Paracrine interactions between primary human macrophages and human fibroblasts enhance murine mammary gland humanization in vivo.

INTRODUCTION: Macrophages comprise an essential component of the mammary microenvironment necessary for normal gland development. However, there is no viable in vivo model to study their role in normal human breast function. We hypothesized that adding primary human macrophages to the murine mammary gland would enhance and provide a novel approach to examine immune-stromal cell interactions during the humanization process. METHODS: Primary human macrophages, in the presence or absence of ectopic estrogen stimulation, were used to humanize mouse mammary glands. Mechanisms of enhanced humanization were identified by cytokine/chemokine ELISAs, zymography, western analysis, invasion and proliferation assays; results were confirmed with immunohistological analysis. RESULTS: The combined treatment of macrophages and estrogen stimulation significantly enhanced the percentage of the total gland humanized and the engraftment/outgrowth success rate. Timecourse analysis revealed the disappearance of the human macrophages by two weeks post-injection, suggesting that the improved overall growth and invasiveness of the fibroblasts provided a larger stromal bed for epithelial cell proliferation and structure formation. Confirming their promotion of fibroblasts humanization, estrogen-stimulated macrophages significantly enhanced fibroblast proliferation and invasion in vitro, as well as significantly increased proliferating cell nuclear antigen (PCNA) positive cells in humanized glands. Cytokine/chemokine ELISAs, zymography and western analyses identified TNFα and MMP9 as potential mechanisms by which estrogen-stimulated macrophages enhanced humanization. Specific inhibitors to TNFα and MMP9 validated the effects of these molecules on fibroblast behavior in vitro, as well as by immunohistochemical analysis of humanized glands for human-specific MMP9 expression. Lastly, glands humanized with macrophages had enhanced engraftment and tumor growth compared to glands humanized with fibroblasts alone. CONCLUSIONS: Herein, we demonstrate intricate immune and stromal cell paracrine interactions in a humanized in vivo model system. We confirmed our in vivo results with in vitro analyses, highlighting the value of this model to interchangeably substantiate in vitro and in vivo results. It is critical to understand the signaling networks that drive paracrine cell interactions, for tumor cells exploit these signaling mechanisms to support their growth and invasive properties. This report presents a dynamic in vivo model to study primary human immune/fibroblast/epithelial interactions and to advance our knowledge of the stromal-derived signals that promote tumorigenesis.

Hornerin, an S100 family protein, is functional in breast cells and aberrantly expressed in breast cancer.

BACKGROUND: Recent evidence suggests an emerging role for S100 protein in breast cancer and tumor progression. These ubiquitous proteins are involved in numerous normal and pathological cell functions including inflammatory and immune responses, Ca(2+) homeostasis, the dynamics of cytoskeleton constituents, as well as cell proliferation, differentiation, and death. Our previous proteomic analysis demonstrated the presence of hornerin, an S100 family member, in breast tissue and extracellular matrix. Hornerin has been reported in healthy skin as well as psoriatic and regenerating skin after wound healing, suggesting a role in inflammatory/immune response or proliferation. In the present study we investigated hornerin's potential role in normal breast cells and breast cancer. METHODS: The expression levels and localization of hornerin in human breast tissue, breast tumor biopsies, primary breast cells and breast cancer cell lines, as well as murine mammary tissue were measured via immunohistochemistry, western blot analysis and PCR. Antibodies were developed against the N- and C-terminus of the protein for detection of proteolytic fragments and their specific subcellular localization via fluorescent immunocytochemisty. Lastly, cells were treated with H(2)O(2) to detect changes in hornerin expression during induction of apoptosis/necrosis. RESULTS: Breast epithelial cells and stromal fibroblasts and macrophages express hornerin and show unique regulation of expression during distinct phases of mammary development. Furthermore, hornerin expression is decreased in invasive ductal carcinomas compared to invasive lobular carcinomas and less aggressive breast carcinoma phenotypes, and cellular expression of hornerin is altered during induction of apoptosis. Finally, we demonstrate the presence of post-translational fragments that display differential subcellular localization. CONCLUSIONS: Our data opens new possibilities for hornerin and its proteolytic fragments in the control of mammary cell function and breast cancer.

Expression of Notch receptors, ligands, and target genes during development of the mouse mammary gland.

