G protein-coupled receptor 137B drives malignant progression and May serves as a diagnostic and prognostic biomarker in esophageal squamous cell carcinoma

Abstract

Background

G protein-coupled receptors (GPCRs) are widely involved in cell signal transduction, and their abnormal activation has been proved to be closely related to the occurrence and development of various cancers. However, the functions of a large number of members in the GPCR family have not been clarified. Among them, the orphan receptor GPR137B is particularly rare, and its role in tumors has been rarely studied systematically.

Methods

The expression of GPR137B in ESCC was confirmed using multi-database analysis and clinical tissue samples, and its prognostic value in ESCC was investigated. The role of GPR137B in ESCC was confirmed through in vivo and in vitro investigations. The probable mechanism of GPR137B in ESCC was investigated by GO and KEGG enrichment analysis and Western blotting. Bioinformatics analysis was employed to investigate the association of immune infiltration, while flow cytometry was utilised to validate the expression of PD-L1. Drug sensitivity analysis investigated the potential of GPR137B as a predictor of drug responsiveness.

Results

GPR137B had elevated levels in ESCC and was associated with unfavourable prognosis. GPR137B may enhance the proliferation, invasion, migration, and tumorigenesis of ESCC cells. GPR137B was associated with the non-classical Wnt/PCP signalling pathway in enrichment analyses. The suppression of GPR137B facilitated the epithelial-mesenchymal transition (EMT) by reducing the expression of Wnt5A, FZD6, and p-JNK, proteins associated with the non-classical Wnt/PCP signalling pathway. The expression of GPR137B was correlated with immune infiltration in ESCC, and flow cytometry indicated a relationship with PD-L1. GPR137B may serve as a prognostic and therapeutic target for ESCC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-026-04229-y.

Introduction

There is a high incidence and significant disease burden of esophageal cancer globally, a common malignant tumor of the gastrointestinal tract, particularly in China, which is often categorized into two subtypes, squamous cell carcinoma and adenocarcinoma, based on histological characteristics, with a notable variation in the prevalence of both pathological forms across different geographical regions. For instance, ESCC continues to represent the predominant type of cases in China and other East Asian nations [1]. Statistics indicate that China constitutes over 50% of the global Esophageal Cancer cases, with an estimated 250,000 to 300,000 new diagnoses annually. Approximately 70% of these individuals are diagnosed with mid- to late-stage illness, leading to an overall five-year survival rate of under 20% [2]. Despite the development and promotion of modern treatments such as minimally invasive surgery (MIE), neoadjuvant immune-combination chemotherapy, and precision radiotherapy, the general prognosis for individuals with esophageal cancer remains bleak [3]. The 5-year survival rate for locally progressed disease patients in China is 30–40%, but advanced disease patients have a survival rate < 20%. The constraints of existing therapeutic approaches underscore the necessity for precise prognostic indicators, the investigation of which is essential for enhancing patient survival.

GPCRs are the most abundant and functionally diverse class of receptors with membranes found thus far, with their encoding genes comprising about 2% of the human genome [4, 5]. These receptors serve a pivotal regulatory function in various physiological processes, including the controlling of smooth and heart muscle contractility, neurotransmitter release, metabolic equilibrium, and blood pressure management [6]. GPCRs are extensively expressed in various tissue cells and are involved in processes like cellular proliferation, differentiation, and migration, while also playing essential roles in critical functions such as embryonic development, angiogenesis, tissue repair, and remodeling [7, 8]. Alterations in GPCR expression or signaling might disturb physiological equilibrium, resulting in chronic inflammation and immune system impairment, and potentially triggering tumor start and progression [9–12]. Recent research has demonstrated that numerous GPCRs are significantly expressed or continuously active in diverse tumor types. Tumour cells can use these receptor-mediated signalling pathways to augment proliferation, inhibit apoptosis, elude immune detection, and facilitate invasion and metastasis [13–18].

GPR137B is an orphan receptor of the GPCR superfamily, and its ligand remains unidentified. There are 399 amino acids in the encoded polypeptide chain, which gives it a molecular weight of about 45 kDa. This receptor has the typical seven transmembrane (7TM) domains and N-terminal extracellular glycosylation alterations, according to the conventional topological attributes of the GPCR family [19, 20]. Research suggests that GPR137B is predominantly localized to the lysosomal membrane and operates as a tissue-specific transmembrane protein. It forms complexes with Rag GTPases, so facilitating their activation and modulating the recruitment and function of the mTORC1 signaling complex [21]. The mTOR pathway is crucial for cellular development, energy metabolism, and tumorigenesis; thus, the regulatory mechanisms involving GPR137B may significantly impact several pathological conditions, including metabolic disorders and cancer [19]. Moreover, GPR137B has a significant level of sequence and structural similarity with its paralogue, GPR137. There is growing evidence that GPR137 plays a role in the increased proliferation, motility, and invasive capacity of tumor cells in a variety of cancer types, such as hepatocellular carcinoma and gastric cancer [22, 23]. Consequently, it is extremely probable that GPR137B is involved in critical regulatory mechanisms of tumorigenesis and progression, although its role in malignant tumors has yet to be systematically clarified.

Consequently, the purpose of this research is to use in vivo and in vitro tests to determine the function and associated mechanisms of GPR137B in ESCC cell proliferation and metastasis, and to elucidate the association between GPR137B expression and ESCC expression features and prognosis.

