KAT2A‐H3K79succ‐Mediated SH2B3 Upregulation Promotes TNBC Metastasis Through Diminishing the Ubiquitin‐Dependent Degradation of Vimentin

ABSTRACT

Triple‐negative breast cancer (TNBC) is the most aggressive subtype among all breast cancer subtypes, attributed to the paucity of available targeted therapeutics. Lysine acetyltransferase 2A (KAT2A), an indispensable catalytic constituent of the transcriptional regulatory complex, has been implicated in the pathogenesis and progression of multiple malignancies. Nevertheless, the role of H3K79 site succinylation (H3K79succ) catalyzed by KAT2A in TNBC remains enigmatic. This study assessed the functional role of KAT2A in TNBC pathogenesis through colony formation assays, CCK‐8 assay, wound healing assays, and transwell invasion assays. BALB/c nude mice were employed to establish models for studying the proliferation and metastasis of TNBC in vivo. Transcriptome sequencing (RNA‐seq) combined with chromatin immunoprecipitation sequencing (ChIP‐seq) and Luciferase reporter assays were conducted to identify the direct transcriptional target of KAT2A‐catalyzed H3K79succ–SH2B adaptor 3 (SH2B3). Mass spectrometry and co‐immunoprecipitation (Co‐IP) assays were designed to investigate the mechanism of SH2B3 involved in promoting aggressiveness. Altogether, our study indicated that KAT2A modulates the malignant phenotype of TNBC by mediating H3K79succ to regulate the transcription of the SH2B3 gene, and the KAT2A/SH2B3/vimentin axis might be the potential molecular targets for diagnosing and treating TNBC.

Lysine acetyltransferase 2A (KAT2A) is a critical catalytic component within transcriptional regulatory complexes and plays a significant role in triple‐negative breast cancer (TNBC). To investigate its underlying mechanism, this study demonstrates that KAT2A catalyzes succinylation at the H3K79 site to promote SH2B3 transcription and enhance its expression. As an adaptor protein, SH2B3 binds to vimentin in TNBC cells and inhibits its TRIM21‐mediated ubiquitination and degradation. These findings identify the H3K79succ/KAT2A/SH2B3/vimentin axis as a key regulatory pathway in TNBC.

1. Introduction

According to global cancer statistics, breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women [1]. Currently, there is a paucity of effective therapeutic targets for triple‐negative breast cancer (TNBC), with a relatively limited range of therapeutic options and a propensity for distant, particularly visceral metastasis. Consequently, this particular subtype presents a significant challenge in clinical treatment and is associated with the poorest prognosis [2]. As a result of the advancement of fundamental research into TNBC, a number of prospective therapeutic targets have been identified. Therefore, chemotherapy combined with immunotherapy, antibody‐drug conjugate (ADC), PARP inhibitors, and androgen receptor antagonists has been gradually applied in clinical practice, and the prognosis of patients with TNBC has been improved. However, some patients still do not benefit from these new treatments [3, 4]. Therefore, it is particularly important to continue to seek potential molecular targets in TNBC and achieve individualized precision treatment for patients with TNBC to improve the prognosis.

Numerous studies have shown that histone modification plays an important role in the development of cancer as an important epigenetic regulatory mechanism [5, 6, 7, 8]. Lysine acetyltransferase 2A (KAT2A), also known as GCN5, is the first discovered histone acetyltransferase (HAT) that is involved in the construction of a variety of transcriptional regulatory complexes and can catalyze various acylation modifications of histones, such as acetylation, propionylation, and butyrylation [9, 10]. KAT2A has also been identified as a nuclear‐localized succinyltransferase in human cell lines [11], where it interacts with the α‐ketoglutarate dehydrogenase (αKGDH) complex at gene promoter regions. Site‐directed mutagenesis studies reveal that tyrosine 645 within a specific structural loop is critical for its preferential binding to succinyl‐CoA [12]. As a succinyltransferase, KAT2A catalyzes succinylation of histone H3 at lysine 79—a modification that occurs predominantly near transcription start sites [13]. Importantly, inhibition of αKGDH complex nuclear translocation or expression of the KAT2A (Tyr645Ala) mutant leads to reduced gene expression, suppressed tumor cell proliferation, and impaired tumor growth [12]. H3K122succ catalyzed by HAT1 significantly activated a large number of gene transcription in HepG2 hepatocellular carcinoma cells and promoted the progression of hepatocellular carcinoma [14]. In pancreatic ductal cancer cells, with high H3K79succ levels in the YWHAZ gene promoter region, tumor‐related protein degradation decreased, and tumor progression accelerated [15]. Studies have shown that in MCF‐7 breast cancer cells, KAT2A/E2F1 is recruited to the UBE2C gene promoter region to regulate the transcription of UBE2C by regulating the H3K9ac level, promoting the proliferation and migration of MCF‐7 cells [16]. Moreover, histone acetyltransferase inhibitors can further inhibit neuroblastoma proliferation by inhibiting KAT2A activity [17]. Lysine acetyltransferase (KAT) plays an important role in a variety of cancers driven by MYC [18]. Among which, it was found that the main component of the Chinese herb oridonin can inhibit the proliferation of breast cancer in vitro and in vivo by inhibiting KAT2A enzyme activity [19] and can also induce apoptosis of colorectal cancer cells [20]. All the above studies suggest that KAT2A can be a potential molecular target for various cancers. However, the roles of KAT2A and its catalyzed H3K79succ in TNBC pathogenesis remain unknown.

SH2B adaptor 3 (SH2B3), also known as lymphocyte adaptor protein (LNK), is mainly expressed in hematopoietic stem cells and lymphocytes [21]. It plays a pivotal role in hematopoietic cells' proliferation and differentiation, vascular endothelial repair, platelet diffusion and adhesion, and cytoskeletal regulation [22]. In the context of blood system diseases, the occurrence and development of leukemia are closely related to the presence of SH2B3 molecular mutations [23]. Functional mutations and single nucleotide polymorphisms (SNPs) of the SH2B3 gene can lead to the abnormal activation of the JAK–STAT signaling pathway, which may represent a novel mechanism of myeloproliferative neoplasms (MPN) [24]. However, the roles of SH2B3 in the occurrence and development of solid tumors are controversial. This molecule has been demonstrated to inhibit the progression of colorectal cancer, lung cancer, and other solid tumors through the regulation of JAK–STAT, Ras/MAPK, P13K, and other signal transduction pathways [25, 26]. However, in melanoma, thyroid cancer, ovarian cancer, and other tumors, upregulated SH2B3 is associated with a poor prognosis for patients [27, 28, 29]. Studies have demonstrated that SH2B3 variants are correlated with an augmented risk of breast cancer development in individuals carrying a BRCA1/2 mutation [30]. Evidence also implies that SH2B3 participates in the progression of TNBC by influencing the JAK/STAT3 pathway [31].

