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The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology.

The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology.

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.

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.

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.

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.

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(1). 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(2,3). 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-kappa B 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.

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(1). 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(2,3). 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-kappa B 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.

Elucidating the mechanism of focal proteolysis at the cell-matrix interface during metastasis is an ongoing challenge. We have completed the dissection of components of proteolytic machinery required for breast cancer cells (metastatic MDA- MB231 and non-metastatic MCF-7) transendothelial migration (TEM). Modulation of individual molecules demonstrates the functional cooperation of furin, cell surface adhesion molecules (alpha(sub v)Beta(sub3), CD44), and matrix metalloproteinases (MMP-2, MMP-9 and MTI-MMP) during the process of TEM. Confocal microscopy shows co-localization of molecules and MMPs critical for TEM, and disruption of these molecules reduces TEM of MDA-MB231 cells. MMP-2 and MTI -MMP localization matches the imprint of spatially restricted fluorogenic gelatin digestion. The digestion occurs in a stage-specific manner and becomes most evident during the migratory phase of tumor cell TEM. Of the above breast cancer cell lines, MDA- MB231 is able to undergo efficient TEM, and MTI-MMP emerges to be one of the key molecules involved for this event. The lack of several key components in MCF-7 cells impairs its ability to transmigrate. We have now extended our analyses of protease activity by these cells in vivo to the mammary gland, illustrating their differential capacity to degrade the matrix components in the transplanted plug. Our data reveal how specific molecular interactions result in a cooperative proteolytic interface at the metastatic tumor cell surface. We have shown this in vitro utilizing the breast tumor cell-endothelial cell microenvironment during TEM, and in vivo in the mammary tissue environment.

Elucidating the degradive effectors responsible for focal proteolysis at the cell-matrix interface during metastasis is an ongoing challenge. We are dissecting the components of the proteolytic machinery required for breast cancer cells (metastatic MDA-MB231 and non-metastatic MCF-7) transendothelial migration and determining the stage of extravasation particularly reliant upon the metalloproteinase activity. Modulation of individual molecules demonstrates the functional cooperation of furin, cell surface adhesion molecules (alphavBeta3, CD44), and matrix metalloproteinases (MMP-2, MMP-9 and MT1 -MMP) during the process of transendothelial migration. Confocal microscopy shows co-localization of molecules critical for MMP cell surface distribution, and disruption of these events and molecules reduces transendothelial migration of MDA-MB231 cells. MMP-2 and MT1 -MMP localization matches the imprint of spatially restricted fluorogenic gelatin digestion. The digestion occurs in a stage-specific manner and becomes most evident during the migratory phase of tumor cell transendothelial migration. Of the above breast cancer cell lines, MDA-MB231 is able to undergo efficient transendothelial migration, and MT1-MMP emerges to be one of the key molecules involved for this event. The lack of several key components in MCF-7 cells impairs its ability to transmigrate. Our data are revealing how specific molecular interactions result in a cooperative proteolytic interface at the tumor cell surface within the breast tumor cell-endothelial cell microenvironment during transendothelial migration, which mimics cancer cell extravasation., The original document contains color images. All DTIC reproductions will be in black and white.

Elucidating the proteolytic effectors responsible for focal proteolysis at the cellular/pericellular matrix interface during metastasis is an on-going challenge. We have begun to dissect the molecular dynamics of proteolysis required for tumor cell transendothelial migration. MDA-MB-231 (metastatic) cells are far more efficient at TEM compared to MCF-7 (non- metastatic) cells. MMP-2, MMP-9, MT1-MMP and TIMP-2 appear active participants during breast cancer TEM. Natural (TIMP-1, TIMP-2) as well as synthetic MMP inhibitors significantly reduced TEM of both MCF-7 and MDA-MB-231 cells. Serine inhibitors also significantly reduce TEM. Furin is detectable intracellularly as well as at the cell surface of MDA-MB-231 cells. Future experiments will focus on identification of molecules that are necessary for complex formation of the MMPs at the cell-matrix interface during TEM.

The bioavailability of IGF-II, a molecule implicated in breast tumorigenesis, was recently shown by our laboratory to be modulated by TlMP-1 through controlling MM P-mediated IGFBP degradation. To investigate the exact role of IGF-II in breast tumorigenesis and examine IGF-II mediated events potentially regulated by TIMP-I expression we generated independent MMTV-IGF-II transgenic mice. Since increased proliferation and/or decreased apoptosis precede tumor development, we investigated the effects of IGF-II overexpression on proliferation during mammary development and its effect on apoptosis during postlactation involution. We found that elevated levels of IGF-II inhibitied mammary epithelial proliferation in vivo. The delay in ductal morphogenesis correlated with an elevation of the phosphatase PTEN, in the transgenic tissue. Higher PTEN levels resulted in reduced phosphorylation of Akt/PKB and a subsequent reduction in cylcin DI protein levels. We have also observed a delay in ductal morphogenesis when the oncogene, erbB2 is overexpressed. IGF-II overexpression also affected epithelial apoptosis in vivo. Specifically, high levels of IGF-II resulted in delayed postlaction involution through inhibiting mammary epithelial apoptosis. This inhibition of apoptosis was associated with a sustained phosphorylation of Akt/PKB in the transgenic tissue. These data demonstrate that an elevation IGF-II can affect the pre-tumoriqenic events, events potentially controllable through manipulating TIMP-I., Original contains color plates: All DTIC reproductions will be in black and white.