Loss of mammary epithelial prolactin receptor delays tumor formation by reducing cell proliferation in low-grade preinvasive lesions.

Top quartile serum prolactin levels confer a twofold increase in the relative risk of developing breast cancer. Prolactin exerts this effect at an ill defined point in the carcinogenic process, via mechanisms involving direct action via prolactin receptors within mammary epithelium and/or indirect action through regulation of other hormones such as estrogen and progesterone. We have addressed these questions by examining mammary carcinogenesis in transplants of mouse mammary epithelium expressing the SV40T oncogene, with or without the prolactin receptor, using host animals with a normal endocrine system. In prolactin receptor knockout transplants the area of neoplasia was significantly smaller (7 versus 17%; P < 0.001 at 22 weeks and 7 versus 14%; P = 0.009 at 32 weeks). Low-grade neoplastic lesions displayed reduced BrdU incorporation rate (11.3 versus 17% P = 0.003) but no change in apoptosis rate. Tumor latency increased (289 days versus 236 days, P < 0.001). Tumor frequency, growth rate, morphology, cell proliferation and apoptosis were not altered. Thus, prolactin acts directly on the mammary epithelial cells to increase cell proliferation in preinvasive lesions, resulting in more neoplasia and acceleration of the transition to invasive carcinoma. Targeting of mammary prolactin signaling thus provides a strategy to prevent the early progression of neoplasia to invasive carcinoma.

Prolactin delays hair regrowth in mice.

Mammalian hair growth is cyclic, with hair-producing follicles alternating between active (anagen) and quiescent (telogen) phases. The timing of hair cycles is advanced in prolactin receptor (PRLR) knockout mice, suggesting that prolactin has a role in regulating follicle cycling. In this study, the relationship between profiles of circulating prolactin and the first post-natal hair growth cycle was examined in female Balb/c mice. Prolactin was found to increase at 3 weeks of age, prior to the onset of anagen 1 week later. Expression of PRLR mRNA in skin increased fourfold during early anagen. This was followed by upregulation of prolactin mRNA, also expressed in the skin. Pharmacological suppression of pituitary prolactin advanced dorsal hair growth by 3.5 days. Normal hair cycling was restored by replacement with exogenous prolactin for 3 days. Increasing the duration of prolactin treatment further retarded entry into anagen. However, prolactin treatments, which began after follicles had entered anagen at 26 days of age, did not alter the subsequent progression of the hair cycle. Skin from PRLR-deficient mice grafted onto endocrine-normal hosts underwent more rapid hair cycling than comparable wild-type grafts, with reduced duration of the telogen phase. These experiments demonstrate that prolactin regulates the timing of hair growth cycles in mice via a direct effect on the skin, rather than solely via the modulation of other endocrine factors.

Estradiol induction of retinoic acid receptors in human breast cancer cells.

Retinoic acid inhibits proliferation and steroid receptor gene expression in human breast cancer cell lines. Retinoic acid receptors (RAR)alpha, -beta, and -gamma are expressed in these cells and the expression of RAR alpha is significantly greater in estrogen receptor (ER)-positive cells. This study was undertaken to determine whether the same relationship between RAR alpha and ER gene expression was present in human breast cancers and to explore the possibility that the higher level of RAR alpha in ER-positive cells was due to estrogen regulation of RAR alpha gene expression. RAR alpha and ER mRNA expression were determined by Northern blot analysis in 116 primary breast tumors; 94 (81%) tumors were ER-positive and of these 87 (93%) were also RAR alpha-positive. The coexpression of ER and RAR alpha was statistically significant (P = 0.0052 by chi 2 contingency analysis). There was also a positive correlation (by linear regression analysis) between the levels of expression of ER and RAR alpha mRNA (r2 = 0.251, P = 0.0001), which confirmed the relationship previously documented in breast cancer cell lines and suggested that RAR alpha expression may be modulated in breast cancer in vivo by estrogens acting via the ER. The ability of estradiol to regulate RAR alpha gene expression was examined in vitro using T-47D cells which had been rendered sensitive to estrogen by repeated passage in steroid-depleted medium. Estradiol increased RAR alpha gene expression, but not that of RAR beta or RAR gamma, in a concentration-dependent manner, with the effect being maximal at 10(-10) M and less marked at higher concentrations. The effect was rapid, being detectable 1 h after and maximal 6 h after treatment with 10(-10) M estradiol. Co-treatment of cells with estradiol and antiestrogens (tamoxifen or ICI 164384, 4 x 10(-7) M for 6 h) inhibited the estradiol induction of RAR alpha gene expression, demonstrating that the effect was ER mediated. The estradiol sensitivity of the effect was underscored by the demonstration that addition of untreated serum to cells growing under steroid-depleted conditions was sufficient to induce maximal RAR alpha gene expression. This effect was totally abolished by addition of ICI 164384. In summary, the demonstration that estradiol increased RAR alpha mRNA levels in breast cancer cells supports the hypothesis that the correlation between RAR alpha and ER gene expression in breast tumors and breast cancer cell lines is due to estradiol augmentation of RAR alpha gene expression.

