Reduced VEPH1 expression is associated with an invasive phenotype and poor prognosis in clear cell renal cell carcinoma

Introduction

Renal cell carcinoma (RCC) is the most common form of kidney cancer and remains a significant cause of cancer-related mortality worldwide (1). Although many renal tumors are detected incidentally at a localized stage, patients with advanced or metastatic disease continue to experience poor outcomes (2). Clear cell renal cell carcinoma (ccRCC), the predominant histological subtype, accounts for approximately 70–85% of RCC cases and is characterized by substantial clinical and molecular heterogeneity (3). Despite advances in surgery, targeted therapy, and immune checkpoint inhibition, reliable molecular biomarkers for individualized prognostic assessment remain limited. These challenges underscore the need to identify robust molecular biomarkers that can improve prognostic stratification and provide insight into the biological processes underlying ccRCC progression.

The VEPH1 gene encodes ventricular zone-expressed pleckstrin homology domain-containing protein 1 (VEPH1), an intracellular adaptor protein capable of modulating several signaling networks, including TGF-β, mTOR, Hippo, FOXO, and AKT signaling (4). Several functional studies support a tumor-suppressive role for VEPH1, although its biological effects appear to be context dependent. Reduced VEPH1 expression promoted mTORC1 signaling by weakening the TSC1–TSC2 association in hepatocellular carcinoma (5), whereas VEPH1 restoration inhibited gastric cancer progression through modulation of Hippo-YAP signaling (6). In ovarian cancer models, VEPH1 expression reduced AKT activation, VEGFA and IL8 expression, and tumor vascularization (7). However, a subsequent study demonstrated that the effects of VEPH1 on EGF/ERBB2 signaling differed according to the molecular background of the ovarian cancer cells (8). The expression pattern, prognostic relevance, and functional role of VEPH1 in ccRCC remain inadequately characterized. In particular, direct evidence regarding the association of VEPH1 with epithelial-mesenchymal transition (EMT)-related molecular changes, migration, and invasion in ccRCC is limited.

EMT is a dynamic cellular program linked with tumor-cell plasticity, migration, and invasion. EMT-related changes commonly include increased expression of mesenchymal markers, such as N-cadherin, and reduced expression of epithelial markers, such as E-cadherin. Previous ccRCC studies have implicated PI3K/AKT and EGFR/ERK signaling in EMT-related marker changes, migration, and invasion (9,10). Because VEPH1 has also been associated with AKT, mTOR, and growth factor signaling in other tumor models, we examined whether VEPH1-associated phenotypic changes in ccRCC cells were accompanied by alterations in AKT and ERK phosphorylation.

In this study, we aimed to systematically investigate the expression and functional relevance of VEPH1 in ccRCC. Through integrative bioinformatics analysis and experimental validation, we evaluated VEPH1 transcript and VEPH1 protein expression, their associations with overall survival, and the functional effects of VEPH1 modulation on tumor-cell migration and invasion. We further examined EMT-related marker expression and assessed whether VEPH1 overexpression was accompanied by changes in AKT and ERK phosphorylation. We hypothesized that reduced VEPH1 expression would be associated with adverse clinical outcomes and that experimental modulation of VEPH1 would alter EMT-related markers and invasive cellular behavior in ccRCC. We present this article in accordance with the MDAR reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0507/rc).

Methods

Clinical sample collection and ethical statement

This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments, and approved by the Ethics Committee of The First Affiliated Hospital of Soochow University (Approval No. 2023-R-165). Written informed consent was obtained from all participants prior to sample collection. A total of 11 pairs of primary ccRCC tissues and matched adjacent non-tumor renal tissues were collected from patients undergoing radical nephrectomy. None of the patients had received preoperative radiotherapy, chemotherapy, or immunotherapy. All tumor diagnoses were confirmed by histopathological evaluation. Immediately after surgical resection, tissue specimens were divided into two portions: one portion was snap-frozen in liquid nitrogen and stored at −80 ℃ for subsequent RNA and protein analyses, whereas the other was fixed in formalin for histological examination.

