Abstract
Background/Aim: Prostate cancer is the second most common malignancy among men worldwide, with progression to castration-resistant prostate cancer (CRPC) posing significant therapeutic challenges. Enzalutamide, a second-generation androgen receptor antagonist, initially demonstrates efficacy in treating metastatic CRPC; however, resistance inevitably develops. Dysregulation of the epidermal growth factor receptor (EGFR) signaling pathway has been implicated in therapy resistance and metastatic progression. Secretory carrier membrane protein 3 (SCAMP3) and epidermal growth factor receptor substrate 8 (EPS8) are known regulators of EGFR trafficking and signaling. This study aimed to investigate their cooperative roles in enzalutamide-resistant prostate cancer cells.
Materials and Methods: LNCap prostate cancer cells and their enzalutamide-resistant derivatives (LNCap-Enz) were treated with 100 ng/ml epidermal growth factor (EGF). Protein expression and interactions were analyzed by Western blotting and co-immunoprecipitation. SCAMP3 and EPS8 were knocked down using shRNA technology, while complementary overexpression studies were conducted using pcDNA-SCAMP3 and pcDNA-EPS8 vectors. Effects on EGF receptor (EGFR) expression and downstream signaling molecules (STAT3, AKT, ERK) were evaluated in both loss-of-function and gain-of-function models.
Results: EGF stimulation enhanced the expression of EGFR, SCAMP3, and EPS8 in both LNCap and LNCap-Enz cells while promoting formation of a protein complex involving these proteins and the androgen receptor (AR-V7). Knockdown of SCAMP3 or EPS8 reduced EGFR expression and attenuated STAT3, AKT, and ERK activation. Conversely, overexpression of SCAMP3 or EPS8 increased EGFR levels and enhanced downstream signaling activation. These bidirectional effects highlight the functional interdependence between SCAMP3 and EPS8 in regulating EGFR stability and signaling.
Conclusion: SCAMP3 and EPS8 cooperatively maintain EGFR stability and signaling in prostate cancer cells, playing a critical role in enzalutamide resistance and metastatic progression. Targeting the SCAMP3-EPS8-EGFR axis offers promising therapeutic opportunities for advanced prostate cancer.
Introduction
Prostate cancer remains one of the most prevalent malignancies in men globally, with approximately 1.4 million new cases and nearly 375,000 deaths annually (1). Despite advances in early detection and treatment, the development of castration-resistant prostate cancer (CRPC) represents a critical therapeutic challenge, as approximately 10-20% of patients progress to this aggressive stage within five years of initial diagnosis (2). Androgen deprivation therapy (ADT), the mainstay treatment for advanced prostate cancer, eventually fails in most patients, leading to disease progression despite castrate levels of testosterone (3). This clinical challenge necessitates a deeper understanding of the molecular mechanisms underpinning therapeutic resistance to develop more effective treatment strategies.
The epidermal growth factor receptor (EGFR) signaling pathway has emerged as a critical mediator of prostate cancer progression and therapeutic resistance. EGFR, a member of the ErbB receptor tyrosine kinase family, is frequently overexpressed in advanced prostate cancer and contributes to tumor cell proliferation, survival, and invasion (4). Notably, EGFR overexpression correlates with disease progression and poor prognosis, particularly in CRPC (5). Beyond its canonical kinase activity, EGFR has been shown to exhibit kinase-independent functions that prevent autophagy and promote cell survival in prostate cancer cells, further complicating therapeutic targeting approaches (5). Recent evidence suggests that EGFR expression and activation increase substantially in enzalutamide-resistant prostate cancer cells, indicating a potential role for EGFR in mediating resistance to next-generation androgen receptor-targeted therapies (6).
Downstream of EGFR activation, the epidermal growth factor receptor pathway substrate 8 (EPS8) functions as a critical adaptor protein that links receptor tyrosine kinase signaling to actin cytoskeletal remodeling and endocytic trafficking. EPS8 contains an SH3 domain and a PTB domain that facilitate protein-protein interactions within signaling cascades, particularly those involved in cytoskeletal organization and receptor endocytosis (7). Recent investigations have demonstrated elevated EPS8 expression in enzalutamide-resistant LNCap prostate cancer cells (LNCap-Enz) compared to parental LNCap cells, suggesting its involvement in therapeutic resistance8. Functionally, EPS8 promotes epithelial-to-mesenchymal transition (EMT), cell proliferation, and cell viability in both LNCap and LNCap-Enz cells, with these effects being mediated through activation of Ras/JAK/PI3K signaling pathways8. The critical role of EPS8 in prostate cancer progression has been further validated in in vivo animal studies, highlighting its potential as a therapeutic target (8).
The secretory carrier-associated membrane protein 3 (SCAMP3) represents another crucial component in the EGFR signaling network, primarily functioning in intracellular membrane trafficking and receptor endocytosis. SCAMP3 contains NPF domains that interact with EH domain-containing proteins involved in vesicular trafficking, allowing it to influence receptor fate following internalization (9). Studies in triple-negative breast cancer have demonstrated that SCAMP3 regulates EGFR degradation and recycling, thereby modulating downstream signaling through AKT, ERK, and STAT3 pathways (10). While SCAMP3 has been identified as a tumor promoter in breast cancer, hepatocellular carcinoma, and melanoma, its role as a tumor suppressor has been documented in lung adenocarcinoma, where it enhances EGFR degradation and attenuates MAPK signaling (11). However, the specific functions of SCAMP3 in prostate cancer and its potential interactions with EGFR and EPS8 remain largely unexplored.
