Pan-cancer RNA sequencing data from 51 datasets were obtained from The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov) and Genotype-Tissue Expression (https://www.gtexportal.org/home/). The data were processed using R software (v.4.2.1) (https://cran.r-project.org/bin/windows/base/old/4.2.1/) and visualized with the ‘ggplot2’ package (v.3.3.6; http://cran.r-project.org/src/contrib/Archive/ggplot2/). Statistical analyses were performed using the Wilcoxon rank-sum test. Additionally, the expression profile of PLEKHA4 in melanoma was examined using the Gene Expression Profiling Interactive Analysis (GEPIA) platform (http://gepia.cancer-pku.cn). Furthermore, the gene expression profiles GSE3189 (20) and GSE8401 (21) were obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), both of which are based on the GPL96 platform. The GSE3189 data were collected from 7 normal skin, 18 nevi and 45 melanoma samples, whereas the GSE8401 data were collected from 31 primary melanoma and 52 melanoma metastasis tissues from patients. Data from the GEO database were downloaded using ‘GEOquery’ (v.2.64.2; http://bioconductor.org/packages/release/bioc/html/GEOquery.html) and were normalized with the ‘normalizeBetweenArrays’ function from the ‘limma’ package (v.3.52.2; http://www.bioconductor.org/packages/release/bioc/html/limma.html). All gene expression data were calibrated, standardized and log₂-transformed. The results were visualized using ‘ggplot2’ (v.3.3.6) and ‘ComplexHeatmap’ (v.2.13.1; http://github.com/jokergoo/complexheatmap). Principal component analysis was performed using R package ggplot2 (https://cran.r-project.org/web/packages/ggplot2/index.html). The expression of PLEKHA4 across various pathological and histological grades was analyzed using R and was visualized with ‘ggplot2’. RNAseq data from TCGA-Skin Cutaneous Melanoma (SKCM) project (portal.gdc.cancer.gov/projects/TCGA-SKCM) was obtained using the STAARpipeline (https://github.com/xihaoli/STAARpipeline) from TCGA database and extracted in transcripts per million (TPM) format. Kyoto Encyclopedia of Genes and Genomes (KEGG) combined with Gene Ontology (GO) analyses were performed using ‘clusterProfiler’ (v.4.4.4; http://bioconductor.org/packages/release/bioc/html/clusterProfiler.html) and GOplot (v.1.0.2; http://cran.r-project.org/web/packages/GOplot/index.html). Correlation analysis was conducted by downloading TCGA-SKCM dataset, as aforementioned. Results were statistically analyzed using Spearman's correlation coefficient. The interaction of PLEKHA4 and other proteins was analyzed using GeneMANIA (http://genemania.org). The overall survival analysis was conducted using the GEPIA platform, incorporating both the Kaplan-Meier method and the log-rank test.

Human melanoma cells A375, A2058 and SK-MEL-3 cells were obtained from the National Collection of Authenticated Cell Cultures. A375, A2058 and SK-MEL-3 cells were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 1% penicillin-streptomycin (both from Biological Industries; Sartorius AG) at 37°C in a 5% CO₂ incubator. For drug treatment, lithium chloride (LiCl; Selleck Chemicals) was added at 5 mM to activate the Wnt/β-catenin pathway, with cells incubated under the same conditions for 12 h at 37°C in a 5% CO₂ incubator. MG132 (cat. no. S2619; Selleck Chemicals) was added at concentrations of 0, 5, 10 and 20 µM and incubated at 37°C in a 5% CO₂ incubator for 24 h to conduct the ubiquitination assay.

A375 and A2058 cells were transduced with PLEKHA4-targeting short hairpin RNA (shRNA) lentiviruses. The shRNA sequences were as follows: shPLEKHA4 [K7453 LV3(H1/GFP&Puro)-PLEKHA4-Homo-2851; target sequence: 5′-GCGAGTCACTCTGCTACAATC-3′], forward, 5′-GATCCGCGAGTCACTCTGCTACAATCTTCAAGAGAGATTGTAGCAGAGTGACTCGCTTTTTTG-3′ and reverse, 5′-AATTCAAAAAAGCGAGTCACTCTGCTACAATCTCTCTTGAAGATTGTAGCAGAGTGACTCGCG-3′; and a negative control (NC) (LV3-shNC; target sequence: 5′-GTTCTCCGAACGTGTCACGT-3′), forward, 5′-GATCCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAACTTTTTTG-3′ and reverse 5′-AATTCAAAAAAGTTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAACG-3′. These lentiviruses were provided by Shanghai GenePharma Co., Ltd.

