ABSTRACT
Aberrant DNA methylation is a hallmark of prostate cancer (PCa) and plays a critical role in sustaining the epigenetic silencing of tumor suppressor genes. However, how oncogenic signaling pathways interface with the DNA methylation machinery remains poorly understood. In this study, we identify AURKA as a key regulator of global DNA methylation in PCa cells. AURKA depletion markedly reduces genomic 5-methylcytosine (5mC) levels, indicating its essential role in DNA methylation maintenance. Mechanistically, AURKA directly interacts with UHRF1-a crucial epigenetic regulator that recruits DNMT1 to hemimethylated DNA, and phosphorylates it at Ser76, thereby enhancing UHRF1 protein stability. AURKA depletion accelerates UHRF1 degradation via the ubiquitin-proteasome pathway, whereas AURKA overexpression stabilizes UHRF1 by suppressing its polyubiquitination. The phospho-deficient UHRF1 S76A mutant exhibits reduced stability and increased polyubiquitination, confirming that phosphorylation at Ser76 is critical for AURKA-mediated UHRF1 stabilization. Functionally, disruption of the AURKA-UHRF1 axis de-represses p16 and p21 and significantly impairs PCa cell proliferation; these effects are contingent upon UHRF1 phosphorylation, as only wild-type UHRF1, not the phospho-deficient S76A mutant, can rescue the observed phenotypic impairments. Collectively, our study reveals a previously unrecognized AURKA-UHRF1 signaling axis that links kinase activity to epigenetic maintenance. By phosphorylating UHRF1 at Ser76 and stabilizing its protein, AURKA sustains global DNA methylation and promotes PCa cell proliferation. These findings provide novel mechanistic insights into PCa biology and suggest that targeting this pathway may offer a promising therapeutic strategy.
KEYWORDS: AURKA, phosphorylation, DNA methylation, UHRF1, cell viability
Introduction
Epigenetic dysregulation is a hallmark of cancer and plays a critical role in tumor initiation, progression, and therapeutic resistance [1]. Among diverse epigenetic modifications, DNA methylation is among the most extensively studied mechanisms, occurring primarily at the 5-position of cytosine (5-methylcytosine, 5mC) within CpG dinucleotides [2]. Aberrant DNA methylation patterns, characterized by global hypomethylation and locus-specific hypermethylation of tumor suppressor genes, are frequently observed in prostate cancer (PCa) and contribute to malignant transformation and disease progression [3,4]. The maintenance of DNA methylation during DNA replication is orchestrated by a coordinated network of epigenetic regulators, including DNMT1 and UHRF1, which ensure the faithful propagation of methylation marks across cell divisions [5,6].
UHRF1, a multifunctional epigenetic integrator, recognizes hemimethylated DNA and recruits DNMT1 to replication forks, thereby coordinating the maintenance of DNA methylation [7]. UHRF1 overexpression has been widely documented across multiple cancer types, including PCa, and is associated with transcriptional silencing of tumor suppressor genes and adverse clinical outcomes [8,9]. The activity and stability of UHRF1 are tightly regulated by post-translational modifications, such as ubiquitination, acetylation, and methylation, which modulate its chromatin binding, enzymatic activity, and protein turnover [10,11]. However, the upstream signaling pathways that dynamically govern UHRF1 function, particularly through phosphorylation-dependent mechanisms, remain poorly understood.
AURKA, a serine/threonine kinase, is well recognized for its essential roles in mitotic progression, centrosome maturation, and spindle assembly [12,13]. Aberrant activation or overexpression of AURKA is frequently observed in PCa and is closely associated with genomic instability, tumor progression, and therapeutic resistance [14,15]. Beyond its established functions in cell cycle regulation, mounting evidence indicates that AURKA also participates in non-mitotic processes, including transcriptional regulation, metabolic reprogramming, and oncogenic signaling [16,17]. Yet, whether AURKA contributes to epigenetic regulation-particularly DNA methylation-remains unexplored.
Recent studies have increasingly highlighted the interplay between kinase signaling pathways and the epigenetic machinery, suggesting that protein kinases may modulate DNA methylation indirectly by regulating key epigenetic factors [18,19]. Given the central role of UHRF1 in DNA methylation maintenance and the oncogenic functions of AURKA in PCa, we hypothesized that AURKA regulates DNA methylation through post-translational modification of UHRF1. Specifically, phosphorylation-dependent regulation of UHRF1 stability could serve as a critical mechanism linking upstream kinase signaling to downstream epigenetic maintenance.
