Epigenetic and Immune Mechanisms Linking Breastfeeding to Lower Breast Cancer Rates

Introduction

The mammary gland is formed through a process known as embryogenesis. During life, the mammary gland undergoes numerous changes, and with age, the ducts undergo elongation and branching within the adipose tissue pad. Pregnancy is a state in which the gland undergoes full development, including the development of lactation-capable alveoli, enabling the process of breastfeeding offspring [1].

The mammary gland can regenerate, in part due to the presence of mammary stem cells (MaSCs). The ability to differentiate into specialized cell lineages and the ability to self-renew are characteristic features of stem cells. MaSCs give rise to epithelial progenitor cells (EPCs), which differentiate into either basal cells or luminal cells [2]. At the onset of lactation, luminal cells are responsible for the production and secretion of milk. In each subsequent pregnancy, MaSCs help secretory alveoli to regenerate [3]. Breast stem cells are under the control of systemic hormones and local stimuli. The review by French and Tornillo indicates that the breast stem cell population is quite heterogeneous, and these cells may have distinct phenotypes and different molecular features depending on their function [4–6].

Research indicates that MaSCs can transform into cancer cells as a result of progression caused by dysregulation, mediated by paracrine factors and the extracellular matrix [3]. One hypothesis suggests that breast cancer stem cells (BCSCs) originate from MaSCs [4]. BCSCs share many commonalities with MaSCs, such as common cell surface markers. The characteristic common markers for MaSC and BCSCs are CD49f, CD44, and protein C receptor (PROCR). Additionally, stem cells derived from humans with the surface marker phenotype Lin – CD10 – – CD24 – PROCR + CD44 + have been detected in breast cancer and in normal mammary gland epithelium [5,7]. BCSCs can regulate neighboring cells, create a nurturing environment, and supply necessary components for cell development. These cells enhance the ability to grow, metastasize, and reproduce, contributing to tumor progression [8].

MaSCs are considered a potential source of the initial transformation events leading to cancer [9]. It is clinically important that BCSCs differentiate more slowly, have less tendency to apoptosis and a higher DNA repair capacity, which makes them more resistant to traditional cancer treatments such as chemotherapy and radiotherapy [10]. Therefore, it is important to review factors that reduce the risk of cancer. Breastfeeding is an important aspect in decreasing cancer susceptibility [11]. It has been shown that longer breastfeeding duration in women decreases the risk of developing breast cancer [12]. Therefore, this article reviews the role of mammary stem cells in breast development, lactation, and breast cancer.

Epidemiology of Breast Cancer

Breast cancer is the most commonly diagnosed cancer in women worldwide, accounting for approximately 24% of all cancer cases in women, ahead of lung cancer [13]. In 2020, approximately 2.3 million new cases were reported, making it the most common cancer in the world [14]. The incidence of breast cancer varies geographically – the highest rates are found in North America, Australia and Western Europe, while the lowest rates are in Asia and Africa. Currently, approximately 80% of BC patients are aged >50 years [15].

Global data reveal significant inequalities in the burden of breast cancer depending on a country’s level of development. In countries with a very high human development index (HDI), 1 in 12 women develop breast cancer in their lifetimes, and 1 in 71 women die from it [16]. In 2020, breast cancer caused 684 996 deaths worldwide [15]. Forecasts predict that by 2030, 2.7 million new cases of breast cancer will be diagnosed annually around the world, and the number of deaths will reach 870 000. This increase is due to many factors, such as an aging population and lifestyle changes, which increase the risk of breast cancer [17].

Breastfeeding is a factor that reduces the likelihood of breast cancer, as every 12 months of breastfeeding reduces the risk of breast cancer by approximately 4% [18].

Development and Changes in Mammary Gland

The mammary gland is a heterogeneous array of tissues, the composition and proportions between them vary depending on factors such as age, physiological status, released hormones (notably estrogen and progesterone), and exogenous influences. Its architectural dynamics fluctuate throughout the menstrual cycle, and crucial transformations occur during puberty, gestation, lactation, and menopause. Complete mammary maturation is exclusively achieved during pregnancy [18,19].