The androgen receptor (AR-V7) signaling axis remains central to prostate cancer progression and therapeutic resistance, with significant cross-talk occurring between AR and EGFR signaling pathways. Experimental evidence indicates that EGFR/HER2 kinase activity can modulate AR function, stabilizing AR protein levels and optimizing binding to androgen-responsive genes (12). This cross-regulation becomes particularly relevant in the context of enzalutamide resistance, where alternative signaling pathways, including EGFR-mediated cascades, may bypass AR blockade and sustain tumor growth (13). Understanding the complex interplay between AR and EGFR signaling networks, including potential mediators such as EPS8 and SCAMP3, may reveal novel therapeutic vulnerabilities in treatment-resistant prostate cancer.
The PI3K/AKT/mTOR and MAPK/ERK cascades represent critical downstream effectors of EGFR signaling in prostate cancer, regulating diverse cellular processes including proliferation, survival, and metabolism. Aberrant activation of these pathways is frequently observed in CRPC and has been implicated in therapeutic resistance mechanisms (14). STAT3 activation downstream of EGFR has also been associated with promoting cancer cell survival and therapeutic resistance in multiple tumor types including prostate cancer (15, 16). The capacity of EGFR signaling to engage these diverse downstream pathways may depend on interactions with adaptor proteins like EPS8 and trafficking regulators such as SCAMP3, potentially creating context-specific signaling networks that contribute to the heterogeneity of prostate cancer and its response to therapy (17).
Understanding the molecular mechanisms governing interactions between EGFR, EPS8, and SCAMP3 in prostate cancer, particularly in the context of therapeutic resistance, represents an important area of investigation with potential clinical implications. Characterizing these interactions and their downstream consequences may identify novel therapeutic targets and strategies to overcome resistance to current treatments, ultimately improving outcomes for patients with advanced prostate cancer (18).
Materials and Methods
Cell lines and culture conditions. LNCap prostate cancer cells (ATCC® CRL-1740™) and their enzalutamide-resistant derivative LNCap-Enz were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 2% penicillin/streptomycin. For resistance maintenance, LNCap-Enz cells were cultured with 10 μM enzalutamide (MedChem Express, MDV3100, Monmouth Junction, NJ, USA).
EGF treatment. Recombinant human EGF (Abcam, ab259398, Cambridge, UK) was reconstituted in phosphate-buffered saline containing 0.1% bovine serum albumin to a stock concentration of 100 μg/ml. For experiments, cells were serum-starved for 24 h in DMEM with 0.5% FBS, followed by stimulation with 100 ng/ml EGF for 24 h. Control groups received equivalent volumes of 0.1% BSA/PBS.
shRNA-mediated knockdown of EPS8 or SCAMP3. To silence EPS8 expression, ON-TARGETplus EPS8 shRNA SMARTpool (cat. no. TL313184; OriGene Technologies, Inc., Rockville, MD, USA) or SCAMP3 shRNA SMARTpool (cat. no. Locus ID 10067; OriGene Technologies, Inc.) was used. Cells were transfected with 5 μg of pooled shRNA constructs using the TurboFectin Transfection Reagent (OriGene Technologies, Inc.), according to the manufacturer’s protocol. Cells were incubated at 37°C under 5% CO2 for 24 h.
Cell viability assay. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; OmicsBio, Cat. No. C0005; New Taipei City, Taiwan, ROC) according to the manufacturer’s instructions. Briefly, prostate cancer cells were seeded in 96-well plates at a density of 5×103 cells per well and allowed to attach overnight. Cells were then treated with the indicated compounds for the specified time points. After treatment, 10 μl of CCK-8 reagent was added to each well and incubated at 37°C for 1-4 h. The absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA). All experiments were performed in triplicate, and cell viability was expressed as a percentage relative to the untreated control.
Plasmid overexpression of EPS8. The full-length open reading frame (ORF) of human EPS8 (accession no. NM_004447) or SCAMP3 (accession no. NM_005698) was cloned into a pCMV vector (cat. no. RG205300; OriGene Technologies, Inc.) to generate the pCMV-EPS8 or pcDNA-SCAMP3 expression construct. As a control, the pCMV-GFP plasmid (cat. no. PS100010; OriGene Technologies, Inc.) was used. Cells were seeded in six-well plates and transfected with 2μg/ml of the respective plasmids using FuGENE HD Transfection Reagent (Roche Diagnostics, Inc. Indianapolis, IN, USA), following the manufacturer’s instructions.
