An enteropathogenic microbial toxin modulates the breast cancer epigenome resulting in concurrent silencing of tumor suppressor genes

Abstract

Background

Recent studies have determined a close association between host microbiota and breast cancer initiation, growth and therapeutic outcomes. We previously uncovered how enterotoxigenic Bacteroides fragilis (ETBF), a gut microbe present in malignant breast tissue, aids breast cancer development and metastatic progression via activating multiple oncogenic pathways and modulating breast tumor microenvironment. Brief exposure to ETBF-secreted toxin, BFT (Bacteroides fragilis toxin), imparts long-term oncogenic, pro-stemness and pro-metastasis memory in breast cancer cells indicating the involvement of epigenetic alterations hence, we aimed to investigate the potential involvement of epigenetics in biological impact of ETBF in breast cancer.

Methods

RNA sequencing and Infinium methylation EPIC microarray were performed in mammary tumors developed from the BFT-exposed breast cancer cells. Alterations in the expression of tumor suppressor genes (TSGs) were assessed using RT-PCR, ICC and IHC. Demethylation agent and HDAC inhibitor were utilized to rescue the expression of BFT-mediated hypermethylated TSGs, and examine the migration/invasion potential of BFT-exposed breast cancer cells.

Results

EPIC methylation microarray analysis of BFT-exposed mammary tumors uncovered 64 differentially methylated significant CpG sites whose analysis led to the identification of 26 hypomethylated and 156 hypermethylated genes. Of the hypermethylated genes, also showing reduced expression in RNA-seq in BFT-exposed group, we identified five important TSGs, namely, NF2, RSK3, FAT4, DCN and DOK2. Survival analysis revealed that decreased expression of these TSGs associated with worse prognosis. While BFT exposure reduced the expression of these TSGs, treatment with Azacytidine or/and Trichostatin A resulted in rescue of the TSGs from BFT-induced repression while mitigating BFT-driven migration and invasion of breast cancer cells.

Conclusions

Collectively, BFT exposure epigenetically modifies the expression of TSGs and impacts migration/invasion potential of breast cancer cells, and treatment with demethylation agent(s) and HDAC inhibitors effectively diminishes the functional impact of BFT.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13058-025-02111-9.

Introduction

The continued worldwide increase in breast cancer incidence underscores the need for determining early molecular events leading to breast cancer initiation and progression in order to develop effective prevention as well as treatment strategies. Recent studies have established a strong association between the breast carcinogenesis and the host microbiota [1, 2]. Dysbiosis of the host physiological microbiota, in terms of abundance and/or composition, has profound effects on breast cancer emergence and malignancy [3]. Through data mining and metagenomic analysis, we identified the increased presence of Bacteroides fragilis, a common colon colonizer, in breast cancer tissues [4]. Enterotoxigenic Bacteroides fragilis (ETBF), a pathogenic strain of gram-negative obligate anaerobe B. fragilis, is an established pro-carcinogen, owing to its secreted virulence factor, B. fragilis toxin (BFT) [5]. Colonization of mammary ducts with ETBF led to the development of early hyperplasia-like features including widespread local inflammation, tissue fibrosis and higher cell proliferation in healthy mice while the tumor-bearing mice exhibited higher tumor growth and metastatic progression [4]. Gut/mammary ducts colonization with ETBF also exerted a profound effect on tumor microenvironment causing early dissemination of cancer cells towards metastasis [6]. Most interestingly, a very brief exposure to BFT imparted breast cancer cells with a long-term oncogenic memory evident in upregulation of multiple oncogenic pathways, stemness and metastasis-related gene networks indicating the involvement of epigenetic regulation.

Epigenetic regulation is a key mechanism governing critical transcription programs [7]. Multiple studies have established the connection between epigenetic dysregulation of oncogenes and tumor suppressors with cancer development and progression [8]. Alterations to the physiological pattern of DNA methylation, one of the most extensively studied epigenetic modifications, are commonly associated with abrogation of cellular and molecular pathways that stimulate the emergence of complex diseases, including breast cancer [9, 10]. A large body of evidence indicates that hypermethylation in the promoters and cytosine guanine dinucleotide-rich (CpG) islands of tumor suppressor genes (TSGs) blocks their transcription, resulting in unhindered cell proliferation [11]. Importantly, increased activity of DNA Methyltransferase 1 (DNMT1) is observed in triple-negative breast cancer compared to the luminal type, which may explain the more metastatic and resistant nature of the former [12]. Several DNA methylation changes serve prognostic value in breast cancer patients [13]. For instance, hypermethylation of BReast CAncer Gene 1 (BRCA1), Phosphatase and TENsin homolog (PTEN), E-cadherin, Glutathione S-Transferase Mu2 (GSTM2) and SuperOxide Dismutase 3 (SOD3) promoters are reported as early events during tumorigenesis and has been correlated with abrogated cellular functions that exacerbate breast cancer progression [14–18]. Such abnormal methylation patterns are associated with chemoresistance, poor prognosis and lower survival among breast cancer patients. Microbial dysbiosis has been implicated in epigenetic modulations mainly via the microbial metabolites. Several bacterial metabolites have been shown to donate methyl groups towards DNA and histone methylation or modulate histone deacetylase (HDAC) and DNMT activity leading to epigenetic modifications of several important genes [19–21]. These studies suggest a close association between microbial dysbiosis and epigenetic regulation although this has not been thoroughly explored in the context of breast cancer.