Cell culture and reagents

Human ccRCC cell lines 786-O (RRID: CVCL_1051) and 769-P (RRID: CVCL_1050), and human embryonic kidney HEK293T cells (Cat. No. GNHu44; RRID: CVCL_0063), were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Gibco BASIC RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. C11875500BT) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were maintained at 37 ℃ in a humidified atmosphere containing 5% CO₂. Key reagents included Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA), the RNAeasy RNA Extraction Kit and radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China), and reverse-transcription and quantitative polymerase chain reaction kits (Thermo Fisher Scientific). Primary antibodies used for western blotting included rabbit anti-VEPH1 (ABclonal, Wuhan, China, Cat. No., 1:1,000), rabbit anti-GAPDH (ABclonal, Cat. No. AC001, 1:10,000), mouse anti-E-cadherin (BD Biosciences, San Jose, CA, USA; Cat. No. 610181, 1:1,000), mouse anti-N-cadherin (BD Biosciences, Cat. No. 610920, 1:1,000), rabbit anti-AKT (Cell Signaling Technology, Danvers, MA, USA; Cat. No. 9272, 1:1,000), rabbit anti-phospho-AKT (Ser473) (Cell Signaling Technology, Cat. No. 4060, 1:2,000), rabbit anti-ERK1/2 (Cell Signaling Technology, Cat. No. 4695, 1:1,000), and rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology, Cat. No. 4370, 1:2,000). Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were obtained from Beyotime Biotechnology and used at a dilution of 1:5,000.

Bioinformatics analysis

Transcriptomic data and corresponding clinical information for The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma (TCGA-KIRC) cohort were obtained from the University of California, Santa Cruz (UCSC) Xena platform. Gene-expression data were normalized using a log₂(TPM + 1) transformation. Differences in VEPH1 transcript expression between tumor and normal renal tissues were analyzed using R software and further examined using the Gene Expression Profiling Interactive Analysis (GEPIA) database. Receiver operating characteristic (ROC) curve analysis was performed to assess the ability of VEPH1 expression to distinguish TCGA-KIRC tumor samples from normal samples.

For the TCGA-KIRC survival analysis, patients were dichotomized at the median VEPH1 expression value into high-expression (n=271) and low-expression (n=270) groups. Overall survival was estimated using the Kaplan-Meier method, and survival distributions were compared using the log-rank test. Subgroup survival analyses according to tumor grade, race, and sex were obtained from the University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN). For these analyses, the portal-defined high-expression and low/medium-expression categories were used. Because the TCGA-KIRC and UALCAN analyses used different grouping criteria, their results were interpreted separately.

Cell transfection and construction of stable cell lines

To generate stable VEPH1-overexpressing cells, the full-length human VEPH1 coding sequence was cloned into the pCDH-CMV-MCS-EF1α-Puro lentiviral expression vector (System Biosciences, Palo Alto, CA, USA; Cat. No. CD510B-1). The recombinant pCDH-VEPH1 construct and corresponding empty-vector control were prepared by Shanghai GeneChem Co., Ltd. (Shanghai, China), and the inserted sequence was verified by Sanger sequencing. Because the recombinant pCDH-VEPH1 construct was custom prepared, a separate commercial catalogue number was not applicable to the completed construct. For lentiviral production, the pCDH-VEPH1 plasmid or empty-vector control was co-transfected into HEK293T cells together with the psPAX2 packaging plasmid and pMD2.G envelope plasmid using Lipofectamine 3000 according to the manufacturer’s instructions. Viral supernatants were collected at 48 and 72 hours after transfection, filtered through a 0.45-µm membrane, and used to transduce 786-O and 769-P cells in the presence of 8 µg/mL polybrene. At 48 hours after transduction, cells were selected with 2 µg/mL puromycin for 7 days to establish stable VEPH1-overexpressing and empty-vector control cell populations. VEPH1 overexpression efficiency was confirmed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting.

For VEPH1 knockdown, two lentiviral short hairpin RNA (shRNA) constructs targeting human VEPH1 (sh-VEPH1-1 and sh-VEPH1-2) and a non-targeting control construct (sh-NC) were synthesized and packaged by Shanghai GeneChem Co., Ltd. These constructs were custom synthesized rather than commercially available products; therefore, standard catalogue numbers were not applicable. Supplier-specific construct identifiers and target sequences were not available in the retained laboratory records. The lentiviral particles were used to transduce 786-O and 769-P cells. At 48 hours after transduction, cells were selected with 2 µg/mL puromycin for 7 days to establish stable cell populations. VEPH1 knockdown efficiency was confirmed by RT-qPCR and western blotting.

