Prolactin Receptor Signaling Research Articles

1335-P: Hepatic Prolactin Receptor Signaling Is Critical for Protecting against Gestational Diabetes

Gestational diabetes mellitus (GDM) is characterized by fasting hyperglycemia and glucose intolerance during pregnancy, but with poorly understood mechanisms. We found that prolactin receptor (Prlr) is markedly upregulated in liver of pregnant female mice. These findings have spurred the hypothesis that hepatic Prlr signaling is pivotal for physiological adaptation of hepatic metabolism to gestation. To address this hypothesis, we generated liver-conditional L-Prlr-KO mice, followed by determination of glucose and lipid metabolism in female L-Prlr-KO vs. age-matched female WT mice prior to and during gestation. Hepatic Prlr depletion did not impact glucose and lipid metabolism in virgin L-Prlr-KO mice. However, pregnant L-Prlr-KO mice developed GDM, as evidenced by the induction of glucose intolerance, fasting hyperglycemia, higher HOMA-IR levels and abnormal glucose-stimulated insulin secretion at gestational day 15.5. We reproduced these findings in female L-Prlr-KO mice in the 1st, 2nd and 3rd pregnancy, with the severity of GDM exacerbated by transient high fat feeding prior to conception in female L-Prlr-KO mice. Hepatic Prlr depletion did not affect litter size, but pups born to L-Prlr-KO mice had significantly lower birth weight. We recapitulated this finding in newborns derived from the 1st, 2nd and 3rd pregnancy in L-Prlr-KO mice. Hepatic Prlr depletion also resulted in increased triglyceride and cholesterol levels in liver, characteristic of nonalcoholic fatty liver disease and consistent with increased insulin resistance in pregnant L-Prlr-KO mice. Multiparous L-Prlr-KO mice developed postpartum prediabetes. Our studies unveiled an unprecedented role of hepatic Prlr signaling in regulating maternal hepatic metabolism to protect against GDM. Disclosure C. Zhu: None. J. Yamauchi: None. C. Pan: None. J. Gao: None. S. Qu: None. R.R. Banerjee: None. H. Dong: None. Funding National Key Research and Development Program of China (2018YFC1314100); National Natural Science Foundation of China (81970677)

Diphenylbutylpiperidine Antipsychotic Drugs Inhibit Prolactin Receptor Signaling to Reduce Growth of Pancreatic Ductal Adenocarcinoma in Mice

Prolactin (PRL) signaling is up-regulated in hormone-responsive cancers. The PRL receptor (PRLR) is a class I cytokine receptor that signals via the Janus kinase (JAK)-signal transducer and activator of transcription and mitogen-activated protein kinase pathways to regulate cell proliferation, migration, stem cell features, and apoptosis. Patients with pancreatic ductal adenocarcinoma (PDAC) have high plasma levels of PRL. We investigated whether PRLR signaling contributes to the growth of pancreatic tumors in mice. We used immunohistochemical analyses to compare levels of PRL and PRLR in multitumor tissue microarrays. We used structure-based virtual screening and fragment-based drug discovery to identify compounds likely to bind PRLR and interfere with its signaling. Human pancreatic cell lines (AsPC-1, BxPC-3, Panc-1, and MiaPaCa-2), with or without knockdown of PRLR (clustered regularly interspaced short palindromic repeats or small hairpin RNA), were incubated with PRL or penfluridol and analyzed in proliferation and spheroid formation. C57BL/6 mice were given injections of UNKC-6141 cells, with or without knockdown of PRLR, into pancreas, and tumor development was monitored for 4 weeks, with some mice receiving penfluridol treatment for 21 days. Human pancreatic tumor tissues were implanted into interscapular fat pads of NSG mice, and mice were given injections of penfluridol daily for 28 days. Nude mice were given injections of Panc-1 cells, xenograft tumors were grown for 2 weeks, and mice were then given intraperitoneal penfluridol for 35 days. Tumors were collected from mice and analyzed by histology, immunohistochemistry, and immunoblots. Levels of PRLR were increased in PDAC compared with nontumor pancreatic tissues. Incubation of pancreatic cell lines with PRL activated signaling via JAK2-signal transducer and activator of transcription 3 and extracellular signal-regulated kinase, as well as formation of pancospheres and cell migration; these activities were not observed in cells with PRLR knockdown. Pancreatic cancer cells with PRLR knockdown formed significantly smaller tumors in mice. We identified several diphenylbutylpiperidine-class antipsychotic drugs as agents that decreased PRL-induced JAK2 signaling; incubation of pancreatic cancer cells with these compounds reduced their proliferation and formation of panco spheres. Injections of 1 of these compounds, penfluridol, slowed the growth of xenograft tumors in the different mouse models, reducing proliferation and inducing autophagy of the tumor cells. Levels of PRLR are increased in PDAC, and exposure to PRL increases proliferation and migration of pancreatic cancer cells. Antipsychotic drugs, such as penfluridol, block PRL signaling in pancreatic cancer cells to reduce their proliferation, induce autophagy, and slow the growth of xenograft tumors in mice. These drugs might be tested in patients with PDAC.