Notch genes play a critical role in mammary gland growth, development and tumorigenesis. In the present study, we have quantitatively determined the levels and mRNA expression patterns of the Notch receptor genes, their ligands and target genes in the postnatal mouse mammary gland. The steady state levels of Notch3 mRNA are the highest among receptor genes, Jagged1 and Dll3 mRNA levels are the highest among ligand genes and Hey2 mRNA levels are highest among expressed Hes/Hey target genes analyzed during different stages of postnatal mammary gland development. Using an immunohistochemical approach with antibodies specific for each Notch receptor, we show that Notch proteins are temporally regulated in mammary epithelial cells during normal mammary gland development in the FVB/N mouse. The loss of ovarian hormones is associated with changes in the levels of Notch receptor mRNAs (Notch2 higher and Notch3 lower) and ligand mRNAs (Dll1 and Dll4 are higher, whereas Dll3 and Jagged1 are lower) in the mammary gland of ovariectomized mice compared to intact mice. These data define expression of the Notch ligand/receptor system throughout development of the mouse mammary gland and help set the stage for genetic analysis of Notch in this context.

Genetic variation in PRL and PRLR, and relationships with serum prolactin levels and breast cancer risk: results from a population-based case-control study in Poland.

INTRODUCTION: Studies suggest that high circulating levels of prolactin increase breast cancer risk. It is unclear if genetic variations in prolactin (PRL) or prolactin receptor (PRLR) genes also play a role. Thus, we examined the relationship between single nucleotide polymorphisms (SNPs) in PRL and PRLR, serum prolactin levels and breast cancer risk in a population-based case-control study. METHODS: We genotyped 8 PRL and 20 PRLR tag SNPs in 1965 breast cancer cases and 2229 matched controls, aged 20-74, and living in Warsaw or Lódz, Poland. Serum prolactin levels were measured by immunoassay in a subset of 773 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) for genotype associations with breast cancer risk were estimated using unconditional logistic regression, adjusted for age and study site. Geometric mean prolactin levels were estimated using linear regression models adjusted for age, study site, blood collection time, and menstrual cycle day (premenopausal women). RESULTS: Three SNPs were associated with breast cancer risk: in premenopausal women, PRLR rs249537 (T vs. C per-allele OR 1.39, 95% CI 1.07 - 1.80, P = 0.01); and in postmenopausal women, PRLR rs7718468 (C vs. T per-allele OR 1.16, 95% CI 1.03 - 1.30, P = 0.01) and PRLR rs13436213 (A vs. G per-allele OR 1.13 95% CI 1.01 - 1.26, P = 0.04). However, mean serum prolactin levels for these SNPs did not vary by genotype (P-trend > 0.05). Other SNPs were associated with serum prolactin levels: PRLR rs62355518 (P-trend = 0.01), PRLR rs10941235 (P-trend = 0.01), PRLR rs1610218 (P-trend = 0.01), PRLR rs34024951 (P-trend = 0.02), and PRLR rs9292575 (P-trend = 0.03) in premenopausal controls and PRL rs849872 (P-trend = 0.01) in postmenopausal controls. CONCLUSIONS: Our data provide limited support for an association between common variations in PRLR and breast cancer risk. Altered serum prolactin levels were not associated with breast cancer risk-associated variants, suggesting that common genetic variation is not a strong predictor of prolactin-associated breast cancer risk in this population.

Cripto-1 is a cell surface marker for a tumorigenic, undifferentiated subpopulation in human embryonal carcinoma cells.

Deregulation of stem cells is associated with the generation and progression of malignant tumors. In addition, genes that are associated with early embryogenesis are frequently expressed in cancer. Cripto-1 (CR-1), a glycosylphosphatidylinositol-linked glycoprotein, is expressed during early embryogenesis and in various human carcinomas. We demonstrated that human embryonal carcinoma (EC) cells are heterogeneous for CR-1 expression and consist of two distinct subpopulations: a CR-1(High) and a CR-1(Low) population. By segregating CR-1(High) and CR-1(Low) populations of NTERA2/D1 EC cells by fluorescence-activated cell sorting, we demonstrated that CR-1(High) cells were more tumorigenic than CR-1(Low) cells by an in vitro tumor sphere assay and by in vivo xenograft formation. The CR-1(High) population was enriched in mRNA expression for the pluripotent embryonic stem (ES) cell genes Oct4, Sox2, and Nanog. CR-1 expression in NTERA2/D1 cells was regulated by a Smad2/3-dependent autocrine loop, by the ES cell-related transcription factors Oct4/Nanog, and partially by the DNA methylation status of the promoter region. These results demonstrate that CR-1 expression is enriched in an undifferentiated, tumorigenic subpopulation and is regulated by key regulators of pluripotent stem cells.

