Purna A. Joshi

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 CD49fhi 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.

Progesterone drives mammary secretory differentiation via RankL-mediated induction of Elf5 in luminal progenitor cells

Progesterone-RankL paracrine signaling has been proposed as a driver of stem cell expansion in the mammary gland, and Elf5 is essential for the differentiation of mammary epithelial progenitor cells. We demonstrate that Elf5 expression is induced by progesterone and that Elf5 and progesterone cooperate to promote alveolar development. The progesterone receptor and Elf5 are expressed in a mutually exclusive pattern, and we identify RankL as the paracrine mediator of the effects of progesterone on Elf5 expression in CD61+ progenitor cells and their consequent differentiation. Blockade of RankL action prevented progesterone-induced side branching and the expansion of Elf5+ mature luminal cells. These findings describe a mechanism by which steroid hormones can produce the expansion of steroid hormone receptor-negative mammary epithelial cells.

RANKL/RANK control Brca1 mutation-driven mammary tumors

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

Summary 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(+)CD49f(hi)) and luminal (CD24(+)CD49f(lo)) 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.

Progesterone Exposure and Breast Cancer Risk: Understanding the Biological Roots

The Women’s Health Initiative (WHI) randomized trials of menopausal hormone therapy have provided insights that have dramatically changed clinical practice and led to reduced breast cancer incidence at a population level. In this issue of JAMA Oncology, Chlebowski et al1 present a detailed analysis of the impact of estrogen plus progesterone (E + P) therapy or estrogen alone on breast cancer incidence during the intervention as well as early (first 2.75 years) and late (beyond 2.75 years) postintervention periods of the WHI trials. While a significant increase in invasive breast cancer risk occurred during the E + P intervention, Chlebowski et al report a “sharp decrease in breast cancer risk” in the early postintervention period for E + P, although risk, as defined by hazard ratios, remained higher than 1, followed by a sustained increased risk higher than 1 in the late postintervention period with a median additional follow-up of 5.5 years (Figures 1 and 3 in the article by Chlebowski et al1). The decrease in risk during the early postintervention compared with the intervention period was postulated to reflect modulatory effects of a changed hormonal environment on preclinical breast cancer lesions. In contrast, the use of estrogen alone was associated with lower breast cancer risk during the intervention, an even lower risk during the early postintervention period with subsequent attenuation of this risk reduction during the late postintervention period. There were suggestions of different patterns of breast cancer subtypes and stage at presentation over time with E + P therapy vs estrogen alone, including a potential increased risk of progesterone receptor (PR)-negative or triple-negative cancers during the intervention and early postintervention period with E + P therapy. The contrast between effects of E + P therapy vs estrogen alone is striking—breast cancer risk is persistently elevated with E + P therapy, while risk is persistently decreased with estrogen alone therapy. Thus, an important message underlying the study is that the progesterone inclusion during a median hormone therapy intervention period of 5.6 years not only increases the breast cancer risk during intervention but results in a continued elevated risk for several years after stopping this regimen. Recent advances in understanding the biological basis of hormone effects on normal mammary epithelial cell populations and breast carcinogenesis shed light on this contrast and provide insight on how progesterone may exert its cancer-promoting effects (Figure). In animal models, E + P therapy, but not estrogen alone, stimulates expansion of the number of mammary stem and progenitor cells, generating new, denser, and more complex mammary morphology that is also recapitulated during the natural progesterone surge of the mouse reproductive cycle.2,3 Estrogen is instrumental for breast development during puberty, but its primary role during adult mammary growth cycles is to induce PR expression to facilitate progesterone’s proliferative effects in this tissue. The human breast also exhibits increased complexity, density, and mitotic activity during the progesterone-high luteal phase of the menstrual cycle.4 Indeed, mammogram diagnostic performance was hindered in the E + P WHI trial in the first years of intervention, likely owing to increased breast density with E + P therapy.5 It is important to consider whether mammary stem and progenitor cells that trigger these morphological changes in the breast underlie the increased risk associated with E + P exposure. Breast cancer animal models incorporating medroxyprogesterone acetate, the same compound used in the WHI trials, have shown its potent capacity to promote mammary tumors through key mitogenic signals that stimulate the mammary epithelium.6 Accumulating evidence suggests that mammary stem cells and progenitors are the likely “cells-oforigin” for different breast cancer subtypes,7,8 and thus research geared toward deciphering the fundamental biological processes of these cells provides important insight into their potential function in breast cancer development. Because mammary stem and progenitor cells are largely hormone receptor negative, the E + P–exposed mammary stem cell niche is thought to drive an increase in their cell number via a paracrine cross talk. Here, PR-positive cells can act as progesterone-sensing cells and deliver mitogenic signals to PR-negative stem/progenitor cells.3,9 This effect of progesterone on the expansion of hormone receptor–negative cells may explain how E + P therapy increases the risk of PR-negative and triple-negative breast cancers seen in the WHI trial reported by Chlebowski et al.1 Hormone receptor– positive tumors observed could stem from a PR-positive cell or a PR-negative progenitor that subsequently acquires hormone receptor expression. It is plausible that a sudden decline in progesterone levels within the stem cell niche would mitigate further development of early cancer lesions that are still dependent on progesterone, resulting in the decreased breast cancer risk during the early postintervention period. However, breast cancer risk still remained elevated (hazard ratio >1), and the cancers that may have formed during this early postintervention period were likely to have already progressed to an advanced preclinical stage prior to stopping treatment and thus no longer Related article page 296 Opinion

