ZDHHC14 enhances P16 stability via palmitoylation to inhibit prostate cancer progression

Introduction

Prostate cancer is one of the most common malignant tumors in men worldwide, and its progression and metastasis pose significant challenges to clinical treatment (1). Tumor suppressor genes play pivotal roles in regulating cell proliferation, migration, and invasion, and their dysregulation is closely linked to tumorigenesis (2). Among these genes, P16 (also known as CDKN2A), which functions in cell cycle arrest and senescence by inhibiting cyclin-dependent kinases, has been extensively studied (3). However, the post-translational regulatory mechanisms of P16, particularly in prostate cancer, remain poorly understood.

Protein palmitoylation, a reversible lipid modification mediated by the ZDHHC family of enzymes, has emerged as a critical regulator of protein stability, localization, and function (4). Dysregulation of palmitoylation is implicated in various cancers (5), but its role in modulating tumor suppressors such as P16 remains underexplored. Previous studies have identified ZDHHC enzymes as potential regulators of cancer-related pathways (6), yet their specific contributions to prostate cancer progression, particularly through interactions with P16, are unclear. Notably, members of the ZDHHC family, such as ZDHHC14, exhibit altered expression in prostate cancer tissues, suggesting their potential as therapeutic targets (7).

Current evidence indicates that CDKN2A expression is frequently downregulated in prostate cancer, leading to uncontrolled cell proliferation and metastasis (8,9). However, the mechanisms underlying P16 destabilization in this context have not been fully elucidated. Post-translational modifications, including ubiquitination and palmitoylation, may critically influence P16 stability and activity (10,11). For instance, palmitoylation has been demonstrated to enhance protein stability by counteracting ubiquitination-mediated degradation in other systems (12). Nevertheless, whether P16 undergoes palmitoylation and how this modification impacts its tumor-suppressive functions in prostate cancer remain unresolved.

This study aimed to investigate the regulatory role of palmitoylation on P16 stability in prostate cancer cells and its functional consequences. Our findings reveal a novel mechanism by which ZDHHC14-mediated palmitoylation sustains P16 stability, thereby exerting tumor-suppressive effects in prostate cancer. This research not only advances the understanding of post-translational regulation of P16 but also highlights ZDHHC14 as a potential therapeutic target for restoring P16 function in cancer therapy. We present this article in accordance with the MDAR reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-552/rc).

Methods

Analysis of ZDHHCs in The Cancer Genome Atlas (TCGA) prostate cancer data

Gene expression and clinical data of prostate cancer (492 tumor tissues and 152 adjacent normal tissues) were downloaded from TCGA database. Differential expression of ZDHHCs was analyzed and visualized using the ggplot2 package in R (version 4.2.1). Survival regression analysis was performed using the survival package in R (version 4.2.1), and receiver operating characteristic (ROC) analysis was conducted with the pROC package.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from tissues or cells using Trizol reagent (Invitrogen, California, USA). RNA concentration and purity were measured using a spectrophotometer. cDNA synthesis was performed with the PrimeScript RT Kit (TaKaRa, Shiga, Japan) according to the manufacturer’s protocol. qRT-PCR was carried out using the SYBR Green PCR Kit (TaKaRa). GAPDH served as the internal reference gene, and relative gene expression was calculated using the 2^−ΔΔCt method. Primer sequences were as follows: ZDHHC14: F-5’-ATCCTGGTCACTAGCGGACT-3’, R-5’-TCTTGGTCGCACATGTCACT-3’; CDKN2A: F-5’-CCGAATAGTTACGGTCGGAGG-3’, R-5’-AATCGGGGATGTCTGAGGGA-3’; GAPDH: F-5’-AATGGGCAGCCGTTAGGAAA-3’, R-5’-GCGCCCAATACGACCAAATC-3’.

Cell culture and transfection

Prostate cancer DU145 and PC3 cells were purchased from ATCC and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Small interfering RNA (siRNA) targeting ZDHHC14 and scramble siRNA (si-SCR) were synthesized by Genepharma (Shanghai, China). Transfection was performed using Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions.