Amplification, expression, and steroid regulation of the preprogalanin gene in human breast cancer.

The GALN gene encodes the preprogalanin protein that is cleaved to liberate the galanin peptide, a neuropeptide and tumor cell mitogen, and the galanin message-associated peptide, which is of unknown function. GALN is located at chromosome 11q13, a frequently amplified locus in diverse tumor types including breast cancer. To determine whether GALN may contribute to the tumor phenotype resulting from 11q13 amplification, we examined GALN amplification and preprogalanin mRNA levels in breast tumors and cell lines. GALN was amplified in a subset of breast tumors and cell lines that carried 11q13 amplifications. Preprogalanin mRNA was expressed in the majority of breast cancer cell lines, but Northern analysis failed to demonstrate a relationship between GALN amplification and preprogalanin mRNA levels. Eight of eight estrogen receptor-positive cell lines expressed detectable preprogalanin mRNA, and further investigation showed that preprogalanin mRNA was increased by treatment with estradiol and progestin and decreased by the removal of serum or treatment with antiestrogens. Thus, GALN amplification is unlikely to contribute to the phenotype conferred by 11q13 amplification in breast cancer, but preprogalanin mRNA is expressed by breast cancer cells and is under steroid hormone control in estrogen receptor-positive cells, opening the wider question of the role of this steroid-regulated neuropeptide in the normal and cancerous breast.

Differential promoter usage in prolactin receptor gene expression: hepatocyte nuclear factor 4 binds to and activates the promoter preferentially active in the liver.

Characterization of the rat PRL receptor (PRLR) gene has revealed three separate untranslated exon 1 sequences, each associated with a different transcription start site and 5'-flanking sequence. We show by RT-PCR that exon 1A is expressed primarily in liver but is also detectable in ovary and mammary gland. Exon 1B expression is observed exclusively in the ovary, whereas exon 1C is expressed in all three tissues. Transient transfection of luciferase reporter constructs containing parts of the 5'-flanking regions (0.3-1.1 kb) of exon 1A, 1B, and 1C, respectively, showed activity of the 1A promoter in Chinese hamster ovary (CHO) cells, the human hepatoma cell line, HepG2, and the rat hepatoma cell line, H4II, which was 10- to 14-fold increased compared with the activity of the promoter-less luciferase vector. No activity of the 1A promoter was detected in the human mammary cell line, T-47D. Relative to a vector containing the Simian virus 40 (SV40) promoter, the 1A promoter had 20% activity in H4II cells and 1-3% activity in CHO and HepG2 cells. The 1B promoter produced a 6.1-fold increase of luciferase activity in CHO cells (approximately 2% of the SV40 promoter), whereas no significant activity was detected in HepG2, H4II, and T-47D cells. The 1C promoter was strongly active in T-47D cells (approximately 64-fold over control) and moderately active in the other cell lines tested (9- to 13-fold over control). 5'-Deletion analysis of the 1A promoter revealed that a fragment containing -83/ +81 bp, relative to the transcription start site, was sufficient to drive transcription in hepatoma cells, whereas this construct was inactive in CHO cells. Cotransfection of CHO cells with the -83/+81 construct and an expression vector encoding the liver-enriched transcription factor, hepatocyte nuclear factor 4 (HNF4), revealed a dose-dependent transactivation of the proximal 1A promoter with a maximal stimulation of approximately 10-fold. Electrophoretic mobility shift assays showed binding of HNF4 to the sequence -14/+24 of the 1A promoter, and mutational analysis revealed that the sequence GGGCAAAGTCA at position +11/+21 is required for this binding. We conclude that the 1A, 1B, and 1C promoters of the PRLR gene are used in a cell type- dependent way that may play a role in differential hormonal regulation of the gene. In particular, we have shown that HNF4 operates on the proximal 1A promoter and may be responsible, in combination with other factors, for the increased activity of this promoter in adult female liver.