Local regulation of human breast xenograft models.

Breast cancer studies implant human cancer cells under the renal capsule, subcutaneously, or orthotopically and often use estrogen supplementation and immune suppressants (etoposide) in xenograft mouse models. However, cell behavior is significantly impacted by signals from the local microenvironment. Therefore, we investigated how the combinatorial effect of the location of injection and procedural differences affected xenograft characteristics. Patient-derived breast cancer cells were injected into mouse abdominal or thoracic mammary glands +/- estrogen and/or etoposide pretreatment. Abdominal xenografts had increased tumor incidence and volume, and decreased latency (P < 0.001) compared to thoracic tumors. No statistically significant difference in tumor volume was found in abdominal xenografts treated +/- estrogen or etoposide; however, etoposide suppressed tumor volume in thoracic xenografts (P < 0.02). The combination of estrogen and etoposide significantly decreased tumor incidence in both sites. In addition, mice treated +/- estradiol were injected orthotopically or subcutaneously with well-characterized breast cancer cell lines (MCF7, ZR75-1, MDA MB-231, or MCF10Ca1h). Orthotopic injection increased tumor volume; growth varied with estrogen supplementation. Location also altered methylation status of several breast cancer-related gene promoters. Lastly, vascularization of orthotopic tumors was significantly enhanced compared to subcutaneous tumors. These data suggest that optimal xenograft success occurs with orthotopic abdominal injections and illustrate molecular details of the compelling influence of the local microenvironment on in vivo models.

Transforming acidic coiled-coil protein-3 (Tacc3) acts as a negative regulator of Notch signaling through binding to CDC10/Ankyrin repeats.

We have identified the transforming acidic coiled-coil protein-3 (Tacc3) as a binding partner for Notch4/Int3 and were able to show that it binds to the intracellular domain (ICD) of all members of the Notch receptor family. Members of the Tacc family reside at the centrosomes and associates with microtubules. Recent studies suggest that Tacc3 also contributes to the regulation of gene transcription. Tacc3 specifically interacts with the Notch4/Int3 CDC10/Ankyrin repeats and to a lesser extent, with residues C-terminal to these repeats in the ICD. Dual label immunofluorescence of mouse mammary tissue shows Tacc3 co-localizes with the Notch3 ICD. Co-immunoprecipitation of endogenous Notch and Tacc3 proteins from NIH3T3 cell extracts, lung and mammary gland confirms that these two proteins interact under physiological conditions. In addition, knock down of Tacc3 in NIH3T3 cells leads to the up-regulation of Hey2, a target gene for Notch signaling. The affinity of Tacc3 binding to Notch4/Int3 ICD is similar to that between Rbpj and Notch4/Int3 ICD. Notch4/Int3 ICD-Tacc3 interaction results in the inhibition of transcription from a Hes1-Luciferase reporter vector in COS-1 cells. The inhibition was reversed in these cells by increasing the levels of Rbpj. Taken together, these results suggest that Tacc3 is a negative regulator of the Notch signaling pathway.

The normal breast microenvironment of premenopausal women differentially influences the behavior of breast cancer cells in vitro and in vivo.