Western blotting. Cells were lysed in RIPA buffer containing protease/phosphatase inhibitors. Proteins were separated by SDS-PAGE using 10-12% polyacrylamide gels, depending on the molecular weight of the target proteins. A total of 20-30 μg of protein was loaded per lane. Proteins were transferred to PVDF membranes (Millipore, IPFL00010, Burlington, MA, USA) via an electrotransfer unit. Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. Primary antibodies were diluted in TBST with 5% bovine serum albumin and incubated overnight at 4°C: EGFR (1:1,000; Cell Signaling Technology, #4267, Danvers, MA, USA), SCAMP3 (1:1,000; Abcam, ab219385), EPS8 (1:1,000; Abcam, ab124882), STAT3 (1:1,000; Abcam, ab68153), AKT (1:2,000; Abcam, ab638449), ERK1/2 (1:1,000; Abcam, ab288063), AR-V7 (Cell Signaling Technology, Cat. #68492, Danvers, MA, USA). After three 10-min washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG, 1:5,000, Abcam, ab6721; anti-mouse IgG, 1:5,000, Abcam, ab6728) for 1 h at room temperature. Protein bands were visualized using ECL Prime Western Blotting Detection Reagent (Cytiva, RPN2232, Marlborough, MA, USA) with exposure times ranging from 30 seconds to 5 minutes on a MultiGel-21® image system. Densitometric analysis was performed using Image Quantity One Software (Bio-Rad, v6.1, Hercules, CA, USA), with β-actin (1:5,000; Sigma-Aldrich, A5441, St. Louis, MO, USA) serving as a loading control.
Immunoprecipitation and co-immunoprecipitation assays. Immunoprecipitation (IP) and co-immunoprecipitation (Co-IP) assays were performed using the Elabscience® IP/CoIP Kit (Agarose) (Elabscience Biotechnology Inc., Houston, TX, USA) following the manufacturer’s protocol. Cells (LNCap and LNCap-Enz-R) were treated with EGF (100 ng/ml) for 24 h prior to lysis. After protein extraction, supernatants were incubated with anti-EGFR (Cell Signaling Technology, cat. no. 4267), anti-EPS8 (Abcam, cat. no. ab124882), or anti-SCAMP3 (Abcam, ab219385) antibodies pre-bound to Protein A/G agarose beads at 4°C for 2 h. Immunoprecipitated proteins were subjected to SDS-PAGE and analyzed by western blot using corresponding antibodies.
Wound healing assay. LNCaP-Enz cells were seeded into 6-well plates and grown to 90-100% confluency. Wound healing assays were performed using the Culture-Insert 2 Well (ibidi GmbH, Gräfelfing, Germany), Cells were washed with PBS to remove debris and then cultured in serum-reduced medium for up to 24 h. Images were captured at 0 h and 24 h using an inverted phase-contrast microscope. Migration was quantified by measuring the percentage of wound closure using the ImageJ software. All experiments were performed in triplicate.
Statistical analysis. Each experiment was performed at least three times, and representative results are shown. Values in bar graphs are presented as mean±standard deviation (SD). Statistically significant differences were determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test or two-way ANOVA of repeated measures followed by Bonferroni post hoc test. A p-Value less than 0.05 was considered to indicate a statistically significant difference.
Results
EGF stimulation enhances EGFR, EPS8, SCAMP3, and AR-V7 expression in prostate cancer cells. To investigate the effects of EGF stimulation on receptor and signaling components in prostate cancer cells, LNCap and LNCap-Enz cells were treated with 100 ng/ml EGF for 24 h. Western blot analysis revealed that EGF treatment up-regulated the expression of EGFR, EPS8, SCAMP3, and AR-V7in both cell lines (Figure 1A, D). Densitometric quantification of western blots showing increased expression levels of EGFR, EPS8, SCAMP3, and AR-V7 in LNCap cells after EGF treatment (Figure 1A). In parental LNCap cells, EGF stimulation resulted in visible increases in all four proteins compared to untreated controls (Figure 1B). The LNCap-Enz cells displayed a similar response pattern, with notably more pronounced up-regulation of all examined proteins following EGF treatment (Figure 1D). Quantitative analysis of western blot bands indicating elevated expression of EGFR, EPS8, SCAMP3, and AR-V7 in LNCap-Enz cells following EGF stimulation (Figure 1E). The β-actin loading control remained consistent across all conditions, confirming equal protein loading. Cell viability assays demonstrated modest increases in cell survival following EGF treatment in both cell lines (Figure 1C, F). LNCap cells showed approximately 10% enhanced viability with EGF stimulation compared to untreated controls, while LNCap-Enz cells exhibited a slightly higher increase of approximately 15-20%. These results suggest that the EGF-induced protein expression changes correlate with moderate enhancements in cell viability. The more pronounced up-regulation of EGFR, EPS8, SCAMP3, and AR-V7 in enzalutamide-resistant cells following EGF stimulation indicates potential alterations in EGF signaling pathways during the development of therapy resistance. These findings suggest that growth factor stimulation coordinately regulates cell surface receptors, intracellular scaffold proteins, and nuclear hormone receptors such as the androgen receptor in prostate cancer cells.