Lrrc55 is a novel prosurvival factor in pancreatic islets.

Pancreatic islets adapt to the increase in insulin demand during pregnancy by upregulating β-cell number, insulin synthesis, and secretion. These changes require prolactin receptor (PrlR) signaling, as mice with PrlR deletion are glucose intolerant with a lower β-cell mass. Prolactin also prevents β-cell apoptosis. Many genes participate in these adaptive changes in the islet, and Lrrc55 is one of the most upregulated genes with unknown function in islets. Because Lrrc55 expression increases in parallel to the increase in β-cell number and insulin production during pregnancy, we hypothesize that Lrrc55 might regulate β-cell proliferation/apoptosis (thus β-cell number) and insulin synthesis. Here, we found that Lrrc55 expression was upregulated by >60-fold during pregnancy in a PrlR-dependent manner, and this increase was restricted only to the islets. Overexpression of Lrrc55 in β-cells had minimal effect on β-cell proliferation and glucose-stimulated insulin secretion but protected β-cells from glucolipotoxicity-induced reduction in insulin gene expression. Moreover, Lrrc55 protects β-cells from glucolipotoxicity-induced apoptosis, with upregulation of prosurvival signals and downregulation of proapoptotic signals of the endoplasmic reticulum (ER) stress pathway. Furthermore, Lrrc55 attenuated calcium depletion induced by glucolipotoxicity, which may contribute to its antiapoptotic effect. Hence our findings suggest that Lrrc55 is a novel prosurvival factor that is upregulated specifically in islets during pregnancy, and it prevents conversion of adaptive unfolded protein response to unresolved ER stress and apoptosis in β-cells. Lrrc55 could be a potential therapeutic target in diabetes by reducing ER stress and promoting β-cell survival.

SAT-163 Prolactin Receptor Signaling Regulates a Pregnancy-Specific Transcriptional Program in Mouse Islets

Pancreatic β-cells undergo profound hyperplasia during pregnancy to maintain maternal euglycemia. Failure to reprogram β-cells into a more replicative state has been found to underlie susceptibility to gestational diabetes (GDM). Our laboratory has recently identified a requirement for prolactin receptor (PRLR) signaling in the metabolic adaptations to pregnancy, where mice lacking β-cell PRLR (βPRLRKO) exhibit a metabolic phenotype consistent with GDM. However, the underlying transcriptional program that is responsible for the PRLR-dependent metabolic adaptations during gestation remains incompletely understood. To identify PRLR signaling gene regulatory networks and target genes within β-cells during pregnancy, we performed a transcriptomic analysis of pancreatic islets isolated from either βPRLRKO mice or littermate controls in late gestation. Gene set enrichment analysis identified Forkhead box protein M1 (Foxm1) and polycomb repressor complex 2 (PRC2) subunits Suz12 and Ezh2 as novel candidate regulators of PRLR-dependent down-regulated and up-regulated genes, respectively. GO-term pathway enrichment revealed both established and novel PRLR signaling target genes that together describe a state of increased cellular metabolism and/or proliferation. In contrast to the requirement for β-cell PRLR signaling in maintaining euglycemia during pregnancy, PRLR target genes were not induced following high-fat-diet feeding. Altogether the current study develops our understanding of genes and transcriptional regulators mediating islet adaptation during pregnancy and suggests pregnancy-specific adaptive mechanisms distinct from those activated by nutritional stress.

