Gut and Breast Microbiota as Endocrine Regulators of Hormone Receptor-positive Breast Cancer Risk and Therapy Response

Despite advances in treatment strategies, breast cancer (BC) remains one of the most prevalent cancers worldwide. Recent studies implicate the gut microbiome as a potential risk factor for BC development. Alterations in gut microbial diversity resulting in dysbiosis have been linked to breast carcinogenesis by modulating host immune responses and inflammatory pathways, favoring tumorigenesis and progression. Moreover, gut microbiota populations are different between women with BC vs those that are cancer free, further implicating the role of the gut microbiome in cancer development. This alteration in gut microbiota is also associated with changes in estrogen metabolism, which strongly correlates with BC development. Gut microbiota that express the enzyme β-glucuronidase (GUS) may increase estrogen bioavailability by deconjugating estrogen-glucuronide moieties enabling reabsorption into circulation. Increased circulating estrogens may, in turn, drive estrogen receptor–positive BC. GUS-expressing microbiota also affect cancer therapy efficacy and toxicity by modifying glucuronide-conjugated drug metabolites. Therefore, GUS inhibitors have emerged as a potential antitumor treatment. However, the effectiveness of GUS inhibitors is still exploratory. Further studies are needed to determine how oral endocrine-targeting therapies may influence or be influenced by the microbiota and how that may affect carcinogenesis initiation and tumor recurrence.

gut microbiome, menopause, breast cancer, estrogen, β-glucuronidase Breast cancer (Bc) is the most commonly diagnosed cancer in women, accounting for 31% of all cancer diagnoses. In 2022 alone, there are an estimated 287 850 new cases and approximately 43 250 BC-related deaths (1). While advances in detection and treatment have reduced BC mortality rates over the past 2 decades, the etiology of BC is still being explored. Given that genetics accounts for only 10% of BCs and as many as 70% of BCs occur in women of average risk, it suggests that in addition to a combination of genetic, epigenetic, and environmental factors are other risk factors leading to carcinogenesis (2, 3). Recent studies have implicated the gut microbiome as a potential risk factor for BC, as well as an explanation for different responses to therapy (4). The human gut microbiome is a complex ecosystem with diverse physiological roles affecting human health and disease. Through various interactions with the host, the gut microbiome modulates several metabolic and immunologic pathways to maintain host homeostasis (5). Host genetics also influence the composition of the gut microbiome, which can further affect metabolic pathways (6, 7). Moreover, a study by Xu et al (8) suggests interactions between host genetics and the gut microbiome may affect human diseases, but further study is needed to determine the exact role in BC. However, alterations in gut microbial diversity are associated with several chronic disease states, including BC. Imbalances in gut microbiota may drive breast carcinogenesis by modulating host immune responses and inflammatory pathways, favoring tumorigenesis and progression (9). Moreover, studies indicate the gut microbiome of women with BC is altered compared to healthy matched controls (10). This alteration in gut microbiota is also associated with changes in estrogen metabolism, which strongly correlates with BC development. Additionally, estrogen and other sex hormones also affect gut microbiota populations (11). Gut microbiota also contribute to drug efficacy by enzymatically transforming drug structure, altering the clinical efficacy of cancer therapeutics (12). Gut Microbiome Homeostasis and Dysbiosis The microbiome encompasses all microorganisms on or within human tissues and biofluids, including bacteria, phages, viruses, fungi, and protozoa (13, 14). Although microbes inhabit numerous body sites, more than 90% of microbes reside in the gastrointestinal (GI) tract, encompassing more than 2000 different species and 12 different phyla (15-18). Most of the gut microbiome is composed of bacteria from 4 major phyla: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria (13, 19, 20). This diversity in microbiota plays an essential role in host physiology, including endogenous synthesis of co-factors for energy metabolism and regulation of gene expression, such as vitamin B (21), metabolism of pollutants (22), development of organized lymphoid tissues and function of immune system cells (5), and biotransformation of drugs (12). The gut