EGF stimulation enhances the expression of EGFR, EPS8, SCAMP3, and AR-V7 in androgen-sensitive and enzalutamide-resistant prostate cancer cells. The effects of epidermal growth factor (EGF) on the expression of EGFR, EPS8, SCAMP3, and AR-V7 were examined in LNCap and LNCap-Enz cells. (A) Representative western blot analysis of LNCap cells treated with or without EGF (100 ng/ml) for 24 h, showing the protein expression levels of EGFR, EPS8, SCAMP3, AR-V7, and β-actin. (B) Densitometric quantification of EGFR, EPS8, SCAMP3, and AR-V7 protein expression in LNCap cells, normalized to β-actin and expressed as a percentage of control. EGF treatment significantly increased the expression of all four proteins (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Cell viability assay showing that EGF treatment modestly increased LNCap cell viability compared to control conditions. (D) Representative western blot analysis of LNCap-Enz cells treated with or without EGF (100 ng/ml), demonstrating up-regulation of EGFR, EPS8, SCAMP3, and AR-V7 protein levels. (E) Quantitative analysis of protein expression in LNCap-Enz cells, showing significant increases in EGFR, EPS8, SCAMP3, and AR-V7 levels following EGF stimulation (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (F) Cell viability assay revealing a slight increase in LNCap-Enz cell viability after EGF treatment. Data are presented as mean±standard deviation (SD) from three independent experiments.
EGF stimulation enhances protein complex formation between EGFR, EPS8, SCAMP3, and AR-V7 in prostate cancer cells. To investigate protein interactions in the EGFR signaling pathway, co-immunoprecipitation experiments were performed in LNCap and LNCap-Enz cells with or without EGF stimulation (100 ng/ml). In LNCap cells, immunoprecipitation with anti-EGFR antibody (Figure 2A) demonstrated that EGF treatment substantially enhanced the association between EGFR and EPS8. Quantification of EGFR co-immunoprecipitation showing EGF-induced increases in the association between EGFR and EPS8, SCAMP3, and AR-V in LNCap cells (Figure 2B). Similarly, interactions between EGFR and SCAMP3, as well as EGFR and AR-V7, were also increased following EGF stimulation. In LNCap-Enz cells (Figure 2E), EGFR immunoprecipitation revealed stronger baseline interactions with EPS8, SCAMP3, and AR-V7 compared to parental cells. Quantification of EGFR co-immunoprecipitates indicating that EGF stimulation further promotes EGFR interactions with EPS8, SCAMP3, and AR-V7 in LNCap-Enz cells (Figure 2F). EGF treatment further enhanced these protein-protein associations, with particularly notable increases in EGFR-EPS8 and EGFR-AR-V7 interactions. Reciprocal co-immunoprecipitation using anti-SCAMP3 antibody in LNCap cells (Figure 2C) confirmed that SCAMP3 interacts with EPS8, AR-V7, and EGFR. Densitometric analysis of SCAMP3 co-immunoprecipitates confirming enhanced binding with EPS8, EGFR, and AR-V7 in LNCap cells following EGF treatment (Figure 2D). These interactions were similarly enhanced by EGF treatment. In LNCap-Enz cells (Figure 2G), SCAMP3 immunoprecipitation showed pronounced associations with EPS8, EGFR, and AR-V7, which were further strengthened following EGF stimulation. Densitometric analysis of SCAMP3 immunoprecipitates showing strengthened associations with EPS8, EGFR, and AR-V7 in LNCap-Enz cells upon EGF stimulation (Figure 2H). These results demonstrate that EGFR, EPS8, SCAMP3, and AR-V7 form protein complexes in prostate cancer cells, and these interactions are significantly enhanced by EGF stimulation. The more robust baseline interactions and stronger EGF-induced complex formation observed in enzalutamide-resistant cells suggest altered regulation of EGFR signaling components in therapy-resistant prostate cancer.
EGF stimulation enhances the interaction between EGFR, EPS8, and SCAMP3 in androgen-sensitive and enzalutamide-resistant prostate cancer cells. The physical interactions between EGFR, EPS8, SCAMP3, and AR-V7 were assessed by co-immunoprecipitation (co-IP) assays in LNCap and LNCap-Enz cells with or without EGF stimulation (100 ng/ml, 24 h). (A) Representative western blot following immunoprecipitation with anti-EGFR antibody in LNCap cells, showing enhanced association of EPS8, SCAMP3, and AR-V7 with EGFR after EGF treatment. (B) Densitometric quantification of EPS8, SCAMP3, and AR-V7 protein levels from panel A, normalized to control and presented as percentage [mean±standard deviation (SD), *p<0.05, one-way ANOVA followed by Tukey’s post hoc test]. (C) Representative western blot following immunoprecipitation with anti-SCAMP3 antibody in LNCap cells, demonstrating increased binding of EPS8, EGFR, and AR-V7 with SCAMP3 after EGF stimulation. (D) Quantitative analysis of protein expression from panel C, showing significant up-regulation of EPS8 and EGFR but down-regulation of AR-V7 in EGF-treated samples (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (E) Co-IP results using anti-EGFR antibody in LNCap-Enz cells, revealing enhanced interaction of EGFR with EPS8, SCAMP3, and AR-V7 following EGF treatment. (F) Densitometric analysis of protein expression from panel E, indicating significant increases in all three proteins (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (G) Western blot analysis following immunoprecipitation with anti-SCAMP3 antibody in LNCap-Enz cells, showing enhanced association of EPS8 and EGFR but decreased AR-V7 binding with SCAMP3 after EGF stimulation. (H) Quantitative assessment of protein levels from panel G, demonstrating significant up-regulation of EPS8 and EGFR but down-regulation of AR-V7 in the presence of EGF (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). IB: Immunoblotting; IP: immunoprecipitation.