Materials and methods

Patient groups and tissue specimens

This research gathered seven pairs of fresh ESCC tumor and adjacent non-cancerous tissue specimens from patients receiving surgical treatment at the Department of Thoracic Surgery, Yancheng Third People’s Hospital (the Sixth Affiliated Hospital of Nantong University) from October 2022 to October 2023. No adjuvant radiotherapy or chemotherapy was given prior to surgery. Post-excision, the tissue samples were promptly preserved at -80 °C for later Western blot and immunohistochemical analysis. Paraffin-embedded tissue blocks containing tumor and adjacent non-cancerous tissue were obtained from 100 ESCC patients who received surgical treatment at Yancheng Third People’s Hospital (the Sixth Affiliated Hospital of Nantong University) from 2013 to 2015. Sections of tumor and neighboring non-cancerous tissue were stained using immunohistochemistry. The criteria for patient inclusion are as follows: (1) Primary patients presenting for an initial diagnosis and treatment at this institution, with postoperative pathological confirmation of ESCC; (2) A clear postoperative pathological report of cancerous tissue, with adjacent tissue being normal esophageal tissue and negative surgical margins; (3) No prior antineoplastic treatment, including radiotherapy, chemotherapy, endocrine therapy or targeted therapy. All patients received standardised postoperative management according to esophageal cancer guidelines. (4) Full, retrievable follow-up records and complete clinical data. Exclusion criteria: (1) Patients with distant metastases from primary lesions; (2) Patients with other systemic tumours or metastatic esophageal carcinoma; (3) Patients with severe cardiovascular disease or other serious primary systemic conditions; (4) Patients with speech or cognitive impairments or psychiatric disorders; (5) Patients with incomplete clinical data or inadequate follow-up records. All enrolled cases underwent a five-year follow-up, yielding comprehensive clinical and prognostic information for each patient. Ethical approval for this study was obtained from the Ethics Committee of Yancheng Third People’s Hospital (LS-2023-93). Every patient or family member gave their written, informed consent before any clinical data was collected or samples were kept.

Expression of GPR137B in ESCA and ESCC

As part of a pan-cancer expression study involving many cancer types, we examined GPR137B expression in tumor tissues using the TIMER database. Researchers used the GEPIA2 [24] and ENCORI [25] databases to compare GPR137B expression in ESCA and normal esophageal tissues. Additionally, the UALCAN online database (https://ualcan.path.uab.edu/) verified GPR137B expression patterns in ESCA compared to normal tissues and studied its putative association with clinical phases [26]. TCGA public database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga), identifying samples with ESCC. Furthermore, three datasets of ESCC—GSE53622, GSE53624, and GSE53625—were acquired to corroborate the disparities in GPR137B expression between esophageal squamous cell cancer and normal esophageal tissue.

Prognostic analysis of GPR137B in ESCA and ESCC

We examined the correlation between GPR137B expression levels and overall survival (OS) in patients with ESCC via the UALCAN online platform. Subsequently, we employed the online Kaplan–Meier Plotter tool (https://kmplot.com/analysis/) [27] to examine the relationship between GPR137B expression and overall survival in patients with ESCC and EAC (esophageal adenocarcinoma). Furthermore, GPR137B expression levels were evaluated concerning patient survival outcomes within the ESCC cohort, utilizing the TCGA database and the GEO dataset (GSE53625). Additionally, we used clinical data to assess GPR137B’s independent prognostic significance in esophageal squamous cell cancer using univariate and multivariate Cox regression models [28].

Differentially expressed genes (DEGs) analysis

In the ESCC cohort, patients were divided into two groups according to their median GPR137B expression level: high-expression and low-expression. R limma (3.60.0) was employed to compare gene transcription levels between groups. This analytical methodology use Empirical Bayes techniques to effectively adjust for gene expression variability, hence enhancing statistical testing in limited sample sizes. Genes exhibiting differential expression were identified using |log2FC| > 1, P < 0.05. The main differentially expressed genes were illustrated in volcano plots and heatmaps utilizing ggplot2 to depict overall expression trends and clustering patterns among expression groups.

Functional enrichment assessment

To better understand GPR137B-related genes’ functions, we utilized ClusterProfiler R to run GO and KEGG pathway enrichment analysis on differentially expressed genes [29]. Using ggplot2, significantly enhanced entries were seen.

Immunohistochemical analysis

This work uses the ESTIMATE algorithm to determine the Immune Score, Stromal Score, and ESTIMATE Score for each sample to assess the relationship between GPR137B and the tumor immune microenvironment in ESCC. This measured immune and stromal abundance [30]. Infiltration proportions of 22 immune cell types were estimated using the CIBERSORT method and the LM22 gene set. Immune cell distribution was evaluated among GPR137B expression groups, and correlation analysis examined the link between GPR137B and immune cells.

Protein extraction and western blot

ESCC tissues and neighboring normal samples were extracted with RIPA buffer (Beyotime, China) and BCA assayed for protein quantities. Equal protein quantities were separated on 12.5% SDS–PAGE gels and transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk at room temperature for 2 h before incubating with primary antibodies against GPR137B (Proteintech, China) and GAPDH overnight at 4 °C. HRP-conjugated secondary antibodies were applied for 1 h in the dark after TBST washes. A Tanon-5200Multi imaging system recorded signals from an ECL detection kit (Ncmbio, China). ImageJ quantified and standardized band intensities to GAPDH.

Immunohistochemistry

After fixation in 10% neutral formaldehyde, tissues were dehydrated by ethanol gradients and embedded in paraffin. Approximately 2 μm thick continuous slices were produced for GPR137B immunohistochemical detection. The sections were treated with xylene to remove wax, graded ethanol to rehydrate, and then antigen retrieval was performed under high pressure and temperature. After inhibiting endogenous peroxidase activity with 3% hydrogen peroxide, normal goat serum was administered for 20 min. At 4 °C overnight, primary antibody against GPR137B (Proteintech, 1:500 dilution) was incubated. Next day, rewarming was followed by 15 min of secondary antibody-HRP incubation. DAB staining lasted 3–5 min, followed by haematoxylin counterstaining, dehydration, clearing, and neutral resin mounting. The final photos were taken with an Olympus BX83 microscope. Results were scored double-blind. In order to calculate the immuno-reactive score (IRS), the formula IRS = SI × PP was used. The intensity score goes from 0 to 3 points, while the proportion of positive cells runs from 0 to 4 points. 0 means negative IRS score, 1–3 means slightly positive, 4–6 means significantly positive, and 7–8 means very positive. Negative and weakly positive results were assigned to the low-expression cohort, while moderately and strongly positive results comprised the high-expression group.