Transcriptional regulation of prolactin receptor gene expression by sodium butyrate in MCF-7 human breast cancer cells.

The prolactin receptor (PRLR) mediates the diverse effects of prolactin, which in the mammary gland include the development of lobuloalveolar structures and increased tumor cell proliferation. Treatment of mammary carcinoma cells with the differentiating agent sodium butyrate (NaB) is known to reduce PRLR binding activity and PRLR gene expression, however the mechanism which mediates these changes is unknown, prompting this investigation. Using MCF-7 human breast cancer cells, assay of the rate of PRLR gene transcription by the nuclear run-on technique indicated that 3 mM NaB reduced PRLR gene transcription by 50% after 3 h of treatment and that this effect was maintained for at least 24 h. The protein synthesis inhibitor cycloheximide failed to abrogate this effect, which indicated that NaB did not require continuing protein synthesis to reduce the rate of PRLR transcription. Measurement of PRLR mRNA stability, using Northern blot analysis at various times after the inhibition of transcription with actinomycin D, showed that NaB treatment did not alter PRLR mRNA half-life. These results indicate that NaB inhibits PRLR gene expression by a transcriptional mechanism that does not require continuing protein synthesis.

Null mutation of the prolactin receptor gene produces a defect in maternal behavior.

We have studied pup-directed maternal behavior in mice carrying a germ line null mutation of the PRL receptor (PRLR) gene. Homozygous mutant and heterozygous mutant nulliparous females show a deficiency in pup-induced maternal behavior. Moreover, primiparous heterozygous females exhibit a profound deficit in maternal care when challenged with foster pups. Morris maze studies revealed normal configural learning in the heterozygous and homozygous animals. Eating, locomotor activity, sexual behavior, and exploration (all processes regulated by the hypothalamus) are normal in PRLR mutant mice. Olfactory function was tested in an aversive conditioning paradigm, results indicating that heterozygous and homozygous PRLR mutant mice are not anosmic. These studies clearly establish the PRLR as a regulator of maternal behavior.

Rescue of preimplantatory egg development and embryo implantation in prolactin receptor-deficient mice after progesterone administration.

PRL, a hormone secreted essentially by the pituitary and other extrapituitary sources such as decidua, has been attributed regulatory roles in reproduction and cell growth in mammals. These effects are mediated by a membrane PRL receptor belonging to the cytokine receptor superfamily. Null mutation of the PRL receptor gene leads to female sterility due to a severely compromised preimplantation development and a complete failure of the implantation of the few embryos reaching the blastocyst stage, strongly implicating PRL in the maternal control of implantation. We measured the hormonal status of -/- mice, which confirmed that the corpus luteum is unable to produce progesterone. Progesterone administration to -/- mice completely rescued the development of preimplantatory eggs and embryo implantation. Pregnancy could be maintained to 19.5 days postcoitum, with about 22% of resulting embryos reaching adulthood. Although progesterone and perhaps PRL appear to facilitate mouse preembryo development throughout the preimplantation stages, other factors as well as a possible direct effect of PRL on the uterus are probably necessary to fully maintain pregnancy. Finally, reduced ductal side-branching in the mammary gland can be rescued by progesterone treatment, but females exhibit reduced alveolar formation. Our model establishes the PRL receptor as a key regulator of reproduction and provides novel insights into the function of lactogenic hormones and their receptor.

Prolactin signaling influences the timing mechanism of the hair follicle: analysis of hair growth cycles in prolactin receptor knockout mice.

