SH3BGRL2 as a vital tumor suppressor and prognostic factor in human esophageal squamous cell carcinoma

Introduction

Esophageal carcinoma is one of the most prevalent malignancies in the world, ranking as the 11th most common tumor type and the 7th leading cause of cancer-related death (1). Notably, China accounts for approximately 44% of all global esophageal cancer cases, making esophageal cancer the 5th leading cause of cancer-related mortality in the country (2). More than 90% of Chinese esophageal cancer cases are esophageal squamous cell carcinoma (ESCC) (3). Despite advancements in therapeutic interventions (4,5), the overall 5-year survival rate of patients with ESCC is only 15–25% (6), indicating a significant lack of effective therapeutic options. Therefore, there is an urgent need to elucidate the pathogenesis of ESCC to develop more efficacious treatment.

The SH3 domain binding glutamate rich protein (SH3BGR) family is a recently identified family of highly conserved small proteins related to the thioredoxin superfamily (7). Several SH3BGR family members has been implicated in cancer (8,9), including SH3BGR in the suppression of cell migration and angiogenesis in Kaposi sarcoma, SH3BGRL as a prognostic biomarker in acute myeloid leukemia (10,11), and SH3BGRL3 as a prognosticator in kidney and bladder cancers (12). However, the role of SH3 domain binding glutamate rich protein-like 2 (SH3BGRL2) in tumorigenesis and progression remains largely unknown. Located on chromosome 6q13-15, SH3BGRL2 consists of four exons and three introns spanning 72 kb of genomic DNA (7). The SH3BGRL2 protein contains a Src homology 3 (SH3) domain and an ENA/VASP Homology 1 (EVH1) domain and is predominantly localized to the nucleus and perinuclear region (7). Since the SH3 and EVH1 domains are central to cell growth, adhesion, and migration, SH3BGRL2 is speculated to participate in various cancer-related biological processes (13,14). Recent studies have reported downregulation of SH3BGRL2 in ESCC (15) and ovarian cancer (16), suggesting that SH3BGRL2 may act as a tumor suppressor. However, the exact role of SH3BRGL2 in ESCC pathogenesis remains unclear.

This study thus investigated the clinical relevance and biological function of SH3BGRL2 in esophageal carcinogenesis and development through RNA- and protein-based gene expression analyses of resected ESCC tumor samples and in vitro and in vivo functional assays using patient-derived cells (PDCs) and PDC xenografts. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1878/rc).

Methods

Tissue samples and cell culture

Human ESCC surgical specimens and adjacent normal tissues were obtained from Zhejiang Cancer Hospital (Hangzhou, China). Samples and clinical data from 247 patients with ESCC who underwent surgical resection between March 2008 and September 2014 at Zhejiang Cancer Hospital were retrospectively reviewed. None of the patients had received preoperative chemotherapy, radiotherapy or immunotherapy. All samples were collected with written informed consent, and the study was approved by the Medical Ethical Committee of Zhejiang Cancer Hospital (No. IRB-2021-248). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The Gene Expression Profiling Interactive Analysis (GEPIA), The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used to analyze SH3BGRL2 expression in ESCC tumor tissues. ESCC patient-derived cells (PDCs) were established and validated by short tandem repeat genotyping as previously described (17). The PDCs were cultured in Dulbecco’s Modified Eagle Medium/F12 supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and penicillin/streptomycin (both from Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells were maintained in a humidified atmosphere containing 5% CO₂ at 37 ℃.

RNA extraction and sequencing

Total RNA was extracted from three pairs of ESCC tumor tissues and adjacent normal tissues or from PDCs through use of TRIzol Reagent (Life Technologies, Thermo Fisher Scientific). Complementary DNA libraries were constructed with the NEBNext super speed RNA library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. Paired-end sequencing with 150 base reads was performed with the HiSeq 4000 platform (Illumina) at Annoroad Gene Technology (Beijing, China). The sequencing reads were aligned with TopHat 2.0.13 software, and differential gene expression analysis was conducted via “DESeq2” package in R version 1.36.0 (The R Foundation for Statistical Computing). Unsupervised clustering was performed with Gene Cluster and TreeView. Gene Ontology (GO) annotation and enrichment analyses were carried out on differentially expressed genes with a false-discovery rate of less than 0.05.

