Acetylation modification of AIM2 by KAT2B suppresses the AKT/Wnt/β-catenin signaling pathway activation and inhibits breast cancer progression

Abstract

Background

The development of breast cancer is known to be greatly influenced by epigenetic changes. The impact of histone acetyltransferase KAT2B on AIM2 and AKT/Wnt/β-catenin signaling have not been studied yet.

Methods

In this study, clinical breast cancer tissue and para-cancer tissue samples were collected from 60 breast cancer patients, and correlations between AIM2 expression and pathological parameters were analyzed. Breast cancer cell lines were obtained for in vitro studies, and AIM2 overexpression or KAT2B knockdown models were constructed. The CCK8 and Edu assay were conducted to measure cell proliferation, and cell invasion was determined by Transwell analysis. For mRNA and protein expression measurement, RT-qPCR and western blotting were utilized, respectively. Co-immunoprecipitation was used to investigate the interaction between KAT2B and AIM2. Animal models were established using BALB/c-nu mice through subcutaneous injection with breast cancer cells transfected with AIM2 K90R mutant vectors. Expression of Ki-67, KAT2B and AIM2 AcK90 was measured using immunohistochemistry.

Results

The clinical samples showed that AIM2 was downregulated in breast cancer tissues and was linked to lymph node metastases and advanced clinical stage. Subsequently, the in vitro studies found that AIM2 exerted a suppressive impact on the growth, spread, and invasion of breast cancer cells. We further demonstrated that KAT2B mediates acetylation of AIM2 at the lysine 90 residue, which suppresses cancer cell growth, invasion, and migration through inhibiting the AKT/Wnt/β-catenin axis. In animal models, we further confirmed that acetylation of AIM2 inhibited the stimulation of the AKT/Wnt/β-catenin axis, thereby suppressing breast cancer growth in vivo. Finally, we proved that the KAT2B and acetylation of AIM2 correlated with the prognosis of clinical breast cancer.

Conclusion

Our study suggests that KAT2B-mediated acetylation of AIM2 can suppress the stimulation of the AKT/Wnt/β-catenin axis, consequently inhibiting breast carcinoma progression.

Highlights

AIM2 downregulation links to poor breast cancer prognosis.

KAT2B acetylates AIM2 to suppress breast cancer progression.

AIM2 acetylation inhibits the AKT/Wnt/β-catenin signaling.

KAT2B and AIM2 acetylation levels correlate with clinical breast cancer prognosis.

Introduction

Breast cancer represents a complicated disease that is influenced by both hereditary and epigenetic mechanisms [1]. Breast cancer can grow and progress due to epigenetic alterations, such as DNA modification and alterations to histone which are important regulators of gene expression [2, 3]. Recent research has highlighted the significant impact of these modifications on genes involved in vital processes including apoptosis, repairs to DNA, and cell cycle control [4]. For instance, in breast cancer, the overexpression of histone methyltransferase EZH2 has been observed to promote tumor growth by silencing tumor suppressor genes [5]. Additionally, methylation of the BRCA1 promoter was proved to link to downregulation of BRCA1 expression, thereby increasing the risk of breast cancer [6].

AIM2 is a cytosolic protein that acts as a sensor for cytosolic DNA and forms inflammasomes to activate caspase 1, thereby facilitating inflammatory cytokines generation [7]. Previous studies revealed AIM2 has a dual function in different cancer types, with its expression level and function varying depending on the specific cancer [8]. In the context of breast cancer, AIM2 has been discovered to enhance breast cancer cell apoptotic rate by upregulating Caspase-3 and DFNA5 [9]. Although AIM2 is downregulated in breast cancer tissues, its post-translational modifications and regulatory mechanisms remain poorly understood. Given that protein acetylation plays a crucial role in tumor progression, we hypothesized that AIM2 may be subject to acetylation, which could influence its tumor-suppressive functions in breast cancer.

KAT2B, a histone acetyltransferase, was reported to be involved in the activation of chromatin and transcription of DNA by acetylating lysine residues on core histones [10]. It controls the expression of genes and participates in a number of cellular activities, including the control of the cell cycle and the repair of damaged DNA [11]. Downregulation of KAT2B has been observed in breast cancer through analysis using the bioinformatics database GEPIA2.0 [12]. Furthermore, the PLMD database predicts acetylation sites on AIM2, suggesting that KAT2B is likely to acetylate and modify AIM2 at sites K90, K343, and K348 [13]. However, the effects of KAT2B on AIM2 acetylation in breast cancer have not yet been investigated.