Our study found that KAT2A was highly expressed in the TNBC population and further confirmed that KAT2A can potentiate TNBC progression in vitro and in vivo. This study aims to explore the regulatory role of histone succinylation on oncogenes and to clarify the potential target gene SH2B3 and its clinical value and related mechanism in TNBC.

2. Materials and Methods

2.1. Patients and Samples

TNBC tumor samples and matched normal breast tissues were collected from 14 TNBC patients who underwent tumor resection at the Fourth Hospital of Hebei Medical University. Fine needle punctures of cancer tissues were collected from 21 patients who underwent neoadjuvant chemotherapy and surgery for TNBC at the Fourth Hospital of Hebei Medical University. Both tumor and non‐tumorous tissues were confirmed histologically. Informed consent was obtained from each patient, and the study protocol that conforms to the ethical guidelines of the Declaration of Helsinki was approved by the Institute Research Ethics Committee of the Fourth Hospital of Hebei Medical University (2021 k‐1053).

2.2. Cell Culture and Transfection

The human TNBC cell lines, MDA‐MB‐231 and MDA‐MB‐468, employed in the experiments were procured from Procell Life Science & Technology (Wuhan, China), and their authenticity was verified through Short Tandem Repeat (STR) analysis. MDA‐MB‐231 and MDA‐MB‐468 cell lines were cultured in DMEM (Gibco, Carlsbad, CA, United States) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, United States) and 1% penicillin–streptomycin (P/S) (NCM Biotech, Suzhou, China) at 37°C in air with 5% CO2. Small interfering RNAs (siRNAs) targeting the mRNA of KAT2A and SH2B3 were synthesized by GenePharma (Shanghai, China). For transient knockdown studies, a final concentration of 60 nM of siRNAs was transfected for 6 h according to Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

KAT2A‐lentivirus (KAT2A) and KAT2A shRNA‐lentivirus (shKAT2A‐1/2), and SH2B3‐lentivirus (SH2B3) and SH2B3 shRNA‐lentivirus (shSH2B3) were produced by GenePharma (Shanghai, China). Stable cell lines were established by infecting cells with lentiviral supernatants for 36 h, followed by selection with 5 μg/mL puromycin (Sigma, St. Louis, USA) for 48 h, in accordance with the manufacturer's instructions. Then, the infected cells were passaged before use after identification by western blot (WB).

2.3. Clone Formation Assay

Cell counting kit‐8 (CCK‐8) assay (Solarbio, Beijing, China) was used to determine the short‐term cell proliferation. Briefly, the cells were seeded onto the 96‐well plates (1000 cells/well). A total of 10 μL of the CCK‐8 solution was added into the fresh culture medium (100 μL) and incubated for 1 h. The optical density was assessed using a microplate reader (TECAN, Männedorf, Switzerland) at 450 nm. Each assay was carried out in triplicate. Cells (800 cells per well) were seeded into a 6‐well plate and incubated for 2 weeks until cell clones appeared. Then, the colonies of cells were fixed with polyoxymethylene and stained with crystal violet solution (Solarbio, Beijing, China).

2.4. Apoptosis Assay

Adherent cells were collected by trypsin digestion without EDTA. Then, the cells were washed twice with PBS and centrifuged at 2000 rpm for 5 min to collect 1–5 × 10⁵cells. Next, the harvested cells were subsequently resuspended with the binding buffer and incubated with Annexin V‐FITC for 10 min, subsequently incubated with Propidium Iodide (PI) for 5 min. Finally, the cells were blended with PBS and detected using flow cytometry within 1 h (ANNEXIN V‐FITC/PI Apoptosis Detection Kit, solarbio, Beijing, China).

2.5. Migration and Invasion Assay

MDA‐MB‐231 and MDA‐MB‐468 cells were maintained in a serum‐free medium for 24 h. For the migration assay, a total of 3 × 10⁴ cells were seeded in a serum‐free medium in the upper chamber (Corning, Beijing, China). Besides, the lower chamber was filled with DMEM containing 10% FBS. The cells were then fixed in 4% paraformaldehyde for 15 min and stained with crystal violet after 48 h. For the invasion assay, the membranes were covered with Matrigel (BD Biosciences, New Jersey, USA). The migrated cells were stained and counted in five different fields.

2.6. Wound Healing Assay

MDA‐MB‐231 and MDA‐MB‐468 were collected, counted, and added to a six‐well plate at 1 × 10⁶ cells per well. The incubator was placed overnight until the cells were attached. Then, the cells were wounded with a sterile plastic tip. Cell migration was observed and photographed by microscopy (OLYMPUS EP50, Tokyo, Japan) at different times.

2.7. RNA Extraction and Quantitative Reverse Transcription‐Polymerase Chain Reaction (qPCR)

Total RNA was isolated from BC tissue and cells using TRIzol (Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions. RNA was reverse transcribed using TransScript SuperMix (TransGen, Beijing, China). qPCR was performed using TransStart Top Green qPCR SuperMix (TransGen, Beijing, China). The levels of candidate mRNAs were normalized by the GAPDH expression. All values were standardized with the 2^−ΔΔCT method. Primers are listed in Table S1.

2.8. Transcriptome Sequencing, Chromatin Immunoprecipitation Sequencing (ChIP‐Seq), and ChIP‐qPCR

RNA‐seq and ChIP‐seq were performed by IGENEBOOK (IGENEBOOK Biotechnology, Wuhan, China). First, total RNA from MDA‐MB‐231 cells was extracted from with or without KAT2A deficiency, which was then subjected to MGI T7. Each RNA sample was designed to have three biological replicates and was finally mixed to analyze further minimizing the variations among samples. The genome (hg38) based on the Hisat2 (version 2.1.0) tool was utilized to map the final transcriptome reads. The gene expression data were further normalized using FPKM (Fragments per kilobase of transcript per million fragments mapped). Differentially expressed genes were identified with edgeR (v3.36.0) with a filter threshold of FDR < 0.05 and |log2FoldChange| > 1. ClusterProfiler (v4.2.2, http://www.bioconductor.org/packages/release/bioc/html/clusterProfiler.html) in R package was employed to perform GO (Gene Ontology, http://geneontology.org/) and KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.genome.jp/kegg/) enrichment analysis. The GO and KEGG enrichment analyses were calculated using hypergeometric distribution with a p‐value cut‐off of 0.05.

