Liver X receptor activation reduces gastric cancer cell proliferation by suppressing Wnt signalling via LXRβ relocalization

Abstract Liver X receptors (LXRs) are involved in various diseases associated with lipid disorders, and in regulating cancer cell proliferation. However, the underlying molecular mechanisms, especially those in gastric cancer (GC) remain to be clarified. In this study, immunohistochemistry analysis revealed that LXRβ was mainly expressed in GC tissue, with less expression in adjacent normal tissues. The LXRβ agonist T0901317 efficiently suppressed the proliferation and colony formation of various GC cell lines. We further showed that LXRβ translocated from the cytoplasm to the nucleus when activated by T0901317. LXRβ nuclear localization suppressed the activation of Wnt signalling and decreased the expression of target genes such as MYC, BMP4, and MMP7 through binding to their promoters. Moreover, we demonstrated that the LXR agonist efficiently suppressed GC tumour growth in a nude mouse xenograft model. Taken together, these results revealed that LXRβ agonist inhibited GC cells proliferation by suppressing Wnt signalling via LXRβ relocalization. The results strongly suggest that LXRβ could be a promising target in GC therapy.


| INTRODUCTION
Liver X receptors (LXRs) are important members of the nuclear receptor (NR) superfamily that are involved in a number of diseases, including lipid disorders, cancer, and neurodegenerative diseases. 1 Two isoforms of LXR, LXRα (NR1H3), and LXRβ (NR1H2), were initially identified as orphan receptors, and each isoform has distinct tissue expression and function. 2,3 LXRβ is ubiquitously expressed in all tissues, and there is accumulating evidence to support that LXRs are involved in a variety of cancers by different mechanisms and are potential targets in cancer therapeutics. 2,4 One of the main mechanisms by which LXR agonist inhibits tumour growth is through inhibition of cell proliferation and induction of cell death. 3 In pancreatic ductal adenocarcinoma (PDAC), Qiang Wang and Fan Feng are co-first author. LXR agonist treatments inhibit cell proliferation, cell-cycle progression, and colony formation, regulating multiple gene networks involved in cell cycle arrest and growth factor signalling. 5 LXR agonists also inhibit cell proliferation and cell cycle arrest in breast cancer cells by regulating hepatic expression of the oestrogen deactivation enzyme. 6 It was also reported that LXR activation leads to cell death through pyroptosis in colon cancer. [7][8][9] Conversely, LXR agonist induces apoptosis in LNCaP cells and reduces the growth of xenograft LNCaP tumours in nude mice. 10 However, whether LXR agonists have an effect on gastric cancer (GC) growth needs to be clarified.
The subcellular localization of LXR is controversial in different cancer cells. It was previously reported that unliganded LXRα mainly localizes the nucleus in a nuclear localization signal-dependent manner, whereas unliganded LXRβ is partially exported from the nucleus. 11,12 In contrast, LXRβ shows predominant cytoplasmic localization in colon cancer cells but not in normal colon mucosa cells. 8,9 Both nuclear and cytoplasmic localization was observed in PDAC samples. 5 In this study, we investigated whether LXR agonists inhibit the growth of GC cells and the underlying mechanism of inhibition. We  The animal experiments were approved by the Ethics Committee of Jiangsu University. For xenograft experiments, 3 × 10 6 SGC cells were subcutaneously injected into 6 to 8-week-old BALB/c athymic nude mice. Two days after cell injection, the mice were intraperitoneally treated with T0901317 (50 μg/g mouse) every 3 days.
Tumour volume was measured every 5 days with a caliper.

| Immunostaining and immunocytochemistry
The cells were seeded on coverslip glasses, fixed in formaldehyde (4% in PBS) and permeabilized with 0.1% Triton X-100 in PBS. Subsequently, the cells were incubated with LXRβ antibodies (Cell Signaling, Boston, MA, USA) for 30 minutes, after which Alexa Fluor 488 goat anti-rabbit IgG (Proteintech, Wuhan, China) was added, and the cells were incubated for 30 minutes. Stained cells were observed under a fluorescence microscope. The relative percent fluorescence was calculated by ImageJ software. Immunocytochemistry were performed as previously described. 13 Briefly, samples were deparaffinized, rehydrated, and incubated in Antigen Retrieval Citra Solution. After being blocked in 5% BSA, the sections were incubated with LXRβ antibodies (1:200 dilution) (Cell Signaling) overnight at 4°C. Then, anti-rabbit IgG SABC and DAB detection kits were used to detect the signals.