A 3rd-generation system facilitated lentiviral transduction. Briefly, 293T cells (National Collection of Authenticated Cell Cultures) were used for viral production, utilizing recombinant shuttle and packaging plasmids (pGag/Pol, pRev and pVSV-G) (Addgene, Inc.). Plasmid concentration and purity were confirmed by ultraviolet absorption, ensuring an A260/A280 ratio between 1.8 and 2.0. The recombinant and packaging plasmids were mixed in an 8:4:4:4 µg ratio (shPLEKHA4 or NC:pGag/Pol:pRev:pVSV-G), added to 293T cells and transfected for 4–6 h at 37°C with 5% CO using EndoFectin^™ MAX (GeneCopoeia, Inc.) The medium was then refreshed and incubation continued for 72 h. The 293T cell cultures were maintained in a 37°C incubator with 5% CO₂ to support optimal growth. After 72 h, the supernatant was collected, filtered through a 0.45-µm filter to remove debris and concentrated by ultracentrifugation at 70,000 × g for 2 h at 20°C. Pellets were resuspended in 100 µl 1X HBSS (Gibco; Thermo Fisher Scientific, Inc.), and another 100 µl was added, yielding a final volume of 200 µl. The solution was vortexed at low speed for 15–30 min, briefly spun, and aliquoted into 20-µl portions for storage at −20°C (up to 1 month) or −80°C (long-term), avoiding more than three freeze-thaw cycles.

Lentiviral infection proceeded immediately post-preparation. The virus titers were: shPLEKHA4 (LV3-PLEKHA4-Homo-2851), 7×10⁸ TU/ml; and shNC (LV3-shNC), 9×10⁸ TU/ml. Multiplicity of infection values for infection were set at 5 and 10 for shPLEKHA4 and shNC in A375 cells, and at 10 and 15 in A2058 cells, respectively. The lentiviruses were added to the cells with 2 µg/ml polybrene to boost infection efficiency and incubation was continued for 24 h. Post-transduction, cells underwent selection with 1 µg/ml puromycin for 7 days, with the same concentration maintained thereafter. Infection efficiency was validated through western blotting, performed immediately after the selection period along with other assays.

To induce PLEKHA4 overexpression in SK-MEL-3 cells, pIRES2-EGFP-PLEKHA4 and the control plasmid (pIRES2-EGFP-empty) were purchased from Shanghai GenePharma Co., Ltd. Transfection was performed using EndoFectin MAX. For transfection in a 6-well plate, 2.5 µg DNA and 5–12.5 µl EndoFectin Max were diluted separately in 125 µl medium. After allowing both solutions to sit for 5 min, they were gently mixed and incubated for 5–20 min to form the DNA-EndoFectin complex. The complex was then added to SK-MEL-3 cells cultured in a 6-well plate, once the cells had reached 80% confluence, and the cells were transfected for 24 h at 37°C. Protein expression was detected 24–48 h post-transfection.

A375 and A2058 cells were seeded in 96-well plates at a density of 3×10³ cells/well and were cultured for 0, 24, 48, 72 and 96 h. Following this, cells were treated with 10 µl Cell Counting Kit (CCK)-8 solution (Shanghai Yeasen Biotechnology Co., Ltd.) and incubated for 1 h at 37°C. Absorbance was then recorded at 450 nm. Each experiment was repeated at least three times.

Transduced A375 and A2058 cells were plated in 6-well plates at a density of 5×10³ cells/well and grown to 90% confluence. Subsequently, the cells were incubated overnight in serum-free medium. The cell monolayers were then mechanically wounded using a 10-µl pipette tip. Images of the wounds were captured at 0 and 24 h using a Nikon Eclipse Ti-S/L100 inverted phase contrast fluorescence microscope (Nikon Corporation) with a 10× objective. The wound was measured by ImageJ bundled with 64-bit Java 8 [version (ij154-win-java8); National Institutes of Health] and calculated with SPSS 29 (IBM Corp.).