RT-qPCR

Total RNA was extracted from tissues and cultured cells using the RNAeasy RNA Extraction Kit (Beyotime Biotechnology) according to the manufacturer’s instructions. RNA concentration and purity were assessed using a spectrophotometer, and 1 µg of total RNA was reverse-transcribed into complementary DNA using a reverse-transcription kit (Thermo Fisher Scientific). Quantitative PCR was performed using SYBR Green Master Mix (manufacturer, city, country) on a Roche LightCycler 480 real-time PCR system (Roche, Basel, Switzerland). The amplification conditions were initial denaturation at 95 ℃ for 5 minutes, followed by 45 cycles of 95 ℃ for 12 seconds and 60 ℃ for 45 seconds. Relative gene expression was calculated using the 2^−ΔΔCt method, with β-actin as the internal reference.

Western blot analysis

Total protein was extracted from cells or tissue specimens using RIPA lysis buffer (Beyotime Biotechnology) supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using a bicinchoninic acid assay. Equal amounts of protein (20–30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk for 1 hour at room temperature and incubated overnight at 4 ℃ with primary antibodies against VEPH1, E-cadherin, N-cadherin, AKT, phosphorylated AKT, ERK, phosphorylated ERK, and GAPDH. After washing with Tris-buffered saline containing Tween-20, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were detected using an enhanced chemiluminescence detection kit and visualized using an Alpha Red imaging system.

Band intensities were quantified using ImageJ software (version 1.54f; National Institutes of Health, Bethesda, MD, USA). VEPH1, E-cadherin, N-cadherin, total AKT, total ERK1/2, phosphorylated AKT, and phosphorylated ERK1/2 protein levels were normalized to GAPDH. For the paired clinical tissue samples, the VEPH1/GAPDH value was calculated separately for each tumor tissue and its matched adjacent non-tumor renal tissue.

Transwell migration and invasion assays

Cell migration and invasion were assessed using Transwell chambers with 8.0-µm pore polycarbonate membranes (BD Biosciences). Cells in the logarithmic growth phase were harvested, washed, and resuspended in serum-free RPMI 1640 medium. For the migration assay, 5×10⁴ cells in 200 µL of serum-free medium were seeded into the upper chamber. For the invasion assay, the upper chamber was precoated with 50 µL of Matrigel (manufacturer, city, country) diluted 1:8 in serum-free medium, and 1×10⁵ cells were subsequently seeded into the chamber. The lower chamber contained 800 µL of RPMI 1640 medium supplemented with 20% fetal bovine serum as a chemoattractant.

After incubation for 24 hours at 37 ℃, non-migrated or non-invaded cells remaining on the upper surface of the membrane were gently removed with a cotton swab. Cells on the lower surface were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.1% crystal violet for 20 minutes. Five randomly selected, non-overlapping microscopic fields were captured from each membrane. Migrated and invaded cells were quantified using the Cell Counter function in ImageJ software. Individual nuclei or clearly distinguishable cell bodies were counted as single cells. In densely stained or overlapping regions, cells were counted based on identifiable nuclear or cellular boundaries and were manually reviewed to minimize duplicate or missed counts. The mean cell count from the five fields was used as the value for each membrane, and each experiment was independently repeated three times.

Statistical analysis

Statistical analyses were performed using SPSS software (version 22.0; IBM Corp., Armonk, NY, USA) and R software 4.3.2. Quantitative data are presented as the mean ± standard deviation. Paired tumor and adjacent non-tumor tissue measurements were compared using a paired two-tailed Student’s t-test. Comparisons between two independent groups were performed using an unpaired two-tailed Student’s t-test. Comparisons among more than two groups were performed using one-way analysis of variance followed by Tukey’s multiple-comparisons test. Kaplan-Meier survival curves were compared using the log-rank test. All statistical tests were two-sided, and P<0.05 was considered statistically significant.

