[Show abstract][Hide abstract] ABSTRACT: Epidemiological studies that have investigated whether dairy (mainly milk) diets are associated with prostate cancer risk have led to controversial conclusions. In addition, no existing study clearly evaluated the effects of dairy/milk diets on prostate tumor progression, which is clinically highly relevant in view of the millions of men presenting with prostate pathologies worldwide, including benign prostate hyperplasia (BPH) or high-grade prostatic intraepithe-lial neoplasia (HGPIN). We report here a unique interventional animal study to address this issue. We used two mouse models of fully penetrant genetically-induced prostate tumori-genesis that were investigated at the stages of benign hyperplasia (probasin-Prl mice, Pb-Prl) or pre-cancerous PIN lesions (KIMAP mice). Mice were fed high milk diets (skim or whole) for 15 to 27 weeks of time depending on the kinetics of prostate tumor development in each model. Prostate tumor progression was assessed by tissue histopathology examination , epithelial proliferation, stromal inflammation and fibrosis, tumor invasiveness potency and expression of various tumor markers relevant for each model (c-Fes, Gprc6a, activated Stat5 and p63). Our results show that high milk consumption (either skim or whole) did not promote progression of existing prostate tumors when assessed at early stages of tumorigenesis (hyperplasia and neoplasia). For some parameters, and depending on milk type, milk regimen could even exhibit slight protective effects towards prostate tumor progression by decreasing the expression of tumor-related markers like Ki-67 and Gprc6a. In conclusion, our study suggests that regular milk consumption should not be considered detrimental for patients presenting with early-stage prostate tumors.
PLoS ONE 05/2015; 10(5). DOI:10.1371/journal.pone.0125423 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Evidence for a role for calcium channel proteins in cell proliferation is numerous suggesting that calcium influx is essential in this physiological process. Several studies in the past thirty years have demonstrated that calcium channel expression levels are determinant in cell proliferation. Voltage-gated, store-operated, second messengers and receptor-operated calcium channels have been associated to cell proliferation. However, the relationship between calcium influx and cell proliferation can be uncoupled in transformed and cancer cells, resulting in an external calcium-independent proliferation. Thus, protein expression could be more important than channel function to trigger cell proliferation suggesting that additional channel functions may be responsible to reconcile calcium channel expression and cell proliferation. When needed, external calcium concentration is obviously important for calcium channel function but it also regulates calcium sensing receptor (CaSR) activity. CaSR can up- or down-regulate cell proliferation depending on physiological conditions. CaSR sensitivity to external calcium is within the 0.5 to 5mM range and therefore, the role of these receptors in cell proliferation must be taken into account. We therefore suggest here that cell proliferation rates could depend on the relative balance between calcium influx and CaSR activation.
European journal of pharmacology 11/2013; 739. DOI:10.1016/j.ejphar.2013.10.072 · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cytokine hormone prolactin has a vast number of diverse functions. Unfortunately, it also exhibits tumor growth promoting properties, which makes the development of prolactin receptor antagonists a priority. Prolactin binds to its cognate receptor with much lower affinity at low pH than at physiological pH and since the extracellular environment around solid tumors often is acidic, it is desirable to develop antagonists that have improved binding affinity at low pH. The pK(a) value of a histidine side chain is ∼6.8 making histidine residues obvious candidates for examination. From evaluation of known molecular structures of human prolactin, of the prolactin receptor and of different complexes of the two, three histidine residues in the hormone-receptor binding site 1 were selected for mutational studies. We analyzed 10 variants by circular dichroism spectroscopy, affinity and thermodynamic characterization of receptor binding by isothermal titration calorimetry combined with in vitro bioactivity in living cells. Histidine residue 27 was recognized as a central hot spot for pH sensitivity and conservative substitutions at this site resulted in strong receptor binding at low pH. Pure antagonists were developed earlier and the histidine mutations were introduced within such background. The antagonistic properties were maintained and the high affinity at low pH conserved. The implications of these findings may open new areas of research in the field of prolactin cancer biology.