Cellular immune fluorescence

In six well plates, place sterile circular coverslips. Incubating the cell suspension in complete medium with 10% FBS for 48–72 h improve cell adherence. Remove the medium and rinse with PBS. After incubating with 4% paraformaldehyde for 30 min, rinse three times with cooled PBS. After 10 min of 0.1%-0.25% Triton X-100, block with 10% goat serum at 37 °C for 30 min to enhance permeability. Day-long incubation of the GPR137B primary antibody at 4 °C. Incubate samples with TRITC-labelled secondary antibodies for one hour in the dark the next day after a PBS wash. Nuclei were DAPI-labeled for 10 min before mounting in anti-fluorescence quenching solution. Final imaging was done with an Olympus BX83 fluorescent microscope with a standard setup for each group.

Cell culture and plasmid transfection

ESCC cell lines KYSE150 and KYSE410 came from Wuhan Pricella. In RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin, cells were cultured at 37 °C with 5% CO₂. Transfection and functional experiments were done on logarithmic-growing cells. GeneChem supplied shRNA targeting GPR137B and a negative control (shNC) with the target sequence 5′-GGGAGAGGAAGGTTATCGTACGA-3′. After reaching 70–80% confluence in 6-well plates, 2.5 µg of plasmid was transfected per well using Lipofectamine 3000. After 6–8 h, cells were cultured for 48 h before being examined in fresh complete medium. The empty vector was used as a control while inserting the GPR137B coding sequence into pcDNA3.1(+). At 70% confluence, Lipofectamine 3000 was used to transfect cells according to the manufacturer’s procedure. The media was switched after six hours, and the cells were cultivated for 24–48 h. Protein expression and functional tests were performed on collected cells.

Cell counting kit-8 (CCK-8) assay

The Cell Counting Kit-8 (Beyotime, China) measured cell proliferation. We incubated KYSE150 and KYSE410 cells at the proper density in 96-well plates at 37 °C with 5% CO₂ for 24, 48, and 72 h. Every well was then treated with 10 µL of CCK-8 reagent, 100 µL of full culture media was added and incubated for around 1 h. To assess cell growth in each group, optical density (OD) values were measured at 450 nm with a microplate reader after the reaction.

Colony formation assay

Six-well plates with three replicate wells for each group were infected with 500–1,000 cells per well. The cells were grown in RPMI-1640 medium supplemented with 10% foetal bovine serum in a CO₂ incubator set at 37 °C for 10–14 days until at least 50 visible clones formed. Two gentle washes with PBS were performed on the cells after the media was removed. For 20 min, the cells were placed in a solution containing 4% paraformaldehyde. The cells were stained with 0.1% crystal violet for 15 min after fixation. Rinsing with running water and air-drying the cells removed excess pigment. ImageJ was used to count colonies in each well using photographs.

Transwell assay

In serum-free RPMI-1640, resuspend KYSE150 and KYSE410 cells. Place around 5 × 10⁴ cells in 200 µL of medium in the Transwell cell insert’s upper chamber. To the lower chamber, add 600 µL of complete medium containing 10% fetal bovine serum. Incubate cells at 37 °C in 5% CO₂ for 24 h. After incubation, remove non-migrated cells from the upper chamber. After fixing with 4% paraformaldehyde for 20 min. For an additional 20 min, cells that were attached to the membrane were stained with 0.5% crystal violet. After rinsing with PBS to remove dye, random fields of view were counted under a microscope. ImageJ was used to count moving cells.

Wound healing test

Mitomycin C (10 µg/mL, Selleck, Shanghai, China) was applied to KYSE150 and KYSE410 cells for 2 h in order to reduce proliferation. A confluent monolayer was established after 18–24 h of growing KYSE150 and KYSE410 cells (5 × 10⁵ cells/well) in 6-well plates. The cells that had detached were scraped in a straight line using a sterile 200 µL pipette tip. The cells were reconstituted with serum-free medium after being washed with PBS 2–3 times. In order to evaluate migration, ImageJ evaluated the same field of vision at 0 and 48 h in terms of gap width.

Flow cytometry

The KYSE150 cells were used to produce sh-GPR137B and sh-NC. The non-specific binding was inhibited after aliquotting the single-cell solution. A FITC-labeled PD-L1 antibody was used for surface membrane marking and on-machine collection (Invitrogen, Thermo Fisher Scientific). FlowJo was employed to screen out detritus and selectively pass single cells via FSC/SSC while maintaining same acquisition parameters. A uniform PD-L1 positive threshold gate was present in the single-cell parent population. To compare the two groups, the fraction of PD-L1⁺ cells was calculated as % of parent (PD-L1⁺/single cells × 100%).

Nude mouse tumour formation assay

The investigation used Yangzhou University 4-week-old female BALB/c nude mice. All animals were kept in a pathogen-free environment with constant temperature of 22 ± 2 °C and humidity of 50–60%, and had unlimited access to food and water. After cell collection, they were resuspended in PBS and calibrated to a density of 2 × 10⁶ cells per 100 µL. Five mice per group received the suspension subcutaneously in the left axilla using a sterile syringe. After 3–4 weeks of continuous monitoring, animals were killed when tumor volumes reached 1000–1500 mm³. Jiangsu Medical Vocational College Ethics Committee recommendations (Approval No.: SYLL-2024-716).

