Epigenetics, Microbiota, and Breast Cancer: A Systematic Review

Citation: Soldado-Gordillo, A.; Álvarez-Mercado, A.I. Epigenetics, Microbiota, and Breast Cancer: A Systematic Review. Life 2024,14, 705. https://doi.org/10.3390/ life14060705 Academic Editor: Paola Nieri Received: 5 April 2024 Revised: 14 May 2024 Accepted: 28 May 2024 Published: 30 May 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). life Systematic Review Epigenetics, Microbiota, and Breast Cancer: A Systematic Review Alba Soldado-Gordillo 1and Ana Isabel Álvarez-Mercado 2,3,4,* 1Department of Biochemistry and Molecular Biology 2, School of Pharmacy, Campus de Cartuja s/n, 18071 Granada, Spain; [email protected].es 2Department Pharmacology, School of Pharmacy, Campus de Cartuja s/n, 18071 Granada, Spain 3Institute of Nutrition and Food Technology “JoséMataix”, Biomedical Research Center, Parque Tecnológico Ciencias de la Salud, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain 4Instituto de Investigación Biosanitaria ibs.GRANADA, Complejo Hospitalario Universitario de Granada, 18071 Granada, Spain *Correspondence: [email protected] Abstract: Breast cancer is the most frequently diagnosed cancer in women worldwide. According to recent studies, alterations in the microbiota and epigenetic modulations are risk factors for this disease. This systematic review aims to determine the possible associations between the intestinal and mammary microbial populations, epigenetic modifications, and breast cancer. To achieve this objective, we conducted a literature search in the PubMed, Web of Science, and Science Direct databases following the PRISMA guidelines. Although no results are yet available in humans, studies in mice suggest a protective effect of maternal dietary interventions with bioactive compounds on the development of breast tumors in offspring. These dietary interventions also modified the gut microbiota, increasing the relative abundance of short-chain fatty acid-producing taxa and preventing mammary carcinogenesis. In addition, short-chain fatty acids produced by the microbiota act as epigenetic modulators. Furthermore, some authors indicate that stress alters the gut microbiota, promoting breast tumor growth through epigenetic and gene expression changes in the breast tumor microenvironment. Taken together, these findings show the ability of epigenetic modifications and alterations of the microbiota associated with environmental factors to modulate the development, aggressiveness, and progression of breast cancer. Keywords: breast cancer; epigenetic; microbiota; nutrition; stress 1. Introduction The term cancer refers to a group of diseases in almost any organ or tissue of the body due to the uncontrolled growth of abnormal cells. Histologically, breast cancer is a type of cancer that develops from breast tissue, including adipose tissue, fibrous tissue, and glandular tissue [1]. Breast cancer is the most diagnosed malignancy in women worldwide, generating 2.3 million new cases each year. It is also the second-leading cause of death in this group, with 666,103 deaths registered in 2022 according to the latest epidemiological surveys provided by the Global Cancer Observatory (GLOBOCAN) [2]. Molecular characterization is crucial in the diagnosis and prognosis of this disease. This type of cancer is molecularly classified according to three main biomarkers: the progesterone receptor, estrogen receptor (ER), and epidermal growth factor receptor 2. The luminal A and luminal B subtypes are ER-positive and constitute about 75% of breast tumors. The triple-negative breast cancer (TNBC) subtype accounts for about 15–20% of breast tumors and lacks all three of the receptors mentioned above [ 3 ] (Figure 1). The prognosis for the different cancer subtypes varies from excellent for luminal subtype A to least favorable for TNBC, which has limited treatment options [4]. Life 2024,14, 705. https://doi.org/10.3390/life14060705 https://www.mdpi.com/journal/life Life 2024,14, 705 2 of 16 Life 2024, 14, x FOR PEER REVIEW 2 of 18 prognosis for the different cancer subtypes varies from excellent for luminal subtype A to least favorable for TNBC, which has limited treatment options [4]. Figure 1. Molecular characterization of breast cancer. Nowadays, the treatment of breast cancer is based on four main strategies: surgery, radiotherapy, systemic treatment, and immunotherapy. Radiotherapy can be used as an adjuvant or palliative therapy. Systemic treatment is administered as adjuvant or