SAT-091 Metabolic Effects of JAK1/2 Inhibition in Patients with Myeloproliferative Neoplasms

Background: Ruxolitinib is a JAK1/2 inhibitor that is FDA approved to treat certain myeloproliferative neoplasms (MPNs), including myelofibrosis (MF) and polycythemia vera (PV). The JAK family kinases are targeted in cancers due to their role in cytokine receptor signal transduction, and are also key intermediates in growth hormone, leptin and prolactin receptor signaling. Clinical trials have reported that ruxolitinib treatment was associated with weight gain, but other metabolic consequences remain unknown. Aim: We aimed to determine the metabolic consequences of JAK1/2 inhibition with ruxolitinib in patients with MPN in the clinical setting. Methods: IRB approval was obtained, and we identified patients from an electronic medical record (EMR) based database who began treatment with ruxolitinib for MPN from December 2011 to December 2016. Data collected from EMRs included age, gender, weight, height, systolic and diastolic blood pressure (SBP, DBP), medications, co-morbidities, random serum glucose, and lipid profile. We compared parameters from baseline (prior to initiating ruxolitinib) to 72±8 weeks after starting treatment. Baseline and 72 week data were compared using the Wilcoxon signed rank test. Results: 129 patients were initially identified, of which 71 had data available for weight, and at least one other metabolic parameter of interest at baseline and 72 weeks. Patient characteristics were as follows: 49.3% male; mean age at initiation of treatment was 64.6±SD11.1 years; indications for treatment were MF (77%), PV(17%), other MPNs (6%). Mean baseline weight was 73.6±16.9kg, and was 78.3±8.9kg at 72 weeks (n=71, p<0.001). Mean body mass index (BMI) at baseline was 25.6±4.6kg/m2, and 27.3±5.4kg/m2 at 72 weeks (n=68, p<0.001).The BMI (kg/m2) distribution for underweight (<18.5), normal (18.5-24.9), overweight (25.0-29.9), and obese (>30) at baseline were: 3%, 43%, 41%, and 13%, respectively; at 72 weeks it was 1%, 40%, 34%, and 28%, respectively (p=0.002). At 72 weeks,19% moved up a BMI class from baseline. Next, we investigated the effect of ruxolitinib treatment on BP and glucose concentrations. SBP was 124±15 mmHg at baseline and 129±18 mmHg at 72 weeks, (p=0.042, n=71). DBP was not different between baseline and 72 weeks. Glucose at baseline was 99±28mg/dL and 101±29mg/dL at 72 weeks (p=0.015, n=59). There was no change in the percent of patients with hyperglycemia (glucose ≥200mg/dL) or on treatment for diabetes at baseline (13.6%) and 72 weeks (13.6%). An insufficient number of patients had lipid data available for analysis. Conclusions: Systemic JAK1/2 inhibition was associated with weight gain, the development of obesity, and increased SBP in this cohort of patients. As pharmacological JAK1 and 2 inhibitors are developed and more widely used, it is important to gain a greater understanding of their long-term metabolic consequences.

Prolactin Receptor Signaling Regulates a Pregnancy-Specific Transcriptional Program in Mouse Islets.

Pancreatic β-cells undergo profound hyperplasia during pregnancy to maintain maternal euglycemia. Failure to reprogram β-cells into a more replicative state has been found to underlie susceptibility to gestational diabetes mellitus (GDM). We recently identified a requirement for prolactin receptor (PRLR) signaling in the metabolic adaptations to pregnancy, where β-cell-specific PRLR knockout (βPRLRKO) mice exhibit a metabolic phenotype consistent with GDM. However, the underlying transcriptional program that is responsible for the PRLR-dependent metabolic adaptations during gestation remains incompletely understood. To identify PRLR signaling gene regulatory networks and target genes within β-cells during pregnancy, we performed a transcriptomic analysis of pancreatic islets isolated from either βPRLRKO mice or littermate controls in late gestation. Gene set enrichment analysis identified forkhead box protein M1 and polycomb repressor complex 2 subunits, Suz12 and enhancer of zeste homolog 2 (Ezh2), as novel candidate regulators of PRLR-dependent β-cell adaptation. Gene ontology term pathway enrichment revealed both established and novel PRLR signaling target genes that together promote a state of increased cellular metabolism and/or proliferation. In contrast to the requirement for β-cell PRLR signaling in maintaining euglycemia during pregnancy, PRLR target genes were not induced following high-fat diet feeding. Collectively, the current study expands our understanding of which transcriptional regulators and networks mediate gene expression required for islet adaptation during pregnancy. The current work also supports the presence of pregnancy-specific adaptive mechanisms distinct from those activated by nutritional stress.