Breast Structure

The female breast consists of parenchyma, divided into several lobes, separated by adipose tissue and connective tissue [20]. The parenchyma contains epithelial cells resting on the basement membrane, forming a network of glandular ducts supported by the mammary fat pad, which is composed of adipocytes. Another function of adipose tissue is to supply the gland with nutrients and immune cells. The maintenance of breast integrity is further delegated to the connective tissue, typified by fibroblasts, forming Cooper’s ligaments. Blood vessels and nerves from the chest wall are also located in this structure [21,22].

Breast Development in the Prenatal Period

The formation of the mammary line in the fetus begins around the 6th week of fetal life, with localized thickenings called mammary placodes appearing around the 7th week. These are the earliest precursors of the mammary gland. These structures later transform into mammary buds, then mammary sprouts (during that period also the mesenchyme changes into the fat pad precursor), and finally into the mature mammary tree, a last step in organogenesis. Unlike later stages of breast development, the early stages are not regulated by hormones [23].

Breast Development in the Postnatal Period

Postnatally, the mammary gland develops similarly in both sexes for about 2 years. Secretory activity begins in the first week of life, manifesting as swelling and redness of the skin above the gland. Yellowish fluid may be expressed from the compressed breast in the 1st and 2nd weeks of life. As the child grows, the amount of progesterone decreases, histologically transitioning from a single layered epithelium to a multilayered one, and the number of ductal branches decreases, reaching a pattern of small clusters of blindly ending ducts by the age of 2 years [24]. The mammary gland remains in this state until puberty, when hormone production in females triggers morphogenesis completion.

Breast Development in Puberty

Initially, there is an increase in gonadotropin levels, leading to estrogen and progesterone production by the ovaries. Alternating hormone concentrations during the menstrual cycle stimulate the development of type 1 lobules, initiating the formation of new alveolar buds, which later transform into type 2 and type 3 lobules. The mammary gland remains in this form until pregnancy occurs [25,26].

Breast Development in Pregnancy

During pregnancy, the female breast undergoes extensive remodeling in preparation for lactation. These changes occur mostly under the control of 2 hormones – progesterone and prolactin. The ductal system, lobules, and alveoli expand. Distal, accessory milk ducts develop, causing the branches to enlarge and become ready for alveolar formation. Epithelium proliferates, generating alveolar buds, which expand into separate alveoli. Intensively developing glandular tissue causes interstitial adipose tissue to disappear due to lack of space. Enhanced vascularization takes place, and by mid-pregnancy each alveolus is enveloped by an extensive network of capillaries [21]. Glandular luminal cells begin synthesizing milk components in small amounts, not leading to lactation. These processes occur throughout pregnancy, with the most extensive changes happening in the first trimester. During and shortly after childbirth, luteal and placental hormones such as human placental lactogen, along with prolactin, initiate the full maturation of secretory alveoli in the breasts. This form of the mammary gland is the most mature and developed [27]. Changes emerging during pregnancy regress shortly after lactation ends and the breast structure returns to its pre-pregnancy state [19].

Around the age of 30 years, involutional changes in the breasts begin. There is a gradual reduction of glandular tissue, partial replacement by adipose tissue, and densification of connective tissue. Breast size decreases due to declining estrogen levels [28] (Figure 1).

Hypotheses on the Protective Mechanisms of Breastfeeding

It has been proven that breastfeeding reduces the risk of developing breast cancer, particularly triple-negative breast cancer and breast cancer in carriers of the BRCA1 mutation [12,25].

There is also a significant correlation between the duration of breastfeeding and cancer development. For every 12 months of breastfeeding, the risk of developing breast cancer decreases by 4.3% [29,30].

This is associated with biological changes occurring in the breast during pregnancy and lactation, rather than socio-economic or environmental factors, as the protective effect of breastfeeding on breast cancer development was found to be consistent regardless of the women’s age, age at first childbirth, economic status of the country, menopausal status, or ethnic group. This is very important, as it was estimated that 4.7% of breast cancer cases in the UK are caused by lack of breastfeeding [12].

Various theories connect breastfeeding with a reduced risk of developing breast cancer. Attention is given to hormonal changes in the breastfeeding woman’s body [31], the elimination of cells with damaged genetic material, increased differentiation of breast cells during breastfeeding [12], reduced serum insulin levels in breastfeeding women, immunological factors, and certain components of milk that may protect the breasts [11,29,31].