In this study, we demonstrate that AURKA is essential for the maintenance of global DNA methylation in PCa cells. Mechanistically, AURKA directly interacts with and phosphorylates UHRF1, thereby enhancing its protein stability by preventing ubiquitin-proteasome-mediated degradation. Functionally, this AURKA-UHRF1 signaling axis preserves DNA methylation, sustains transcriptional repression of tumor suppressor genes, and promotes malignant cellular phenotypes. Our findings reveal a novel link between mitotic kinase signaling and epigenetic regulation, providing new insights into the molecular basis of PCa progression and suggesting potential therapeutic strategies targeting this pathway.
Results
Inhibition of AURKA reduces global DNA methylation in prostate cancer cells
To determine whether AURKA participates in the regulation of DNA methylation in prostate cancer (PCa), we treated androgen-independent, AR-positive C4-2 cells and AR-negative PC3 cells with Alisertib, a highly selective AURKA kinase inhibitor, and assessed global DNA methylation status by DNA dot blot using an anti-5mC antibody. Unexpectedly, Alisertib markedly reduced global DNA methylation levels in a dose-dependent manner (Figure 1(A,B)). In parallel, we knocked down AURKA in C4-2 and PC3 cells using two independent small interfering RNAs (siRNAs) (Figure 1(C)) and subsequently examined the impact of AURKA depletion on global DNA methylation by DNA dot blot and quantitative enzyme-linked immunosorbent assay (ELISA). Consistent with the pharmacological inhibition data, siRNA-mediated AURKA depletion significantly reduced global DNA methylation (Figure 1(D-F)). Importantly, since Alisertib specifically inhibits AURKA catalytic activity without eliminating the protein itself, these complementary results demonstrate that global DNA methylation maintenance depends on AURKA kinase activity rather than merely on AURKA protein presence.
Figure 1.
Silencing of AURKA decreased the global DNA methylation level in prostate cancer (PCa) cells. (A-B) PCa cells were treated with different doses of DMSO or Alisertib for 72 h, and the genomic DNA was then prepared. The levels of 5mC were assessed by DNA dot blotting, and the dot intensities were quantified. (C) Cells were transfected with either a scrambled siRNA or two distinct AURKA siRnas for 72 hours. The levels of AURKA protein were evaluated via western blotting. The genomic DNA of C4-2 and PC3 cells was prepared, and the levels of 5-methylcytosine (5mC) were determined by DNA dot blotting (D-E) or enzyme-linked immunosorbent assay (ELISA). (F) Data are presented as the mean ± standard deviation (SD) of each group from three independent experiments. **p < 0.01.
AURKA physically interacts with UHRF1 and phosphorylates it at Ser76
The maintenance of global DNA methylation is intricately linked to the activity of core epigenetic regulators, rather than to direct modification of DNA by upstream kinases. To explore this relationship, we sought to identify epigenetic factors that physically interact with AURKA and could mediate its effect on DNA methylation. We performed immunoprecipitation followed by mass spectrometry (IP-MS) analysis in PC3 cells expressing Flag-tagged AURKA. Among the identified AURKA-interacting proteins, UHRF1 emerged as a prominent candidate with a well-established role in DNA methylation regulation (Figure 2(A)). Given the crucial function of UHRF1 in coordinating maintenance DNA methylation through DNMT1 recruitment, we hypothesized that AURKA modulates DNA methylation indirectly by regulating UHRF1 function.
Figure 2.
AURKA physically interacts with the UHRF1 protein and induces its phosphorylation. (A) Immunoprecipitation was conducted using PC3 cells expressing AURKA-Flag, and the experimental samples were analyzed via mass spectrometry. (B-C) Cell lysates underwent immunoprecipitation with either anti-AURKA or anti-UHRF1 antibody. Subsequently, the immunoprecipitated protein was subjected to immunoblotting with the specified antibodies. (D) Recombinant GST and GST-UHRF1 proteins were purified. The protein interaction between UHRF1 and AURKA was then investigated through a GST-pull down assay. (E) Bacterial-expressed GST-UHRF1 was subjected to an in vitro kinase assay, and the phosphorylation level of UHRF1 was evaluated using an anti-phosphoserine antibody. The loading controls are presented in the bottom panel (stained by Coomassie blue). (F) Schematic representation of the putative AURKA-mediated phosphorylation sites in the UHRF1 protein. (G) Bacterial-expressed GST-UHRF1 wild-type or mutants were subjected to an in vitro kinase assay, and the phosphorylation level of UHRF1 was evaluated using an anti-phosphoserine antibody. The loading controls are presented in the bottom panel (stained by Coomassie blue). (H) Cells were transfected with plasmids expressing AURKA-Flag and UHRF1-His wild type and mutants. UHRF1 was immunoprecipitated with anti-His antibody, and the total phosphorylation level was measured with an anti-phosphoserine antibody.
