br Introduction Breast cancer is a heterogeneous disease

Introduction Breast cancer is a heterogeneous disease characterized by at least eighteen histopathological and six distinct molecular subtypes, making it extremely difficult to treat (Sørlie et al., 2001). This phenotypic diversity is considered to reflect the epithelial lineage from which a given breast cancer originates (Sørlie et al., 2001; Lim et al., 2009). Although the molecular understanding of the earliest events contributing to breast cancer remains unclear, it is increasingly apparent that a small population of self-renewing epithelial glucagon receptor in the mammary glands (designated mammary stem cells; MaSC) can undergo oncogenic transformations, thereby, endowing these transformed cells (designated cancer stem cells, CSC) with tumor-initiating properties and the ability to persist in tumors as a distinct population (Al-Hajj et al., 2003; Stingl et al., 2006; Clarke, 2008; Visvader, 2009). CSC and their normal MaSC counterparts are considered to share regulatory programs for their maintenance and propagation. Signaling pathways mediated by Wnt, Notch, Hedgehog, and phosphatidylinositol 3-kinase/Akt have been identified as critical for growth control of normal MaSC and when de-regulated, may underlie aberrant CSC self-renewal and multi-differentiation (Li et al., 2003; Liu et al., 2006; Farnie and Clarke, 2007; Korkaya et al., 2009). Given recent findings of the dynamic plasticity of normal SC and CSC-like cells (Chaffer et al., 2011); that both luminal and myoepithelial (basal) cells can equally contribute to epithelial regeneration (Van Keymeulen et al., 2011); and that luminal and basal epithelial signatures are tightly maintained by autocrine and paracrine-signaling mechanisms (Balko et al., 2012), the identification of novel factors that alter the expansion and biology of epithelial subpopulations has significant implications for breast cancer prevention and treatment. Diet and lifestyle are highly modifiable determinants of breast cancer risk. In support of this, clinical and epidemiological investigations have demonstrated an inverse association between high soy food intake and breast cancer susceptibility (Fink et al., 2007; Wu et al., 2008), with the strongest and most consistent association demonstrated when dietary intake occurred during early childhood than at any other life stage (Korde et al., 2009). Nevertheless, the molecular mechanisms underlying the linkage between diet and breast cancer risk remain poorly understood, in part because translating epidemiological data to clinical (human) studies and equating these with experimental evidence from animal models of breast cancer has not been completely straightforward. Previously, we showed that dietary intake of soy-rich foods reduced chemically-induced mammary tumor formation in rodent models, partly through inhibition of Wnt-signaling and upregulation of PTEN expression (Simmen et al., 2005; Dave et al., 2005; Su et al., 2007; Su and Simmen, 2009; Rahal and Simmen, 2010). Using the human breast cancer cell lines MCF-7 and MDA-MB-231 that display a subpopulation of cells with SC-like properties, we further showed that the soy isoflavone genistein can limit the expansion and differentiation potential of this subpopulation, as measured by their decreased ability to form mammospheres when cultured under non-adherent conditions (Montales et al., 2012). Since PTEN and Wnt signaling pathways are functionally linked (Li et al., 2003; Korkaya et al., 2009) and constitute key regulators of mammary SC and CSC biology (Liu et al., 2004; Farnie and Clarke, 2007; Zhou et al., 2007; Korkaya et al., 2009), our findings raise the intriguing possibility that epithelial cells with mammosphere-forming activities, suggestive of regenerative potential, are early in vivo targets of dietary factors for anti-tumor protection. Little is known about the influence of diet on specific mammary epithelial populations in vivo. To investigate this, we evaluated the relative frequency and functional behavior of distinct epithelial subpopulations in pre-neoplastic (hyperplastic) mammary tissues of MMTV-Wnt1-transgenic mice (Tsukamoto et al., 1988) by using well-characterized cell-surface antigens for fluorescence-activated cell sorting (FACS) and soy-based foods as dietary paradigm. Wnt1-Tg female mice are prone to spontaneous mammary tumors due to SC expansion (Cho et al., 2008; Vaillant et al., 2008). Hyperplastic mammary glands from these mice display increased repopulating activity while resultant mammary tumors show enhanced accumulation of CSC with tumor-initiating ability. We show here that the frequency of CD29hiCD24+Lin− epithelial cells, unlike that of the CD29loCD24+Lin− is reduced in hyperplastic mammary glands of Wnt1-Tg mice exposed to dietary soy protein isolate (SPI) beginning at weaning and throughout lifetime when compared to those of mice fed the control diet. We also show that the reduction of this epithelial subpopulation with dietary SPI exposure is associated with decreased self-renewal capacity and lower outgrowth potential of mammary epithelial cells and reduced mammary tumor incidence. Gene expression profiling of this mammary epithelial subpopulation further revealed a suppressed transcriptional network important to inflammation, cytokine and chemokine signaling, and proliferation. Our results provide evidence for the functional impact of diet on specific epithelial subpopulations that may relate to breast cancer risk and suggest that diet-regulated cues can be further explored for breast cancer risk assessment and prevention.