Progesterone induces adult mammary stem cell expansion.

Reproductive history is the strongest risk factor for breast cancer after age, genetics and breast density. Increased breast cancer risk is entwined with a greater number of ovarian hormone-dependent reproductive cycles, yet the basis for this predisposition is unknown. Mammary stem cells (MaSCs) are located within a specialized niche in the basal epithelial compartment that is under local and systemic regulation. The emerging role of MaSCs in cancer initiation warrants the study of ovarian hormones in MaSC homeostasis. Here we show that the MaSC pool increases 14-fold during maximal progesterone levels at the luteal dioestrus phase of the mouse. Stem-cell-enriched CD49fhi cells amplify at dioestrus, or with exogenous progesterone, demonstrating a key role for progesterone in propelling this expansion. In aged mice, CD49fhi cells display stasis upon cessation of the reproductive cycle. Progesterone drives a series of events where luminal cells probably provide Wnt4 and RANKL signals to basal cells which in turn respond by upregulating their cognate receptors, transcriptional targets and cell cycle markers. Our findings uncover a dynamic role for progesterone in activating adult MaSCs within the mammary stem cell niche during the reproductive cycle, where MaSCs are putative targets for cell transformation events leading to breast cancer.

Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer.

Breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe. The Women's Health Initiative and the Million Women Study have shown that hormone replacement therapy is associated with an increased risk of incident and fatal breast cancer. In particular, synthetic progesterone derivatives (progestins) such as medroxyprogesterone acetate (MPA), used in millions of women for hormone replacement therapy and contraceptives, markedly increase the risk of developing breast cancer. Here we show that the in vivo administration of MPA triggers massive induction of the key osteoclast differentiation factor RANKL (receptor activator of NF-κB ligand) in mammary-gland epithelial cells. Genetic inactivation of the RANKL receptor RANK in mammary-gland epithelial cells prevents MPA-induced epithelial proliferation, impairs expansion of the CD49f(hi) stem-cell-enriched population, and sensitizes these cells to DNA-damage-induced cell death. Deletion of RANK from the mammary epithelium results in a markedly decreased incidence and delayed onset of MPA-driven mammary cancer. These data show that the RANKL/RANK system controls the incidence and onset of progestin-driven breast cancer.

The mammary stem cell conundrum: is it unipotent or multipotent?

Exploring the normal biology and regulation of stem cells has the promise to yield insights into the etiological roots and survival of breast cancer cells. Many studies have supported the existence of a multipotent mammary stem cell that regenerates all aspects of glandular development. However, Van Keymeulen and colleagues (2011) illustrated the presence of lineage-restricted unipotent stem cells that self-renew and collaborate in postnatal mammary development, whereas multipotent stem cells were found only during embryonic mammogenesis. This prompts a re-evaluation of currently accepted mammary stem cell dynamics and conceivably its impact on the evolution of different breast cancer subtypes.

PDGFRα+ stromal adipocyte progenitors transition into epithelial cells during lobulo-alveologenesis in the murine mammary gland.

The mammary gland experiences substantial remodeling and regeneration during development and reproductive life, facilitated by stem cells and progenitors that act in concert with physiological stimuli. While studies have focused on deciphering regenerative cells within the parenchymal epithelium, cell lineages in the stroma that may directly contribute to epithelial biology is unknown. Here we identify, in mouse, the transition of a PDGFRα+ mesenchymal cell population into mammary epithelial progenitors. In addition to being adipocyte progenitors, PDGFRα+ cells make a de novo contribution to luminal and basal epithelia during mammary morphogenesis. In the adult, this mesenchymal lineage primarily generates luminal progenitors within lobuloalveoli during sex hormone exposure or pregnancy. We identify cell migration as a key molecular event that is activated in mesenchymal progenitors in response to epithelium-derived chemoattractant. These findings demonstrate a stromal reservoir of epithelial progenitors and provide insight into cell origins and plasticity during mammary tissue growth.