To validate this interaction, we performed endogenous co-immunoprecipitation (co-IP) assays in PC3 cells. Reciprocal co-IP using anti-AURKA or anti-UHRF1 antibodies confirmed a robust interaction between endogenous AURKA and UHRF1 (Figure 2(B,C)). Furthermore, GST pull-down assays using purified recombinant proteins demonstrated a direct interaction between AURKA and UHRF1 in vitro (Figure 2(D)). In vitro kinase assays further confirmed that AURKA directly phosphorylates UHRF1 (Figure 2(E)).
We next predicted candidate AURKA phosphorylation sites on UHRF1. Based on the AURKA consensus sequence R/K-X-S/T-I/V/L [20], Ser76 was identified as the sole predicted AURKA phosphorylation site (Figure 2(F)). We therefore generated a Ser76-to-Ala (S76A) mutant and repeated the in vitro kinase assay. AURKA significantly enhanced the phosphorylation of wild-type UHRF1, whereas phosphorylation was abolished in the S76A mutant (Figure 2(G)). To further confirm AURKA-dependent Ser76 phosphorylation in cells, we co-transfected plasmids encoding AURKA-Flag and either wild-type UHRF1-His or the S76A mutant into PC3 cells, immunoprecipitated UHRF1 with an anti-His antibody, and assessed phosphorylation using an anti-pan-phospho-Ser antibody. Two key observations were made: (i) the S76A mutant exhibited a significantly decreased basal phosphorylation level compared with wild-type UHRF1; and (ii) AURKA overexpression elevated the phosphorylation of wild-type UHRF1 but failed to enhance phosphorylation of the S76A mutant. These data confirm that Ser76 is the exclusive AURKA phosphorylation site on UHRF1 (Figure 2(H)).
AURKA regulates UHRF1 protein stability in a phosphorylation-dependent manner
To determine whether AURKA regulates UHRF1 expression, we first assessed UHRF1 protein levels after pharmacological inhibition of AURKA with Alisertib or siRNA-mediated AURKA depletion. Both Alisertib treatment and AURKA knockdown significantly reduced endogenous UHRF1 protein abundance in C4-2 and PC3 cells (Figure 3(A,B)). Conversely, ectopic overexpression of AURKA resulted in a substantial increase in UHRF1 protein levels (Figure 3(C)), indicating that AURKA positively regulates UHRF1 protein abundance. To determine whether this regulation occurs at the transcriptional or post-transcriptional level, we measured UHRF1 mRNA levels by quantitative RT-PCR. Neither AURKA depletion nor overexpression significantly altered UHRF1 mRNA levels (Figure 3(D,E)), suggesting that AURKA regulates UHRF1 primarily through a post-transcriptional mechanism.
Figure 3.
AURKA regulates UHRF1 protein stability in a phosphorylation-dependent manner. (A) PCa cell was treated with different doses of DMSO or Alisertib for 72 h, UHRF1 protein was assessed by immunoblotting. (B) C4-2 and PC3 cells were transfected with scrambled or two different AURKA siRnas. The levels of UHRF1 protein were assessed by immunoblotting. (C) Cells were transfected with the pCMV-AURKA-Flag plasmid, and the levels of UHRF1 protein were assessed by immunoblotting. (D) AURKA was depleted in PC3 cells. The mRNA levels of the AURKA and UHRF1 genes were analyzed by RT-PCR. ACTIN was used as an internal control. (E) Cells were transfected with the pCMV-AURKA-Flag plasmid, and the mRNA levels of the AURKA and UHRF1 genes were analyzed by RT-PCR. ACTIN was used as an internal control. (F) AURKA-depleted PC3 cells were treated with cycloheximide (CHX, 50 μg/ml) for the indicated times, and the endogenous UHRF1 protein levels were monitored by immunoblotting. The protein levels were quantified by grayscale analysis. (G) Cells were transfected with the pCMV-AURKA-Flag plasmid and then treated with CHX for the indicated times, and the endogenous UHRF1 protein levels were monitored by immunoblotting. The protein levels were quantified by grayscale analysis. (H) PC3 cells were transfected with the plasmids expressing UHRF1(WT)-His and UHRF1(S76A)-His for 60 h, and then were treated with CHX. The cells were harvested at the indicated time points. UHRF1 protein was analyzed by immunoblotting and was quantified by grayscale analysis.
