Downregulation of uncoupling protein 1 by hypermethylation in gastric cancer activates Rap1 signaling

Abstract

BACKGROUND

Uncoupling protein 1 (UCP1) plays a pivotal role in modulating energy expenditure and maintaining metabolic homeostasis within brown and beige adipocytes. It has also been implicated in tumorigenesis.

AIM

To investigate the expression and function of UCP1 in gastric cancer (GC).

METHODS

UCP1 protein expression in 211 GC tissues was examined using immunohistochemistry. Bisulfite sequencing PCR (BSP) was used to detect the methylation status of the UCP1 promoter in GC cell lines and tissues. The relationship between UCP1 expression and clinicopathological parameters was analyzed. CCK8, scratch, transwell, and flow cytometry assays were carried out to analyze the proliferation, migration, invasion, and apoptosis of GC cell lines after knockdown or overexpression of UCP1 in vitro. A nude mouse tumor xenograft model was used to investigate the function of UCP1 in vivo. RNA sequencing, Kyoto Encyclopedia of Genes and Genomes analysis, and Rap1 pull-down assays were performed to identify the pathway associated with UCP1.

RESULTS

Loss of UCP1 was significantly associated with gender, poor differentiation, and advanced TNM stage of GC. Hypermethylation of UCP1 was confirmed in GC cells and tumor tissues by BSP. Overexpression of UCP1 suppressed GC cell proliferation, migration, and invasion, and it promoted apoptosis in vitro. UCP1 overexpression also suppressed GC tumor growth in vivo. Moreover, overexpression of UCP1 in GC cells resulted in a significant decrease in active Rap1 protein levels, whereas downregulation of UCP1 markedly enhanced Rap1 activity.

CONCLUSION

UCP1 downregulation in GC through promoter hypermethylation is related to the progression of GC, indicating that UCP1 plays a role as a tumor suppressor in GC. It regulates Rap1 signaling and may be a potential therapeutic target in GC.

Key Words: Uncoupling protein 1; Gastric cancer; Hypermethylation; Rap1 signaling

Core Tip: Uncoupling protein 1 (UCP1) plays a significant role in regulating energy expenditure and metabolic homeostasis in brown and beige adipocytes. Hypermethylation of UCP1 was confirmed in gastric cancer (GC) cells and tumor tissues. Overexpression of UCP1 suppressed GC cell proliferation, migration, and invasion, and it promoted apoptosis in vitro. Overexpression of UCP1 also suppressed GC tumor growth in vivo. The level of active Rap1 protein in GC cells was significantly decreased with UCP1 overexpression and significantly increased after UCP1 knockdown. UCP1 plays a role as a tumor suppressor in GC; it regulates Rap1 signaling and may be a potential therapeutic target in GC.

INTRODUCTION

Metabolic reprogramming, a hallmark of malignancy, plays a significant role in tumor initiation, progression, metastasis, resistance to chemotherapy, and recurrence[1,2]. Lipid metabolic reprogramming, in particular, is a crucial feature of cancer, as it is essential for promoting cancer cell proliferation and progression[3,4]. Epidemiological studies have reported a high incidence of gastric cancer (GC) in overweight individuals, prompting scholars to acknowledge the growing importance of fatty acid metabolism in the pathogenesis and advancement of GC[5]. Chi et al[6] found that the lipid metabolism regulated by PHTF2 was of great significance in the diagnosis and treatment of GC. Zhao et al[7] showed that the HKDC1/G3BP1-PRKDC regulatory axis induced GC metastasis and chemoresistance via reprogramming lipid metabolism. These findings imply that fatty acid metabolism may play a role in the development and progression of GC.