Transduced A375 and A2058 cells were plated in 6-well plates at a density of 5×10³ cells/well and were cultured for 7 days. After incubation, the cells were rinsed twice with PBS at room temperature, fixed with 4% paraformaldehyde for 15 min and stained with Giemsa (both from Beijing Solarbio Science & Technology Co., Ltd.) for 20 min at room temperature. The cells were then washed twice with PBS. Colonies, defined as groups of ≥50 cells, were counted using ImageJ software [bundled with 64-bit Java 8 (ij154-win-java8), National Institutes of Health].

Cell samples were transferred to a 1.5-ml centrifuge tube and lysed with DB lysis buffer (8 M urea, 100 mM TEAB, pH 8.5). The solution was then alkylated with sufficient iodoacetamide. Protein quantity was assessed using a Bradford protein quantitation kit. The proteins underwent enzymatic hydrolysis and salt removal. Subsequently, the samples were processed with tandem mass tag (TMT) labeling reagent (Thermo Fisher Scientific Inc.). The separated peptides were analyzed by Q Exactive^™ HF-X mass spectrometer, with an ion source of Nanospray Flex^™ (ESI), spray voltage of 2.3 kV and ion transport capillary temperature of 320°C. The full scan ranged from 350 to 1,500 m/z with a resolution of 60,000 (at m/z 200), the automatic gain control (AGC) target value was 3×10⁶ and the maximum ion injection time was 20 msec. The 40 most abundant precursors in the full scan were selected and fragmented by higher energy collisional dissociation and analyzed in tandem mass spectrometry, where resolution was 45,000 (at m/z 200) for 10 plex, the AGC target value was 5×10⁴, the maximum ion injection time was 86 msec, the normalized collision energy was set at 32%, the intensity threshold was 1.2×10⁵, and the dynamic exclusion parameter was 20 sec. The spectra from each run were analyzed individually against the UniProt database (https://www.uniprot.org/) using Proteome Discoverer (Thermo Fisher Scientific, Inc.). Liquid chromatography analysis was performed with the EASY-nLC^™ 1200 UHPLC system (Thermo Fisher Scientific, Inc.). Identified peptide-spectrum matches and proteins were retained with a false discovery rate (FDR) of no more than 1.0%. The protein quantitation results were then statistically analyzed using unpaired Student's t-test. Proteins that showed significant differences in quantity between the experimental and control groups (P<0.05 and |log2FC|>0.5) were defined as differentially expressed proteins. Finally, KEGG analysis was utilized to analyze the protein families and pathways.

Cells were trypsinized without EDTA, centrifuged at 300 × g for 5 min at 4°C, washed twice with chilled PBS, and resuspended in 100 µl 1X Binding Buffer (Shanghai Yeasen Biotechnology Co., Ltd.). Annexin V-FITC and PI Staining Solution (Shanghai Yeasen Biotechnology Co., Ltd.) were added, and the cells were incubated in the dark at room temperature for 10–15 min. Subsequently, 400 µl 1X Binding Buffer (Shanghai Yeasen Biotechnology Co., Ltd.) was added on ice. Samples were analyzed on a CytoFLEX SRT flow cytometer (Beckman Coulter, Inc.) within 1 h, with FlowJo V8 (FlowJo; BD Biosciences) used for data analysis.

The ubiquitination assay was performed using the Ubiquitylation Assay Kit (cat. no. ab139467; Abcam). Protein lysates from A375shNC, A375shPLEKHA4, A375shPLEKHA4, A2058shNC, A2058shPLEKHA4 and A2058shPLEKHA4 cells following treatment with 0, 5, 10 and 20 µM MG132 were subjected to the ubiquitination assay. To prepare the reaction, the E1 stock solution was first diluted 1:20 in 1X ubiquitylation buffer and 5 µl was added to a 50-µl reaction solution (including E1 Enzyme, E2 Enzyme, Ubiquitin, 10X Ubiquitylation Buffer, ATP Solution, E3 Enzyme). Next, the E2 stock solution was diluted 1:40 in 1X ubiquitylation buffer and 5 µl was added to the reaction solution, and ubiquitin was diluted 1:20 in 1X ubiquitylation buffer and 5 µl was added to the reaction solution. Subsequently, 3.5 µl 10X ubiquitylation buffer and 5 µl 2 mM ATP solution were added to the reaction solution, with the amount depending on the substrate concentration, and the volume was made up to 48.5 µl with water. Finally, 1.5 µl 0.05 mg/ml E3 was added to generate a final volume of 50-µl reaction mixture), which was incubated at room temperature for 1 h. After incubation, western blot analysis was performed.