| Western blot analysis
Cells were lysed in RIPA lysis buffer according to the manufacturer's protocol ((BOSTER, Wuhan, China). Immunoblot analyses were conducted as previously described. 5  body. An enhanced chemiluminescence detection system was used to detect the signals.

| Proliferation and colony formation assay
For analysis of cell proliferation, cells were cultured in 96-well plates overnight and, then treated with LXR agonists. After treatment, WST-1 reagents were added to the cell culture medium, and the cells were incubated for 2 hours. The absorption at 450 nm was determined using a BioTek microplate reader.
For BrdU assays, cells grown in 6-well plates were incubated with 10 μmol L −1 T0901317 for 48 hours. Then, the cells were incubated with 1 mg/mL BrdU for 48 hours. Cells were fixed, permeabilized, and blocking with 3% BSA for 1 hour. Next, the cells were incubated with anti-BrdU antibody at 4°C overnight. Then, FITClabelled anti-rat IgG was added for 1 hour. Cells were observed under a fluorescence microscope and counted.
For colony formation assays, 2 × 10 2 cells were seeded in 10-cm plates and treated with the LXR agonist for 10 days.
Then, the cells were washed, fixed with 4% formaldehyde, stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA) and counted under a microscope.

| Chromatin immunoprecipitation assay
ChIP assays were performed using the ChIP Assay Kit (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. Briefly, chromatin was cross-linked at RT for 10 minutes with 1% formaldehyde in medium; 200 μL lysis buffer was added, and the supernatant was suspended in elution buffer. Immunoprecipitation was performed for 8 hours at 4°C with anti-LXRβ antibodies (1:50 dilution) (Cell Signaling) or normal IgG as a negative control. Protein A agarose was added for 1 hour at 4°C to collect the protein-antibody complexes. After washing, immunocomplexes were eluted in elution buffer. Finally, the free precipitated DNA was purified with a PCR Purification Kit (BBI). PCR was performed using ExTaq (Takara, Kyoto, Japan) following the manufacturer's protocol. The primer sequences were 5′-TTGCTGGGTTATTTTAATCAT-3′ and 5′-ACTGTTTGACAAACCGCATCC-3′ for the c-Myc promoter and 5′-GATACCTATGAGAGCAGTCA-3′ and 5′-CTGCTAGTGACTGCA-GAAAT-3′ for the MMP7 promoter.

| LXRβ is elevated in gastric cancer tissues
We first examined LXRβ expression in GC tissues and adjacent normal tissues in clinical samples. Immunohistochemical staining revealed that LXRβ was mainly expressed in GC tissues ( Figure 1B and D), with less expression in adjacent normal tissues ( Figure 1A and C). LXRβ were mainly expressed in the cytoplasm in GC tissues and adjacent normal tissues. (Figure 1). Next, we analysed LXRβ expression in 55 paired tumour and normal tissues from the Oncomine dataset (https://www.oncomine.org). As shown in Figure (Table 1). Taken together, these data suggest that LXRβ expression is increased in GC tissues.

| LXRβ is mainly expressed in the cytoplasm in human gastric cancer cell lines
We then utilized Western blot analysis to evaluate LXRβ expression levels in different human GC cell lines. As expected, LXRβ protein was expressed in all the examined human GC cell lines, including AGS, AZ521, SGC, BGC, and MGC cells (Figure 2A and B). The subcellular localization of LXRβ in these cell lines was examined by immunofluorescence staining. LXRβ was detected in both the nucleus and cytoplasm ( Figure 2C and D), but its expression was much higher in the cytoplasm than in the nucleus, especially in the SGC and BGC cell lines (approximately 60%-80% cytoplasmic LXRβ) ( Figure 2D).

| LXR agonist reduces the growth of gastric
cancer cells (H) AGS cells were stimulated with or without 5 μmol L −1 T0901317 for 24 h, and invasion ability was quantified. Data are shown as the mean ± standard deviation (SD). *P < 0.05 (two-tailed Student's t-test) ( Figure 3B). We then examined whether inhibiting cell proliferation contributed to the reduction in GC cell numbers. The BrdU assay showed that cell proliferation was significantly decreased after treatment with 5 μmol L −1 T0901317 for 72 hours ( Figure 3C). We then examined the effect of T0901317 on the proliferation and colony formation of GC cell lines in colony formation assays. As shown in Figure 3D and T0901317 also the inhibited the invasion capacity of AGS cells ( Figure 3H). Taken together, these data suggest that LXR agonists reduce GC cell growth by inhibiting proliferation.