[Show abstract][Hide abstract] ABSTRACT: Human prolactin (PRL) is currently viewed as a hormone of pituitary origin, whose production (i.e. serum levels) is controlled by dopamine, whose biological actions relate exclusively to lactation and reproductive functions, for which any genetic disorder is yet to be identified, and whose unique associated pathology is hyperprolactinemia. Both experimental studies and human sample/cohort-based investigations performed during the past decade have considerably widened our perception of PRL biology: i) there are now strong epidemiological arguments supporting the fact that circulating PRL is a risk factor for breast cancer, ii) in addition to the endocrine hormone, locally produced PRL has been documented in several human tissues; there is increasing evidence supporting the tumor growth potency of local PRL, acting via autocrine/paracrine mechanisms, in both rodent models, and human breast and prostate tumors, iii) the first functional germinal polymorphisms of the PRL receptor were recently identified in patients presenting with breast tumors, which involve single amino acid substitution variants exhibiting constitutive activity, iv) human PRL analogs have been engineered, which were shown in experimental models to down-regulate the effects triggered by local PRL (competitive antagonism) or by the constitutively active receptor variants (inverse agonism). The aim of this review is to discuss these novel concepts in PRL biology, including their potential pathophysiological outcomes.
Journal of Endocrinology 04/2010; 206(1):1-11. DOI:10.1677/JOE-10-0069 · 3.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of prolactin (PRL) and its receptor (hPRLR) in promoting breast tumors is debated. We recently identified a gain-of-function hPRLR variant (I146L) in four women with multiple breast fibroadenomas (MFA) and no control subject.
The specific aims were to describe this cohort of women presenting with MFA to identify and functionally characterize germline variants of hPRL/hPRLR genes and compare phenotypes of all patients.
Ninety-five patients prospectively underwent clinical examination, breast ultrasonography, magnetic resonance imaging, and hormonal evaluation of gonadal and lactotrope functions. We analyzed hPRL/hPRLR coding sequences and made comparisons with a control population of 194 women. Functional characterization of hPRLR variants was performed. Pathology and immunochemistry were systematically carried out after surgical removal of tumors.
One third of patients had a family history of breast disease. No hormonal imbalance was observed, except 30.7% of explosive stimulated PRL. Prolactin receptor variants were identified in exon 5 (I76V: 10 patients, eight controls) and exon 10 (one patient, no control). Both I146L and I76V variants exhibited constitutive activity. Pathology showed common fibroadenomas and identified six benign phyllodes tumors. Estrogen and progesterone receptors were detected in 85 and 98% of samples, respectively. Ki-67 median staining was less than 5%. No phenotypic difference was observed between carriers and noncarriers of either hPRLR variant.
We present the largest population with MFA ever described, 15% of which had a hPRLR exhibiting basal activity in vitro. This questions the involvement of the hPRLR in MFA etiology and the potential relevance of therapeutic inhibition of PRLR signaling in patients.
