Structure-activity study on the LH- and FSH-releasing and anticancer effects of gonadotropin-releasing hormone (GnRH)-III analogs

GnRH-III was reported to have selective FSH-releasing activity in rats and significant anticancer potency on human breast cancer cells. To improve either of these effects, 14 analogs were synthesized and investigated for FSH/LH stimulation and breast cancer inhibition. Analogs with single amino acid changes in positions 5-7 or 10 showed small or no difference in the FSH- or LH-releasing activity compared with GnRH-III but their anticancer potency decreased significantly. Modification of the terminal amino acids, side chain cyclization at the 6-8 regions, or combined amino acid changes at positions 4, 6 and/or 8 resulted in the decrease of both effects. Gonadotropin-releasing activity of Arg(8)-GnRH-III was improved 3-11-fold. A copolymer conjugate of GnRH-III showed 2-3-fold anticancer activity while losing endocrine potency. CONCLUSION: The activation of GnRH-receptors on pituitary and breast cancer cells requires a specific structure and/or conformation that makes possible to improve the anticancer selectivity of GnRH analogs.     

Control of aromatase activity in breast tumours: the role of the immune system

Cytokines such as interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF), have been identified as important regulators of aromatase activity in fibroblasts derived from normal and malignant breast tissues, and may play an important role in controlling aromatase activity in breast tumours. The major source of such cytokines within breast tumours remains to be established but macrophages and lymphocytes, which can infiltrate tumours, have been identified as a potential source of aromatase stimulatory cytokines. To obtain further insight into the possible role played by the immune system in cancer development, and in particular its ability to regulate aromatase activity via cytokine production, we have obtained peripheral blood monocytes and lymphocytes from an immunosuppressed kidney transplant recipient, receiving cyclosporin A therapy, and a woman with breast cancer. Monocytes and lymphocytes were stimulated with lipopolysaccharide (LPS), and the conditioned medium (CM) collected from these cells was tested for its ability to stimulate aromatase activity in fibroblasts derived from normal breast tissue from a woman undergoing lumpectomy for the removal of a breast tumour. The white blood cell count was lower for the immunosuppressed patient, mainly because of the reduction in the number of monocytes and lymphocytes. The ability of CM from the monocytes and lymphocytes of the immunosuppressed patient to stimulate aromatase activity was significantly reduced (68% and 82% for monocytes and lymphocytes, respectively) compared with that of CM from the cells of the woman with breast cancer. It is possible, therefore, that immunosuppression, which has been found to be associated with a reduction in the incidence of de novo breast cancer in kidney transplant recipients, may exert its effect by inhibiting cytokine production by the cells of the immune system and thus oestrogen synthesis. In contrast to the stimulatory effects that TNF has on aromatase activity in breast fibroblasts, in MCF-7 breast cancer cells, which possess low aromatase activity, it reduced activity. However, the extent of inhibition of aromatase activity in these epithelial cells was much lower than the marked stimulation which it can induce in breast fibroblasts.     

Gonadotropin-Releasing Hormone Agonists Sensitize,and Resensitize,Prostate Cancer Cells to Docetaxel in a p53-Dependent Manner

Gonadotropin-releasing hormone (GnRH) receptors are expressed in prostate cancer, specifically in the most aggressive stage of the tumor (castration-resistant prostate cancer, CRPC) for which the standard treatment, docetaxel-based chemotherapy, can only improve the median survival time by few months. We previously showed that GnRH agonists exert an antitumor activity in CRPC cells; however, a link between GnRH receptors and the apoptotic machinery remains to be defined. Aim of this study was to evaluate whether, in CRPC cells, GnRH agonists might affect the expression/activity of apoptosis-related proteins and might sensitize, or resensitize, cancer cells to chemotherapeutics. We demonstrated that, in p53-positive DU145 cells, GnRH agonists: a) increase the expression of the proapoptotic protein Bax; this effect is mediated by the phosphorylation (activation) of p53, triggered by the p38 MAPK; b) potentiate the antiproliferative/proapoptotic activity of docetaxel; c) resensitize docetaxel-resistant cells to the antitumor activity of the cytotoxic drug. These data indicate that GnRH agonists sensitize and, more importantly, resensitize DU145 CRPC cells to chemotherapy in a p53-dependent manner. To confirm the crucial role of p53 in the activity of GnRH agonists, experiments were performed in p53-null PC3 cells. We found that GnRH agonists fail to increase Bax expression and do not potentiate the cytotoxic activity of docetaxel. These results may provide a rationale for novel combination treatment strategies, especially for docetaxel-resistant CRPC patients expressing a functional p53 protein.     

