Cellular distribution of growth hormone-releasing hormone receptor in human reproductive system and breast and prostate cancers

Growth hormone releasing hormone receptor (GHRH-R) mRNA and protein was first localized to the anterior pituitary gland, consequent with the action of its ligand on GH synthesis and release. Subsequent studies found GHRH-R also expressed in the hypothalamus and in systemic tissues including those of the reproductive system. In the present work, we studied the distribution of GHRH-R in human reproductive system of males and females by immunohistochemical method. GHRH-R immunostaining was localized in male reproductive system: Leydig cells, Sertoli and basal germ cells of the seminiferous tubules and prostate secretory cells. GHRH-R immunostaining was also demonstrated in the ovary: oocytes, follicular cells, granulosa, thecal and corpus luteum cells. Endometrial glands, placenta and normal mammary glands also showed GHRH-R immunostaining. Our results demonstrate the localization of GHRH-R in the reproductive system, which may mediate the direct action of GHRH in these tissues. Moreover, GHRH-R was demonstrated in prostate and breast carcinomas, opening a variety of possibilities for the use of GHRH antagonists in the treatment of prostatic and mammary tumors.     

RB1 and p53 at the crossroad of EMT and triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a heterogeneous disease that includes Basal-like and Claudin-low tumors. The Claudin-low tumors are enriched for features associated with epithelial-to-mesenchymal transition (EMT) and possibly for tumor initiating cells. Primary TNBCs respond relatively well to conventional chemotherapy; however, metastatic disease is virtually incurable. Thus, there is a great interest in identifying specific therapeutic targets for TNBC. The tumor suppressor RB1 is frequently lost in Basal-like breast cancer. To test for a causative role of RB1 gene loss in BC and for its effect on specific subtypes, we deleted mouse Rb in mammary stem/bipotent progenitor cells. This led to diverse mammary tumors including TNBC, with a subset of the latter containing p53 mutations and exhibiting features of Basal-like BC or EMT. Combined mutation of Rb and p53 in mammary stem/bipotent progenitors induced EMT type tumors. Here, we review our findings and those of others, which connect Rb and p53 to EMT in TNBC. Furthermore, we discuss how by understanding this circuit and its vulnerabilities, we may identify novel therapy for TNBC.     

Atrazine binds to F1F0-ATP synthase and inhibits mitochondrial function in sperm

Atrazine is a widely used triazine herbicide. Although controversy still exists, a number of recent studies have described its adverse effects on various animals including humans. Of particular interest is its effects on reproductive capacity. In this study, we investigated the mechanisms underlying the adverse effects of atrazine, with a focus on its effects on sperm. Here we show evidence that mitochondrial F₁F₀-ATP synthase is a molecular target of atrazine. A series of experiments with sperm and isolated mitochondria suggest that atrazine inhibits mitochondrial function through F₁F₀-ATP synthase. Moreover, affinity purification using atrazine as a ligand demonstrates that F₁F₀-ATP synthase is a major atrazine-binding protein in cells. The inhibitory activity against mitochondria and F₁F₀-ATP synthase is not limited to atrazine but is likely to be applicable to other triazine-based compounds. Thus, our findings may have wide relevance to pharmacology and toxicology.     

The BRG1 chromatin remodeler protects against ovarian cysts, uterine tumors, and mammary tumors in a lineage-specific manner

The BRG1 catalytic subunit of SWI/SNF-related complexes is required for mammalian development as exemplified by the early embryonic lethality of Brg1 null homozygous mice. BRG1 is also a tumor suppressor and, in mice, 10% of heterozygous (Brg1(null/+)) females develop mammary tumors. We now demonstrate that BRG1 mRNA and protein are expressed in both the luminal and basal cells of the mammary gland, raising the question of which lineage requires BRG1 to promote mammary homeostasis and prevent oncogenic transformation. To investigate this question, we utilized Wap-Cre to mutate both Brg1 floxed alleles in the luminal cells of the mammary epithelium of pregnant mice where WAP is exclusively expressed within the mammary gland. Interestingly, we found that Brg1(Wap-Cre) conditional homozygotes lactated normally and did not develop mammary tumors even when they were maintained on a Brm-deficient background. However, Brg1(Wap-Cre) mutants did develop ovarian cysts and uterine tumors. Analysis of these latter tissues showed that both, like the mammary gland, contain cells that normally express Brg1 and Wap. Thus, tumor formation in Brg1 mutant mice appears to be confined to particular cell types that require BRG1 and also express Wap. Our results now show that such cells exist both in the ovary and the uterus but not in either the luminal or the basal compartments of the mammary gland. Taken together, these findings indicate that SWI/SNF-related complexes are dispensable in the luminal cells of the mammary gland and therefore argue against the notion that SWI/SNF-related complexes are essential for cell survival. These findings also suggest that the tumor-suppressor activity of BRG1 is restricted to the basal cells of the mammary gland and demonstrate that this function extends to other female reproductive organs, consistent with recent observations of recurrent ARID1A/BAF250a mutations in human ovarian and endometrial tumors.     