Cellular functions such as cell growth, differentiation, and death are tightly affected by the AKT/Wnt/β-catenin system [14]. This pathway is abnormally activated in many cancer types, including breast cancer, and it aids in the onset and evolution of the illness by encouraging cell proliferation and preventing apoptosis [15]. Bim, a protein that promotes apoptosis, has been discovered to be downregulated by the AKT signaling pathway, increasing the survival of breast cancer cells [16]. Additionally, stimulation of the Wnt/β-catenin axis promotes self-renewal of breast cancer stem cells and facilitates cancer growth [17].

Based on the above information, our hypothesis suggests that KAT2B-mediated acetylation of AIM2 can suppress the activated AKT/Wnt/β-catenin axis, consequently inhibiting the progression of breast cancer. This hypothesis aims to present a viable biological target and alternative strategy for clinical therapy of breast cancer.

Methods

Clinal samples

Totally 60 patients with breast cancer voluntarily participated in the research after providing written informed consent and receiving ethical approval. The patients were fully informed about the study’s purpose and potential risks. Inclusion criteria required patients diagnosed with breast cancer, without prior treatment, and willing to participate. Patients with other types of cancer or a history of previous cancer treatment were excluded. The hospital’s institutional review board granted the study procedure their approval. During surgery, tissues of breast cancer and adjacent samples were isolated. The collected samples were anonymized to ensure patient confidentiality and promptly preserved in RNAlater solution (Thermo Fisher Scientific, Waltham, MA, USA) at -80 °C to maintain RNA integrity until further use. In addition, clinical and pathological parameters of the patients were collected, and Chi-square test was implemented to assess the relationships between AIM2 level and pathological characteristics. Kaplan-Meier method was implemented for assessing the effects of AIM acetylation on survival over time.

Cell culture

Human normal breast epithelial cell line MCF10A and breast cancer cell lines (MCF7, T47D, BT474, MDA-MB-231, and BT549) were obtained from ATCC and grown in grown in Ham’s F12 medium/DMEM (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. A humidified environment with 5% CO₂ was used for cell maintaining at 37 °C. The cultivated mediums was refreshed every three days, and cells were passaged when confluence reached 80%.

Vectors

Overexpression plasmids for AIM2 (oe-AIM2) and the KR mutants (K90R, K343R, K348R), or siRNA targeting KAT2B (si-KAT2B) were synthesized via the manufacturer (GeneChem Co., Ltd., Shanghai, China). Flag-AIM2, HA-p300, HA-CBP, HA-TIP60, HA-KAT2A, HA-KAT2B were provided by Institute of Biochemistry and Cell Biology, Shanghai, China.

Cell transfection

Breast cancer cells were inoculated into a 6-well plate, and transfection of vectors was implemented by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), following the protocol of manufacturers. After 48 h of transfection, cells were collected for next investigations. Transfection efficacy was confirmed by quantitative real-time PCR (RT-qPCR) or western blotting to determine the levels of AIM2 or KAT2B. Transfection of negative control cells was implemented with empty vectors (oe-NC) or non-targeting siRNA (si-NC).

Cell proliferation

Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8, Solarbio, Beijing, China). Breast cancer cells were seeded in a 96-well plate (2000 cells/well) and incubated for 24, 48, and 72 h. Absorbance at 450 nm was measured using a microplate reader (BioTek, Winooski, VT, USA). Additionally, Edu staining was performed by incubating cells with Edu solution (Solarbio, Beijing, China), fixing with 4% paraformaldehyde, and staining with DAPI. The ratio of Edu-positive cells was analyzed using ImageJ software. Each experiment was conducted in triplicate.