microbiome's composition is determined by internal and external factors, including age, race, and maternal colonization, as well as host genetics and environmental exposures to xenobiotics and antibiotics (3). In a symbiotic state, host-microbe interactions are critical for maintaining homeostasis in the gut. This confers health benefits to the host, including inhibition of pathogens and prevention of carcinogenesis (9). However, perturbation of the microbiome (resulting in dysbiosis) can lead to the development of autoimmune, inflammatory, and malignant diseases, including BC (18, 23, 24). Dysbiosis contributes to carcinogenesis by increasing the number of harmful bacteria in the gut, leading to the release of enterotoxins, like lipopolysaccharide (LPS). Specifically, dysbiosis results in alterations in the Firmicutes/Bacteroidetes ratio. An increase in Firmicutes to Bacteroidetes is associated with obesity (25), while a decrease in Firmicutes to Bacteroidetes is associated with inflammatory bowel disease (26). Dysbiosis has also been shown to affect zonula occludens toxin levels, which causes zonula occludens (ZO-1) tight junction protein to disengage from the tight junctional complex (27). This leads to increased permeability and induces the production of immunosuppressive proteins. Thus, increased intestinal permeability and increased circulating LPS due to dysbiosis increase low-grade inflammation, which increases the risk of BC development (28, 29). Gut Dysbiosis and Cancer Microbial dysbiosis results in chronic inflammation, inflammation-mediated carcinogenesis processes, and immune invasion favoring tumorigenesis (30). The microbiome drives carcinogenesis by stimulating host cells to proliferate, affecting programmed cell death, regulating the immune response, and altering the metabolism of indigestible dietary components, xenobiotics, and pharmaceuticals (28, 29). Furthermore, diet-induced dysbiosis has been linked to many key changes in gut populations, suggesting that diet-induced changes may transform healthy gut microbiota into a disease-inducing state (31). Western diets, high in fat and sugar, affect both GI tract metabolism and immune homeostasis, contributing to disease states (31, 32). For example, studies in mice fed a high-fat, Western-style diet show an absence in the gut barrier–protecting Bifidobacteria (33). Moreover, diet-induced dysbiosis results in an increased Firmicutes/Bacteroidetes ratio (the 2 dominant phyla in the gut) and is associated with pathological conditions, including cancers (28, 34). In a chemical carcinogen-induced murine model, fecal microbiota transplantation (FMT) from the high-fat diet (HFD)-fed mice to control diet–fed mice increased mammary tumorigenesis, resulting in shifted gut microbiota populations. Both consumption of the HFD and fecal transplantation from HFD-fed mice to control diet–fed mice increased the Firmicutes/Bacteroidetes ratio, consistent with a dysbiotic gut microbiome. However, FMT from control diet–fed mice to HFD-fed mice did not affect Bacteroidetes or Firmicutes phyla proportional abundance, but did lead to shifts in certain genera (35). Shifts in the Firmicutes/Bacteroidetes ratio contribute to carcinogenesis by increasing the number of harmful bacteria in the gut, leading to the release of enterotoxins, like LPS, and chronic low-grade inflammation (28, 29) Obesity is associated with an increased risk of at least 13 different cancers, including postmenopausal BC, endometrial, colorectal, and ovarian cancer (36). Moreover, approximately 50% of breast carcinomas are associated with obesity and poor diet (37-39). Obesity is associated with an increased incidence both of estrogen receptor (ER)-positive postmenopausal breast cancer and triple-negative breast cancer (TNBC) (40). Hossain et al (41) found that TNBC tumor-bearing obese mice had reduced alpha diversity compared to tumor-bearing lean mice. Metagenomics analysis at the phyla level displayed a decrease in Bacteroides in obese mice, irrespective of tumor status, consistent with an increase in Firmicutes/Bacteroidetes ratio. Gut microbiota variations have also been identified in patients with benign compared with malignant tumors, suggesting that the microbiome profile could be associated with the malignancy of the tumor (42). Variations in gut microbial diversity have been observed in healthy women vs those with BC (43, 44). One study showed that fecal microbiota in postmenopausal BC patients exhibited less diversity and differential proportional abundances compared to healthy controls (10). Specifically, higher abundances of Clostridia, Enterobacterium, Lactobacilli, Bacteroides, and