Drug sensitivity analysis

The ‘oncoPredict’ package in R was used to estimate medication sensitivity in ESCC patient samples to analyze inter-individual therapy response. To find out how sensitive certain samples were, we calculated the half-maximal inhibitory concentration (IC₅₀) values for every drug. IC₅₀ is a key metric for measuring drug inhibitory efficacy, representing the concentration required to achieve 50% inhibition of a biological process in vitro. In oncological research, lower IC₅₀ values typically reflect a more intense tumour cell response to drugs, indicating greater cellular sensitivity and more pronounced therapeutic effects. This analysis can identify potential therapeutic agents that are highly sensitive to specific molecules, providing a basis for developing personalised treatment regimens.

Statistical analysis

R and GraphPad Prism were used for statistical analysis. The mean ± SD is used to display the data. The two-tailed When comparing two groups, the Student’s t-test was utilised. When comparing three or more groups, the one-way ANOVA was employed. Finally, a post hoc multiple comparison test was executed. A statistically significant result was defined as P < 0.05. A minimum of three repetitions of each experiment were carried out.

Results

Expression of GPR137B in ESCA and ESCC

In the TIMER pan-cancer study, GPR137B expression levels were shown to be considerably greater in different types of tumors as compared to normal tissues (P < 0.05) (Fig. 1A). Breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colorectal adenocarcinoma (COAD), esophageal carcinoma (ESCA, including ESCC and EAC subtypes), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), gastric adenocarcinoma (STAD), thyroid carcinoma (THCA), and uterine endometrial carcinoma (UCEC) were the most common types of cancers with abundant expression. An integrated analysis of online databases, specifically GEPIA2 (Fig. 1B), ENCORI (Fig. 1C), and UALCAN (Fig. 1D), found that normal esophageal tissue and esophageal cancer tissue had significantly different levels of GPR137B expression. GPR137B expression was markedly increased in tumor tissues, showing a statistically significant difference when compared to normal tissues (P < 0.05). The differential expression levels of GPR137B across different clinical stages of ESCC were analyzed using the UALCAN database. The results demonstrated a significant increase in GPR137B expression in ESCA tissues relative to normal tissues across all stages (Stage 1–4) (P < 0.05), with the most pronounced difference observed in Stage 3 (P < 0.0001). Comparisons across stages revealed a statistically significant difference in expression between stages 2 and 3 (P < 0.05) (Fig. 1D). The expression of GPR137B consistently rises throughout the progression of esophageal cancer and may correlate with disease stage. We examined GPR137B expression in ESCC by analyzing samples from the TCGA and GEO databases, specifically GSE53622, GSE53624, and GSE53625. In contrast to normal tissues, esophageal squamous cell carcinoma tissues showed a considerable increase in GPR137B expression. (P < 0.001) (Fig. 1E).

Fig. 1.

(A) TIMER database analysis showing GPR137B expression across a spectrum of tumor types. (B) GEPIA2 results illustrating GPR137B upregulation in ESCA relative to normal tissues (*P < 0.05). (C) ENCORI-based comparison revealing significant elevation of GPR137B in ESCA samples (****P < 0.0001). (D) UALCAN analysis depicting tumor–normal differences in GPR137B expression and its stage-specific variation in ESCA (****P < 0.0001). (E) TCGA and GEO datasets collectively confirming increased GPR137B expression in ESCC (***P < 0.001; ****P < 0.0001)

Expression of GPR137B in human ESCC tissue and intracellular expression

To corroborate the conclusions suggested by the bioinformatics analysis, we examined the expression levels of GPR137B in human ESCC tissues and cells. Western blot examination of seven paired fresh human ESCC tissue samples demonstrated that GPR137B expression was markedly elevated in squamous cell carcinoma tissue compared to neighboring normal tissue (P < 0.05) (Fig. 2A). Notably, immunohistochemical results corroborated this finding. In ESCC tissue, GPR137B was predominantly localised in the cytoplasm and cell membrane, exhibiting a brownish-yellow granular staining pattern and significantly higher expression levels than in normal esophageal mucosal epithelium. By contrast, only faint positive or negative signals were observed in normal tissue (Fig. 2B). Moreover, cellular immunofluorescence studies indicated that GPR137B is mostly localized to the plasma membrane of the ESCC cell lines KYSE150 and KYSE410. This discovery further corroborates the concept that GPR137B is a membrane-bound receptor protein. (Fig. 2C).

Fig. 2.

(A) Western blot study of GPR137B expression in seven pairs of fresh ESCC and normal tissues. (B) Immunohistochemical labeling confirmed GPR137B expression in ESCC tumors and surrounding normal lung adenocarcinoma tissues. (C) Immunofluorescence assay of GPR137B expression distribution in KYSE150 and KYSE410 cells

Potential association of GPR137B with ESCC prognosis

Possible correlation of GPR137B with ESCC prognosis Initially, we conducted an exploratory evaluation of the prognostic correlation of GPR137B using available databases. The UALCAN data indicated that elevated expression of GPR137B correlated with reduced overall survival in ESCA patients (P < 0.05) (Fig. 3A). The Kaplan–Meier Plotter analysis indicated that this association was predominantly observed in the ESCC cohort (P < 0.05), but no significant difference was detected in the EAC group (P > 0.05) (Fig. 3B). Consistent outcomes were likewise observed in the TCGA-ESCC and GSE53625 cohorts (Fig. 3C–D). Cox regression analysis indicated that GPR137B expression and stage were significantly correlated with overall survival in univariate analysis; following multivariate analysis, GPR137B and stage remained statistically significant (Fig. 3E). We incorporated 100 examples of ESCC paired tissues at our centre for immunohistochemistry validation. The expression of GPR137B protein was markedly elevated in cancer tissues compared to surrounding tissues (55% high expression versus 45% low expression) (Fig. 3F). The elevated expression was strongly associated with T stage, N stage, and clinical stage, but not with gender or age (Table 1). Results from a survival analysis indicated that elevated GPR137B expression (High vs. Low) was substantially associated with a reduced overall survival rate. Considering the inherent overlap between Stage and T/N, elevated GPR137B expression remained statistically significant after the multivariate Cox model incorporated GPR137B, T stage, N stage, age, and gender. T3–4 compared to T1–2 and N1–3 compared to N0 (Table 2). The results of several cohorts consistently show that high expression of GPR137B may be linked to a poor prognosis for ESCC, and the correlation stays even after the existing clinical variables are taken into account. However, some important clinical variables, like treatment, recurrence, and comorbidity, have not been fully included, and the predictive value is still low. Its prognostic value needs to be further confirmed in a larger, independent external cohort with more complete clinical information.