Cell invasion

For assessing the invasive potential of breast cancer cells, Transwell invasion assays (Corning, NY, USA) were employed. Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was applied to the upper chamber of the Transwell insert, and DMEM/F12 medium with 10% FBS was placed in the lower chamber. Breast cancer cells were seeded into the upper chamber (1 × 10⁵ cells/well) in serum-free DMEM/F12 medium. After 24 h of incubation, non-invading cells on the top layer of the insert were eliminated. The bottom surface’s invasive cells were stained with 0.1% crystal violet after being fixed with 4% paraformaldehyde. Subsequently, the number of invading cells was calculated in five randomly chosen fields per well using a microscope (Olympus, Tokyo, Japan). For each circumstance, three separate trials were conducted.

RT-qPCR

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using the PrimeScript RT reagent kit (Takara Bio, Shiga, Japan). RT-qPCR was performed with SYBR Premix Ex Taq II (Takara Bio) on a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA). The relative expression of genes was calculated using the 2^−ΔΔCt method and normalized to β-actin. The primers used were:

AIM2-F: 5’-AGGCTGCTACAGAAGTCTGTCC-3’.

AIM2-R: 5’-TCAGCACCGTGACAACAAGTGG-3’.

KAT2B-F: 5’-GAAGAGAACAGAAGCTCCAGG-3’.

KAT2B-R: 5’-GCAATTGGTAAAGACTCGCTG-3’.

β-actin-F: 5’-TGGCACCACACCTTCTACAA-3’.

β-actin -R: 5’-CCAGAGGCGTACAGGGATAG-3’.

Western blotting

Cells and tissues were lysed using RIPA buffer (Beyotime, Shanghai, China) with protease and phosphatase inhibitors. Protein concentrations were measured using a BCA protein assay kit (Beyotime). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat milk and incubated with primary antibodies against AIM2 (ab93015, 1:1000), KAT2B (ab56275, 1:1000),

p-AKT (ab38449, 1:1000), AKT (ab8805, 1:1000), p-GSK3β (ab68476, 1:1000), GSK3β (ab32391, 1:1000), β-catenin (ab32572, 1:1000) or β-actin (ab8226, 1:1000). After washing, membranes were incubated with HRP-conjugated secondary antibodies and visualized using an ECL detection system (Thermo Fisher Scientific, Waltham, MA, USA). Band intensity was quantified using ImageJ software.

Co-immunoprecipitation (Co-IP)

To investigate the relations regarding KAT2B and AIM2, Co-IP was performed. In brief, cells were treated by cell transfection (Flag-AIM2 and related KR mutants, HA-p300, HA-KAT2A, HA-KAT2B, HA-TAP60, HA-CBP) according to various groups. 48 h post transfection, the extracted proteins were incubated with primary antibodies against KAT2B (ab176316, 1:1000) or AIM2 (ab93015. 1:1000) overnight at 4 °C with gentle shaking. Following that, The mixture was then combined with Protein A/G magnetic beads (Bimake, Houston, TX, USA) and incubated for 2 h. The beads were then washed three times with cold PBS and eluted with 2× SDS loading buffer (Beyotime, Shanghai, China). The eluted proteins were visualized through western blotting, as described above. The specificity of the Co-IP was confirmed by using IgG as a negative control.

Acetylation analysis

The HEK293 cells were used for in vitro acetylation analysis as previously described [18]. Briefly, the cells were transfected with oe-AIM2 plasmids. For inhibiting HDAC activity, 48 h post transfection, cells were treated with 1 µM Trichostatin A (TSA) and 5 mM Nicotinamide (NAM) for 24 h. Then cell lysates were obtained as described above, pre-cleared with Protein A/G agarose beads and incubated overnight with a pan-acetyl-lysine antibody. Immunocomplexes were captured with Protein A/G beads, washed, and analyzed by Western blotting using anti-AIM2 antibody.

Moreover, for detection of acetylation of AIM2, the anti-AIM2 AcK90 antibody was synthesized by PTM Biolabs, Zhejiang, China. The acetylated AIM2 levels in cells or tissues were determined by western blotting as described above or immunohistochemistry (IHC) below, respectively.

Animal model

Athymic nude mice (BALB/c-nu, 5–6 weeks old) were procured from Shanghai Laboratory Animal Center (SLAC, Shanghai, China) and kept in the institution’s animal facility under specified pathogen-free conditions. All animal experiments adhered to the regulations for animal experiments of the institution and received approval from the Animal Care and Use Committee.