Given that AURKA directly phosphorylates UHRF1, we investigated whether AURKA affects UHRF1 protein stability. Cycloheximide (CHX) chase assays demonstrated that AURKA depletion significantly accelerated UHRF1 protein degradation, shortening its half-life (Figure 3(F)). In contrast, ectopic expression of AURKA significantly prolonged the UHRF1 half-life (Figure 3(G)), supporting a critical role for AURKA in maintaining UHRF1 protein stability.
To directly evaluate the requirement for phosphorylation in this process, we compared the protein stability of wild-type UHRF1 with that of the phospho-deficient S76A mutant. Notably, the S76A mutant displayed significantly reduced stability compared with wild-type UHRF1 (Figure 3(H)), phenocopying the effect of AURKA depletion. Collectively, these findings demonstrate that AURKA maintains UHRF1 protein stability in a phosphorylation-dependent manner.
AURKA-mediated phosphorylation protects UHRF1 from ubiquitin-proteasome-dependent degradation
Since AURKA regulates UHRF1 protein stability without altering its mRNA levels, we next investigated whether UHRF1 undergoes proteasomal degradation upon AURKA inhibition. Treatment with Alisertib in combination with MG132, a specific proteasome inhibitor, effectively rescued UHRF1 protein levels (Figure 4(A)). Similarly, siRNA-mediated AURKA depletion induced endogenous UHRF1 degradation, and this degradation was also reversed by MG132 (Figure 4(B)). These results indicate that the reduction in UHRF1 levels following AURKA inhibition is mediated by proteasomal degradation.
Figure 4.
AURKA-induced phosphorylation prevents UHRF1 protein degradation through the ubiquitin-proteasome pathway. (A) PC3 cell was treated with Alisertib for 48 h, followed by 50 μM MG132 for 6 h. UHRF1 protein was assessed by immunoblotting. (B) Cells were transfected with scrambled or AURKA siRnas for 72 h and then treated with 50 μM MG132 for 6 h. The protein levels of AURKA and UHRF1 were assessed by immunoblotting. (C) the plasmids expressing HA-ubiquitin and UHRF1-His together with AURKA siRnas were transfected into cells for 48 h and then treated with 50 μM MG132 for 6 h. UHRF1 protein was immunoprecipitated with an anti-His antibody, and the polyubiquitylated UHRF1 was assessed with an anti-HA antibody. (D) the plasmids expressing HA- ubiquitin, UHRF1-His, and AURKA-Flag were transfected into cells for 48 h and then treated with 50 μM MG132 for 6 h. UHRF1 protein was immunoprecipitated with an anti-His antibody, and the polyubiquitylated UHRF1 was assessed with an anti-HA antibody. (E) Cells stably expressing HA ubiquitin were transfected with the indicated plasmids for 48 h and then treated with 50 μM MG132 for 6 h. UHRF1 was immunoprecipitated with an anti-His antibody, and the polyubiquitylated UHRF1 protein was detected by an anti-HA antibody.
To directly assess whether AURKA modulates UHRF1 ubiquitination, we performed ubiquitination assays in PCa cells. Immunoprecipitation of UHRF1 followed by immunoblotting for ubiquitin revealed that AURKA knockdown significantly increased the levels of polyubiquitinated UHRF1 (Figure 4(C)). Conversely, ectopic AURKA expression markedly reduced UHRF1 polyubiquitination (Figure 4(D)), indicating that AURKA negatively regulates UHRF1 ubiquitination. Since Ser76 is the primary AURKA phosphorylation site, we next investigated whether this phosphorylation is required for AURKA to suppress UHRF1 ubiquitination. Consistent with our hypothesis, the phospho-deficient S76A mutant exhibited significantly higher polyubiquitination than wild-type UHRF1 (Figure 4(E)). These results establish that AURKA-mediated phosphorylation of UHRF1 at Ser76 is critical for preventing ubiquitin-dependent proteasomal degradation.
AURKA sustains DNA methylation maintenance through UHRF1 phosphorylation
UHRF1, an essential cofactor, recruits DNMT1 to replication forks and preferentially converts hemimethylated CpGs to fully methylated status during DNA replication [7]. To determine whether AURKA-induced UHRF1 phosphorylation affects the UHRF1-DNMT1 interaction, we assessed the impact of AURKA on this interaction by co-IP. AURKA knockdown markedly attenuated the interaction between UHRF1 and DNMT1 (Figure 5(A)). Moreover, the S76A mutation significantly weakened the UHRF1-DNMT1 interaction compared with wild-type UHRF1 (Figure 5(B)). Given that AURKA both maintains UHRF1 protein stability and enhances the UHRF1-DNMT1 interaction, we next determined the functional implications of UHRF1 phosphorylation for DNA methylation maintenance.