Briefly, genetically transduced or transfected A375, A2058 and SK-MEL-3 cells were seeded in 6-well plates at a density of 1×10⁶ cells/well and were cultured in DMEM supplemented with 10% FBS until they reached ~90% confluence. Protein lysates were extracted using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.), and nuclear proteins were isolated with the Nuclear and Cytoplasmic Extraction Kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Protein concentration was assessed using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Proteins (30 µg/lane) were separated by SDS-PAGE on 8–10% gels, transferred to PVDF membranes and blocked with 5% non-fat dry milk (MilliporeSigma) for 1 h at room temperature, before overnight incubation with primary antibodies at 4°C. The primary antibodies used included PLEKHA4 (cat. no. NBP1-56679; Novus Biologicals; Bio-Techne), β-actin (cat. no. GB12001-100; Wuhan Servicebio Technology), phosphorylated (p)-p38 MAPK (cat. no. AF4001; Affinity Biosciences), p38 MAPK (cat. no. AF6456; Affinity Biosciences), p-JNK1/2/3 (cat. no. AF3318; Affinity Biosciences), JNK1/2/3 (cat. no. AF6318; Affinity Biosciences), p-cJUN (cat. no. AF3095; Affinity Biosciences), c-Jun (cat. no. AF6090; Affinity Biosciences), p-MEK1/2 (cat. no. AF8035; Affinity Biosciences), MEK (cat. no. AF6385; Affinity Biosciences), p-ERK (cat. no. AF1015; Affinity Biosciences), ERK1/2 (cat. no. AF0155; Affinity Biosciences), COX2 (cat. no. AF7003; Affinity Biosciences), p-NF-κB p65 (cat. no. AF2006; Affinity Biosciences), NF-kB p65 (cat. no. AF5006; Affinity Biosciences), cleaved-caspase-3 (cat. no. AF2006; Affinity Biosciences), caspase-3 (cat. no. AF6311; Affinity Biosciences) cMyc (cat. no. 10828-1-AP; Proteintech Group), ubiquitin polyclonal antibody (cat. no. 10201-2-AP; Proteintech Group), β-catenin (cat. no. AF6266; Affinity Biosciences), p-GSK3β (cat. no. sc-373800; Santa Cruz Biotechnology), GSK3β (cat. no. sc-377213; Santa Cruz Biotechnology), cyclin D1 (cat. no. AF0931; Affinity Biosciences) and Lamin B1 (cat. no. sc-374015; Santa Cruz Biotechnology), each diluted 1:1,000 in Primary Antibody Dilution Buffer (Beyotime Institute of Biotechnology). Membranes were then incubated with HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (cat. nos. RGAR001 and RGAM001; Proteintech Group, Inc.) diluted 1:5,000 in Tris-buffered saline-0.1% Tween 20 for 2 h at room temperature. Blots were visualized with ECL Western Blotting Substrate (Beijing Solarbio Science & Technology Co., Ltd.) using the Shenhua Science Technology Co., Ltd. system. Protein grayscale values were semi-quantified with ImageJ (version: ij154-win-java8). Each experiment was performed at least three times.

Upon reaching a confluence of ~90%, cells were lysed with RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) and subjected to co-immunoprecipitation to investigate protein interactions. Protein concentrations were determined using the Bradford assay. A cMyc (cat. no. 10828-1-AP; Proteintech Group, Inc.) antibody was diluted to a final concentration of 10 µg/ml with whole cell lysates free of debris (to clear debris, the lysates were centrifuged at 20,000 × g for 20 min at 4°C, and the sediments were discarded). Subsequently, 400 µl whole lysate-antibody complex was added to 25 µl Protein A/G Magnetic Beads (cat. no. HY-K0202; MedChemExpress). The mixture was centrifuged at 120 × g for 30 sec at 4°C and the supernatant was discarded. The beads were washed 3–4 times with 1 ml RIPA lysis buffer, and underwent further centrifugation at 500–1,000 rpm for 30 sec at 4°C before the supernatant was discarded. Two elutions of the pellet were performed using 40 µl 0.10 M glycine and 0.05 M Tris-HCl (pH 1.5–2.5) elution buffer with 500 mM NaCl. The eluates were then combined and neutralized with 10X PBS buffer (pH 6.8–7.2) to a final concentration of 1X. Finally, the collected eluates were analyzed by western blotting to identify specific protein interactions.