For establishing the breast cancer xenograft model, subcutaneous injection with 1 × 10⁷ breast cancer cells into the mice right flank was performed. The mice were randomly assigned into three groups (n = 5 in each): Control group, AIM2^WT group, and AIM2^KR group. Cells in AIM2^WT or AIM2^KR group were respectively infected with AIM2 wide-type or AIM2 K90R lentiviruses constructed by GeneChem Co., Ltd., Shanghai, China as per the protocol of manufacturers, after which the cells were screened by puromycin. Every four days, calipers were used to measure tumor growth, and the tumor volume was computed by the formula V = π/6 × L × W², where V represents volume, L is length, and W is width. After 28 days, the mice were euthanized by CO₂ asphyxiation, and the tumors were excised for subsequent experiments.

Immunohistochemistry (IHC)

Paraffin-embedded tumor tissues were sectioned (5 μm thick) and processed for IHC. Sections were deparaffinized, rehydrated, and antigen retrieval was performed. After blocking endogenous peroxidase activity, sections were incubated overnight at 4 °C with primary antibodies against Ki-67 (ab15580, 1:1000), KAT2B (ab176316, 1:1000) and AIM2 AcK90 antibody (PTM Biolabs, Zhejiang, China). The following day, sections were incubated with biotinylated secondary antibodies and an avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Visualization was done using a DAB substrate (Vector Laboratories) and counterstaining with hematoxylin. Ki-67 positive cells were counted using a microscope (Olympus, Tokyo, Japan) and the staining index was calculated.

To generate the K90 acetylation-specific antibody, we synthesized a peptide corresponding to the acetylation site at lysine 90 (K90) of the AIM2 protein, which was acetylated at the lysine residue. This peptide was conjugated to keyhole limpet hemocyanin (KLH) as a carrier protein and used to immunize rabbits. Multiple immunization boosts were performed, and the serum was collected after sufficient immune response was generated. For antibody purification, serum was subjected to affinity chromatography using an immobilized peptide corresponding to the K90 acetylated site. The purified antibodies were assessed for specificity via Western blotting, immunohistochemistry (IHC), and peptide competition assays.

Statistical analysis

The SPSS 22.0 software (SPSS, Chicago, IL) was used to analyze and show all data as mean standard deviation (SD). Student’s t-test was used to assess differences between two groups, while one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test were used to analyze differences between several groups. Statistics were considered significant for P values under 0.05.

Results

AIM2 is downregulated in breast cancer tissues and is related with poor prognosis

RT-qPCR and Western blotting were conducted to check the expression of AIM2 in breast cancer patients tissues. As depicted in Fig. 1A and B, AIM2 level was significantly lower in breast cancer tissues than para-cancer tissues. Further confirmation was obtained by performing RT-qPCR and Western blotting in MCF10A, MCF7, T47D, BT474, MDA-MB-231, and BT549 cells. AIM2 expression was significantly decrease in breast cancer cells when compared to MCF10A cells (Fig. 1C and D). Among these cells, MCF7 and MDA-MB-231 exhibited the most pronounced decrease in AIM2 expression levels, thus being selected for subsequent experiments. To assess the clinical relevance of AIM2 level in breast cancer, the connections between AIM2 levels and clinicopathological parameters was analyzed. As illustrated in Table 1, low AIM2 expression levels were obviously related with lymph node metastases, advanced clinical stage, HER2 status and Ki-67, indicating AIM2 may be used as a predictive marker.

Fig. 1

AIM2 expression is decreased in breast cancer tissues and cells. (A) RT-qPCR analysis and (B) Western blot analysis of AIM2 expression in breast cancer tissues and adjacent non-cancerous tissues (representative results from a total of 60 cases). β-actin served as a loading control. (C) RT-qPCR analysis and (D) Western blot analysis of AIM2 expression in normal breast epithelial cells (MCF10A) and breast cancer cell lines (MCF7, T47D, BT474, MDA-MB-231, BT549). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. *P < 0.05, **P < 0.01

Table 1 Correlations between AIM2 protein expression and clinical characteristics in breast cancer patients (n = 60)

AIM2 exerts inhibitory effects on proliferation, migration, and invasion of breast cancer cells

As demonstrated in Fig. 2A and B, AIM2 overexpression significantly elevated AIM2 levels in both cells. Subsequently, the impacts of AIM2 overexpression on cell viability were assessed by CCK-8 and Edu kits. As depicted in Fig. 2C and D, AIM2 overexpression significantly inhibited the growth of both cells. Transwell experiments were used to examine how AIM2 overexpression affected cell migration and invasion. Overexpressed AIM2 significantly suppressed cancer cells migration and invasion (Fig. 2E). Therefore, above findings imply that AIM2 may hold promise as a molecular target for breast cancer therapy.