For the ChIP‐seq experiment, the MDA‐MB‐231 cells were cross‐linked and lysed, and sonication was used to shear the DNA fragments into 200–500 bp. The ChIP‐seq assays were conducted via Active Motif using the KAT2A antibody (Cat#3305, CST, MA, USA). Trimmomatic (version 0.36) was used to filter out low‐quality reads. Clean reads were mapped to the GRCh38.94 genome by Bwa (version 0.7.15). Samtools (version 1.3.1) was used to remove potential PCR duplicates. MACS2 software (version 2.1.1.20160309) was used to call peaks by default parameters (bandwidth, 300 bp; model fold, 5, 50; q‐value, 0.05). If the midpoint of a peak is located closest to the TSS of one gene, the peak will be assigned to that gene. HOMER (version 3) was used to predict motif occurrence within peaks with default settings for a maximum motif length of 12 base pairs. ClusterProfiler (http://www.bioconductor.org/packages/release/bioc/html/clusterProfiler.html) in R package was employed to perform GO (Gene Ontology, http://geneontology.org/) and KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.genome.jp/kegg/) enrichment analysis. The GO and KEGG enrichment analyses were calculated using hypergeometric distribution with a q‐value cut‐off of 0.05.

For the ChIP‐qPCR assays, qPCR was conducted following the ChIP assay. Relative occupancy of the promoter region of SH2B3 was normalized to the normal rabbit IgG (Cat#2729, CST, MA, USA).

2.9. Western Blotting

The cells were collected and lysed using RIPA lysates with a protease inhibitor (Solarbio, Beijing, China). Then, the protein samples were loaded into 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, MA, USA). After blocking with 5% non‐fat dry milk for 1 h at room temperature (21°C–25°C), the membranes were incubated with the specific primary antibody overnight. After washing in TBST four times in 20 min, the membranes were then incubated with the secondary antibodies (peroxidase‐labeled anti‐mouse and anti‐rabbit antibodies) for 1 h. Briefly, the primary antibodies were utilized at a dilution according to the manual, while the secondary antibodies were diluted 1:10000. After washing in TBST four times in 40 min, the membranes were finally visualized in an NcmECL SuperUltra Reagent (NCM Biotech, Suzhou, China). The detailed information of the primary antibodies was summarized as follows: KAT2A (Cat#3305, CST, MA, USA), H3K79succ (PTM‐412, PTM BIO, Hangzhou, China), SH2B3 (#DF9898, Affinity Biosciences, USA), H3 (17168–1‐AP, Proteintech, Wuhan, China), E‐cadherin (20874–1‐AP, Proteintech, Wuhan, China), N‐cadherin (22018–1‐AP, Proteintech, Wuhan, China), vimentin (60330–1‐Ig, Proteintech, Wuhan, China), GAPDH (10494–1‐AP, Proteintech, Wuhan, China).

2.10. Animal Experiments

The 4–6‐week‐old BALB/c Nude mice were purchased from Mu Beijing Vital River Laboratory Animal Technology Co (Beijing, China), which were maintained in our institutional pathogen‐free mouse facilities. Briefly, 6 × 10⁶ indicated MDA‐MB‐231 cells were suspended in 150 μL of PBS buffer and injected into the flanks of male nude mice. Tumor growth of mice was monitored every 5 days and the maximum diameter of the mouse tumor was no more than 15 mm, for a total period of 30 days. At the end of 8 weeks, all mice were sacrificed, and in vivo solid tumors were dissected and analyzed. Luciferase‐labeled MDA‐MB‐231 cells were injected systemically via the tail vein (2 × 10⁶ cells/per mouse) to generate a metastatic model to mimic breast cancer lung metastasis. Evidence of lung metastasis was determined in real time by luciferase‐based noninvasive bioluminescence imaging using the IVIS Spectrum CT Imaging System (PerkinElmer, Massachusetts, US). All experimental protocols were reviewed and approved by the Ethics Review Committee for Animal Experimentation of Hebei Medical University.

2.11. Immunofluorescence (IF)

The cells were inoculated onto a sterile lid of a 6‐well culture plate. After 24 h, the cells were fixed with 4% paraformaldehyde and washed three times using PBS. Subsequently, 0.1% Triton X‐100 permeated the cells at room temperature for 10 min and was sealed at room temperature with an Immunostaining blocking solution (P0102, Beyotime, Shanghai, China) for 1 h. The cells were then submerged and incubated at 4°C overnight. After extensive washing, they were incubated with FITC or enzyme‐labeled secondary antibody at room temperature for 1 h. The nuclei were stained with DAPI at room temperature for 10 min. Finally, fluorescence was observed under a laser scanning confocal microscope (LSM900 Airyscan2 ZEISS, Oberkochen, Germany).

2.12. Coimmunoprecipitation (Co‐IP)

CoIP was performed using Dynabeads Protein G Immunoprecipitation Kit (10007D, Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's protocols. Add antibody (typically 2 μg) diluted in 200 μL of Ab Binding and Washing Buffer to 30 μL magnetic beads, which were pretreated with Ab Binding and Washing Buffer and incubated at 4°C for 2 h in a flip mixer. Proteins extracted from cells using RIPA lysates with protease inhibitor (Solarbio, Beijing, China) were fully suspended with the antibody‐magnetic‐bead complexes and incubated at 4°C overnight in a flip mixer. After thorough washing, add 20 μL Elution Buffer and 10 μL of premixed NuPAGE LDS sample, and heat for 10 min at 70°C. The supernatant was subjected to Western blot analysis.

2.13. Immunohistochemical (IHC) Staining and Scoring

Human TNBC specimens and the database were obtained from the fourth Hospital of Hebei Medical University. The detailed clinical pathological data were scored on the basis of the tumor classification of the American Joint Committee on Cancer (AJCC) tumor staging system. The complete data of patient characteristics are shown in Table S2. Immunohistochemistry assay was performed as previously described33. Human TNBC tissues were stained with antibodies against KAT2A (Cat#3305, CST, MA, USA) and SH2B3 (#DF9898, Affinity Biosciences, USA). The IHC scores were assessed by two independent authors blinded to section treatment. The tissue sections were quantitatively scored according to the staining intensity (0, no signal; 1, weak; 2, moderate; and 3, strong) and percentage of positive cells (1, 0%–25%; 2, 26%–50%; 3, 51%–75%; and 4%, > 75%). The scores ranged from 0 to 12, as described previously. The specimens with a score > 5 were classified as high level while those with a score ≤ 4 were classified as low level.

2.14. Statistical Analysis

Statistical significance (p‐value) was calculated using a two‐sided unpaired student's t‐test, and a two‐sided ANOVA was performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001). The cell culture experiment was conducted at least three times independently. In addition to animal experiments, all experiments were conducted at least three times independently.