Escherichia coli were observed in BC patients compared to healthy controls (10). Zhu et al (43) analyzed gut microbiota diversity by comparing premenopausal Bc patients, healthy premenopausal controls, postmenopausal Bc patients, and healthy postmenopausal controls and found that Bc status, menopause status, and age were significant factors influencing gut microbial diversity. Specifically, 45 species significantly differed between postmenopausal BC patients and postmenopausal controls. Thirty-eight species were enriched in postmenopausal BC patients, including E coli, Klebsiella sp _1_1_55, and Prevotella amnii. Seven species were reduced, including Porphyromonas uenonis, Eubacterium eligens, and Lactobacillus vaginalis. Conversely, no significant differences in gut microbiota were observed between premenopausal Bc patients and premenopausal controls (43). Specific gut microbiota that are overabundant in early BC patients compared to healthy individuals are also associated with a worse prognosis (44). Metagenomic analysis of fecal microbiota from early Bc patients pre (n = 76) and post chemotherapy (n = 45), enrolled in the CANTO (CANcer Toxicities, NCT01993498) prospective study revealed differences in β-diversity, which is the measure of similarity or dissimilarity between 2 communities, according to tumor size (pT1), Scarff, Bloom, and Richardson grade (Gr1/2 vs Gr3), axillary node involvement, and TNM staging (stage I vs stage II-III) (44). A current clinical trial (NCT03885648) investigates the association of mammary and gut microbiota with BC risk and how exposure to environmental contaminants may alter microbiota to affect cancer risk (45). Metabolomic analysis of fecal and breast tissue samples from women aged 25 to 70 diagnosed with BC will be performed to determine shifts in microbiota populations. Estrogens, estrogen metabolites, and environmental contaminants, such as endocrine disruptors, will also be analyzed. Results will reveal the contribution of bacteria, archaea, viruses, and fungi along with their alteration by environmental contaminants on the risk of BC (45). Our group is currently investigating the effects of oral aromatase inhibitor treatment on gut microbiota shifts in BC patients (NCT05030038). Results from this clinical trial will reveal the effect of aromatase inhibitor on gut microbial diversity as well as bacterial-derived bioactive compounds and glucuronidated sex hormones that may affect BC treatment. The breast has a distinct microbiome, which studies have indicated can be shifted by tumors and diet (46-48). A diverse microbial population has been detected in multiple tissue sites around the breast, including areas close to the nipple and toward the chest wall. Regardless of sample location, various bacterial populations were detected across the breast tissue (46). Dysbiosis of the breast microbiome can lead to chronic inflammation, inflammation-mediated carcinogenesis processes, and immune invasion of the breast tissue (30, 49). Microbial differences in the breast tissue of patients with BC compared to healthy matched controls have been observed (10, 30, 50). Patients with early BC display a relative enrichment in Firmicutes with an associated depletion in Bacteroidetes compared to healthy women (51). Urbaniak et al (50) found higher abundances of Enterobacteriaceae and Staphylococcus in BC patients compared to healthy controls. These taxa are associated with DNA double-strand breaks, the most detrimental type of DNA damage. Bacillus, which has carcinogenic effects, was also elevated in women with BC compared to healthy controls. Differences in breast microbial diversity between women with benign vs malignant BC have also been identified. Proteobacteria, Firmicutes, and Actinobacteria abundances were significantly higher in breast tissue taken from women with malignant BC than from women with benign BC (52). Furthermore, reduced Lactobacillus abundance in breast tissue from patients with malignant breast tumors compared to benign breast tumors was observed (48). Hieken et al (53) showed that increased breast stromal fat percentage is associated with malignant disease. Moreover, the increased stromal fat percentage was associated with increased Staphylococcus abundance. These results suggest a link between breast tissue stromal alterations and the breast tissue microbiome. HFD-induced dysbiosis has been linked to breast carcinogenesis through changes in breast microbiota populations as well (35). Our group showed a bacterial microbiome population both in tumor and breast tissue in human, nonhuman primate (NHP), and murine models shifted