Adipose tissue—predominantly comprising adipocytes and pre-adipocytes—plays various physiological roles in adult humans[8]. White adipose tissue, the most prevalent type, is involved in energy storage, metabolism, insulin sensitivity, and inflammation[9]. Conversely, brown adipose tissue (BAT) contains brown adipocytes with a high mitochondrial content, resulting in its distinctive brownish color. BAT is predominantly found in small depots located in the cervical, supraclavicular, axillary, and paravertebral regions in adult humans[10]. Its primary function includes energy expenditure, and it may be involved in metabolic disorders such as diabetes and obesity[11]. Emerging evidence indicates that BAT may also contribute to tumor progression and could be a factor in the pathogenesis of cancer cachexia[9,12]. The brown fat phenotype is principally distinguished by the presence of uncoupling protein 1 (UCP1), which induces a proton leak in the mitochondrial respiratory chain, thereby resulting in the dissipation of energy as heat rather than the production of ATP[13]. Consequently, this phenotype is closely linked to energy expenditure and metabolism. There is also evidence that UCPs are involved in various biological processes in carcinogenesis, such as proliferation, differentiation, apoptosis, and chemotherapy resistance[14-17]. In non-small cell lung cancer, UCP1 is highly expressed with increased glucose absorption[18]. However, UCP1 is downregulated in colorectal cancer and associated with a better prognosis[19]. As a fundamental element in lipid catabolism and non-shivering thermogenesis, UCP1 has been found to induce a reduction in clear cell renal cell carcinoma, promoting lipid browning and hindering tumor advancement[20,21]. These inhibitory effects are similarly present in the breast cancer cell line HCC1806[22]. In ALDH-positive breast cancer stem cells, UCP1 can reduce the inhibitory effect of Snail on glucose metabolism enzyme FBP1, thereby suppressing the tumor progression[23]. However, the expression and effects of UCP1 in GC have not been clearly reported.

In this study, we investigated the expression of UCP1 in GC and the association of UCP1 expression with clinicopathological features. The status of CpG island methylation in the UCP1 promoter was analyzed. In vitro and in vivo experiments were subsequently performed to explore the function of UCP1 in GC cells. Finally, we conducted RNA sequencing to analyze the molecules involved in the regulation of UCP1 in GC.

MATERIALS AND METHODS

Patients and samples

In total, 211 formalin-fixed paraffin-embedded (FFPE) GC tissues were obtained from the Department of Pathology at The Affiliated Jiangning Hospital of Nanjing Medical University. The median age was 66 years (range, 45-85 years), and most of the patients were male (75.4%). The percentages of patients with stage I, II, III, and IV disease at surgery were 19.4%, 43.1%, 23.2%, and 14.2%, respectively. Detailed information on the patients can be found in Table 1. The patients did not receive any treatment before the operation. Ethical approval for this retrospective study was granted by the Medical Ethics Committee of The Affiliated Jiangning Hospital of Nanjing Medical University, with a waiver of the requirement for patient informed consent in accordance with the institutional review board’s protocols for retrospective data analyses.

Table 1 Clinicopathological features of the 211 patients in the present study.

Clinicopathological features		n (%)
Sex	Male	159 (75.4)
	Female	52 (24.6)
Age (years)	< 65	97 (46.0)
	≥ 65	114 (54.0)
Depth of invasion	T1	15 (7.1)
	T2	26 (12.3)
	T3	101 (47.9)
	T4	69 (32.7)
Differentiation	Well	48 (22.7)
	Moderate	69 (32.7)
	Poor	94 (44.5)
Lymph node metastasis	No	66 (31.3)
	Yes	145 (68.7)
TNM stage	I	41 (19.4)
	II	91 (43.1)
	III	49 (23.2)
	IV	30 (14.2)

Immunohistochemical staining

The FFPE tissues were sliced into 5-μm-thick sections. The sections were deparaffinized with xylene and rehydrated in decreasing concentrations of ethanol and then were heated in a microwave for 5 minutes in a 10 mmol/L sodium citrate buffer for antigen retrieval. Next, they were incubated with a UCP1 antibody (23673-1-AP; Proteintech, China; 1:1000 dilution) at 4 °C overnight. After washing with phosphate-buffered saline (PBS), a peroxidase-conjugated secondary antibody (Dako; Agilent Technologies, Inc.) was added. Finally, color was developed using a Pierce DAB Substrate Kit (34002, Invitrogen; Thermo Fisher Scientific, Inc.). The stained sections were examined by two pathologists, and any differences were resolved through discussion. The staining intensity criteria for positive cells were as follows: 0 (no staining), 1 (light yellow staining), 2 (brown staining), and 3 (dark brown staining). Scores of 0 and 1 were defined as negative, while scores of 2 and 3 were defined as positive expression.