Figure 5.
AURKA-induced phosphorylation of UHRF1 is essential for the maintenance of global DNA methylation. (A) C4-2 cell was co-transfected with the indicated plasmids and siRnas for 72 h, UHRF1 was immunoprecipitated with anti-His antibody, and DNMT1 was analyzed by immunoblotting. (B) C4-2 cell was transfected with the plasmids expressing UHRF1(WT)-His and UHRF1(S76A)-His mutants as indicated for 72 h. UHRF1 was immunoprecipitated with anti-His antibody, and DNMT1 was assessed by western blot. (C) C4-2 and PC3 cells with UHRF1 depletion were transfected with the specified plasmids for 72 hours, after which the levels of UHRF1 proteins were detected via western blotting. Genomic DNA from C4-2 and PC3 cells was prepared, and the levels of 5-methylcytosine (5mC) were evaluated through DNA dot blotting (D-E) and enzyme-linked immunosorbent assay (ELISA) (F). Data are presented as the mean ± standard deviation (SD) of each group from three independent experiments. **p < 0.01.
We reconstituted UHRF1-depleted C4-2 and PC3 cells with either wild-type UHRF1 or the phospho-deficient S76A mutant (Figure 5(C)). Global DNA methylation analysis revealed that restoration of wild-type UHRF1 effectively rescued the reduction in 5mC levels caused by UHRF1 depletion, as measured by DNA dot blot and ELISA-based quantification. In contrast, re-expression of the S76A mutant failed to restore global 5mC levels, despite being expressed at comparable levels (Figure 5(D-F)). The consistent results obtained from complementary methylation assays imply that phosphorylation of UHRF1 at Ser76 is functionally indispensable for maintaining genomic DNA methylation downstream of AURKA. These findings identify UHRF1 phosphorylation as the critical molecular event by which AURKA sustains global DNA methylation in PCa cells.
AURKA sustains tumor suppressor gene repression and promotes malignant phenotypes through UHRF1 phosphorylation
Given that AURKA-mediated phosphorylation of UHRF1 is critical for DNA methylation maintenance, we next examined the biological consequences of disrupting this signaling axis in PCa cells. Genetic perturbation of the AURKA-UHRF1 pathway resulted in significant de-repression of tumor suppressor genes. Specifically, UHRF1 depletion or abrogation of UHRF1 phosphorylation led to marked upregulation of p16 and p21 expression in both C4-2 and PC3 cells (Figure 6(A,B)), consistent with the loss of epigenetic repression.
Figure 6.
The phosphorylation of UHRF1 by AURKA was crucial for maintaining cell viability. The C4-2 or PC3 cells with UHRF1 depletion were transfected with the specified plasmids. (A-B) The expression levels of the UHRF1, p16, and p21 genes were analyzed via reverse-transcription polymerase chain reaction (RT-PCR). ACTIN was employed as an internal control. (C-D) MeDIP data showing the methylation modification levels of the promoters in the p21 and p16. (E-F) The cells were seeded at a density of 1 × 104 cells in triplicate 60-mm plates. The cells were trypsinized and counted at the designated time points. (G-H) A colony formation assay was conducted, and the number of clones was quantified. The data represent the mean ± SD of each group from three independent experiments. **p < 0.01.
We next investigated the effect of UHRF1 phosphorylation on the methylation status of the p16 and p21 promoters. Compared with wild-type UHRF1, the phospho-deficient S76A mutant significantly reduced methylation levels at both promoters (Figure 6(C,D)). Subsequently, we performed rescue experiments to assess the functional impact of AURKA-UHRF1 signaling on malignant phenotypes. Reconstitution with wild-type UHRF1 effectively restored repression of the tumor suppressor genes p16 and p21, as well as cell proliferation and colony-forming capacity, in UHRF1-depleted cells. In contrast, the phospho-deficient S76A mutant failed to rescue these phenotypes (Figure 6(E-H)). Collectively, these results demonstrate that disruption of the AURKA-UHRF1 phosphorylation axis leads to de-repression of p16 and p21 and impairs PCa cell proliferation, and that these effects are contingent upon UHRF1 phosphorylation at Ser76.
Discussion
In this study, we reveal a previously unrecognized epigenetic function of AURKA in prostate cancer (PCa), demonstrating that AURKA sustains global DNA methylation through phosphorylation-dependent stabilization of UHRF1. Mechanistically, AURKA directly interacts with UHRF1 and phosphorylates it at Ser76, thereby preventing its ubiquitin-proteasome-mediated degradation. This stabilization of UHRF1 is essential for maintaining genomic 5-methylcytosine (5mC) levels, preserving transcriptional repression of tumor suppressor genes, and promoting malignant phenotypes in PCa cells. These findings establish an AURKA-UHRF1 signaling axis that functionally connects mitotic kinase activity to epigenetic maintenance.