Tissue microarray

Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples were subjected to hematoxylin and eosin (H&E) staining, and the diagnosis of ESCC was independently verified by two senior pathologists. FFPE blocks from 247 cases of esophageal cancer and paired normal samples were used for the construction of tissue microarrays. Specifically, H&E-stained slides from each section of esophageal cancer tissues were reviewed, and one representative tumor area (2 mm in diameter) and the corresponding normal tissue (2 mm in diameter) were excised from the FFPE tissue blocks. Sections of 3 µm thickness were prepared with a microtome for immunohistochemical (IHC) analysis.

Immunohistochemistry

Standard IHC was performed with a primary antibody against human SH3BGRL2 (#21944-1-AP; Proteintech, Rosemont, IL, USA) at a dilution of 1:200. This was followed by incubation with a secondary antibody and staining with a DAB kit (#GK500705; Genetech, Beijing, China). IHC staining was independently assessed by two experienced pathologists. The immunoreactive score, reflecting the proportion of positively stained cells, was categorized as follows: 1, <25%; 2, 25–50%; 3, 51–75%; and 4, >75%. Staining intensity was graded as follows: 0, no staining; 1, weak; 2, moderate; and 3, strong. A semiquantitative scoring criterion was used, where the staining index (values 1–7) was calculated by adding the staining intensity and the proportion of positive cells. Cases were divided into two groups: low expression (score 0–3) and high expression (score 4–7).

Western blot analysis

Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA). The membranes were blocked in 5% bovine serum albumin for 1 hour at room temperature. The membranes were then incubated with the following primary antibodies overnight at 4 ℃: β-actin [1:1,000; #4967; Cell Signaling Technology (CST), Danvers, MA, USA], β-tubulin (1:1,000; #2146; CST), SH3BGRL2 (1:1,000; #21944-1-AP; Proteintech), and early growth response 1 (EGR1; 1:500, #4154; CST). The membranes were washed three times with Tris-buffered saline with Tween20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000) for 1 hour at room temperature. The membranes were washed three times with TBST and visualized via enhanced chemiluminescence (Thermo Fisher Scientific).

RNA extraction and quantitative reverse transcription-polymerase chain reaction

Total RNA was extracted with a RNeasy mini kit (Qiagen, Hilden, Germany). The RNA concentration was measured with the Nanodrop 2000 (Thermo Fisher Scientific). Complement DNA was synthesized with the QuantiTect Reverse Transcription Kit (Qiagen) and the related primers (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The sequences of primers were as follows: GAPDH forward, 5'-GTGTCGCTGTTGAAGTCAGAG-3'; GAPDH reverse, 5'-CATCAAGAAGGTGGTGAAGCAG-3'; SH3BGRL2 forward, 5'-GCGACCGATACTGTGGAGATTAT-3'; SH3BGRL2 reverse, 5'-CATCTTCCACTCTTCTTCTCTAAGGTTC-3'; EGR1 forward, 5'-GGTCAGTGGCCTAGTGAGC-3'; and EGR1 reverse 5'-GTGCCGCTGAGTAAATGGGA-3'.

Cell transfection

Small interfering RNAs (siRNAs) synthesized by GenePharma (Shanghai, China) or a nontargeting siRNA control were transfected into ESCC PDCs via the Hiperfect transfection reagent (Qiagen) according to the manufacturer’s instructions. Cells were collected 48 hours after transfection and used for subsequent experiments as specified. Additionally, short hairpin RNA (shRNA) lentiviral vectors targeting the SH3BGRL2 or SH3BGRL2-FLAG plasmids were used to establish stably silenced or overexpressing SH3BGRL2 PDCs, respectively. The SH3BGRL2 shRNA sequences used in this study were 5'-GCAAGAUGUGGUUAGAUUUTT-3' (shSH3BGRL2-1) and 5'-GCAUCAAAGGCAGAACCUUTT-3' (shSH3BGRL2-2). Scramble and shRNA lentiviral particles were purchased from GenePharma and packaged in PDCs. Recipient cell lines were exposed to conditioned medium containing viruses supplemented with 5 µg/mL of polybrene for 48 hours. Transfected cells were selected with puromycin to generate stable cell lines with SH3BGRL2 knockdown and overexpression.