Modulation of oestrone sulphatase activity in breast cancer cell lines by growth factors

Currently there is much interest in the role that growth factors may play in the development of human breast tumours. We have shown previously that growth factors secreted by breast tumours may influence the activity of oestradiol hydroxysteroid dehydrogenase, the enzyme which catalyses the interconversion of oestrone (E₁) and oestradiol. As the formation of E₁ from its sulphate (E₁S) by oestrone sulphatase may be quantitatively more important than production from androstenedione via aromatase, we have studied the effect of insulin-like growth factor-1 (IGF-I) and basic fibroblast growth factor (bFGF) on oestrone sulphatase activity in the hormone-dependent MCF-7 and the hormone-independent MDA-MB-231 breast cancer cell lines. In both these cell types, bFGF (1–200 ng/ml) and IGF-I (25–200 ng/ml) significantly stimulated oestrone sulphatase activity in a dose-dependent manner (by 8–60%) after 48 h. Additionally, cycloheximide significantly inhibited (by 90–120%) this stimulation of oestrone sulphatase activity by the two growth factors in both MCF-7 and MDA-MB-231 cells. Basal oestrone sulphatase activity was higher in the oestrogen receptor, ER - ve MDA-MB-231 cells than in the ER + ve MCF-7 breast cancer cells. We conclude that these growth factors, believed to be secreted by breast tumours, may induce enzymes of oestrogen synthesis and hence increase local production of oestrogens.     

Role, mechanism of action and application of gonadoliberins in reproductive processes

Gonadoliberin (gonadotropin releasing hormone, GnRH) plays a central role in the regulation of reproductive functions as it regulates the release of both luteinizing hormone (LH) and follicle stimulating hormone (FSH). The isolation and structure determination of GnRH opened the possibility of its use for influencing reproductive processes. This possibility initiated a rapid development in the design of potent and long-acting GnRH agonists and antagonists. The most important structural modifications of GnRH leading to superagonists are the D-amino acid substitutions in position 6 combined with Pro9-ethylamide or azaGly10 at the C-terminus. We have synthesized several superagonists of GnRH according to these substitution principles. Furthermore, our L-isoaspartyl modification in position 6, as a new approach to GnRH agonist design, also resulted in superactive analogs. The recently discovered sequences of non-mammalian GnRH-s opened new routes for us to synthesize species specific GnRH agonists. All three groups of the above mentioned GnRH analogs have been successfully used for the treatment of sexual disorders of different animals (cattle, pigs, rabbits, etc.). Ovulation synchronization and a 30% increase in the fertility rate could be achieved by using GnRH agonists in cattle breeding. Analogs derived from species specific sequences could be applied for the induced artificial propagation of fish even out of the spawning season. It is known that superactive GnRH analogs can suppress the growth of certain hormone-dependent tumours. In vitro and in vivo tests of our analogs showed promising antitumour activity in breast cancer which might be explained by the mechanism of desensitization. Almost a hundred antagonist analogs of GnRH have been developed in our laboratory. The most effective ones contain 4 or 5 D-amino acids, and one of them is even orally active. The inhibition of ovulation can also be achieved by the administration of GnRH superagonists. This phenomenon might also be explained by the desensitization of LH-release. Radioactive analogs specifically labeled with tritium in different amino acid residues have been synthesized and used for studying tissue distribution and biodegradation of gonadoliberins. Analogs containing a photoreactive group have been prepared and applied for the trials of GnRH receptor isolation.     

Estrogen and progesterone receptor status in primary breast cancer--a study of 11,273 patients from the year 1990 to 2002

The aim of this study was to gain insight of the breast cancer hormone receptor status of our patients, its stratification according to age as well as its changes during the period of 13 years. 11,273 patients with primary breast cancer from several towns in Croatia were included in this study. Patients' tumour specimens were collected from 1990 to 2002 and were analysed on estrogen (ER) and progesterone (PR) receptors in the Laboratory of the Department of Medical Oncology, University Hospital Centre Zagreb. More than half of our breast cancer patients had ER positive tumours (54.3%). We observed ER + tumours increased with age continuously, with highest percentage in the age group of 70 to 79 years (68.1%). Similarly, proportion of PR + tumours was higher in the older age groups, being the highest between 40 and 49 years (55.9%). During 13 years of the study, the increase in frequency and proportion of ER + tumours was observed (from 52% in 1990 to 62% in 2002) and decrease of PR + tumours (56% to 53%). We confirm previous findings that the risk of hormone dependent breast cancer increases with aging. Risk of ER + breast cancer increased for 10% from 1990 to 2002 and PR + tumours decreased for 3.5% in the same period.     

