RESUMO
BACKGROUND: Permixon®, the hexanic lipidosterolic extract of saw palmetto Serenoa repens (LSESr), has shown properties that highlight its benefit in the management of benign prostate hyperplasia (BPH). To address its actual anti-inflammatory potency, we used a unique pro-inflammatory mouse model of prostate hyperplasia involving prostate-specific over-expression of prolactin transgene (Pb-Prl). METHODS: Six month-old Pb-Prl males were administered with Permixon® per os at the daily dose of 100 mg/kg for 28 days. Body and prostate weights were measured weekly and at sacrifice, respectively. Prostate histology was carefully assessed by a pathologist and detailed quantifications of epithelial and stromal compartments were performed using image analysis software. Luminal cell proliferation index was determined using Ki-67 immunostaining, and apoptosis using Bax/Bcl2 mRNA ratio. Tissue inflammation and fibrosis were assessed by histological analyses then quantified using CD45 immunostaining and picrosirius staining, respectively. Expression profiling of selected pro-inflammatory cytokines, chemokines, and chemokine receptors was performed by quantitative RT-PCR. RESULTS: In this model, Permixon® significantly decreased tissue weight and proliferation index specifically in the ventral lobe. Although treatment had no noticeable effect on epithelial histology of any lobe, it markedly reduced the histological hallmarks of inflammation in all lobes. This was confirmed by the global down-regulation of prostate pro-inflammatory cytokine profile, with significant reduction of CCR7, CXCL6, IL-6, and IL-17 expression. CONCLUSIONS: In this mouse model of prostate hyperplasia, Permixon® exerted potent anti-inflammatory properties in the whole prostate while anti-androgenic effects were lobe-specific, suggesting that distinct LSESr components may be involved in these effects. Our results support the beneficial role of Permixon® treatment for BPH. The relevance of CCR7, CXCL6, IL-6, and IL-17 as potential biomarkers to follow up BPH inflammatory status needs to be assessed.
Assuntos
Apoptose/efeitos dos fármacos , Inflamação/tratamento farmacológico , Extratos Vegetais/farmacologia , Hiperplasia Prostática/tratamento farmacológico , Serenoa/química , Animais , Citocinas/genética , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/patologia , Antígeno Ki-67/genética , Masculino , Camundongos , Camundongos Transgênicos , Tamanho do Órgão/efeitos dos fármacos , Hiperplasia Prostática/imunologia , Hiperplasia Prostática/patologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Proteína X Associada a bcl-2/genéticaRESUMO
The anterior pituitary is permanently regulated by processes of apoptosis and proliferation in order to maintain tissue homeostasis. Several factors have been implicated in this regulation and lately, prolactin (PRL) has been included into that list. However, since PRL is secreted by anterior pituitary lactotropes, the actual outcome of its autocrine/paracrine actions on pituitary cells has remained difficult to assess. The availability of the pure PRL receptor antagonist Del1-9-G129R-hPRL has been helpful to circumvent this problem. While PRL has been traditionally associated with increased cell proliferation, recent studies revealed that this hormone actually induces apoptosis and decreases proliferation of anterior pituitary cells, by mechanisms involving the PRL receptor. The aim of this short review is to overview our current understanding of the regulation of pituitary homeostasis by PRL. Moreover, studies involving Del1-9-G129R-hPRL have helped anticipate to what extent future treatments involving PRL receptor inhibitors may interfere with processes regulated by PRL at the central level.
Assuntos
Homeostase/fisiologia , Hipófise/fisiologia , Prolactina/fisiologia , Receptores da Prolactina/antagonistas & inibidores , Animais , Homeostase/efeitos dos fármacos , Humanos , Hipófise/efeitos dos fármacos , Prolactina/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores da Prolactina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
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 â¼6.8 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.
