Barbara K. Vonderhaar

Establishing a Framework for the Functional Mammary Gland: From Endocrinology to Morphology

From its embryonic origins, the mammary gland in females undergoes a course of ductal development that supports the establishment of alveolar structures during pregnancy prior to the onset of lactogenesis. This development includes multiple stages of proliferation and morphogenesis that are largely directed by concurrent alterations in key hormones and growth factors across various reproductive states. Ductal elongation is directed by estrogen, growth hormone, insulin-like growth factor-I, and epidermal growth factor, whereas ductal branching and alveolar budding is influenced by additional factors such as progesterone, prolactin, and thyroid hormone. The response by the ductal epithelium to various hormones and growth factors is influenced by epithelial–stromal interactions that differ between species, possibly directing species-specific morphogenesis. Evolving technologies continue to provide the opportunity to further delineate the regulation of ductal development. Defining the hormonal control of ductal development should facilitate a better understanding of the mechanisms underlying mammary gland tumorigenesis.

Cellular expression of growth hormone and prolactin receptors in human breast disorders

Growth hormone (GH) and prolactin (PRL) exert their regulatory functions in the mammary gland by acting on specific receptors. Using isotopic in situ hybridization and immunohistochemistry, we have localized the expression of hGH receptor (hGHR) and hPRL receptor (hPRLR) in a panel of human breast disorders. Surgical specimens from adult females included normal breast, inflamatory lesions (mastitis) benign proliferative breast disease (fibroadenoma, papilloma, adenosis, epitheliosis), intraductal carcinoma or lobular carcinoma in situ, and invasive ductal, lobular or medullary carcinoma. Cases of male breast enlargement (gynecomastia) were also studied. In situhybridization analysis demonstrated the co‐expression of hGHR and hPRLR mRNA in all samples tested. Epithelial cells of both normal and tumor tissues were labelled. Quantitative estimation of receptor mRNA levels was regionally measured in areas corresponding to tumor cells and adipose cells from the same section. It demonstrated large individual variation and no correlation emerged according to the histological type of lesion. Receptor immunoreactivity was detected both in the cytoplasm and nuclei or in the cytoplasm alone. Scattered stromal cells were found positive in some cases, but the labeling intensity was always weaker than for neoplastic epithelial cells. Our results demonstrate the expression of the hGHR and hPRLR genes and their translation in epithelial cells of normal, proliferative and neoplastic lesions of the breast. They also demonstrate that stromal components express GHR and PRLR genes. Thus the putative role of hGH or hPRL in the progression of proliferative mammary disorders is not due to grossly altered levels of receptor expression. Int. J. Cancer (Pred. Oncol.) 79:202–211, 1998.© 1998 Wiley‐Liss, Inc.

CD44posCD49fhiCD133/2hi Defines Xenograft-Initiating Cells in Estrogen Receptor–Negative Breast Cancer

Defining the populations of tumor-initating cells that are present in tumors is a first step in developing therapeutics to target these cells. We show here that both CD44(pos)CD24(neg) and CD44(pos)CD24(pos) cell populations in estrogen receptor (ER) alpha-negative breast tumors are tumorigenic in murine xenograft models. We also describe a third population of xenograft-initiating cells (XIC) enriched in CD44(pos)CD49f(hi)CD133/2(hi) cells that display heightened tumorigenicity, self-renewal in vivo, and the capacity to give rise to functional and molecular heterogeneity. Consistent with their capacity for self-renewal, these cells express elevated levels of Sox2, Bmi-1, and/or Nanog and their CpG islands are hypermethylated relative to nontumorigenic cells. These differences in methylome regulation may be responsible for the dramatic functional differences between the two populations. The identification of CD44(pos)CD49f(hi)CD133/2(hi) XIC in ER-negative tumors may lead to expanded understanding of these tumors and ultimately the development of therapeutics designed to specifically target the cells.

Dynamic regulation of CD24 and the invasive, CD44posCD24neg phenotype in breast cancer cell lines.

IntroductionThe invasive, mesenchymal phenotype of CD44posCD24neg breast cancer cells has made them a promising target for eliminating the metastatic capacity of primary tumors. It has been previously demonstrated that CD44neg/lowCD24pos breast cancer cells lack the ability to give rise to their invasive CD44posCD24neg counterpart. Here we demonstrate that noninvasive, epithelial-like CD44posCD24pos cells readily give rise to invasive, mesenchymal CD44posCD24neg progeny in vivo and in vitro. This interconversion was found to be dependent upon Activin/Nodal signaling.MethodsBreast cancer cell lines were sorted into CD44posCD24pos and CD44posCD24neg populations to evaluate their progeny for the expression of CD44, CD24, and markers of a mesenchymal phenotype. The populations, separated by fluorescence activated cell sorting (FACS) were injected into immunocompromised mice to evaluate their tumorigenicity and invasiveness of the resulting xenografts.ResultsCD24 expression was dynamically regulated in vitro in all evaluated breast cancer cell lines. Furthermore, a single noninvasive, epithelial-like CD44posCD24pos cell had the ability to give rise to invasive, mesenchymal CD44posCD24neg progeny. Importantly, this interconversion occurred in vivo as CD44posCD24pos cells gave rise to xenografts with locally invasive borders as seen in xenografts initiated with CD44posCD24neg cells. Lastly, the ability of CD44posCD24pos cells to give rise to mesenchymal progeny, and vice versa, was blocked upon ablation of Activin/Nodal signaling.ConclusionsOur data demonstrate that the invasive, mesenchymal CD44posCD24neg phenotype is under dynamic control in breast cancer cell lines both in vitro and in vivo. Furthermore, our observations suggest that therapies targeting CD44posCD24neg tumor cells may have limited success in preventing primary tumor metastasis unless Activin/Nodal signaling is arrested.