neoadjuvant and includes chemotherapy, endocrine hormone therapy, and biological or targeted therapy [5]. Breast cancer is a multifactorial disease [6]. Some of the risk factors are passive (those that patients merely experience passively), such as genetic predisposition, and others are active risks that are therefore preventable and modifiable (e.g., dietary patterns, obesity, or stress) [1]. Numerous studies have related several dietary compounds with protective effects against multiple cancers, including breast cancer. For instance, the maternal diet contributes to these benefits in a transgenerational manner [7]. It is believed that events occurring during early development, including maternal nutrition, have an important impact on the health of offspring and the progression of breast cancer [8]. Stress is also an environmental factor that influences the development of breast cancer and aggravates the disease. Chronic stress is detrimental to long-term health because of the constant release of hormones such as cortisol. Given that stress is increasingly inevitable, this factor has become the subject of many studies [9]. In addition to these well-defined risk factors, recent studies suggest that epigenetic modifications and changes in the microbiota may be involved in the development of breast tumors [10]. The microbiota is the set of microbes that reside in our organism. Different microbiota ecosystems are located in various body parts, with the gut microbiota standing out quantitatively [6]. The function of the gut microbiota is to maintain an active balance with the host, performing local and remote tasks in several physiological processes. However, when the balance of this commensal community is disrupted, a phenomenon known as dysbiosis can be involved in the development of various human diseases, including cancer [11]. Everyone’s gut microbiota is unique and is determined by genetic and lifestyle factors (among other factors). This high variation between individuals makes the definition of dysbiosis challenging. Microbial dysbiosis occurs when the microbial community of an organ or tissue is abnormally composed or maladapted and has recently been implicated Figure 1. Molecular characterization of breast cancer. Nowadays, the treatment of breast cancer is based on four main strategies: surgery, radiotherapy, systemic treatment, and immunotherapy. Radiotherapy can be used as an adjuvant or palliative therapy. Systemic treatment is administered as adjuvant or neoadjuvant and includes chemotherapy, endocrine hormone therapy, and biological or targeted therapy [5]. Breast cancer is a multifactorial disease [ 6 ]. Some of the risk factors are passive (those that patients merely experience passively), such as genetic predisposition, and others are active risks that are therefore preventable and modifiable (e.g., dietary patterns, obesity, or stress) [1]. Numerous studies have related several dietary compounds with protective effects against multiple cancers, including breast cancer. For instance, the maternal diet contributes to these benefits in a transgenerational manner [ 7 ]. It is believed that events occurring during early development, including maternal nutrition, have an important impact on the health of offspring and the progression of breast cancer [ 8 ]. Stress is also an environmental factor that influences the development of breast cancer and aggravates the disease. Chronic stress is detrimental to long-term health because of the constant release of hormones such as cortisol. Given that stress is increasingly inevitable, this factor has become the subject of many studies [9]. In addition to these well-defined risk factors, recent studies suggest that epigenetic modifications and changes in the microbiota may be involved in the development of breast tumors [10]. The microbiota is the set of microbes that reside in our organism. Different microbiota ecosystems are located in various body parts, with the gut microbiota standing out quantitatively [ 6 ]. The function of the gut microbiota is to maintain an active balance with the host, performing local and remote tasks in several physiological processes. However, when the balance of this commensal community is disrupted, a phenomenon known as dysbiosis can be involved in the development of various human diseases, including cancer [11]. Everyone’s gut microbiota is unique and is determined by genetic and lifestyle factors (among other factors). This high variation between individuals makes the