The role and proposed mechanism by which oestradiol 17β-hydroxysteroid dehydrogenase regulates breast tumour oestrogen concentrations

Synthesis of the biologically active oestrogen, oestradiol, within breast tumours makes an important contribution to the high concentrations of oestrogens which are present in malignant breast tissues. In breast tumours, oestrone is preferentially converted to oestradiol by the Type I oestradiol 17β-hydroxysteroid dehydrogenase (E2DH). Several growth factors, such as insulin-like growth factor Type I, and cytokines, such as Tumour Necrosis Factor (TNF), have been shown to stimulate E2DH activity in MCF-7 breast cancer cells. As little is known about the regulation of Type I E2DH expression and activity in other breast cancer cell lines, the expression and activity of this enzyme was examined in other oestrogen receptor positive and also oestrogen receptor negative breast cancer cell lines. As it is possible that E2DH activity may be limited by co-factor availability, the effects of exogenous co-factors on enzyme activity in these cell lines was also investigated. For T47D and BT20 breast cancer cells, the addition of exogenous co-factors was found to enhance enzyme activity. TNF, in addition to stimulating E2DH activity in MCF-7 cells, also increased activity in T47D and MDA-MB-231 cells, although to a lesser extent than in MCF-7 cells. An investigation of signalling pathways involved in the regulation of E2DH activity revealed that stimulation of both the protein kinase C (PKC) and PKA pathways may be involved in regulation of E2DH activity. As several growth factors and cytokines have now been found to be involved in regulating E2DH activity, the role that macrophages and lymphocytes have in supplying these factors and the mechanism by which these factors may stimulate tumour growth, is also reviewed.     

Reduction in serum IL-6 after vacination of breast cancer patients with tumour-associated antigens is related to estrogen receptor status

Elevated serum IL-6 concentrations have been associated with poor prognosis in a variety of cancers, and decreases in serum IL-6 concentrations have been reported after chemotherapy. We have demonstrated that serum IL-6 concentrations are elevated in breast cancer patients [normal women 0.7 +/- 2.5 pg/ml (n=36), breast cancer patients 38.3 +/- 138.7 pg/ml (n = 111)]. After vaccination of breast cancer patients with a combination of tumour-associated antigens and biological adjuvants (IL-2 and GM-CSF), the concentration of IL-6 decreased significantly (P<0.05) to 8.1 +/- 14.6 pg/ml (n=85). Other studies have shown that oestrogen suppresses IL-6 production in oestrogen receptor positive breast cancer cells. We have demonstrated that the decrease in IL-6 associated with vaccination is related to the oestrogen receptor status of the tumours from breast cancer patients, as a decrease in IL-6 from 124.0 +/- 267.5 pg/ml (n=26) to 6.2 +/- 11.0 pg/ml (n=34) only occurs in patients with oestrogen receptor negative tumours. The IL-6 concentration in breast cancer patients with oestrogen receptor positive tumours remained unchanged (9.5 pg/ml before vaccination, and 9.3 pg/ml after vaccination). These results suggest that postmenopausal women with oestrogen receptor negative breast cancers, who do not respond well to either hormonal therapy with tamoxifen or adjuvant chemotherapy, may have a significant response to vaccination with autologous tumour-associated antigens.     