Fig. 3.

(A) UALCAN survival curves demonstrating that ESCA patients with higher GPR137B expression exhibit poorer OS (P < 0.05). (B) KM Plotter analysis of ESCC and EAC showing the prognostic effect of GPR137B expression, with a significant impact observed in ESCC (P < 0.05). (C) TCGA-ESCC cohort verification of the association between elevated GPR137B and reduced OS (P < 0.05). (D) Independent validation of this survival trend using GEO dataset GSE53625 (P < 0.05). (E) Forest plot of univariate and multivariate Cox regression analysis of ESCC patients in TCGA. (F) Survival analysis of GPR137B expression in ESCC patients based on immunohistochemical grouping (n = 100)

Table 1.

Clinical-Pathological characteristics and GPR137B expression in one hundred cases of ESCC

Clinicopathological Features	n	GPR137B High expression(n = 55) Low expression(n = 45)		P value#
Gender				0.412
Male  Femal	6040	35 20	25 20
Age (years)				0.700
>60  ≤60	7327	41 14	32 13
T stage				0.030*
T1/T2  T3/T4	5743	26 29	31 14
N stage				< 0.001*
N0  N1/N2/N3	34 66	8 47	26 19	**
Clinical stage				< 0.001*
I/II  III/IV	3862	11 44	27 18	**

#: Chi-square test; *P < 0.05, ***P < 0.001

Table 2.

OS at five years in patients with ESCC: univariate and multivariate analysis

Characteristic	Univariate analysis			Multivariate analysis
	HR	95%CI	P	HR	95%CI	P
GPR137B expression High vs. Low
	2.398	1.524 ~ 3.774	< 0.001***	2.336	1.368 ~ 4.000	0.002**
Gender
Male vs. Female	0.973	0.634 ~ 1.517	0.903
Age (years)
>60 vs. ≤ 60	1.012	0.620 ~ 1.652	0.962
T stage
T1 ~ 2 vs. T3 ~ 4	5.025	3.165 ~ 7.937	< 0.001***	6.098	3.650 ~ 10.204	< 0.001***
N stage
N0 vs. N1 ~ 3	2.933	1.789 ~ 4.808	< 0.001***
Clinical stage
I/II vs. III/IV	3.322	2.045 ~ 5.405	< 0.001***

HR, hazard ratio; CI, confidence interval; *P < 0.05, **P < 0.01, *** P < 0.001

Effects of GPR137B on ESCC cell proliferation and invasive migration

We developed knockdown groups (sh-GPR137B) and their negative controls (sh-NC) and overexpression groups (OE-GPR137B) and their empty vector controls (OE-NC) to determine GPR137B’s biological role in ESCC cells. These were then added to KYSE150 and KYSE410 ESCC cells. Western blot examination showed that sh-GPR137B was significantly downregulated while OE-GPR137B was significantly upregulated. Figure 4A shows that the transfection technique was successfully implemented for functional evaluations. Assays for colony formation and CCK-8 cell viability were used to evaluate the impact of GPR137B on the proliferation of ESCC cells. (Figure 4B and C). The findings showed that GPR137B knockdown significantly reduced KYSE150 and KYSE410 cell proliferation, whereas overexpression drastically boosted it. Transwell invasion and wound-healing assays assessed GPR137B’s cell migration and invasion regulation. The sh-GPR137B group had less cells move through the membrane in Transwell. The OE-GPR137B group showed a significant increase, demonstrating that GPR137B overexpression increases ESCC cell invasiveness (Fig. 4D). The scratch assay showed that GPR137B knockdown lowered cell migration velocity and overexpression improved wound healing (Fig. 4E). The results show that GPR137B significantly increases ESCC cell proliferation, migration, and invasion. This suggests GPR137B is a pro-carcinogenic agent that promotes ESCC.

Fig. 4.

(A) Western blot confirms efficient GPR137B knockdown and overexpression in KYSE150 and KYSE410 cells. (B) CCK-8 assay showing proliferation curves at 0, 24, 48, and 72 h post-knockdown and overexpression of GPR137B in KYSE150 and KYSE410 cells. (C) Clonogenic assay verifying the impact of GPR137B knockdown and overexpression on proliferation capacity in KYSE150 and KYSE410 cells. (D) Transwell assay verifying the impact of GPR137B knockdown and overexpression on invasion capacity in KYSE150 and KYSE410 cells. (E) The effect of GPR137B knockdown and overexpression on migration ability in KYSE150 and KYSE410 cells was verified by scratch assay after treatment with mitomycin C

Effects of GPR137B on ESCC tumour growth

Subcutaneously implanting cell lines with stable GPR137B knockdown (sh-GPR137B group), empty vector control cells (Control group), and GPR137B overexpression cells (OE-GPR137B group) into nude mice created a tumor model (Fig. 5A). Tumor volume rise was considerably reduced in the sh-GPR137B group, while the OE-GPR137B group developed the fastest (Fig. 5B). From day 15, the OE-GPR137B group had larger tumor volumes than the control group, while the sh-GPR137B group had smaller tumors (P < 0.001). By day 24, the tumor volume averaged 1000 mm³ in the OE-GPR137B group, 700 mm³ in the control group, and 400 mm³ in the sh-GPR137B group. The final tumor weight (Fig. 5C) showed a substantial increase in the OE-GPR137B group compared to the control group (P < 0.001), whereas the sh-GPR137B group showed a significant decrease (P < 0.001). GPR137B overexpression significantly increases tumor growth and proliferation in ESCC cells, while knockdown reduces these processes in vivo. This suggests a major oncogenic involvement in ESCC progression.