Results

VEPH1 expression and overall survival in the TCGA-KIRC cohort

To investigate the expression profile of VEPH1 in ccRCC, transcriptomic data from the TCGA-KIRC cohort were analyzed. VEPH1 transcript expression was significantly reduced in ccRCC tissues compared with normal tissues (Figure 1A). Consistent results were observed using the GEPIA database, further supporting the lower expression of VEPH1 in ccRCC (Figure 1A). To evaluate the ability of VEPH1 expression to distinguish TCGA-KIRC tumor samples from normal samples, ROC curve analysis was performed. VEPH1 expression demonstrated the ability to distinguish tumor from normal samples within the TCGA-KIRC dataset, with an area under the curve (AUC) of 0.900 (95% CI: 0.871–0.929) (Figure 1B). Patients were dichotomized at the median VEPH1 expression value into high-expression (n=271) and low-expression (n=270) groups. Kaplan-Meier survival analysis showed that patients with low VEPH1 expression had significantly poorer overall survival than those with high VEPH1 expression (log-rank P=3.43×10⁻⁵) (Figure 1C).

Figure 1 VEPH1 transcript expression and overall survival in the TCGA-KIRC cohort. (A) Comparison of VEPH1 transcript expression between normal renal tissues and clear cell renal cell carcinoma tissues using unpaired TCGA-KIRC samples, paired TCGA-KIRC samples, and the GEPIA database. In the GEPIA panel, the red box represents ccRCC tumor tissues (n=523), whereas the gray box represents normal renal tissues (n=72). The corresponding group labels are also shown directly below the box plots. (B) Receiver operating characteristic curve showing the ability of VEPH1 transcript expression to distinguish TCGA-KIRC tumor samples from normal samples (AUC 0.900; 95% CI: 0.871–0.929). (C) Kaplan-Meier analysis of overall survival in patients dichotomized at the median VEPH1 transcript-expression value into high-expression (n=271) and low-expression (n=270) groups. *P<0.05, ***P<0.001. AUC, area under the curve; ccRCC, clear cell renal cell carcinoma; CI, confidence interval; FPR, false positive rate; GEPIA, Gene Expression Profiling Interactive Analysis; KIRC, kidney renal clear cell carcinoma; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; TPR, true positive rate.

Exploratory subgroup survival analyses using UALCAN

The association between VEPH1 expression and overall survival in ccRCC was further explored using the UALCAN database. Patients were categorized using the UALCAN portal-defined high-expression and low/medium-expression groups. Stratified survival analyses were performed across different clinicopathological subgroups. Overall survival differed among the combined VEPH1 expression and tumor-grade subgroups (P<0.0001) (Figure 2A). When stratified by race (African American, Asian, and Caucasian), no statistically significant survival difference was observed among the combined VEPH1 expression and race subgroups (P=0.28; Figure 2B). The high-expression Asian subgroup included only three patients, and its survival curve was difficult to distinguish because it substantially overlapped with another survival trajectory. The sex-stratified analysis did not show a statistically significant survival difference among the combined VEPH1 expression and sex subgroups (P=0.07; Figure 2C). In the overall UALCAN cohort, high VEPH1 expression was associated with longer overall survival than low/medium expression (P=0.01; Figure 2D). The grade-, race-, and sex-stratified analyses were considered exploratory, particularly because several subgroup categories contained small numbers of patients.

Figure 2 Exploratory subgroup analyses of VEPH1 transcript expression and overall survival in the UALCAN TCGA-KIRC cohort. (A) Overall survival among combined VEPH1 expression and tumor-grade subgroups (P<0.0001). (B) Overall survival among combined VEPH1 expression and race subgroups (P=0.28). The high-expression Asian subgroup comprised only three patients. Its survival curve is included in the plot but is difficult to distinguish because it is largely superimposed on another survival trajectory. The corresponding curve has therefore been identified directly in Panel B. (C) Overall survival among combined VEPH1 expression and sex subgroups (P=0.07). (D) Overall survival according to the UALCAN-defined high-expression and low/medium-expression groups (P=0.01). Patients were categorized using the expression groups defined by the UALCAN portal. The grade-, race-, and sex-stratified analyses were considered exploratory, particularly for categories containing small numbers of patients. KIRC, kidney renal clear cell carcinoma; TCGA, The Cancer Genome Atlas; UALCAN, University of Alabama at Birmingham Cancer Data Analysis Portal.