Pituitary PRL regulates seasonal hair follicle growth cycles in many mammals. Here we present the first evidence implicating PRL in the nonseasonal, wave-like pelage replacement of laboratory mice. In this study we show that messenger RNA transcripts encoding the one long and two short forms of PRL receptor are present in the skin of adult and neonate mice. The receptor protein was immunolocalized to the hair follicle as well as the epidermis and sebaceous glands. Furthermore, PRL messenger RNA was detected within skin extracts, suggesting a possible autocrine/paracrine role. Analysis of the hair growth phenotype of PRL gene-disrupted mice (PRLR(-/-)) revealed a change in the timing of hair cycling events. Although no hair follicle development differences were noted in PRLR(-/-) neonates, observations of the second generation of hair growth revealed PRLR(-/-) mice molted earlier than wild types (PRLR(+/+)). The advance was greater in females (29 days) than in males (4 days), resulting in the elimination of the sexual dimorphism associated with murine hair replacement. Heterozygotes were intermediate between PRLR(-/-) and PRLR(+/+) mice in molt onset. Once initiated, the pattern and progression of the molt across the body were similar in all genotypes. Although all fiber types were present and appeared structurally normal, PRLR(-/-) mice had slightly longer and coarser hair than wild types. These findings demonstrate that PRL has an inhibitory effect on murine hair cycle events. The pituitary PRL regulation of hair follicle cycles observed in seasonally responsive mammals may be a result of pituitary PRL interacting with a local regulatory mechanism.

Osteoblasts are a new target for prolactin: analysis of bone formation in prolactin receptor knockout mice.

Bone development is a multistep process that includes patterning of skeletal elements, commitment of hematopoietic and/or mesenchymental cells to chondrogenic and osteogenic lineages, and further differentiation into three specialized cell types: chondrocytes in cartilage and osteoblasts and osteoclasts in bone. Although PRL has a multitude of biological actions in addition to its role in the mammary gland, very little is known about its effect on bone. Mice carrying a germline null mutation for the PRL receptor gene have been produced in our laboratory and used to study the role of PRL in bone formation. In -/- embryos, we observed an alteration in bone development of calvaria. In adults, histomorphometric analysis showed that the absence of PRL receptors leads to a decrease in bone formation rate using double calcein labeling and a reduction of bone mineral density, measured by dual energy x-ray absorptiometry. In addition, serum estradiol, progesterone, testosterone, and PTH levels were analyzed. We also established that osteoblasts, but not osteoclasts, express PRL receptors. This suggests that an effect of PRL on osteoblasts could be required for normal bone formation and maintenance of bone mass. Thus, the PRL receptor knockout mouse model provides a new tool to investigate the involvement of PRL in bone metabolism.

Coexpression and cross-regulation of the prolactin receptor and sex steroid hormone receptors in breast cancer.

The sex steroid hormones and PRL interact synergistically to control the neoplastic growth of the mammary gland. The basis for this hormonal synergy is unknown, but may involve cellular coexpression of the sex steroid and PRL receptors, coupled with receptor cross-regulation. To examine this hypothesis the expression of the sex steroid and PRL receptors was examined in 20 human breast cancer cell lines and 123 primary breast cancers. Regulation of sex steroid receptors by PRL and of the PRL receptor by sex steroids was examined in T-47D and MCF-7 breast cancer cells. Northern analysis of the breast cancer cell lines and tumors indicated that the PRL receptor and the sex steroid receptors were coexpressed. The level of PRL receptor expression in the breast cancer cell lines was linearly related to that of the estrogen and progesterone receptors, but not to that of the androgen receptor. In MCF-7 and T-47D cells, acute treatment with progestins and androgens and long term treatment with estrogens increased PRL receptor levels. Analysis of sex steroid receptor messenger ribonucleic acid and binding activity showed that acute PRL treatment produced a time- and concentration-dependent increase in progesterone receptor and a decrease in androgen receptor. These results indicate that receptors for sex steroids and PRL are coexpressed and are cross-regulated, providing a potential mechanism for the observed synergy among estrogen, progesterone, and PRL in the control of tumor growth.

Long and short forms of the ovine prolactin receptor: cDNA cloning and genomic analysis reveal that the two forms arise by different alternative splicing mechanisms in ruminants and in rodents.

Prolactin is a pituitary hormone that binds to specific receptors in numerous tissues. Depending on the size of their cytoplasmic domain, long and short prolactin receptors (l-PR, s-PR) have been described. Up to now, s-PR were found in rodents only. We report here the cloning of full-length coding sequences for short and long ovine prolactin receptors (s-oPR, l-oPR). The only difference between s- and l-oPR coding sequences was, respectively, the presence or absence of a 39 base pair insert at the beginning of the cytoplasmic domain, with two contiguous inframe stop codons at its 3' end. Sequence comparison revealed that the alternative splicing producing s- and l-oPR was different from that of rodents, although the resulting proteins were very similar. PCR experiments on ovine genomic DNA showed that the 39 base pair insert was directly linked to the downstream exon, and separated from the upstream exon by an 800 base pair intron. Thus, the alternative splicing used a single intron with one 5' and two 3' sites. The same organization was found in bovine and caprine genomes, suggesting that this feature is general in ruminants and different from rodents, which use mutually exclusive exons to produce s-PR and l-PR.