Protective Effects of Hormones on Breast Tissue

Breast maturation is already linked to hormones – estrogens, gonadotropins, and the transcriptional regulator FOXP1 – that stimulate stem cells for morphogenesis and also for remodeling after birth [32]. All of these factors lead to rearrangement and proliferation of mammary cells, leading to formation of terminal end-joining compounds (TEBs) at the end of the mammary ducts. In pregnancy, branching takes place, directed specifically by the hormone’s progesterone and prolactin [19].

There is also a strong relationship between breast cancer development and ovarian hormonal activity, related to menarche, childbirth, and late menopause [33].

During lactation, there is a significant increase in prolactin levels, which is responsible for milk production and suppressing ovulation, delaying its recurrence [12]. Suppression of ovulation leads to decreased cumulative exposure to estrogen, which promotes initiation and growth of certain types of breast cancer. Therefore, breastfeeding reduces exposure to the oncogenic effects of estrogens by inhibiting breast cell proliferation and lowering the potential for malignant transformation [12].

During breastfeeding, oxytocin is also released, which is a hormone that may protect against breast cancer due to its influence on cell differentiation and growth in breast tissue [11,29].

Another noteworthy ovarian hormone is progesterone, recognized as a potent mitogen in the breasts [34]. This is evidenced by the fact that women using hormone replacement therapy containing both estrogen and progestins had a higher risk of developing breast cancer than women using estrogen-only therapy [35]. Additionally, it was observed that during the luteal phase of the menstrual cycle, when progesterone levels are highest, mammographic breast density is elevated [36]. Knowing that ovarian hormonal activity decreases during lactation, resulting in lower progesterone levels, it can be concluded that breastfeeding provides protection against breast cancer [37].

A significant reduction in insulin levels was also observed in breastfeeding women compared to non-breastfeeding women [11,29], as well as a decrease in the insulin-like growth factor IGF-1, which may affect anti-apoptotic activity and proliferation of cancer cells, protecting breast cells during lactation [31,38].

Protective Effect of Cellular Changes – Increased Differentiation of Breast Cells

Around the perinatal period, the secretory units of the mammary glands are numerous and well-differentiated. These secretory unit cells have more efficient DNA repair capabilities through the excision of DNA damaged by genetic mutations and have a longer cell cycle than before pregnancy [29].

During lactation, there are also cyclic structural changes in the mammary glands: milk production begins, ducts expand, and involution of the glands occurs after lactation ends. This allows the removal of mutated or potentially damaged cells from breast tissue, which could undergo malignant transformation in the future [11,29,31].

Protective Effect of Breast Milk Components

Breast milk contains various components such as antibodies, cytokines, and immune cells, which support the infant’s immune system [39]. It is suggested, however, that they may also have a protective effect on the mother’s breast tissue by identifying and removing mutated or potentially cancerous cells in the breast. It is also suspected that substances in milk can modulate immune responses and inflammation throughout the mother’s body, thereby inhibiting tumorigenesis. However, research in this area is not yet sufficient [29].

Among the components of human milk is alpha-lactalbumin, which, when combined with oleic acid, forms a complex known as HAMLET (Human Alpha-lactalbumin Made Lethal to Tumor cells). This complex can induce apoptosis in cancer cells without affecting healthy, differentiated cells, leading to its protective role against development of breast cancer [11].

Lactoferrin contained in breast milk, which is regulated by miR-214, binds to DNA sequences of health-promoting bacteria in breast milk, developing due to the presence of oligosaccharides, and inhibits methylation-induced NF-κB pathway activation [40]. Methylation of the p53 suppressor gene was higher in premenopausal women who had never breastfed than in women who had breastfed [41]. Table 1 summarizes the plausible hypotheses presented on reducing breast cancer risk with breastfeeding.

Breast Stem Cells: Characteristics, Phenotype, Signaling Pathways, and Their Association with Breast Cancer

Stem cells are pluripotent cells that can differentiate into any cell type, giving rise to a variety of tissues. Stem cells in the mammary gland are derived from the embryonic leaf [42]. To fully characterize the mammary gland, we begin by describing MaSCs. These are cells that give rise to 2 lines of mammary gland epithelium – progenitor cells and basal cells. Progenitor cells are either ductal or alveolar cells. Basal cells contain a rich population of stem cells and myoepithelial cells necessary for milk secretion during lactation. MaSCs have Lin, CD24+, and CD29+ surface markers [43].