3. Results

3.1. KAT2A Is Highly Expressed in TNBC and Correlated With Poor Prognosis of Patients With TNBC

Previous research has established a correlation between aberrant KAT2A expression and aggressive phenotypes in various tumors. In our study, we utilized transcriptional expression data from The Cancer Genome Atlas (TCGA) and translational expression data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC), accessed via the UALCAN online platform (https://ualcan.path.uab.edu/analysis‐prot.html). Analysis of the TCGA pan‐cancer transcriptome data revealed that KAT2A transcriptional expression levels were significantly upregulated in 15 types of tumor tissues compared to normal tissues, with a statistical significance of p < 0.001 (Figure 1A). Despite these findings, the specific role of KAT2A in breast cancer (BC) remains insufficiently understood. To elucidate its role in BC, we analyzed expression data from the TCGA‐BC cohort, discovering that KAT2A expression was significantly elevated in 1097 tumor samples compared to 114 normal samples, with p < 0.001 (Figure 1B). Additionally, increased KAT2A expression was observed in tumor tissues from another BC cohort—GSE211729 (Figure 1C). We further conducted a comprehensive analysis of KAT2A expression levels across various subtypes of BC. Our findings indicated that the differences in KAT2A expression between Luminal and triple‐negative breast cancer (TNBC) subtypes were more pronounced than those observed in normal populations, with statistical significance (p < 0.001; Figure 1D). To corroborate these results, we examined KAT2A protein expression levels using data from the CPTAC tumor protein database. The CPTAC data revealed a significant elevation of KAT2A protein levels in four cancer types compared to adjacent non‐cancerous tissues, with the most notable increase observed in BC (p < 0.001; Figure 1E,F). Furthermore, analysis of KAT2A protein data from The Cancer Genome Atlas, accessed via the TRGAted online platform (https://nborcherding.shinyapps.io/TRGAted/), demonstrated that patients exhibiting high KAT2A levels experienced poorer survival outcomes compared to those with lower levels (p < 0.01; Figure 1G). Consistent with these findings, Western blot analysis of human fresh TNBC tissues from our hospital revealed that KAT2A protein levels were significantly elevated in 12 out of 14 cases (85.7%) compared to paired normal breast tissues (Figure 1H).

FIGURE 1.

KAT2A was highly expressed in TNBC and had prognostic significance. (A) KAT2A expression in different tumors and normal tissues were analyzed via the ULCAN‐TCGA platform. (B, C) The expression of KAT2A in the BC tumor samples versus normal tissues in the TCGA‐BC cohort and GSE211729 dataset. (D) The expression levels of KAT2A in TCGA‐BC tissue samples in different subtypes. (E) The protein levels of KAT2A in pan‐cancer tissue and normal tissue samples were analyzed via the ULCAN‐CPTAC platform. (F) The protein levels of KAT2A in CPTAC‐BC tissue samples in different subtypes. (G) Kaplan–Meier analysis indicated that patients with high KAT2A levels suffered from worse survival outcomes versus those with low KAT2A levels (Log‐rank test p < 0.05). (H) The protein levels of KAT2A were assessed in TNBC tumor tissues and matched normal breast tissues through Western blot (N = 14). *p < 0.05, **p < 0.01, ***p < 0.001.

3.2. KAT2A Accelerates TNBC Cells Growth and Metastatic In Vitro and In Vivo

To elucidate the function of KAT2A in TNBC cells, we employed two distinct KAT2A shRNAs to achieve knockdown in MDA‐MB‐231 and MDA‐MB‐468 cell lines (Figure S1A). The CCK‐8 assays demonstrated that KAT2A knockdown significantly suppressed TNBC cell proliferation compared to the control group, a finding that was consistent across both cell lines (Figure 2A). Furthermore, the absence of KAT2A also reduced the clonogenic capacity of TNBC cells (Figure 2B). In contrast, the overexpression of KAT2A substantially enhanced both the proliferation and clonogenic potential of TNBC cells (Figure S1B,C). Additionally, we performed tumor xenograft studies to evaluate the oncogenic functions of KAT2A in vivo, observing that KAT2A depletion significantly hindered tumor growth compared to the control group (Figure 2C). Subsequent investigations into the impact of KAT2A on cell apoptosis in TNBC cells indicated that KAT2A silencing exerted minimal effects on apoptotic processes in these cells (Figure S1D). Regarding the influence of KAT2A on the migratory capabilities of TNBC cells, wound‐healing assays revealed that KAT2A overexpression significantly accelerated cell migration, whereas KAT2A knockdown markedly impeded migratory efficiency (Figure S1E). Additionally, Transwell assays indicated that the downregulation of KAT2A significantly diminished the migration and invasion capacity of TNBC cells, and the enforced expression of KAT2A markedly enhanced the migration and invasion capacity of TNBC cells (Figure 2D,E; Figure S1F). The epithelial‐mesenchymal transition (EMT) is intricately linked to various facets of BC progression, including invasion, metastasis formation, and resistance to therapy [32]. Consequently, we conducted an in‐depth investigation into the influence of KAT2A on pivotal molecules involved in the EMT process. Our findings revealed that the knockdown of KAT2A resulted in the downregulation of N‐cadherin and vimentin protein levels, thereby inhibiting EMT in TNBC cells (Figure 2F). Tumor metastasis is a multifaceted process that is closely associated with the cytoskeleton [33]. Our study confirmed that the knockdown of KAT2A can partially reduce cytoskeletal formation in TNBC cells (Figure 2G). Furthermore, in vivo assays were performed to evaluate the impact of KAT2A on the later stages of metastasis of TNBC. Luciferase‐labeled TNBC cells with modified KAT2A expression were developed and administered via tail vein injection into BALB/c nude mice. After 7 weeks, it was observed that the knockdown of KAT2A significantly inhibited lung metastasis in the MDA‐MB‐231 metastatic model (Figure 2H). Overall, these findings underscore the pivotal role of KAT2A in facilitating the malignant phenotype of TNBC.

FIGURE 2.

KAT2A promoted malignant phenotype of TNBC cells in vivo and in vitro. (A) CCK‐8 and (B) colony‐formation assays assessed the effects of KAT2A knockdown on the proliferation of MDA‐MB‐231 and MDA‐MB‐468 cells. (C) KAT2A‐knockdown tumors showed a significant decrease in tumor volume compared with control tumors. Data are presented as the mean ± S.D. of three mice for each group (6 tumors). (D, E) Transwell migration and invasion assays were performed to detect changes in cell migration and invasion rates after KAT2A knockdown (Scale bars, 100 μm. Data are presented as mean ± S.D. ***p < 0.001). (F) Western blot analysis of E‐cadherin, N‐cadherin and vimentin in TNBC cells with KAT2A knockdown or overexpression. (G) Images of F‐actin staining of MDA‐MB‐231 and MDA‐MB‐468 cells inhibited by KAT2A knockdown (Scale bars, 20 μm, green for F‐actin staining and blue for DAPI staining). (H) The representative pictures of bioluminescence imaging (BLI) of mice 7 weeks after portal vein injection of MDA‐MB‐231 cells with KAT2A knockdown or vector. *p < 0.05, ***p < 0.001.