RANKL/RANK control Brca1 mutation- .

Breast cancer is the most common female cancer, affecting approximately one in eight women during their life-time. Besides environmental triggers and hormones, inherited mutations in the breast cancer 1 (BRCA1) or BRCA2 genes markedly increase the risk for the development of breast cancer. Here, using two different mouse models, we show that genetic inactivation of the key osteoclast differentiation factor RANK in the mammary epithelium markedly delayed onset, reduced incidence, and attenuated progression of Brca1;p53 mutation-driven mammary cancer. Long-term pharmacological inhibition of the RANK ligand RANKL in mice abolished the occurrence of Brca1 mutation-driven pre-neoplastic lesions. Mechanistically, genetic inactivation of Rank or RANKL/RANK blockade impaired proliferation and expansion of both murine Brca1;p53 mutant mammary stem cells and mammary progenitors from human BRCA1 mutation carriers. In addition, genome variations within the RANK locus were significantly associated with risk of developing breast cancer in women with BRCA1 mutations. Thus, RANKL/RANK control progenitor cell expansion and tumorigenesis in inherited breast cancer. These results present a viable strategy for the possible prevention of breast cancer in BRCA1 mutant patients.

RANK Signaling Amplifies WNT-Responsive Mammary Progenitors through R-SPONDIN1.

Systemic and local signals must be integrated by mammary stem and progenitor cells to regulate their cyclic growth and turnover in the adult gland. Here, we show RANK-positive luminal progenitors exhibiting WNT pathway activation are selectively expanded in the human breast during the progesterone-high menstrual phase. To investigate underlying mechanisms, we examined mouse models and found that loss of RANK prevents the proliferation of hormone receptor-negative luminal mammary progenitors and basal cells, an accompanying loss of WNT activation, and, hence, a suppression of lobuloalveologenesis. We also show that R-spondin1 is depleted in RANK-null progenitors, and that its exogenous administration rescues key aspects of RANK deficiency by reinstating a WNT response and mammary cell expansion. Our findings point to a novel role of RANK in dictating WNT responsiveness to mediate hormone-induced changes in the growth dynamics of adult mammary cells.

A Progesterone-CXCR4 Axis Controls Mammary Progenitor Cell Fate in the Adult Gland.

Progesterone drives mammary stem and progenitor cell dynamics through paracrine mechanisms that are currently not well understood. Here, we demonstrate that CXCR4, the receptor for stromal-derived factor 1 (SDF-1; CXC12), is a crucial instructor of hormone-induced mammary stem and progenitor cell function. Progesterone elicits specific changes in the transcriptome of basal and luminal mammary epithelial populations, where CXCL12 and CXCR4 represent a putative ligand-receptor pair. In situ, CXCL12 localizes to progesterone-receptor-positive luminal cells, whereas CXCR4 is induced in both basal and luminal compartments in a progesterone-dependent manner. Pharmacological inhibition of CXCR4 signaling abrogates progesterone-directed expansion of basal (CD24+CD49fhi) and luminal (CD24+CD49flo) subsets. This is accompanied by a marked reduction in CD49b+SCA-1- luminal progenitors, their functional capacity, and lobuloalveologenesis. These findings uncover CXCL12 and CXCR4 as novel paracrine effectors of hormone signaling in the adult mammary gland, and present a new avenue for potentially targeting progenitor cell growth and malignant transformation in breast cancer.

Fully interlocking: a story of teamwork among breast epithelial cells.

In this issue of Developmental Cell, Forster et al. (2014) show that the basal myoepithelial cell layer directs the final maturation of the adjacent luminal cell sheet during pregnancy. Do all mammary epithelial cells both give and take instructions from others to create the milk production machinery?

BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival.