Fig. 2

AIM2 overexpression inhibits proliferation, migration, and invasion of MDA-MB-231 and MCF7 cells. (A) RT-qPCR analysis of AIM2 expression. (B) Western blot analysis of AIM2 expression in MDA-MB-231 and MCF7 cells. (C) CCK-8 and (D) Edu assays showing reduced proliferation upon AIM2 overexpression. (E) Transwell assays demonstrating that AIM2 overexpression inhibits migration and invasion of breast cancer cells. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. *P < 0.05, **P < 0.01

AIM2 undergoes acetylation at the lysine 90 residue mediated by KAT2B

Since AIM2 expression is reduced in breast cancer, we next sought to investigate whether its acetylation status is also altered. Our analysis revealed significantly lower AIM2 acetylation levels in breast cancer tissues compared to normal tissues (Fig. 3A), suggesting a potential regulatory mechanism. To further explore the functional role of AIM2 acetylation, we utilized an overexpression model to rapidly assess the phenotypic effects of this modification. As depicted in Fig. 3B, AIM2 was found to be acetylated in HEK293 cells treated with the pan-HDAC inhibitor TSA and NAM. HDACs not only regulate the acetylation of histones but also catalyze the deacetylation of non-histone proteins [19]. To further verify this hypothesis, we conducted immunoprecipitation, revealing the interaction between AIM2 and KAT2B (Fig. 3C). Knockdown of KAT2B significantly reduced the acetylations of AIM2 (Fig. 3D), and co-immunoprecipitation demonstrated the KAT2B and AIM2 binding relations (Fig. 3E). Moreover, utilizing the GPS-PAIL database, we predicted three potential acetylation sites on AIM2 (K90, K343, and K348) that could be modulated by KAT2B. To determine the critical acetylation site, each of the three lysine residues was mutated to arginine (R), and the acetylation levels were analyzed. As demonstrated in Fig. 3F, only the K90R mutation significantly reduced the acetylation level of AIM2, highlighting K90 as the major acetylation site on AIM2. Furthermore, in cells overexpressing KAT2B, the acetylation levels of these mutants were considerably lower than that of wild-type AIM2 (Fig. 3G). Moreover, we confirmed the specificity of the K90 acetylation-specific antibody. The results revealed a clear band corresponding to the acetylated K90 form of wild-type AIM2 (Fig. 3H). In contrast, no band was observed when the K90R mutant was tested, demonstrating the loss of acetylation at this site. Additionally, there were no detectable signals in unrelated control proteins (Fig. 3H). Collectively, these findings indicate that AIM2 undergoes acetylation, and this dynamic post-translational modification is facilitated by the acetyltransferase KAT2B. Notably, our results suggest that K90 is the primary acetylation site on AIM2, offering a potential therapeutic target for diseases associated with AIM2 dysregulation.

Fig. 3

Acetylation of AIM2 mediated by KAT2B. (A) Lower AIM2 acetylation levels in breast cancer tissues compared to normal tissues (B) Western blot analysis showing AIM2 acetylation in HEK293 cells treated with TSA (1 µM) and NAM (5 mM) for 24 h. Immunoprecipitation was performed using a pan-acetyl-lysine antibody (deacetylase inhibitors were used to inhibit deacetylases only in this specific experiment). (C) Co-immunoprecipitation assay confirming the interaction between AIM2 and KAT2B. (D) Western blot analysis showing that KAT2B knockdown (si-KAT2B) reduces AIM2 acetylation. (E) Co-immunoprecipitation assay validating the interaction between KAT2B and AIM2. (F) Analysis of AIM2 acetylation levels upon mutation of lysine residues. (G) Reduced acetylation levels of AIM2 mutants in cells overexpressing KAT2B. (H) Validation of the K90 acetylation-specific antibody using wild-type AIM2 and K90R mutant AIM2 proteins. The band intensity showing a significant difference between WT and K90R mutant AIM2, confirming antibody specificity for the acetylation site. *P < 0.05, **P < 0.01