In this study, we evaluated the impact of BFT on the global methylation profile in breast cancer cells and its functional implications. EPIC methylation microarray analysis revealed differential methylation patterns in BFT-exposed breast cancer cells, comprising of multiple hyper and hypomethylated genes. Among the hypermethylated genes, we found five critical TSGs namely, Neurofibromatosis type 2 (NF2), FAT atypical cadherin 4 (FAT4), Ribosomal S6 Kinase 3 (RSK3), Decorin (DCN), and Docking Protein 2 (DOK2). RNA sequencing revealed a significant downregulation in their expression in the BFT-exposed group, which correlated with their hypermethylated status. Additionally, treating breast cancer cells with DNMT or HDAC inhibitors, either alone or in combination, reverted the BFT-mediated repression of TSG expression and mitigated BFT-associated pro-cancer features, such as elevated migration and invasion potential. Overall, our evidences put forth BFT as a modulator of the epigenome which can potentiate breast cancer owing to the suppression of multiple TSGs.

Materials and methods

Cell lines, Bacteroides fragilis toxin, reagents and animal experiments

Human breast cancer cell lines MCF7, HCC1806, HCC1937 and MDA-MB231 were procured from the ATCC and maintained at 37 °C in 5% CO₂ and 95% humidity. Experiments were conducted within 10 to 20 passages from thawing of the cell lines. Authentication for all cells was performed using short tandem repeat testing. The MycoAlert Detection Kit (Lonza, LT07-218) was regularly executed for mycoplasma detection. Bacteroides fragilis toxin (BFT) was HPLC purified from ETBF culture supernatants (kindly gifted by Dr. Cynthia L. Sears, SOM, Johns Hopkins) as previously elaborated [22] and 5 nmol/L (100 ng/mL) concentration of purified BFT was used for treating cells in media containing 2% serum for the indicated durations. Inhibitors DNA methyltransferase inhibitor, 5-AZAcytidine (Sigma, USA), and histone deacetylase inhibitor, Trichostatin A (Sigma, USA) were used for the study. Zebularine was also procured from Sigma, USA. NOD/SCID mice (female, 6–8 weeks old) were acquired from Sidney Kimmel Comprehensive Cancer Center (SKCCC) animal facility and maintained in house. Exponentially growing control MCF7 cells and MCF7 cells treated with 5 nmol/L BFT for 72 h (5 × 10⁷ cells in 100 µL matrigel), were implanted in the fourth mammary fat pad on either side. After eight weeks, tumors were collected, dissociated into single cells, counted and reinjected into the fourth mammary gland of a fresh set of SCID mice for a limited dilution assay. After an additional eight weeks, tumor samples were collected for the RNA-sequencing and methylation analysis. Human HER2-overexpressing transgenic mice in FVB background (HuHER2) that spontaneously develop mammary tumors and lung metastases were generously gifted by Robert Ivkov, SKCCC, Johns Hopkins. These mice were given antibiotic cocktail (clindamycin 0.1 g/L and streptomycin 5 g/L) in water bottles (Hospira and Amresco) for 7 days followed by oral infection with ∼10⁸ CFU of ETBF in 1X PBS. Sham control mice were administered with 1X PBS. All animal experiments were performed according to the JHU ACUC.

EPIC microarray

Infinium methylation EPIC microarray analysis was performed on control and BFT-treated tumor samples. Library preparation was performed as per Illumina EPIC array protocol. (Illumina, San Francisco USA). Further, libraries were processed through Illumina microarray. Raw data was collected in red and green files. EPIC array provides CpG sites in and outside of CpG islands and differentially methylated regions in tumor versus normal sets. Data analysis Raw data of EPIC array was analyzed through R-based ChAMP tool version 2.26 [23]. ChAMP is an integrated data analysis pipeline for filtering low-quality probes, detecting differentially methylated positions (DMPs), and identifying differentially methylated regions (DMRs). Expression profiles of TSGs were identified in our previously published RNA sequencing DEG results [4]. To analyze the expression profile in public databases, a web-based UALCAN database was used.