Cell lines

Three GC cell lines (BGC-823, SGC-7901, and AGS) and HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured and maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, United States) supplemented with 10% fetal bovine serum (FBS, Gibco, United States) and 1% penicillin/streptomycin (Sangon, Shanghai, China) at 37 °C and 5% CO₂ in an incubator.

Bisulfite sequencing PCR assay

A bisulfite sequencing PCR (BSP) assay was performed to evaluate the methylation status of UCP1 in the GC cell lines and FFPE samples. An EZ DNA Methylation-Gold Kit (ZYMO Research, CA, United States) was used for the bisulfite conversion of DNA. The forward primer sequence was 5’-AAGGGAAAGGAATTTTTTTTATTTT-3’, and the reverse sequence was 5’-CCCCTCTACCTAACTACCTAAAACC-3’. The thermal cycling program for the PCR was as follows: Denaturation at 94 °C for 10 minutes; 40 cycles at 94 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 1 minute; and a final extension step at 72 °C for 5 minutes. Sanger sequencing was used to determine the methylation status.

5-Aza-CdR treatment

BGC-823, SGC-7901, and AGS cells were treated with 5-Aza-CdR (5-Aza; Sigma, Saint Louis, MO, United States). Briefly, the cells were cultured in 96-well plates overnight and treated with 5-AZA dissolved in PBS for 48 hours at a concentration of 5 mmol/L. The control was treated with PBS. The expression level of UCP1 in these cells was subsequently analyzed via reverse-transcription PCR (RT-PCR).

RT-PCR

Total RNA was extracted using an RNA-extraction kit (Sangon, Shanghai, China) and reverse-transcribed. The prepared complementary DNA (cDNA) was then amplified. The primers used were obtained from Sangon (Shanghai, China). PCR was performed on the obtained cDNA using a TaKaRa Ex Taq (Takara Bio, Dalian, China) with the following PCR primers: Forward 5’-GCTCCAGGTCCAAGGTGAATGC-3’ and reverse 5’-TGCCACTCCTCCAGTCGTTAGA-3’. The thermal cycling procedure was as follows: 40 cycles of 30 seconds at 95 °C, 30 seconds at 60 °C, and 60 seconds at 72 °C. PCR products were analyzed via 2% agarose gel electrophoresis.

Cell transfection

For the knockdown study, 5 × 10⁵ cells were transfected with 100 nM UCP1-siRNA or control siRNA for 48 hours using Lipofectamine transfection reagent (Thermo Fisher, 168 Third Avenue Waltham, MA, United States 02451) following the manufacturer’s protocol. siRNAs were designed and purchased from Sangon (Shanghai, China). The sequences of the siRNAs used were as follows: (1) siUCP1: Sense 5’-CAAUGAAUGUGUUCACUAACG-3’ and antisense 5’-UUAGUGAACACAUUCAUUGCA-3’; and (2) siNC: Sense 5’-UUCUCCGAACGUGUCACGUUA-3’ and antisense 5’-ACGUGACACGUUCGGAGAACG-3’.

For the overexpression study, the UCP1 gene was ligated into pLVX-IRES-puro to construct a UCP1-overexpressing plasmid. The pLVX-IRES-puro and pRUF-IRES-puro-UCP1 constructs were transfected into the HEK293T viral packaging cell line together with the psPAX2 and pMD2. G plasmids. After 48 hours of culture, puromycin (1 μg/mL) was added to the medium. After screening with 1 μg/mL puromycin three times to determine which cells had undergone successful transfection, DMEM containing 10% FBS was added for 24 hours, after which the cells were collected in culture flasks for subsequent experiments.

CCK8 assay

Cell proliferation was measured using a CCK-8 Kit (Sangon, Shanghai, China). A total of 2 × 10³ cells were seeded in 96-well plates with 100 μL of the medium, 10 μL of CCK-8 solution was added to the wells, and the medium was replaced with fresh medium following plating. After 4 hours, the medium was scanned by a microplate reader at OD450.