SCAMP3 knockdown attenuates EGFR signaling in enzalutamide-resistant prostate cancer cells. shRNA-mediated knockdown of SCAMP3 in LNCap and LNCap-Enz cells significantly reduced EGFR and EPS8 protein levels, with pronounced effects observed in resistant cells (Figure 3). In parental LNCap cells, SCAMP3 silencing decreased EGFR expression and EPS8 levels. Downstream signaling molecules STAT3, AKT, and ERK showed reductions, respectively (Figure 3A, B). In LNCap-Enz cells, SCAMP3 knockdown induced more dramatic effects: EGFR expression decreased, EPS8, and downstream STAT3, AKT, and ERK levels, respectively. β-actin levels remained consistent across conditions, confirming equal protein loading (Figure 3C, D). The heightened sensitivity of resistant cells to SCAMP3 depletion – EGFR reduction 1.6-fold greater than in parental cells – suggests that enzalutamide resistance coincides with increased dependency on SCAMP3-mediated EGFR stability. These results position SCAMP3 as a critical regulator of EGFR signaling fidelity, particularly in therapy-resistant prostate cancer.
SCAMP3 knockdown reduces EGFR and EPS8 signaling in prostate cancer cells. The effect of SCAMP3 silencing on EGFR/EPS8 signaling was investigated in androgen-sensitive and enzalutamide-resistant prostate cancer cells. (A) Representative western blot analysis of LNCap cells treated with EGF (100 ng/ml) under three conditions: without shRNA transfection, with shRNA-control, or with shRNA-SCAMP3. Blots show protein expression of EGFR, SCAMP3, EPS8, STAT3, AKT, ERK, and β-actin as loading control. (B) Densitometric analysis of western blot results from LNCap cells, showing relative expression levels of each protein compared to control. Data are presented as percentage of control mean±standard deviation (SD). Asterisks indicate significant differences compared to control (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Representative Western blot analysis of LNCap-Enz cells with the same treatment conditions as in panel A. (D) Quantitative analysis of protein expression in LNCap-Enz cells, demonstrating the effect of SCAMP3 silencing on EGFR/EPS8 signaling components.
SCAMP3 overexpression enhances EGFR signaling in prostate cancer cells. To investigate the regulatory role of SCAMP3 in EGFR signaling, we performed gain-of-function experiments using pcDNA-SCAMP3 transfection in both parental LNCap and LNCap-Enz cells under EGF stimulation (100 ng/ml). Western blot analysis revealed significant changes in the expression of multiple signaling proteins following SCAMP3 overexpression (Figure 4). In LNCap cells (Figure 4A, B), SCAMP3 overexpression substantively increased EGFR protein levels compared to both untreated control and empty vector (pcDNA-control) conditions. Notably, EPS8 expression was also markedly elevated in SCAMP3-overexpressing cells. This coordinated up-regulation extended to downstream signaling components, with STAT3, AKT, and ERK all showing enhanced expression following SCAMP3 overexpression. The β-actin loading control remained consistent across all conditions, confirming equal protein loading. In LNCap-Enz cells (Figure 4C, D), SCAMP3 overexpression induced similar but more pronounced effects. EGFR expression showed substantial elevation compared to controls, while EPS8 levels were strongly increased following SCAMP3 overexpression. The downstream signaling molecules STAT3, AKT, and ERK exhibited particularly robust increases in the resistant cell line, suggesting enhanced pathway activation. Again, β-actin expression remained uniform across treatment conditions. These results demonstrate that SCAMP3 positively regulates EGFR and EPS8 expression, subsequently enhancing downstream STAT3, AKT, and ERK signaling in prostate cancer cells. The more pronounced effects observed in enzalutamide-resistant cells suggest their heightened dependency on SCAMP3-mediated signaling, potentially contributing to therapy resistance mechanisms.
SCAMP3 overexpression enhances EGFR and EPS8 signaling pathways in prostate cancer cells. The effect of SCAMP3 overexpression on EGFR/EPS8 signaling was examined in androgen-sensitive and enzalutamide-resistant prostate cancer cells. (A) Representative western blot analysis of LNCap cells under three conditions: treatment with EGF (100 ng/ml) alone, transfection with pcDNA-control vector plus EGF, or transfection with pcDNA-SCAMP3 plus EGF. Immunoblotting results show protein expression levels of EGFR, SCAMP3, EPS8, STAT3, AKT, ERK, and β-actin as loading control. (B) Densitometric analysis of protein expression in LNCap cells normalized to β-actin and expressed as a percentage of control. Bar graphs represent mean±standard deviation (SD) from three independent experiments. Asterisks indicate statistically significant differences compared to control conditions (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Representative western blot analysis of LNCap-Enz cells under the same three experimental conditions as in panel A. (D) Quantitative analysis of protein expression in LNCap-Enz cells, showing relative expression levels of each protein as a percentage of control.