Protein isolation and western blotting

Whole cell lysates were prepared using modified RIPA buffer containing 2 mM phenylmethanesulfonyl fluoride, 2 mM NaF, 1 mM Na₃VO₄ and protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). Protein concentration was determined with Bradford reagent (Bio-Rad, USA). Equal amount of protein lysates was subjected to SDS-PAGE followed by transfer onto polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk for 1 h, incubated with specific primary (Table 1) and horseradish peroxidase-conjugated secondary antibodies and developed employing enhanced chemiluminescence (Radiance Plus Substrate, Azure Biosystems).

Table 1

Antibody	Company	Catalog number	Secondary
NF2	Santa Cruz	sc-55,575	Mouse
RSK3	Santa Cruz	sc-517,283	Mouse
FAT4	Abcam	ab130076	Rabbit
DCN2	Thermo	PA5-13538	Rabbit

RT-PCR

Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. One microgram of total RNA was used to synthesize cDNA with an iScript cDNA Synthesis Kit (Bio-Rad, CA, USA). RT-PCR was performed and imaged using the Gel Doc image system (Bio-Rad, CA, USA). For quantitative Real-time PCR, the SYBR GREEN Master Mix (Applied Biosystems, Foster City, CA, USA) was employed as per the manufacturer’s protocol with GAPDH as internal control and target genes listed in Table 2.

Table 2

Primer name	Forward primer (5’-3’)	Reverse primer (5’-3’)	Product size (bp)	Annealing temperature (°C)
Human NF2	ACCGTTGCCT CCTGACATAC	TCGGAGTTC TCATTGTGCAG	234	60
Human FAT4	ATGATACGGG GTGGATTTCA	GCACTCATG TGGCTTTGAGA	181	60
Human RSK3	GGCTCTCCT TCCTCACACAG	CCACTGCAG ATTGGGAGAAT	205	60
Human DCN2	AGAAAGGG CATATGCACACC	CCCTCACAGT CACTGCAGAA	153	60
Human DOK2	TTCTGCTG GTGACTCCTCCT	TTGTCCTCTG CCCTAAATGC	246	60
Human GAPDH	AATCCCAT CACCATCTTCCA	TGGACTCCA CGACGTACTCA	82	58–60

Immunocytochemistry (ICC)

Cells (5 × 10⁴) were seeded into 4-well chambered slides (Nunc, Rochester, NY) followed by appropriate treatment. Thereafter, cells were fixed with 4% para-formaldehyde and permeabilized with citrate buffer, followed by blocking and overnight incubation with primary antibody in 3% BSA at 4°C. For secondary antibody incubation, cells were incubated with Alexa Fluor-tagged secondary antibody (Molecular Probe, USA). Images of slides were captured with Nikon spinning disc confocal microscope at the Johns Hopkins SOM Microscope facility at 40× magnification using an oil immersion objective lens. Further images were processed with ImageJ tool.

Immunohistochemistry

Tumor tissue sections were fixed in 10% formalin, paraffin-embedded and sectioned. IHC analyses of the tissue sections was carried out with anti-NF2, anti-RSK3, anti-FAT4 and anti-DCN2 antibodies, followed by incubation with HRP-conjugated secondary antibodies and developed using DAB peroxidase substrate kit (SK-4100, Vector Laboratories, CA). Images were captured with a Leica microscope at 20× magnification. Images were analyzed by Aperio ImageScope Software, Leica. Quantification of micrographs and IHC was done using Leica Aperio ImageScope, Leica Biosystems.

Transwell migration assay

Briefly, 1 × 10⁴ cells were seeded in serum-free media in the upper chamber of transwell inserts in 12-well plates. The lower chambers were filled with serum-supplemented media and incubated for defined durations. Migrated cells were fixed in formalin and stained with 0.05% crystal violet. Excess cells were removed using a damp cotton swab and cells on the bottom surface of the inserts were imaged using microscope, quantified using Leica ImageScope software and graphically presented.

Scratch migration assay

Cells were seeded in each well of the ibidi Culture-Insert 2 well in 35 mm cell culture dishes overnight and allowed to form monolayer. Following day, culture-inserts were removed using sterile tweezers. Cell monolayer was washed with PBS to remove any cell debris and fresh medium added with appropriate treatments. Mitomycin C (final concentration of 10 µg/ml) was added to the cells at this point to prevent cell proliferation. Cells were photographed at defined time intervals. Migration distance was quantified using Leica ImageScope software.