The Journal of Clinical Endocrinology and Metabolism 11/2009; 95(1):271-9. DOI:10.1210/jc.2009-1494 · 6.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gonadectomy induces in certain inbred stains of mice adrenal hyperplasia and tumorigenesis, originating from the putative subcapsular stem/progenitor cell layer. This response is apparently triggered by the elevated post-gonadectomy levels of luteinising hormone (LH), followed by ectopic upregulation of adrenal LH/chorionic gonadotrophin (CG) receptors (Lhcgr). The clear strain dependence of this adrenal response to gonadectomy prompted us to study its genetic basis. Tumorigenic DBA/2J and non-tumorigenic C57BL/6J mice, as well as their F2 and backcrosses, were studied by whole genome linkage analysis. Gonadectomy induced similar upregulation of adrenal Lhcgr in both parental strains and their crosses, irrespective of the tumour status, indicating that ectopic expression of this receptor is not the immediate cause of tumours. Linkage analysis revealed one major significant quantitative trait locus (QTL) for the tumorigenesis on chromosome 8, modulated by epistasis with another QTL on chromosome 18. Hence, post-gonadectomy adrenal tumorigenesis in DBA/2J mice is a dominant trait, not a direct consequence of adrenal Lhcgr expression, and is driven by a complex genetic architecture. A promising candidate gene in the tumorigenesis linkage region is Sfrp1 (secreted frizzled-related protein 1), a tumour suppressor gene, which was down-regulated in the neoplastic tissue. Our findings may have relevance to the human pathogenesis of macronodular adrenal hyperplasia and postmenopausal adrenocortical tumours. A distinctly different adrenal response was observed in TG mice overexpressing LH or CG, or a constitutively activated form of the follicle-stimulating hormone receptor (Fshr). These mice developed perimedullary hyperlasia of foamy multinucleated cells, reminding of macrophages and filled with lipofuscin. Similar response was observed in TG mice overexpressing aromatase (CYP19). The cause of this response is not related to direct LH/CG action, but merely to adrenal response to chronically elevated oestrogen levels. This phenotype is reminiscent of the rare 'black adenomas' of the human adrenal cortex.
[Show abstract][Hide abstract] ABSTRACT: The human adrenal cortex expresses low levels of luteinizing hormone/chorionic gonadotropin receptors (LHCGR), a characteristic gonad-specific G-protein coupled receptor (GPCR). LHCGR levels increase in the adrenal cortex after exposure to chronically elevated gonadotropins (e.g. after gonadectomy). In fact, heightened ectopic LHCGR levels are observed in a subclass of human adrenocortical tumors, and gonadotropin-responsive adrenocortical hyperplasia and tumors occur in several animal species. These findings suggest that adrenocortical responsiveness to LH/CG might be a physiological phenomenon that is amplified in the presence of elevated gonadotropin levels. Such increased gonadotropin action can induce pathologies ranging from adrenocorticotropic hormone (ACTH)-independent Cushing syndrome to malignant adrenal tumors. The authors review the current information on adrenocortical responses to gonadotropins in experimental animals and humans.
Trends in Endocrinology and Metabolism 10/2008; 19(7):231-8. DOI:10.1016/j.tem.2008.06.003 · 9.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is currently no known genetic disease linked to prolactin (Prl) or its receptor (PrlR) in humans. Given the essential role of this hormonal system in breast physiology, we reasoned that genetic anomalies of Prl/PrlR genes may be related to the occurrence of breast diseases with high proliferative potential. Multiple fibroadenomas (MFA) are benign breast tumors which appear most frequently in young women, including at puberty, when Prl has well-recognized proliferative actions on the breast. In a prospective study involving 74 MFA patients and 170 control subjects, we identified four patients harboring a heterozygous single nucleotide polymorphism in exon 6 of the PrlR gene, encoding Ile(146)-->Leu substitution in its extracellular domain. This sole substitution was sufficient to confer constitutive activity to the receptor variant (PrlR(I146L)), as assessed in three reconstituted cell models (Ba/F3, HEK293 and MCF-7 cells) by Prl-independent (i) PrlR tyrosine phosphorylation, (ii) activation of signal transducer and activator of transcription 5 (STAT5) signaling, (iii) transcriptional activity toward a Prl-responsive reporter gene, and (iv) cell proliferation and protection from cell death. Constitutive activity of PrlR(I146L) in the breast sample from a patient was supported by increased STAT5 signaling. This is a unique description of a functional mutation of the PrlR associated with a human disease. Hallmarks of constitutive activity were all reversed by a specific PrlR antagonist, which opens potential therapeutic approaches for MFA, or any other disease that could be associated with this mutation in future.