Assuntos
Prolactina/metabolismo , Receptores da Prolactina/antagonistas & inibidores , Receptores da Prolactina/metabolismo , Calorimetria , Dicroísmo Circular , Histidina/química , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Prolactina/genética , Ligação Proteica , Conformação Proteica , Receptores da Prolactina/química , Receptores da Prolactina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
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.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroadenoma/genética , Fibroadenoma/metabolismo , Mutação de Sentido Incorreto , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismo , Adulto , Estudos de Casos e Controles , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Éxons/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Humanos , Imuno-Histoquímica , Estudos Prospectivos , Receptores da Prolactina/agonistas , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologiaRESUMO
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.
Assuntos
Receptores da Prolactina/antagonistas & inibidores , Animais , Neoplasias da Mama/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológicoRESUMO
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.
Assuntos
Neoplasias do Córtex Suprarrenal , Gônadas/fisiopatologia , Hiperplasia , Hipófise/fisiopatologia , Neoplasias do Córtex Suprarrenal/patologia , Neoplasias do Córtex Suprarrenal/fisiopatologia , Animais , Estrogênios/metabolismo , Humanos , Hiperplasia/patologia , Hiperplasia/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Orquiectomia , OvariectomiaRESUMO
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.
Assuntos
Neoplasias do Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/metabolismo , Gonadotropina Coriônica/metabolismo , Hormônio Luteinizante/metabolismo , Animais , HumanosRESUMO
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.
Assuntos
Desenho de Fármacos , Prolactina/metabolismo , Receptores da Prolactina/antagonistas & inibidores , Receptores da Prolactina/metabolismo , Receptores da Somatotropina/antagonistas & inibidores , Receptores da Somatotropina/metabolismo , Animais , Sítios de Ligação , Humanos , Mutação/genética , Receptores da Prolactina/química , Receptores da Prolactina/genéticaRESUMO
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.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Epigênese Genética/fisiologia , Genômica , Ovariectomia , Neoplasias das Glândulas Suprarrenais/fisiopatologia , Animais , Feminino , Ligação Genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Obesidade/genética , Obesidade/fisiopatologia , Fenótipo , Pós-Menopausa , Locos de Características Quantitativas , Receptores do LH/genética , Especificidade da EspécieRESUMO
Active surveillance has emerged as an alternative to immediate treatment for men with low-risk prostate cancer. Accordingly, identification of environmental factors that facilitate progression to more aggressive stages is critical for disease prevention. Although calcium-enriched diets have been speculated to increase prostate cancer risk, their impact on early-stage tumors remains unexplored. In this study, we addressed this issue with a large interventional animal study. Mouse models of fully penetrant and slowly evolving prostate tumorigenesis showed that a high calcium diet dramatically accelerated the progression of prostate intraepithelial neoplasia, by promoting cell proliferation, micro-invasion, tissue inflammation, and expression of acknowledged prostate cancer markers. Strikingly, dietary vitamin D prevented these calcium-triggered tumorigenic effects. Expression profiling and in vitro mechanistic studies showed that stimulation of PC-3 cells with extracellular Ca2+ resulted in an increase in cell proliferation rate, store-operated calcium entry (SOCE) amplitude, cationic channel TRPC6, and calcium sensing receptor (CaSR) expression. Notably, administration of the active vitamin D metabolite calcitriol reversed all these effects. Silencing CaSR or TRPC6 expression in calcium-stimulated PC3 cells decreased cell proliferation and SOCE. Overall, our results demonstrate the protective effects of vitamin D supplementation in blocking the progression of early-stage prostate lesions induced by a calcium-rich diet. Cancer Res; 77(2); 355-65. ©2016 AACR.