Validation of VEPH1 expression in paired ccRCC tissues

To validate the expression pattern of VEPH1 in clinical samples, 11 pairs of pathologically confirmed ccRCC tissues and matched adjacent non-tumor renal tissues were analyzed. All samples were obtained from patients undergoing surgical resection at The First Affiliated Hospital of Soochow University. RT-qPCR analysis demonstrated that VEPH1 transcript expression was significantly lower in ccRCC tissues than in matched adjacent non-tumor renal tissues (P<0.01) (Figure 3A). Consistently, western blotting showed reduced VEPH1 protein expression in tumor tissues. Densitometric analysis, with VEPH1 normalized to GAPDH, confirmed significantly lower VEPH1 protein expression in ccRCC tissues than in matched adjacent non-tumor renal tissues (P<0.01) (Figure 3B,3C).

Figure 3 Validation of VEPH1 expression in paired ccRCC clinical tissues. (A) Relative VEPH1 mRNA expression in ccRCC tissues and matched adjacent non-tumor renal tissues. (B) Representative western blot images of VEPH1 protein expression in 11 matched tissue pairs. (C) Densitometric quantification of VEPH1 protein expression normalized to GAPDH. Data are presented as paired measurements. **P<0.01. ccRCC, clear cell renal cell carcinoma; mRNA, messenger RNA; N, adjacent non-tumor renal tissue; T, ccRCC tissue.

VEPH1 overexpression is associated with EMT-related marker changes in ccRCC

To investigate the functional role of VEPH1 in ccRCC, stable VEPH1-overexpressing (VEPH1-OE) 786-O and 769-P cell lines were established. Western blot analysis was performed to evaluate the expression of EMT-related markers, including E-cadherin and N-cadherin. Compared with the corresponding empty-vector control group within each cell line, VEPH1 overexpression resulted in a significant decrease in N-cadherin expression and a corresponding increase in E-cadherin expression (all P<0.01) (Figure 4A-4D). These findings suggest that VEPH1 overexpression is associated with a shift toward a more epithelial-like marker profile in ccRCC cells.

Figure 4 Effect of VEPH1 overexpression on EMT-related marker expression in ccRCC cells. (A,C) Representative western blot images of VEPH1, E-cadherin, and N-cadherin protein expression in 786-O and 769-P cells. (B,D) Densitometric quantification of VEPH1, E-cadherin, and N-cadherin expression normalized to GAPDH. All comparisons were performed within each cell line relative to its corresponding Vector control; the 786-O and 769-P blots were generated in separate experiments and were not intended for direct comparison of basal protein expression between the two cell lines. Data are presented as the mean ± SD from three independent experiments. **P<0.01; ***P<0.001. ccRCC, clear cell renal cell carcinoma; EMT, epithelial-mesenchymal transition; OE, overexpression; SD, standard deviation.

VEPH1 knockdown is associated with EMT-related marker changes in ccRCC

To further evaluate the role of VEPH1, stable VEPH1-knockdown cell lines (sh-VEPH1-1 and sh-VEPH1-2) were established in 786-O and 769-P cells. Western blot analysis was performed to assess the expression of EMT-related markers. E-cadherin was evaluated as an epithelial marker, whereas N-cadherin was evaluated as a mesenchymal marker. Compared with the corresponding sh-NC control within each cell line, partial VEPH1 knockdown resulted in a significant increase in N-cadherin expression and a concomitant decrease in E-cadherin expression (all P<0.01) (Figure 5A-5D). Similar trends were observed in both 786-O and 769-P cell lines. These findings indicate that partial VEPH1 knockdown is associated with a more mesenchymal-like marker profile in ccRCC cells.

Figure 5 Effect of partial VEPH1 knockdown on EMT-related marker expression in ccRCC cells. (A,C) Representative western blot images of VEPH1, E-cadherin, N-cadherin, and GAPDH in 786-O and 769-P cells following VEPH1 knockdown. (B,D) Densitometric quantification of VEPH1, E-cadherin, and N-cadherin protein expression normalized to GAPDH. E-cadherin was assessed as an epithelial marker, whereas N-cadherin was assessed as a mesenchymal marker. Comparisons were performed within each cell line relative to the corresponding sh-NC control. The 786-O and 769-P western blots were generated in separate experiments and were intended for within-cell-line comparisons; therefore, absolute band intensities should not be used to compare basal protein expression between the two cell lines. Data are presented as the mean ± SD from three independent experiments. **P<0.01; ***P<0.001. ccRCC, clear cell renal cell carcinoma; EMT, epithelial-mesenchymal transition; NC, negative control; SD, standard deviation; sh, short hairpin.