Investigation of the role of prolactin in the development and function of the lacrimal and harderian glands using genetically modified mice.

PURPOSE: To determine whether prolactin receptor is essential for normal development and function of the lacrimal gland and whether hyperprolactinemia can alter lacrimal development. METHODS: Lacrimal gland morphology and function were examined in two genetic mouse models of prolactin action: a prolactin receptor knockout model that is devoid of prolactin action and a transgenic model of hyperprolactinemia. RESULTS: Image analysis of lacrimal and Harderian gland sections was used to quantify glandular morphology. In females, lacrimal acinar area decreased by 30% and acinar cell density increased by 25% over control subjects in prolactin transgenic animals, but prolactin receptor knockout mice showed no changes. In males, transgenic animals showed no changes, but prolactin receptor knockout mice showed a 5% reduction in acinar area and an 11% increase in acinar cell density, which was lost after castration. The morphology of the Harderian glands underwent parallel changes but to a lesser degree. A complete loss of porphyrin accretions was seen in the Harderian glands of male and female knockout animals. No differences in tear protein levels were seen in knockout animals by two-dimensional gels. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the level of secretory component and IgA in knockout mouse tears remained unchanged. There was no change in the predisposition of the 129 mouse strain to conjunctivitis in the knockout animals. CONCLUSIONS: Prolactin plays a small role in establishing the sexual dimorphism of male lacrimal glands. In females, hyperprolactinemia causes a hyperfemale morphology, suggesting a role in dry eye syndromes. Prolactin is required for porphyrin secretion by the Harderian gland but plays no essential role in the secretory immune function of the lacrimal gland.

Inverse regulation of oestrogen receptor and epidermal growth factor receptor gene expression in MCF-7 breast cancer cells treated with phorbol ester.

In human breast cancer cell lines, an inverse relationship exists between the basal levels of oestrogen receptor (ER) and epidermal growth factor receptor (EGF-R) gene expression. In addition, the tumour-promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) inhibits ER and stimulates EGF-R expression in MCF-7 breast cancer cells. This study aimed to define further the potential mechanisms involved in the modulation of ER and EGF-R gene expression by TPA. ER mRNA levels were reduced after 3 h and declined to 30% of control between 12 and 72 h after exposure to 10 nM TPA. This decrease in mRNA levels was preceded by an apparent fall in ER transcription rate. There was no effect on the stability of ER mRNA following pretreatment for 3-24 h with TPA, supporting the conclusion that the fall in ER mRNA levels was predominantly due to a decrease in ER transcription rate. Levels of EGF-R mRNA increased 10-fold by 12 h due predominantly to an increased transcription rate. The TPA-induced decrease in ER mRNA was unaffected by the simultaneous administration of the protein synthesis inhibitor cycloheximide, whereas the increase in EGF-R mRNA was inhibited by co-incubation with cycloheximide. These data indicate a requirement for continuing protein synthesis for the TPA effect on EGF-R but not on ER mRNA levels. Because the modulation of ER and EGF-R gene expression by TPA is likely to involve the protein kinase C (PKC) signal transduction pathway, the effects of other known activators of PKC were investigated. The non-phorboid tumour promoter mezerein modulated ER (an 80% decrease) and EGF-R (a 20-fold increase) mRNA levels in a similar manner to TPA. In contrast, neither 1,2-dioctanoyl-sn-glycerol (DiC8) nor 1-oleoyl-2-acetyl-sn-glycerol (OAG), both permeant analogues of the endogenous physiological activators of PKC, affected ER and EGF-R mRNA levels. These latter results were not due to a lack of efficacy because a single administration of DiC8 was as effective as TPA in inducing c-fos mRNA at 30 min. However DiC8 was less active in the later induction of c-myc mRNA. These data demonstrate reciprocal regulation of ER and EGF-R gene expression by TPA, involving effects on transcriptional events, which appear to be mediated by sustained activation of PKC.