Assuntos
Cálcio/toxicidade , Colecalciferol/farmacologia , Dieta/efeitos adversos , Neoplasias da Próstata/patologia , Receptores de Detecção de Cálcio/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Linhagem Celular Tumoral , Suplementos Nutricionais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Canal de Cátion TRPC6 , Regulação para CimaRESUMO
CONTEXT: In a cohort of 95 women with multiple breast fibroadenomas (MFAs), we recently identified patients harboring germline heterozygous variants of the prolactin receptor (PRLR) exhibiting constitutive activity (PRLRI146L and PRLRI176V). OBJECTIVE: This study sought to better delineate the potential role of PRLR gain-of-function variants in benign and malignant mammary tumorigenesis. DESIGN: This was an observational study and transgenic mouse model analysis. SETTING: The study took place at the Department of Endocrinology, Reproductive Disorders and Rare Gynecologic Diseases, Pitié Salpêtrière, Paris, and Inserm Unit 1151, Paris. PATIENTS OR OTHER PARTICIPANTS: We generated a second MFA cohort (n = 71) as well as a group of control subjects (n = 496) and a cohort of women with breast cancer (n = 119). We also generated two transgenic mouse models carrying the coding sequences of human PRLRI146L or PRLRWT. INTERVENTION: We aimed to determine the prevalence of PRLR variants in these three populations and to uncover any association of the latter with specific tumor pattern, especially in patients with breast cancer. RESULTS: This study did not highlight a higher prevalence of PRLR variants in the MFA group and in the breast cancer group compared with control subjects. Transgenic mice expressing PRLRI146L exhibited very mild histological mammary phenotype but tumors were never observed. CONCLUSION: PRLRI146L and PRLRI176V variants are not associated with breast cancer or MFA risk. However, one cannot exclude that low but sustained PRLR signaling may facilitate or contribute to pathological development driven by oncogenic pathways. Long-term patient follow-up should help to address this issue.
Assuntos
Neoplasias da Mama/genética , Fibroadenoma/genética , Receptores da Prolactina/genética , Adolescente , Adulto , Animais , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Adulto JovemRESUMO
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 intraepithelial 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 tumorigenesis 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.
Assuntos
Progressão da Doença , Leite/metabolismo , Neoplasias da Próstata/patologia , Animais , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Dieta , Modelos Animais de Doenças , Fibrose , Hipertrofia , Inflamação/patologia , Masculino , Camundongos , Invasividade Neoplásica , Tamanho do Órgão , Próstata/patologia , Aumento de PesoRESUMO
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.
Assuntos
Prolactina/química , Prolactina/farmacologia , Sequência de Aminoácidos , Diferenciação Celular , Humanos , Dados de Sequência Molecular , Prolactina/análogos & derivados , Prolactina/metabolismo , Deleção de SequênciaRESUMO
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 5 mM 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.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Animais , Ciclo Celular , Proliferação de Células , Humanos , Receptores de Detecção de Cálcio/metabolismoRESUMO
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.
Assuntos
Prolactina/fisiologia , Animais , Neoplasias da Mama/sangue , Neoplasias da Mama/epidemiologia , Dopamina , Feminino , Humanos , Hiperprolactinemia , Masculino , Mutação , Prolactina/química , Prolactina/genética , Neoplasias da Próstata , Receptores da Prolactina/química , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismo , Fatores de Risco , Fator de Transcrição STAT5RESUMO
BACKGROUND: 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. OBJECTIVES: 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. DESIGN: 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. RESULTS: 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. CONCLUSION: 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.
Assuntos
Neoplasias da Mama/genética , Fibroadenoma/genética , Receptores da Prolactina/genética , Adolescente , Adulto , Neoplasias da Mama/diagnóstico por imagem , Estudos de Casos e Controles , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Fibroadenoma/diagnóstico por imagem , Frequência do Gene , Mutação em Linhagem Germinativa , Humanos , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Polimorfismo de Nucleotídeo Único , Prolactina/genética , Radiografia , Receptores da Prolactina/fisiologia , Análise Serial de Tecidos , Ativação Transcricional/genética , Ultrassonografia , Adulto JovemRESUMO
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.
Assuntos
Prolactina/análogos & derivados , Prolactina/farmacologia , Receptores da Prolactina/antagonistas & inibidores , Antineoplásicos/farmacologia , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Prolactina/antagonistas & inibidores , Prolactina/fisiologia , Prolactina/uso terapêutico , Receptores da Prolactina/metabolismoRESUMO
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.
Assuntos
Bioensaio/métodos , Fibroblastos/fisiologia , Prolactina/agonistas , Prolactina/antagonistas & inibidores , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Fibroblastos/citologia , Humanos , Luciferases/genética , Camundongos , Prolactina/fisiologia , Proteínas Recombinantes , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
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.