Cell proliferation and colony formation

Cells were seeded in 96-well plates at a density of 1×10³ cells per well and cultured for 1, 2, 3, or 4 days at 37 ℃. The Cell Counting Kit-8 (CCK-8, Beyotime Bio-technology, Shanghai, China) cell viability assay was performed as per kit instructions. Briefly, cells were incubated with 100 µL of CCK-8 solution (1:10 dilution) at 37 ℃ for 1 hours. The optical density was measured at 450 nm. Each sample was analyzed in triplicate. For the colony formation assay, 2,000 cells were seeded in a six-well plate. After 2 weeks, cells were fixed, stained with 0.1% crystal violet, and photographed.

Xenograft tumor and growth

The animal experiment was approved by the Animal Committee of Zhejiang Cancer Hospital (No. 2024-09-022), in compliance with national guidelines for the care and use of animals. A protocol was prepared before the study without registration. For the tumor growth model, ESCC PDCs (1×10⁷) were injected subcutaneously into the flanks of female BALB/c nude mice (4 weeks old, weighing 17–20 g), purchased from GemPharmatech Co., Ltd (Nanjing, China). The experimental animals were randomly divided into an experimental group (n=8) and a control group (n=8) via a random number table. Tumor size was monitored every 3 days, and the tumor growth curve and animal weight were recorded accordingly. Tumor volume was calculated as follows: tumor volume = length × width²/2. The mice were killed via CO₂ inhalation (70% v/v and 2 L/min flow rate) in a prefilled chamber after 4 weeks. Tumor tissues were excised, weighed, photographed, and then fixed with 4% paraformaldehyde at room temperature for IHC staining.

Statistical analysis

Statistical analysis was performed with SPSS 23.0 software (IBM Corp., Armonk, NY, USA). For comparisons, the Chi-squared (χ²), Student t-test and one-way analysis of analysis (ANOVA) were used as appropriate. Data from a minimum of three independent experiments are expressed as the mean ± standard deviation (SD). Survival curves were calculated via the Kaplan-Meier method, and differences were evaluated with the log-rank test. The Cox proportional hazards model was used to determine risk factors, which were initially identified through univariate analysis. Statistical significance was indicated by a two-tailed P value <0.05.

Results

SH3BGRL2 was downregulated in ESCC tissues

RNA sequencing was used to detect the differentially expressed messenger RNAs (mRNAs) between three pairs of resected ESCC tumor samples and adjacent normal tissues. Analysis of differentially expressed genes revealed 283 shared upregulated genes and 173 shared downregulated genes (fold change >2). Among them, SH3BGRL2 was identified as the one of most strongly downregulated genes in all three tumor tissues [average log (fold change) =−2.8; P<0.001; Figure 1A]. Analysis of the GEPIA and GEO datasets (GSE23400, GSE17351, and GSE45670) also revealed decreased SH3BGRL2 expression in ESCC tumor tissues (P<0.01, P<0.0001, P<0.05, and P<0.0001, respectively; Figure 1B,1C).

Figure 1 mRNA and protein expression levels and prognostic significance of SH3BGRL2 in ESCC. (A) Differentially expressed mRNAs between ESCC tumor samples and adjacent normal tissues identified by RNA sequencing (T: tumor tissue; N: normal tissue). (B,C) SH3BGRL2 expression levels in ESCC as analyzed via the GEPIA database (P<0.01) and GEO database (both P<0.05, GSE23400, GSE17351, and GSE45670). (D) Representative tissue microarray images of SH3BGRL2 staining via immunohistochemistry (×200): positive SH3BGRL2 expression in tumor tissues (a) and normal tissues (b) and negative SH3BGRL2 expression in tumor tissues (c) and normal tissues (d). (E) Percentages of SH3BGRL2-positive samples in tumor and nontumor esophageal tissues (31.2% vs. 51.0%; P<0.001). (F,G) Kaplan-Meier curves showing the disease-free survival (F) or overall survival (G) of patients with ESCC and higher SH3BGRL2 expression and in those with lower SH3BGRL2 expression. Error bars represent the standard deviation of the mean. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 (Student t-test, one-way ANOVA). ANOVA, analysis of analysis; DFS, disease-free survival; ESCC, esophageal squamous cell carcinoma; GEO, Gene Expression Omnibus; GEPIA, Gene Expression Profiling Interactive Analysis; OS, overall survival; SH3BGRL2, SH3 domain binding glutamate rich protein-like 2.