Peptides and antitumor activity. Development and investigation of some peptides with antitumor activity

We developed a group of synthetic analogs of GnRH and Somatostatin to inhibit the tumor growth of different kind. The GnRH analogs decreasing the gonadotroph and steroid hormone levels act on the hormone dependent tumors and influence their growth. One of the most effective antitumor analog was patented under the name FOLLIGEN which inhibited the breast cancer caused by DMBA in rats without any side-effects. Other inhibitory analogs of GnRH with long-lasting effect were effective in the treatment of breast, ovary and prostate tumors. Another analog [alpha-Asp(DEA)]6,Gln8-hGnRH showed a very low endocrine but high antitumor effect in both in vitro and in vivo experiments. Its tritium labeled derivative exhibited specific binding sites on human tumor cell lines. We synthesized the analogs of GnRH-III with effective selective antitumor activity which does not alter the ovarian cycle of rats but inhibits the colony-formation of human breast cancer cell lines and has a significant antiproliferative effect. We also synthesized conjugates of potent GnRH analogs with a branched chain polylysine backbone which induce a 33-35% decrease of cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines and 45-50% inhibition of cell proliferation. Another conjugate decreased the tumor growth of MDA-MB-231 xenografts by 80% in a treatment of 9 weeks and even tumor free animals could be found among the ones treated. Using these radiolabeled peptide hormone analogs we found that human tumor cell lines and xenografts specifically bind the GnRH conjugates. We also synthesized a series of Somatostatin analogs which inhibit tyrosine kinases and the growth of several breast, prostate and colon tumor cell lines. One of our best analogs was a heptapeptide, TT-232, which strongly inhibited the tyrosine kinase activity and the cell-proliferation in different colon tumor cells. However, it did not inhibit the growth hormone release either in vitro or in vivo from rat pituitary cells. The TT-232 was found to be effective on 60 human tumor cell lines, it significantly inhibited the tumor growth on different animal tumor models, and induced apoptosis, as a result of which some animals became tumor free. The TT-232 inhibited the tumor growth of PC3 prostate xenografts with 60% and caused a 100% survival of mice 60 days after the transplantation. It is being preclinically tested at present. We have shown that the new GnRH analogs acting without any hormonal effect and the Somatostatin analogs with strong antitumor and tyrosine kinase inhibitory activity but no hormonal effect may represent a breakthrough in the research of the antitumor peptides, having direct effect on tumor cells.     

Human breast cancer cell lines and tissues express tartrate-resistant acid phosphatase (TRAP)

Tartrate-resistant acid phosphatase (TRAP) is expressed by osteoclasts, macrophages and dendritic cells. TRAP has been identified in a wide variety of tissues, however, its biological function is not fully understood. Serum TRAP is a marker of diseases involving excessive bone resorption including metastatic bone disease in breast cancer patients and can be used to monitor responses to treatment. Our aim in this study was to determine whether TRAP is expressed by human breast tumours. Four breast cancer cell lines were assayed for TRAP activity. MDA-MB-435, the most tumourigenic line, had an activity twofold higher than the other cell lines. Immunohistochemistry using a TRAP specific antibody confirmed that both cell lines and human breast tumours express TRAP. Expression was absent in benign tissues and abundant in more aggressive tumours. This work suggests that tumour derived TRAP contributes to the raised enzyme activity found in the serum of breast cancer patients.     

Estrogen receptor status of breast cancer in Ontario

Data from a number of studies of breast cancer have suggested that after the ages associated with the menopause the rates of estrogen-receptor-positive tumours increase with age, whereas the rates of estrogen-receptor-negative tumours do not. Previous investigators studied cases in specific treatment centres, so there was a possibility that the findings were influenced by differences in patterns of case referral by age. A review of all the cases of breast cancer diagnosed in Ontario women in 1981 and assayed for estrogen receptors, however, confirmed the earlier findings. The results showed that the incidence of estrogen-receptor-positive and estrogen-receptor-negative tumours increased at about the same rate before age 45, but thereafter an increase in incidence was seen only for estrogen-receptor-positive tumours. These differences in patterns of incidence suggest the possibility that the two types of tumour may have different etiologic factors.     

Prognostic factors in human primary breast cancer: comparison of c-myc and HER2/neu amplification.

Amplification of oncogenes in primary tumours may have prognostic and/or therapeutic significance for patients with breast cancer. We have studied HER2/neu and c-myc amplification together with steroid receptors in human primary breast tumours and related the outcome with (relapse-free) survival. A strong inverse correlation was found between HER2/neu amplification and the presence of oestrogen and progesterone receptors. Actuarial 5-years survival showed that breast cancer patients with c-myc amplification in their primary tumours experience a shorter relapse-free survival, especially in node-negative and in receptor-positive tumours, whereas HER2/neu amplification may be of prognostic value for overall survival in receptor-negative tumours. Overall, in our hands, c-myc amplification appeared to be a more potent prognosticator than HER2/neu amplification in human primary breast cancer.     