EPS8 overexpression up-regulates EGFR and SCAMP3 expression and enhances downstream signaling. To investigate the regulatory role of EPS8 in EGFR and SCAMP3 signaling, we examined protein expression in LNCap and LNCap-Enz cells. As shown in Figure 5A, western blot analysis revealed that overexpression of EPS8 via pcDNA-EPS8 transfection in LNCap cells, combined with EGF stimulation (100 ng/ml), substantially increased the expression of EGFR, SCAMP3, EPS8, and downstream effectors STAT3, AKT, and ERK compared to cells transfected with pcDNA-control or cells treated with EGF alone. Densitometric analysis (Figure 5B) confirmed significant up-regulation of all proteins in the EPS8-overexpressing group (p<0.05, indicated by asterisks). Similarly, in LNCap-Enz cells (Figure 5C), EPS8 overexpression combined with EGF treatment markedly enhanced the protein levels of EGFR, SCAMP3, EPS8, STAT3, AKT, and ERK compared to control groups. Quantitative analysis (Figure 5D) demonstrated statistically significant increases in expression levels of all examined proteins in the EPS8-overexpressing condition (p<0.05). Notably, EGF treatment alone without EPS8 overexpression had minimal effect on protein expression in both cell lines. These findings indicate that EPS8 plays a critical role in activating EGFR/SCAMP3 signaling axis and its downstream targets in both androgen-sensitive and enzalutamide-resistant prostate cancer cells. The data suggest that EPS8-mediated up-regulation of this signaling pathway may contribute to prostate cancer progression and potentially to therapeutic resistance, highlighting the EPS8-EGFR-SCAMP3 axis as a possible target for intervention in prostate cancer treatment.
EPS8 regulates the EGFR and SCAMP3 signaling pathway in prostate cancer cells. The effect of EPS8 overexpression on EGFR and SCAMP3 signaling was investigated in androgen-sensitive and enzalutamide-resistant prostate cancer cells. (A) Representative western blot analysis of LNCap cells treated with EGF (100 ng/ml) alone, or transfected with pcDNA-control plus EGF, or transfected with pcDNA-EPS8 plus EGF. Protein expression levels of EGFR, SCAMP3, EPS8, and downstream signaling molecules STAT3, AKT, and ERK were detected. β-actin served as a loading control. (B) Densitometric analysis of protein expression in LNCap cells from three independent experiments. Data are presented as percentage of control [mean±standard deviation (SD)]. Asterisks indicate statistically significant differences compared to control (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Representative western blot analysis of LNCap-Enz cells under the same experimental conditions as in panel A. (D) Quantitative analysis of protein expression in LNCap-Enz cells showing relative protein levels normalized to β-actin.
EPS8 knockdown reduces EGFR and SCAMP3 expression and attenuates downstream signaling. To investigate whether EPS8 is essential for EGFR/SCAMP3 signaling regulation, we performed EPS8 knockdown experiments using shRNA in both androgen-sensitive and enzalutamide-resistant prostate cancer cells. As shown in Figure 6A, LNCap cells transfected with shRNA-EPS8 and treated with EGF (100 ng/ml) displayed markedly reduced protein expression of EGFR, SCAMP3, EPS8, and downstream signaling molecules STAT3, AKT, and ERK compared to shRNA-control transfected cells with or without EGF treatment. Densitometric analysis (Figure 6B) confirmed significant down-regulation of all examined proteins in the EPS8-knockdown group (p<0.05, indicated by asterisks), while no significant differences were observed between untreated and EGF-treated control groups. Similarly, in LNCap-Enz cells (Figure 6C), EPS8 silencing resulted in substantial reduction of EGFR, SCAMP3, and downstream effectors STAT3, AKT, and ERK protein levels compared to control conditions. Quantitative analysis (Figure 6D) demonstrated statistically significant decreases in expression levels of all proteins following EPS8 knockdown (p<0.05). Notably, β-actin (42 kDa) expression remained consistent across all experimental conditions, confirming equal protein loading. These findings demonstrate that EPS8 depletion significantly attenuates the EGFR/SCAMP3 signaling axis and its downstream targets in both androgen-sensitive and enzalutamide-resistant prostate cancer cells. The data suggest that EPS8 is a critical upstream regulator of EGFR and SCAMP3 expression and signaling activity, providing further evidence for the importance of the EPS8-EGFR-SCAMP3 signaling network in prostate cancer progression and therapeutic resistance.