Effect of VEPH1 on ccRCC cell migration and invasion

Transwell assays were performed to evaluate the effects of VEPH1 on the migratory and invasive capacities of ccRCC cells. Cells were grouped as Vector, VEPH1 overexpression (VEPH1-OE), sh-NC, sh-VEPH1-1, and sh-VEPH1-2. In 786-O cells, VEPH1 overexpression significantly reduced the number of migrated cells compared with the Vector group (P<0.001), whereas VEPH1 knockdown resulted in increased migration relative to the sh-NC group (P<0.001). Similarly, invasion assays showed fewer invasive cells in the VEPH1-OE group and increased invasion in the VEPH1 knockdown groups (P<0.001) (Figure 6A-6E). Consistent results were observed in 769-P cells, where VEPH1 overexpression was associated with reduced migration and invasion, while VEPH1 knockdown enhanced these cellular behaviors (Figure 7A-7E). Collectively, VEPH1 overexpression reduced, whereas partial VEPH1 knockdown increased, the migration and invasion of ccRCC cells under the experimental conditions used.

Figure 6 Effect of VEPH1 overexpression and partial knockdown on the migration and invasion of 786-O cells. (A) Representative images of Transwell migration and invasion assays following VEPH1 overexpression or knockdown (crystal violet staining). (B) Quantification of migrated cells in the Vector and VEPH1-OE groups. (C) Quantification of invaded cells in the Vector and VEPH1-OE groups. (D) Quantification of migrated cells in the sh-NC, sh-VEPH1-1, and sh-VEPH1-2 groups. (E) Quantification of invaded cells in the sh-NC, sh-VEPH1-1, and sh-VEPH1-2 groups. Cells were counted in five randomly selected, non-overlapping fields per membrane using ImageJ-assisted manual counting. Each point represents one independent experiment, and data are presented as the mean ± SD (n=3). Scale bars, 100 μm. ***P<0.001. NC, negative control; OE, overexpression; SD, standard deviation; sh, short hairpin.

Figure 7 Effect of VEPH1 overexpression and partial knockdown on the migration and invasion of 769-P cells. (A) Representative images of Transwell migration and invasion assays following VEPH1 overexpression or knockdown (crystal violet staining). (B) Quantification of migrated cells in the Vector and VEPH1-OE groups. (C) Quantification of invaded cells in the Vector and VEPH1-OE groups. (D) Quantification of migrated cells in the sh-NC, sh-VEPH1-1, and sh-VEPH1-2 groups. (E) Quantification of invaded cells in the sh-NC, sh-VEPH1-1, and sh-VEPH1-2 groups. Cells were quantified in five randomly selected, non-overlapping fields per membrane using ImageJ software. In densely stained fields, ImageJ-assisted manual counting was performed based on identifiable nuclear or cellular boundaries. Data are presented as the mean ± SD from three independent experiments. Scale bars, 100 μm. ***P<0.001. NC, negative control; OE, overexpression; SD, standard deviation; sh, short hairpin.

Association of VEPH1 overexpression with AKT and ERK phosphorylation

To examine whether VEPH1 overexpression was accompanied by changes in AKT and ERK phosphorylation, phosphorylated and total AKT and ERK1/2 protein levels were evaluated in 786-O cells. Western blotting showed that VEPH1 overexpression was associated with significantly lower phosphorylated AKT and phosphorylated ERK1/2 levels, whereas total AKT and total ERK1/2 levels were not significantly altered (Figure 8A,8B). These findings indicate that VEPH1 overexpression was associated with reduced AKT and ERK phosphorylation in 786-O cells.

Figure 8 Exploratory association of VEPH1 overexpression with AKT and ERK phosphorylation in 786-O cells. (A) Representative western blot images of VEPH1, phosphorylated AKT1/2/3, total AKT1/2/3, phosphorylated ERK1/2, total ERK1/2, and GAPDH in Vector and VEPH1-OE cells. (B) Densitometric quantification of VEPH1, phosphorylated AKT1/2/3, total AKT1/2/3, phosphorylated ERK1/2, and total ERK1/2 protein levels normalized to GAPDH. Data are presented as the mean ± SD from three independent experiments. These findings demonstrate an association only and do not establish that AKT or ERK signaling mediates the EMT-related marker, migration, or invasion changes observed following VEPH1 modulation. ns, not significant; **P<0.01. OE, overexpression; SD, standard deviation.