Proceedings of the National Academy of Sciences 09/2008; 105(38):14533-8. DOI:10.1073/pnas.0800685105 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is increasing evidence that prolactin (PRL) and growth hormone (GH) act as growth-promoters of breast tumors. Recent arguments have accumulated to suggest that when they are locally-produced within the mammary tissue, these hormones, acting by an autocrine-paracrine mechanism may have enhanced, or even specific functions compared to endocrine PRL and GH. Classical drugs blocking pituitary hormone production (dopamine and somatostatin analogs) are ineffective on extrapituitary expression of PRL/GH genes, therefore the undesirable effects of these locally-produced hormones remain a target of interest for alternative strategies. This has encouraged the development of competitive PRL and/or GH receptor antagonists, which involve engineered variants of natural receptor ligands (PRL or GH) aimed at blocking receptor activation rather than hormone production in peripheral tissues. This article overviews the rational design of this new class of molecules, their specific molecular features (receptor specificity, biological properties, etc.) and whenever available, the data that have been obtained in cell or animal models of breast cancer.
Journal of Mammary Gland Biology and Neoplasia 04/2008; 13(1):105-17. DOI:10.1007/s10911-008-9066-8 · 4.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Postgonadectomy adrenocortical tumorigenesis is a strain-specific phenomenon in inbred mice, assumed to be caused by elevated LH secretion and subsequent ectopic LH receptor (LHR) overexpression in adrenal gland. However, the molecular mechanisms of this cascade of events remain unknown. In this study, we took advantage of the mouse strain dependency of the phenotype to unravel its genetic basis. Our results present the first genome-wide screening related to this pathology in two independent F2 and backcross populations generated between the neoplastic DBA/2J and the nonsusceptible C57BL/6J strains. Surprisingly, the postgonadectomy elevation of serum LH was followed by similar up-regulation of adrenal LHR expression in both parental strains and their crosses, irrespective of their tumor status, indicating that it is not the immediate cause of the tumorigenesis. Linkage analysis revealed one major significant locus for the tumorigenesis on chromosome 8, modulated by epistasis with another quantitative trait locus on chromosome 18. Weight gain, a secondary phenotype after gonadectomy, showed a significant but separate quantitative trait locus on chromosome 7. Altogether, postgonadectomy adrenocortical tumorigenesis in DBA/2J mice is a dominant trait that is not a direct consequence of adrenal LHR expression but is driven by a complex genetic architecture. Analysis of candidate genes in the tumorigenesis linkage region showed that Sfrp1 (secreted frizzled-related protein 1), a tumor suppressor gene, is differentially expressed in the neoplastic areas. These findings may have relevance to the human pathogenesis of macronodular adrenal hyperplasia and adrenocortical tumors in postmenopausal women and why some of them develop obesity.
[Show abstract][Hide abstract] ABSTRACT: There is a large body of literature showing that prolactin (PRL) exerts growth-promoting activities in breast cancer, and possibly in prostate cancer and prostate hyperplasia. In addition, increasing evidence argues for the involvement of locally produced (autocrine) PRL, perhaps even more than pituitary-secreted (endocrine) PRL, in tumor growth. Because dopamine analogs are unable to inhibit PRL production in extrapituitary sites, alternative strategies need investigation. To that end, several PRL receptor antagonists have been developed by introducing various mutations into its natural ligands. For all but one of these analogs, the mechanism of action involves a competition with endogenous PRL for receptor binding. Such compounds are thus candidates to counteract the undesired actions of PRL, not only in tumors, but also in dopamine-resistant prolactinomas. In this review, we describe the different versions of antagonists that have been developed, with emphasis on the controversies regarding their characterization, and the limits for their potential development as a drug. The most recently developed antagonist, Delta1-9-G129R-hPRL, is the only one that is totally devoid of residual agonistic activity, meaning it acts as pure antagonist. We discuss to what extent this new molecule could be considered as a lead compound for inhibiting the actions of human PRL in the above-mentioned diseases. We also speculate on the multiple questions that could be addressed with respect to the therapeutic use of PRL receptor antagonists in patients.