| LXR agonist induces LXRβ subcellular relocalization in gastric cancer cells
Under unstimulated activation, LXRβ was mainly localized in the cytoplasm of GC cells ( Figure 2C and D). When AGS, AZ521, SGC, and MGC cells were treated with the LXR agonist T0901317 for 12 hours, LXRβ relocalized to the nucleus (Figure 4A). As shown in Figure 4B, approximately 80% and 60% AGS and AZ521 cells, respectively, showed nuclear localization after T0901317 treatment. Nuclear relocalization of LXRβ also significantly increased in SGC and BGC cells after T0901317 treatment ( Figures 2D and 4B).

| LXR agonist suppresses Wnt signalling in gastric cancer cells
We then examined the mechanisms by which the LXR agonist inhibits proliferation of GC cells. As LXRβ relocalized to the nucleus after activation, we investigated whether LXRβ is involved in activating the Wnt signalling pathway. TOP/FOP reporter assays were conducted to examined Wnt signalling activation in a β-catenindependent manner. 14 As AGS and AZ521 cells showed greater nuclear localization of LXRβ after treatment, we examined Wnt signalling activation in these two cell lines. As shown in Figure 4C, the luciferase reporter analysis revealed that Wnt signalling activity was significantly inhibited in both AGS and AZ521 cells pretreated with the LXR agonist T0901317. Notably, Wnt activation levels decreased in accordance with the concentration of T0901317 ( Figure 4C).
Next, we assessed whether the LXR agonist affects expression of Wnt signalling target genes, such as MYC, BMP4, and MMP7, 15 in GC cells. MYC, BMP4, and MMP7 expression was decreased in AGS cells treated with T0901317 ( Figure 4D). The protein expression level of MYC was also slightly increased in AGS cells after treatment ( Figure 4E). We then performed chromatin immunoprecipitation (ChIP) assays to determine whether LXRβ is recruited to the promoters of Wnt target genes. As shown in Figure 4F Taken together, these data suggest that the LXR agonist inhibits GC cell proliferation by suppressing the activation of Wnt signalling.

| LXR agonist suppresses tumour growth in nude mice
To determine whether the LXR agonist suppresses tumour growth in vivo, we ascertained the effect of T0901317 on the growth of xenografts in nude mice. As shown in Figure 5A, tumour weight in nude mice was significantly reduced after treatment with T0901317 for 15 days. Moreover, Ki67 expression in tumour cells was significantly decreased in mice treated with T0901317 for 15 days ( Figure 5B). Furthermore, the tumour volume of T0901317-treated mice was significantly lower than that of control mice ( Figure 5C and 5D). We also examined protein expression of LXRβ, MYC,

| DISCUSSION
In this study, we demonstrated that LXRβ was strongly expressed in GC tissues from clinical samples at both the mRNA and protein levels. Interestingly, LXRβ expression was much weaker in adjacent normal tissue than in GC tissue ( Figure 1). These findings indicate that LXRβ is abnormally expressed in GC tissues from patients, which is consistent with the findings of a previous report. 5 In   9,16 suggesting that LXRβ could be a promising target in cancer therapy. We also found that subcutaneous injection of an LXR agonist into nude mice suppressed human GC xenograft growth.
The localization of LXRβ in cells of different cancer types is controversial. It was reported that unliganded LXRβ is mainly localized in the nucleus. 11 However, in colon cancer, unliganded LXRβ shows a predominant cytoplasmic localization. 9 Here, we revealed that LXRβ had different subcellular localization patterns during activation. Unliganded LXRβ was mainly expressed in the cytoplasm, whereas liganded LXRβ showed nuclear localization after activation in human GC cells (Figures 2 and 4). The different subcellular localization of LXRβ correlated with the function of LXRβ in inhibition GC cell growth. There results suggest that the differential localization of LXRβ in cancer cells is critical for inducing cell death or inhibiting cellular growth.
The Wnt signalling pathway plays a critical role in GC initiation and progression. Several key components of the Wnt pathway have been reported to be overexpressed in gastric carcinogenesis. 17 In this study, we demonstrated that LXRβ translocated into the nucleus and suppressed Wnt signalling through recruitment to the promoters of Wnt target genes. The expression levels of Wnt target genes, including MYC, BMP4, and MMP7, were significantly suppressed after treatment with T0901317. MYC is required for the activation of the majority of Wnt target genes in colon carcinogenesis. 18 In agreement with our result, LXRβ activation controls MYC gene expression in colon cancer and prostate cancer. 7,19,20 BMP4 expression level was also suppressed in colon cancer and MEFs after stimulation with an LXR agonist. 21,22 This suppression is associated with activation of Wnt signalling. 23 Although a previous report showed that LXRβ interacts with β-catenin in colon cancer cells, 20  This study suggests that LXRβ is a potential target in cancer therapy.