Apoptosis

Flow cytometry was utilized to measure the percentage of apoptotic GC cells. The cells were stained with an Annexin V-FITC and PI Kit (Sangon, Shanghai, China). The cells were divided into live, early apoptotic, late apoptotic, and necrotic groups.

Wound-healing assay

For the wound-healing assay, the cells were seeded in a 6-cm dish and grown until close to 90% or more confluence. A sterile pipette tip (200 μL) was used to longitudinally scratch the monolayer cell plane to create a wound. After washing with PBS, the cell debris was removed, and the cells were cultivated in a medium without FBS. Finally, we obtained images at 0 and 12 hours using an inverted microscope (magnification: 100 ×; Olympus Corporation, Tokyo, Japan). We evaluated the cell migration capacity according to the scratch area measured with ImageJ software. All experiments were repeated at least three times.

Invasion and migration assays

Transwell migration assays were completed on 8-μm-pore membranes in 24-well plates (Corning, NY, United States). The upper chamber was covered, either with or without Matrigel, and a cell suspension containing 2% FBS and 2 × 10⁵ cells was added to it. Next, 600 mL of medium containing 10% FBS was added to the lower chamber. Transwell invasion assays were performed by precoating the upper membrane with 40 μL of matrix glue (Corning, NY, United States). After 24 hours, the cells that did not migrate were removed from the top of the membrane with a cotton swab, and the migrated cells were then fixed, stained with crystal violet, and observed under a microscope (magnification: 200 ×; Olympus Corporation). The average value of at least six visual fields was taken, and the experiment was repeated three times.

Animal models

To construct a subcutaneous tumor model in nude mice, 5 × 10⁶ BGC-823 GC cells were injected into the right axilla of each female nude mouse. The mice were randomly divided into the UCP1 overexpression group (n = 5) and control group (n = 5), and the mice were kept under sterile conditions, receiving sterile nutrition and water. Tumor growth was monitored by living imaging. After 4 weeks, the nude mice were killed, their subcutaneous tumors were removed, and the weights and volumes of the removed tumors were measured; 4% paraformaldehyde was subsequently used to store the tumors. The fixed tumor tissues were then embedded in paraffin, sectioned, and stained with H&E.

RNA sequencing

BGC-823 cells were stably transfected with PLVX-Puro-shUCP1 or an empty vector. Total RNA was isolated and purified using TRIzol (Sangon, Shanghai, China). Then, a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, United States) was used to determine the quantity and purity of the total RNA. The captured mRNA was fragmented at 94 °C for 5-7 minutes using a magnesium ion fragmentation kit (NEBNext^® RNA Fragmentation Module, article no. E6150S, United States). cDNA was synthesized from the segmented RNA using Invitrogen SuperScript™ II Reverse Transcriptase (No. 1896649, CA, United States). An Illumina NovaSeq™ 6000 (LC Biotechnology Co., Ltd., Hangzhou, China) was used for double-terminal sequencing in the PE150 sequencing mode, in line with standard methods. Cutadapt (https://cutadapt.readthedocs.io/en/stable, version 1.9) was used to remove the joint plane raw data processing software. A significant difference was analyzed between samples, and the multiple of the difference [fold change (FC) > 1, with a P value < 0.05] defined differentially expressed genes, which were analyzed for enrichment using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (https://www.genome.jp/kegg/pathway.html).

Western blot

Protein expression was examined through western blot analysis. Briefly, cells were harvested and lysed with RIPA buffer (Beyotime, China), and the cleared lysates (30-50 μg/well) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4%-20% gel) and transferred onto nitrocellulose membranes. After blocking for 1 hour at room temperature in Tris-buffered saline (pH 7.4) supplemented with 0.1% Tween 20 (TBST) containing 5% nonfat dry milk, the membranes were incubated with primary rabbit anti-Rap1A/Rap1B (CST, 2399; diluted 1:500). The membranes were subsequently washed in TBST and incubated with an anti-rabbit secondary antibody (CST, 7074; diluted 1:5000) at room temperature for 1 hour. The bands were visualized with an ECL Plus chemiluminescence reagent kit (ACE, China).