AURKA has been well characterized as a serine/threonine kinase essential for mitotic progression, centrosome maturation, and chromosomal stability [21]. However, emerging evidence suggests that AURKA also exerts non-mitotic functions that contribute to tumorigenesis [22]. Our study significantly broadens this paradigm by identifying AURKA as a critical regulator of DNA methylation. Unlike canonical epigenetic enzymes such as DNMT1, AURKA does not directly catalyze DNA methylation but rather modulates the stability of the core epigenetic machinery. This indirect regulatory mechanism reveals a new dimension of crosstalk between kinase signaling and epigenetic regulation. Most DNA methylation occurs on newly replicated strands during S phase, and AURKA inhibition leads to cell cycle arrest [7,21]. However, reconstitution with wild-type UHRF1 effectively restored DNA methylation levels in UHRF1-depleted cells, whereas the phospho-deficient S76A mutant failed to do so (Figure 5(C-F)). These results indicate that, although AURKA may influence DNA methylation indirectly through cell cycle regulation, it primarily maintains DNA methylation through phosphorylation of UHRF1.
Aberrant DNA methylation, accompanied by altered gene expression patterns, is a fundamental feature of cancer, characterized by regional hypermethylation of gene promoters and silencing of tumor suppressor genes (TSGs) across virtually all human cancer types. UHRF1 is a well-established master coordinator of DNA methylation maintenance, primarily through its capacity to recruit DNMT1 to hemimethylated DNA during replication [23]. Dysregulation of UHRF1 is frequently observed in cancers and is closely associated with global DNA hypermethylation and silencing of TSGs [24]. While previous studies have implicated post-translational modifications such as ubiquitination and acetylation in controlling UHRF1 function, the role of phosphorylation in regulating its stability has remained poorly understood [25]. Our findings identify UHRF1 as a direct substrate of AURKA and reveal phosphorylation as a critical determinant of UHRF1 protein stability. Importantly, we demonstrate that AURKA-mediated phosphorylation of UHRF1 suppresses its polyubiquitination and subsequent proteasomal degradation. Proteasome inhibition and ubiquitination assays support a model in which phosphorylation serves as a protective signal that shields UHRF1 from degradation, consistent with emerging concepts in protein homeostasis whereby phosphorylation can counteract ubiquitin-mediated turnover by interfering with E3 ligase recognition or enhancing protein folding and stability. Although the specific E3 ligase responsible for UHRF1 degradation in this context remains to be identified, our data provide a robust foundation for future investigations into the interplay between phosphorylation and ubiquitination in regulating UHRF1 dynamics.
Functionally, phosphorylation-dependent stabilization of UHRF1 is demonstrated to be essential for maintaining global DNA methylation. Rescue experiments demonstrate that only wild-type UHRF1, not the phospho-deficient mutant, can restore 5mC levels in UHRF1-depleted cells, emphasizing the significance of this post-translational modification in sustaining epigenetic integrity. Our MeDIP data further link UHRF1 phosphorylation to locus-specific methylation changes at the p16 and p21 promoters, showing that the phospho-deficient S76A mutant significantly reduces promoter methylation at both genes (Figure 6(C,D)). However, we wish to note an important distinction between our global and locus-specific methylation findings. While global 5mC measurements (DNA dot blot and ELISA) capture bulk genomic methylation changes, they do not identify which specific regions are affected. Global hypomethylation likely impacts numerous genomic loci beyond p16 and p21, and the observed phenotypic impairments may involve de-repression of additional genes that were not examined in this study. Disruption of the AURKA-UHRF1 axis leads to de-repression of genes such as p16 and p21 and impairs cell proliferation and clonogenic capacity; however, the causal relationship between promoter demethylation and gene de-repression remains to be fully established. We acknowledge that p16/p21 de-repression may not be exclusively attributable to loss of promoter methylation, as alternative mechanisms-including AURKA-dependent cell cycle perturbation, cellular stress responses, or genomic instability-may also contribute. Formal proof of causation would require targeted demethylation or methylation restoration experiments at specific promoters, which are beyond the scope of this study. Accordingly, we present the relationship between DNA methylation loss and tumor suppressor de-repression as a mechanistic hypothesis supported by correlative evidence, rather than a definitive causal mechanism. These results provide a mechanistic understanding of how AURKA contributes to tumor progression beyond its classical role in cell cycle regulation.