Western blot

Cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China), and total protein concentration was quantified using a bicinchoninic acid (BCA) assay kit (Sangon Biotech, Shanghai, China). Equal amounts of protein (20 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, Massachusetts, USA). After blocking with 5% non-fat milk for 1 h, membranes were incubated overnight at 4 ℃ with primary antibodies against P16 (Abcam, Shanghai, China, ab270058) and β-actin (Abcam, ab179467), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam) for 2 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) substrate (Beyotime Biotechnology).

Colony formation assay

Cells were seeded into 6-well plates at a density of 1×10³ cells/well and cultured in a 37 ℃, 5% CO₂ incubator. The medium was partially replaced every 3 days. After 14 days, colonies were fixed, stained, and counted under a microscope. Clusters containing ≥10 cells were defined as colonies.

Transwell assay

Matrigel-coated Transwell chambers (Corning, New York, USA) were used to assess cell invasion. PC3 and DU145 cells (5×10⁴ cells/well) were seeded into the upper chamber with serum-free medium, while complete medium containing 10% FBS was added to the lower chamber. After 24 h, cells that invaded through the membrane were fixed, stained with crystal violet, and counted under a microscope (5 random fields per chamber).

Co-immunoprecipitation (Co-IP)

PC3 and DU145 cells were lysed with RIPA buffer 24 h post-transfection. Total protein (500 µg) was incubated with P16 antibody (Abcam, ab270058), GAPDH (Abcam, ab128915) and ZDHHC14 (Thermo Fisher Scientific, MA, USA, PA5-49367) overnight at 4 ℃, followed by incubation with Protein A/G magnetic beads for 2 h. The immunoprecipitated complexes were washed, resolved by SDS-PAGE, and analyzed via Western blot to detect ZDHHC14 and palmitoylation levels.

Statistical analysis

Data were analyzed using SPSS 26.0 (IBM Corp., Chicago, USA). Each group had three replicates, comparisons between two groups were performed using Student’s t-test, with P<0.05 considered statistically significant. Graphs were generated using GraphPad Prism 9.0 (GraphPad Software, San Diego, USA).

Results

Overexpression of CDKN2A inhibits prostate cancer cell proliferation and invasion

To investigate the functional role of CDKN2A in prostate cancer, CDKN2A was overexpressed in PC-3 and DU-145 cell lines via plasmid transfection. qRT-PCR and Western blot analyses confirmed a significant upregulation of both mRNA and protein levels of CDKN2A in transfected cells compared to controls (Figure 1A). The impact of CDKN2A overexpression on cell proliferation was evaluated using a colony formation assay. As shown in Figure 1B, CDKN2A-overexpressing cells exhibited a marked reduction in colony formation capacity compared to cells transfected with control plasmids. Furthermore, Transwell assays demonstrated that CDKN2A overexpression significantly suppressed the invasive ability of PC-3 and DU-145 cells (Figure 1C).

Figure 1 Overexpression of CDKN2A in prostate cancer cell lines PC-3 and DU-145. (A) Upregulation of both mRNA and protein expression levels of CDKN2A. (B) Reduced cell proliferation ability of PC-3 and DU-145 cells following CDKN2A overexpression. Cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Images were captured under a light microscope at ×100 magnification. (C) Decreased invasive capacity of PC-3 and DU-145 cells after CDKN2A overexpression. Invaded cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Representative fields were photographed under a light microscope at ×200 magnification. ***, P<0.001.

Palmitoylation at Cys 72 enhances P16 protein stability

To investigate the role of palmitoylation in regulating P16 stability, prostate cancer cells were treated with the palmitoylation inhibitor 2-bromopalmitate (2-BP). qRT-PCR analysis revealed that 2-BP treatment at varying concentrations had no significant effect on P16 mRNA levels (Figure 2A). Endogenous and exogenous Co-IP assays confirmed that P16 undergoes palmitoylation, and this modification was markedly reduced by 2-BP (Figure 2B). Using the GPS-Palm online prediction tool (http://gpspalm.biocuckoo.cn/), we identified a potential palmitoylation site at Cys 72 of P16 (Figure 2C). Site-directed mutagenesis (C72S) significantly diminished P16 palmitoylation (Figure 2D), and the palmitoylation agonist hexadecyl acetyl phosphate (HAM) failed to restore palmitoylation levels in mutant cells (Figure 2E).