3.3. KAT2A Alters the Oncogenic Transcriptome in TNBC

To elucidate the underlying mechanisms by which KAT2A influences the progression of TNBC, we employed RNA sequencing technology to analyze the differential transcriptome between control and KAT2A‐knockdown MDA‐MB‐231 cells. Our analysis revealed 857 differentially expressed genes (DEGs) with a significance threshold of |Log (fold change)| > 1 and false discovery rate (FDR) < 0.01 (Figure 3A,B). Gene ontology (GO) analysis indicated that these DEGs were predominantly enriched in pathways associated with DNA transcription, highlighting the oncogenic role of KAT2A in TNBC (Figure 3C). Furthermore, our analysis revealed that the differentially expressed molecules in TNBC cells after KAT2A knockdown were significantly enriched in inflammatory pathways. This observation is consistent with previous studies demonstrating that KAT2A regulates inflammatory responses via epigenetic mechanisms, thereby affecting the progression of conditions such as acute lung injury and rheumatoid arthritis [34, 35]. However, the precise mechanisms through which KAT2A modulates inflammation to facilitate TNBC progression remain to be fully elucidated. To identify genes regulated by KAT2A‐mediated H3K79succ, we conducted ChIP‐seq in MDA‐MB‐231 cells utilizing anti‐KAT2A and anti‐H3K79succ antibodies. We subsequently mapped the binding peaks of KAT2A and H3K79succ to identify the nearest genes and cross‐referenced them with the DEGs, resulting in the identification of five target genes (Figure 3D and Table S2). Notably, among these genes, SH2B3 exhibited the highest correlation with KAT2A at the mRNA level, as determined through analysis of the TCGA database via the GEPIA 2 online tool (http://gepia2.cancer‐pku.cn/#index) (Figure 3E and Figure S22). We conducted a more detailed analysis of the transcript levels of these genes in TNBC cell lines (Figure 3F). The association between KAT2A and SH2B3 protein levels was corroborated by WB, which demonstrated that SH2B3 protein levels increased or decreased in response to the upregulation or downregulation of KAT2A, respectively (Figure 3G). Collectively, through high‐throughput sequencing and subsequent validation experiments, we identified SH2B3 as a critical downstream target of KAT2A‐mediated H3K79succ, warranting further investigation.

FIGURE 3.

KAT2A altered oncogenic transcriptome and mainly activates downstream SH2B3 expression. (A, B) The heatmap and volcano plot revealed the differentially expressed genes (DEGs) between KAT2A knockdown and WT control MDA‐MB‐231 cells. (C) Enrichment analysis for representative GO_BP and KEGG pathways in downregulation genes in MDA‐MB‐231 cells with KAT2A knockdown. (D) Venn diagram showing the overlap of KAT2A‐occupied gene promoters (purple), H3K79succ‐occupied gene promoters (yellow) in MDA‐MB‐231 cells and downregulated genes in MDA‐MB‐231 following KAT2A knockdown treatment (green). (E) Positive correlation between KAT2A and SH2B3 in the TCGA database from GEPIA2. (F) The mRNA levels of SH2B3, FAM105A and ZFP36L2 in KAT2A knockdown TNBC cells. (G) The protein levels of SH2B3 in MDA‐MB‐231 and MDA‐MB‐468 cells with KAT2A knockdown or overexpression. *p < 0.05, **p < 0.01.

3.4. KAT2A‐Mediated H3K79 Succinylation Activates the Transcription of SH2B3 in TNBC

Initially, immunofluorescence analysis revealed a substantial co‐localization of KAT2A and H3K79succ within the nuclei of TNBC cells (Figure 4A). Subsequent dual‐luciferase reporter assays corroborated the binding affinity of KAT2A to the SH2B3 promoter (Figure 4B). To further elucidate the specific epigenetic regulation of SH2B3 via KAT2A‐mediated H3K79succ, we performed ChIP‐qPCR analysis in TNBC cells utilizing H3K79succ antibodies. The findings indicated that the deletion of KAT2A markedly diminished the binding levels of H3K79succ within the SH2B3 promoter regions spanning −1372 to −1439, −1248 to −1240, and −386 to −468 (Figure 4C,D). Moreover, the exogenous administration of sodium succinate‐coenzyme A (succinyl‐CoA) and succinate resulted in a dose‐dependent enhancement of both transcriptional and expression levels of SH2B3 in TNBC cells (Figure 4E,F). Notably, the addition of succinyl‐CoA and succinate not only augmented the level of H3K79succ modification catalyzed by KAT2A (Figure 4G) but also activated transcription within the SH2B3 promoter region (Figure 4H). Succinyl donors are mainly derived from the three major cellular metabolic pathways, some of which are derived from the conversion of the fatty acid metabolite propionyl coenzyme A [36]. Therefore, we mimicked the high‐fat environment by adding oleic acid to the cells (Figure S3A) and found that the addition of oleic acid promoted the level of KAT2A‐catalyzed H3K79succ and increased the expression of SH2B3 (Supplementary Figure S3B,C). Rescue experiments showed that the knockdown of KAT2A and SH2B3 could reverse some of the oleic acid‐induced migration and invasion of TNBC cells (Figure S3D,E).

FIGURE 4.

KAT2A‐H3K79succ mediated SH2B3 upregulation. (A) Immunofluorescence showed the nuclear localization of KAT2A and H3K79succ in MDA‐MB‐231 and MDA‐MB‐468 cells. (B) KAT2A significantly increased the SH2B3 promoter activity. (C, D) KAT2A knockdown decreased the enrichment of H3K79succ on the different regions of the SH2B3 promoter. (E) Sodium succinate‐coenzyme A and succinate increased the transcriptional levels of SH2B3. (F) Sodium succinate‐coenzyme A and succinate increased the protein levels of SH2B3 by improving KAT2A‐mediated H3K79succ. (G) Immunofluorescence showed the protein levels of KAT2A and H3K79succ in MDA‐MB‐231 treated with 4 μM succinyl‐CoA and 2 M succinate. (H) Succinyl‐CoA (4 μM) and succinate (2 M) significantly increased the SH2B3 promoter activity. *p < 0.05, **p < 0.01, ***p < 0.001.