BACKGROUND: Breast cancer studies frequently focus on the role of the tumor microenvironment in the promotion of cancer; however, the influence of the normal breast microenvironment on cancer cells remains relatively unknown. To investigate the role of the normal breast microenvironment on breast cancer cell tumorigenicity, we examined whether extracellular matrix molecules (ECM) derived from premenopausal African-American (AA) or Caucasian-American (CAU) breast tissue would affect the tumorigenicity of cancer cells in vitro and in vivo. We chose these two populations because of the well documented predisposition of AA women to develop aggressive, highly metastatic breast cancer compared to CAU women. METHODS: The effects of primary breast fibroblasts on tumorigenicity were analyzed via real-time PCR arrays and mouse xenograft models. Whole breast ECM was isolated, analyzed via zymography, and its effects on breast cancer cell aggressiveness were tested in vitro via soft agar and invasion assays, and in vivo via xenograft models. Breast ECM and hormone metabolites were analyzed via mass spectrometry. RESULTS: Mouse mammary glands humanized with premenopausal CAU fibroblasts and injected with primary breast cancer cells developed significantly larger tumors compared to AA humanized glands. Examination of 164 ECM molecules and cytokines from CAU-derived fibroblasts demonstrated a differentially regulated set of ECM proteins and increased cytokine expression. Whole breast ECM was isolated; invasion and soft agar assays demonstrated that estrogen receptor (ER)-, progesterone receptor (PR)/PR- cells were significantly more aggressive when in contact with AA ECM, as were ER+/PR+ cells with CAU ECM. Using zymography, protease activity was comparatively upregulated in CAU ECM. In xenograft models, CAU ECM significantly increased the tumorigenicity of ER+/PR+ cells and enhanced metastases. Mass spectrometry analysis of ECM proteins showed that only 1,759 of approximately 8,000 identified were in common. In the AA dataset, proteins associated with breast cancer were primarily related to tumorigenesis/neoplasia, while CAU unique proteins were involved with growth/metastasis. Using a novel mass spectrometry method, 17 biologically active hormones were measured; estradiol, estriol and 2-methoxyestrone were significantly higher in CAU breast tissue. CONCLUSIONS: This study details normal premenopausal breast tissue composition, delineates potential mechanisms for breast cancer development, and provides data for further investigation into the role of the microenvironment in cancer disparities.

Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies.

BACKGROUND: Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR) are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. METHODS: Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. RESULTS: We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76%) of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72%) and 27% had only low levels of expression. CONCLUSIONS: Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in treatment of this disease by providing new reagents to study the protein expression of the human PRLR.

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

INTRODUCTION: The 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. METHODS: Breast 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. RESULTS: CD24 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. CONCLUSIONS: Our 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.

Identification of a regulatory loop for the synthesis of neurosteroids: a steroidogenic acute regulatory protein-dependent mechanism involving hypothalamic-pituitary-gonadal axis receptors.

Brain sex steroids are derived from both peripheral (primarily gonadal) and local (neurosteroids) sources and are crucial for neurogenesis, neural differentiation and neural function. The mechanism(s) regulating the production of neurosteroids is not understood. To determine whether hypothalamic-pituitary-gonadal axis components previously detected in the extra-hypothalamic brain comprise a feedback loop to regulate neuro-sex steroid (NSS) production, we assessed dynamic changes in expression patterns of steroidogenic acute regulatory (StAR) protein, a key regulator of steroidogenesis, and key hypothalamic-pituitary-gonadal endocrine receptors, by modulating peripheral sex hormone levels in female mice. Ovariectomy (OVX; high serum gonadotropins, low serum sex steroids) had a differential effect on StAR protein levels in the extrahypothalamic brain; increasing the 30- and 32-kDa variants but decreasing the 37-kDa variant and is indicative of cholesterol transport into mitochondria for steroidogenesis. Treatment of OVX animals with E(2), P(4), or E(2) + P(4) for 3 days, which decreases OVX-induced increases in GnRH/gonadotropin production, reversed this pattern. Suppression of gonadotropin levels in OVX mice using the GnRH agonist leuprolide acetate inhibited the processing of the 37-kDa StAR protein into the 30-kDa StAR protein, confirming that the differential processing of brain StAR protein is regulated by gonadotropins. OVX dramatically suppressed extra-hypothalamic brain gonadotropin-releasing hormone 1 receptor expression, and was further suppressed in E(2)- or P(4)-treated OVX mice. Together, these data indicate the existence of endocrine and autocrine/paracrine feedback loops that regulate NSS synthesis. Further delineation of these feedback loops that regulate NSS production will aid in developing therapies to maintain brain sex steroid levels and cognition.

Historical perspectives of prolactin and growth hormone as mammogens, lactogens and galactagogues--agog for the future!