Discussion

VEPH1 encodes an 833-amino-acid protein. The gene is located on the negative strand of chromosome 3 (3q25.31–q25.32). Structurally, the VEPH1 gene spans over 274 kb and consists of 15 exons and 14 introns, with its coding region extending from exon 3 to exon 15. The C-terminus of VEPH1 contains a conserved pleckstrin homology domain, which is known to mediate membrane recruitment of signaling molecules. In addition, its unique N-terminal region enables VEPH1 to function as an adaptor or docking protein, facilitating protein-protein interactions and modulating intracellular signaling pathways. Through these structural features, VEPH1 plays an important role in regulating cellular processes and signal transduction (11,12). VEPH1 is highly expressed in human kidney, pancreas, spleen, and embryonic brain tissues (13), and has been implicated in the regulation of multiple signaling pathways, including TGF-β, mTOR, FOXO, Hippo, and AKT (4).

Accumulating evidence suggests that VEPH1 is involved in tumor progression across multiple cancer types. Nie et al. (6) demonstrated that VEPH1 suppressed gastric cancer progression and regulated EMT-related features through modulation of Hippo-YAP signaling. Dong et al. (5) reported that reduced VEPH1 expression leads to activation of the mTORC1 pathway in hepatocellular carcinoma, highlighting its potential therapeutic relevance. Ragazzon et al. (14) further showed that VEPH1 expression is markedly decreased in aggressive adrenocortical carcinomas and is associated with poor clinical outcomes. Despite these findings, the role of VEPH1 in ccRCC has remained unclear.

In the present study, integrative analysis of TCGA-KIRC data revealed that VEPH1 transcript expression was significantly lower in ccRCC tissues than in normal renal tissues. Survival analysis further demonstrated that lower VEPH1 expression was associated with poorer overall survival. ROC analysis showed that VEPH1 expression distinguished tumor samples from normal samples within the TCGA-KIRC dataset, with an AUC of 0.900. These findings support an association between reduced VEPH1 expression and adverse clinical outcomes in ccRCC; however, independent-cohort and multivariable validation are required before VEPH1 can be considered an established prognostic biomarker.

To further validate these observations, we analyzed clinical samples and in vitro models. Both RT-qPCR and western blotting confirmed reduced VEPH1 transcript and VEPH1 protein expression, respectively, in ccRCC tissues. Functional studies demonstrated that VEPH1 overexpression was associated with increased E-cadherin and decreased N-cadherin expression, whereas partial VEPH1 knockdown produced the opposite pattern within each cell line. In parallel, Transwell assays showed that VEPH1 overexpression reduced, whereas partial VEPH1 knockdown enhanced, the migration and invasion of ccRCC cells. Together, these findings indicate that experimental modulation of VEPH1 is associated with changes in selected EMT-related markers and cellular migration and invasion in ccRCC.

Baseline E-cadherin abundance was not directly compared between 786-O and 769-P cells in this study. The western blots for the two cell lines were generated in separate experiments and were intended to evaluate changes relative to the corresponding control within each cell line. Previous studies have shown that von Hippel-Lindau (VHL) loss and hypoxia-inducible factor (HIF) activation can repress E-cadherin expression in renal carcinoma models, including VHL-deficient cells (15,16). Therefore, differences in absolute band intensity between the 786-O and 769-P panels should not be interpreted as evidence of similar basal E-cadherin expression. A direct comparison would require lysates from both cell lines to be analyzed on the same membrane under identical exposure conditions.

EMT is a critical biological process that contributes to tumor invasion and metastasis by enabling epithelial cells to acquire mesenchymal characteristics (17,18). Among the signaling pathways implicated in EMT regulation, the PI3K/AKT axis plays a central role (19,20). In ccRCC, activation of the PI3K/AKT/mTOR pathway has been associated with EMT-related phenotypic changes, including reduced E-cadherin expression and increased expression of mesenchymal markers (10). In osteosarcoma, Fan et al. (21) reported that miR-23b-3p promoted malignant cellular behavior by targeting VEPH1 and modulating PI3K/AKT signaling.