Matrigel invasion assay

Invasion was determined using the 24-well BD BioCoat Matrigel invasion chambers (BD Biosciences, San Jose, CA). Inserts were rehydrated using serum free media for at least 2 h. A total of 1 × 10⁵ cells were seeded in the upper inserts in 0.5 ml of serum free medium while the bottom wells were filled with complete media. Cells were allowed to invade towards the complete medium through the matrigel-coated membrane for 48 h. Non-invading cells were removed using a damp cotton swab while the invading cells that adhered to the bottom surface of the insert were fixed in formalin, stained with 0.05% crystal violet stain and imaged using microscope, quantified using Leica ImageScope software and graphically presented.

Survival analysis

Recurrence-free survival analysis for genes was performed using KM Plotter (https://kmplot.com/analysis/) for the breast cancer gene chip. KM plotter uses the log-rank test to assess the difference between survival curves, and the hazard ratio (HR) with its 95% confidence interval is provided to quantify risk. We selected the default parameter for recurrence-free survival (Split patients by median) and selected the green probes for genes which have a high number of patients.

Statistical analysis

GraphPad Prism 10 was used for statistical analysis, and student’s t-test was performed for each experiment based on sample size to get the p-values. Experiments were performed three times in triplicates.

Results

Bacteroides fragilis toxin exposure modulates the methylation landscape in breast cancer

We recently identified BFT as a potent carcinogenic driver for the initiation, growth and metastatic progression of breast cancer through modulation of key oncogenic pathways, including the β-catenin and Notch1 signaling cascade [4]. Interestingly, a short 72 h exposure to BFT imparted a long-term oncogenic memory in breast cancer cells [4], indicating the involvement of epigenetic alterations. To query, whether BFT is indeed an epigenetic modifier, secondary tumors (formed in the limited-dilution assay using 5-million or 5-thousand primary tumor-dissociated cells) from the BFT-pretreated (72 h) MCF7 group and control group were subjected to EPIC methylation microarray. Of note, genome-wide DNA methylation analysis allows quantitative determination of the total methylation level and a comprehensive coverage of CpG islands, enhancer regions, open chromatin sites and other important regions of the methylome. Sample distribution is listed in Supplementary Fig. 1. The obtained microarray data was analyzed with Chip Analysis Methylation (ChAMP) analysis pipeline, which detects differentially methylated regions (DMRs), finding differentially methylated positions (DMPs). We found 64 differentially-methylated (DM) significant CpG sites between the secondary tumors from the BFT-pretreated MCF7 group and control group (p-value < 0.05) (Fig. 1a, Supplementary Table 1). Further analysis of these sites led to the identification of 26 hypomethylated genes and 156 hypermethylated genes in the secondary tumors from the BFT-pretreated MCF7 group (Fig. 1b, Supplementary Table 2). Hyper- and hypo-methylation of gene promoters regulate their expression through chromatin accessibility [24]. Out of the 156 hypermethylated genes, five genes, NF2, FAT4, RSK3, DCN, and DOK2 are established tumor suppressor genes (TSGs) [25–29]. DNA methylation level is reportedly higher in breast cancer cells and hypermethylation of TSGs facilitates diverse neoplastic phenotypes, including uncontrolled cell proliferation and metastasis [11, 30]. Therefore, we hypothesized that BFT may promote hypermethylation of these TSGs resulting in reduced expression, which may culminate in downstream dysregulation of normal cellular physiology, resulting in oncogenesis. To examine the gene expression profile of these TSGs, we queried our RNA-sequencing data obtained from the secondary tumors from BFT-pretreated MCF7 group and control group [4]. NF2, FAT4, RSK3, DCN, and DOK2 are highlighted in the volcano plot (Fig. 1c). Hypermethylation and reduced expression of NF2, FAT4, RSK3, DCN, and DOK2 was noted in the BFT-pretreated tumor group in comparison to the control group (Fig. 1d). Collectively, our analysis revealed that BFT-mediated hypermethylation associate with a noticeable inhibition in the expression of TSGs in breast tumor samples.

Fig. 1.

BFT treatment modulates methylation and expression of tumor suppressor genes in breast cancer cells. (a) A total of 64 differentially methylated regions (DMRs) are identified between tumors derived from BFT-pretreated (100ng/ml of BFT treatment for 72 h) MCF7 breast cancer cells versus untreated control MCF7 cells using EPIC methylation microarray. Heatmap shows hyper- and hypo-methylated CpGs. (b) The differentially methylated genes corresponding to these DMRs are presented in the waterfall plot. (c) Volcano plot depicts the differentially expressed genes from the RNA seq analysis of tumors derived from BFT-pretreated MCF7 vs. untreated control MCF7 cells. (d) Heatmap shows hypermethylation (EPIC methylation microarray) and expression (RNA-seq) levels of five tumor suppressor genes. BFT-5 M (tumors derived from 5 million BFT-treated MCF7 cells), BFT-5 K (tumors derived from 5 thousand BFT-treated MCF7 cells)

Reduced expression of NF2, FAT4, RSK3, DCN, and DOK2 is associated with disease progression and poor survival in breast cancer patients