by diet. Our group has shown in an NHP model that the consumption of a Mediterranean diet led to a 10-fold increase in breast Lactobacillus abundance compared to Western-diet–fed NHP (47). FMT from HFD-fed mice to control diet–fed mice increased mammary tumorigenesis, resulting in shifted breast microbiota populations in a chemical carcinogen-induced BC murine model (35). Intracellular microbiota have been detected in many cancer types, including lung, bladder, gastric, prostate, melanoma, glioblastoma multiforme, and BCs (54-56). Using the spontaneous mouse breast–tumor MMTV-PyMT model, intratumoral microbiota modulates the cellular cytoskeleton and cell viability, playing a crucial role in tumor metastasis (57). Specifically, intratumoral bacteria in circulating tumor cells promote host-cell survival by enhancing resistance to fluid shear stress through reorganization of the actin cytoskeleton. Moreover, the administration of selected bacteria strains isolated from tumor-resident microbiota, including S xylosus, L animalis, and S cuniculi, promoted metastasis both in MMTV-PyMT and MMTV-WNT tumor models. However, metastatic potential differed between models (57). Breast tumor tissue taken from Bc patients has higher abundances of Methylobacterium radiotolerans and lower abundances of Sphinogomomas yanoikuyae compared with matched healthy tissues (58). S yanoikuyae, which may have probiotic functions, express glycosphingolipids that serve as activators of invariant natural killer T (iNKT) cells. iNKT cells play an essential role in tumor immunosurveillance, immunity, and BC metastasis (58-60). This study also reported an inverse correlation between bacterial load and BC disease stage. Quantifying total bacterial DNA load demonstrated that stage 1 patients had the highest copy number of bacterial DNA compared to stage 2 and 3 BC patients. This difference in bacterial load was also related to a reduced expression of antibacterial response genes in advanced-stage BC patients (58). Innate immune sensors, including toll-like receptors 2, 5, and 9, and antimicrobial response effectors IL-12A, BPI, and MPO were expressed at lower levels in breast tumors than in healthy breast tissue (58). Dysbiosis has been shown to lower circulating lymphocytes and increase the neutrophil-to-lymphocyte ratio, which has been associated with decreased survival in women with BCs (9). Streptococcus and Propionibacterium abundances have been shown to correlate positively with T-cell activation-related genes (61). These results demonstrate that human breast microbiota populations are associated with immunological signatures in BC and healthy tissue. Taken together, these findings suggest certain bacterial species may contribute to maintaining healthy breast tissue by stimulating resident immune cells, such as iNKT cells. However, dysbiosis may reduce the abundance of beneficial bacteria, such as S yanoikuyae, and result in a cellular environment conducive to breast carcinogenesis (58). Gut Microbiome and Menopause Menopausal status was shown to shift the gut microbiome, leading to a reduction in bacterial diversity and richness (62). Postmenopausal women have a higher Firmicutes/Bacteroidetes ratio than premenopausal women, suggesting menopausal changes may contribute to dysbiosis and BC progression (43, 62). Furthermore, several studies indicate that this ratio may contribute to estrogen metabolism homeostasis, as shifts in this ratio have been identified between premenopausal and postmenopausal women and women with high and low estrogen levels (62-64). However, a study found that postmenopausal women (n = 24) had a lower Firmicutes/Bacteroidetes ratio compared to body mass index (BMI)-matched premenopausal women (n = 24), suggesting that factors other than changes due to menopause affect the gut microbiome (63). Shifts at the family and genus level have also been observed between premenopausal and postmenopausal women. Shin et al (64) found a significant decrease in Veillonellaceae family abundance and a significant increase in Lachnospiraceae in postmenopausal women. Members of the Lachnospiraceae family are among the leading producers of short-chain fatty acids (SCFAs), including butyrate. Butyrate reduces inflammation through the differentiation of regulatory T cells (65). At the genus level, differential abundances of Roseburia, SCFA-producing commensal bacteria thought important in metabolic and endocrine disease, have been reported in postmenopausal women. One study found lower abundances of Roseburia spp. in postmenopausal women, which may be due to changes in metabolism (63). Another study found higher abundances