A short form of the prolactin (PRL) receptor is able to rescue mammopoiesis in heterozygous PRL receptor mice

The heterozygous prolactin (PRL) receptor (PRLR +/-) mouse fails to develop a fully functional mammary gland at the end of the first pregnancy and shows markedly impaired lobuloalveolar development and milk secretion in young females. The PRLR is expressed ubiquitously, with various proportions of long and short isoforms in different tissues. Conflicting data have appeared on the putative role of the receptor short forms, with both agonist and antagonistic actions proposed. To assess whether the mouse PR-1 short isoform of the PRLR is potentially able to transduce a signal, we overexpressed it in heterozygous mice and investigated its effect on the rescue of mammary development. PRLR+/- mice were not able to develop a functional mammary gland, but restoration of mammary alveolar development and an increase in the expressions of casein and whey acidic protein genes were observed in transgenic PRLR+/- mice expressing the short form of the PRLR, leading to a complete rescue of mammary gland development and function in young females. These results demonstrate that PR-1, the short form of the PRLR, can improve mammary development in PRLR+/- mice, which compensates for the haploinsufficiency of the receptor long form; this effect is probably caused by accelerated proliferation and an activation of the PRLR signaling cascade, resulting in activation of target genes involved in mammary development and milk synthesis.     

Estrogen receptor alpha is required for mammary development and the induction of mammary hyperplasia and epigenetic alterations in the aromatase transgenic mice

Aromatase transgenic mice exhibit hyperplastic and dysplastic changes, attesting to the importance of local estrogen in breast carcinogenesis. These mice also show increased levels of the estrogen receptor and β (ER, ERβ) suggesting that this receptor may play an important role in the initiation of estrogen-mediated mammary hyperplasia observed in these mice. To address the specific role of ER in the mammary development and in the induction of estrogen-mediated hyperplasia in aromatase transgenic mice, we have generated MMTV-aromatase × ER knockout cross (referred as aromatase/ERKO). Even though ERβ is expressed in aromatase/ERKO mice, lack of ER leads to impaired mammary growth in these mice. The data suggest that ER plays an important role in the mammary gland development as well as in the induction of mammary hyperplasia in aromatase transgenic mice. Lack of ER expression in the aromatase/ERKO mice resulted in a decrease in the expression of Cyclin D1, PCNA and TGFβ relative to the aromatase parental strain. The studies involving aromatase/ERKO mice show that lack of ER results in impaired mammary development even in the presence of continuous tissue estrogen, suggesting estrogen/ER-mediated actions are critical for mammary development and carcinogenesis.     

The int-2 gene product acts as an epithelial growth factor in transgenic mice.

The induction of mammary tumors by mouse mammary tumor virus (MMTV) is thought to occur through proviral activation of one or more cellular genes. One of these, int-2, encodes a 27 kd protein which exhibits striking homology to the basic fibroblast growth factor family. To assess directly the role of the int-2 protein in cell proliferation, we have established transgenic mice which carry the int-2 gene driven by the MMTV promoter/enhancer. Expression of the int-2 gene in female transgenic mice results in pronounced mammary gland hyperplasia. Interestingly, expression of the MMTV-int-2 transgene in the prostate gland of male carriers results in a benign, but dramatic, epithelial hyperplasia similar to benign prostatic hypertrophy (BPH), a common but poorly understood disorder in human populations. Together, these results indicate that the int-2 product can act as a potent growth factor in these epithelial tissues.     

Development of mammary hyperplasia and neoplasia in MMTV-TGF alpha transgenic mice

To study the role of transforming growth factor alpha (TGF alpha) in normal mammary development and mammary neoplasia in vivo, we have generated transgenic mice in which a human TGF alpha cDNA is expressed under the control of the MMTV enhancer/promoter. Overexpression of TGF alpha in the mammary epithelium, as confirmed by in situ hybridization and immunohistochemistry, is associated with hyperplasia of alveoli and terminal ducts in virgin female and pregnant transgenic mice. A range of morphologic abnormalities including lobular hyperplasia, cystic hyperplasia, adenoma, and adenocarcinoma is seen in mammary tissue of transgenic females. In contrast, no morphologic abnormalities are seen in transgenic males in spite of TGF alpha overexpression in salivary glands and reproductive organs. TGF alpha can therefore act as an oncogene in vivo and appears to predispose mammary epithelium to neoplasia and carcinoma.     