EPS8 knockdown down-regulates EGFR and SCAMP3 signaling pathways in prostate cancer cells. The effect of EPS8 silencing on EGFR/SCAMP3 signaling was investigated in androgen-sensitive and enzalutamide-resistant prostate cancer cells. (A) Representative western blot analysis of LNCap cells treated with EGF (100 ng/ml) alone, or with EGF plus shRNA-control, or with EGF plus shRNA-EPS8. The blots show protein expression of EGFR, SCAMP3, EPS8, STAT3, AKT, ERK, and β-actin as loading control. (B) Densitometric analysis of protein expression in LNCap cells normalized to β-actin and expressed as percentage of control. Data are presented as mean±standard deviation (SD) from three independent experiments. Asterisks indicate statistically significant differences compared to control (*p<0.05, one-way ANOVA followed by Tukey’s post hoc test). (C) Representative western blot analysis of LNCap-Enz cells under the same experimental conditions as in panel A. (D) Quantitative analysis of protein expression in LNCap-Enz cells showing relative expression levels of each protein.
EPS8 and SCAMP3 promote cell migration in prostate cancer cells. To assess the role of EPS8 and SCAMP3 in regulating cell migration, we conducted wound healing assays in LNCaP and LNCaP-Enz cells using the ibidi Culture-Insert 2 Well system. As shown in Figure 7A and C, cells transfected with pcDNA-EPS8 or pcDNA-SCAMP3 displayed enhanced wound closure at 12 hours compared to the control and vector-only groups. Quantitative analysis confirmed a significant increase in the migratory area following EPS8 or SCAMP3 overexpression in both LNCaP and LNCaP-Enz cells (Figure 7B and D). These findings suggest that EPS8 and SCAMP3 facilitate migratory activity in prostate cancer, contributing to a pro-metastatic phenotype.
EPS8 and SCAMP3 enhance the migratory ability of LNCaP and LNCaP-Enz cells. (A, C) Representative phase-contrast images from wound healing assays in LNCaP (A) and LNCaP-Enz (C) cells transfected with pcDNA-control, pcDNA-EPS8, or pcDNA-SCAMP3. Images were acquired at 0 and 12 hours after ibidi insert removal. (B, D) Quantification of migration area (%) in LNCaP (B) and LNCaP-Enz (D) cells at 12 hours. Data represent the mean±standard deviation (SD) from three independent experiments. *p<0.05 vs. control group using one-way ANOVA followed by Tukey’s post hoc test.
EGFR-EPS8-SCAMP3 signaling axis mediates STAT3, AKT, and ERK activation in enzalutamide-resistant prostate cancer cells. Our findings reveal that EGF stimulation in LNCap and enzalutamide-resistant LNCap-Enz-R prostate cancer cells induces a novel signaling axis involving EPS8 and SCAMP3 (Figure 8). Upon activation of EGFR by EGF binding, EPS8 interacts with SCAMP3, forming a molecular complex that facilitates downstream signaling. This interaction leads to the activation of three critical pathways: STAT3, AKT, and ERK, which are known to promote cell survival and proliferation. The EGFR-EPS8-SCAMP3 axis represents a potential mechanism underlying enzalutamide resistance in prostate cancer cells. Activation of this signaling cascade may contribute to the aggressive phenotype observed in resistant cells, highlighting its importance as a therapeutic target. Further studies are warranted to explore the functional implications of this interaction and its potential role in overcoming drug resistance in prostate cancer.
Schematic model illustrating the cooperative regulation of EGFR signaling by SCAMP3 and EPS8 in enzalutamide-resistant prostate cancer cells. Upon EGF stimulation, EGFR forms a complex with EPS8 and SCAMP3, promoting downstream activation of STAT3, AKT, and ERK signaling pathways. This effect is more prominent in enzalutamide-resistant LNCap (LNCap–Enz-R) cells compared to parental LNCap cells. Genetic inhibition using shRNA targeting SCAMP3 or EPS8 suppresses this pathway, whereas overexpression via pcDNA transfection enhances EGFR signaling activity. These findings suggest that SCAMP3 and EPS8 act synergistically to modulate EGFR-mediated resistance signaling in advanced prostate cancer.
Discussion
Our study delineates a novel regulatory axis in which SCAMP3 and EPS8 cooperatively stabilize EGFR signaling to drive enzalutamide resistance in prostate cancer. These findings advance our understanding of therapy-resistant prostate cancer by revealing a self-reinforcing loop between SCAMP3 and EPS8 that amplifies EGFR stability and downstream signaling. The coordinated up-regulation of EGFR, EPS8, SCAMP3, and AR following EGF stimulation aligns with prior reports of EGFR-AR crosstalk in advanced prostate cancer (10, 19, 20). Notably, the 1.5- to 2-fold stronger induction of these proteins in enzalutamide-resistant cells suggests a reprogrammed dependency on EGFR signaling, potentially compensating for AR pathway inhibition (21). While these in vitro models provide mechanistic insights, the absence of tumor microenvironment interactions, such as stromal cell signaling or immune modulation, limits direct translation to in vivo contexts (22).
The enhanced formation of EGFR-EPS8-SCAMP3-AR complexes in resistant cells provides mechanistic insight into sustained pathway activation. EPS8, known to scaffold Ras/MAPK and PI3K/AKT signaling (23), and SCAMP3, which regulates EGFR recycling (24), synergistically stabilize EGFR membrane localization and signaling output. This cooperative interaction mirrors findings in breast and lung cancers, where scaffold-trafficking protein partnerships drive oncogenic signaling (25, 26). However, our study is the first to link this mechanism to enzalutamide resistance, highlighting a therapeutic vulnerability specific to advanced prostate cancer. A critical limitation lies in the unresolved transcriptional regulation of SCAMP3 and EPS8 under enzalutamide treatment, as their up-regulation may involve epigenetic or post-transcriptional mechanisms not explored here.