Figure 2 Palmitoylation modification affects the protein stability of P16. (A) mRNA expression of P16 remained unaffected by varying concentrations of 2-BP. (B) P16 protein undergoes palmitoylation, and treatment with the palmitoylation inhibitor 2-BP significantly reduced this modification. (C) Prediction of palmitoylation sites on P16 protein. (D) Palmitoylation of P16 was markedly diminished by the C72S point mutation. (E) The palmitoylation agonist HAM failed to elevate P16 palmitoylation. (F) Inhibition of palmitoylation did not alter the intracellular localization of P16. (G) 2-BP at various concentrations significantly suppressed P16 protein levels. Cells were stained with anti-P16 primary antibody followed by fluorescent secondary antibody, and nuclei were counterstained with DAPI. Images were obtained using a fluorescence microscope at ×400 magnification. (H) Ubiquitination of P16 increased substantially upon 2-BP treatment. (I) Protein stability of P16 was significantly reduced following 2-BP administration. ***, P<0.001. 2-BP, 2-bromopalmitate; CHX, cycloheximide; DAPI, 4’,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; HAM, hexadecyl acetyl phosphate; WT, wild type.

Since palmitoylation may influence protein localization or stability, immunofluorescence was performed to assess P16 subcellular localization. As shown in Figure 2F, inhibition of palmitoylation did not alter P16 localization. However, Western blot analysis demonstrated that 2-BP treatment dose-dependently reduced P16 protein levels (Figure 2G). Further studies revealed that 2-BP treatment increased ubiquitination of P16 (Figure 2H) and decreased its protein stability (Figure 2I). These findings collectively indicate that palmitoylation at Cys 72 suppresses ubiquitination-mediated degradation, thereby enhancing the half-life and stability of P16.

ZDHHC14 enhances P16 expression via palmitoylation

To identify the palmitoyltransferase responsible for P16 modification, we analyzed the expression of ZDHHC family members in prostate cancer and their correlation with patient survival using the Gene Expression Profiling Interactive Analysis (GEPIA) 2.0 database (http://gepia2.cancer-pku.cn/). As shown in Figure 3A, the mRNA levels of ZDHHC1, ZDHHC3, ZDHHC4, ZDHHC12, ZDHHC14, and ZDHHC20 were significantly downregulated in prostate cancer tissues, suggesting their potential tumor-suppressive roles. Survival analysis revealed that high ZDHHC14 expression positively correlated with prolonged patient survival, whereas ZDHHC3 expression showed a negative correlation (Figure 3B).

Figure 3 Expression of ZDHHC family members in prostate cancer and their impact on patient survival. (A) Analysis of ZDHHC family member expression levels. (B) Correlation between ZDHHC family member expression and patient survival curves. ***, P<0.001. CI, confidence interval; HR, hazard ratio; TPM, transcripts per million.

To validate the functional role of ZDHHC14, two specific siRNAs targeting ZDHHC14 were designed and transfected into PC-3 and DU145 cells. qRT-PCR confirmed efficient knockdown of ZDHHC14 mRNA (Figure 4A,4B), with no effect on P16 mRNA levels. However, Western blot analysis demonstrated that ZDHHC14 knockdown significantly reduced both ZDHHC14 and P16 protein expression (Figure 4C,4D). In addition, knockdown of ZDHHC14 also decreased the palmitoylation of P16 (Figure 4E).

Figure 4 ZDHHC14 regulates P16 protein via post-translational modification. (A,B) Knockdown of ZDHHC14 significantly reduced ZDHHC14 mRNA levels but did not affect P16 mRNA expression. (C,D) Both ZDHHC14 and P16 protein levels were significantly decreased upon ZDHHC14 knockdown. (E) The palmitoylation of P16 was detected by Western blot. ***, P<0.001.

Computational molecular docking was performed to predict the interaction interface between ZDHHC14 and P16 (Figure 5A). Exogenous Co-IP using Flag-tagged P16 and HA-tagged ZDHHC14 plasmids confirmed their physical interaction (Figure 5B). Endogenous Co-IP further validated this interaction in prostate cancer cells (Figure 5C). These results collectively indicate that ZDHHC14 interacts with P16, mediates its palmitoylation, and enhances protein stability and half-life, thereby suppressing prostate cancer progression.

