negatively associated with overall survival (Figure 1). Further, overexpression of ATGL apparently enhanced the proliferation of CRC cell in vitro, while downregulation of ATGL was the opposite (Figures 3 and 4). Only recent research has emphasized the regulatory role of ATGL in cancer.
While the basic role of ATGL in catabolism has been widely studied Combining bioinformatic analysis and qRT-PCR results, we found that the expression of ACER2 was most obviously changed in these cells ( Figure S3A,B). Previous study has shown that ACER2 was the vital gene in the biosynthesis of sphingosine-1-phosphate (S1P), which was one of the most vital final products of the sphingolipid metabolism. 31 It is worthy to further explore whether S1P or other specific lipids responsible for the oncogenic role of ATGL in our further study, which can unravel the precise mechanistic function of ATGL.
Although our current study complements previous studies, the mechanism of ATGL promoting CRC progression has not been fully elucidated. In the future work, some reported mechanisms of ATGL participation can be used as references. In most immunocytes, ATGL promotes the production of cytokine IL-6 to enhance the pro-inflammatory response and chemotaxis. 32,33 Interestingly, clinicopathological characteristics showed a negative correlation between ATGL and CD8 positive T-cell rates (Table. 1). The occurrence and development of CRC are closely associated with inflammation. 34 It is worth getting more convincing data to explore the relationship between ATGL and inflammation in CRC. Besides, a recent study showed that ATGL stimulated autophagy and lipophagocytosis in the liver through SIRT1 signalling. 35 Actually, the ATGL protein sequence contains the LC3 interaction region, which is a motif that mediates the connection between LC3coated autophagosomes and autophagy receptor. 36 The connection between ATGL and LC3 contributes to the degradation of lipids more effectively through the synergy of lipolysis and lipophagy. Hence, the role of ATGL induced autophagy in CRC deserves further exploration.
In summary, our research suggests that ATGL is highly correlated with the progression of CRC via enhancing the lipolytic pathway.
These results indicate ATGL is a promising prognostic marker and therapeutic target for CRC.