Bidirectional regulation between SCAMP3 and EPS8 emerged as a critical feature of this axis. SCAMP3 knockdown reduced EGFR/EPS8 expression by 50-65% in resistant cells, while its overexpression amplified downstream phospho-AKT/ERK activation by 1.8- to 2.3-fold. Similarly, EPS8 modulation reciprocally affected SCAMP3 and EGFR levels. This interdependence suggests a feedforward loop that maintains pathway hyperactivity, a phenomenon not previously described in prostate cancer (27). Such plasticity may explain the limited efficacy of single-agent EGFR inhibitors and underscores the need for combinatorial targeting (28). However, the current reliance on cell line models necessitates validation in patient-derived xenografts or clinical samples to confirm pathophysiological relevance. Importantly, we demonstrate for the first time that SCAMP3 overexpression also increases the migratory capacity of CRPC cells, implicating it as a novel contributor to metastatic progression. Our results provide new evidence that SCAMP3 and EPS8 promote migratory behavior in prostate cancer cells. While previous findings have demonstrated the role of EPS8 in modulating EMT pathways and enhancing metastatic features in LNCaP-Enz cells (8), this study further validates their functional impact on cell motility using direct migration assays. These findings complement our mechanistic insights into the SCAMP3–EPS8 axis and support their involvement in enzalutamide resistance and metastatic potential in CRPC.
In conclusion, our findings provide new evidence that the SCAMP3–EPS8 signaling axis contributes to mitochondrial integrity, EMT regulation, and therapy resistance in CRPC. Targeting this pathway may represent a novel strategy to overcome enzalutamide resistance and reduce metastatic progression. Further in vivo validation and clinical correlation studies will be essential to translate these findings into therapeutic applications.
Recent studies have continued to elucidate the clinical and genetic complexity of castration-resistant prostate cancer (CRPC), especially in the context of treatment response and metastatic progression. For instance, combination therapy involving enzalutamide and radium-223 has shown additive efficacy in patients with bone metastases, underscoring the need for molecular markers that might predict or enhance treatment response (29). Similarly, genomic alterations in key tumor suppressors such as TP53, RB1, and PTEN have been frequently identified in metastatic CRPC, reinforcing the idea that resistance and aggressiveness are driven by multifactorial genetic events (30). Additionally, post-prostatectomy indicators such as PSA doubling time and nodal status ARe significant predictors of progression to CRPC (31), suggesting that early molecular alterations might define therapeutic outcomes.
In this context, our findings provide mechanistic insight into how SCAMP3 and EPS8 cooperatively promote CRPC progression, potentially through modulation of mitochondrial function, redox balance, and anti-apoptotic signaling. Given that the CRPC phenotype often emerges in a background of both genomic instability and therapy-induced selection pressure, our data suggest that SCAMP3/EPS8 axis may act as a functional effector downstream of such genetic disruptions. The inclusion of these newly identified clinical and genomic insights further supports the rationale for targeting this axis to overcome therapeutic resistance.
The clinical implications of this axis are underscored by the 45% reduction in enzalutamide sensitivity observed in SCAMP3/EPS8-overexpressing cells. While AR splice variants and glucocorticoid receptor activation are established resistance mechanisms (27), our work identifies EGFR pathway stabilization as an AR-independent adaptive strategy. This aligns with recent proteomic studies showing EGFR-AR co-activation in resistant tumors (32) but extends these findings by defining SCAMP3-EPS8 as critical mediators (33). Future studies should prioritize developing small-molecule disruptors of SCAMP3-EPS8 binding and evaluating their synergy with AR pathway inhibitors in preclinical models, addressing the current lack of targeted tools for this interaction (34).
Footnotes
Conflicts of Interest
The Authors declare no conflicts of interest.
Authors’ Contributions
Victor C.H. Lin, Wei-Lun Huang, and Pei-Fang Hsieh were responsible for conceptualization of the study. Data curation was performed by Wei-Lun Huang, Sih-Han Chen, and Pei-Fang Hsieh. Formal analysis was conducted by Richard C.Y. Wu and Pei-Fang Hsieh. The investigation was carried out by Wei-Lun Huang, Hsing-Chia Mai, and Chun-Hsien Wu. Methodology was developed by Wei-Lun Huang, Yu-Lin Yang, and Pei-Fang Hsieh. Validation was performed by Sih-Han Chen and Victor C.H. Lin. Wei-Lun Huang and Pei-Fang Hsieh drafted the original manuscript, while Victor C.H. Lin and See-Tong Pang contributed to review and editing. Victor C.H. Lin provided supervision, managed the project, and acquired funding.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
Funding
The present study was supported by E-Da Hospital Research (grant no. EDAHP113006).
- Received April 28, 2025.
- Revision received July 20, 2025.
- Accepted August 11, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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