In our study, VEPH1 overexpression was associated with reduced phosphorylated AKT and phosphorylated ERK1/2 levels in 786-O cells, whereas total AKT and total ERK1/2 levels were not significantly altered. These observations indicate an association between VEPH1 overexpression and reduced AKT and ERK phosphorylation. Although AKT and ERK signaling has been implicated in EMT-related processes and tumor progression, the present findings do not establish that these pathways mediate the effects of VEPH1 in ccRCC. Accordingly, the AKT and ERK findings should be interpreted as preliminary signaling associations rather than evidence of pathway dependency; the experimental limitations underlying this interpretation are detailed below. Overall, our findings suggest that VEPH1 is associated with tumor progression-related phenotypes in ccRCC and may have potential relevance as a prognostic indicator.

Limitations

This study has several limitations that should be acknowledged. First, the clinical validation was based on a relatively small sample size (n=11) derived from a single center, which may limit the generalizability of the findings. Second, the signaling investigation remains preliminary. AKT and ERK phosphorylation was examined only after VEPH1 overexpression in 786-O cells, and corresponding knockdown, pathway-inhibition, and rescue experiments were not performed. Therefore, the present data do not establish a causal role for AKT or ERK signaling in the observed cellular effects. In addition, the shRNA constructs produced only partial VEPH1 depletion; stronger loss-of-function approaches, including CRISPR/Cas9-mediated gene disruption, could provide additional validation. Third, the assessment of EMT was limited to two protein markers, E-cadherin and N-cadherin, which may not fully capture the complexity of EMT-related phenotypic changes. A transcriptome-based analysis of the association between VEPH1 and a broader panel of epithelial and mesenchymal markers in the TCGA-KIRC cohort was not included and should be considered in future studies. In addition, all functional experiments were conducted in vitro, and the absence of in vivo validation limits the translational relevance of the findings. Finally, multivariate survival analyses and independent cohort validation were not performed, which limits conclusions regarding the independent prognostic value of VEPH1 expression.

Future perspectives

Future studies should aim to address these limitations and further elucidate the role of VEPH1 in ccRCC. Validation in larger, multicenter patient cohorts, including independent external cohorts, together with multivariable survival analysis, is required to determine whether VEPH1 transcript expression has independent prognostic value. Mechanistic studies should assess AKT and ERK phosphorylation following VEPH1 knockdown and incorporate pathway-specific inhibitors and rescue experiments to determine whether AKT or ERK signaling mediates the observed cellular effects. More robust loss-of-function approaches, including CRISPR/Cas9-mediated disruption of VEPH1, may provide additional validation of the effects observed following partial shRNA-mediated knockdown. Comprehensive profiling of EMT, including additional markers such as vimentin, Snail, and ZEB1, TCGA-KIRC-based correlation analysis of epithelial and mesenchymal markers, and phenotypic characterization, will provide a more complete understanding of the biological effects associated with VEPH1. Moreover, in vivo models are needed to evaluate the impact of VEPH1 on tumor growth and metastasis. Finally, the therapeutic relevance of VEPH1-associated signaling should be evaluated only after pathway dependency and antitumor effects have been confirmed in mechanistic and in vivo studies.

Conclusions

In this study, we systematically investigated the expression and functional relevance of VEPH1 in ccRCC. Integrated bioinformatics analyses and experimental validation demonstrated lower VEPH1 transcript and VEPH1 protein expression in ccRCC tissues, whereas lower VEPH1 transcript expression was associated with poorer overall survival. Functional assays revealed that VEPH1 modulation was associated with changes in EMT-related markers, as well as altered migration and invasion of ccRCC cells. Furthermore, VEPH1 overexpression was associated with reduced AKT and ERK phosphorylation in 786-O cells. Collectively, these findings support an association between VEPH1 and invasive cellular phenotypes in ccRCC. However, given the limited clinical sample size, lack of independent-cohort validation, and absence of pathway-inhibition, rescue, and in vivo validation, further studies are required to clarify its biological functions and clinical relevance.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0507/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-0507/dss

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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. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The First Affiliated Hospital of Soochow University (No. 2023-R-165), and written informed consent was obtained from all individual participants.

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