Male nude mice (average weight, 14 g; age, 4 weeks) were obtained from the Experimental Animal Center of Yanbian University (Yanji, China) and were randomly divided into two groups (n=5/group): The shNC and shPLEKHA4 groups. The mice were kept in a pathogen-free environment at 25°C with 30% humidity, under a 12-h light/dark cycle, and were provided unrestricted access to cobalt-60-sterilized feed and autoclaved water. Animal health and behavior were monitored daily. Each mouse was injected subcutaneously with 200 µl of a solution containing 5×10⁶ A375 cells transduced with either shNC or shPLEKHA4 in the right flank. Before the subcutaneous injection, mice were given 50 mg/kg sodium pentobarbital via the intraperitoneal route as an anesthetic to minimize suffering. Tumor growth was tracked every 2 days. The humane endpoints were as follows: Euthanasia was performed if tumor weight surpassed 10% of body weight or tumor diameter exceeded 20 mm; however, none of these criteria were met before day 15 when all mice were sacrificed. The two groups of total 10 mice (n=5/group) were euthanized, and no mice were euthanized or found dead prior to day 15. Mice were euthanized by cervical dislocation after anesthesia with an intravenous dose of 70 mg/kg sodium pentobarbital, with death confirmed by cessation of respiration and heartbeat for >5 min.

Subcutaneous tumors were fixed in 10% formalin at room temperature for 24 h, dehydrated with graded ethanol and embedded in paraffin. Tumor samples were then sectioned into 4-µm slices, baked at 56°C overnight, and stained with an H&E Stain kit (Beijing Solarbio Science & Technology Co., Ltd.), following the manufacturer's instructions with all procedures conducted at room temperature (25°C). Briefly paraffin was removed by immersing the slides in xylene for two to three changes, each lasting 5 min and rehydration was achieved through a graded ethanol series. The slides were then rinsed in distilled water for 1–2 min. Hematoxylin staining was conducted at room temperature for 5–10 min, followed by rinsing in running tap water for 5 min. Excess hematoxylin was removed using 1–2 dips in acid alcohol, and the slides were immediately rinsed again under running tap water. A bluing reagent (0.1% ammonia solution) was then applied for 1–2 min and the sections were immersed in eosin staining solution for 1–3 min, followed by a final rinse in running tap water for 5 min. Images were captured using a Nikon Eclipse Ti-S/L100 inverted phase contrast fluorescent microscope with a 10× objective.

Statistical analysis was performed using an unpaired Student's t-test for two-group comparisons, and one-way ANOVA with Tukey's post hoc test for multiple group comparisons. SPSS 26.0 (IBM Corp.) was used for data analysis, with results presented as the mean ± SD. Each experiment was repeated at least three times. P<0.05 was considered to indicate a statistically significant difference.

The expression of PLEKHA4 in different human cancer tissues was assessed using RNA sequencing data. PLEKHA4 was widely expressed in nearly all tissue types, with notably higher expression in melanoma tissues than in normal skin tissues from healthy individuals (Fig. 1A and B). This expression was also confirmed by GEPIA in which tumor tissues displayed a distinct gene expression pattern compared to that in normal skin tissues from healthy individuals (Fig. 1C). Subsequently, gene expression profiles from the GSE3189 and GSE8401 datasets were retrieved from the GEO database. As shown in Fig. 1D, tumor tissues displayed a distinct gene expression pattern compared to than in normal skin tissues from healthy individuals. The heatmap and volcano plots highlighted distinct genes in melanoma tissues (Fig. 1E and F), with the heatmap specifically indicating the upregulation of PLEKHA4 (Fig. 1E). Furthermore, as depicted in Fig. 2A, metastatic tumor tissues exhibited a pronounced gene expression profile relative to primary tumor tissues. The volcano plot and heatmap in Fig. 2B and C revealed distinct genes in melanoma metastatic tissues; however, PLEKHA4 was not among them, suggesting that PLEKHA4 may not be involved in melanoma metastasis. Additionally, PLEKHA4 expression across different pathological stages showed no significant variation (Fig. 2D-G), and its expression was not directly associated with overall survival outcomes (Fig. 2H), further suggesting that PLEKHA4 may not be involved in metastasis. To further explore the role of PLEKHA4 in melanoma, KEGG combined with GO analyses were performed. The results highlighted an upregulation of ‘beta-catenin binding’, suggesting activation of the Wnt signaling pathway in melanoma tissues (Fig. 1G). Additionally, the heatmap across different pathological stages (Fig. 2D) revealed high expression levels of PLEKHA4 along with key MAPK signaling genes (MAPK1, MAPK3 and MAPK12), and Wnt signaling genes (CTNNB1 and GSK3B).

