Introduction

Although there has been notable progress in the early detection and treatment of breast cancer, the considerable heterogeneity and aggressiveness of the disease make detection and treatment challenging [1,2,3]. As a result, breast cancer remains the leading cause of cancer-related death in women, second only to lung cancer [4,5,6]. The adoption of immune checkpoint blockade (ICB) therapy, which has demonstrated early success in treating melanoma and lung cancer, has been relatively slow. Therefore, it is crucial to develop innovative strategies and therapies to improve patient survival rates [7, 8]. Among the recently identified tumour characteristics in 2022, nonmutation epigenetic reprogramming has emerged as a novel feature [9]. Notably, cancer is a complex disease involving both genetic and epigenetic alterations. Epigenetic mechanisms exert crucial influences on multiple aspects of cancer biology, including the promotion of primary tumour growth and invasion, as well as the regulation of immune responses within the tumour microenvironment [10,11,12]. These mechanisms involve intricate interactions between DNA methylation and histone modification. Among the various classes of enzymes involved in epigenetic regulation, DNA methyltransferase (DNMT) and histone deacetylase (HDAC) have emerged as particularly significant. In fact, targeted drugs, such as the DNMT inhibitor decitabine [13, 14] and the HDAC inhibitor vorinostat (SAHA), have been developed to specifically target these enzymes [15].

Within mammals, there are four distinct members of the DNMT family, namely, DNMT1, DNMT3A, DNMT3B, and DNMT3L [16]. The overexpression of these DNMTs facilitates the methylation of cytosine within CpG islands, thereby leading to the suppression of tumour suppressor genes (TSGs) in human cancer [17]. This phenomenon contributes to the initiation and progression of cancer. Conversely, DNMT inhibitors (DNMTis) reactivate TSGs [18], augment tumour immunogenicity, and induce the secretion of cytotoxic cytokines by various immune cells, including macrophages, natural killer (NK) cells, and CD8+ T cells. Ultimately, these actions promote the death of tumour cells. The upregulation of the MHC I class molecules has been observed. Presently, the primary mechanism through which DNMTis exert their immune function is by targeting cancer cells through the induction of viral mimicry. This is achieved by reducing the methylation level of the endogenous retrovirus (ERV) gene promoter and activating the expression of endogenous retroviral elements [19,20,21]. Consequently, the intracellular levels of double-stranded RNA (dsRNA) are increased, leading to an inflammatory immune response. This response further promotes the production of type I and type III interferons and induces the production of a variety of interferon-stimulated genes (ISGs) and cytokines [19, 22, 23], thereby inhibiting tumour cell immune escape.

HDACs, a group of epigenetic enzymes, are intricately associated with tumorigenesis. In conjunction with histone acetyltransferases (HATs), HDACs govern the equilibrium of histone and protein acetylation through the processes of lysine acetylation and deacetylation [24]. HDACs exert significant regulatory effects on numerous key cellular processes, such as cell proliferation, cellular differentiation, and programmed cell death [25]. Furthermore, the expression and activity of HDACs are frequently disrupted in cancer, whereby HDACs counteract lysine acetylation on histones, thereby inducing chromatin remodelling and altering the expression of TSGs [26, 27], ultimately contributing to increased susceptibility to carcinogenesis. Inhibiting HDACs can result in the release of oncogenic transcription suppressors, leading to cell cycle arrest and apoptosis. Moreover, HDAC inhibitors (HDACis) upregulate the expression of T-cell chemokines, namely, CCL5 and CXCL10, thereby establishing a direct correlation with increased T-cell infiltration within tumours [S1.

Flow cytometry analysis

MDA-MB-231 and HCC1937 were treated with either vehicle or 15a for 24 or 48 h. Using Cell Cycle and Apoptosis Kit (Yesen) and Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime), the samples were made according to the instructions. CytoFLEX flow cytometry was used to detect the number of cells in different cycles and the level of apoptosis.

RNA extraction and quantitative real-time PCR assay (qRT‒PCR)

According to the instructions, total RNA was extracted from the cells using the Eastep Super total RNA extraction kit, and the extracted total RNA was quantifiable by Nanodrop spectrophotometer with acceptable purity. For qRT‒PCR, the total RNA was reverse-transcribed by Bio-Rad PCR after different volume samples and 4 μL Hifair® III 1st Strand cDNA Synthesis SuperMix reagent were added according to the calculation (reversing 1 μg of RNA). Using 2 μL cDNA, 10 μL Hieff® qPCR SYBR Green Master Mix (Low Rox Plus), and 0.5 μL forward and reverse primers, using 7500 Fast system under 20 μL volume reaction condition, qPCR was performed using 20 μg cDNA at 95 °C, 5 min, 95 °C, 10 s 60 °C 30 s 40 cycles. The primers used in this study are listed in the Supplementary Table S2.