Expression of Erk5 in Early Stage Breast Cancer and Association with Disease Free Survival Identifies this Kinase as a Potential Therapeutic Target

Background

Breast cancer is the most common neoplasia in women. Even though advances in its treatment have improved disease outcome, some patients relapse. Therefore, attempts to better define the molecular determinants that drive breast cancer cell proliferation may help in defining potential therapeutic targets. Mitogen-activated protein kinases (MAPK) play important roles in tumorigenesis. One of them, Erk5, has been linked to the proliferation of breast cancer cells in vitro. Here we have investigated the expression and prognostic value of Erk5 in human breast cancer.

Methodology/Principal Findings

Animal and cellular models were used to study Erk5 expression and function in breast cancer. In 84 human breast tumours the expression of Erk5 was analyzed by immunohistochemistry. Active Erk5 (pErk5) was studied by Western blotting. Correlation of Erk5 with clinicopathological parameters and with disease-free survival in early stage breast cancer patients was analyzed. Expression of Erk5 was detected in most patients, and overexpression was found in 20%. Active Erk5 was present in a substantial number of samples, as well as in tumours from an animal breast cancer model. Overexpression of Erk5 was associated with a decrease in disease-free survival time, which was independent of other clinicopathological parameters of prognosis. Transient transfection of a short hairpin RNA (shRNA) targeting Erk5, and a stable cell line expressing a dominant negative form of Erk5 (Erk5^AEF), were used to investigate the influence of Erk5 on drugs used in the clinic to treat breast tumours. We found that inhibition of Erk5 decreased cancer cell proliferation and also sensitized these cells to the action of anti-HER2 therapies.

Conclusions/Significance

Overexpression of Erk5 is an independent predictor of disease-free survival in breast cancer, and may represent a future therapeutic target.     

Nestin expression associates with poor prognosis and triple negative phenotype in locally advanced (T4) breast cancer

Nestin, an intermediate filament protein, has traditionally been noted for its importance as a neural stem cell marker. However, in recent years, expression of nestin has shown to be associated with general proliferation of progenitor cell populations within neoplasms. There is no reported study addressing nestin expression in T4 breast cancer patients. Thus, the aim of the present study was to investigate, through immunohistochemistry, the expression and distribution of nestin in T4 breast cancer, in order to determine its association with clinical and pathological parameters as well as with patients' outcome. Nestin was detectable in tumoral cells and in endothelial cells of blood microvessels, and it is significantly expressed in triple-negative and in inflammatory breast cancer (IBC) subgroups of T4 breast tumours. The Kaplan-Meier analysis showed that the presence of nestin in tumoral cells significantly predicted poor prognosis at 5-years survival (P=0.02) and with borderline significance at 10-years of survival (P=0.05) in T4 breast cancer patients. On the basis of these observations, we speculate that nestin expression may characterize tumours with an aggressive clinical behavior, suggesting that the presence of nestin in tumoral cells and vessels may be considered an important factor that leads to a poor prognosis. Further studies are awaited to define the biological role of nestin in the etiology of these subgroups of breast cancers.     

Pyrrolidon carboxypeptidase activities in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-ovary axes of rats with mammary gland cancer induced by N-methyl nitrosourea.

Pyrrolidon carboxypeptidase is an omega-peptidase that hydrolyses N-terminal pyroglutamyl residues from biologically active peptides such as gonadotropin-releasing and thyrotrophin-releasing hormones. We previously described a decrease in both rat and human pyrrolidon carboxypeptidase activity with breast cancer, suggesting that gonadotropin-releasing hormone may be an important local intracrine, autocrine and/or paracrine hormonal factor in the pathogenesis of breast cancer while playing a role in the tumoral process. However, the other susceptible substrate of pyrrolidon carboxypeptidase, thyrotrophin-releasing hormone, may also be modified with breast cancer, supporting an association between breast cancer and thyroid disorders. The present work analyses soluble and membrane-bound pyrrolidon carboxypeptidase activities in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-ovary axes in N-methyl nitrosourea-induced breast cancer in rats. Our aim was to determine the possible relationship between gonadotropin-releasing hormone and thyrotrophin-releasing hormone regulation through pyrrolidon carboxypeptidase activity. We propose that pyrrolidon carboxypeptidase activity dysregulation at various local and systemic levels may participate in the initiation, promotion and progression of breast cancer induced in rat by N-methyl nitrosourea through the increase in gonadotropin-releasing hormone. Since pyrrolidon carboxypeptidase activity also acts on thyrotrophin-releasing hormone, the dysregulation of this enzyme's activity could indirectly affect hypothalamus-pituitary-thyroid axis function, and thus potentially represent a link between the diseases of thyroid and breast cancer.     