To further evaluate SH3BGRL2 expression at the protein level, we collected tumor tissue and paired adjacent normal tissue samples from a large cohort of 247 patients with ESCC. The clinicopathological characteristics of these enrolled patients are summarized in Table 1. The median age was 60 (range, 42–79) years, and most patients were male (n=220, 89.1%) and younger than 65 years (n=187, 75.7%). A total of 186 (75.3%) patients had a drinking history, 194 (78.5%) had smoking experience, and 66 (26.7%) had family history of cancer. The primary tumor was commonly located in the middle (n=150; 60.7%) and lower (n=80, 32.4%) esophagus. Moreover, 9 (3.6%) cases had well-differentiated tumor, 173 (70.7%) moderately differentiated, and 65 (26.3%) poorly differentiated. According to the eighth edition of the American Joint Committee on Cancer staging system, 3 (1.2%) patients had stage II disease and 244 (98.8%) had stage III disease.

IHC staining of SH3BGRL2 was performed on tumor and normal tissues from these 247 patients with ESCC. Representative images of SH3BGRL2-positive and -negative samples are shown in Figure 1D. SH3BGRL2 was predominantly localized in the cell nucleus. Moreover, consistent with the RNA-sequencing data, the percentage of SH3BGRL2-positive samples was significantly lower among ESCC tissues (77/247, 31.2%) as compared with paired adjacent normal tissues (126/247, 51.0%) (P<0.001; Figure 1E). However, no significant associations were observed between SH3BGRL2 expression and patient age, gender, tumor location, pathological differentiation, disease stage, family history, smoking, or alcohol exposure (P>0.05; Table 1).

High SH3BGRL2 protein level predicted favorable prognosis in patients with ESCC

The correlation between SH3BGRL2 protein level and survival outcomes was subsequently evaluated in the 247 patients with ESCC. The median follow-up time for this cohort was 68.9 (range, 0.1–120.1) months. Kaplan-Meier analysis revealed that patients with ESCC and elevated SH3BGRL2 expression had significantly prolonged disease-free survival (DFS) compared to those with significantly lower SH3BGRL2 expression (median DFS: 47.1 vs. 18.2 months; P=0.02, Figure 1F). More importantly, higher SH3BGRL2 expression was also associated with significantly more favorable overall survival (OS) (median OS: 60.8 vs. 20.6 months; P=0.003; Figure 1G). As shown in Table 2, multivariate analysis indicated that SH3BGRL2 expression was an independent prognostic factor in patients with resectable ESCC in terms of DFS [hazard ratio (HR) =0.62, 95% confidence interval (CI): 0.43–0.91; P=0.02] and OS (HR =0.55, 95% CI: 0.37–0.81; P=0.002). Collectively, our findings, corroborated by results from public datasets, suggested that SH3BGRL2 is a prognostic marker in ESCC and may play a role in inhibiting ESCC progression.

SH3BGRL2 inhibited cell proliferation of ESCC PDCs

To elucidate the role of SH3BGRL2 in ESCC progression, we first assessed SH3BGRL2 expression in PDCs. RNA sequencing and Western blot analyses showed that SH3BGRL2 mRNA and protein expression were markedly downregulated in most PDCs (Figure 2A). We then established SH3BGRL2 knockdown in ESCC cell lines ZEC043, ZEC056, and ZEC145 (Figure 2B,2C) and overexpression in ZEC014, another ESCC cell line (Figure 2D).