Repeated exposure of C3H/HeJ mice to ultra-wideband electromagnetic pulses: lack of effects on mammary tumors

It has been suggested that chronic, low-level exposure to radiofrequency (RF) radiation may promote the formation of tumors. Previous studies, however, showed that low-level, long-term exposure of mammary tumor-prone mice to 435 MHz or 2450 MHz RF radiation did not affect the incidence of mammary tumors. In this study, we investigated the effects of exposure to a unique type of electromagnetic energy: pulses composed of an ultra-wideband (UWB) of frequencies, including those in the RF range. One hundred C3H/HeJ mice were exposed to UWB pulses (rise time 176 ps, fall time 3.5 ns, pulse width 1.9 ns, peak E-field 40 kV/m, repetition rate 1 kHz). Each animal was exposed for 2 min once a week for 12 weeks. One hundred mice were used as sham controls. There were no significant differences between groups with respect to incidence of palpated mammary tumors, latency to tumor onset, rate of tumor growth, or animal survival. Histopathological evaluations revealed no significant differences between the two groups in numbers of neoplasms in all tissues studied (lymphoreticular tissue, thymus, respiratory, digestive and urinary tracts, reproductive, mammary and endocrine systems, and skin). Our major finding was the lack of effects of UWB-pulse exposure on promotion of mammary tumors in a well-established animal model of mammary cancer.     

Study of the growth factors for the mammary gland: Epidermal growth factor and mesenchyme-derived growth factor

The physiological importance of EGF in the development of the mouse mammary gland during pregnancy and in spontaneous mammary tumorigenesis has been documented by a series of experimental results presented herein. In our study, we have taken a variety of experimental approaches including radioimmunoassay of EGF in the submandibular gland and plasma, mammary gland organ/cell culture, EGF receptor assay, sialoadenectomy and treatment with EGF and EGF antibodies to assess the role of EGF in the mammary gland. In particular, studies employing sialoadenectomy and EGF replacement have provided valuable information concerning the function of EGF in the body. These studies are possible in the mouse system because the submandibular gland serves as a major source of circulating EGF and also because purified mouse EGF is available commercially. Our work on the biological, endocrinological, and physiological aspects of EGF in normal and neoplastic growth of the mammary gland should be useful for the study of the regulation of mammary gland growth at the molecular level as well as for clinical investigations of mammary tumors. Finally, our findings of a mammary growth factor in embryonic mesenchymal cultures suggest the possible involvement of paracrine growth factor(s) in mammary cell growth. Further progress in this area is needed for better understanding of the complex process of mammary gland growth and development.     

SCE analysis in G2 lymphocyte prematurely condensed chromosomes after exposure to atrazine: the non-dose-dependent increase in homologous recombinational events does not support its genotoxic mode of action

Several studies have been carried out to evaluate the mutagenic and carcinogenic potential of atrazine, the most prevalent of triazine herbicides classified as a "possible human carcinogen". The majority of these studies have been negative but positive responses have been also reported including mammary tumors in female Sprague-Dawley rats. Sister chromatid exchanges (SCEs) caused by the presence of DNA lesions at the moment of DNA replication have been extensively used for genotoxicity testing, but for non-cytotoxic exposures to atrazine controversial results have been reported. Even though exposures to higher concentrations of atrazine could provide clear evidence for its genotoxicity, conventional SCE analysis at metaphase cells cannot be used because affected cells are delayed in G2-phase and do not proceed to mitosis. As a result, the genotoxic potential of atrazine may have been underestimated. Since clear evidence has been recently reported relating SCEs to homologous recombinational events, we are testing here the hypothesis that high concentrations of atrazine will cause a dose-dependent increase in homologous recombinational events as quantified by the frequency of SCEs analyzed in G2-phase. Towards this goal, a new cytogenetic approach is applied for the analysis of SCEs directly in G2-phase prematurely condensed chromosomes (PCCs). The methodology enables the visualization of SCEs in G2-blocked cells and is based on drug-induced PCCs in cultured lymphocytes. The results obtained for high concentrations of atrazine do not demonstrate a dose-dependent increase in homologous recombinational events. They do not support, therefore, a genotoxic mode of action. However, they suggest that an important part in the variation of SCE frequency reported by different laboratories when conventional SCE analysis is applied after exposure to a certain concentration of atrazine, is due to differences in cell cycle kinetics of cultured lymphocytes, rather than to a true biological variation in the cytogenetic end point used.     