From a therapeutic perspective, our study suggests that targeting AURKA may exert dual antitumor effects by simultaneously disrupting mitotic progression and reversing epigenetic repression. Several AURKA inhibitors have been developed and evaluated in clinical trials; however, their efficacy as monotherapies has been limited [26]. Our findings raise the possibility that combining AURKA inhibition with epigenetic therapies, such as DNA methylation inhibitors, may enhance therapeutic responses by synergistically reactivating tumor suppressor pathways. Furthermore, given the central role of UHRF1 in multiple cancer types, the AURKA-UHRF1 axis may represent a broadly relevant vulnerability in oncology.
Despite these advances, several limitations merit consideration. First, the current study primarily relies on in vitro PCa models, and in vivo validation will be essential to confirm the physiological relevance of this pathway; future studies employing xenograft models or genetically engineered mice will be important to establish the in vivo significance of our findings. Second, although we identify phosphorylation as a key regulatory mechanism, the precise structural basis by which phosphorylation stabilizes UHRF1 remains to be elucidated. Third, the upstream signals that regulate AURKA activity in the context of epigenetic control are not fully defined and may involve oncogenic signaling pathways or cellular stress responses. Fourth, while our MeDIP data link UHRF1 phosphorylation to promoter methylation at p16 and p21, the causal chain from promoter demethylation to gene de-repression to phenotypic impairment has not been formally demonstrated, and additional genes beyond p16 and p21 likely contribute to the observed phenotypes.
In summary, this study reveals a novel mechanism by which AURKA maintains DNA methylation and promotes PCa progression through phosphorylation-dependent stabilization of UHRF1. These findings not only expand the functional repertoire of AURKA but also provide new insights into the integration of kinase signaling and epigenetic regulation in cancer. Targeting this newly identified axis may offer promising opportunities for therapeutic intervention in PCa.
Materials and methods
Cell culture
Human prostate cancer cell lines C4-2 was obtained from FENGHUI Biotechnology Corporation (Changsha, China) and PC3 was obtained from National Collection of Authenticated Cell Cultures (Shanghai, China). These cell lines were cultured under the recommended conditions and maintained in either RPMI-1640 or Ham’s F-12K medium. Both media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cultures were incubated at 37°C in a humidified incubator containing 5% CO2. Cell line authentication was performed by short tandem repeat (STR) profiling, and all cultures were routinely tested and confirmed to be free of mycoplasma contamination.
Antibodies and chemicals
The antibodies and chemicals employed in this study included anti-Flag (Sigma, F1804; Cell Signal Technology, CST, 14,793), anti-His (Genscript, A00186), anti-phosphoserine (Santa Cruz Biotechnology, Santa, sc-81516), anti-AURKA (CST, 14,475; Proteintech, 66,757–1-Ig), anti-UHRF1 (Proteintech, 21,402–1-AP; Santa, sc-373750), anti-HA (CST, 3724), anti-5-Methylcytosine (Active motif, 39,649; CST, 28,692), and anti-β-actin (Abclonal, AC004). Cycloheximide (CHX) was procured from CST, and the proteasome inhibitor MG132 was obtained from Chemicals Selleck (Selleck, Shanghai) and utilized at the specified concentrations.
siRNA transfection and plasmid overexpression
Small interfering RNAs (siRNAs) targeting AURKA and a non-targeting control siRNA were transfected into cells using a lipid-based transfection reagent according to the manufacturer’s instructions (Mirus, Madison, WA). For overexpression experiments, cells were transfected with plasmids encoding Flag-tagged AURKA or UHRF1 constructs using standard protocols. Wild-type and phosphorylation-deficient mutants of UHRF1 were generated by site-directed mutagenesis and verified by DNA sequencing. The siRNA sequences used for AURKA knockdown were (5’-3’): AURKA#1 (GAGUCUACCUAAUUCUGGA), AURKA#2 (CCUGUCUUACUGUCAUUCG).
Immunoblotting (Western blot)
Cells were lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined, and equal amounts of protein were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Polyvinylidene Fluoride (PVDF) membranes. Membranes were blocked and incubated with primary antibodies against AURKA, UHRF1, ubiquitin, and loading controls, followed by appropriate HRP-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence.
DNA dot blot assay
Genomic DNA was extracted using a commercial DNA isolation kit (CoWin Biosciences, Beijing, China). Equal amounts of denatured DNA were spotted onto nitrocellulose membranes and cross-linked. Membranes were blocked and incubated with anti-5-methylcytosine (5mC) antibody, followed by Horseradish Peroxidase (HRP)-conjugated secondary antibody. Signals were detected using chemiluminescence.