of Roseburia after menopause (62). Differences in Roseburia abundance may be due to differences in BMI. The first study looked only at lean women, while the second study included only women with a BMI greater than 25 (62, 63). Changes in estrogen metabolism due to menopause are also linked to changes in the gut microbiome (43, 63, 64). Nonovarian estrogens, such as those produced by adipose tissue, are inversely associated with fecal microbiome alpha diversity and richness (66). Increased Firmicute abundance is associated with elevated estrogen levels, which is consistent with the increased Firmicutes/Bacteroidetes ratio observed in menopausal women. Flores et al (67) found in postmenopausal women but not premenopausal women that urinary levels of estrone and estrone metabolites were directly associated with fecal microbiome richness. Specifically, nonovarian estrogens were significantly associated with Clostridia, the most abundant family of Firmicutes. The ratio of estrogen metabolites to parent estrogens in postmenopausal women was directly associated with Clostridiales order and Ruminococcaceae family relative abundance. In contrast, the Bacteroides genus was inversely associated with this ratio (66). An inverse relationship between urinary estrogens and total fecal estrogen levels has also been identified (67). Specifically, deconjugated fecal estrone was inversely correlated with total urine estrone levels. Conjugated fecal estrogens were not significantly associated with total urinary estrogen levels (67). Enzymatic activity of fecal β-glucuronidase (GUS) was also inversely correlated with deconjugated and conjugated fecal estrogens (67). The correlation between estrone levels and gut microbial diversity suggests that more estrogens are excreted through feces when bacterial diversity and enzymatic activity are low (67). Estrobolome The gut microbiome modulates metabolic and immunologic pathways through metabolites, immunologic messengers, and hormonal intermediates. Through these different pathways, the gut microbiota may affect the development of BC (10, 18, 68, 69). Specifically, the gut microbiota has been shown to modulate estrogen homeostasis through the “estrobolome,” which is the totality of enteric bacterial genes whose products can metabolize estrogens (10, 70). In premenopausal women, the ovaries use cholesterol derived from saturated fats for estrogen synthesis. In postmenopausal women, adipose tissue, adrenal glands, and other organs convert circulating androgens into estrogens using the aromatase enzyme (71). Circulating estrogens and their metabolites are conjugated in the liver through methylation, glucuronidation, or sulfonation to allow for biliary excretion (72). Conjugated estrogens are then excreted in bile, urine, and feces. Approximately 50% of estrogen metabolites, exclusively in conjugated form, are present in bile (73). However, only about 7% of these conjugated estrogen metabolites are found in excreted feces, indicating that the bulk of these compounds are hydrolyzed into free estrogenic compounds and reabsorbed in the intestines (74). Conjugated estrogens and estrogen metabolites can be deconjugated by certain microbiota species. These microbiota species contain GUS, an enzyme that removes the inactivating glucuronic acid from estrogen and releases the newly deconjugated estrogen into the GI lumen (70, 75). Bacteria with GUS activity can deconjugate estrogen metabolites (estradiol-17-glucuronide and estrone-3-glucuronide) from bile acids to elevate bioavailability (18, 76). Treatment with antibiotics significantly enhanced the concentration of conjugated estrogen metabolites in the feces, demonstrating this pathway's reliance on gut microflora populations (77). Once active, free estrogen enters the enterohepatic circulation enabling it to bind both to ERα and ERβ, thus eliciting its physiological effects (Fig. 1) (70, 75). Consequently, endogenous estrogen is a major factor in postmenopausal BC development, as 70% of BCs are ER positive (78). β-Glucuronidase Structure and Estrogen Reactivation Studies have shown that gut microbial GUS enzymes have unique structures and functions (79). To date, 279 and 444 distinct GUS proteins from human and mouse gut microbiomes have been identified (79, 80). GUS proteins can be divided into 6 structural classes based on different active sites: loop 1 (L1), mini-loop 1 (mL1), loop 2 (L2), mini-loop 2 (mL2), mini-loop 1,2 (mL12), and no loop (NL). Different microbial phyla demonstrate loop structure preference. For example, GUS from Firmicutes phylum display loop 1, mini-loop 1, and no loop structures, while Bacteroidetes