There have been increasing reports of BCSCs causing resistance to treatment. BCSCs have either the CD24+ and CD44+ marker, depending on the mutation. Cancer stem cells (CSCs) can differentiate into any cell type and can self-renew, and therefore can cause tumor recurrence and metastasis [44]. CSCs can arise from new MaSCs by acquiring genetic mutations or arise from differentiated tumor cells that change their phenotype to a cancer stem cell-like phenotype [7]. They were first detected in leukemia. The classical theory of CSCs defines them as subpopulations of constantly renewing, long-lived, malignant cells that maintain a constant but low level of unrestricted proliferation [45].

In the mammary gland, mammary stem cells play a crucial role. CSCs do not have specifically defined surface antigens – these vary depending on the type of tumor. CD44+, CD24+, and high expression of aldehyde dehydrogenase have been proposed as markers for human BCSCs [46,47].

The CD44+ and CD24− phenotype is associated with higher grade of adenocarcinoma and the CD44+ and CD24− phenotype with lower grade, indicating that both markers are useful for assessing tumor behavior [48]. Other additional MaSC-specific surface markers (eg, CD133+, CD61+, and CD29+) have also been characterized [32].

The mesenchymal cells surrounding the epithelial bud accumulate and transform to form the mammary mesenchyme, which is a layer of fibroblastic cells. Importantly, factors secreted by the mesenchyme have a major impact on the mammary lineage, promoting differentiation toward the mammary epithelium [47].

Transplantation studies indicate that MaSCs lead to formation of 2 types of mammary epithelium – lumen progenitor cells and basal cells. Lumen progenitor cells can be divided into those that are restricted to ductal or follicular cells. Basal cells contain an enriched population of stem/progenitor cells and myoepithelial cells, which are essential for milk secretion during lactation [25].

Tracing of MaSC cell lines during adolescence and adulthood has identified stem cells located in the TEB; they are typically dispersed in the mammary epithelium as a result of TEB elongation. As the TEB elongates and bifurcates, MaSCs lose their ability to divide asymmetrically to form clonal clusters. In the human mammary gland, subpopulations of cells with immobile MaSCs, but with zones of proliferation of heterogeneous stem cells, have been localized, which has important implications for breast cancer development [49].

Lobular ducts are the main site of origin of breast cancers, as they contain MaSCs, which are the main source of proliferation [12]. The initial characterization of human mammary stem cells (hMaSCs) involved separating human mammary epithelial cells (HMECs) based on the expression levels of MUC1, CD10, and epithelial cell adhesion molecule (EpCAM). The specific population marked by MUC1lo/med, CD10med/hi, and EpCAMhi showed a high capacity for generating heterogeneous colonies [50]. Furthermore, cells with high CD44 expression and low CD24 expression, which resemble stem cells, can be generated from non-stem cells by induction of epithelial-mesenchymal transition (EMT). This suggests that cells must lose their epithelial characteristics to acquire stem cell properties. This may be consistent with the hypothesis that MaSCs are located at the leading edge of developing organoids, as mesenchymal features may be necessary for invasion into the surrounding matrix [6]. Additionally, previous studies indicated that MaSC populations can be distinguished based on epithelial and mesenchymal markers, suggesting that they also differ in activation of signaling pathways. RNA sequencing of single human MaSC cells, separated by ALDH+ and CD44+/CD24− markers, revealed differences in gene expression. ALDH+ cells had higher expression of WNT2 (wingless Int 2), IGF1 (insulin-like growth factor 1), and Notch ligand DLL1 (delta like canonical Notch ligand 1), while CD44+/CD24− cells had higher levels of Wnt (wingless-related integration site) pathway proteins such as DKK3 (Dickkopf-related protein 3), CCND2 (cyclin D2), PRICKLE2 (prickle planar cell polarity protein 2), and DRAXIN (dorsal inhibitory axon guidance protein) [7]. There are currently 2 theories about the origin of BCSCs – the first suggests that BCSCs are derived from MaSCs, while the second suggests that BCSCs originate from more differentiated progenitor cells that acquire the ability to self-renew through genetic and epigenetic reprogramming [51,52]. STs can transform into cancer cells without interfering with the genetic material [53]. Interestingly, the pathways that maintain the stem cell phenotype are the same for MaSCs and BCSCs – Hedgehog, Notch, or Wnt – so if a mutation occurs in MaSC, they can develop into BCSC [44].