definition of dysbiosis challenging. Microbial dysbiosis occurs when the microbial community of an organ or tissue is abnormally composed or maladapted and has recently been implicated as a key factor in the onset and progression of cancer. Indeed, some authors have suggested that altering the composition of the gut microbiota may promote the development and aggressiveness of extraintestinal tumors and contribute to the generation of hyperplastic and neoplastic lesions in the mammary glands [12]. Life 2024,14, 705 3 of 16 Although the gut microbiota has received the most research interest concerning its connection to cancer, other anatomical sites have also been examined, including the mammary glands. Even though initially conceived as a sterile site, it has recently been suggested that the microbial populations of breast tissue may be involved in the initiation and progression of breast cancer [13]. In addition, the microbiota may play a destructive or protective role in the development of breast cancer mediated by epigenetic regulation [ 8 ]. Epigenetics consists of various biological processes that affect gene expression, resulting in heritable phenotype or gene activity changes without altering the underlying DNA sequence. Covalent post-translational modifications of histones, DNA methylation, and modification of non-coding RNAs, such as miRNAs, are essential epigenetic mechanisms in biological processes such as cell replication, survival, division, and regulation of gene expression. However, disrupting these epigenetic modulations can lead to the activation of oncogenic transcriptional pathways and alterations in the function of genes implicated in mammary tumor development [14]. While miRNAs can regulate gene expression by degrading multiple mRNAs and interfering with the translation that regulates tumor cell survival and multiplication [ 15 ], we will focus on DNA methylation and post-translational modification of histones because of their importance during early mammalian development. DNA methylation consists of adding a methyl group to the fifth carbon position of cytosine, mainly at the cytosine-guanine dinucleotides, through the action of DNA methyltransferase (DNMT) enzymes [ 16 ]. On the other hand, histones are proteins susceptible to post-translational modifications, including methylation and demethylation. However, it is their acetylation and deacetylation that have attracted the most interest in the microbiological area of studying breast cancer [16,17]. The individualized epigenome is initiated during early development by establishing unique epigenetic marks through epigenetics reprogramming. These epigenetic signatures persist throughout life and can even be passed on to offspring through germline epigenetic inheritance. This provides a reliable mechanism for transcriptional regulation of genes across generations [8]. It is becoming increasingly clear that the origins of breast cancer can be traced back to early maternal and fetal lifestyles. In contrast to the genome, epigenomes are particularly sensitive to environmental factors and can be dysregulated during early development. One of the environmental stimuli that has the greatest impact on the fetal epigenome is the nutritional status of the mother, in part because maternal nutrition is the only source of nutrients during this period [8]. Intestinal microorganisms can ferment dietary fiber to produce low molecular weight bioactive compounds, such as short-chain fatty acids (SCFAs), which may be involved in epigenetic processes, including at extraintestinal sites. Many advances suggest that dysregulation of the epigenome may also be involved in the pathogenesis of mammary neoplasia [ 15 ]. Disruption of the metabolomic profile of gut and blood metabolites has been implicated in this effect. These blood metabolites may subsequently mediate epigenetic and gene expression changes in the breast tumor microenvironment which promote breast cancer development [3]. In this regard, in recent years, the relationship between the gut microbiota and epigenetic DNA modifications and breast cancer has become of great interest in biomedical research [ 10 ]. This review aims to identify possible associations between gut and breast microbial populations, epigenetic modifications, and breast cancer risk and progression. 