Oxidative stress plays an important role in cancer development and treatment. Recent data implicate the tumor suppressor BRCA1 in regulating oxidative stress, but the molecular mechanism and the impact in BRCA1-associated tumorigenesis remain unclear. Here, we show that BRCA1 regulates Nrf2-dependent antioxidant signaling by physically interacting with Nrf2 and promoting its stability and activation. BRCA1-deficient mouse primary mammary epithelial cells show low expression of Nrf2-regulated antioxidant enzymes and accumulate reactive oxygen species (ROS) that impair survival in vivo. Increased Nrf2 activation rescues survival and ROS levels in BRCA1-null cells. Interestingly, 53BP1 inactivation, which has been shown to alleviate several defects associated with BRCA1 loss, rescues survival of BRCA1-null cells without restoring ROS levels. We demonstrate that estrogen treatment partially restores Nrf2 levels in the absence of BRCA1. Our data suggest that Nrf2-regulated antioxidant response plays a crucial role in controlling survival downstream of BRCA1 loss. The ability of estrogen to induce Nrf2 posits an involvement of an estrogen-Nrf2 connection in BRCA1 tumor suppression. Lastly, BRCA1-mutated tumors retain a defective antioxidant response that increases the sensitivity to oxidative stress. In conclusion, the role of BRCA1 in regulating Nrf2 activity suggests important implications for both the etiology and treatment of BRCA1-related cancers.

Active allies: hormones, stem cells and the niche in adult mammopoiesis.

Adult stem cells are recruited in response to specific physiological demands to regenerate, repair or maintain essential cellular components of tissues, while preserving self-renewal capacity. Signals that activate adult stem cells are not simply cell autonomous and stem cells are part of a larger dynamic framework, the stem cell 'niche', which integrates systemic and local cues to sustain stem cell functionality. The mammary stem cell niche responds readily to hormonal stimuli, generating pertinent signals that activate stem cells, culminating in stem cell expansion and tissue growth. We review here current knowledge of the mammary stem cell niche with attention to the potent stimulation rendered by ovarian hormones, relevant cellular and molecular players, and the implication of a deregulated niche, for breast cancer risk.

Loss of Alx4, a stromally-restricted homeodomain protein, impairs mammary epithelial morphogenesis.

Postnatal development of the mammary gland is determined by reciprocal interactions between the ductal epithelia and adjacent stroma. Alx4 is a mesenchymally restricted homeodomain transcription factor expressed in a number of developing tissues, including skin appendages such as hair follicles, whiskers and teeth. We show here that Alx4 is expressed in a subset of ERalpha-expressing mammary stromal cells adjacent to terminal end buds and alveoli during puberty and pregnancy, respectively. Alx4 expression is induced in mammary stromal cells at the onset of puberty and can be induced in prepubescent mice by administration of 17beta-estradiol. In order to determine the role of Alx4 during mammary gland development, we characterized mammary gland development of mice homozygous for the null allele of Alx4, lst(D). Mammary glands from animals lacking Alx4 activity exhibit profound alterations in ductal morphogenesis. Overall development is delayed, ducts being grossly distorted in size and structure. Terminal end buds are also disoriented, displaying aberrant architecture during bifurcation. Despite the developmental delay, the ductal network typically reaches the limits of the fat pad. However, during puberty and in the adult virgin mice, the frequency and density of branch points is significantly reduced. We show further that the defective ductal morphogenesis is due to defects in stromal cells. Specifically, when injected into the cleared fat pad of wild-type recipients, mixed populations of wild-type epithelial cells and Alx4-deficient stromal cells give rise to retarded ductal morphogenesis. Wild-type stromal cells mixed with Alx4-deficient epithelial cells result in normal progression of ductal development. Defective branching morphogenesis in Alx4-deficient females is not due to a loss in expression of HGF, since the level of HGF message in mammary stromal cells is similar in mutant and wild-type littermates. MMP3 is similarly expressed while a 40% increase in MMP2 and a 50% decrease in MMP9 message levels in Alx4-deficient mice relative to their wild-type littermates is observed. Thus, the activity of the stromally restricted homeodomain factor, Alx4, is required for normal branching morphogenesis of the ductal epithelia during pubescent mammary gland development.