Integration of the DNA of mouse mammary tumor virus in virus-infected normal and neoplastic tissue of the mouse.

We have used restriction endonucleases which cleave the DNA of mouse mammary tumor virus (MMTV) at one site (Eco RI) and several sites (Pst I, Sac I and Bam HI) to study infection and mammary tumorigenesis in mice. Proviruses acquired during infection of BALB/c mice foster-nursed by virus-producing C3H females can be distinguished from the MMTV proviruses endogenous to uninfected BALB/c mice by the nature of the fragments generated with Pst I and Bam HI. Using this assay, we show that lactating mammary glands as well as mammary tumors from BALB/cfC3H mice have acquired MMTV DNA, and that a minimum of approximately 10% of normal glandular cells can be infected. The new proviruses appear to be linked to cellular DNA of mammary tumors and infected lactating mammary glands within a limited region (0.2 x 10(6) daltons) of the viral DNA; the location of this region, based upon mapping studies with unintegrated MMTV DNA, suggests that the orientation of these proviruses is colinear with linear DNA synthesized in infected cells and thus approximately colinear with the viral RNA. Comparisons of many mammary tumors and studies of lactating mammary glands with a high proportion of independently infected cells indicate that a large number of sites in the cellular genome can accommodate a new provirus; the acquired proviruses are rarely, if ever, found in tandem with each other or with endogenous proviruses. We cannot, however, distinguish between random integration and integration into a large number of preferred sites in the host genome. Since Eco RI and Bam HI cleavage of DNA from each mammary tumor generates a unique set of viral-specific fragments, we propose that the tumors are composed principally of cells derived from a subset of the many infected cells in a mammary gland; this proposal is supported by our finding that Eco RI digestion of DNA from several transplants of a primary tumor yields the pattern characteristic of the primary tumor.     

Acute involution in the tammar wallaby: identification of genes and putative novel milk proteins implicated in mammary gland function

Marsupials provide a suitable alternative model to studying mammary gland involution. They have evolved a different reproductive strategy from eutherians, giving birth to an altricial young and secreting milk that changes in composition during lactation. In this study, we used a marsupial-specific EST microarray to identify 47 up-regulated genes during mammary gland involution in the tammar wallaby (Macropus eugenii). These include the pro-apoptotic tumour necrosis factor receptor superfamily 21 (TNFRSF21) gene, whose expression in the mammary gland has not previously been reported. Genes encoding putative novel milk proteins which may protect the mammary gland from infection were also found to be up-regulated, such as amiloride binding protein 1 (ABP1), complement component 1QB (C1QB), complement component 4A (C4A) and colony stimulating factor 2 receptor β (CSF2Rβ). Our results show that the marsupial reproductive strategy was successfully exploited to identify genes and putative novel milk proteins implicated in mammary gland involution.     

Zinc accumulation in N-methyl-N-nitrosourea-induced rat mammary tumors is accompanied by an altered expression of ZnT-1 and metallothionein

Zinc is essential for cell proliferation. Several human studies have shown that in breast cancer tissues, zinc concentration expressed on a per tissue weight basis is higher than that in normal breast tissues. However, the mechanisms involved are unknown. N-methyl-N-nitrosourea (MNU)-induced rat mammary tumorigenesis is one of the most widely used rodent mammary tumorigenesis models for studying human breast cancer due to their similarities in hormone dependency, pathogenesis, histological classification, and immunocytochemical markers. This study was to establish if there was an accumulation of zinc in MNU-induced rat mammary tumors and, if there was, to explore the possible mechanisms involved. Sprague-Dawley rats were sham-treated or MNU-treated (50 mg/kg; n = 12) for 100 days. In MNU-induced mammary tumors (mammary tumors), zinc concentration expressed on a per dry weight basis was 12 times of that in normal mammary glands. Moreover, the mRNA level of ZnT-1 (a transporter involved in zinc efflux) in mammary tumors was reduced by 55% as compared with that in normal mammary glands. The mRNA level of Nramp2 (a divalent cation importer) and ZnT-4 (another transporter involved in zinc efflux) was unaffected by MNU-induced mammary tumorigenesis. The mRNA and protein levels of metallothionein (a putative zinc storage protein) in mammary tumors were 1.3 and 3.5 times of that in normal mammary glands, respectively. Collectively, our observations showed that zinc is accumulated in MNU-induced rat mammary tumors and this accumulation is accompanied by an altered expression of ZnT-1 and metallothionein, suggesting that zinc homeostasis might be altered in MNU-induced rat mammary tumorigenesis. Because zinc is essential to cell proliferation and cell proliferation is increased in mammary tumors, zinc accumulation is likely a part of an integrated effort to ensure sufficient zinc supply to sustain tumor growth.     