2. Materials and Methods 2.1. Search Strategy and Selection Criteria This systematic review is based on the relevant literature in PubMed, Web of Science, and Science Direct databases. These systematic searches were conducted from February 2023 to June 2023, following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. To use a controlled vocabulary, and to make sure that the terminology used is the commonly accepted English terminology to denote the Life 2024,14, 705 4 of 16 concepts under study, the search strategy was performed using the following Medical Subject Headings (MeSH) terms: (“breast cancer” OR “breast neoplasms” [Title/Abstract] OR “breast tumors” [Title/Abstract]) AND (“microbiota” [Title/Abstract] OR “dysbiosis” [Title/Abstract]) AND (“epigenetics” [Title/Abstract] OR “epigenetic mechanisms” [Title/Abstract] OR “histone post-translational modification” [Title/Abstract] OR “DNA methylation” [Title/Abstract]) AND (“nutrition” [Title/Abstract] OR “maternal nutrition” [Title/Abstract]) AND (“stress” [Title/Abstract]). We included novel articles addressing the association between breast cancer, microbiota, or epigenetic mechanisms. The exclusion criteria included studies not published in English or before January 2019, studies focused on cancers other than breast cancer, and reviews or other works that did not provide original data. 2.2. Data Extraction To extract data from the included studies, we relied on key information that allowed us to find associations between breast cancer, epigenetic mechanisms, and microbial communities in the gut or breast. As mentioned above, we focused on data from breast cancer cases, although some articles also included data from other cancer types. There were no restrictions on the study design. Both in vitro and in vivo tests in animal models and human clinical trials were included. Clinical trial data included both diseased and healthy women of all ages, due to the greater impact of breast cancer on women. Ethnicity and the type of cancer were also not mutually exclusive. On the other hand, although we are aware of the recent changes in the taxonomy and nomenclature of bacteria, we have retained the nomenclature used by the authors to improve the traceability of the works included in this review. 2.3. Quality Assessment The quality assessment and selection were performed by two authors (A.I.Á.-M. and A.S.G., who independently worked according to the main criteria of Population, Intervention, Comparison, and Outcome (PICO)) (Table 1). Table 1. Population, Intervention, Comparison, and Outcome (PICO) criteria for inclusion of studies. Parameter Inclusion Criteria Population Studies performed in cells and animals, including humans (women) with breast cancer. Intervention Eligible interventions included tests associated with the Chao1 and Shannon indices in humans, the most widely used indices for quantifying species’ biodiversity and dietary interventions in animals. Comparison Healthy women and animals without breast cancer. Outcome Diagnostic accuracy of indices of microbial diversity and relative microbial abundance and the preventive effect of maternal dietary interventions. 3. Results A total of 2015 publications were initially identified. After eliminating duplicates and applying the eligibility criteria described above, the search was reduced to 557. To continue the selection, the titles and abstracts of the articles were reviewed to decide whether the information contained in the articles was relevant to the aim of this review. At this stage, 158 articles were excluded. When the relevance of the articles was not clear from the abstract, these studies were selected, and the full text was assessed. In the end, 20 studies were included. The PRISMA flowchart is shown in Figure 2. Life 2024,14, 705 5 of 16 Life 2024, 14, x FOR PEER REVIEW 5 of 18 3. Results A total of 2015 publications were initially identified. After eliminating duplicates and applying the eligibility criteria described above, the search was reduced to 557. To continue the selection, the titles and abstracts of the articles were reviewed to decide whether the information contained in the articles was relevant to the aim of this review. At this stage, 158 articles were excluded. When the relevance of the articles was not clear from the abstract, these studies were selected, and the full text was assessed. In the end, 20 studies were included. The PRISMA flowchart is shown in Figure 2. Figure 2. PRISMA flowchart. Main Outcomes The study of parental nutritional status and diet (Table 2) has gained importance in recent years because of its impact on the health of offspring, revealing the relationship between these variables and