Pancreatic prolactin receptor signaling regulates maternal glucose homeostasis.

Prolactin (PRL) signaling has been implicated in the regulation of glucose homeostatic adaptations to pregnancy. In this report, the PRL receptor (Prlr) gene was conditionally disrupted in the pancreas, creating an animal model which proved useful for investigating the biology and pathology of gestational diabetes including its impacts on fetal and placental development. In mice, pancreatic PRLR signaling was demonstrated to be required for pregnancy-associated changes in maternal β cell mass and function. Disruption of the Prlr gene in the pancreas resulted in fewer insulin producing cells, which failed to expand appropriately during pregnancy resulting in reduced blood insulin levels and maternal glucose intolerance. This inability to sustain normal blood glucose balance during pregnancy worsened with age and a successive pregnancy. The etiology of the insulin insufficiency was attributed to deficits in regulatory pathways controlling β cell development. Additionally, the disturbance in maternal blood glucose homeostasis, was associated with fetal overgrowth and dysregulation of inflammation and prolactin-associated transcripts in the placenta. Overall, these results indicate that the PRLR, acting within the pancreas, mediates maternal pancreatic adaptations to pregnancy and therefore its dysfunction may increase a woman's chances of becoming glucose intolerant during pregnancy.

Identification of Novel Small Molecule Kinase Antagonists of the Prolactin Receptor In Breast Cancer

Breast cancer remains to be a prevalent malignancy, as an estimated 231,840 women were diagnosed in the US in 2015. Furthermore, cancer cells have developed chemoresistance, necessitating the pursuit of innovative targets. Prolactin (PRL) acting via the prolactin receptor (PRLR) is an anti‐apoptotic hormone in breast cancer. Extensive research indicates that PRLR activation in breast cancer is associated with enhanced tumor growth, invasiveness, metastasis, and resistance to chemotherapy.To identify novel antagonists of the PRLR, a 340,000 small molecule library was scanned using high throughput screening and computational modeling techniques (in‐silico docking) to observe binding potential to the PRLR. Kd and IC50 values were found using cell based assays for testing ligand binding efficiency in T47D and Ba/F3 cell lines. 2 molecules, SMI 1 and SMI 6, were identified as potential inhibitors of the PRLR. Inhibitor cytotoxicity was tested using an LDH assay, utilizing a 468 cancer cell line, to ensure drug viability as a potential treatment. An invasion assay was produced to test cell invasion when exposed to 1nM prolactin in a 468 cell line. Immunoprecipitation and western blotting were used to observe downregulation of the PRLR signaling pathway. Cancer cell proliferation after inhibitor exposure was calculated using flow cytometry.Results indicated SMI 1 and 6 had minimal toxicity, ensuring both molecules' capabilities to be therapeutic agents. Furthermore, both inhibitors reduced cell invasion, and cell proliferation when exposed to prolactin, compared to exposure to prolactin only. Additionally, the inhibitors also appear to downregulate the STAT‐5 signaling pathway. Thus, both these small molecule antagonists hold great promise in targeting the prolactin receptor as potential cancer therapeutic drugs of the future.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting the Prolactin Receptor Signaling Using an Antipsychotic Drug to Suppress Pancreatic Cancer