Mouse prolactin receptor gene: genomic organization reveals alternative promoter usage and generation of isoforms via alternative 3'-exon splicing.

In rodents, the prolactin receptor is expressed as multiple isoforms with identical extracellular and membrane-proximal region sequences but with different 3' sequences, encoding different cytoplasmic regions, and different 5' untranslated region (UTR) sequences. These divergent sequences could be the result of multiple prolactin receptor genes or of a single gene which displays alternative promoter usage and 3'-exon splicing. To investigate the molecular basis for these observations, we have cloned and determined the organization of the mouse prolactin receptor gene. Genomic DNA cloning allowed the arrangement of promoters 1A, 1B, and 1C to be determined. 5'-RACE-PCR from mouse liver identified two novel 5' prolactin receptor sequences, indicating that the gene has at least five different promoters, four of which are active in liver. The remaining nonvariable 5' UTR is encoded by a separate exon (exon 2), while a further 11 coding exons follow, the last 4 of which are alternatively spliced to produce the four isoforms of the receptor. Functional units were found to be exon specific. Thus, the multiple prolactin receptor isoforms are the product of a single gene of >120 kb which displays multiple promoter usage and 3'-exon splicing.

The effect of progestins on prolactin receptor gene transcription in human breast cancer cells.

The sex steroid hormone progesterone modulates the developmental and lactogenic activity of prolactin in the mammary gland. Regulation of the level of prolactin receptor (PRLR) provides one possible mechanism by which this may occur, prompting this investigation of the molecular mechanisms involved in progestin regulation of prolactin receptor levels. Treatment of T-47D and MCF-7 human breast cancer cells with 10 nM of the synthetic progestin ORG 2058 for 24 hr resulted in an increase in all four PRLR mRNA transcripts detected. The effect of ORG 2058 was shown in T-47D cells to be time- and concentration-dependent and resulted in an approximate two-fold increase in PRLR mRNA after 24 hr of treatment with 10 nM or 100 nM ORG 2058. Nuclear run-on assays indicated that ORG 2058 increased the rate of T-47D PRLR gene transcription at all times between 1 hr and 28 hr of treatment. The protein synthesis inhibitors cycloheximide and puromycin abrogated the induction of PRLR gene transcription at 1 hr and 2 hr, which demonstrated that on-going protein synthesis was required for the ORG 2058 effect and suggested that progestins may exert some transcriptional effects via the induction of an intermediary protein. These experiments demonstrated that progestin induced a transcriptionally based increase in PRLR gene expression and provided a mechanism by which progesterone may modulate the mitogenic activity of prolactin during mammary gland development.

Prolactin: a hormone at the crossroads of neuroimmunoendocrinology.

Prolactin (PRL), secreted by the pituitary, decidua, and lymphoid cells, has been shown to have a regulatory role in reproduction, immune function, and cell growth in mammals. The effects of PRL are mediated by a membrane-bound receptor that is a member of the superfamily of cytokine receptors. Formation of a trimer, consisting of one molecule of ligand and two molecules of receptor, appears to be a necessary prerequisite for biological activity. The function of these receptors is mediated, at least in part, by two families of signaling molecules: Janus tyrosine kinases (JAKs) and signal transducers and activators of transcription (STATs). To study these receptors, we have used two approaches: mutational analysis of their cytoplasmic domains coupled with functional tests and inactivation (knockout) of the receptor gene by homologous recombination in mice. We have produced mice by gene targeting in embryonic stem cells carrying a germline null mutation of the prolactin receptor gene. Heterozygous (+/-) females show almost complete failure to lactate, following their first, but not subsequent pregnancies. Homozygous (-/-) females are infertile as a result of multiple reproductive abnormalities, including ovulation of premiotic oocytes, reduced fertilization of oocytes, reduced preimplantation oocyte development, lack of embryo implantation, and the absence of pseudopregnancy. Half of the homozygous males are infertile or show reduced fertility. In view of the wide-spread distribution of PRL receptors, other phenotypes including those on the immune system, are currently being evaluated in -/- animals. This study establishes the prolactin receptor as a key regulator of mammalian reproduction and provides the first total ablation model to further study the role of the prolactin receptor and its ligands.

Mammary gland development and the prolactin receptor.