[Show abstract][Hide abstract] ABSTRACT: The N-terminus is the most divergent region within the prolactin (PRL)/placental lactogen (PL)/growth hormone (GH) family. Since all of these ligands are able to activate the lactogen receptor, it has been usually assumed that the N-terminus plays no major role in biological actions of any family member. In this study, we generated several analogs of human PRL in which the N-terminus was truncated by 9 and iteratively up to the 14 first residues. Truncation did not alter protein folding, and it even decreased the formation of PRL aggregates that appear during the purification of refolded protein. Removal of the entire N-terminal loop (14 residues) decreased the affinity for the receptor by two-three-fold, and reduced the ability of the hormone to activate the human lactogen receptor. In contrast, removal of 13 or less residues improves receptor activation since these analogs are able to produce supra-maximal activities in a transcriptional bioassay, or in proliferation assays exhibit dose-response curves that are less bell-shaped, which reflects enhanced stabilization of receptor dimers. Altogether, these data suggest that the N-terminus of PRL is actually slightly detrimental to bioactivity, but may be required for other properties of the hormone.
[Show abstract][Hide abstract] ABSTRACT: Prolactin (PRL) promotes tumor growth, as recently highlighted by the spontaneous appearance of prostate hyperplasia and mammary neoplasia in PRL transgenic mice. Increasing experimental evidence argues for the involvement of autocrine PRL in this process. Human (h)PRL receptor antagonists have been developed to counteract these undesired proliferative actions of PRL. However, all PRL receptor antagonists obtained to date exhibit partial agonism, limiting their therapeutic use as full antagonists. This is the case for the first generation antagonists (the prototype of which is G129R-hPRL) that we developed ten years ago, which display antagonistic activity in some, but not all in vitro bioassays, and fail to inhibit PRL activity in transgenic mice expressing this analog. We recently developed new human PRL antagonists devoid of agonistic properties, and therefore able to act as pure antagonists. This was demonstrated using several in vitro bioassays, including assays able to detect extremely low levels of receptor activation. These new compounds also act as pure antagonists in vivo, as demonstrated by their ability to competitively inhibit PRL-triggered signaling cascades in various target tissues (liver, mammary gland and prostate). Finally, using transgenic mice specifically expressing PRL in the prostate, which have constitutively activated signaling cascades and prostate hyperplasia, these new PRL analogs are able to completely revert PRL-activated events to basal levels. These second generation antagonists are good candidates to be used as inhibitors of the growth-promoting actions of hPRL.
[Show abstract][Hide abstract] ABSTRACT: Prolactin (PRL) promotes tumor growth in various experimental models and leads to prostate hyperplasia and mammary neoplasia in PRL transgenic mice. Increasing experimental evidence argues for the involvement of autocrine PRL in this process. PRL receptor antagonists have been developed to counteract these undesired proliferative actions of PRL. However, all forms of PRL receptor antagonists obtained to date exhibit partial agonism, preventing their therapeutic use as full antagonists. In the present study, we describe the development of new human PRL antagonists devoid of agonistic properties and therefore able to act as pure antagonists. This was demonstrated using several in vitro bioassays, including highly sensitive assays able to detect extremely low levels of receptor activation. These new compounds also act as pure antagonists in vivo, as assessed by analyzing their ability to competitively inhibit PRL-triggered signaling cascades in various target tissues (liver, mammary gland, and prostate). Finally, by using transgenic mice expressing PRL specifically in the prostate, which exhibit constitutively activated signaling cascades paralleling hyperplasia, we show that these new PRL analogs are able to completely revert PRL-activated events. These second generation human PRL antagonists are good candidates to be used as inhibitors of growth-promoting actions of PRL.
