br Materials and methods br Results

Materials and methods
Results
Discussion To the best of our knowledge, the present study constitutes the first report describing diet-mediated inhibition of the expansion of the MaSC-enriched (CD29hiCD24+Lin−) epithelial subpopulation in vivo that may relate to decreased susceptibility to mammary tumor formation. Studies to date have largely demonstrated dietary factor effects on MaSCs in vitro using established breast cancer cell lines (Kakarala et al., 2010; Montales et al., 2012). Using Wnt1-Tg mice, which develop spontaneous mammary tumors previously shown to originate from dysregulation of mammary stem/progenitor hydroxypropyl beta cyclodextrin (Li et al., 2003; Liu et al., 2004), we show that diet may modify mammary tumor risk and outcome in part, by influencing the expansion of a distinct epithelial subpopulation with self-renewal capacity. The reduction in MaSC-enriched population in the hyperplastic mammary glands by dietary soy (by ~40%) was recapitulated in a similar reduction in the CSC (Thy+) population (by ~60%) but not in the luminal subpopulation. While speculative, the lack of dietary SPI effects on the luminal subpopulations could mean that soy-associated bioactive factors may (directly or indirectly) preferentially target cells that give rise to tumors with non-luminal A molecular subtype (Sørlie et al., 2001). We further show that the limited expansion of the basal (CD29hiCD24+Lin−) epithelial subpopulation with dietary SPI is associated with suppression of pro-inflammatory cytokine, chemokine and proliferative gene expression in this epithelial subpopulation and with reductions in systemic levels of the steroid hormones estradiol and progesterone and the cytokine IL-6, all of which are considered to promote breast cancer risk. Our collective data suggest that diet-regulated hormonal cues can impact MaSC self-renewal programs and by so doing, limit the transformation of MaSC to cancer stem cells for tumor initiation. A major feature of the SPI-altered gene network in the CD29hiCD24+Lin− epithelial subpopulation is a suppressed cytokine and chemokine transcriptional signature. Based on previously reported signaling pathways involving cytokines in this gene signature, we identify IL6 as a candidate target of SPI effects within the MaSC population. By reducing systemic levels and MaSC-enriched production of IL6, dietary SPI may effectively block a number of critical pathways in tumor initiation and subsequent tumor survival and metastasis. While the mechanistic association between SPI-induction of IL6 and inhibition of MaSC expansion was not evaluated in the present study, our findings are consistent with previous reports that IL6 promotes breast CSC self-renewal (Iliopoulos et al., 2011) and fosters the positive feedback loop between MaSCs and breast CSCs (Iliopoulos et al., 2011). Moreover, our findings that SPI diet reduced the expression of a preponderance of genes with pro-inflammatory activities in the CD29hiCD24+Lin− epithelial subpopulation highlight the functional importance of controlling the inflammatory milieu for breast cancer prevention (Cabodi and Taverna, 2010). Further, they support the clinical importance of an extensive search for bioactive food components with anti-inflammatory activities for further investigations into dietary cues for breast cancer prevention. Our findings of decreased systemic progesterone, taken together with the observed reduction of the MaSC-enriched (CD29hiCD24+Lin−) epithelial subpopulation and lower tumor incidence with dietary SPI intake, are consistent with the reduced incidence of mammary tumors in mice null for progesterone receptor when administered with the chemical carcinogen DMBA (Lydon et al., 1999). Moreover, the transient breast cancer risk that lasts for a few years after a full-term pregnancy (Brit et al., 2007) has been attributed to the expansion of mammary stem cells during this period of high progesterone exposure (Tiede et al., 2009). Similarly, the lower levels of estradiol-17β in Wnt1-Tg mice fed SPI are consistent with high circulating estrogens as a risk factor for breast cancer (Litton et al., 2012). While MaSCs lack expression of receptors for estrogen and progesterone (Asselin-Labat et al., 2006, 2010; Joshi et al., 2010), they are subject to estrogen- and progesterone-induced expansion involving the stem cell niche via paracrine signaling. The pathway(s) linking the decline in systemic levels of progesterone, estrogen and IL6 by dietary SPI with the reduction in the frequency of a distinct epithelial subpopulation with renewal capacity remains to be determined and is the subject of current investigations in our group.