Figure 2 SH3BGRL2 inhibited the proliferation of ESCC PDCs. (A) RNA sequencing and western blot analysis of SH3BGRL2 expression levels in different ESCC PDCs. Western blot assays validated the efficiencies of SH3BGRL2 knockdown in ZEC043, ZEC056, and ZEC145 cells (B,C) and overexpression in ZEC014 cells (D). CCK-8 and colony formation with crystal violet staining assays analyzed cell proliferation in ZEC043, ZEC056, ZEC145 cells (E-H), and ZEC014 cells (I). Each picture represents a well of a 6-well plate. Data are presented as the mean ± standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001 (Student t-test). The grayscale analysis values of western blot bands are listed below the bands. CCK-8, Cell Counting Kit-8; ESCC, esophageal squamous cell carcinoma; OD, optical density; PDC, patient-derived cell line; SH3BGRL2, SH3 domain binding glutamate rich protein-like 2.

Transient SH3BGRL2 knockdown with siRNA-mediated silencing of SH3BGRL2 significantly increased the PDC proliferation in ZEC043, ZEC056, and ZEC145 cells according to both cell viability and colony formation assays (Figure 2E-2G). Stable SH3BGRL2 knockdown via two shRNAs also induced cell proliferation and colony formation assays in ZEC145 cells (Figure 2H). Moreover, ZEC014 cells overexpressing SH3BGRL2 showed greater proliferative potential than did ZEC014 cells transfected with vehicle plasmid (Figure 2I).

SH3BGRL2 knockdown promoted tumor growth in vivo

To further investigate the role of SH3BGRL2 in ESCC tumorigenesis, SH3BGRL2 knockdown and vector control ZEC-145 cells were subcutaneously injected into BALB/c nude mice. Tumors derived from SH3BGRL2-silenced cells grew significantly faster than did those derived from control vector cells (Figure 3A,3B). Four weeks after inoculation, tumors from SH3BGRL2-silenced cells were significantly larger and heavier than those from vector control cells (Figure 3C). Collectively, these data demonstrate that SH3BGRL2 inhibits the proliferation of ESCC cells both in vitro and in vivo.

Figure 3 SH3BGRL2 suppressed the growth of ESCC PDCs in vivo. (A) Representative images of BALB/c nude mice subcutaneously injected with vector control (upper row) or SH3BGRL2 knockdown ZEC-145 cells (lower row). (B) Analysis of tumor volume of mice measured weekly (n=8 per group). (C) Analysis of tumor weight of xenograft tumors 4 weeks after tumor inoculation (n=8 per group). Data are presented as the mean ± standard deviation. *, P<0.05 (Student t-test and Chi-squared test). ESCC, esophageal squamous cell carcinoma; PDC, patient-derived cell line; SH3BGRL2, SH3 domain binding glutamate rich protein-like 2.

SH3BGRL2 downregulation was associated with the expression and transcriptional activity of EGR1

To elucidate the mechanism by which SH3BGRL2 inhibits ESCC proliferation, RNA sequencing was performed in SH3BGRL2-silenced ZEC-145 cells and control vector cells. Investigation of differentially expressed genes revealed that EGR1 expression was upregulated in both SH3BGRL2-silenced shRNA-1 and shRNA-2 cells (fold change >1.5; Figure 4A,4B). Analysis of the transcription factors associated with differentially expressed genes revealed significant enrichment of the C2H2 zinc finger transcription factor family (Figure 4C). Within this family, EGR1 exhibited markedly elevated expression levels in SH3BGRL2-silenced cells. Examination of the TCGA database also indicated a significant negative correlation between EGR1 and SH3BGRL2 expression in ESCC tissues (P<0.001; Figure 4D). Furthermore, quantitative reverse transcription-PCR (qRT-PCR) confirmed that EGR1 mRNA expression was increased in SH3BGRL2-knockdown cells (P<0.001; Figure 4E). In line with this, EGR1 protein levels were increased in SH3BGRL2-knockdown ZEC-145 cells (Figure 4F) and decreased in SH3BGRL2-overexpressing ZEC-014 cells (Figure 4G). Overall, these findings suggest that SH3BGRL2 functions as a tumor suppressor in ESCC, likely through inhibiting EGR1-mediated gene transcription.