Pancreatic cancer (PCa) is deadly cancer with an abysmal five‐year survival rate of &lt; 6%. PCa has a bad prognosis and is associated with resistance to standard of care chemotherapeutic gemcitabine and high recurrence rate after surgery. Unfortunately, gemcitabine remains the drug of choice for metastatic pancreatic adenocarcinoma, with minor improvements in survival rate. Hence, there is a need for novel therapeutic interventions for the treatment of PCa. Here we report that prolactin receptor (PRLR) is overexpressed in both PCa tissues and in cell lines. When it is bound by the ligand prolactin (PRL), various downstream events such as JAK‐STAT and ERK‐MAPK signaling pathways are activated. In PCa cell lines, PRL treatment induced dose‐ and time‐dependent STAT3, and ERK1/2 phosphorylation. Moreover, while PRL did not affect PCa cell proliferation, it significantly enhanced pancosphere formation suggesting that PRL affects stem cells. We manipulated PRLR levels using the CRISPR/Cas9 system. This resulted in the lower colony and spheroid formation capacity, decreased migration in vitro as well as reduced tumor‐forming capacity in an orthotopic model of PCa in C57BL/6 mice. Based on these results, we propose that PRL signaling is a novel target for therapeutic intervention. Currently, there are no known small molecules targeting PRL‐PRLR signaling, and X‐ray crystal structure of the intracellular domain of PRLR is not available. Hence, we obtained a homology model for the C‐terminal intracellular region of the receptor and performed a virtual screening for small molecules in silico. In this, we demonstrated that penfluridol (a first‐generation antipsychotic drug) interacts with the JAK2 binding site of the receptor. Penfluridol is known for its long half‐life and is used in the treatment of chronic schizophrenia. We experimentally confirmed binding of penfluridol to PRLR in the cells by using the cellular thermal shift assay (CETSA). Penfluridol treatment decreased PRL‐induced STAT3 and ERK phosphorylation, spheroid formation as well as the migration of PCa cells. We observed a significant increase in autophagy based on increased levels of LC3B, p62 and enhanced expression of autophagy‐related genes ATG‐5, −7 and −12 after penfluridol treatment. Penfluridol treatment also significantly decreased tumor burden in orthotopic PCa model and induced the expression of autophagic related proteins. Taken together, these results suggest that PRL‐PRLR signaling is important for PCa growth and repurposing penfluridol to target PRL‐signaling an effective therapeutic targeting strategy for treating the disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ETS transcription factor ELF5 induces lumen formation in a 3D model of mammary morphogenesis and its expression is inhibited by Jak2 inhibitor TG101348

The loss of expression of a single gene can revert normal tissue to a malignant phenotype. For example, while normal breast has high lumenal expression of CEACAM1, the majority of breast cancers exhibit the early loss of this gene with the concurrent loss of their lumenal phenotype. MCF7 cells that lack CEACAM1 expression and fail to form lumena in 3D culture, regain the normal phenotype when transfected with CEACAM1. In order to probe the mechanism of this gain of function, we treated these cells with the clinically relevant Jak2 inhibitor TG101348 (TG), expecting that disruption of the prolactin receptor signaling pathway would interfere with the positive effects of transfection of MCF7 cells with CEACAM1. Indeed, lumen formation was inhibited, resulting in the down regulation of a set of genes, likely involved in the complex process of lumen formation. As expected, inhibition of the expression of many of these genes also inhibited lumen formation, confirming their involvement in a single pathway. Among the genes identified by the inhibition assay, ETS transcription factor ELF5 stood out, since it has been identified as a master regulator of mammary morphogenesis, and is associated with prolactin receptor signaling. When ELF5 was transfected into the parental MCF7 cells that lack CEACAM1, lumen formation was restored, indicating that ELF5 can replace CEACAM1 in this model system of lumenogenesis. We conclude that the event(s) that led to the loss of expression of CEACAM1 is epistatic in that multiple genes associated with a critical pathway were affected, but that restoration of the normal phenotype can be achieved with reactivation of certain genes at various nodal points in tissue morphogenesis.

Prolactin receptor targeting in breast and prostate cancers: New insights into an old challenge.