Loss or reduced expression of TSGs directly/indirectly facilitates cancer progression, and this may serve as a potent mechanism for microbial dysbiosis-mediated carcinogenesis. Hence, we questioned if the TSGs identified in our methylation array and RNA-seq data exhibit any clinical relevance in breast cancer patients. As depicted in Fig. 2a, analysis of the TCGA dataset revealed that, with the exception of NF2, all other TSGs (FAT4, RSK3, DCN, and DOK2) had a notably reduced expression in primary tumors in comparison to normal tissue across breast cancer patients. Moreover, FAT4, DCN, and RSK3 exhibited a significant decline in their expression with progressing stages of breast carcinoma, suggesting that downregulation of these TSGs may be closely associated with enhanced disease progression in breast cancer patients (Fig. 2b). Prognostic relevance of these TSGs was investigated by performing recurrence-free survival analysis for all five TSGs using the Kaplan-Meier Plotter (kmplot.com/analysis). Our results clearly demonstrated that the decreased expression of either NF2, FAT4, RSK3, DCN or DOK2 was significantly associated with poor recurrence-free survival of breast cancer patients (Fig. 2c). Overall, these results underline the clinical relevance of the TSGs that exhibit increased methylation and reduced expression upon BFT exposure.

Fig. 2.

BFT-modulated tumor suppressor genes associate with better prognosis in breast cancer patients. (a) Plots show expression profile of NF2, FAT4, DCN, RSK3, and DOK2 in normal versus primary breast tumor samples in TCGA cohort. (b) Plots show expression profile of NF2, FAT4, DCN, RSK3, and DOK2 in different stages of breast tumors using the TCGA dataset. (c) Kaplan-Meier curves show recurrence free survival of breast cancer patients with high or low mean expression of NF2, FAT4, DCN, RSK3, and DOK2

Expression of TSGs is considerably suppressed in breast cancer cells exposed to Bacteroides fragilis toxin or enterotoxigenic Bacteroides fragilis (ETBF)

Methylation array revealed the hypermethylation of five TSGs while RNA-seq results showed their reduced expression in breast cancer cells upon BFT exposure. To further validate these findings, we treated HCC1806 cells with BFT for different durations and examined the expression of the TSGs. qRT-PCR analysis showed a notable reduction in the expression of NF2, FAT4, DCN2, and RSK3, (Fig. 3a). DOK2 did not exhibit a consistent pattern in its transcript level following BFT treatment, therefore, we did not proceed with this particular TSG for our subsequent experiments. We queried the prognostic relevance of these TSGs on recurrence-free-survival (RFS). Our results clearly demonstrated that the combined decreased expression of NF2, FAT4, RSK3, DCN or DOK2 was significantly associated with poor RFS of breast cancer patients (Fig. 3b). Moreover, immunofluorescence staining corroborated these findings and showed a clear decrease in the expression of NF2, FAT4, DCN2, and RSK3 in the BFT-exposed cells compared to control cells after 24 h of treatment (Fig. 3c, Supplementary Fig. 2a-d). Together, our results suggested that BFT exposure led to a significant inhibition of expression of NF2, FAT4, DCN2, and RSK3 in breast cancer cells. Additionally, immunohistochemical analysis of tumors from BFT-pretreated MCF7 group compared to control group revealed a marked decrease in the expression of NF2, FAT4, DCN2, and RSK3 in the BFT group compared to control (Fig. 3d, Supplementary Fig. 2e). Next, we investigated the impact of ETBF exposure on the TSG expression in mammary tumors. Towards this, immunohistochemical analysis was performed using mammary tumor tissues from MMTV.f.HuHer2 mice, a human Her2 overexpressing spontaneous breast cancer model, orally gavaged with ETBF (10⁸ CFU) or sham controls. Mammary tumors from mice harboring gut ETBF colonization demonstrated a notable reduction in the expression of the TSGs relative to vehicle group (Fig. 3e, Supplementary Fig. 2f). These in vivo results corroborated our in vitro and in silico findings.

Fig. 3.