PLEKHA4 knockdown experiments were conducted in A375 and A2058 melanoma cell lines; notably, a significant reduction in PLEKHA4 protein expression was detected in the A375 and A2058 cells lines following shRNA transduction (Fig. 3A). Subsequent assessments through CCK-8, colony formation and wound healing assays were conducted to evaluate cell proliferation and migration. Knockdown of PLEKHA4 resulted in the inhibition of cell proliferation, colony formation and migration (Fig. 3B-D). Subsequently, subcutaneous tumor growth models were established to assess the effect of PLEKHA4 on tumor development. Notably, PLEKHA4 knockdown significantly reduced tumor growth (Fig. 4A-C). Tumors in the A375shPLEKHA4 group grew more slowly than those in the A375shNC group (Fig. 4B), and tumor weight in the A375shPLEKHA4 group was lower compared to that in the A375shNC group (Fig. 4C). Following tumor collection, histological analysis was conducted through H&E staining. Cells in the shPLEKHA4 group exhibited lighter staining, were smaller and showed greater heterogeneity compared with those in the shNC group (Fig. 4D).

TMT proteomics analysis has evolved as an important technology for investigating abnormal signaling and therapeutic responses in cancer. As shown in Fig. 5A, shPLEKHA4-transduced A375 cells exhibited a distinct proteomic profile compared with those transduced with shNC. Subsequently, KEGG enrichment analysis was conducted using the TMT proteomics data. As depicted in Fig. 5B, the differentially expressed proteins were enriched in the ‘MAPK signaling pathway’. Utilizing GeneMANIA, the interactions of PLEKHA4 with proteins in the MAPK pathway were further explored (Fig. 5C). PLEKHA4 was found to have physical interactions with MAPK1 (ERK), MAPK9 (JNK) and MAP2K2 (MEK2). Additionally, correlation analysis unveiled a positive relationship between PLEKHA4 expression and MAPK1 (ERK2), MAPK3 (ERK1) and JUN (cJUN) (Fig. 5D-F) Although the r-values were <0.3, the P-values indicated statistical significance, suggesting that the relationship is unlikely to be random and warrants further investigation. Weak correlations may still hold biological significance, especially in multi-factorial systems where individual contributions are inherently subtle. Additionally, this is a gene-level correlation, and further validation at the protein level through western blotting is necessary to confirm these findings. Subsequent validation through western blotting demonstrated that following PLEKHA4 knockdown, the levels of p-P38, p-JNK, p-cJUN, p-MEK and p-ERK were diminished (Fig. 6A). In addition, an overexpression assay was conducted to examine the effects of PLEKHA4 overexpression on cells with low PLEKHA4 levels. The results indicated that PLEKHA4 overexpression successfully upregulated MAPK signaling in SK-MEL-3 cells (Fig. 6B). Collectively, these results indicated that PLEKHA4 may affect the MAPK pathway in melanoma.

Quantitative analysis of alterations in the A375 cell proteome following PLEKHA4 gene silencing was conducted using mass spectrometry (Fig. 7A and B). A total of 6,206 proteins were quantified, with 133 peptides that reached an FDR-corrected P<0.05; among these, 23 were upregulated >5-fold and 111 were downregulated <5-fold in the shPLEKHA4 group compared with in the shNC group (Table SI). Notably, these dysregulated peptides encompassed sites known to regulate various apoptotic and proliferative molecules. Specifically, the substantial reduction of caspase-3 in PLEKHA4knockdown cells, was associated with apoptosis (Table SI). Additionally, a significant reduction was observed in COX2 levels in PLEKHA4 knockdown cells (Table SI), consistent with the findings from GeneMANIA (Fig. 5C). Subsequent expression correlation analysis revealed a correlation between NFKB1 (p65) and CASP3 (with genes in the MAPK pathways at the mRNA level, consistent with the outcomes depicted in GeneMANIA (Fig. 7C). To confirm the aforementioned results, western blotting was conducted to assess protein levels in shNC cells and PLEKHA4 knockdown cells. The results showed that the protein levels of COX2 and p-p65 were reduced, whereas the levels of cleaved caspase-3 were increased (Fig. 7D). Subsequently, flow cytometry was performed to compare the apoptotic effects between the shNC and shPLEKHA4 groups. The findings indicated an elevation in apoptosis in the shPLEKHA4 cells (Fig. 7E).