Prolactin (PRL), synthesized by the anterior pituitary and to a lesser extent by numerous extrapituitary tissues, affects more physiological processes than all other pituitary hormones combined. This hormone is involved in > 300 separate effects in various vertebrate species where its role has been well documented. The initial step in its action is the binding to a specific membrane receptor which belongs to the superfamily of class 1 cytokine receptors. The function of this receptor is mediated, at least in part, by two families of signaling molecules: Janus kinases and signal transducers and activators of transcription. PRL-binding sites have been identified in a number of cells and tissues of adult animals. Disruption of the gene for the PRL receptor has provided a new animal model with which to better understand the actions of PRL on mammary morphogenesis and mammary gland gene expression. The recent availability of genetic mouse models provides new insights into mammary developmental biology and how the action of a hormone at specific stages of development can have effects later in life on processes such as mammary development and breast cancer initiation and progression.

RNAi-mediated stathmin suppression reduces lung metastasis in an orthotopic neuroblastoma mouse model.

Metastatic neuroblastoma is an aggressive childhood cancer of neural crest origin. Stathmin, a microtubule destabilizing protein, is highly expressed in neuroblastoma although its functional role in this malignancy has not been addressed. Herein, we investigate stathmin's contribution to neuroblastoma tumor growth and metastasis. Small interfering RNA (siRNA)-mediated stathmin suppression in two independent neuroblastoma cell lines, BE(2)-C and SH-SY5Y, did not markedly influence cell proliferation, viability or anchorage-independent growth. In contrast, stathmin suppression significantly reduced cell migration and invasion in both the neuroblastoma cell lines. Stathmin suppression altered neuroblastoma cell morphology and this was associated with changes in the cytoskeleton, including increased tubulin polymer levels. Stathmin suppression also modulated phosphorylation of the actin-regulatory proteins, cofilin and myosin light chain (MLC). Treatment of stathmin-suppressed neuroblastoma cells with the ROCKI and ROCKII inhibitor, Y-27632, ablated MLC phosphorylation and returned the level of cofilin phosphorylation and cell invasion back to that of untreated control cells. ROCKII inhibition (H-1152) and siRNA suppression also reduced cofilin phosphorylation in stathmin-suppressed cells, indicating that ROCKII mediates stathmin's regulation of cofilin phosphorylation. This data demonstrates a link between stathmin and the regulation of cofilin and MLC phosphorylation via ROCK. To examine stathmin's role in neuroblastoma metastasis, stathmin short hairpin RNA (shRNA)\luciferase-expressing neuroblastoma cells were injected orthotopically into severe combined immunodeficiency-Beige mice, and tumor growth monitored by bioluminescent imaging. Stathmin suppression did not influence neuroblastoma cell engraftment or tumor growth. In contrast, stathmin suppression significantly reduced neuroblastoma lung metastases by 71% (P<0.008) compared with control. This is the first study to confirm a role for stathmin in hematogenous spread using a clinically relevant orthotopic cancer model, and has identified stathmin as an important contributor of cell invasion and metastasis in neuroblastoma.

c-Myc and Her2 cooperate to drive a stem-like phenotype with poor prognosis in breast cancer.

The HER2 (ERBB2) and MYC genes are commonly amplified in breast cancer, yet little is known about their molecular and clinical interaction. Using a novel chimeric mammary transgenic approach and in vitro models, we demonstrate markedly increased self-renewal and tumour-propagating capability of cells transformed with Her2 and c-Myc. Coexpression of both oncoproteins in cultured cells led to the activation of a c-Myc transcriptional signature and acquisition of a self-renewing phenotype independent of an epithelial-mesenchymal transition programme or regulation of conventional cancer stem cell markers. Instead, Her2 and c-Myc cooperated to induce the expression of lipoprotein lipase, which was required for proliferation and self-renewal in vitro. HER2 and MYC were frequently coamplified in breast cancer, associated with aggressive clinical behaviour and poor outcome. Lastly, we show that in HER2(+) breast cancer patients receiving adjuvant chemotherapy (but not targeted anti-Her2 therapy), MYC amplification is associated with a poor outcome. These findings demonstrate the importance of molecular and cellular context in oncogenic transformation and acquisition of a malignant stem-like phenotype and have diagnostic and therapeutic consequences for the clinical management of HER2(+) breast cancer.