3.5. SH2B3 Deletion Reversed the Proliferative Effect of KAT2A on TNBC

To elucidate the functional role of SH2B3 in TNBC, we employed specific shRNA targeting SH2B3 mRNA to achieve knockdown of SH2B3 in TNBC cells. The results of the CCK‐8 assays (Figure 5A and Figure S4A) indicated that depletion of SH2B3 significantly reduced the cells' proliferative capacity, whereas overexpression of SH2B3 enhanced proliferation. However, flow cytometry analysis revealed no significant impact on apoptosis rates (Figure S4B). Furthermore, transwell assays demonstrated that SH2B3 knockdown resulted in diminished migratory and invasive capabilities compared to the control group, while SH2B3 overexpression facilitated increased cell migration and invasion (Figure 5B and Figure S4C). To investigate whether KAT2A inhibits TNBC metastasis through SH2B3, we ectopically overexpressed KAT2A in TNBC cells with stable KAT2A expression and simultaneously knocked down SH2B3 using siRNA. Our findings revealed that SH2B3 knockdown significantly mitigated the effects of KAT2A overexpression in TNBC cells, suggesting that KAT2A relies on SH2B3 to enhance TNBC proliferation, migration, invasion, and cytoskeleton formation (Figure 5C–F).

FIGURE 5.

SH2B3 deletion reversed the proliferative effect of KAT2A on TNBC cells. (A) CCK‐8 assays assessed the effects of SH2B3 on the proliferation of MDA‐MB‐231 cells. (B) Transwell migration and invasion assays were performed to detect changes in cell migration and invasion rates after SH2B3 knockdown or overexpression (Scale bars, 100 μm. Data are presented as mean ± S.D. ***p < 0.001). (C) Representative pictures and quantification analysis of cell proliferation of KAT2A‐overexpressed TNBC cells transfected with the SH2B3 siRNAs or controls. **p < 0.01, ***p < 0.001. (D) The scratch assay was used to detect the effect of KAT2A‐overexpressed MDA‐MB‐231 cells transfected with the SH2B3 siRNAs or controls. (Scale bars, 200 μm. Data are presented as mean ± S.D. ***p < 0.001). (E) Transwell invasion assays were performed to detect changes in cell invasion rates of KAT2A‐overexpressed TNBC cells transfected with the SH2B3 siRNAs or controls. (Scale bars, 100 μm. Data are presented as mean ± S.D. ***p < 0.001). (F) Images of F‐actin staining of KAT2A‐overexpressed MDA‐MB‐231 cells transfected with the SH2B3 siRNAs or controls. (Scale bars, 20 μm, green for F‐actin staining and blue for DAPI staining).

3.6. SH2B3 Promotes TNBC Progression by Diminishing Ubiquitin‐Dependent Degradation of Vimentin

SH2B3 plays a complex and context‐dependent role in tumorigenesis and cancer progression. Its function exhibits marked tissue specificity and pathway dependency, with tumor‐suppressive or oncogenic effects dictated by tumor type, genetic background, and microenvironmental cues [23]. In hematologic malignancies, SH2B3 acts as a tumor suppressor, constraining disease progression. In contrast, its role in solid tumors is multifaceted [27, 29, 31]. For instance, in lung cancer, SH2B3 exerts anti‐tumor effects by binding to JAK2 and inhibiting the JAK2/STAT3 signaling axis [26]. Conversely, in melanoma, elevated SH2B3 expression promotes tumor progression by interacting with STAT1 and suppressing the IFN/STAT1 pathway [28], highlighting its dual regulatory capacity in different malignancies.

To elucidate the mechanism of SH2B3, we conducted a CoIP experiment combined with mass spectrometry (MS) to identify potential gene interactions with SH2B3 (Figure 6A). Our findings revealed that vimentin interacts with SH2B3 and exhibits significant correlations with it, as confirmed by CoIP‐WB (Figure 6B) and immunofluorescence (IF) assays (Figure 6C). Based on these observations, we concentrated our investigation on vimentin within TNBC cells. Vimentin is a well‐established factor associated with metastasis and plays a crucial role in the progression of various malignancies, including prostate and breast cancer [37]. To ascertain whether SH2B3 regulates vimentin expression, we initially assessed vimentin mRNA levels using qPCR. Notably, neither overexpression nor knockdown of SH2B3 significantly affected vimentin transcript levels, thereby excluding transcriptional regulation by SH2B3 (Figure 6D; Figure S5A). Consequently, we hypothesized that SH2B3 might facilitate vimentin degradation at the post‐translational level. To investigate this, we treated TNBC cells with cycloheximide (CHX) to inhibit protein synthesis and subsequently measured the half‐life of vimentin protein. In a notable observation, MDA‐MB‐231 and MDA‐MB‐468 cells with downregulated SH2B3 expression demonstrated a significantly reduced half‐life of vimentin compared to control cells (Figure 6E). However, the application of the proteasome inhibitor MG132 mitigated the decrease in vimentin levels induced by SH2B3 knockdown, reinforcing the involvement of SH2B3 in the proteasome‐mediated degradation of vimentin (Figure 6F). Through CoIP‐MS analysis, we identified an interaction between vimentin and the ubiquitinating enzyme TRIM21 in MDA‐MB‐231 cells (Figure 6G). Moreover, SH2B3 knockdown was found to enhance vimentin ubiquitination while reducing its interaction with TRIM21 in MDA‐MB‐231 cells and MDA‐MB‐468 cells (Figure 6H and Supplementary Figure S5B). These results suggest that SH2B3 competitively binds to vimentin, thereby interfering with TRIM21‐mediated ubiquitination. As a result, decreased ubiquitination of vimentin leads to its stabilization, culminating in increased vimentin protein levels in TNBC cells. Within the intracellular environment, vimentin assembles into filamentous structures that are essential for cytoskeletal organization and the preservation of cellular architecture, thus playing a critical role in tumorigenesis and metastasis [36, 37]. Consequently, the knockdown of SH2B3 in TNBC cells led to a marked decrease in cytoskeletal extension, as evidenced by immunofluorescence staining (Figure S5C).

FIGURE 6.