Signaling Pathways and Their Association with Breast Cancer

There are several signaling pathways at work in stem cells: sonic hedgehog (SHH), wingless Int, and Notch, whose function is to mediate stem cell self-renewal; interestingly, they are also found in human tumors [54]. The Wnt pathway can be divided into canonical β-catenin-dependent and non-canonical β-catenin-independent pathways, specifically Wnt calcium and planar cell polarity (PCP) [55–58]. Studies have shown that in breast cancer cells, Wnt5a levels are significantly reduced, which stimulates activation of the canonical Wnt pathway and further tumor growth [59]. It has also been observed that increased expression of non-canonical Wnt5a and the receptor for it, Frizzled Class Receptor 2 (Fzd2), is present in metastatic cell lines [60]. The abnormal Wnt β-catenin signaling pathway stimulates renewal of cancer stem cells, causing their proliferation. The pathway is activated when the Wnt ligand binds to the Frizzeld receptor and low-density lipoprotein receptor-related proteins (LRP 5/6). This results in activation of Disheveled (Dvl) and displacement of glycogen synthase kinase (GSK-3β) from the APC/Axin complex [61], which entails activation and stabilization of α-catenin in the cytoplasm. Activated α-catenin is transported to the cell nucleus, where it initiates transcription of genes responsible for cell proliferation. In addition, in breast cancer cells, the level of secreted frizzled-related protein (SFRP1) serving as a negative regulator of this cascade decreases, and this involves an increase in c-Myc expression [41].

C-Myc is a regulator of the tumor microenvironment, and is especially rich in tumor-associated fibroblasts. Mammary epithelial cells with increased c-Myc expression secrete insulin-like growth factor 1 and 2, which stimulate fibroblast proliferation and tumor growth [62]. In addition, processes such as angiogenesis and evasion of the host immune system are regulated by c-Myc. Increased c-Myc levels result in increased expression of vascular endothelial growth factor (VEGF) via activation of the b-catenin pathway [63].

The last cascade, the Notch cascade, is involved in angiogenesis, proliferation and stem cell renewal. When ligands, delta proteins, bind to the Notch receptor, they cause glycosylation and cleavage into a Notch intracellular domain (NICD) and a Notch extracellular domain (NECD). The Notch ligand of one cell binds to the NECD present on the other cell, forming a complex that is cleaved by ADAM/TACE, resulting in NEXT-Notch extracellular truncation.

NEXT is cleaved by γ-secretase, releasing NICD [64], which is transported to the cell nucleus, where it binds to the suppressor of hairless coregulatory protein lag-1 (CSL), which initiates transcription of the c-Myc and HER2 genes [65]. Inhibition of Notch was reported to prevent tumor recurrence and delay tumor growth in 67% of cases [66].

There is much debate about abnormal DNA methylation of progenitor genes. DNA methylation in the mammary gland in a mouse model is affected by hormonal changes. DNA bisulphate sequencing in parturient and non-pregnant mice showed increased methylation of IGFR, supporting the hypothesis that suppression of the IGF signaling pathway prevents breast cancer [49]. In addition, pregnancy had a marked effect on the mammary epithelial stem cell population, and many of the changes were related to the down-regulation of genes necessary for proper stem cell function as a result of methylation of substances such as SHH. A study of 733 patients found that methylation of the Forkhead box protein A1 (FOXA1) gene was increased in non-breastfeeding women, and FOXA1 protein expression was higher in non-breastfeeding women than breastfeeding women. FOXA1 regulates luminal gene expression in stem cells and represses the basal phenotype, linking this mechanism associated with reproduction and the occurrence of breast cancer [67,68].