Mammary tumorigenesis and metastasis caused by overexpression of insulin receptor substrate 1 (IRS-1) or IRS-2

Insulin receptor substrates (IRSs) are signaling adaptors that play a major role in the metabolic and mitogenic actions of insulin and insulin-like growth factors. Reports have recently noted increased levels, or activity, of IRSs in many human cancers, and some have linked this to poor patient prognosis. We found that overexpressed IRS-1 was constitutively phosphorylated in vitro and in vivo and that transgenic mice overexpressing IRS-1 or IRS-2 in the mammary gland showed progressive mammary hyperplasia, tumorigenesis, and metastasis. Tumors showed extensive squamous differentiation, a phenotype commonly seen with activation of the canonical beta-catenin signaling pathway. Consistent with this, IRSs were found to bind beta-catenin in vitro and in vivo. IRS-induced tumorigenesis is unique, given that the IRSs are signaling adaptors with no intrinsic kinase activity, and this supports a growing literature indicating a role for IRSs in cancer. This study defines IRSs as oncogene proteins in vivo and provides new models to develop inhibitors against IRSs for anticancer therapy.     

Activation of Wnt signaling by chemically induced dimerization of LRP5 disrupts cellular homeostasis

Wnt signaling is crucial for a variety of biological processes, including body axis formation, planar polarity, stem cell maintenance and cellular differentiation. Therefore, targeted manipulation of Wnt signaling in vivo would be extremely useful. By applying chemical inducer of dimerization (CID) technology, we were able to modify the Wnt co-receptor, low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5), to generate the synthetic ligand inducible Wnt switch, iLRP5. We show that iLRP5 oligomerization results in its localization to disheveled-containing punctate structures and sequestration of scaffold protein Axin, leading to robust β-catenin-mediated signaling. Moreover, we identify a novel LRP5 cytoplasmic domain critical for its intracellular localization and casein kinase 1-dependent β-catenin signaling. Finally, by utilizing iLRP5 as a Wnt signaling switch, we generated the Ubiquitous Activator of β-catenin (Ubi-Cat) transgenic mouse line. The Ubi-Cat line allows for nearly ubiquitous expression of iLRP5 under control of the H-2K(b) promoter. Activation of iLRP5 in isolated prostate basal epithelial stem cells resulted in expansion of p63(+) cells and development of hyperplasia in reconstituted murine prostate grafts. Independently, iLRP5 induction in adult prostate stroma enhanced prostate tissue regeneration. Moreover, induction of iLRP5 in male Ubi-Cat mice resulted in prostate tumor progression over several months from prostate hyperplasia to adenocarcinoma. We also investigated iLRP5 activation in Ubi-Cat-derived mammary cells, observing that prolonged activation results in mammary tumor formation. Thus, in two distinct experimental mouse models, activation of iLRP5 results in disruption of tissue homeostasis, demonstrating the utility of iLRP5 as a novel research tool for determining the outcome of Wnt activation in a precise spatially and temporally determined fashion.     

Prolactin Induces Apoptosis of Lactotropes in Female Rodents

Anterior pituitary cell turnover occurring during female sexual cycle is a poorly understood process that involves complex regulation of cell proliferation and apoptosis by multiple hormones. In rats, the prolactin (PRL) surge that occurs at proestrus coincides with the highest apoptotic rate. Since anterior pituitary cells express the prolactin receptor (PRLR), we aimed to address the actual role of PRL in the regulation of pituitary cell turnover in cycling females. We showed that acute hyperprolactinemia induced in ovariectomized rats using PRL injection or dopamine antagonist treatment rapidly increased apoptosis and decreased proliferation specifically of PRL producing cells (lactotropes), suggesting a direct regulation of these cell responses by PRL. To demonstrate that apoptosis naturally occurring at proestrus was regulated by transient elevation of endogenous PRL levels, we used PRLR-deficient female mice (PRLRKO) in which PRL signaling is totally abolished. According to our hypothesis, no increase in lactotrope apoptotic rate was observed at proestrus, which likely contributes to pituitary tumorigenesis observed in these animals. To decipher the molecular mechanisms underlying PRL effects, we explored the isoform-specific pattern of PRLR expression in cycling wild type females. This analysis revealed dramatic changes of long versus short PRLR ratio during the estrous cycle, which is particularly relevant since these isoforms exhibit distinct signaling properties. This pattern was markedly altered in a model of chronic PRLR signaling blockade involving transgenic mice expressing a pure PRLR antagonist (TG^{Δ1–9-G129R-hPRL}), providing evidence that PRL regulates the expression of its own receptor in an isoform-specific manner. Taken together, these results demonstrate that i) the PRL surge occurring during proestrus is a major proapoptotic signal for lactotropes, and ii) partial or total deficiencies in PRLR signaling in the anterior pituitary may result in pituitary hyperplasia and eventual prolactinoma development, as observed in TG^{Δ1–9-G129R-hPRL} and PRLRKO mice, respectively.     