Differential gene signatures in rat mammary tumors induced by DMBA and those induced by fractionated gamma radiation

The aim of this work was to identify specific genes involved in rat mammary tumors induced by dimethylbenz(a)anthracene (DMBA) or radiation. More TUNEL- and PCNA-positive cells were present in mammary tumors induced by radiation than in tumors induced by DMBA, whereas DNA damage responses like p53 accumulation and histone H2AX phosphorylation were higher in DMBA-induced tumors, even though the pathology was similar in both types of tumors. cDNA microarray and real-time RT-PCR analysis of radiation- or DMBA-induced tumor tissues, revealed that stanniocalcin 2 (Stc2), interferon regulatory factor 1 (Irf1), interleukin 18 binding protein (Il18bp), and chloride channel calcium activated 3 (Clca3) were expressed in both, and that arachidonate 5-lipoxygenase activating protein 1 (Alox5ap) and cathepsin S (Ctss) were expressed only in radiation-induced tumors. No DMBA-specific gene signatures were found. Soft agar growth assays were carried out to identify the carcinogenic features of these specific genes. Cells stably transfected with Alox5ap, Ctss, Stc2, Irf1, Il18bp and Clca3 showed morphological changes compared to controls. These findings indicate different gene alterations in carcinogen- or radiation-induced mammary tumors with similar pathological stages.     

Exploring the Gain of Function Contribution of AKT to Mammary Tumorigenesis in Mouse Models

Elevated expression of AKT has been noted in a significant percentage of primary human breast cancers, mainly as a consequence of the PTEN/PI3K pathway deregulation. To investigate the mechanistic basis of the AKT gain of function-dependent mechanisms of breast tumorigenesis, we explored the phenotype induced by activated AKT transgenes in a quantitative manner. We generated several transgenic mice lines expressing different levels of constitutively active AKT in the mammary gland. We thoroughly analyzed the preneoplastic and neoplastic mammary lesions of these mice and correlated the process of tumorigenesis to AKT levels. Finally, we analyzed the impact that a possible senescent checkpoint might have in the tumor promotion inhibition observed, crossing these lines to mammary specific p53(R172H) mutant expression, and to p27 knock-out mice. We analyzed the benign, premalignant and malignant lesions extensively by pathology and at molecular level analysing the expression of proteins involved in the PI3K/AKT pathway and in cellular senescence. Our findings revealed an increased preneoplastic phenotype depending upon AKT signaling which was not altered by p27 or p53 loss. However, p53 inactivation by R172H point mutation combined with myrAKT transgenic expression significantly increased the percentage and size of mammary carcinoma observed, but was not sufficient to promote full penetrance of the tumorigenic phenotype. Molecular analysis suggest that tumors from double myrAKT;p53(R172H) mice result from acceleration of initiated p53(R172H) tumors and not from bypass of AKT-induced oncogenic senescence. Our work suggests that tumors are not the consequence of the bypass of senescence in MIN. We also show that AKT-induced oncogenic senescence is dependent of pRb but not of p53. Finally, our work also suggests that the cooperation observed between mutant p53 and activated AKT is due to AKT-induced acceleration of mutant p53-induced tumors. Finally, our work shows that levels of activated AKT are not essential in the induction of benign or premalignant tumors, or in the cooperation of AKT with other tumorigenic signal such as mutant p53, once AKT pathway is activated, the relative level of activity seems not to determine the phenotype.     

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.     

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.     

The mammary tumor response in triazine-treated female rats: a threshold-mediated interaction with strain and species-specific reproductive senescence.