the occurrence of chronic human disorders and diseases. In line with these findings, some authors pointed to epigenetic mechanisms as a way in which parental nutrition influences offspring disease development in adulthood [18,19]. The results of several studies showed that changes in DNA methylation patterns in breast tissue are diet-dependent, with DNA hypermethylation predominating in the breast tissue of rodents with obesity compared with rodents subjected to calorie restriction, according to a study by Bowers et al. (2022) [20]. Furthermore, research by Li et al. (2020) [21] and Arora et al. (2022) [19] showed the protective effect of maternal feeding enriched in bioactive components such as sulforaphane (SFN) in broccoli sprouts (BSps) against the development of breast cancer in the offspring through significant transcriptional reduction of important enzymes, such as DNMTs and histone deacetylases (HDACs), which are involved in epigenetic modifications. The same protective effect against breast cancer through histone acetylation and DNA methylation was observed in the study by Abbas et al. (2021) [7], which was based on a diet rich in canola oil. Likewise, Chen et al. (2022) [18] showed that the epigenetic protective effect against breast cancer from genistein (GE) in transgenic soy depends on the time of maternal exposure to the dietary component. Figure 2. PRISMA flowchart. Main Outcomes The study of parental nutritional status and diet (Table 2) has gained importance in recent years because of its impact on the health of offspring, revealing the relationship between these variables and the occurrence of chronic human disorders and diseases. In line with these findings, some authors pointed to epigenetic mechanisms as a way in which parental nutrition influences offspring disease development in adulthood [18,19]. The results of several studies showed that changes in DNA methylation patterns in breast tissue are diet-dependent, with DNA hypermethylation predominating in the breast tissue of rodents with obesity compared with rodents subjected to calorie restriction, according to a study by Bowers et al. (2022) [ 20 ]. Furthermore, research by Li et al. (2020) [ 21 ] and Arora et al. (2022) [ 19 ] showed the protective effect of maternal feeding enriched in bioactive components such as sulforaphane (SFN) in broccoli sprouts (BSps) against the development of breast cancer in the offspring through significant transcriptional reduction of important enzymes, such as DNMTs and histone deacetylases (HDACs), which are involved in epigenetic modifications. The same protective effect against breast cancer through histone acetylation and DNA methylation was observed in the study by Abbas et al. (2021) [ 7 ], which was based on a diet rich in canola oil. Likewise, Chen et al. (2022) [ 18 ] showed that the epigenetic protective effect against breast cancer from genistein (GE) in transgenic soy depends on the time of maternal exposure to the dietary component. Life 2024,14, 705 6 of 16 Table 2. Summary of the studies addressing nutritional status and epigenetic modifications in breast cancer. Authors Study Design Model Sample Size Intervention Key Findings Li et al., 2020 [21] In vivo Her 2/neu female mice that develop ER (−) tumors N = 42 broods Mothers treated with BSps from 3 weeks of age until the weaning of their offspring. ↓The development of mammary tumors in offspring. Methylated histone H3K9 was enriched in the promoter regions of tumor suppressor genes. Arora et al., 2022 [19]In vivo SV40 female mice that develop ER (−) tumors N = 40 broods Mothers treated with BSps from 4 weeks of age until the weaning of their offspring. Preventive effects of breast cancer in offspring. Histone acetylation and global DNA methylation were affected. SFN downregulated HDAC expression, leading to an increase in histone acetylation. Abbas et al., 2021 [7]In vivo BALB/c female mice N = 200 broods Mothers treated with canola oil during gestation and lactation of their offspring. Epigenetic modifications that contributed to the activation of pathways that suppressed cell proliferation in tumorigenesis, with ↑survival, ↓tumor size, and ↓mortality. Chen et al., 2022 [18]In vivo SV40 female mice that develop mammary tumors spontaneously N = 30 broods Mothers treated with GE from 4 weeks of age until the weaning of their offspring. Tumor demethylation in the progeny. Chen et al., 2022 [8]In vivo Her 2/neu and SV40 female mice that develop ER (−) mammary tumors spontaneously N = 