Figure 5 ZDHHC14 mediates palmitoylation of P16 protein. (A) Computer-simulated molecular docking revealed interaction sites and modes between ZDHHC14 and P16. (B) Exogenous Co-IP confirmed the interaction between P16 and ZDHHC14. (C) Endogenous Co-IP further validated the physical interaction between ZDHHC14 and P16. Co-IP, co-immunoprecipitation.

Discussion

This study elucidates the tumor-suppressive role of CDKN2A in prostate cancer and reveals a novel regulatory mechanism mediated by ZDHHC14-dependent palmitoylation, which enhances P16 stability and function. Our findings not only reinforce the established role of CDKN2A in suppressing cancer progression but also provide critical insights into the post-translational regulation of this key tumor suppressor.

Consistent with prior studies demonstrating P16’s role in cell cycle arrest and tumor suppression across multiple cancers (13,14), our data indicate that CDKN2A overexpression significantly inhibits proliferation and invasion in prostate cancer cell lines (PC-3 and DU-145). These results align with P16’s canonical function in blocking CDK4/6 activity, thereby preventing Rb phosphorylation and G1/S transition (15). However, the observed reduction in metastatic potential via Transwell assays suggests that CDKN2A may exert broader anti-tumor effects beyond cell cycle regulation, potentially involving pathways such as epithelial-mesenchymal transition (EMT) or matrix metalloproteinase (MMP) inhibition.

A pivotal discovery in this study is the identification of palmitoylation as a critical post-translational modification regulating P16 stability. We demonstrated that palmitoylation at Cys 72 suppresses ubiquitination, thereby prolonging P16’s half-life. This finding adds a new layer to the understanding of P16 regulation, which has primarily focused on transcriptional silencing via promoter hypermethylation or mutations in cancers (16,17). Notably, the specificity of this modification was confirmed using the palmitoylation inhibitor 2-BP and site-directed mutagenesis (C72S). The observed increase in P16 ubiquitination upon palmitoylation inhibition suggests that palmitoylation either competes with ubiquitination for lysine residues or structurally shields ubiquitination sites. This interplay resembles the regulatory dynamics observed in other tumor suppressors, such as PTEN (18), highlighting a conserved mechanism for stabilizing critical proteins in cancer.

Our data further identify ZDHHC14 as the primary palmitoyltransferase responsible for P16 modification. The correlation between ZDHHC14 downregulation and poor patient prognosis underscores its tumor-suppressive role in prostate cancer. Mechanistically, ZDHHC14 knockdown reduced both P16 expression and stability, while molecular docking and Co-IP confirmed their direct interaction. These results position ZDHHC14 as a key upstream regulator of P16, offering a potential therapeutic avenue to restore P16 function in cancers with ZDHHC14 loss. Interestingly, the lack of impact on P16 mRNA levels upon ZDHHC14 knockdown suggests that ZDHHC14 regulates P16 exclusively at the post-translational level, contrasting with other ZDHHC family members that may influence transcription or splicing (19,20).

Therapeutically, targeting the ZDHHC14-P16 axis could represent a strategy to counteract P16 loss in prostate cancer. Small-molecule agonists of ZDHHC14 or palmitoylation mimetics might stabilize P16, thereby inhibiting tumor progression. However, there are limitations in this study. All experiments were conducted in vitro, and future work should validate these findings in vivo using xenograft models or patient-derived organoids. Additionally, the functional consequences of ZDHHC14-mediated palmitoylation in other cancer types remain unexplored. It is also unclear whether other ZDHHC enzymes compensate for ZDHHC14 loss in advanced tumors, which may explain the heterogeneity in clinical outcomes.

Conclusions

In summary, our study establishes ZDHHC14-dependent palmitoylation as a novel mechanism stabilizing P16 in prostate cancer. By linking post-translational modification to P16’s tumor-suppressive activity, these findings expand the understanding of P16 regulation and highlight ZDHHC14 as a potential prognostic marker and therapeutic target. Future research should focus on translating these insights into preclinical models and exploring synergistic strategies to reactivate P16 in malignancies with epigenetic or post-translational silencing.

Acknowledgments

The authors thank the reviewers for their helpful comments on this report.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-552/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-552/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-552/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-552/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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

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