RNA binding protein RALY promotes Protein Arginine Methyltransferase 1 alternatively spliced isoform v2 relative expression and metastatic potential in breast cancer cells

Aberrant expression of Protein Arginine Methyltransferases (PRMTs) has been observed in several cancer types, including breast cancer. We previously reported that the PRMT1v2 isoform, which is generated through inclusion of alternative exon 2, is overexpressed in breast cancer cells and promotes their invasiveness. However, the precise mechanism by which expression of this isoform is controlled and how it is dysregulated in breast cancer remains unknown. Using a custom RNA interference-based screen, we identified several RNA binding proteins (RBP) which, when knocked down, altered the relative abundance of the alternatively spliced PRMT1v2 isoform. Amongst the top hits were SNW Domain containing 1 (SNW1) and RBP-associated with lethal yellow mutation (RALY), which both associated with the PRMT1 pre-mRNA and upon depletion caused an increase or decrease in the relative abundance of PRMT1v2 isoform mRNA and protein. Most importantly, a significant decrease in invasion was observed upon RALY knockdown in aggressive breast cancer cells, consistent with targeting PRMT1v2 directly, and this effect was rescued by the exogenous re-expression of PRMT1v2. We show that SNW1 expression is decreased, while RALY expression is increased in breast cancer cells and tumours, which correlates with decreased patient survival. This work revealed crucial insight into the mechanisms regulating the expression of the PRMT1 alternatively spliced isoform v2 and its dysregulation in breast cancer. It also provides proof-of-concept support for the development of therapeutic strategies where regulators of PRMT1 exon 2 alternative splicing are targeted as an approach to selectively reduce PRMT1v2 levels and metastasis in breast cancer.     

Immunocytochemical and histochemical analyses of gonadotrophin releasing hormone,tyrosine hydroxylase,and cytochrome oxidase reactivity within the hypothalamus of chicks showing early sexual maturation

In order to determine the changes in gonadotrophin releasing hormone (GnRH) and dopaminergic activity within the brain during the onset of sexual precocity, a Halasz-like knife was developed to produce discrete parasagittal cuts in 2-week-old male broiler chicks. At 5 weeks of age, sexually precocious respondents were selected on the basis of advanced secondary sex characteristics and randomly paired with sham-operated controls. Each pair of birds was perfused with heparinized saline followed by 4% paraformaldehyde. Sections 40 m thick, obtained throughout the hypothalamus, were immunostained with either anti-GnRH or anti-tyrosine hydroxylase (TH) to ascertain dopaminergic activity. Alternate sections from each pair of brains were also treated with cytochrome oxidase to determine metabolic activity levels or with Nissl stain to localize the knife cuts. Analysis revealed an increase in GnRH immunoreactivity within the bed nucleus of the pallial commissure (nCPa) and paraventricular nucleus (PVN), as well as the median eminence (ME). An increase in TH immunoreactivity was observed in the nucleus intramedialis (nI). Also an increase in metabolic activity was seen in the PVN as revealed by cytochrome oxidase reactivity. It is hypothesized that during the onset of puberty there is an increase in immunoreactive GnRH cell numbers as a result of a decrease in the inhibition of the GnRH system, possibly involving the nI and PVN. The source of the dopamine reported in the ME could be from the nI and other nearby nuclei. Dopamine from the tubero-infundibular area may be one of the putative neurotransmitters responsible for the increased activity of GnRH within the ME of chicks showing precocious puberty.     

Do estrogens always increase breast cancer risk?

The etiology of breast cancer is closely linked to the female hormone estrogen, with high life-time exposure being suggested to increase breast cancer risk [Nature 303 (1983) 767]. However, there appears to be a disparity between studies attempting to establish an association between high estrogen levels and breast cancer risk. This disparity becomes smaller by taking into consideration a timing factor, and we propose that estrogens can increase, decrease, or have no effect on breast cancer risk, depending on the timing of estrogen exposure. We further propose that the timing of estrogenic exposures may play at least as important a role in affecting breast cancer risk as life-time exposure.