A Coaching-Based Leadership Program for Women Postdoctoral Fellows at the National Cancer Institute that Cultivates Self-confidence and Persistence in STEMM.

Despite making strides in gender equality, women in Science, Technology, Engineering, Mathematics, and Medicine (STEMM) continue to face a persistent underrepresentation in leadership roles. In an effort to reverse this trend, the National Cancer Institute created the Sallie Rosen Kaplan (SRK) Postdoctoral Fellowship, a year-long coaching-based leadership training program. The SRK program aims to empower women to explore careers across a broad range of fields, including academia, industry, and government, and to excel in leadership positions in those fields. Analyzing a decade of data from 111 participants, we describe the positive impact of the SRK program on participant's self-reported capabilities. Increased self-confidence, improved time management and work/life balance, enhanced goal-setting and attainment skills, and strengthened communication and relationship-building abilities are highlighted as statistically significant outcomes. Moreover, the program's emphasis on coaching, mentorship, peer cohort support, and building lasting professional relationships also contributed to high ratings for satisfaction and value of the program. Successful programs like SRK can serve as a model for institutions striving to close gender gaps in leadership.

Building Capacity for Global Cancer Research: Existing Opportunities and Future Directions.

Cancer incidence and mortality are increasing in low- and middle-income countries (LMICs), where more than 75% of global cancer burden will occur by the year 2040. The primary drivers of cancer morbidity and mortality in LMICs are environmental and behavioral risk factors, inadequate prevention and early detection services, presence of comorbidities, and poor access to treatment and palliation. These same drivers also contribute to marked cancer health disparities in high-income countries. Studying cancer in LMICs provides opportunities to better understand and address these drivers to benefit populations worldwide, and reflecting this, global oncology as an academic discipline has grown substantially in recent years. However, sustaining this growth requires a uniquely trained workforce with the skills to pursue relevant, rigorous, and equitable global oncology research. Despite this need, dedicated global cancer research training programs remain somewhat nascent and uncoordinated. In this paper, we discuss efforts to address these gaps in global cancer research training at the US National Institutes of Health.

Mechanisms of transient signaling via short and long prolactin receptor isoforms in female and male sensory neurons.

Prolactin (PRL) regulates activity of nociceptors and causes hyperalgesia in pain conditions. PRL enhances nociceptive responses by rapidly modulating channels in nociceptors. The molecular mechanisms underlying PRL-induced transient signaling in neurons are not well understood. Here we use a variety of cell biology and pharmacological approaches to show that PRL transiently enhanced capsaicin-evoked responses involve protein kinase C ε (PKCε) or phosphatidylinositol 3-kinase (PI3K) pathways in female rat trigeminal (TG) neurons. We next reconstituted PRL-induced signaling in a heterologous expression system and TG neurons from PRL receptor (PRLR)-null mutant mice by expressing rat PRLR-long isoform (PRLR-L), PRLR-short isoform (PRLR-S), or a mix of both. Results show that PRLR-S, but not PRLR-L, is capable of mediating PRL-induced transient enhancement of capsaicin responses in both male and female TG neurons. However, co-expression of PRLR-L with PRLR-S (1:1 ratio) leads to the inhibition of the transient PRL actions. Co-expression of PRLR-L deletion mutants with PRLR-S indicated that the cytoplasmic site adjacent to the trans-membrane domain of PRLR-L was responsible for inhibitory effects of PRLR-L. Furthermore, in situ hybridization and immunohistochemistry data indicate that in normal conditions, PRLR-L is expressed mainly in glia with little expression in rat sensory neurons (3-5%) and human nerves. The predominant PRLR form in TG neurons/nerves from rats and humans is PRLR-S. Altogether, PRL-induced transient signaling in sensory neurons is governed by PI3K or PKCε, mediated via the PRLR-S isoform, and transient effects mediated by PRLR-S are inhibited by presence of PRLR-L in these cells.

Outcome evaluation of the National Cancer Institute career development awards program.

The National Cancer Institute (NCI) career development (K) awards program supports investigators to develop their cancer research programs and achieve independence. The NCI Center for Cancer Training conducted a K program evaluation by analyzing outcomes of awardees and individuals who applied to the program but were not funded. The evaluation covered seven NCI mechanisms (K01, K07, K08, K11, K22, K23, and K25) between 1980 and 2008. Descriptive statistics and regression modeling were performed on the full cohort (n = 2,893 individuals, 4,081 K applications) and a comparison cohort described herein. K awardees proportionately received more subsequent NIH grants and authored more publications, and time to first R01 grant was unaffected. Of those not pursuing research, K awardees were more likely to participate in activities signaling continued scientific engagement. The NCI K program had a positive impact, not only on participants' biomedical research careers but also on achieving outcomes significant to the scientific enterprise.

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.

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.

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.

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.

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.

Persistent organic pollutants and obesity: are they potential mechanisms for breast cancer promotion?

Dietary ingestion of persistent organic pollutants (POPs) is correlated with the development of obesity. Obesity alters metabolism, induces an inflammatory tissue microenvironment, and is also linked to diabetes and breast cancer risk/promotion of the disease. However, no direct evidence exists with regard to the correlation among all three of these factors (POPs, obesity, and breast cancer). Herein, we present results from current correlative studies indicating a causal link between POP exposure through diet and their bioaccumulation in adipose tissue that promotes the development of obesity and ultimately influences breast cancer development and/or progression. Furthermore, as endocrine disruptors, POPs could interfere with hormonally responsive tissue functions causing dysregulation of hormone signaling and cell function. This review highlights the critical need for advanced in vitro and in vivo model systems to elucidate the complex relationship among obesity, POPs, and breast cancer, and, more importantly, to delineate their multifaceted molecular, cellular, and biochemical mechanisms. Comprehensive in vitro and in vivo studies directly testing the observed correlations as well as detailing their molecular mechanisms are vital to cancer research and, ultimately, public health.