Activation ofraf-1, MEK, and MAP kinase in prolactin responsive mammary cells

SummaryThe polypeptide hormone prolactin (Prl), acting through its cell surface receptors, promotes growth and differentiation in normal and malignant breast cells. We demonstrate herein that two Prl-responsive cell lines, NOG-8 normal mouse mammary epithelial and T47D human breast cancer cells, respond to Prl by rapid and transient activation of a series of kinases.Raf-1 was activated within 2–5 min of Prl treatment. This was followed rapidly by activation of MEK (MAP kinase kinase) and MAP kinase activity in these cells. Increased MAP kinase activity was accompanied by tyrosine phosphorylation of both the 42 kDa and 44 kDa isoforms. The tyrosine kinase inhibitors genestein and tyrphostin blocked the increase in MAP kinase activity as well as Prl induced growth of the T47D cells. These results indicate that the Prl receptor, after binding to Prl in mammary cells, activates theraf-MEK-MAP kinase pathway for signal transduction leading to mitogenesis.

Prolactin as a mitogen in mammary cells

Prolactin (PRL) acts as both a mitogen and a differentiating agent in the breast. The decision to respond to PRL as a mitogen by breast cells depends on the hormonal milieu in which the epithelial cell resides. In addition, PRLs action on the breast is regulated (1) at the level of the hormone itself; (2) at the receptor level; (3) at the level of selection of signaling pathway; and, (4) by combinations of these aspects. The development of cell lines containing only one class of the PRL receptors and showing qualitative differences in response and signaling pathways will help in understanding the pleiotropic nature of PRL action.

Transcriptional and spatiotemporal regulation of prolactin receptor mRNA and cooperativity with progesterone receptor function during ductal branch growth in the mammary gland

Ductal branching within the mammary gland is stimulated by prolactin (PRL) and progesterone (P) acting through their receptors (PRLR and PR). Analysis of mammary gland PRLR expression revealed increasing expression of the long form (L‐PRLR) and two of the three short forms (S1‐ and S3‐PRLR) during puberty that became maximal late in pubescence and early gestation, then declined during gestation. By contrast, S2‐PRLR mRNA levels remained constant. Examination of stromal PRLR revealed the consistent expression of L‐PRLR mRNA. By contrast, S1‐PRLR was present only in the mammary fat pad of neonates, whereas high neonatal expression of S2‐PRLR became undetectable during puberty. Stromal expression of S3‐PRLR decreased to low levels during puberty and was undetectable during lactation and involution. Exogenous PRL stimulated DNA synthesis in both epithelial and adjacent stromal cells in vivo. Distribution of PRLR mRNA in mammary epithelium was homogeneous before puberty and heterogeneous during puberty, gestation, and early lactation. A mutual role for PRLR and PR was suggested wherein PR mRNA increased beyond 6 weeks to maximal levels during puberty and gestation then became undetectable during lactation. In situ hybridization revealed that PR mRNA distribution is homogeneous in the ductal epithelium before 6 weeks and heterogenous during puberty and gestation and that PRLR and PR are similarly distributed in the ductal epithelium. Neither hormone stimulated DNA synthesis in mammary glands of ovariectomized females while their effects interacted markedly. These results demonstrate differential PRLR transcription by epithelial and stromal cells and a similar distribution of PRLR and PR that may facilitate the interaction between P and PRL during ductal branching in the mammary gland.

Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We show that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and β-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.

Prolactin: the forgotten hormone of human breast cancer.

Prolactin (PRL) is both a mitogen and a differentiating agent in the mammary gland. It has been shown to be involved in mammary cancer development in rodents, but in human breast cancer, its role has long been overlooked. Three criteria are applied to demonstrate PRLs involvement in this disease: (1) PRL receptors are present in human breast cancer cells, (2) human breast cancer cells in culture respond to PRL as a mitogen, and (3) PRL is synthesized by human breast cancer cells and inhibition of the binding of PRL to its receptors inhibits cell growth.

Prolactin-induced expression of vascular endothelial growth factor via Egr-1

Angiogenesis is a dynamic process regulated by both local and systemic factors. Among these is vascular endothelial growth factor (VEGF), a potent effector of angiogenesis and vascular permeability. Previously we showed that VEGF is temporally and spatially regulated in the mouse mammary gland during development and lactation. Given the functions of prolactin (PRL) during these stages and the supporting role of the vasculature, we investigated the regulation of VEGF by PRL. Treatment of HC11 mouse mammary epithelial and Nb2 rat lymphoma cells with PRL induced VEGF expression. Deletion and mutation analysis identified a GC-rich region in the proximal region of the VEGF promoter that constitutively bound Sp1 and PRL-induced Egr-1. These sites conferred PRL-responsiveness leading to increased VEGF transcription. The induction of VEGF by PRL was PRL receptor-, Jak2- and MAP kinase kinase-dependent. Our results indicate that PRL induces VEGF expression through Egr-1, and implicates VEGF as an intermediary of PRL-regulated angiogenesis.