75 broods Mothers treated with GE from 4 weeks of age until weaning of their calves (Ma-LT-GE), GE from gestation to weaning of their offspring (Ma-ST-GE), and offspring treated with GE postnatally (from 4 weeks of age to the end of the experiment) (Post-GE). Ma-ST-GE: ↓protection against breast cancer compared with Ma-LT-GE. Ma-LT-GE: ↑expression of Trp63,↓than that of Myc. Results of chemoprevention similar to Post-GE. Bowers et al., 2022 [20]In vivo C57BL/5 female mice N = 100 broods Mothers subjected to weight loss regimes 5 days a week for 10 weeks. Caloric restriction: ↓predominance to hypermethylation of DNA. Four of six genes that were differentially methylated and differentially expressed were also differentially expressed without being methylated. BSps = broccoli sprouts; ER ( − ) = estrogen receptor negative; GE = genistein; HDACs = histone deacetylases; Ma-ST-GE = short-term maternal treatment with genistein; Ma-LT-GE = long-term maternal treatment with genistein; N = number; Post-GE = postnatal dietary exposure of offspring to genistein; SFN = sulforaphane; ↑= increase; ↓= decrease. Stress has been proposed as another active environmental factor modulating epigenetic regulation in breast cancer. Studies by Cui et al. (2022) [ 9 ] showed that stress induces gene expression and significant differential methylation of two genes, Tbc1d9 and Cdh10, which are related to breast cancer prognosis and survival. The in vitro study carried out by Intabli et al. (2023) [ 3 ] to observe the influence of cortisol, a glucocorticoid released in stressful situations, on the development of breast cancer showed an epigenetic alteration characterized by decreased methylation levels in the promoter regions of several breast cancer suppressor genes (Figure 3and Table 3). Life 2024,14, 705 7 of 16 Life 2024, 14, x FOR PEER REVIEW 7 of 18 Stress has been proposed as another active environmental factor modulating epigenetic regulation in breast cancer. Studies by Cui et al. (2022) [9] showed that stress induces gene expression and significant differential methylation of two genes, Tbc1d9 and Cdh10, which are related to breast cancer prognosis and survival. The in vitro study carried out by Intabli et al. (2023) [3] to observe the influence of cortisol, a glucocorticoid released in stressful situations, on the development of breast cancer showed an epigenetic alteration characterized by decreased methylation levels in the promoter regions of several breast cancer suppressor genes (Figure 3 and Table 3). Figure 3. Altered gut-derived metabolites mediate epigenetic and gene expression changes in the breast tumor microenvironment, promoting breast cancer development. Table 3. Summary of the studies addressing stress and epigenetic modifications in breast cancer. Authors Study Design Model Sample Size Intervention Key Findings Cui et al., 2022 [9] In vivo BALB/c mice subjected to stress and not stressed N = 12 Mice subjected to chronic stress by restriction 2 h per day for 10 consecutive days. ↑ expression of the Cdh10 gene and ↓ expression of the Tbc1d9 in TNBC, with ↓ survival and worse prognosis of breast cancer. Intabli et al., 2023 [3] In vitro MDA-MB-231 (TNBC) y MCF-7 cells N/R Growth medium with fresh cortisol for 20 days. ↓ levels of methylation in the promoter regions of several tumor suppressor genes and loss of global DNA methylation. N = number; N/R = not reported; TNBC = triple-negative breast cancer; ↑ = increase; ↓ = decrease. The microbiota is considered an additional organ in the body. It has been observed that the composition of the gut and mammary microbiota may differ between patients diagnosed with breast cancer and healthy subjects due to a disruption in the balance of this commensal community (Table 4). This imbalance would result in an altered or dysbiotic state in the microbiota that could be implicated in the etiology of cancer, influencing the disease’s prevention, diagnosis, and prognosis [6,12,13,22]. Figure 3. Altered gut-derived metabolites mediate epigenetic and gene expression changes in the breast tumor microenvironment, promoting breast cancer development. Table 3. Summary of the studies addressing stress and epigenetic modifications in breast cancer. Authors Study Design Model Sample Size Intervention Key Findings Cui et al., 2022 [9] In vivo BALB/c mice subjected to stress and not stressed N = 12 Mice subjected to chronic stress by restriction 2 h per day for 10 consecutive days. ↑expression of the Cdh10 gene and ↓expression of the