In the era of precision medicine, the identification of new targets is a constant challenge to improve cancer therapy. Preclinical investigations, epidemiological studies and analyses of tissue specimens from patients strongly support the contribution of prolactin receptor (PRLR) signaling to breast and prostate tumorigenesis and cancer progression. Although a clear causative link with mutations of the genes encoding prolactin or its receptor is lacking, increased PRLR signaling in these cancers can be assessed by the overexpression of cognate proteins and is often confirmed by over-activation of downstream signaling effectors. Nevertheless, the PRLR neutralizing antibody LFA102 tested recently in a Phase I trial in advanced, PRLR-positive prostate cancer and breast cancer patients failed to provide any clinical benefit. This underlines the need to better understand the actual impact of PRLR signaling on the progression of these cancers. Canonical PRLR-triggered signaling cascades include STAT5A/B, ERK1/2, PI3K/Akt, FAK and Src family kinases. Recent studies suggested that the nature and the outcome of PRLR signaling might be markedly different in breast than in prostate cancer. In the latter, like in many organs, PRLR/STAT5 signaling acts as a pro-tumorigenic pathway. In particular, it promotes the amplification of treatment-resistant prostate stem/progenitor cells, predicts early cancer recurrence and favors metastatic dissemination. In contrast, PRLR/STAT5 signaling was recently proposed to prevent breast cancer cell dissemination and to predict favorable clinical outcomes. While there is no evidence that pathways other than STAT5 are activated by prolactin in the prostate, these alternate signaling cascades may be primarily responsible for the pro-tumorigenic effects of prolactin in breast cancer. If these conclusions are confirmed in future studies, the therapeutic targeting of PRLR signaling in breast and prostate cancer may warrant the development of organ-specific strategies.

Nectin-4 co-stimulates the prolactin receptor by interacting with SOCS1 and inhibiting its activity on the JAK2-STAT5a signaling pathway

Cell-surface cytokine receptors are regulated by their cis-interacting stimulatory and inhibitory co-receptors. We previously showed that the Ig-like cell-adhesion molecule nectin-4 cis-interacts with the prolactin receptor through the extracellular region and stimulates prolactin-induced prolactin receptor activation and signaling, resulting in alveolar development in the mouse mammary gland. However, it remains unknown how this interaction stimulates these effects. We show here that the cis-interaction of the extracellular region of nectin-4 with the prolactin receptor was not sufficient for eliciting these effects and that the cytoplasmic region of nectin-4 was also required for this interaction. The cytoplasmic region of nectin-4 directly interacted with suppressor of cytokine signaling 1 (SOCS1), but not SOCS3, JAK2, or STAT5a, and inhibited the interaction of SOCS1 with JAK2, eventually resulting in the increased phosphorylation of STAT5a. The juxtamembrane region of nectin-4 interacted with the Src homology 2 domain of SOCS1. Both the interaction of nectin-4 with the extracellular region of the prolactin receptor and the interaction of SOCS1 with the cytoplasmic region of nectin-4 were required for the stimulatory effect of nectin-4 on the prolactin-induced prolactin receptor activation. The third Ig-like domain of nectin-4 and the second fibronectin type III domain of the prolactin receptor were involved in this cis-interaction, and both the extracellular and transmembrane regions of nectin-4 and the prolactin receptor were required for this direct interaction. These results indicate that nectin-4 serves as a stimulatory co-receptor for the prolactin receptor by regulating the feedback inhibition of SOCS1 in the JAK2-STAT5a signaling pathway.

Prolactin receptor in breast cancer: marker for metastatic risk.

Prolactin and prolactin receptor signaling and function are complex in nature and intricate in function. Basic, pre-clinical and translational research has opened up our eyes to the understanding that prolactin and prolactin receptor signaling function differently within different cellular contexts and microenvironmental conditions. Its multiple roles in normal physiology are subverted in cancer initiation and progression, and gradually we are teasing out the intricacies of function and therapeutic value. Recently, we observed that prolactin has a role in accelerating the time to bone metastasis in breast cancer patients and identified the mechanism by which prolactin stimulated breast cancer cell-mediated lytic osteoclast formation. The possibility that the prolactin receptor is a marker for metastasis, and specifically bone metastasis, is one that may have to be put into the context of the different variants of prolactin, different prolactin receptor isoforms and intricate signaling pathways that are regulated by the microenvironment. The more complete the picture, the better one can test biomarker identity and design clinical trials to test therapeutic intervention. This review will cover the recent advances and highlight the complexity of prolactin receptor biology.