Detection of active Rap1

Active Rap1 (GTP-bound) was detected using an active Rap1 detection kit (Cell Signaling Technology, United States). All steps were performed in accordance with the manufacturer’s instructions. Briefly, 750 μg of protein lysate obtained from GC cells was combined with GST-RalGDS-RBD in a spin cup placed within a collection tube. Following this, the spin cups were subjected to incubation at a temperature of 4 °C for a duration of 1 hour with gentle rocking. The GTP-bound Rap1 (active Rap1) protein was extracted. Finally, the eluted samples were subjected to western blotting.

Statistical analysis

IBM SPSS Statistics (version 24.0; Armonk, NY, United States) and GraphPad Prism 8 software (GraphPad Software, Inc., La Jolla, CA, United States) were used to analyze the data for this study. A t-test was used to compare two groups. The χ² test and Fisher's exact test were employed to examine relationships between expression levels and clinicopathological parameters, with a two-tailed P value < 0.05 denoting statistical significance.

RESULTS

Differential expression of UCP1 in GC

The UCP1 protein expression was assessed in the GC FFPE samples using immunohistochemical staining. The UCP1 protein was differentially expressed in GC; namely, it was positively expressed in normal mucosa, and negatively, moderately, or positively expressed in tumors (Figure 1). According to their immunohistochemical scoring (Table 2), the cohort of 211 GC patients comprised a UCP1-negative group (n = 76) and a UCP1-positive group (n = 135). UCP1 expression level was significantly correlated with gender (P = 0.037), tumor differentiation (P < 0.001), and TNM stage (P < 0.001); was associated with tumor invasion depth (P = 0.084) and lymph node metastasis (P = 0.074); and was not associated with patient age (P = 0.787; Table 2). Similarly, multivariate logistic regression showed that UCP1 expression was significantly inversely associated with tumor differentiation (OR = 0.446, P = 0.020) and TNM stage (OR = 0.233, P < 0.001; Table 3).

Figure 1 Uncoupling protein 1 was differentially expressed in gastric cancer formalin-fixed paraffin-embedded tissues as revealed by immunohistochemistry staining. A: Uncoupling protein 1 was positively expressed in normal gastric tissues; B-D: It was negatively (B) expressed in some of gastric cancer (GC) tissues (76 out of 211), and moderately (C) and strongly (D) expressed in some of GC tissues (135 out of 211). Image magnification: 200 ×.

Table 2 Relationship between uncoupling protein 1 expression and the clinicopathological features of gastric cancer.

Characteristics		UCP1 expression		P value	HR (95%CI)
		Negative	Positive
Sex	Male	51	108	0.037a	0.839 (0.702-1.003)
	Female	25	27
Age (years)	≥ 65	42	72	0.787	1.036 (0.802-1.339)
	< 65	34	63
Depth of invasion	T1/T2	10	31	0.084	0.573 (0.298-1.103)
	T3/T4	66	104
Differentiation	Well	9	39	< 0.001b	0.613 (0.451-0.831)
	Moderate	21	48
	Poor	46	48
Lymph mode metastasis	No	18	48	0.074	0.666 (0.419-1.058)
	Yes	58	87
TNM stage	I/II	30	102	< 0.001b	0.522 (0.389-0.701)
	III/IV	46	33

^aP < 0.05.

^bP < 0.01.

χ² test was performed. UCP1: Uncoupling protein 1; OR: Odds ratio; 95%CI: 95% confidence interval.

Table 3 Multivariate logistic regression analysis of the association between uncoupling protein 1 methylation and clinicopathological characteristics in patients with gastric cancer.