[Show abstract][Hide abstract] ABSTRACT: The reference bioassay for lactogens is the Nb2 cell proliferation assay, whose extreme sensitivity allows the detection of very low amounts of lactogenic activity in biologic fluids. The use of rat Nb2 cells raises the problem of species specificity when analyzing lactogens of other origin, including human lactogenic hormones for which no reference bioassay currently exists. In this article, we describe two new homologous bioassays for human lactogens. One is a transcriptional bioassay generated by stably transfecting 293 human embryonic kidney fibroblasts using two plasmids, encoding the human prolactin receptor (hPRLR) and the PRL-responsive lactogenic hormone response element luciferase reporter gene. The second is a proliferation assay obtained by stably transfecting Ba/F3 cells with a plasmid encoding the hPRLR. We provide characterization of the various clones or cell populations that were isolated, and we describe experiments that were performed to achieve optimized protocols for both bioassays. These new assays were compared with other cells types exhibiting well-recognized PRL-mediated responses (proliferation of Nb2 or of human breast tumor cell lines), using various lactogen analogs. This comparative analysis provides strong evidence that the intrinsic characteristics of each bioassay dramatically affect the biologic properties attributed to the lactogen of interest. Depending on the assay, a given analog can exhibit agonistic or antagonistic properties. We hypothesize that in addition to species specificity, assay sensitivity is the key parameter in directing the apparent bioactivity of lactogens. Of course, in the end, it will be necessary to confirm the agonistic or antagonistic properties of the tested analogs, in vivo.
[Show abstract][Hide abstract] ABSTRACT: For many years, our group has been involved in the development of human PRL antagonists. In two recent publications, S179D-human PRL, a human PRL analog designed to mimic a putative S179-phosphorylated human PRL, was reported to be a highly potent antagonist of human PRL-induced proliferation and signaling in rat Nb2 cells. We prepared this analog with the aim of testing it in various bioassays involving the homologous, human PRL receptor. In our hands, S179D- human PRL was able to stimulate 1) the proliferation of rat Nb2 cells and of human mammary tumor epithelial cells (T-47D), 2) transcriptional activation of the lactogenic hormone response element-luciferase reporter gene, and 3) activation of the Janus kinase/signal transducer and activator of transcription and MAPK pathways. Using the previously characterized antagonist G129R-human PRL as a control, we failed to observe any evidence for antagonism of S179D-human PRL toward any of the human PRL-induced effects analyzed, including cell proliferation, transcriptional activation, and signaling. In conclusion, our data argue that S179D-human PRL is an agonist displaying slightly reduced affinity and activity due to local alteration of receptor binding site 1, and that the antagonistic properties previously attributed to S179D-human PRL cannot be confirmed in any of the assays analyzed in this study.
[Show abstract][Hide abstract] ABSTRACT: The involvement of human prolactin (hPRL) in breast cancer has been recently reconsidered based on its autocrine/paracrine proliferative effect described in human mammary tumor epithelial cells. Therefore, there is growing interest in the development of potent hPRL antagonists that may inhibit this effect. We previously designed hPRL analogs displaying antagonistic properties in a human transcriptional bioassay. We now report that the most potent of those analogs, G129R-hPRL, antagonizes all hPRL-induced effects analysed in various breast cancer cell lines, including cell proliferation. The analog per se lacks intrinsic agonistic activity on PRL receptor-activated signaling cascades, cell proliferation and apoptosis, indicating that its mode of action only occurs through competitive inhibition of hPRL. We provide some molecular basis of this antagonistic effect by demonstrating that G129R-hPRL competitively inhibits hPRL-activation of the JAK-STAT and MAPK pathways, two signaling cascades involved in the mitogenic effect of hPRL in mammary epithelial cells. This competitive inhibition persists for at least 48 h, as evidenced by long term analysis of STAT5b activation or of progression through cell cycle. These results are the first demonstration at the molecular level that hPRL antagonists interfering with receptor dimerization disrupt signaling events in breast cancer cells, which prevents hPRL-induced cell proliferation.