Gain-of-function Prolactin Receptor Variants Are Not Associated With Breast Cancer and Multiple Fibroadenoma Risk.

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). This study sought to better delineate the potential role of PRLR gain-of-function variants in benign and malignant mammary tumorigenesis. This was an observational study and transgenic mouse model analysis. The study took place at the Department of Endocrinology, Reproductive Disorders and Rare Gynecologic Diseases, Pitié Salpêtrière, Paris, and Inserm Unit 1151, Paris. 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. 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. 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. 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.

Maternally involved galanin neurons in the preoptic area of the rat.

Recent selective stimulation and ablation of galanin neurons in the preoptic area of the hypothalamus established their critical role in control of maternal behaviors. Here, we identified a group of galanin neurons in the anterior commissural nucleus (ACN), and a distinct group in the medial preoptic area (MPA). Galanin neurons in ACN but not the MPA co-expressed oxytocin. We used immunodetection of phosphorylated STAT5 (pSTAT5), involved in prolactin receptor signal transduction, to evaluate the effects of suckling-induced prolactin release and found that 76% of galanin cells in ACN, but only 12% in MPA were prolactin responsive. Nerve terminals containing tuberoinfundibular peptide 39 (TIP39), a neuropeptide that mediates effects of suckling on maternal motivation, were abundant around galanin neurons in both preoptic regions. In the ACN and MPA, 89 and 82% of galanin neurons received close somatic appositions, with an average of 2.9 and 2.6 per cell, respectively. We observed perisomatic innervation of galanin neurons using correlated light and electron microscopy. The connection was excitatory based on the glutamate content of TIP39 terminals demonstrated by post-embedding immunogold electron microscopy. Injection of the anterograde tracer biotinylated dextran amine into the TIP39-expressing posterior intralaminar complex of the thalamus (PIL) demonstrated that preoptic TIP39 fibers originate in the PIL, which is activated by suckling. Thus, galanin neurons in the preoptic area of mother rats are innervated by an excitatory neuronal pathway that conveys suckling-related information. In turn, they can be topographically and neurochemically divided into two distinct cell groups, of which only one is affected by prolactin.

Unexpected severe consequences of Pikfyve deletion by aP2- or Aq-promoter-driven Cre expression for glucose homeostasis and mammary gland development.

Systemic deficiency of PIKfyve, the evolutionarily conserved phosphoinositide kinase synthesizing cellular PtdIns5P and PtdIns(3,5)P2 and implicated in insulin signaling, causes early embryonic death in mice. In contrast, mice with muscle‐specific Pikfyve disruption have normal lifespan but exhibit early‐age whole‐body glucose intolerance and muscle insulin resistance, thus establishing the key role of muscle PIKfyve in glucose homeostasis. Fat and muscle tissues control postprandial glucose clearance through different mechanisms, raising questions as to whether adipose Pikfyve disruption will also trigger whole‐body metabolic abnormalities, and if so, what the mechanism might be. To clarify these issues, here we have characterized two new mouse models with adipose tissue disruption of Pikfyve through Cre recombinase expression driven by adipose‐specific aP2‐ or adiponectin (Aq) promoters. Whereas both mouse lines were ostensibly normal until adulthood, their glucose homeostasis and systemic insulin sensitivity were severely dysregulated. These abnormalities stemmed in part from accelerated fat‐cell lipolysis and elevated serum FFA. Intriguingly, aP2‐Cre‐PIKfyvefl/fl but not Aq‐Cre‐PIKfyvefl/fl females had severely impaired pregnancy‐induced mammary gland differentiation and lactogenesis, consistent with aP2‐Cre‐mediated Pikfyve excision in nonadipogenic tissues underlying this defect. Intriguingly, whereas mammary glands from postpartum control and Aq‐Cre‐PIKfyvefl/fl mice or ex vivo mammary gland explants showed profound upregulation of PIKfyve protein levels subsequent to prolactin receptor activation, such increases were not apparent in aP2‐Cre‐PIKfyvefl/fl females. Collectively, our data identify for the first time that adipose tissue Pikfyve plays a key role in the mechanisms regulating glucose homeostasis and that the PIKfyve pathway is critical in mammary epithelial differentiation during pregnancy and lactogenesis downstream of prolactin receptor signaling.