Around 80 years ago researchers first established that the pituitary gland regulates mammary gland function as demonstrated by the ability of its extracts to promote both mammogenesis and lactogenesis in animal models. Little did they realize that in fact two hormones, prolactin (PRL) and growth hormone (GH), were contributing to these effects. By the mid 1930s PRL had been purified as a distinct lactogen, while the galactopoietic effect of GH was confirmed after its purification in the 1940s. Interest in these hormones initially centered about their potential for increasing milk production, while in the latter half of the twentieth century it became obvious that these hormones also had the potential to influence mammary cancer development. During the past 50 years large strides have been made into understanding how these hormones signal to, and within, cells of the mammary gland, paralleling rapid developments in the fields of cellular and molecular biology. In compiling this review we have summarized the progress that has been made to date regarding roles for these hormones in the mammary gland, with a goal of ensuring that some of the seminal literature is not diluted or forgotten. In doing so it is clear that there are lessons to be learned from past experiences, where new methods and technologies will continue to present exciting new opportunities to revisit lingering questions regarding these fascinating hormones and this fascinating organ.

Estrogen stimulates transcription from the human prolactin distal promoter through AP1 and estrogen responsive elements in T47D human breast cancer cells.

Human prolactin (hPRL) is a pleiotropic and versatile hormone that exercises more than 300 biological activities through binding to its cognate receptors. Recently, multiple studies have implicated hPRL in the development of human breast cancer. As a target of hPRL, both normal and neoplastic human breast cells also synthesize and secrete hPRL, which therefore establishes an autocrine/paracrine action loop in the mammary gland. In contrast to the extensive studies of regulation of hPRL expression in the pituitary gland, regulation of hPRL in mammary tissue and human breast cancer cells has not been extensively addressed. Extrapituitary PRL expression is primarily regulated by a distal promoter located 5.8 kb upstream to the pituitary promoter. As a result of alternative promoter usage, extrapituitary PRL is regulated by different signalling pathways and different hormones, cytokines or neuropeptides compared to regulation in the pituitary. Here, we present evidence that shows estrogen directly induces hPRL gene expression in T47D human breast cancer cells. We have identified a functional, non-canonical estrogen responsive element (ERE) and an AP1 site located in the hPRL distal promoter. Gel shift and chromatin immunoprecipitation assays demonstrated that both estrogen receptor (ER)alpha and ERbeta directly bind to the ERE. However, only ERalpha interacts with AP1 proteins that bind to the AP1 site in the hPRL distal promoter. Promoter-reporter gene studies demonstrate that both ERE and AP1 sites are required for full induction of the promoter activity by estradiol. Our studies suggest that the interactions between estrogens, ERs, the ERE and AP1 transcription factors in regulation of autocrine/paracrine PRL in the human breast may be critical for oncogenesis and may contribute to progression of breast cancer.

S179D prolactin increases vitamin D receptor and p21 through up-regulation of short 1b prolactin receptor in human prostate cancer cells.

In this study, we further investigated the mechanisms by which pseudophosphorylated prolactin (S179D PRL) inhibits the growth of human prostate cancer cells. When treated with S179D PRL for 3 days, LnCAP cells responded by increasing expression of the vitamin D receptor (VDR) and the cell cycle regulatory molecule, p21, whereas PC3 and DU145 cells did not. After 5 days of treatment, both PC3 and DU145 cells responded. Untreated LnCAP cells express the short 1b form (SF1b) of the human prolactin receptor, but DU145 and PC3 cells express only low amounts of this receptor until elevated by treatment with S179D PRL. DU145 and PC3 cells become sensitive to the negative effects of S179D PRL on cell number after induction of the SF1b. Transfection of either SF1b or SF1a into PC3 or DU145 cells made them sensitive to S179D PRL in the 3-day time frame, a finding that was not duplicated by transfection with the long form of the receptor. Treatment of LnCAP cells with S179D PRL increased long-term activation of extracellular signal-regulated kinase 1/2 (ERK1/2). This did not occur in PC3 and DU145 cells until transfection with SF1a/SF1b. Blockade of ERK signaling eliminated S179D PRL-stimulated expression of the VDR and p21 in LnCAP cells and transfected PC3 and DU145 cells. We conclude that initiation of alternative splicing to produce SF1b, and subsequent altered signaling, contribute to the growth inhibitory mechanisms of S179D PRL. This is the first indication of a role for short prolactin receptors in the regulation of cell proliferation.