Given the upregulation of Wnt signaling-related proteins observed in melanoma (Fig. 2D), a correlation analysis focusing on β-catenin was initially performed. The analysis revealed significant correlations between CTNNB1 and MAPK1, MAPK8, and MAPK14, highlighting a potential interaction between β-catenin and these MAPK family members (Fig. 8A). Subsequently, Wnt signaling proteins (p-GSK3β, β-catenin and cyclin D1) were examined and their levels were shown to be reduced following PLEKHA4 knockdown (Fig. 8B). The nuclear translocation of β-catenin is crucial for initiating gene transcription and promoting tumorigenesis. Upon PLEKHA4 knockdown, β-catenin levels decreased in both total and nuclear fractions, while cytosolic β-catenin levels significantly increased (Fig. 8B and C). These findings indicated that PLEKHA4 may promote the nuclear translocation of β-catenin. To further investigate its effect on the Wnt/β-catenin pathway, melanoma cells were treated with the Wnt signaling activator LiCl following PLEKHA4 knockdown. Western blotting revealed that LiCl reversed the changes induced by PLEKHA4 knockdown (Fig. 8D). Additionally, an overexpression assay in SK-MEL-3 cells demonstrated that PLEKHA4 overexpression successfully upregulated Wnt/β-catenin signaling proteins (Fig. 8E). These findings suggested that PLEKHA4 may regulate Wnt/β-catenin signaling in melanoma cells.

cMyc is dysregulated in ~70% of human cancer cases, with substantial evidence linking aberrant cMyc expression to both tumor initiation and maintenance (18). GeneMANIA results indicated that cMyc has physical interactions with multiple members in MAPK pathway (Fig. 5C). Subsequently, a correlation analysis was performed and it was revealed that MYC was positively correlated with MAPK1 and MAPK14 (Fig. 9A). Subsequent western blot analysis was conducted to investigate cMyc expression following the inhibition of MAPK signaling in PLEKHA4 knockdown melanoma cells. PLEKHA4 knockdown markedly reduced cMyc and ubiquitin expression at the protein level (Fig. 9B). cMyc, as an unstable protein, has a half-life of <30 min in non-transformed cells and is rapidly degraded mainly by the ubiquitin-proteasome pathway (19). To examine whether cMyc was stabilized through the ubiquitin-proteasome pathway, shPLEKHA4 melanoma cells were treated with different concentrations of the proteasome inhibitor MG132 (0, 5, 10 and 20 µM). cMyc protein expression was rescued by different concentrations of MG132 (Fig. 9C), suggesting that cMyc degradation could be inhibited via the ubiquitin-proteasome pathway. A higher concentration of MG132 resulted in a higher level of cMyc protein. Subsequently, a ubiquitination assay was employed to detect the influence of PLEKHA4 on cMyc ubiquitination. As revealed in Fig. 9D, MG132 markedly upregulated cMyc polyubiquitination, with higher MG132 concentrations resulting in elevated levels. Furthermore, co-immunoprecipitation was conducted to test cMyc polyubiquitination. As shown in Fig. 9E, cMyc and ubiquitin were detected; however, PLEKHA4 was not detected. This suggests that the PLEKHA4 does not directly interact with cMyc polyubiquitination; however, knockdown of PLEKHA4 resulted in decreased cMyc expression and its ubiquitination, thus suggesting that PLEKHA4 may regulate cMyc and its polyubiquitination indirectly, possibly through another molecule. In Fig. 9E, although PLEKHA4 was knocked down, MG132 still increased cMyc ubiquitination in its absence, thus indicating that while PLEKHA4 may influence cMyc ubiquitination, other pathways or factors may also be involved in regulating this process. Collectively, these results strongly suggested that cMyc is degraded and ubiquitinated by the proteasome system, which may be related to molecules other than PLEKHA4.