BFT treatment inhibits NF2, FAT4, DCN2 and RSK3 expression in breast cancer cells and mammary tumor samples. (a) qRT-PCR analysis of NF2, FAT4, DCN2 and RSK3 in HCC1806 cells following treatment with 100 ng/ml of BFT for the indicated timepoints. Graphical data represent mean ± SD from three independent experiments. Statistical significance was calculated using student’s two-tailed t-test. * p-value < 0.05. (b) Kaplan-Meier curves show Recurrence-free-survival of breast cancer patients with high or low mean combined expression of NF2 (218915_at), RPS6KA2/RSK3 (212912_at), DCN (209335_at), DOK2 (214054_at), and FAT4 (219427_at). (c) Representative immunostaining images for NF2, FAT4, DCN2 and RSK3 in HCC1806 cells treated with 100 ng/ml of BFT for 24 h. Nuclei are counterstained with DAPI. (d, e) Representative IHC images of NF2, FAT4, DCN2 and RSK3-stained tissue sections from mammary tumors formed in NOD/SCID mice implanted with either untreated or BFT-treated (72 h) MCF7 cells (d), and from spontaneous mammary tumors developed in MMTV.f.HuHer2 mice subjected to oral gavage with either vehicle or ETBF (10⁸ CFU) (e). Graphs show the quantification of the intensity of expression of respective TSGs from each experimental group. Scale bar, 100 μm. Graphical data represent mean ± SD from two different animals from each experimental group

Treatment with demethylation agent and/or histone deacetylase inhibitor effectively reverses the effect of BFT in breast cancer cells

Our aforementioned findings collectively indicated that BFT promotes hypermethylation of the TSGs, which, subsequently, leads to reduced expression of the TSGs in breast cancer cells, thus suggesting BFT-mediated epigenetic modulation of these genes. DNA methylation and histone modifications regulate gene expression via chromatin modifications and are intimately associated with the etiology of various cancers [31]. Earlier studies have shown that DNA methyltransferase 1 (DNMT1) inhibition and/or HDAC inhibition successfully leads to increased expression of genes that are epigenetically silenced [32, 33]. Hence, we proposed that, if BFT reduced expression of the TSGs in breast cancer as a consequence of epigenetic dysregulation (such as, DNA methylation or histone deacetylation), then, the use of DNMT and/or HDAC inhibitors could potentially reverse this impact. To address this, we treated HCC1806 cells with a DNMT inhibitor, 5-Azacytidine (Aza) [34] and an HDAC inhibitor, Trichostatin A (TSA) [35, 36] alone and in combination. Intriguingly, our results demonstrated that while BFT exposure led to a considerable decline in the expression of NF2, FAT4, DCN and RSK3, treatment with 5-Azacytidine and/or Trichostatin A successfully reversed this effect for all the TSGs tested (Fig. 4). This important observation supported our hypothesis that BFT exposure indeed reduced the expression of TSGs in breast cancer through epigenetic remodeling and disrupting the epigenetic silencing restored the physiological expression of these TSGs in breast cancer cells.

Fig. 4.

Treatment with demethylation agent and/or HDAC inhibitor reverses BFT-mediated inhibition of NF2, FAT4, DCN2 and RSK3. qRT-PCR analysis of NF2, FAT4, DCN2 and RSK3 in HCC1806 cells following treatment with 2.5 µM of 5-azacytidine (Aza) and/or 100 ng/ml of trichostatin A (TSA) for 48 h prior to exposure to 100 ng/ml of BFT for 24 h. Graphical data represent mean ± SD from two independent experiments. Statistical significance was calculated using student’s two-tailed t-test. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001

BFT-stimulated cellular migration and invasion are mitigated in the presence of the DNMT1 and HDAC inhibitors

BFT is well-established as a pro-oncogenic toxin that induces migration and invasion potential of breast cancer cells [4]. To understand whether DNMT1 and HDAC inhibitors can effectively block the functional impact of BFT on breast cancer cells, we treated BFT-exposed cells with Aza and/or TSA and queried their migration and invasion potential. Scratch migration assay in HCC1806 cells revealed a faster wound-closure in the presence of BFT, implying enhanced cell migration. On the contrary, treatment with either Aza or TSA or Aza + TSA resulted in a delayed wound closure indicating mitigated migration in BFT-exposed cells (Fig. 5a). Similar results were noted in additional breast cancer cell lines, MCF7, HCC1937 and MDAMB231, where BFT exposure increased cell migration and treatment with Aza, TSA or Aza + TSA blocked BFT-induced migration (Supplementary Fig. 3a-c). Next, we tested Zebularine, another known DNMT inhibitor and demethylation agent with higher efficacy and reduced toxicity [37], for its effectiveness against BFT-mediated increased migration of breast cancer cells. MCF7 and HCC1937 cells exhibited increased migration upon BFT exposure as expected, which is effectively inhibited with zebularine treatment (Supplementary Fig. 4). Accordingly, BFT treatment led to higher migration and invasion of MCF7 and HCC1806 breast cancer cells while these oncogenic features were diminished following treatment with either Aza or TSA or their combination in both the cell lines (Fig. 5b, c, Supplementary Fig. 5a, b). This trend was also observed in terms of HCC1937 cell migration (Supplementary Fig. 5c). Our collated evidences, thus, strengthen the notion that BFT exposure epigenetically modifies the expression of TSGs and impacts migration/invasion potential of breast cancer cells, and treatment with demethylation agent(s) and HDAC inhibitors effectively reduces the functional impact of BFT.