The RANKL signaling axis is sufficient to elicit ductal side-branching and alveologenesis in the mammary gland of the virgin mouse

Receptor of Activated NF-κB Ligand (RANKL) is implicated as one of a number of effector molecules that mediate progesterone and prolactin signaling in the murine mammary epithelium. Using a mouse transgenic approach, we demonstrate that installation of the RANKL signaling axis into the mammary epithelium results in precocious ductal side-branching and alveologenesis in the virgin animal. These morphological changes occur due to RANKL-induced mammary epithelial proliferation, which is accompanied by increases in expression of activated NF-kB and cyclin D1. With age, prolonged RANKL exposure elicits limited mammary epithelial hyperplasia. While these transgenics exhibit RANKL-induced salivary gland adenocarcinomas, palpable mammary tumors are not observed due to RANKL-suppression of its own signaling receptor (RANK) in the mammary epithelium. Together, these studies reveal not only that the RANKL signaling axis can program many of the normal epithelial changes attributed to progesterone and prolactin action in the normal mammary gland during early pregnancy, but underscore the necessity for tight control of this signaling molecule to avoid unwarranted developmental changes that could lead to mammary hyperplasia in later life.     

Stage-sensitive blockade of pituitary somatomammotrope development by targeted expression of a dominant negative epidermal growth factor receptor in transgenic mice

The epidermal growth factor receptor (EGFR) and its ligands EGF and transforming growth factor-alpha (TGF alpha) are expressed in the anterior pituitary, and overexpression of TGF alpha in the lactotrope cells of the pituitary gland in transgenic mice results in lactotrope hyperplasia and adenomata, suggesting a role for EGFR signaling in pituitary cell proliferation. To address the role of EGFR signaling in pituitary development in vivo, we blocked EGFR signaling in transgenic mice using the dominant negative properties of a mutant EGFR lacking an intracellular protein kinase domain (EGFR-tr). We directed EGFR-tr expression to GH- and PRL- producing cells using GH and PRL promoters, and a tetracycline-inducible gene expression system, to allow temporal control of gene expression. EGFR-tr overexpression in GH-producing cells during embryogenesis resulted in dwarf mice with pituitary hypoplasia. Both somatotrope and lactotrope development were blocked. However, when EGFR-tr overexpression was delayed to the postnatal period either by directing its expression with the PRL promoter or by delaying the onset of induction with tetracycline in the GH cells, no specific phenotype was observed. Lactotrope hyperplasia during pregnancy also occurred normally in the PRL-EGFR-tr mice. These data suggest that EGFR signaling is required for the differentiation and/or maintenance of somatomammotropes early in pituitary organogenesis but not later in life. (Molecular Endocrinology 15: 600-613, 2001)     

Changes in gene expression during the development of mammary tumors in MMTV-Wnt-1 transgenic mice

Background

In human breast cancer normal mammary cells typically develop into hyperplasia, ductal carcinoma in situ, invasive cancer, and metastasis. The changes in gene expression associated with this stepwise progression are unclear. Mice transgenic for mouse mammary tumor virus (MMTV)-Wnt-1 exhibit discrete steps of mammary tumorigenesis, including hyperplasia, invasive ductal carcinoma, and distant metastasis. These mice might therefore be useful models for discovering changes in gene expression during cancer development.

Results

We used cDNA microarrays to determine the expression profiles of five normal mammary glands, seven hyperplastic mammary glands and 23 mammary tumors from MMTV-Wnt-1 transgenic mice, and 12 mammary tumors from MMTV-Neu transgenic mice. Adipose tissues were used to control for fat cells in the vicinity of the mammary glands. In these analyses, we found that the progression of normal virgin mammary glands to hyperplastic tissues and to mammary tumors is accompanied by differences in the expression of several hundred genes at each step. Some of these differences appear to be unique to the effects of Wnt signaling; others seem to be common to tumors induced by both Neu and Wnt-1 oncogenes.

Conclusion

We described gene-expression patterns associated with breast-cancer development in mice, and identified genes that may be significant targets for oncogenic events. The expression data developed provide a resource for illuminating the molecular mechanisms involved in breast cancer development, especially through the identification of genes that are critical in cancer initiation and progression.     