Clinicopathological characteristics	OR	95%CI	P value
Sex (male vs female)	1.958	0.958-4.001	0.065
Age (≥ 65 years vs < 65 years)	0.870	0.467-1.622	0.662
Depth of invasion (T3/T4 vs T1/T2)	1.677	0.592-4.751	0.330
Differentiation (well & moderate vs poor)	0.446	0.227-0.880	0.020a
Lymph mode metastasis (yes vs no)	1.057	0.455-2.455	0.898
TNM Stage (III and IV vs I/II)	0.233	0.115-0.469	< 0.001b

^aP < 0.05.

^bP < 0.001.

OR: Odds ratio; 95%CI: 95% confidence interval.

Methylation of UCP1 in GC cell lines and FFPE samples

To ascertain the potential regulatory role of DNA methylation in UCP1 expression, the methylation status of the UCP1 promoter region was assessed in the GC cell lines and FFPE samples using BSP. The findings revealed a state of hypermethylation within the UCP1 promoter in BGC-823, SGC-7901, and AGS cells, aligning with the observed diminished UCP1 expression in these cell lines (Figure 2A and B). Moreover, bisulfite sequencing analysis demonstrated an inverse correlation between UCP1 methylation status and its expression level in FFPE samples, indicating that DNA methylation plays a role in regulating UCP1 expression (Figure 2C-E).

Figure 2 Uncoupling protein 1 expression is silenced by promoter hypermethylation in gastric cancer. A: The parts of nucleotide sequences of a CpG island in the Uncoupling protein 1 (UCP1) promoter region including seven CG sites. The upper line shows nucleotide sequences in the GenBank, and the lower line shows those after treatment with sodium bisulfite; B: Bisulfite sequencing PCR analysis of UCP1 methylation status in gastric cancer (GC) cell lines. Methylated DNA was detected in all three GC cell lines (methylated C is indicated with a blue arrow). The methylation status of UCP1 in GC tissues was consistent with the UCP1 expression level; C: Unmethylated CG sites (orange arrow) were detected in GC tissue with positive UCP1 expression; D: Both unmethylated and methylated CG sites (green arrow) were detected in GC tissues with moderate expression of UCP1; E: Methylated CG sites (blue arrow) were detected in GC tissues with negative expression of UCP1; F: The expression of UCP1 in all GC cell lines was restored after 5-Aza treatment. UCP1 mRNA expression levels (right) with or without 5-Aza treatment in SGC-7901, AGS, and BGC-823 cells were detected using reverse-transcription PCR, compared with the house-keeping gene GAPDH as internal control (left). UCP1: Uncoupling protein 1.

DNA hypermethylation can potentially be reversed through the use of DNA-demethylating agents. In this study, gastric cells were treated with 5-Aza, and the expression of the UCP1 gene was assessed through RT-PCR analysis. Treatment with 5 μmol/L 5-Aza significantly elevated UCP1 mRNA levels (Figure 2F). These results confirm that the loss of UCP1 expression can be attributed to the UCP1 promoter methylation.

UCP1 plays a role of a tumor suppressor in GC

Three GC cell lines (SGC-7901, BGC-823, and AGS) were transfected with UCP1 siRNA or UCP1-overexpressing plasmid for loss-of-function or gain-of-function assays, respectively. UCP1 knockdown or overexpression was verified by RT-PCR (Supplementary Figure 1). The proliferation was enhanced in the UCP1-knockdown cell lines relative to the control cell lines, providing evidence that UCP1 inhibits tumor growth (Figure 3A). Knockdown of UCP1 led to a significant decrease in both early and late apoptotic rates in human GC cell lines relative to control siRNA (Figure 3B). UCP1 knockdown significantly enhanced the ability of GC cells to migrate to the scratched area compared with the control group (Figure 3C and D). The transwell assay revealed a significant increase in migration following UCP1 siRNA transfection (Figure 3E and F). Overexpression of UCP1 in GC cell lines resulted in the opposite effect (Figure 4).

Figure 3 Knockdown of uncoupling protein 1 promoted gastric cancer cell proliferation, migration, and invasion, and inhibited apoptosis. A-F: Knockdown of Uncoupling protein 1 with siRNA significantly promoted gastric cancer cell proliferation (A), migration (C and D), and invasion (E and F) compared with negative control groups, and inhibited apoptosis (B). Image magnification: 200 ×. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. UCP1: Uncoupling protein 1; Ctrl: Control; OE: Overexpression.