SH2B3 promoted vimentin stabilization by weakening its ubiquitination. (A) Proteins immunoprecipitated with anti‐SH2B3 were separated by SDS‐PAGE from MDA‐MB‐231 cells. Bands close to 35–70 kDa were manually excised, identified by mass spectrometry, and analyzed for specific proteins. (B) CoIP using antibodies against SH2B3 and vimentin revealed the exogenous interaction between SH2B3 and vimentin. (C) Representative co‐staining images of SH2B3 and vimentin in MDA‐MB‐231 and MDA‐MB‐468 cells. (D) qPCR of vimentin levels in MDA‐MB‐231 cells with SH2B3 downregulation or upregulation. (E, F) WB of vimentin levels in TNBC cells with transfected shCtrl and shSH2B3 with CHX and MG132 treatment. (G) CoIP‐MS determined that vimentin bound to the ubiquitinating enzyme TRIM21 in MDA‐MB‐231 cells. (H) Co‐IP tests were carried out to examine the ubiquitination of vimentin protein and the interaction between vimentin and TRIM21 in TNBC cells transfected with shSH2B3 with MG132 treatment. ** p < 0.01, *** p < 0.001.

3.7. KAT2A and SH2B3 Affect the Carboplatin Sensitivity of TNBC Cells

KAT2A has been implicated in drug resistance across multiple cancer types [38, 39]. For instance, in prostate cancer cells, KAT2A mediates the acetylation of the androgen receptor (AR), facilitating its translocation from the cytoplasm to the nucleus. This process enhances the transcriptional activity of AR target genes, including prostate‐specific antigen (PSA), ultimately conferring resistance to abiraterone in PCa cells [40]. Intriguingly, our findings revealed that TNBC patients with low expression of KAT2A and SH2B3 exhibited a higher likelihood of achieving pathological complete response (pCR) following neoadjuvant chemotherapy which contains carboplatin, suggesting a potential association between KAT2A/SH2B3 and carboplatin chemosensitivity (Figure 7A). Based on these observations, we hypothesized that KAT2A and its downstream transcriptional regulator SH2B3 might also modulate carboplatin resistance in TNBC cells. To test this hypothesis, we first assessed the impact of KAT2A and SH2B3 knockdown on carboplatin sensitivity using CCK‐8 cytotoxicity assays. The results demonstrated a significant reduction in the IC50 values of carboplatin upon KAT2A or SH2B3 depletion, indicating enhanced drug sensitivity in TNBC cells (Figure 7B). To further validate these findings, we treated MDA‐MB‐231 and MDA‐MB‐468 cells with carboplatin at concentrations of 25 μM and 15 μM, respectively, in DMEM complete medium for 96 h. Flow cytometry analysis revealed that KAT2A or SH2B3 knockdown significantly promoted carboplatin‐induced apoptosis, underscoring their functional role in regulating carboplatin efficacy in TNBC (Figure 7C). These data collectively showed that inhibition of KAT2A/SH2B3 enhanced the antitumor effects of carboplatin in TNBC.

FIGURE 7.

KAT2A/SH2B3 affected the carboplatin sensitivity of TNBC cells. (A) H&E and IHC staining of KAT2A and SH2B3 protein in tumor tissues from TNBC patients before receiving neoadjuvant chemotherapy. (N = 21, Scale bar, 50 μm). (B) Carboplatin dose–response curves in MDA‐MB‐231 and MDA‐MB‐468 cells transfected with shKAT2A or shSH2B3. (C) Annexin V‐FITC and PI staining showing apoptosis in TNBC cells treated with 15 μM carboplatin in MDA‐MB‐231 and 8 μM carboplatin in MDA‐MB‐468 cells. *p < 0.05, **p < 0.01.

4. Discussion

Our study elucidated that KAT2A modulates SH2B3 expression through the catalysis of H3K79 succinylation, thereby influencing the malignant phenotypic progression of TNBC. Mechanistically, we demonstrated that SH2B3 impedes the ubiquitination and subsequent degradation of vimentin by disrupting its interaction with the ubiquitinating enzyme TRIM21. This finding represents the first evidence of a succinylation‐mediated regulatory mechanism in TNBC oncogenesis. Furthermore, we established that the KAT2A‐SH2B3 axis affected the sensitivity of TNBC cells to carboplatin and was clinically associated with patient prognosis in the neoadjuvant TNBC cohort, highlighting its potential as a therapeutic target.

KAT2A is a vital histone acetyltransferase within the GNAT (GCN5‐related N‐acetyltransferase) superfamily, playing a crucial role in epigenetic regulation by facilitating various acylation modifications of histones [9, 10]. This enzyme is predominantly expressed in tissues such as the heart, muscle, liver, brain, and kidney, with significant enrichment in mitochondrial compartments, where it is indispensable for mitochondrial function and cellular metabolism [41]. Recent evidence suggests that KAT2A is frequently overexpressed across multiple cancer types, with its elevated expression being strongly correlated with increased tumor aggressiveness. Consequently, the pharmacological inhibition of KAT2A, for instance, through the small‐molecule inhibitor CPTH2, has been proposed as a potential therapeutic approach [38, 42]. Through an integrated analysis of TCGA transcriptomic data and CPTAC proteomic data using the UALCAN platform, we identified a significant upregulation of KAT2A in TNBC. Importantly, high KAT2A expression was associated with poor clinical outcomes in TNBC patients, prompting further investigation into its functional role. Our experimental validation has substantiated that KAT2A enhances the malignant phenotypes of TNBC cells in both in vitro and in vivo settings. These findings build upon previous pan‐cancer studies by elucidating the oncogenic role of KAT2A specifically in breast cancer, thereby reinforcing its potential as a comprehensive tumor biomarker and a promising target for therapeutic intervention.

Many studies have demonstrated that KAT2A, a versatile histone acyltransferase, governs gene transcription by catalyzing diverse acylation modifications of histones and facilitates the occurrence and development of numerous cancers [16]. Former studies primarily concentrated on the acetylase function of KAT2A; for instance, KAT2A facilitated the emergence and progression of hepatocellular carcinoma, gastric cancer, nasopharyngeal carcinoma, renal clear cell carcinoma, and other tumors by catalyzing H3K9ac [43, 44, 45, 46]. Since the confirmation of the widespread presence of succinylation modification in organisms [47], KAT2A, as a histone succinylation modifier, has also been discovered to regulate the function of histone succinylation modification for promoting gene transcription [11]. For example, KAT2A further promotes the advancement of pancreatic cancer by catalyzing H3K79succ to boost 14–3‐3ζ transcription [15]. In our study, we identified a significant correlation between KAT2A protein levels and patient prognosis within the TNBC population. This association was further substantiated through both in vivo and in vitro experiments, corroborating previous findings regarding the oncogenic role of KAT2A in various cancers. However, the specific mechanism by which KAT2A functions as a succinylase in TNBC has not been thoroughly investigated. To address this gap, we employed ChIP‐seq analysis to demonstrate that H3K79succ, catalyzed by KAT2A, exerts transcriptional regulation over multiple molecules in TNBC cells, with a particular focus on SH2B3. Our findings elucidate the role of H3K79succ catalyzed by KAT2A in the progression of TNBC.