Effects of raloxifene on circulating prolactin and estradiol levels in premenopausal women at high risk for developing breast cancer.

BACKGROUND: Prolactin is a peptide hormone necessary for normal breast development that may contribute to breast tumorigenesis. Estrogen is a significant positive regulator of prolactin synthesis; therefore, raloxifene, a selective estrogen receptor modulator under study as a breast cancer prevention agent, may modulate both estradiol and prolactin levels by inhibiting estradiol from binding to its receptor. METHODS: Premenopausal women at increased risk for invasive breast cancer participated in a pilot chemoprevention trial and were given 60 mg raloxifene daily for 24 months. Fasting serum samples collected at baseline and after 12 months on drug were used to measure circulating prolactin, estradiol, and sex hormone binding globulin (SHBG) levels. RESULTS: Of the 27 subjects who completed 12 months of raloxifene, 23 had paired prolactin samples, and 20 had paired estradiol and SHBG samples. Prolactin levels did not significantly change with raloxifene treatment, but SHBG levels increased (mean change = 7.3 nmol/L; P = 0.0001; 95% confidence interval, 3.9-10.7). Estradiol (mean change = 42 pg/mL; P = 0.048; 95% confidence interval, 1-84 pg/mL) levels were elevated when comparing 15 of the 20 women with paired estradiol measurements who also had both of these samples taken during the early follicular phase of the menstrual cycle. CONCLUSIONS: This report is the first to examine the long-term effects of raloxifene on prolactin, estradiol, and SHBG levels in premenopausal women who are also at increased risk for developing invasive breast cancer. Raloxifene had no significant effect on prolactin levels but did increase estradiol and SHBG measurements.

Unmodified prolactin (PRL) and S179D PRL-initiated bioluminescence resonance energy transfer between homo- and hetero-pairs of long and short human PRL receptors in living human cells.

We have used bioluminescence resonance energy transfer (BRET) to examine the interaction between human prolactins (PRLs) and the long (LF) and two short isoforms (SF1a and SF1b) of the human PRL receptor in living cells. cDNA sequences encoding the LF, SF1a, and SF1b were subcloned into codon-humanized vectors containing cDNAs for either Renilla reniformis luciferase (Rluc) or a green fluorescent protein (GFP(2)) with a 12- or 13-amino acid linker connecting the parts of the fusion proteins. Transfection into human embryonic kidney 293 cells demonstrated maintained function of Rluc and GFP(2) when linked to the receptors, and confocal microscopy demonstrated the localization of tagged receptors in the plasma membrane by 48 h after transfection. All three tagged receptors transduced a signal, with the LF and SF1a stimulating, and SF1b inhibiting, promoter activity of an approximately 2.4-kb beta-casein-luc construct. Both unmodified PRL (U-PRL) and the molecular mimic of phosphorylated PRL, S179D PRL, induced BRET with all combinations of long and short receptor isoforms except SF1a plus SF1b. No BRET was observed with the site two-inactive mutant, G129R PRL. This is the first demonstration, 1) that species homologous PRL promotes both homo- and hetero-interaction of most long and short PRLR pairs in living cells, 2) that both U-PRL and S179D PRL are active in this regard, and 3) that there is some aspect of SF1a-SF1b structure that prevents this particular hetero-receptor pairing. In addition, we conclude that preferential pairing of different receptor isoforms is not the explanation for the different signaling initiated by U-PRL and S179D PRL.

Beta protein 1 homeoprotein induces cell growth and estrogen-independent tumorigenesis by binding to the estrogen receptor in breast cancer.