Gestational Diabetes Mellitus From Inactivation of Prolactin Receptor and MafB in Islet β-Cells.

β-Cell proliferation and expansion during pregnancy are crucial for maintaining euglycemia in response to increased metabolic demands placed on the mother. Prolactin and placental lactogen signal through the prolactin receptor (PRLR) and contribute to adaptive β-cell responses in pregnancy; however, the in vivo requirement for PRLR signaling specifically in maternal β-cell adaptations remains unknown. We generated a floxed allele of Prlr, allowing conditional loss of PRLR in β-cells. In this study, we show that loss of PRLR signaling in β-cells results in gestational diabetes mellitus (GDM), reduced β-cell proliferation, and failure to expand β-cell mass during pregnancy. Targeted PRLR loss in maternal β-cells in vivo impaired expression of the transcription factor Foxm1, both G1/S and G2/M cyclins, tryptophan hydroxylase 1 (Tph1), and islet serotonin production, for which synthesis requires Tph1. This conditional system also revealed that PRLR signaling is required for the transient gestational expression of the transcription factor MafB within a subset of β-cells during pregnancy. MafB deletion in maternal β-cells also produced GDM, with inadequate β-cell expansion accompanied by failure to induce PRLR-dependent target genes regulating β-cell proliferation. These results unveil molecular roles for PRLR signaling in orchestrating the physiologic expansion of maternal β-cells during pregnancy.

Targeting Prolactin Receptor (PRLR) Signaling in PRLR-Positive Breast and Prostate Cancer.

In this issue of The Oncologist, Agarwal et al. report negative results from a phase I trial of LFA102. Although “negative” in terms of antitumor activity, the study provides useful pharmacokinetic and pharmacodynamic information. Future trials evaluating PRLR blockers alone and in combination with other agents may still be warranted in patients with breast and prostate cancer.

A Novel Nectin-mediated Cell Adhesion Apparatus That Is Implicated in Prolactin Receptor Signaling for Mammary Gland Development

Mammary gland development is induced by the actions of various hormones to form a structure consisting of collecting ducts and milk-secreting alveoli, which comprise two types of epithelial cells known as luminal and basal cells. These cells adhere to each other by cell adhesion apparatuses whose roles in hormone-dependent mammary gland development remain largely unknown. Here we identified a novel cell adhesion apparatus at the boundary between the luminal and basal cells in addition to desmosomes. This apparatus was formed by the trans-interaction between the cell adhesion molecules nectin-4 and nectin-1, which were expressed in the luminal and basal cells, respectively. Nectin-4 of this apparatus further cis-interacted with the prolactin receptor in the luminal cells to enhance the prolactin-induced prolactin receptor signaling for alveolar development with lactogenic differentiation. Thus, a novel nectin-mediated cell adhesion apparatus regulates the prolactin receptor signaling for mammary gland development.

Germline Prolactin Receptor Mutation Is Not a Major Cause of Sporadic Prolactinoma in Humans

Background/Aims: No genetic anomalies specifically predisposing humans to prolactinomas have so far been identified. The prolactin receptor (PRLR) is a good candidate, however, as Prlr knockout mice develop prolactinomas, and a case of familial hyperprolactinemia has been linked to PRLR mutation. The main objective of this study was to detect germline PRLR mutations in patients with sporadic prolactinomas unrelated to AIP or MEN1 mutation. Methods: We sequenced all PRLR exons and intron-exon junctions on genomic DNA from 88 patients with a median age of 24 years. Results: We identified 4 PRLR variations (p.Ile76Val, p.Ile146Leu, p.Glu108Lys and p.Glu554Gln) in 16 patients. One patient had the rare variant p.Glu554Gln in the heterozygous state. Another patient had the extremely rare p.Glu108Lys variant described here for the first time. The other 2 variants (p.Ile76Val and p.Ile146Leu) are relatively common in the general population. All these 4 variants have been functionally tested in vitro and have no effect on PRLR expression, localization and signaling after prolactin stimulation. Conclusion: Inactivating germline variations of PRLR are not associated with sporadic prolactinoma in this series. Nevertheless, somatic disruption of PRLR has not been excluded in this subset of pituitary tumors.