Fig. 5.

Epigenetic modifiers abrogate BFT-triggered increased migration and invasion of breast cancer cells. HCC1806 cells were treated with 2.5 µM of 5-azacytidine (Aza) and/or 100 ng/ml of trichostatin A (TSA) either alone or in combination with 100 ng/ml of BFT. (a) Representative images of cells undergoing scratch migration assay for 12 h. Graphical data represent mean ± SD from three independent experiments. Statistical significance was calculated using student’s two-tailed t-test. * p-value < 0.05, ** p-value < 0.01. (b) Representative images of cells undergoing transwell migration assay for 48 h. Graphical data represent mean ± SD from three independent experiments. Statistical significance was calculated using student’s two-tailed t-test. *** p-value < 0.001. (c) Representative images of cells undergoing Matrigel invasion assay for 48 h. Graphical data represent mean ± SD from three independent experiments. Statistical significance was calculated using student’s two-tailed t-test. *** p-value < 0.001.d

Discussion

Connecting microbial dysbiosis and epigenetic modifications, the results presented here are the first to demonstrate a direct role for ETBF-BFT exposure on distinct epigenetic alterations in breast cancer. We found 64 differentially-methylated significant CpG sites in breast tumors derived from breast cancer cells briefly exposed to BFT and further analysis of these sites led to the identification of 26 hypomethylated genes and 156 hypermethylated genes in the BFT group. While no study has reported a microbes-methylation link in breast cancer, it has been shown how epigenetic landscape in colorectal cancer (CRC), modulated by microbes, is linked with CRC pathogenesis. Patients harboring Helicobacter pylori or Epstein Barr virus infection exhibit hypermethylation of DNA in the gastric mucosa. Also, significant associations between the colonization with Fusobacterium nucleatum (F. nucleatum) or B. fragilis and the presence of CIMP (CpG island methylator phenotype) in CRC patients were observed [38, 39]. In fact, CRC samples found to harbor higher accumulation of F. nucleatum or B. fragilis or K. pneumoniae termed bacterial “Superhigh” also showed higher hypermethylation with distinct patterns in multiple CpGs [40]. In a clinical study involving 1232 CRC cases, higher DNA amounts of B. fragilis and ETBF in tumor tissues significantly correlated with CIMP-high CRC [41]. Allen et al. demonstrated a cross-talk between BFT-induced chromatin accessibility and gene expression changes that favors colon tumorigenesis [42]. A specific subtype of colorectal cancer which is characterized with distinct methylation events shows differential colonization with microbes, hence presenting an association between microbiota and methylation in cancer [43]. BFT mediates activation of inflammatory signaling pathways, such as NF-κB, which has been associated with increased activity of DNA methyltransferases (DNMTs) and subsequent promoter hypermethylation in cancer cells [44, 45]. Though no direct connection has been reported between BFT and DNMT, inflammatory cytokines may mediate the effects of microbial toxins on methyl binding proteins. ETBF administration in mouse model of colitis showed that ETBF-BFT axis induces inflammation and oxidative stress leading to prominent alterations in several epigenetic modifiers such as SIRT1 and EZH2 favoring aberrant methylation [46]. Methyl-CpG binding proteins (MBPs) recognize methylated CpG sites and regulate transcriptional repression of TSGs [47, 48], few reports have shown the direct/indirect impact of microbes on DNA methylation in colorectal cancer [49]. These previous studies, predominantly focusing on colon cancer, presented a connection between microbial dysbiosis and epigenetic landscape of cancer cells. Our study focused on breast cancer cells and showed the modulation in DNA methylation, and uncovered hypermethylation and diminished expression of distinct TSGs upon BFT exposure.

Among the hypermethylated genes showing reduced expression, we found five critical TSGs namely, NF2, FAT4, RSK3, DCN, and DOK2 in the secondary tumors from the BFT-pretreated MCF7 group compared to the control group. Of importance, lower levels of these TSGs strongly correlated with poor outcome among breast cancer patients. All these TSGs are known to play diverse roles in regulating cellular growth, morphology, communication and motility. Naturally, abrupt expression of these genes is expected to favor uncontrolled cell division and, subsequently, cancer. NF2 is known for its role in inhibiting cell proliferation and oncogene-induced transformations, and mutation/loss of expression of NF2 protein is directly responsible for tumorigenesis, although its molecular mechanism remains to be explored [50]. Loss of FAT4 expression, which is a cadherin-associated protein, as a downstream effect of its promoter methylation is reported in a significant fraction of primary breast tumors, implying it as a candidate TSG in breast cancer [26]. DCN is a proteoglycan that blocks tumor cell invasion and growth via accelerated destabilization of E-cadherin and inhibition of the EGFR/ERK signaling pathway in models of inflammatory breast carcinoma [51]. Downregulation of RSK3, a serine/threonine kinase also known as RPS6KA2, is associated with disease progression and metastasis in breast cancer [52]. DOK2 modulates multiple pathways including PI3K/Akt/mTOR or Ras/MAPK/ERK and functions as a tumor suppressor gene in multiple cancers [53]. Our study is the first to indicate a potential relationship between the microbiota and dysregulation of these TSGs that serve important roles in breast cancer development.