5mC quantification by ELISA
Global DNA methylation levels were quantified using a 5mC ELISA-based assay kit according to the manufacturer’s instructions (Epigentek Inc, Farmingdale, NY). Briefly, genomic DNA was bound to assay wells and incubated with capture and detection antibodies specific for 5mC. Absorbance was measured, and relative methylation levels were calculated.
Immunoprecipitation (IP) and co-immunoprecipitation (Co-IP)
Cells were lysed in IP lysis buffer, and clarified lysates were incubated with specific antibodies against AURKA or UHRF1 overnight at 4°C, followed by incubation with protein A/G agarose beads (MedChemExpress, Shanghai, China). Immunocomplexes were washed, eluted, and analyzed by immunoblotting. Reciprocal Co-IP experiments were performed to validate protein-protein interactions.
MeDIP
MeDIP analysis was performed using a MeDIP Kit (Active Motif) according to the manufacturer’s instructions. Briefly, cells with UHRF1 depletion were transfected with the specified plasmid. At 90% cell confluence, DNA was collected, restriction enzyme digestion, and incubated with antibodies on a rotator at 4 ◦C overnight, and precipitated with magnetic beads. DNA fragments were eluted and purified used for quantitative PCR (qPCR). The binding efficiency was calculated according to the manufacturer’s instructions. The primers are listed in Table S1.
Mass spectrometry analysis
PC3 cells expressing Flag-tagged AURKA were subjected to immunoprecipitation using anti-Flag beads. Eluted protein complexes were separated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify AURKA-interacting proteins.
GST (glutathione-S-transferase) pull-down assay
Recombinant GST-tagged UHRF1 and His-tagged AURKA proteins were expressed in bacteria and purified. GST pull-down assays were performed by incubating GST fusion proteins with AURKA, followed by precipitation with glutathione beads. Bound proteins were analyzed by immunoblotting.
In vitro kinase assay
AURKA was incubated with GST-UHRF1 in kinase buffer containing ATP. Reactions were terminated, and phosphorylation levels were analyzed by immunoblotting using phospho-serine specific antibodies. Site-directed mutants of UHRF1 were used to identify phosphorylation sites.
Cycloheximide chase assay
To assess protein stability, cells were treated with cycloheximide to inhibit protein synthesis. Cells were harvested at indicated time points, and UHRF1 protein levels were analyzed by Western blotting to determine protein half-life.
Ubiquitination assay
Cells were transfected with indicated constructs and treated with MG132 prior to harvest. Cell lysates were subjected to immunoprecipitation with anti-His antibody, followed by immunoblotting to detect polyubiquitinated UHRF1.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted using e RNAiso Plus and reverse-transcribed into cDNA (Takara Bio. Beijing, China). The mRNA level of genes was measured by an applied biosystems qTOWER3G Real-Time PCR system (Analytik Jena, Germany) according to the manufacturer’s instructions. Primers for RT-PCR were listed in Table S1.
Statistical analysis
All experiments were performed with at least three independent biological replicates. Data are presented as mean ± standard deviation (SD). Statistical significance was determined using Student’s t-test as appropriate. A p-value < 0.05 was considered statistically significant [27].
Supplementary Material
Acknowledgments
Conceptualization: L.X. Investigation: X.Q., P.Y.C., Y.B.Y. Writing-original draft: X.Q., P.Y.C. Resources: L.X. Supervision: L.X. Funding acquisition: L.X., P.Y.C. Writing-review & editing: L.X.
Funding Statement
This work was supported by the National Natural Science Foundation of China (NSFC 82303867 and NSFC 81572542); National Natural Science Foundation of Guangdong Province [Grant number 2026A1515012742]; Project of Joint cultivation Fund of Guangdong Pharmaceutical University-Songgang People Hospital, Baoan District, Shenzhun (SGYY2024A005); Key Engineering Team, Discipline Cultivation Projects, Guangdong Pharmaceutical University (2024ZZ10); Basic Research Program of Guangzhou City (SL2024A04J00837).
Disclosure statement
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics declaration: not applicable.
Data availability statement
The datasets generated during the current study are available in the Mendeley Data repository (DOI: 10.17632/3kj8mrccmx.1). Further information is available from the corresponding author upon reasonable request.
Supplementary Information
Supplemental data for this article can be accessed online at https://doi.org/10.1080/15592294.2026.2719245
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets generated during the current study are available in the Mendeley Data repository (DOI: 10.17632/3kj8mrccmx.1). Further information is available from the corresponding author upon reasonable request.