GUS display loop 1 structure (79, 80). The structural diversity among GUS enzymes indicates that species may exert substrate preference (81). GUS with mL1, L2, mL2, and NL active sites process polysaccharides with glucuronic acid, while L1, mL1, and L2 enzymes with longer loops process small glucuronide substrate more effectively (79). Consistently, Ervin et al (76) analyzed 35 human GUS enzymes’ ability to deconjugate 2 distinct estrogen glucuronides, estrone-3-glucuronide and estradiol-17-glucuronide to estrone and estradiol, respectively. Seventeen of the GUS enzymes could reactivate estrone-3-glucuronide and estradiol-17-glucuronide. Furthermore, the crystal structure analysis showed that estrogen glucuronides belong to small-molecule glucuronides and therefore are more likely to be processed by GUS with longer loops, such as L1 and mL1 (76). Sui et al (81) further demonstrated that 18 of the 35 GUS enzymes identified by Ervin and colleagues (76) were from the Firmicutes phyla, and 11 could deconjugate estrone-3-glucuronide and estradiol-17-glucuronide. However, of the 13 GUS enzymes from the Bacteroidetes phylum, only 2 displayed weak deconjugation activity. These results suggest that Firmicutes GUS containing L1 and mL1 active sites are the primary source for estrogen deconjugation. Due to the role of the gut microbiome in estrogen metabolism, dysbiosis leading to alterations in estrogen levels has been identified as a potential risk factor for ER-positive BC. A study that evaluated the association between the fecal microbiome and urinary estrogens in healthy postmenopausal women observed that microbial diversity is positively associated with the ratio of estrogen metabolites to parent estrogens. These findings suggest that variability in gut microbial diversity is associated with differences in estrogen metabolism and thus circulating estrogen levels (66). Another study investigated the gut microbial diversity differences between postmenopausal women with BC and age-matched healthy women. They reported that the fecal microbiota was less diverse and compositionally different in the postmenopausal women with BC compared to the age-matched healthy women. They also noted that the BC patients had higher levels of systemic estrogens; however, this was not statistically significant (10). Overall, lower microbial diversity, correlated with higher levels of circulating estrogens, is associated with a higher risk of BC (67, 82). Adiposity has also been associated with increased circulating estrogen levels, leading to higher BC risk, particularly in postmenopausal women (83). A meta-analysis of 50 prospective observational studies confirmed a relationship between adult weight gain in women and BC risk. For each 5-kg increase in adult weight gain, there was an 11% increase in postmenopausal BC risk (83). In postmenopausal women, excess adiposity may contribute to increased circulating estrogens through peripheral aromatization of androgens, insulin resistance (IR), and reduced production of hepatic hormone-binding proteins, thereby increasing total and bioavailable estrogens (84). Diet is a major determinant of gut microbial diversity and therefore plays a critical role in bacterial GUS activity and associated estrogen metabolism (81). The effects of diet on gut microbiota and estrogen metabolism have been implicated as potential BC risk factor. Vegetarian women display increased fecal excretion of conjugated estrogens, leading to decreased circulating free estrogen compared to omnivorous women (85). Specifically, Goldin and colleagues (85) found that women fed a vegetarian high-fiber, moderate-fat (30% of calories) diet had triple the concentration of fecal estrogen and 15% to 20% lower serum estrogen concentrations compared to age-matched women fed a low-fiber, high-fat (40%) “Western diet.” Diets rich in fats also promote the production and excretion of bile acids used for fat digestion and absorption. Certain gut bacteria convert secondary bile acids into deoxycholic and lithocholic acids, metabolites that promote the growth of Proteobacteria species (such as E coli), contributing to dysbiosis. E coli produce potent GUS, deconjugating estrogens in the intestine and contributing to higher circulating free estrogen levels (86). Fecal bacterial GUS activity is also significantly lower in women consuming a vegetarian diet than in nonvegetarian women (85). Creekmore et al (80) demonstrated in a murine model that diet influences GUS-expressing bacteria abundances. HFD-fed mice displayed increased levels of Firmicutes and decreased levels of Bacteroidetes. Changes in microbial abundance with