Pregnancy-Induced Changes in Breast Stem Cells and Their Protective Role

Research and observations indicate that early-age pregnancy reduces the number or function of breast stem cells. This was discovered by analysis of epidemiological data and the study of mouse mammary glands, which are structurally similar to human mammary glands. These studies on mice have shown that pregnancy at an earlier age leads to a reduction in the quantity of mammary gland lobules by up to 50% [69]. Stem cells, due to their low differentiation, may provide a basis for carcinogenesis. Hence, hypotheses are put forward about the protective role of early-age pregnancy in breast cancer risk [70].

However, the protective effect of pregnancy against breast cancer does not occur immediately; directly after pregnancy, there is a transient increase in the risk of breast cancer, which results from changes associated with remodeling and maturation of the mammary gland for lactation, as well as from the possible influence of various hormones during pregnancy and immediately afterward [71].

A prospective Norwegian study [72] suggests that early pregnancy is associated with a lower risk of transiently increased breast cancer risk. This effect is most noticeable in women over 30 years of age, and the authors emphasize the complex nature of the influence of reproduction in women on risk of breast cancer. They examined the impact of timing and intervals between pregnancies on cancer risk, finding that immediately after childbirth, there is a transient increase in risk, especially noticeable when the first pregnancy occurs at a later age (>30 years). However, in the long term, multiparous women who have had more births tend to have lower risk of breast cancer compared to nulliparous women. However, much remains unclear on this topic, so the authors of the study stressed the need for further research and analysis [73].

Some studies question the changes in the function of stem cells in the mammary gland. Despite their decrease in quantity with subsequent pregnancies, they still retain their properties, regenerative abilities, and ability to further divide [74]. These conclusions were drawn based on observations of stem cells in mice, their totipotency, and the presence of growths within the mammary gland in older multiparous mice [65].

On the other hand, other authors [75] showed that early pregnancy, and the associated remodeling of the mammary gland related to lactation preparation, causes changes in the properties of breast stem cells in their signaling pathways. This results from increased expression of genes responsible for cell differentiation and decreased Wnt signaling, which are responsible for potential carcinogenic transformation. These data are very promising for the long-term protective anti-cancer properties [76,77].

Pregnancy affects not only cells but also genes and their expression, causing breast stem cells during the pregnancy to increase in number, and during deplete during lactation. These changes are permanent [77].

Under the influence of estrogen and progesterone during pregnancy, cells in the mammary gland, including breast stem cells, undergo remodeling and differentiation. Epigenetic processes involve chromatin remodeling, DNA remodeling, and changes in histones. As a result of the transition from type 1 stem cells to type 2 stem cells, their specific genomic signature changes. Type 1 stem cells are characterized by other markers, for example CD44 antigen, Integrin, alpha 6 (ITGA6), and Keratin 19 (KRT19), while type 2 stem cells are characterized by other markers such as Inhibin, alpha (INHA), p53 protein, and whey acidic protein (WAP). In abnormal gene expression, type 1 stem cells are associated with cancer transformation, and the genomic signature of type 2 stem cells may have a protective effect and provide resistance to cancer development. In nulliparous women, type 1 predominates. Conversely, in women who have had 1 or more pregnancies, type 2 predominates, which is perceived as more differentiated and resistant to carcinogenic transformation. Even after menopause, when there is involution of the mammary gland, type 2 stem cells of the breast predominate in multiparous women. It is still unclear whether type 1 stem cells are the main element underlying carcinogenic transformation under the influence of various harmful factors, but this provides strong evidence for the protective action of pregnancies and the need for further research and observation of breast stem cells [33].

An analysis of data from the multicenter clinical control study – the Cancer and Steroid Hormone Study – involving 4599 women aged 20–55 years with a preliminary diagnosis of breast cancer, confirms the importance of age at the first full-term pregnancy, but that breastfeeding and the number of births have an equally significant impact. Lower breast cancer risk was observed in women who breastfed their child compared to women who had a full-term pregnancy and gave birth but did not breastfeed [78].

Reports suggest that shorter breastfeeding duration is associated with an increased risk of triple-negative breast cancer, characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) (triple-negative breast cancer ER− PR− HER2−) [79].

Conversely, an article [80] based on a multicenter clinical control study on breast cancer, demonstrated a significant impact of breastfeeding duration on the protective anti-cancer effect, both for estrogen receptor-positive (ER+) and progesterone receptor-positive (PR+) receptor-positive tumors, as well as ER− and PR-negative tumors [81].