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.

Abstract B023: Identifying molecular programs of progesterone-driven mammary stem cell expansion

Lifetime exposure to ovarian hormones plays a crucial role in determining a woman9s risk for breast cancer: the risk of developing breast cancer is positively correlated with the number of ovarian-hormone-dependent menstrual cycles. Progesterone is an ovarian steroid hormone that peaks during the luteal phase of the female menstrual cycle. Recent research has revealed that progesterone is a key mediator of cellular changes in the mammary gland that likely underlies the correlation between ovarian hormones and breast cancer risk. These studies have shown that progesterone can exert mitogenic effects through paracrine signaling between specific mammary epithelial populations and control mammary stem cell (MaSC) expansion. Such findings provide new insights into MaSC dynamics and underscore the involvement of ovarian hormones in regulating fundamental mammary epithelial changes. Given the differentiation potential of luminal progenitors and MaSCs, they are the proposed cellular targets of transformation in breast cancer. Although it is known that progesterone can induce luminal and basal cell expansion, the underlying mechanisms driving hormone action within the different cellular compartments (basal, luminal and stromal) of the mammary gland are yet to be defined. Therefore, I hypothesize that the transcriptional response to progesterone will reveal important paracrine signaling pathways involved in MaSC changes. To test my hypothesis, mRNA expression profiles were generated from the different mammary cellular compartments under defined hormone treatments. More specifically, basal, luminal and stromal cells were FACS purified after 2 weeks of hormone stimulation with progesterone, estrogen, progesterone plus estrogen, or vehicle control and subjected to microarray analyses using the Agilent platform. To analyze this microarray data, an optimal pre-processing method was generated before any downstream analysis. After pre-processing, significantly altered genes under each hormone treatment were investigated and cross compared within different cellular compartments. Our lab is interested in examining progesterone-mediated ligand and receptor expression changes in the different epithelial compartments that may play a paracrine role in altering the MaSC population. Once significantly altered ligand-receptor pairs are identified, I will validate specific pathways through both in vivo and in vitro experiments utilizing knockout mice to test their functional significance and investigate the effects of aberrant signaling in these pathways in cell culture assays. Progesterone is believed to play a crucial role in MaSC regulation and this might in part explain why a greater number of reproductive cycles and hormone replacement therapy using progestins contribute to a higher risk of developing breast cancer. MaSCs are postulated to be involved in breast cancer initiation, hence elucidating the mechanisms that induce MaSC expansion will allow us to identify putative targets that can be harnessed to control stem/progenitor cells and limit cellular transformation. Citation Format: Yu-Jia Shiah, Purna A. Joshi, Alexander G. Beristain, Michelle Chan-Seng-Yue, Paul C. Boutros, Rama Khokha. Identifying molecular programs of progesterone-driven mammary stem cell expansion. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B023.