Transwell evaluation

The 8 μm pore membrane chamber (Corning) was placed in a 24-well plate, and the cells with a density of 4 × 105 were placed in the chamber. The 24-well plate was added with 1 mL medium containing either vehicle or different concentrations of 15a, which was treated for 48 h. After the chamber was washed three times with 1× PBS, adding 1 mL Bourn’s Tissue fixative into the chamber and fixing for 1 h. Then washing three times with 1× PBS after fixing, add 1 mL crystal violet solution dyeing solution for 1 h, and finally wash three times with 1× PBS, wipe the chamber gently with a cotton swab, and then take images with an inverted fluorescence microscope after drying. Three fields of view were taken from each sample.

Colony-forming assay

MDA-MB-231 and HCC1937 cells were seeded in 6-well plates at a density of 300 cells/mL and treated with either vehicle or 15a for 14–21 d, replacing the medium with fresh medium every 72 h. Subsequently, the formed colonies were fixed with 1 mL Bourn’s Tissue fixative for 1 h, washed three times with 1× PBS after fixation, added 1 mL crystal violet solution staining solution for 1 h, and finally washed three times with 1× PBS, dried and then photographed with an inverted fluorescence microscope. Three fields of view were taken for each sample.

Wound healing assay

MDA-MB-231 and HCC1937 cells were cultured in 6-well plates at a density of 2 × 105 cells/mL. Upon reaching confluence, the cells were subjected to serum starvation using FBS-free medium for a duration of 12 h. Prior to cell seeding, three parallel lines were marked on the bottom of the 6-well plates. Subsequently, wounds were induced by vertically scratching the cell monolayer with 200 µL sterile pipette tips along the aforementioned lines. Following a thorough washing and medium replacement, the cells were exposed to either a control solution or varying concentrations of 15a. The wounds adjacent to the lines were captured through photography at the initial time point and 48 h following the act of scratching, employing an inverted fluorescence microscope equipped with a 10× objective lens. For each sample, three distinct fields of view were documented.

Thermal shift assay

The cells resuspended in 1× protease inhibitor was subjected to freezing using liquid nitrogen and subsequent thawing at 37 °C in a water bath. Once approximately 60% of the cells were thawed, they were transferred to ice to continue thawing, repeating this process three times. The soluble proteins in the supernatant were separated from the cell precipitation by centrifugation at 20,000× g for 20 min at 4 °C. The resulting supernatants were then treated with both a vehicle and 15a. Following a 5 min incubation at room temperature with either a vehicle or drugs, the lysates were partitioned into multiple 50 µL aliquots within new 200 µL PCR tubes. These aliquots were then individually heated at varying temperatures for 3 min using a thermal cycler, followed by a 3 min cooling period at room temperature. Subsequently, the heated lysates underwent centrifugation at 20,000× g for 20 min at 4 °C to separate soluble proteins from precipitated proteins. The resulting soluble proteins were utilized for western blotting, with their protein concentration determined through the employment of a BCA assay.

Transient transfection cell assay

Cells were initially plated at a confluency range of 50% to 60% in 60 mm plates and incubated for a duration of 24 h. Subsequently, the cells were transiently transfected using Lipofectamine RNAiMAX Transfection Reagent, employing two sets of pooled siRNAs per gene, specifically DNMT1, HDAC1, MDA5, RIG-I and MAVS, which were procured from GenePharma. To serve as a control, nonspecific scrambled siRNA was utilized. Following a 24-hour period of transfection, the cells were lysed for the purpose of immunoblotting analysis. The gene sequence can be found in Supplementary Table S3.

Construction of stable cells

Extraction of plasmid: take a conical bottle and pour 100 mL LB medium, seal with tissue culture sealing film and rubber band, and then sterilize with high-pressure steam. After cooling to room temperature, ampicillin was added (final concentration was 100 μg/mL), 500 μL of sequenced bacterial solution was added, and the bed was shaken at 37 °C at 200× g overnight. After the bacterial solution was cloudy, the plasmid was extracted in large quantities (Beyotime), and the gene sequence was shown in Supplementary Table S4. (2) Packaged virus: HEK-293T cells were incubated in DMEM medium at 37 °C. The mixture (4 μg target plasmid, 1 μg pMD2.G envelope plasmid, 3 μg psPAX2 package plasmid and 10 μL (1 mg/mL) PEI (Beyotime, C0537) was added when the density of cells was 80%–90%. The mixture was prepared with FBS-free medium to 400 μL and then left for 10 min. Drips were added to 293 T cells in 10 cm petri dish. After 6–8 h, the medium was discarded and was added into 5 mL DMEM medium. After 24 h, the venom was collected and was centrifuged for 1000× g for 5 min, and the supernatant was divided into 1 mL tubes and stored at −80 °C. (3) Infected cells: the target cells were spread in a 6 cm dish and incubated overnight at 37 °C. When the cell density was 80–90%, the old medium was discarded and 1 mL of disease venom and 1 μL polybrene (8 mg/mL, Beyotime, C0351) were added. After 4 h, the disease venom was sucked away and added to 3 mL of complete medium, or directly added to 2 mL of complete medium. In order to screen out the successfully infected cells, complete medium containing puromycin was added to the next passage and subsequent culture for culture, and after the state of the target cells was stable, western blot assay and qRT‒PCR assay were used to verify whether the knockdown was carried out.