KAT2B regulates AIM2 acetylation to influence the AKT/Wnt/β-catenin pathway and breast Cancer cell proliferation, migration, and invasion

The expression of KAT2B was further analyzed in breast cancer tissues and cells. Compared with normal tissues, the mRNA and protein levels of KAT2B were significantly decreased in breast cancer tissues (Figure S2A and S2B). Similarly, in comparison with the human normal breast epithelial cell line MCF10A, the mRNA and protein levels of KAT2B were notably downregulated in all five breast cancer cell lines tested: MCF7, T47D, BT474, MDA-MB-231, and BT549 (Figure S2C and S2D). These results indicate that KAT2B expression is significantly reduced in breast cancer at both the mRNA and protein levels, suggesting its potential involvement in breast cancer progression. To further confirm the regulatory role of KAT2B in AIM2 acetylation and its downstream effects, we conducted KAT2B knockdown experiments. Compared to the oe-NC + si-NC group, overexpression of AIM2 (oe-AIM2 + si-NC) significantly suppressed the AKT/Wnt/β-catenin signaling pathway, leading to a reduction in breast cancer cell proliferation, migration, and invasion. Conversely, knockdown of KAT2B alone (oe-NC + si-KAT2B) enhanced the AKT/Wnt/β-catenin pathway, promoting tumor cell proliferation and metastatic potential. Importantly, when AIM2 was overexpressed in the context of KAT2B knockdown (oe-AIM2 + si-KAT2B), the tumor-suppressive effects of AIM2 overexpression were reversed, supporting the notion that KAT2B-mediated AIM2 acetylation is required for its inhibitory role in breast cancer progression (Fig. 4A–E). These findings provide strong evidence that KAT2B regulates AIM2 acetylation, which in turn modulates breast cancer cell growth and invasion via the AKT/Wnt/β-catenin axis.

Fig. 4

KAT2B regulates AIM2 acetylation and its effects on the AKT/Wnt/β-catenin pathway and breast cancer progression. (A) CCK-8 assay and (B) Edu staining showing that AIM2-induced suppression of proliferation is reversed by KAT2B knockdown. (C) Transwell migration and invasion assays showing that AIM2-induced suppression is reversed by KAT2B knockdown. (D) RT-qPCR and (E) Western blot analysis showing that knockdown of KAT2B in AIM2-overexpressing cells reverses the suppressive effect of AIM2 acetylation. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. *P < 0.05, *P < 0.01

Acetylation of AIM2 at K90 suppresses cancer cell proliferation, invasion, and migration by inhibiting the AKT/Wnt/β-catenin axis

To examine the functional significance of AIM2 acetylation, a K90R mutant was generated to disrupt the acetylation modification of AIM2. Comparing the K90R mutant to wild-type AIM2, it was discovered that the latter promoted cell proliferation, invasion, and migration (Fig. 5A-C). To elucidate the putative mechanism regarding the anti-tumor properties of AIM2 acetylation, the AKT/Wnt/β-catenin axis was investigated, as it was reported to be vital for cancer cell proliferation, survival, and metastasis. As illustrated in Fig. 5D, the K90R mutant significantly stimulated the AKT/Wnt/β-catenin axis, as evidenced by increased expression of phosphorylated AKT and GSK3β, and enhanced level of β-catenin. Overall, our data suggest that acetylation of AIM2 at K90 inhibits cancer cell growth, invasion, and migration by suppressing the AKT/Wnt/β-catenin axis. The evidences offer fresh insights into the AIM2 functions and propose that targeting AIM2 acetylation may hold therapeutic potential for cancer treatment.