Prolactin and transforming growth factor-beta signaling exert opposing effects on mammary gland morphogenesis, involution, and the Akt-forkhead pathway

Both prolactin (PRL) and TGF-beta regulate cell survival in mammary epithelial cells, but their mechanisms of interactions are not known. In primary mammary epithelial cells and the HC11 mouse mammary epithelial cell line, PRL prevented TGF-beta-induced apoptosis, as measured by terminal deoxynucleotidyltransferase dUTP nick-end labeling staining and caspase-3 activation. This effect depended on phosphatidyl inositol triphosphate kinase (PI3K). PI3K activates a downstream serine/threonine kinase, Akt; therefore, we investigated the role of Akt in the interaction between PRL and TGF-beta signaling. Akt activity was inhibited by TGF-beta over a 20- to 60-min time course. In TGF-beta-treated cells, PRL disinhibited Akt in a PI3K-dependent manner. Expression of dominant negative Akt blocked the protective effect of PRL in TGF-beta-induced apoptosis. Transgenic mice overexpressing a dominant-negative TGF-beta type II receptor (DNIIR) in the mammary epithelium undergo hyperplastic alveolar development, and this effect was PRL dependent. Involution in response to teat sealing was slowed by overexpression of DNIIR; furthermore, Akt and forkhead phosphorylation increased in the sealed mammary glands of DNIIR mice. Thus, Akt appears to be an essential component of the interaction between PRL and TGF-beta signaling in mammary epithelial cells both in vitro and in vivo.     

Increased apoptosis of periprostatic adipose tissue in VDR null mice

The vitamin D receptor (VDR) is a member of the steroid/retinoid receptor superfamily of nuclear receptors that controls mineral ion homeostatis and has potential tumor-suppressive functions for various cancer types, specifically prostate cancer. A VDR ablated transgenic animal model (VDDRII, vitamin D-dependent rickets type II) has been developed and the animals typically have various diseases including, hypocalcemia, hyperparathyroidism, rickets, osteomalacia, and alopecia. This transgenic mouse system provides us with a model to decipher the influences of the VDR on prostatic growth and function. VDRs are abundant both in prostatic epithelial and stromal cells, and vitamin D signaling can be studied in this model. Although, there were no gross differences between the prostate tissue of the experimental and control groups, VDR null mice showed fat necrosis and individual cell apoptosis in the periprostatic adipose tissue. This indicates a possible role of VDR in the signaling pathways resulting the prostate. This may be particularly attractive for VDR targets for the inhibition of cancer progression using VD(3) and its analogs as potential chemo-preventive agents.     

Integrin-interacting protein Kindlin-2 induces mammary tumors in transgenic mice

Kindlin-2, an integrin-interacting protein, regulates breast cancer progression. However, currently, no animal model to study the role of Kindlin-2 in the carcinogenesis of mammary gland is available. We established a Kindlin-2 transgenic mouse model using a mammary gland-specific promoter, mammary tumor virus (MMTV) long terminal repeat (LTR). Kindlin-2 was overexpressed in the epithelial cells of the transgenic mice. The mammary gland ductal trees were found to grow faster in MMTV-Kindlin-2 transgenic mice than in control mice during puberty. Kindlin-2 promoted mammary gland growth as indicated by more numerous duct branches and larger lumens, and more alveoli were formed in the mammary glands during pregnancy under Kindlin-2 overexpression. Importantly, mammary gland-specific expression of Kindlin-2 induced tumor formation at the age of 55 weeks on average. Additionally, the levels of estrogen receptor and progesterone receptor were decreased, whereas human epidermal growth factor receptor 2 and β-catenin were upregulated in the Kindlin-2-induced mammary tumors. These findings demonstrated that Kindlin-2 induces mammary tumor formation via activation of the Wnt signaling pathway.

     

Inducible dimerization of FGFR1: development of a mouse model to analyze progressive transformation of the mammary gland

To develop an inducible and progressive model of mammary gland tumorigenesis, transgenic mice were generated with a mouse mammary tumor virus-long terminal repeat-driven, conditional, fibroblast growth factor (FGF)-independent FGF receptor (FGFR)1 (iFGFR1) that can be induced to dimerize with the drug AP20187. Treatment of transgenic mice with AP20187 resulted in iFGFR1 tyrosine phosphorylation, increased proliferation, activation of mitogen-activated protein kinase and Akt, and lateral budding. Lateral buds appeared as early as 3 d after AP20187 treatment and initially consisted of bilayered epithelial cells and displayed apical and basolateral polarity appeared after 13 d of AP20187 treatment. Invasive lesions characterized by multicell-layered lateral buds, decreased myoepithelium, increased vascular branching, and loss of cell polarity were observed after 2-4 wk of treatment. These data indicate that acute iFGFR1 signaling results in increased lateral budding of the mammary ductal epithelium, and that sustained activation induces alveolar hyperplasia and invasive lesions.