Fig. 5.

(A) Subcutaneous tumours in xenograft mouse models of KYSE150 cells transfected with GPR137B in Sh, Control, and OE groups. (B) Effect of GPR137B expression on tumour volume in nude mice. (C) Effect of GPR137B expression levels on tumour weight in nude mice. (D) Volcano plot of differential analysis of GPR137B in ESCC (patients were divided into high expression group and low expression group according to the median expression of GPR137B). (E) GO and KEGG analysis significantly enriched pathways. (F)Western blot analysis of non-classical Wnt/PCP-JNK signaling pathway-related proteins (WNT5A, FZD6, p-JNK) and EMT-related proteins (E-cadherin, Vimentin, ZEB1)

GPR137B regulates non-classical Wnt/PCP signaling and promotes EMT in ESCC cells

Patients from the TCGA cohort were categorised into high and low expression groups based on the median expression levels of GPR137B in ESCC tumour tissues. Subsequently, differential expression analysis was conducted on the two groups, resulting in the creation of the volcano plot (Fig. 5D) and heat map (Fig. S1A). A total of 222 genes were upregulated, whereas 150 genes were downregulated. Enrichment analysis of differentially expressed genes indicated significant involvement in Wnt signalling processes, particularly highlighting the enrichment of planar cell polarity (PCP)-related entries, which included typical PCP components such as WNT5A and FZD6 (Fig. 5E). Subsequently, we conducted a correlation analysis on these two representative molecules, revealing that GPR137B exhibited a significant positive correlation with both WNT5A and FZD6 (both P < 0.05) (Figure S1B), indicating that GPR137B may be associated with the activation characteristics of the Wnt/PCP-related signalling axis. To determine whether GPR137B controls the non-classical Wnt/PCP pathway, we performed Western blot analysis in ESCC cells (Fig. 5F). The results demonstrated that following GPR137B knockdown, p-JNK, FZD6, and WNT5A levels were significantly lower than those in the control group. It appears that GPR137B may regulate the activation of signals associated with Wnt and PCP-JNK. When we additionally searched for markers associated with EMT, we discovered that E-cadherin levels increased significantly, whereas Vimentin and ZEB1 levels decreased significantly, when GPR137B was switched off. This provides more evidence that GPR137B could be useful during EMT. The findings demonstrate that GPR137B has the ability to activate the non-classical Wnt/PCP-JNK signalling pathway, facilitating EMT in ESCC cells and increasing the likelihood of tumour malignancy.

Potential association of GPR137B with immune-related features of ESCC

We utilised the ESTIMATE technique to examine the relationship between GPR137B and immunity in ESCC, assessing the components of the tumour microenvironment across different GPR137B expression groups (Fig. 6A). The results demonstrated that the ImmuneScore, StromalScore, and ESTIMATEScore of the GPR137B high expression group were significantly higher than those of the low expression group (P < 0.001), suggesting a relationship between its expression level and the alterations in immune and matrix components of tumour tissue. We employed the CIBERSORT method once more to analyse the quantities of 22 immune cell types. The inferred ratios of regulatory T cells (Tregs) and quiescent dendritic cells were increased in the cohort with high GPR137B expression (Fig. 6B). association analysis revealed a significant positive association between GPR137B expression and the estimated fraction of resting dendritic cells (Fig. 6C-D). The aforementioned data indicate that GPR137B may be associated with significant characteristics of the ESCC immunological microenvironment. This conclusion mostly relies on computational logic. Considering that ESTIMATE/CIBERSORT primarily offers inferential data at the tissue level, we additionally enhanced the assessment of immune checkpoint-related phenotypes at the tumour cell level. Flow cytometry was employed to ascertain PD-L1 expression on the cell membrane (Fig. 6E). The findings indicated that, utilising identical gating strategies and acquisition parameters, the percentage of PD-L1⁺ cells (% of parent) within the singlet population was markedly reduced in the GPR137B knockdown group compared to the control group, implying that GPR137B may influence the PD-L1 immune checkpoint-related phenotype at the tumour cell level. In conclusion, the findings from cell experiments indicate a potential association between GPR137B and immune-related phenotypes in ESCC, influencing PD-L1 expression in tumour cells. However, the implications for immune cell infiltration at the tissue level and its possible regulatory effects on immunity require further validation.

Fig. 6.

(A) ESCC tumor microenvironment scores plotted against GPR137B expression levels. (B) Immune infiltration comparison illustrating how immune cell composition varies with GPR137B expression. (C) Analysis of the association between GPR137B and infiltration abundance of individual immune cell subsets. (D) Correlation analysis of GPR137B and Dendritic cells resting. (E) Flow cytometry was used to detect the proportion of PD-L1 positive cells in the GPR137B knockdown group and the control group

GPR137B anti-tumor drug analysis and prediction

At now, radiation, chemotherapy, and immunotherapy remain the primary therapeutic modalities for individuals diagnosed with esophageal squamous cell carcinoma (ESCC). To investigate potential individualised treatment indicators and assess variations in medication response among distinct genomic subtypes, we conducted a computational analysis of drug sensitivity in ESCC patients utilising publicly available transcriptome data (Fig. 7A). We employed a predictive IC₅₀ model to assess the relationship between GPR137B expression and drug sensitivity metrics. The findings indicated that the anticipated IC₅₀ values for the remaining five medications (AC220, Crizotinib, GNF-2, Rapamycin, and TAE684), except NU-7441, exhibited a strong negative correlation with GPR137B expression; specifically, samples with elevated GPR137B expression were associated with reduced expected IC₅₀ values (Fig. 7B). The aforementioned results indicate that GPR137B may be associated with particular drug responses; however, this conclusion is derived from a computational prediction model primarily intended for hypothesis generation, and its potential translational significance requires further validation through in vitro drug sensitivity assays or clinical evidence.