Immunofluorescence (IF)

Breast cancer cells with a density of 50–60% were cultured in confocal petri dish (Nest, KH-TB-A1111) for 48 h, then fixed with −20 °C pre-cooled ice methanol at room temperature for 10 min, and washed three times with 1× PBS after fixation. Goat serum blocking buffer (1× PBS/5% normal serum / 0.3% Triton X-100) was added, enclosed at room temperature for 1 h, washed three times with 1× PBS, STAT1 (1:200) and J2 (1:200) antibody, incubated overnight at 4 °C, recovered primary antibody on the second day, washed three times with 1× PBS, and added rabbit secondary antibody (1:200) or mouse secondary antibody (1:50), after incubation at room temperature for 1 h, wash three times with 1× PBS, add DAPI anti-fluorescence quencher (Yesen, 36308ES20), and incubate in dark light for 5 min before taking photos. The primary and secondary antibodies were formulated with a dilute release buffer (1× PBS / 1% BSA / 0.3% Triton X-100).

Methylation-specific PCR (MSP) analysis

Cells were subjected to treatment with a vehicle and different concentrations of 15a. The Blood/Cell/Tissue Genomic DNA Extraction Kit (Tangen) was used to extract DNA from these cells. Subsequently, the DNA was processed using the D5005/EZDNAMethylation-GoldKit kit (Zymo Research) and the methylation-specific PCR kit (Tangen). During this process, unmethylated cytosine was converted to uracil, while methylated cytosine remained unaltered. Based on this alteration in the DNA bases, primers targeting methylated and unmethylated sequences were designed and amplified using PCR.

In vivo functional assays

A total of 2 × 105 4T1 cells were subcutaneously inoculated into female BALB/c wild-type mice aged 6–8 weeks, which were obtained from Guangdong Pharmaceutical Company. Once the tumour volume reached 80–100 mm3, the mice were randomly assigned to two groups, with five mice in each group. The groups were administered either a vehicle (10% DMSO, 45% PEG300, and 45% 1× PBS) or 15a (dissolved in vehicle) intravenously once daily. Tumour size was measured using vernier calipers every three days. The mean tumour volume for each group was calculated using the formula (length × width × width)/2 and expressed in cubic millimeters. The animals were killed on day 21, and the removed tumour tissue was weighed and photographed. For in vivo combination therapy, mice were treated with vehicle or 15a alone injected on once every three days or in combination with anti-PD-L1 antibody (BioXcell, 100 µg injected on days 7, 10, 13). The experiments were conducted in accordance with the regulations of the Agency’s Animal Management and Use Committee and were approved by the Committee.

Hematoxylin and eosin (HE) staining

The organ specimens, comprising the heart, liver, spleen, lung, and kidney, were subjected to a series of procedural steps, followed by staining with the HE staining kit (Biosharp).

Immunohistochemistry (IHC)

The tumour specimen underwent a series of procedures, including dewaxing and washing with 1× PBS, treatment with 3% H2O2 for 20 min to prevent endogenous peroxidase activity, heat-induced epitope retrieval at EDTA (pH 9.0) for 2 min, and incubation in a blocking buffer containing 10% goat serum for 30 min at room temperature. Subsequently, the sections were exposed to primary antibodies against PD-L1 (CST, 64988, 1:200) and CD8α (CST, 98941, 1:200) overnight at 4 °C, followed by incubation with secondary antibodies (MaxVision) for 1 h at room temperature. Finally, the sections were developed using diaminobenzidine (DAB). The IHC profiler plugin in Image J software was utilized to automatically analyze the percentage and staining intensity of positive cells in each image [55, 56].

Database analysis

Gene correlation analysis was retrieved from the TCGA database (https://portal.gdc.cancer.gov/) RNA expression profiles and matched clinicopathological information of breast cancer patients. Spearman correlation analysis was conducted on DNMT1 and HDAC1 in tumor and non-tumor groups using R4.2.1 to explore the relationship between DNMT1 and HDAC1. A statistically significant difference was defined as a difference with a p-value < 0.05. Kaplan–Meier overall survival curves of human breast tumors generated using Kaplan–Meier Plotter according to DNMT1 and HDAC1 gene expression levels. The R package ggplot2 was used to plot gene difference analysis, correlation analysis and Kaplan–Meier survival curves.

Statistical analysis

All experiments requiring statistical analysis were independently conducted with a minimum of three replications. The experimental data are presented as the mean value ± standard deviation (SD) or standard error of the mean (SEM). Statistical analysis was carried out using GraphPad Prism 8.0. Differences were assessed an unpaired Student’s t test for two-sample comparisons and one-way analysis of variance (ANOVA) for multiple-sample comparisons. A statistically significant difference was defined as a p-value < 0.05. Spearman correlation analysis was employed for assessing correlations between DNMT1 and HDAC1 expression levels. Kaplan–Meier survival curves were analyzed using the Mantel–Cox log-rank test. Inclusion/exclusion criteria were all pre-established and no samples or animals were excluded from the analysis.