Triazine herbicides are among the most heavily used agricultural pesticides. Although they possess a very low acute toxicity in animals, a mammary tumor response has been consistently observed in Sprague-Dawley (SD) female rats following chronic oral dosing of atrazine and simazine at and above maximum tolerated doses. However, a substantial collection of detailed research has clearly shown that triazines are not genotoxic or mutagenic, nor do they possess estrogenic agonist activity that might promote mammary tumor growth. Examination of estrous cycling records of atrazine-treated SD rats revealed a premature appearance of persistent estrous episodes, beyond the prevalent occurrence normally seen in untreated, aging SD rats. A significant correlation has been found between early or severe estrous cycle disruption of atrazine-treated rats and the early appearance of mammary tumors. In studies using SD female rats fed atrazine for 6 months, then ovariectomized and administered an estrogen-containing silastic s.c. implant, a deficient luteinizing hormone surge was observed at a 400 parts per million (ppm) dose, but not at 25 or 50 ppm. Because SD rats exhibiting persistent estrus also have a prolonged elevation of estrogen secretion, it is proposed that the triazine-associated mammary tumor response is promoted by the test animal's own estrogen from ovarian follicles that fail to ovulate because gonadotropin surge sufficiency is blocked by the high dose of herbicide. It is further proposed that, because reproductive senescence in SD rats is fundamentally different from menopause in women, the animal response to dosing, as well as the enormous requisite dosing level, establishes a safety margin of very low risk to human health from this mode of action.     

Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2

The prognosis of breast cancer in young women is influenced by reproductive history. Women diagnosed within 5 years postpartum have worse prognosis than nulliparous women or women diagnosed during pregnancy. Here we describe a mouse model of postpartum breast cancer that identifies mammary gland involution as a driving force of tumor progression. In this model, human breast cancer cells exposed to the involuting mammary microenvironment form large tumors that are characterized by abundant fibrillar collagen, high cyclooxygenase-2 (COX-2) expression and an invasive phenotype. In culture, tumor cells are invasive in a fibrillar collagen and COX-2-dependent manner. In the involuting mammary gland, inhibition of COX-2 reduces the collagen fibrillogenesis associated with involution, as well as tumor growth and tumor cell infiltration to the lung. These data support further research to determine whether women at high risk for postpartum breast cancer would benefit from treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) during postpartum involution.     

Molecular Mechanism of SLC5A8 Inactivation in Breast Cancer

SLC5A8 is a putative tumor suppressor that is inactivated in more than 10 different types of cancer, but neither the oncogenic signaling responsible for SLC5A8 inactivation nor the functional relevance of SLC5A8 loss to tumor growth has been elucidated. Here, we identify oncogenic HRAS (HRAS^G12V) as a potent mediator of SLC5A8 silencing in human nontransformed normal mammary epithelial cell lines and in mouse mammary tumors through DNMT1. Further, we demonstrate that loss of Slc5a8 increases cancer-initiating stem cell formation and promotes mammary tumorigenesis and lung metastasis in an HRAS-driven murine model of mammary tumors. Mammary-gland-specific overexpression of Slc5a8 (mouse mammary tumor virus-Slc5a8 transgenic mice), as well as induction of endogenous Slc5a8 in mice with inhibitors of DNA methylation, protects against HRAS-driven mammary tumors. Collectively, our results provide the tumor-suppressive role of SLC5A8 and identify the oncogenic HRAS as a mediator of tumor-associated silencing of this tumor suppressor in mammary glands. These findings suggest that pharmacological approaches to reactivate SLC5A8 expression in tumor cells have potential as a novel therapeutic strategy for breast cancer treatment.     

A mouse model to dissect progesterone signaling in the female reproductive tract and mammary gland

Considering the regulatory complexities of progesterone receptor (PR) action throughout the female reproductive axis and mammary gland, we generated a mouse model that enables conditional ablation of PR function in a spatiotemporal specific manner. Exon 2 of the murine PR gene was floxed to generate a conditional PR allele (PR^flox) in mice. Crossing the PR^flox/flox mouse with the ZP3‐cre transgenic demonstrated that the PR^flox allele recombines to a PR null allele (PR^d). Mice homozygous for the recombined null PR allele (PR^d/d) exhibit uterine, ovarian, and mammary gland defects that phenocopy those of our previously described PR knockout (PRKO) model. Therefore, this conditional mouse model for PR ablation represents an invaluable resource with which to further define in a developmental and/or reproductive stage‐specific manner the individual and integrative roles of distinct PR populations resident in multiple progesterone‐responsive target sites. genesis 48:106–113, 2010. © 2009 Wiley‐Liss, Inc.