Fig. 7.

Drug sensitivity prediction based on public database (A) IC50 values of different drugs in GPR137B high and low expression groups. (B) Correlation analysis of drug IC50 value and GPR137B gene expression

Discussion

As a family of membrane receptors that are extensively involved in diverse biological activities, GPCRs play a pivotal regulatory role in maintaining normal physiological homeostasis. This includes mediating neurotransmitter release, regulating metabolic balance and participating in the regulation of cardiovascular function [31–33]. However, aberrations in GPCR expression or signalling can also lead to pathological responses, such as chronic inflammation and immune dysregulation [10–12]. Recent research on the biological functions of GPCRs has increasingly concentrated on oncology, revealing that several GPCRs facilitate numerous malignant characteristics in tumours. GPR137 exerts a pro-cancer influence across various tumours, including gastric cancer, leukaemia, and osteosarcoma, primarily through the PI3K/AKT and Hippo-YAP/TAZ signalling pathways, alongside the modulation of the cell cycle and apoptosis, thereby enhancing the malignant characteristics of tumour cells [22, 34, 35]. GPR37 facilitates cell survival and augments anti-apoptotic capacity via MAPK-p38 signalling in colorectal cancer [36]; activation of GPR120 stimulates the secretion of factors such as VEGF and IL-8, thereby advancing the epithelial-mesenchymal transition (EMT) in breast cancer [37]; furthermore, GPR132 is intricately associated with metastasis and the remodelling of the immune microenvironment in breast cancer. It can facilitate the interaction between tumour cells and macrophages via the lactic acid-related axis, induce the polarisation of M2 macrophages, and expedite lung metastasis. Inhibiting GPR132 diminishes the immunosuppressive milieu and mitigates tumour malignancy progression [38]. GPR133 exhibited specific traits of suppressing proliferation and triggering apoptosis in lung cancer [39]. Consequently, GPCRs exhibit mechanistic diversity and complexity in tumours, regulating distinct malignant behaviours of tumour cells, with indirect effects through multiple route mechanisms and interactions with the immunological milieu. Among the GPCR family, GPR137B is one of the few orphan receptors. Evidence suggests it may regulate mTORC1 signalling and affect the Rag-mediated nutrition sensing mechanism; as a result, it is linked to lysosomal function and autophagy homeostasis maintenance [19]. Along with the aforementioned signs, there is also a lack of comprehensive research on its biological effects, especially in tumours. At first, this study combined public records with centre clinical samples, and it showed that GPR137B was significantly up-regulated in ESCC, which was associated with negative clinical features and poor survival rates, suggesting that it might have predictive value.

The upregulation of GPR137B significantly enhances the proliferation, migration, and invasion of ESCC cells, as demonstrated through in vitro functional assays and in vivo tumorigenesis studies, while concurrently promoting and accelerating tumour growth in vivo. To investigate the role of GPR137B in ESCC, we identified the pivotal Wnt signalling pathway prevalent in tumours using enrichment analysis, revealing a substantial positive association with the important molecules WNT5A and FZD6 of the non-classical Wnt/PCP pathway via correlation analysis. Cellular processes such as migration, proliferation, differentiation, polarity, metabolism, and survival are tightly regulated by the Wnt signalling pathway, an essential and extremely conserved network of cellular communication. This pathway mainly includes the standard Wnt/β-catenin pathway as well as the non-classical pathways, such as Wnt/PCP and Wnt/Ca²⁺ [40]. Tumours often have an abnormal activation of the classic Wnt/β-catenin pathway, which leads to the stabilisation of β-catenin. This stabilised β-catenin then moves to the nucleus to start transcription, which promotes malignant traits and increases invasion and metastasis. There has been relatively little research into the non-classical Wnt pathway, which controls cell polarity and skeletal development through Wnt/PCP and affects the cancer microenvironment through Wnt/Ca²⁺ via calcium signalling [41, 42]. Important components of the non-canonical Wnt/PCP pathway include FZD6 and WNT5A. After binding to FZD6, WNT5A can activate PCP signals that are downstream of it. This activates the JNK branch pathway and modifies the activity of several transcription factors. By coordinating EMT-related phenotypic changes and the overexpression of matrix breakdown molecules, the axis can help cancer cells behave malignantly. For non-classical Wnt/PCP signals that encourage tumour invasion and metastasis, the WNT5A-FZD6-JNK axis is considered a major effector route [40–42]. Lower levels of WNT5A, FZD6, and p-JNK were observed in GPR137B knockdown, suggesting that GPR137B may be involved in maintaining non-classical Wnt/PCP-JNK signalling. This work suggests that GPR137B may be involved in maintaining the activity of non-classical Wnt/PCP-JNK signalling, as knocking it down significantly decreased the levels of WNT5A, FZD6, and p-JNK. An increase in E-cadherin and a decrease in Vimentin and ZEB1 were found in the identification of EMT-related proteins, suggesting that the EMT process may be facilitated. This data provides support for the idea that the GPR137B-Wnt/PCP pathway may play a role in the progression of ESCC and lays the groundwork for future research into its potential as a therapeutic target.