Fig. 5

Impact of AIM2 acetylation on cancer cell proliferation, invasion, and migration through the AKT/Wnt/β-catenin axis. (A) CCK-8 assay and (B) Edu staining revealing increased proliferation in cells expressing the K90R AIM2 mutant. (C) Transwell assay demonstrating enhanced migration and invasion of cells expressing the K90R AIM2 mutant. (D) Western blotting showing activated AKT/Wnt/β-catenin axis in cells expressing the K90R AIM2 mutant. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. *P < 0.05, **P < 0.01

Acetylation of AIM2 inhibits the activation of the AKT/Wnt/β-catenin axis, thereby suppressing breast cancer growth in vivo

For assessing the impact of AIM2 acetylation on breast cancer growth, the xenograft tumor model was constructed. The mice were assigned into 3 groups: Control, AIM2^WT (the same as AIM2 overexpression), and AIM2^KR. As illustrated in Fig. 6A-B, the AIM2^KR group exhibited significantly enhanced tumor volume and weight when compared to the AIM2^WT group. To delve into the potential mechanism of the anti-tumor ability of AIM2 acetylation, the levels of Ki67, a well-established growth marker, were examined in tumor tissues. As demonstrated in Fig. 6C, the AIM2^KR group displayed markedly higher Ki67 expression levels in comparison to the AIM2^WT group. Additionally, the AKT/Wnt/β-catenin signaling pathway was assessed in tumor tissues via Western blotting. AIM2 wild-type overexpression reduced AKT/Wnt/β-catenin pathway activation, but this effect was lost in the K90R mutant, indicating that AIM2 acetylation at K90 is required for its inhibitory function (Fig. 6D). These results collectively demonstrate that acetylation of AIM2 impedes the upregulation of the AKT/Wnt/β-catenin axis, leading to the suppression of breast cancer growth in vivo.

Fig. 6

Effects of AIM2 acetylation on breast cancer growth in vivo. (A) Tumor volume growth curve showing increased tumor volume in mice injected with AIM2^KR cells compared to AIM2^WT cells. (B) Tumor weight showing significantly increased tumor weight in mice injected with AIM2^KR cells compared to AIM2^WT cells. (C) Immunohistochemistry analysis of Ki67 expression demonstrating higher proliferation in tumors derived from AIM2^KR cells. (D) Western blotting revealing activated AKT/Wnt/β-catenin axis in tumors derived from AIM2^KR cells. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. *P < 0.05, **P < 0.01

The acetylation of AIM2 and KAT2B and correlates with the prognosis of clinical breast cancer

We then investigated the relationship between AIM2 (K90) acetylation levels and the incidence or metastasis of human tumors. The acetylation levels of AIM2 (K90) in human breast cancer tissue samples were analyzed through IHC staining, revealing significantly decreased acetylation levels of AIM2 (K90) in breast cancer tissues (Fig. 7A and S1). Since AIM2 (K90) acetylation is facilitated by KAT2B in cells, we further explored the potential association between these factors and the incidence or metastasis of human breast cancer. KAT2B protein expression were subsequently examined, highlighting significantly lower level of KAT2B in breast cancer tissues than in corresponding para-cancer tissues (Fig. 7A and S1). Moreover, low levels of KAT2B protein and AIM2 (K90) acetylation were related with a poor prognosis in breast cancer patients (Fig. 7B). These results emphasize that AIM2 (K90) acetylation, mediated by KAT2B, inhibits breast cancer development, progression, and metastasis.

Fig. 7

Association between KAT2B and AIM2 acetylation and clinical prognosis in breast cancer. (A) Immunohistochemistry analysis showing decreased AIM2 (K90) acetylation levels and KAT2B expression in breast cancer tissues compared to para-cancer tissues. The K90 acetylation-specific antibody was used to detect AIM2 acetylation at the K90 site. (B) Kaplan-Meier survival analysis demonstrating the association of low KAT2B and AIM2 (K90) acetylation levels with poor prognosis in breast cancer patients. The analysis was performed using the log-rank test. *P < 0.05, **P < 0.01

Discussion

The AIM2’s functions in breast cancer have been extensively investigated in recent years. A previous study demonstrated a correlation between reduced level of AIM2 in breast cancer tissues and poor prognosis [20]. Additionally, Shah et al. revealed a link between the absence of AIM2 and the development of colorectal cancer, with overexpression of AIM2 significantly suppressing the growth of BRAF-mutant colorectal cancer cells [21]. It was demonstrated that AIM2 expression can suppress the levels of the anti-apoptotic protein Bcl-xL, thereby influencing breast cancer cell survival and proliferation through apoptosis [22]. Consistently, our study further supports these findings by demonstrating significantly decreased levels of AIM2 in breast cancer tissues, while upregulation of AIM2 inhibits proliferation and invasion of breast cancer cells in vitro. Furthermore, this research explores the inhibitory effects of AIM2 in breast cancer by investigating the connections between AIM2 acetylation and breast cancer progression.