Figure 4 SH3BGRL2 inhibited the EGR1 expression of ESCC cells. (A) Differentially expressed genes between SH3BGRL2-silenced and vector control ZEC145 cells. (B) Elevated mRNA expression of EGR1 in SH3BGRL2-silenced ZEC145 and vector control cells, as indicated in orange borders. (C) Transcription factors associated with differentially expressed genes between SH3BGRL2-silenced and vector control ZEC145 cells. Results show the significant enrichment of the C2H2 zinc finger transcription factor family (zf-C2H2). X-axis: the number of genes in each transcription factor family. (D) The significant negative correlation between EGR1 and SH3BGRL2 expression in ESCC tissues as analyzed via TCGA database (P<0.001). (E) Quantitative reverse transcription-PCR confirmed that EGR1 mRNA expression was increased in SH3BGRL2-knockdown cells (P<0.001). (F,G) Protein expression of EGR1 in SH3BGRL2-silenced ZEC145 cells and SH3BGRL2-overexpressing ZEC014 cells and corresponding vector control cells. Data are presented as the mean ± standard deviation. The grayscale analysis values of western blot bands are listed below the bands. **, P<0.01; ***, P<0.001 (Student t-test). EGR1, early growth response 1; ESCC, esophageal squamous cell carcinoma; SH3BGRL2, SH3 domain binding glutamate rich protein-like 2; TCGA, The Cancer Genome Atlas; TPM, transcripts per million.

Discussion

In this study, we generated clinical evidence indicating downregulated SH3BGRL2 mRNA and protein expression in ESCC tumors and the association between high SH3BGRL2 expression and prolonged OS and DFS in patients with resectable ESCC. Functional assays revealed that SH3BGRL2 inhibits the proliferation of ESCC cells in vitro and in vivo. Our results highlight the critical role of SH3BGRL2 as a tumor suppressor and prognostic biomarker in ESCC.

Our RNA-sequencing and IHC analyses both demonstrated significant downregulation of SH3BGRL2 in ESCC tissues as compared to paired adjacent normal tissues (P<0.01). Decreased SH3BGRL2 expression has been reported in a number of cancer types, including anaplastic thyroid carcinoma (18), chemotherapy-resistant ovarian cancer (16), glioblastoma (19), and endometrial stromal cell cancer (20). In an mRNA analysis of 179 ESCC tissues, the expression of SH3BGRL2 was downregulated (15). Moreover, in another study, whole-genome sequencing of DNA and RNA in ESCC samples from 94 Chinese patients indicated a positive correlation of SH3BGRL2 copy number amplifications with SH3BGRL2 mRNA level (R>0.3; P<0.05) (21). Taken together, our results were consistent with previous findings and suggest that SH3BGRL2 is weakly expressed in ESCC and may be involved in ESCC progression.

The mechanisms underlying SH3BGRL2 downregulation in ESCC remains unclear. Li et al. discovered SH3BGRL2-mediated proliferation suppression in breast cancer cells, in which SH3BGRL2 expression could be transcriptionally activated by transforming growth factor-β1 (TGF-β1) (22). However, we found TGF-β1 was highly expressed and negatively correlated with SH3BGRL2 expression in an esophageal cancer dataset in TCGA database (Figure S1). Therefore, different from breast cancer, TGF-β1 may not be a driver of SH3BGRL2 expression in esophageal cancer. Additionally, epigenetic modifications, such as DNA methylation or histone acetylation, or posttranscriptional regulation by microRNAs, might potentially contribute to the reduced expression of SH3BGRL2 in ESCC, which warrants further investigation.