Expression of Beta Protein 1 (BP1), a homeotic transcription factor, increases during breast cancer progression and may be associated with tumor aggressiveness. In our present work, we investigate the influence of BP1 on breast tumor formation and size in vitro and in vivo. Cells overexpressing BP1 showed higher viability when grown in the absence of serum (p < 0.05), greater invasive potential (p < 0.05) and formed larger colonies (p < 0.004) compared with the controls. To determine the influence of BP1 overexpression on tumor characteristics, MCF-7 cells transfected with either empty vector (V1) or overexpressor plasmids (O2 and O4) were injected into the fat pads of athymic nude mice. Tumors grew larger in mice receiving O2 or O4 cells than in mice receiving V1 cells. Moreover, BP1 mRNA expression levels were positively correlated with tumor size in patients (p = 0.01). Interestingly, 20% of mice injected with O2 or O4 cells developed tumors in the absence of estrogen, while no mice receiving V1 cells developed tumors. Several mechanisms of estrogen independent tumor formation related to BP1 were established. These data are consistent with the fact that expression of breast cancer anti-estrogen resistance 1 (BCAR1) was increased in O2 compared to V1 cells (p < 0.01). Importantly, O2 cells exhibited increased proliferation when treated with tamoxifen, while V1 cells showed growth inhibition. Overall, BP1 overexpresssion in MCF-7 breast cancer cells leads to increased cell growth, estrogen-independent tumor formation, and increased proliferation. These findings suggest that BP1 may be an important biomarker and therapeutic target in ER positive breast cancer.

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.

Local insulin-like growth factor-II mediates prolactin-induced mammary gland development.

Prolactin (PRL) is a major determinant of mammary epithelial cell proliferation during alveolar development in sexually mature and pregnant mice. To date, it has not been clear whether PRL effects these responses alone or by also invoking the action of autocrine/paracrine growth factors. In this study, we provide evidence that part of the effect of PRL on mammary gland growth is mediated by IGF-II. During sexual maturity and in early pregnancy, the level of IGF-II mRNA in the mammary gland was increased concurrent with increased PRL receptor expression. The level of IGF-II mRNA was reduced in mammary tissue from PRL receptor-/- mice during early pregnancy, and explants of mouse mammary gland and HC11 mammary epithelial cells both increased their expression of IGF-II after exposure to PRL in vitro. These findings coincided with the demonstration that IGF-II stimulated alveolar development in mammary glands in whole organ culture. PRL was most efficacious in stimulating IGF-II gene transcription from promoter 3 of the mouse IGF-II gene in vitro. Insight into the mechanism by which PRL induced IGF-II expression was provided by the fact that it was blocked by the Jak2 inhibitor AG490 and the MAPK inhibitor PD98059. Finally, induction of insulin receptor substrate (IRS)-1 in the mammary glands of PRL-treated mice and induction of IRS-1 and IRS-2 after treatment with PRL plus progesterone indicates that these molecules are induced by PRL as potential signaling intermediates downstream from IGF-I/insulin receptors. Together, these data demonstrate a role for IGF-II as a mediator of PRL action in the mouse mammary gland during ductal branching and alveolar development.

Disruption of steroid and prolactin receptor patterning in the mammary gland correlates with a block in lobuloalveolar development.

Targeted deletion of the bZIP transcription factor, CCAAT/enhancer binding protein-beta (C/EBPbeta), was shown previously to result in aberrant ductal morphogenesis and decreased lobuloalveolar development, accompanied by an altered pattern of progesterone receptor (PR) expression. Here, similar changes in the level and pattern of prolactin receptor (PrlR) expression were observed while screening for differentially expressed genes in C/EBPbeta(null) mice. PR patterning was also altered in PrlR(null) mice, as well as in mammary tissue transplants from both PrlR(null) and signal transducer and activator of transcription (Stat) 5a/b-deficient mice, with concomitant defects in hormone-induced proliferation. Down-regulation of PR and activation of Stat5 phosphorylation were seen after estrogen and progesterone treatment in both C/EBPbeta(null) and wild-type mice, indicating that these signaling pathways were functional, despite the failure of steroid hormones to induce proliferation. IGF binding protein-5, IGF-II, and insulin receptor substrate-1 all displayed altered patterns and levels of expression in C/EBPbeta(null) mice, suggestive of a change in the IGF signaling axis. In addition, small proline-rich protein (SPRR2A), a marker of epidermal differentiation, and keratin 6 were misexpressed in the mammary epithelium of C/EBPbeta(null) mice. Together, these data suggest that C/EBPbeta is a master regulator of mammary epithelial cell fate and that the correct spatial pattern of PR and PrlR expression is a critical determinant of hormone-regulated cell proliferation.