Tbc1d9 in TNBC, with ↓ survival and worse prognosis of breast cancer. Intabli et al., 2023 [3]In vitro MDA-MB-231 (TNBC) y MCF-7 cells N/R Growth medium with fresh cortisol for 20 days. ↓ levels of methylation in the promoter regions of several tumor suppressor genes and loss of global DNA methylation. N = number; N/R = not reported; TNBC = triple-negative breast cancer; ↑= increase; ↓= decrease. The microbiota is considered an additional organ in the body. It has been observed that the composition of the gut and mammary microbiota may differ between patients diagnosed with breast cancer and healthy subjects due to a disruption in the balance of this commensal community (Table 4). This imbalance would result in an altered or dysbiotic state in the microbiota that could be implicated in the etiology of cancer, influencing the disease’s prevention, diagnosis, and prognosis [6,12,13,22]. Life 2024,14, 705 8 of 16 Table 4. Summary of the studies on microbial dysbiosis and breast cancer.. Authors Study Design Sample Sample Size Method of Detection Key Findings Klann et al., 2020 [13] Clinical trial Observational Case-control Samples of bilateral breast tumors from patients with cancer and breast samples from healthy subjects. N = 46 Sequencing of the hypervariable regions V1–V2 of the 16 s rRNA. ↑uniformity and richness are bacterial in the normal breasts of healthy women. Hoskinson et al., 2022 [23] Clinical trial Observational Case-control Samples of breast tumors from patients with cancer and breast samples from healthy subjects. N = 159 Sequencing based on the 16 s rRNA gene. ↑bacterial richness in samples of healthy breasts. The existence of a bacterial signature before the development of the tumor was maintained in cancerous tissues. ↓ functionality of the bacteriome in women with cancer. Bobin-Dubigeon et al., 2021 [12] Clinical trial Observational Case-control Stool samples from female patients with breast cancer (early, untreated, before treatment) and stool samples from healthy subjects. N = 55 Sequencing of the hypervariable regions V3–V4 of the 16 s rRNA. ↓bacterial diversity, ↑abundance of Firmicutes, Clostridium cluster XIVa, and Clostridium cluster IV, ↓abundance of Bacteroidetes, Butyricimonas sp., Odoribacter sp., and Coprococcus sp. in breast cancer patients. Caleça et al., 2023 [6] Clinical trial Observational Case-control Stool samples from female breast cancer survivors and stool samples from healthy controls. N = 314 Sequencing of the amplicon of the V4 region of the 16 s rRNA gene. Control group: ↑bacterial diversity. ↑F/B ratio, and abundance of Clostridum perfringers,Escherichia coli, and Akkermansia muciniphila. Byrd et al., 2021 [22] Clinical trial Observational Case-control Stool samples from Ghanaian women diagnosed with breast cancer or non-malignant breast disease and stool samples from healthy women. N = 895 Sequencing of the amplicon of the V4 region of the 16 s rRNA gene. Different diversity in the control group. Bacteroidetes were consistently positively associated with breast cancer in contrast to Romboutsia and Coprococcus 2. Aarnoutse et al., 2021 [24] Clinical trial Observational Case-control Stool samples from postmenopausal women with ER (+) breast cancer and stool samples from healthy women. N = 148 Sequencing of the amplicon of the V4 region of the 16 s rRNA gene. ↑abundance of Veillonellaceae and Dialister in patients scheduled for systematic adjuvant treatment. Rosean et al., 2019 [25]In vivo C57BL/6 female mice. N/R Mice fed with an antibiotic cocktail for 14 days. Dysbiosis eater preset: ↑ dissemination of tumor cells and ↑ inflammation in the breast tissue. Chen et al., 2022 [18]In vivo SV40 female mice spontaneously developing mammary tumors and wild-type C57BL/6J female mice. N = 30 broods Mothers treated with GE from 4 weeks of age until weaning of their offspring. Alteration of the gut microbial community and ↑relative abundance of Allobaculum, Bifidobacterium, and Bacteroidetes in offspring. Cui et al., 2022 [9] In vivo BALB/c mice subjected to stress and not stressed. N = 12 Mice subjected to chronic stress by restriction 2 h per day for 10 consecutive days. ↓F/B ratio and ↑abundance of Rhodospirillales and Clostridiales. ER (+) = estrogen receptor positive; F/B = Firmicutes/Bacteroidetes; N = number; N/R = not reported; ↑= increase; ↓= decrease. It has recently been observed that one of the possible mechanisms by which the microbiota affects our health is interference with normal epigenetic control mechanisms. SCFAs