Figure 4 Uncoupling protein 1 overexpression. A-F: Uncoupling protein 1 overexpression significantly inhibited gastric cancer cell proliferation (A), migration (C and E), invasion (D and F), and induced apoptosis (B). Image magnification: 200 ×. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Ctrl: Control; OE: Overexpression.

Overexpression of UCP1 suppresses tumor growth in vivo

To examine the function of UCP1 in vivo, stable UCP1-overexpressing GC cells were implanted into nude mice (n = 5 for each group). Subcutaneous masses became detectable 10 days after injection of the tumor cells, with live imaging confirming the successful establishment of the nude mouse xenograft tumor model for GC. By the third week, all mice in the UCP1-overexpression group displayed notable inhibition of tumor growth, as evidenced by bioluminescence intensity (P < 0.001; Figure 5A). The mice were euthanized at the end of the third week, and the tumor tissues were then stripped and weighed. As demonstrated in Figure 5B and C, the overexpression of UCP1 remarkably reduced the tumor volume and weight (P < 0.001). In particular, H&E staining of the tumor tissue isolated from the nude mouse xenograft tumor model confirmed decreased nuclear division after UCP1 overexpression, indicating that antisense UCP1 has an antiproliferative effect (Figure 5D). The results of the in vivo tumor growth experiment indicated that overexpression of UCP1 inhibited in vivo tumor development in the xenograft mouse model.

Figure 5 Uncoupling protein 1 overexpression inhibited tumor growth in vivo. A: In vivo live imaging of mice with a subcutaneously grafted tumor; B: Representative photographs of tumor size in each group; C: Analysis of tumor weight and size in the Uncoupling protein 1 (UCP1)-overexpression and control groups; D: Grafted tumors were subjected to H&E staining. The results indicated that the number of mitotic cells (orange arrow) significantly decreased in UCP1-overexpressed gastric cancer cells compared with control cells (P < 0.001; Image magnification: 400 ×). ^aP < 0.001. Ctrl: Control; OE: Overexpression.

UCP1 regulates Rap1 signaling activity

To further explore the regulatory effect of UCP1 knockdown on GC cells, RNA-sequencing analysis was performed. Compared with the control group, the UCP1-knockdown group had 3335 upregulated genes and 2713 downregulated genes (Padj < 0.05, |log2FC| > 1, Figure 6A). The Gene Ontology analysis terms showing significant differences were related to peptidyl-proline dioxygenase activity, G-protein-coupled amine receptor activity, ephrin receptor binding, thioesterase binding, and transmembrane-ephrin receptor activity (Supplementary Figure 2). For the KEGG pathway, Rap1 signaling pathway, Hippo signaling pathway, and cAMP signaling pathway were significantly enriched (Figure 6B).

Figure 6 Uncoupling protein 1 regulates the Rap-1 signaling pathway in gastric cancer cells. A: Volcano plot for differential gene expression; B: The Kyoto Encyclopedia of Genes and Genomes enrichment of the upregulated genes in the low- Uncoupling protein 1 (UCP1)-expression group; C: Western blot for active and total Rap1 in gastric cancer cells with UCP1 knockdown (left) or overexpression (right). UCP1: Uncoupling protein 1; Ctrl: Control; OE: Overexpression.

We further detected Rap1 activity in the cells subjected to UCP1 overexpression or knockdown. As shown in Figure 6C, the level of active Rap1 protein in GC cells was significantly decreased after UCP1 overexpression and significantly increased after UCP1 knockdown. The total Rap1 protein was unchanged after overexpression or knockdown of UCP1. Thus, UCP1 regulates the Rap1 signal in GC.

DISCUSSION

In this study, we evaluated the expression profile and clinical significance of UCP1 in GC and further explored its possible function in GC cells. To the best of our knowledge, this is the first study of UCP1 in GC.