An analysis of 47 epidemiological studies from 30 countries, in addition to the previously mentioned protective effect of pregnancy and number of births on breast cancer development, shows reduced breast cancer risk in breastfeeding women. It is also emphasized that a longer breastfeeding duration, especially every 12 months of breastfeeding, reduced the relative risk of cancer by 4.3%, and with subsequent births this decrease was even greater (7%) [82].

Comparing breastfeeding women to non-breastfeeding women indicates the protective effect of lactation against breast cancer. Researchers also emphasize that breastfeeding duration is very important, with longer durations providing better protective properties [83].

Breast milk may contain breast stem cells, suggesting that they are excreted from the mother’s body. Thus, breastfeeding likely reduces the number of cells susceptible to potential carcinogenic transformation [84]. However, research on this mechanism is very limited. Further observation of the effect of lactation on the pool of breast stem cells on the development of specific tumor subtypes is essential [85].

The importance of the breastfeeding period may indicate changes in the population of progenitor cells within the breast. A significant challenge remains in determining the minimum breastfeeding time that can induce cell differentiation and provide a protective anti-cancer effect. The limitation of studies lies in the lack of precision in identifying stem cells and/or progenitors in the mammary gland [79].

The molecular profile of the human mammary gland is also being studied, where a reduced number of CD44+ progenitor cells associated with breast stem cells has been demonstrated. There is evidence that pregnancy and lactation alter signaling pathways by downregulating genes related to important functions of breast stem cells. However, carriers of BRCA1/BRCA2 gene mutations did not have lower risk of breast cancer. A positive correlation was observed only in women who had multiple pregnancies [86].

Another study notes that breastfeeding may protect against breast cancer in carriers of the BRCA1 mutation, which was not observed in BRCA2 carriers [87].

Due to the decrease in the number of breast stem cells associated with pregnancy and lactation, there is a need for better understanding of the mechanisms of carcinogenic transformation within these stem cells, and above all, for determining the effects of various carcinogenic factors on these cells [70].

Because of the aforementioned reports on the influence of stem cells on breast cancer development, new therapeutic goals are emerging, focusing on reducing the number of unnecessary stem cells and progenitors. This is particularly important in breast cancer prevention for women at high risk to improve their quality of life [36]. Table 2 summarizes the impact of pregnancy and breastfeeding on breast stem cells.

Future Directions: Mammary Stem Cells as a Potential Target for Therapy

MaSCs have an important association with breast cancer and they play a crucial role in its initiation and progression, suggesting that targeting these cells may also be important in cancer therapy. Progesterone stimulates stem cells proliferation, and population-based studies showed that anti-progesterone substances, selective progesterone receptor modulators, and paracrine effector antagonists decrease the population of stem cells and have anti-tumor effect [36]. There are also studies focused on immunotherapy and aiming immunological cells to affect mammary stem cells and causing their depletion. That action could potentially enhance the specificity of cancer therapies [88]. Stem cells could also be engineered to deliver therapeutic substances directly to tumor tissue, which might limit adverse effects and enhance therapeutic effects [10]. Some studies showed the effects of targeting tumor surface antigens such as CD 133 and CD44 and the Notch and Hedgehog signaling pathways [89,90].

Conclusions

This article has reviewed the roles of mammary stem cells in breast development, lactation, and development of breast cancer with future potential for targeted therapies directed at this cell population. Breast stem cells are the starting point of carcinogenesis and breastfeeding is an important factor in reducing the risk of BC. There are many theories as to why breastfeeding reduces the risk of developing cancer, from the most well-known hormonal theory, through immunological theory, to the components of breast milk that activate apoptosis in cancer cells. Many studies confirmed the relationship between breastfeeding and a lower risk of breast cancer, but it requires more research. Reducing the risk of breast cancer may be related to reducing their number and activity while maintaining their biological function. However, research into the processes of carcinogenesis per se is also necessary, regardless of whether or not it is stem cells that undergo neoplastic transformation under the influence of favorable factors.

The link between breast stem cells, breastfeeding, and breast cancer risk reduction holds great promise, especially for development of new cancer therapies. It is important to develop therapies that are effective and help patients have a higher quality of life.