diet were also associated with alterations in GUS structural loops, with high NL levels and reduced L2 levels in the HFD-fed mice. The concomitant occurrence of HFD-induced dysbiosis, obesity, and increased bacterial GUS activity, with the associated increased circulating estrogen levels, may be responsible for a 20% higher risk of developing BC (9). Gut Microbiome and Cancer Therapeutics Studies have shown that the gut microbiome modulates the host response to chemotherapeutic drugs through direct and indirect chemical signaling cascades. Microbiota play a substantial role in cancer therapy by modulating drug efficacy, abolishing the anticancer effect, and mediating toxicity (87). Specifically, the gut microbiome affects chemotherapy toxicity and efficacy through several key mechanisms, including metabolism, immunomodulation, translocation, enzymatic degradation, and a reduction in diversity (88). For example, doxorubicin, an anthracycline used to treat TNBC, induces translocation, the process in which bacteria cross the gut barrier to extraintestinal sites, which contributes to the morbidity of cancer therapy (88). Bacterial GUS also limit the efficacy of cancer therapeutics by reversing the glucuronidation process. Glucuronidation inactivates and detoxifies drugs and other molecules by increasing their water solubility to promote their removal through the kidneys or GI tract (89). GUS bacteria remove the glucuronic acid moiety, regenerating the original molecule marked initially for elimination in the gut. The reactivated molecule can then reenter the circulation (90). The anticancer prodrug irinotecan, used to treat colorectal and pancreatic cancers, is the archetype of bacterial GUS-induced drug toxicity. SN-38, the active form of irinotecan, is inactivated in the liver via glucuronidation, which is reversed by GUS bacteria, reforming SN-38, which causes GI toxicities, including diarrhea (90). Inhibitors for bacterial GUS alleviated SN-38 toxicity in mice (86). Given the role of microbial GUS activity on drug toxicity, further studies are needed to determine if GUS inhibitors can improve the efficacy of other anticancer drugs. As previously mentioned, GUS bacteria participate in deconjugating estrogens, allowing estrogens to enter circulation. However, the effect of exogenous estrogen administration on the gut microbiome and GUS activity is relatively unknown. Chen et al (91) determined the effects of conjugated estrogens (Ce) and Bazedoxifene (BZA) on the gut microbiome. Using an ovariectomized mouse model, they found that while Ce + BZA treatment did not affect the overall gut microbial community, fecal GUS activity was significantly reduced and positively correlated with Lactobacillaceae family abundance. Using an in vitro model, this study also found tamoxifen, and 4-hydroxytamoxifen, the active metabolite of tamoxifen, are potential GUS bacteria substrates (91). The authors of this study suggest that gut microbial metabolism of endogenous, exogenous, and even synthetic estrogens may produce novel ER ligands, which may affect an individual's response to estrogen replacement therapy and other hormonal therapies (82, 91). Further study is needed to determine whether oral endocrine therapies used to treat ER-positive BC, including tamoxifen, alter and/or are metabolized by gut microbiota and how that may affect cancer recurrence. A recent study demonstrated that low serum concentrations of active tamoxifen metabolites are associated with poor patient outcomes in adjuvant-treated BC patients (92). β-Glucuronidase Inhibitors Gut microbial GUS enzymes increase estrogen bioavailability and may contribute to estrogen-driven breast carcinogenesis; therefore, inhibiting bacterial GUS activity through GUS inhibitors may elicit an antitumor response (see Fig. 1). Ervin et al (76) demonstrated in vitro the GUS inhibitor UNC10201652, which selectively inhibits loop 1 group of GUS enzymes, potently inhibits both estrone-3-glucuronide and estradiol-17-glucuronide reactivation by several microbial loop 1 GUS enzymes, including Eubacterium eligens and Clostridium perfringens. However, UNC10201652 did not inhibit the activities of the loop 1 GUS enzymes from Faecalibacterium prausnitzii or Lactobacillus rhamnosus, suggesting that differences in amino acid structure in the loop 1 region may result in variations of UNC10201652 potency (93, 94). In an MMTV-PyMT transgenic mouse model, which initially develops ER-positive tumors, UNC10201652 treatment resulted in no differences in tumor size or multiplicity compared to untreated controls. These results suggest UNC10201652 may be insufficient to block