Progesterone receptor activates Msx2 expression by downregulating TNAP/Akp2 and activating the Bmp pathway in EpH4 mouse mammary epithelial cells.

Previously we demonstrated that EpH4 mouse mammary epithelial cells induced the homeobox transcription factor Msx2 either when transfected with the progesterone receptor (PR) or when treated with Bmp2/4. Msx2 upregulation was unaffected by Wnt inhibitors s-FRP or Dkk1, but was inhibited by the Bmp antagonist Noggin. We therefore hypothesized that PR signaling to Msx2 acts through the Bmp receptor pathway. Herein, we confirm that transcripts for Alk2/ActR1A, a non-canonical BmpR Type I, are upregulated in mammary epithelial cells overexpressing PR (EpH4-PR). Increased phosphorylation of Smads 1,5, 8, known substrates for Alk2 and other BmpR Type I proteins, was observed as was their translocation to the nucleus in EpH4-PR cells. Analysis also showed that Tissue Non-Specific Alkaline Phosphatase (TNAP/Akp2) was also found to be downregulated in EpH4-PR cells. When an Akp2 promoter-reporter construct containing a ½PRE site was transfected into EpH4-PR cells, its expression was downregulated. Moreover, siRNA mediated knockdown of Akp2 increased both Alk2 and Msx2 expression. Collectively these data suggest that PR inhibition of Akp2 results in increased Alk2 activity, increased phosphorylation of Smads 1,5,8, and ultimately upregulation of Msx2. These studies imply that re-activation of the Akp2 gene could be helpful in downregulating aberrant Msx2 expression in PR+ breast cancers.

Effects of age and parity on mammary gland lesions and progenitor cells in the FVB/N-RC mice.

The FVB/N mouse strain is extensively used in the development of animal models for breast cancer research. Recently it has been reported that the aging FVB/N mice develop spontaneous mammary lesions and tumors accompanied with abnormalities in the pituitary glands. These observations have a great impact on the mouse models of human breast cancer. We have developed a population of inbred FVB/N mice (designated FVB/N-RC) that have been genetically isolated for 20 years. To study the effects of age and parity on abnormalities of the mammary glands of FVB/N-RC mice, twenty-five nulliparous and multiparous (3-4 pregnancies) females were euthanized at 16-22 months of age. Examination of the mammary glands did not reveal macroscopic evidence of mammary gland tumors in either aged-nulliparous or multiparous FVB/N-RC mice (0/25). However, histological analysis of the mammary glands showed rare focal nodules of squamous changes in 2 of the aged multiparous mice. Mammary gland hyperplasia was detected in 8% and 71% of the aged-nulliparous and aged-multiparous mice, respectively. Epithelial contents and serum levels of triiodothyronine were significantly higher in the experimental groups than the 14-wk-old control mice. Immuno-histochemical staining of the pituitary gland pars distalis showed no difference in prolactin staining between the control and the aged mice. Tissue transplant and dilution studies showed no effect of age and/or parity on the ability of putative progenitor cells present among the injected mammary cells to repopulate a cleared fat pad and develop a full mammary gland outgrowth. This FVB/N-RC mouse substrain is suitable to develop mouse models for breast cancer.

Progesterone induces expression of the prolactin receptor gene through cooperative action of Sp1 and C/EBP.

Prolactin (Prl) and progesterone (P) cooperate synergistically during mammary gland development and tumorigenesis. We hypothesized that one mechanism for these effects may be through mutual induction of receptors (R). EpH4 mouse mammary epithelial cells stably transfected with PR-A express elevated levels of PrlR mRNA and protein compared to control EpH4 cells that lack the PR. Likewise, T47D human breast cancer cells treated with P overexpress the PrlR and activate PrlR promoter III. PrlR promoter III does not contain a classical P response element but contains several binding sites for transcription proteins, including C/EBP, Sp1 and AP1, which may also interact with the PR. Using promoter deletion and site directed mutagenesis analyses as well as gel shift assays, cooperative activation of the C/EBP and adjacent Sp1A, but not the Sp1B or AP1, sites by P is shown to confer P responsiveness leading to increased PrlR transcription.

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.

Effects of neonatal exposure to diethylstilbestrol, tamoxifen, and toremifene on the BALB/c mouse mammary gland.

In this study, we compared the long-term effects of neonatal exposure to diethylstilbestrol (DES, 0.0125-50 microg), tamoxifen (TAM, 0.0125-50 microg), and toremifene (TOR, 53 microg) on mammary gland development and differentiation. Allometric growth of the mammary ducts was stimulated by neonatal DES exposure (12.5 microg) and impaired by exposure to TAM (25 microg). Neonatal treatment with high doses of DES resulted in mammary ducts that displayed extensive dilatation and precocious lactogenesis in postpubertal, nulliparous females. Initiation of this precocious differentiation coincided with the absence of corpora lutea, increased levels of serum prolactin (PRL), and the induction of Prl mRNA expression within the mammary glands. Neonatal exposure to 1.25 microg TAM increased alveolar development in postpubertal, nulliparous females similar to that recorded in females treated with low doses of DES. Lower doses of TAM did not affect alveolar development, whereas branching morphogenesis and alveolar development were impaired by higher doses. Increased alveolar development in females exposed to 1.25 microg TAM was associated with elevated serum progesterone (P) and increased alveolar development in response to exogenous P. Taken together, our findings demonstrate that neonatal exposure to both DES and TAM exerts long-lasting effects on the proliferation and differentiation of the mammary glands in female BALB/c, primarily as the result of endocrine disruption.