SH2B3, a member of the SH2B family of connexins, plays a role in suppressing cancer in a variety of hematologic tumors [23]. Nevertheless, the role of SH2B3 in solid tumors is highly debatable. For instance, low expression of SH2B3 in colorectal cancer and overexpression of SH2B3 can inhibit the invasion of colorectal cancer cells [25]. In lung cancer, SH2B3 further inhibits lung cancer progression by binding to JAK2 to inhibit JAK2/STAT3 signaling [26], yet high expression of SH2B3 in melanoma can promote tumor progression by inhibiting the IFN‐STAT pathway in combination with STAT1 [28]. SH2B3 has been demonstrated to promote cancer in ovarian cancer in vitro and in vivo [27]. These studies suggest that SH2B3, as an adaptor protein, possesses no enzymatic activity of its own, and its role in tumor cells depends on the protein molecules to which the molecule binds. Therefore, we verified that SH2B3 can bind vimentin in TNBC cells through the CoIP experiment, and further research findings indicated that SH2B3 maintained the stability of vimentin molecules by influencing the Trim21‐mediated ubiquitination degradation of vimentin. It has been confirmed that SH2B3 can promote the progression of TNBC through the JAK/STAT3 and ERK1/2 pathways [31], and on this basis, our study adds a new mechanism through which SH2B3 plays a cancer‐promoting role in TNBC.

Regarding the effect of KAT2A/SH2B3 on the chemotherapy outcome of TNBC patients, there were only 16 clinical samples, and the small number was not persuasive. Additionally, in the cell experiment, we only conducted drug sensitivity experiments on KAT2A/SH2B3 to carboplatin, and further experiments on the drug sensitivity of other types of chemotherapy drugs are necessary to enhance the study on the mechanism of chemotherapy application of KAT2A/SH2B3 in TNBC patients.

5. Conclusion

KAT2A has been extensively investigated as a multifunctional acylation modification enzyme in tumors, and small molecule drugs targeting this target have emerged in the basic experimental field. Our research elucidates the mechanism of KAT2A in TNBC cells for the first time. KAT2A catalyzes succinylation at the H3K79 site to regulate SH2B3 transcription and further enhance its expression. SH2B3 functions as an adaptor protein that binds to vimentin in TNBC cells, preventing vimentin from associating with ubiquitinating enzymes TRIM21. This interaction inhibits the ubiquitination‐mediated degradation of vimentin within TNBC cells. Meanwhile, oleic acid can elevate the H3K79succ level of TNBC cells, facilitating the H3K79succ levels and the expression of SH2B3, driving the progression of TNBC. Additionally, we discovered that KAT2A/SH2B3 can impact the carboplatin drug sensitivity of TNBC cells and influence the efficacy of neoadjuvant chemotherapy in TNBC patients. Based on our extensive findings, KAT2A demonstrates significant potential as a biomarker for the diagnosis and therapeutic interventions of triple‐negative breast cancer.

Author Contributions

Xiaohui Shi and Ran Hao participated in the completion of most of the experiments and supervised the manuscript writing. Xiaoran Wang, Yiwei Lu, and Huanqi Ji performed the animal experiments. Xiaohui Shi was involved in the analysis of the data, and Sheng Ma and Xuehua Liu provided the experimental technical support. Yixin Qi and Jie Hu evaluated the results, guided the experimental designs, and reviewed and revised the manuscript. All authors have reviewed and accepted the published version of the manuscript.

Ethics Statement

Clinical samples were obtained from the Department of Breast Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, China. This project has received medical ethics support (2022KY205). All animal procedures were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Hebei Medical University (IACUC‐Hebum‐20 240 803).

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1: Supporting Information.

Figure S1: KAT2A regulates the malignant phenotype of TNBC cells. (A) Upregulation and downregulation of KAT2A was confirmed by qRT‐PCR and WB. (B) CCK‐8 and (C) Colony‐formation assays assessed the effects of KAT2A on the proliferation of TNBC cells. (D) The effect of KAT2A on cell apoptosis was detected by flow cytometry. (E) The scratch assay was used to detect the effect of KAT2A on the migration ability of TNBC cells. (Scale bars, 200 μm. Data are presented as mean ± S.D. ***p < 0.001). (F) Transwell migration and invasion assays were performed to detect changes in cell migration and invasion rates after KAT2A overexpression. (Scale bars, 100 μm. Data are presented as mean ± S.D. ***p < 0.001).

Figure S2: The correlation of gene expression was analyzed by GEPIA 2 in TCGA database.

Figure S3: Oleic acid elevates the H3K79succ level of TNBC cells and facilitates the H3K79succ levels and the expression of SH2B3. (A) Oil red staining was used to determine the lipid status of TNBC cells after the addition of oleic acid. (B) OA increased the transcriptional levels of SH2B3. (C) OA increased the protein levels of SH2B3 by improving KAT2A‐mediated H3K79succ. (D‐E) Knockdown of KAT2A or SH2B3 suppresses the OA‐induced enhancement of invasive capacity in MDA‐MB‐231 and MDA‐MB‐468 cells. (**p < 0.01, ***p < 0.001).

Figure S4: KAT2A‐SH2B3 regulates the malignant phenotype of TNBC cells. (A) CCK‐8 assays assessed the effects of SH2B3 on the proliferation of TNBC cells. (B) The effect of SH2B3 on cell apoptosis was detected by flow cytometry. (C) Transwell migration and invasion assays were performed to detect changes in cell migration and invasion rates after SH2B3 knockdown or overexpressed. (Scale bars, 100 μm. Data are presented as mean ± S.D. ***p < 0.001).

Figure S5: SH2B3 affects the ubiquitination and degradation of vimentin. (A) qRT‐PCR of vimentin levels in MDA‐MB‐468 cells with SH2B3 downregulation or upregulation. (B) Co‐IP tests were carried out to examine the ubiquitination of vimentin protein and the interaction between vimentin and TRIM21 in MDA‐MB‐468 cells transfected with shSH2B3 with MG132 treatment. (C) Images of F‐actin staining of TNBC cells with the SH2B3 knockdown. (Scale bars, 20 μm, green for F‐actin staining and blue for DAPI staining).

Table S1: Gene‐specific primers used for qRT–PCR.

Table S2: The target genes identified through the integration of ChIP‐seq and RNA‐seq data.

Shi X., Hao R., Yang Z., et al., “ KAT2A‐H3K79succ‐Mediated SH2B3 Upregulation Promotes TNBC Metastasis Through Diminishing the Ubiquitin‐Dependent Degradation of Vimentin,” The FASEB Journal 39, no. 20 (2025): e71140, 10.1096/fj.202502178R.

Data Availability Statement

Privacy/ethical restrictions.