The immunological milieu surrounding tumours is strongly associated with cancer growth. This study analysed publically accessible data to determine the effect of GPR137B on the tumour microenvironment of esophageal squamous cell carcinoma (ESCC). Samples with high levels of GPR137B expression had an inferred proportion of Tregs and resting dendritic cells that was significantly higher, showing a positive correlation with the assumed proportion of resting dendritic cells. Cancer immunological microenvironment is significantly impacted by changes in regulatory T cell (Treg) and dendritic cell (DC) status. Tregs are a type of immunosuppressive T cell subset that can inhibit CD8⁺ T cells and NK cell effector capabilities by secreting inhibitory cytokines like IL-10 and TGF-β and by regulating co-stimulation through CTLA-4. They also make it easier to recruit and maintain myeloid cells, which inhibit the immune system and make it harder for the body to detect and eliminate tumours [43–45]. The effectiveness of antigen presentation and the amplitude of the initial T cell response are directly impacted by the level of maturation and activation of dendritic cells. When dendritic cells are in their “resting” state, they often release pro-inflammatory signals and insufficient co-stimulatory molecules. This leads to less than ideal T cell activation or the development of immunological tolerance. This tolerance can then be used to help expand or maintain regulatory T cells, which in turn strengthens the network that suppresses the immune system. Our results suggest that increased GPR137B expression is associated with an immunosuppressive milieu in ESCC, wherein Tregs are enriched and resting DCs are increased. This points to an immunological condition where antigen presentation is limited and effector T cells are not adequately activated, which could allow tumours to evade the immune system and develop. Furthermore, we found that ESCC cell PD-L1 on-cell membrane proportions dropped following GPR137B knockdown, lending more credence to its role in intrinsic tumour cell immune checkpoint-related phenotypes. An important immunosuppressive mechanism within the tumour immunological microenvironment (TME) is the PD-L1 immune checkpoint pathway. The cancer cell-produced PD-L1 can bind to the effector T cell PD-1, lowering the activation, proliferation, and cytotoxic function of T cells. This leads to functional exhaustion, reduced anti-tumor immunity, and easier immune evasion [46, 47]. It is common for PD-L1-related traits to accompany the increase of immunosuppressive cell populations in the tumour microenvironment in this setting. While preliminary evidence suggests a potential association between GPR137B and an immunosuppressive tumour microenvironment and immune checkpoint phenotype in ESCC, further experimental validation is needed to determine the exact causal relationship and real impact.

Currently, the treatment methods for ESCC predominantly involve radiotherapy, chemotherapy, and immunotherapy in clinical practice. This work identified drug sensitivity from public transcriptome data via a computational algorithm. Upon assessing the correlation between GPR137B expression levels and the predicted IC₅₀, we noted that the predicted IC₅₀ values for AC220, Crizotinib, GNF-2, Rapamycin, and TAE684 were predominantly lower in samples exhibiting elevated GPR137B expression, with the exception of NU-7441, indicating a potential association between GPR137B and drug responsiveness. This finding is derived from the “predictive” outcomes of in vitro drug sensitivity, which primarily serve to generate hypotheses. The potential of GPR137B as a candidate biomarker for drug response prediction requires validation within the ESCC model of GPR137B function, and necessitates further corroboration through comprehensive mechanistic studies and clinical evidence.

There are still some limitations in this study that need further attention. First, the sample size of patients included in this study is still relatively limited, which affects the statistical efficacy of prognostic analysis and the universality of results to a certain extent. It is necessary to expand the clinical cohort and related variables to verify its stability in the future. Secondly, the immune and drug levels are mainly based on bioinformatics prediction. Although it provides directional clues for its potential immune function, there is still a lack of systematic experimental evidence, and it is still necessary to clarify it thru specific related experiments.

Conclusion

In conclusion, our data suggests that GPR137B could be a biomarker for ESCC patients with a poor prognosis, and that high GPR137B expression is associated with tumour invasion and proliferation. Additionally, GPR137B is important in regulating the non-classical Wnt/PCP signalling pathway and driving EMT, as well as ESCC cell proliferation, migration, and invasion, and tumour formation, according to the study. By controlling PD-L1 checkpoint expression on ESCC cells, it might potentially influence the immunological microenvironment as well. The results of these investigations might shed light on the causes and potential treatments for ESCC.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Rongqi Guo conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and tables, authored or reviewed drafts of the paper, and approved the final draft. Yangyang Li performed the experiments, analyzed the data, prepared figures and tables, and approved the final draft. Weisong Zhang , Hao wang and Yihao Wang collected clinical data and tissue specimens. Zhongquan Yi, Xia Li and Jianxiang Song conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.All authors contributed to the article and approved the submitted version.

Funding

Special Research Fund for Clinical Medicine, Nantong University, 2023(2023JZ022). 2021 Jiangsu Provincial Health and Health Commission Medical Research Guidance Project (Z2021087). Nantong University’s 2023 Academic Level Research Project (Special Project of Yancheng Third Institute) YXY-Z2023008.

Data availability

Data supporting the results of this study may be obtained by contacting the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

This study was approved by the Ethical Review Committee of the Sixth Hospital Affiliated to Nantong University (Yancheng Third People’s Hospital) (LS-2023-93). Informed consent was obtained from all participants and/or their legal guardians. All animal experiments were conducted in strict accordance with the Regulations on the Management of Laboratory Animals and adhered to the internationally recognized ethical guidelines for the care and use of laboratory animals. The study protocol was reviewed and approved by the Ethics Committee of Jiangsu Medical Vocational College (Approval No.: SYLL-2024-716). All efforts were made to minimize animal suffering and to reduce the number of animals used.

Consent to participate

Written informed consent to participate in this study was obtained from all individual participants (or their legal guardians).

Consent for publication

Written informed consent for publication of anonymized clinical data and any accompanying images was obtained from all individual participants (or their legal guardians).