Metabolic reprogramming plays a significant role in the pathogenesis of cancer, with tumor cells adapting their lipid-related metabolic pathways and nutritional structures to facilitate metastasis[24]. It has been demonstrated that gastric tumor cells can modulate the function of stromal or immune cells via lipid metabolism, thereby resulting in immune evasion and immunosuppression, ultimately contributing to therapy resistance and cancer relapse[25,26]. Therefore, targeting the lipid metabolism of tumor cells and reducing their lipid availability may present novel therapeutic strategies for the treatment of GC. UCP1 plays a significant role in thermogenesis in brown and beige adipocytes[27]. Our data indicate that UCP1 is differentially expressed in GC, with decreased expression of UCP1 being associated with tumor invasive depth, lymph node metastasis, poor differentiation, and advanced TNM stage. The results of the experiments in vitro and in vivo suggested that UCP1 plays a role of a tumor suppressor in GC.

Methylation has been reported as one of the mechanisms that regulate gene expression, and aberrant DNA methylation has been linked to the oncogenesis of multiple cancers[28,29]. Prior research demonstrated reduced CpG methylation at the CRE3 site within the UCP1 enhancer in brown adipocytes[30]. Promoter methylation contributes to UCP1 silencing in white adipose tissue, but its function in GC has not been investigated thus far[30,31]. We detected hypermethylation of UCP1 in GC cells, and 5-Aza treatment significantly restored UCP1 expression. UCP1 was hypermethylated in gastric tumor tissues and was related to the expression level of UCP1.

KEGG analysis revealed that the Rap1 signaling pathway was enriched in UCP1-knockdown cells. Rap1 is a small GTPase of the RAS superfamily that often switches between GTP-bound and -unbound states to activate downstream targets to convey biological signals and execute physiological functions[32,33]. Rap1 also serves as a transcriptional regulator that controls the capacity of downstream metabolic pathways critical for metabolic maturation[34,35]. According to Martínez et al[36], in the absence of Rap1, mice develop obesity, glucose intolerance, and hepatic steatosis. Rap1, a key regulator of cell adhesion and integrin function, promotes cancer progression by initiating and sustaining ERK signaling. Activation of Rap1 signaling has been confirmed in various malignancies, including prostate cancer[37], breast cancer[38], ovarian cancer[39], colon cancer[40], and non-small cell lung cancer[41]. Therefore, targeting the Rap1 pathway represents a promising strategy to improve antitumor outcomes. Rap1 has also been reported to play a role in GC; research by Li et al[42] indicates that Rap1 may be involved in TRF2-mediated resistance to etoposide in GC cells. Moreover, a study by Ma et al[43] found that F2RL3 regulates the angiogenesis and epithelial-mesenchymal transition of GC cells through the Rap1/MAPK pathway, thereby influencing the onset and progression of GC. In the present study, Rap1 was identified as the downstream molecule of UCP1 in GC cells, and the results were verified by western blotting. Further studies of the regulation and downstream targets of Rap1 are warranted. In the future, detailed research on the effect of UCP1/Rap1 on metabolism in GC should be conducted.

This study has several limitations. First, the RNA-seq analysis was performed using only a single pair of samples without biological replicates, significantly compromising statistical power and reproducibility. Second, the proposed mechanistic relationship between UCP1 and Rap1 relies solely on indirect assessments of Rap1 activity, lacking a direct validation of molecular interactions. Future studies employing mechanistic approaches, such as protein-protein interaction assays, are warranted to delineate whether UCP1-Rap1 crosstalk occurs through direct binding or via intermediary molecules. Finally, while UCP1 is a mitochondrial protein in BAT known to regulate thermogenesis by dissipating energy as heat, research on its roles in GC biology—particularly its effects on energy metabolism, ROS homeostasis, and apoptosis—remain in an early exploratory phase, with its underlying mechanisms yet to be fully elucidated.

CONCLUSION

In the present study, we found that UCP1 was downregulated in GC due to the hypermethylation of its promoter. UCP1 plays a role of a tumor suppressor in regulating cell viability, proliferation, and migration. UCP1 might participate in the tumorigenesis of GC by regulating the Rap1 signaling pathway and may serve as a potential therapeutic target for GC.