produced by the gut microbiota from the fermentation of dietary fiber showed satisfactory results in the treatment and prevention of breast cancer, especially sodium butyrate (BS) and sodium propionate (PS), as reported by Semaan et al. (2020), Sharma et al. (2022), and Chen et al. (2022) [ 26 ] (Figure 4). Furthermore, according to Cui et al. (2022) [ 9 ], stress-induced taxonomic perturbations of the gut microbiome altered the metabolomic profile of intestinal and serum metabolites across the brain-gut axis, which are metabolites that may mediate epigenetic and gene expression changes at different locations in our body, including the mammary gland (Table 5). Life 2024,14, 705 9 of 16 Life 2024, 14, x FOR PEER REVIEW 10 of 18 It has recently been observed that one of the possible mechanisms by which the microbiota affects our health is interference with normal epigenetic control mechanisms. SCFAs produced by the gut microbiota from the fermentation of dietary fiber showed satisfactory results in the treatment and prevention of breast cancer, especially sodium butyrate (BS) and sodium propionate (PS), as reported by Semaan et al. (2020), Sharma et al. (2022), and Chen et al. (2022) [26] (Figure 4). Furthermore, according to Cui et al. (2022) [9], stress-induced taxonomic perturbations of the gut microbiome altered the metabolomic profile of intestinal and serum metabolites across the brain-gut axis, which are metabolites that may mediate epigenetic and gene expression changes at different locations in our body, including the mammary gland (Table 5). Figure 4. Short-chain fatty acids produced by the gut microbiota from the fermentation of dietary fibers positively impact the treatment and prevention of breast cancer. Figure 4. Short-chain fatty acids produced by the gut microbiota from the fermentation of dietary fibers positively impact the treatment and prevention of breast cancer. Table 5. Summary of the studies addressing epigenetic modulations in microbiota-sensitive breast cancer. Authors Study Design Model Sample Size Intervention Key Findings Semaan et al., 2020 [26]In vitro MCF-7 cell line N/R Incubation with BS or PS for 24–96 h at different concentrations. BS effect was more powerful than PS. Low-to-medium levels of BS and PS: blockade of MCF-7 cells in G1. High levels of BS and PS: induced cell apoptosis in MCF-7. Sharma et al., 2022 [14]In vitro Cell line MDA-MB-231 (TNBC) and MCF-7 (ER (+)) N/R Individual and combined doses of GE, BS, and SFN for 3 days. SFN, BS, and GE combined: ↓ enzymatic activity of HDACs and DNMTs, ↓expression of EZH2 and SUVH39H1, and ↓ synergistically the cell viability in MCF-7 and MDA-MB-231. Chen et al., 2022 [18]In vivo SV40 female mice that develop mammary tumors spontaneously and wild-type female C57BL/6J mice N = 30 broods Mothers treated with GE from 4 weeks of age until weaning of their offspring. GE: ↑SCFAs, ↓tumor proliferation, and ↓gene expression tumors. Sharma et al., 2020 [27]In vivo Her 2/neu female mice N = 120 Mice treated with BSps and GTPs from 3 weeks of age to adulthood and mothers treated with BSps and GTPs during their gestation and lactation. Group nurtured from conception: ↑isobutyrate and propionate. Cui et al., 2022 [9]In vivo BALB/c mice N = 12 Mice subjected to chronic stress by restriction 2 h per day for 10 consecutive days. Stress caused taxonomic perturbations of the gut microbiome that altered the metabolomic profile of gut and serum metabolites. BS = sodium butyrate; BSps = broccoli sprouts; DNMTs = DNA methylTransferases; ER (+) = positive estrogen receptor; Ge = genistein; GTPs = green tea polyphenols; HDACs = histone deacetylases; N = number; N/R = not reported; PS = sodium propionate; SCFAs = short-chain fatty acids; SFN = sulforaphane; ↑ = increase; ↓= decrease. 4. Discussion This systematic review synthesized evidence from 20 studies investigating the relationship between epigenetics, microbiota, and breast cancer. The analysis of the main Life 2024,14, 705 16 of 16 27. Sharma, M.; Arora, I.; Stoll, M.L.; Li, Y.; Morrow, C.D.; Barnes, S.; Berryhill, T.F.; Li, S.; Tollefsbol, T.O. Nutritional combinatorial impact on the gut microbiota and plasma short-chain fatty acids levels in the prevention of mammary cancer in Her 2/neu estrogen receptor-negative transgenic mice. PLoS ONE 2020,15, e0234893. [CrossRef] [PubMed] 28. Paul, B.; Barnes, S.; Demark-Wahnefried, W.; Morrow, C.; Salvador, C.; Skibola, C.; Tollefsbol, T.O. 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