the reactivation of estrogens by GUS that potentially participate in tumorigenesis in this model (76). Therefore, GUS inhibitors with broader active site loop specificity may be more efficient at preventing estrogen reactivation and tumor growth. The use of GUS inhibitors, while promising, does have some limitations. As previously mentioned, tamoxifen competes for GUS activity, suggesting tamoxifen is reactivated in the gut (91). Reactivation of tamoxifen may result in increased serum active tamoxifen metabolites, improving ER-positive BC patient outcomes (91). Inhibiting GUS activity may reduce tamoxifen serum levels and worsen patient prognosis (92). Future Prospective: Role of the Gut Microbiome in Other Hormone-related Diseases In addition to BC, alterations in the gut microbiome increase the risk of other estrogen-related diseases, including polycystic ovary syndrome (PCOS) and endometriosis. Gut microbiota dysbiosis is linked to PCOS through IR. Alterations in microbial diversity result in disrupted intestinal epithelial cell permeability, allowing LPS leakage across the gut mucosa into circulation. Activation of the immune system due to circulating LPS may interfere with the function of the insulin receptor, promoting IR and, ultimately, follicular development (95). Furthermore, gut microbiota composition in patients with PCOS is significantly altered compared with women without PCOS, with PCOS patients displaying an increased Bacteroidetes/Firmicutes ratio (96-99). Endometriosis is associated with alterations in several microbial populations, including increases in Proteobacteria, Enterobacteriaceae, Streptococcus spp., and E coli (100-102). Furthermore, Lactobacillus abundance is depleted in women with endometriosis (103). However, oral administration of Lactobacillus has ameliorated endometriosis-associated pain in women (104). Oral combined hormonal contraceptives are associated with a minor decrease in gut microbiota diversity and differences in the abundance of several bacterial taxa in healthy premenopausal women (105). Given the role of gut microbiota in several hormone-related diseases, further study is needed to determine the interactions between microbial populations and estrogen.

The gut microbiome influences breast carcinogenesis both by direct and indirect mechanisms. Recently, the role of the gut microbiome in endogenous and exogenous estrogen metabolism has been suggested as a risk factor for hormonal BC. Specifically, the role of GUS-expressing bacteria has been identified as a potential factor driving ER-positive BC. GUS inhibitors have therefore been proposed as a possible treatment. However, the effect of GUS inhibitors on endocrine-targeting BC therapy efficacy is still unknown. Further studies are needed to determine how oral endocrine-targeting therapies may influence or be influenced by the microbiota, whether direct bug-drug interactions exist, and how these activities may affect carcinogenesis initiation and tumor recurrence. Financial Support This work was supported in part by a Department of Defense (DOD) Congressionally Directed Medical Research Program (CDMRP) Breast Cancer Research Program (BCRP) Breakthrough Level 2 Grant (W81XWH-22-1-0055 and W81XWH-20-1-0014 to K.L.C.). A.A.A. is supported by an National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (NIAID) T32 Training Program in Immunology and Pathogenesis (5T32AI007401). Abbreviations - BC breast cancer - BMI body mass index - BZA bazedoxifene - CE conjugated estrogens - ER estrogen receptor - FMT fecal microbiota transplantation - GI gastrointestinal - GUS β-glucuronidase - HFD high-fat diet - iNKT invariant natural killer T - IR insulin resistance - L1 loop 1 - L2 loop 2 - LPS lipopolysaccharide - mL1 mini-loop 1 - mL2 mini-loop 2 - mL12 mini-loop 1,2 - NHP nonhuman primate - NL no loop - PCOS polycystic ovary syndrome - SCFA short-chain fatty acid - TLR toll-like receptor - TNBC triple-negative breast cancer Contributor Information Alana A Arnone, Department of Surgery, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA; Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157, USA. Katherine L Cook, Department of Surgery, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA; Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA; Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA. Author Contributions Both authors contributed equally to this work. Disclosures The authors have nothing to disclose. Data Availability Data sharing is not applicable to this article as no data sets were generated or analyzed during the present study.

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