KAT2B participates in the acetylation of various proteins and gene expression regulation. Recent studies have implicated KAT2B in the acetylation modification of various proteins and its association with cancer [23]. Guo et al. suggested that histone acetylation modifications significantly affect breast cancer aggressiveness and therapeutic potential [24]. Dysregulated KAT2B has also been reported to strongly influence tumor proliferation and metastasis in breast cancer through controlling genes involved in cell cycle and epithelial-mesenchymal transition [25, 26]. Consistent with these findings, the present study firstly demonstrates that KAT2B interacts with AIM2 for acetylation modification in breast cancer cells. The inhibitory effect of KAT2B-mediated AIM2 acetylation on breast cancer progression further supports the emerging understanding of KAT2B as a critical regulator in breast cancer.

While our study provides new insights into AIM2 acetylation at K90 and its role in breast cancer progression, key questions remain regarding whether the K90R mutation’s effects are solely due to loss of acetylation or other structural changes. The mutation may alter AIM2’s conformation, disrupting critical protein-protein interactions and indirectly affecting its tumor-suppressive role via the AKT/Wnt/β-catenin pathway. Additionally, epistatic interactions between K90R and KAT2B knockdown warrant further investigation. If K90R is epistatic to KAT2B depletion, it may suggest a broader regulatory network, whereas a K90Q (acetylation mimic) mutant rescuing KAT2B knockdown effects would provide stronger evidence for acetylation-dependent regulation. While these experiments are beyond the scope of this study, they represent critical next steps. Despite these limitations, our findings support AIM2 acetylation as a key tumor-suppressive mechanism, and future site-directed mutagenesis, interaction studies, and structural analyses (e.g., X-ray crystallography or cryo-EM) will be needed to fully elucidate the molecular significance of K90 acetylation in AIM2 function.

We propose that the apparent contradiction between reduced AIM2 expression and its functional suppression through acetylation may be explained by the regulatory role of acetylation at the post-translational level. Even with lower AIM2 expression in breast cancer tissues, acetylation at K90 could modulate AIM2’s activity by altering its interaction with other proteins or its cellular localization, thus suppressing its tumor-suppressive functions. Additionally, the reduced expression of AIM2 may work in concert with acetylation and other cancer-associated factors to inhibit AIM2’s function, creating a permissive environment for tumor progression. This suggests that AIM2’s activity is regulated not only by its expression levels but also by acetylation, highlighting a complex layer of regulation that warrants further investigation.

The AKT/Wnt/β-catenin axis has been studied in various cancers, while its aberrant activation has been implicated in promoting tumor proliferation and metastases. A recent study found that stimulation of the AKT/Wnt/β-catenin axis promotes breast cancer cell growth and invasion by upregulating the level of cyclin D1 and c-Myc [27]. It has also been reported that AKT-targeted therapy holds promise as an approach to overcome drug resistance in breast cancer [28]. Similarly, other research has shown that targeting the Wnt/β-catenin axis could be effective in inhibiting breast cancer progression [29]. In this research, we not only confirmed the activated AKT/Wnt/β-catenin axis in breast cancer cells but also investigated the novel link between AIM2 acetylation and the suppression of this pathway.

In conclusion, our study demonstrated that KAT2B inhibited the activated AKT/Wnt/β-catenin axis through acetylation modification of AIM2, leading to the suppression of breast cancer progression. These findings enhance our comprehension on the biological mechanisms underlying breast cancer through a comprehensive investigation of AIM2, KAT2B, and the AKT/Wnt/β-catenin pathway, providing an alternative approach for clinical treatment. However, it is important to acknowledge that future research should aim to explore additional mechanisms and relevant signaling pathways involved in the regulation of AIM2 and KAT2B and investigate the therapeutic potential of targeting AIM2 acetylation in preclinical and clinical settings.