Few recent studies have indicated how host microbiota may serve as an environmental modulator in the epigenetic regulation through microbial metabolites. Commensal microbes generally produce short chain fatty acids like folate, propionate, butyrate and acetate. Among these, butyrate is recognized as an HDAC inhibitor and has been shown to rescue epigenetically silenced genes, such as p21 and BAK, which exert profound effects on cancer therapy [54]. In addition, butyrate suppresses EGF and HIF-1α in cancer cells and has been implicated in suppression of angiogenesis [55]. Many of the microbial metabolites alter the DNA or histone modifications via influencing the availability of chemical donors [56] while others such as folate and B vitamins donate methyl groups. Several other metabolites including biotin are directly or indirectly involved in DNA and histone methylation. Though these studies suggest the implications of host microbiota in epigenetic reprogramming, there is not enough evidence to attest that microbiota-mediated epigenetic alterations can stimulate breast oncogenesis. Additional research including multiple mice models and clinical samples is needed to decipher the finer details of the connection between microbial dysbiosis and methylation modifications in breast cancer.

DNMT and HDAC inhibitors comprise of an attractive class of epigenetic drugs that function by reversing any aberrant epigenetic modification, commonly encountered in cancer cells, including breast cancer. Early-phase clinical trials are investigating the efficacy of such drugs in reducing breast cancer, particularly among patients with a genetic predisposition, such as patients harboring BRCA mutation. We found that treatment of breast cancer cells with DNMT inhibitor, azacytidine, or HDAC inhibitor, TSA, either as monotherapy or as a combinatorial approach, successfully reverted the BFT-mediated inhibition of TSGs expression. While BFT exposure caused a significant increase in breast cancer cell migration and invasion, treatment with these drugs led to a pronounced mitigation in this pro-oncogenic phenotype. DNMT inhibitors like azacytidine (AZA) and its deoxyl-derivative are FDA-approved to treat patients with hematological malignancies while their antitumor efficacy in solid tumors is undergoing clinical trials [57, 58]. Recent years have witnessed a number of promising new drugs, which target the epigenome. Zebularine, an analogue of oligodeoxynucleotide duplex containing 2-H pyrimidinone, acts as a potent antitumor therapeutic and is characterized by its superior stability, minimal toxicity, and oral bioavailability. Zebularine has been reported to induce impressive anticancer effects in breast cancer even at low doses and imparts additive effects when used in combination with other DNMT inhibitors [59]. Our results showed a clear reduction in BFT-mediated enhanced breast cancer cell migration following Zebularine treatment, thus indicating, for the first time, its correlation with a pro-cancerous microbe.

Conclusions

Altogether, our study revealed a novel association between BFT and TSG hypermethylation in breast cancer. We showed, for the first time, BFT functions as a prime epigenetic modulator to inhibit TSG expression in breast cancer, thus disrupting their downstream effector mechanisms, culminating in neoplastic growth. The evidences, thus, provide a deeper understanding of the role of microbiota, in particular, ETBF, in abrogating epigenetic mechanisms in breast cancer. Additionally, the findings herein indicate that epigenome modulating drugs, such as azacytidine and TSA, represent an important class of chemotherapeutic agents and a promising intervention strategy for treating breast cancer harboring microbial dysbiosis.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

DV: Conceptualization, Analysis, Investigation, Data Curation, contributed to all figures, Writing-Original Draft Preparation; DN: Conceptualization, Analysis, Investigation, Data Curation, contributed to all figures, Writing-Original Draft Preparation; SP: Conceptualization, Analysis, Investigation, Data Curation, contributed to all figures; AS: Experimentation Supplementary Figure 4. DS: Supervision, Conceptualization, Analysis, Writing: Reviewing and Editing.

Funding

This work was supported by the Breast Cancer Research Foundation (BCRF) 90047965, CDMRP DOD BCRP (BC191572, BC210668) and The Fetting Fund for Cancer Prevention to Dipali Sharma.

Data availability

The Methylation EPIC array raw data will be available on request.

Declarations

Ethics approval and consent to participate

All animal studies were conducted in accordance with the guidelines of Johns Hopkins University Animal Care and Use Committee and are reviewed by Johns Hopkins ACUC. No Human data are reported here.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.