Limitations of current ESCC prognostic markers hinder their widespread clinical application. In addition to decreased expression in tumor tissues, high SH3BGRL2 expression conferred a favorable prognosis in patients with ESCC. Multivariate analysis suggested SH3BGRL2 to be an independent prognostic factor for OS and DFS. Consistent with this, SH3BGRL2 expression has been reported to be downregulated in clear-cell renal cell carcinoma (ccRCC) tissues, with patients with lower expression levels of SH3BGRL2 having a worse OS and DFS (23). In addition, SH3BGRL2 has been shown to be weakly expressed in glioblastoma multiforme and associated with poor patient prognosis (19). In triple-negative breast cancer, SH3BGRL2 emerged as a key factor in the prognostic model, associated with markedly longer OS (24). However, the prognostic value of SH3BGRL2 in ESCC has not been previously reported on, making our study a significant contribution to the field. SH3BGRL2 expression level may serve as a valuable prognostic indicator for patients with ESCC and facilitate the development of novel treatment options.

We further found that SH3BGRL2 played a crucial role in inhibiting the proliferative capacity of ESCC. In vitro and in vivo, ESCC PDC proliferation was suppressed by SH3BGRL2 knockdown and promoted by SH3BGRL2 overexpression. suggesting SH3BGRL2 as a key regulator of ESCC cell growth and proliferation. These results were consistent with a study on ccRCC, in which SH3BGRL2 inhibited tumor cell growth and metastasis through the hippo/TEAD1/Twist1 pathway (23). Moreover, SH3BGRL2 significantly inhibited glioblastoma multiforme cell growth, and migration in vitro as well as tumor growth in vivo (19). In our study, we found upregulation of EGR1, a transcription factor of the C2H2 zinc finger family, in SH3BGRL2-silenced PDCs and xenograft models and decreased EGR1 protein level in SH3BGRL2-overexpressing cells. EGR1 was also enriched in transcription factors associated with the differentially expressed genes between SH3BGRL2-knockdown and control ESCC cells. EGR1 is involved in various cellular processes including cell proliferation, differentiation, and apoptosis (25-27). Previous studies have demonstrated that EGR1 is involved in the regulation of TFEB and PTEN pathways in several cancers (27-29). And EGR1 has been shown to drive the migration and invasion of ESCC cells (30). Therefore, as our study generated evidence for an association between SH3BGRL2 level and EGR1 expression and transcription activity, the SH3BGRL2–EGR1 axis may have therapeutic potential in ESCC. Further research is warranted to validate the functional relationship between SH3BGRL2 and EGR1.

Our study involved several limitations that should be acknowledged. First, the number of tissue samples subjected to RNA sequencing (n=3) was relatively small. Second, we employed a single-center design, reducing the representativeness of our patient sample. Third, our key finding was the identification of SH3BGRL2’s role in ESCC tumorigenesis. However, the upstream and downstream relationships of SH3BGR2 relevant to the inhibition of ESCC proliferation need to be further clarified.

Conclusions

In this study, SH3BGRL2 expression was significantly decreased in ESCC and was associated with poor prognosis in patients with resectable ESCC. SH3BGRL2 inhibited ESCC growth and proliferative in vitro and in vivo, likely via blocking EGR1 signaling. Collectively, our findings highlight the critical role of SH3BGRL2 in the pathogenesis and prognosis of ESCC, providing the basis for deeper insights into the underlying mechanisms and potential therapeutic targets in ESCC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1878/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1878/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1878/prf

Funding: This work was supported by National Natural Science Foundation of China (No. 82203817 to R.Z.), Zhejiang Provincial Natural Science Foundation of China (No. LQ24H160038 to K.C.), and Medical Health Science and Technology Project of Zhejiang Province (Nos. 2023KY601 to K.C., 2024KY809 to K.C., 2022RC016 to F.Z.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1878/coif). K.C. reports Zhejiang Provincial Natural Science Foundation of China (Grant LQ24H160038), and Medical Health Science and Technology Project of Zhejiang Province (Nos. 2023KY601, 2024KY809). R.Z. reports National Natural Science Foundation of China (No. 82203817). F.Z. reports Medical Health Science and Technology Project of Zhejiang Province (No. 2022RC016). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was approved by the Medical Ethical Committee of Zhejiang Cancer Hospital (No. IRB-2021-248). All samples were collected with written informed consent. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The animal experiment was approved by the Animal Committee of Zhejiang Cancer Hospital (No. 2024-09-022), in compliance with national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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