A Novel Mechanism of hPRL-G129R, a Prolactin Antagonist, Inhibits Human Breast Cancer Cell Proliferation and Migration.

Prolactin (PRL) and its receptor, PRLR, are closely related to the occurrence and development of breast cancer. hPRL-G129R, an hPRLR antagonist, has been found to induce apoptosis in breast cancer cells via mechanisms currently unknown. Recent studies have indicated that PRLR exhibits dual functions based on its membrane/nucleus localization. In that context, we speculated whether hPRL-G129R is a dual-function antagonist. We studied the internalization of the hPRLR-G129R/PRLR complex using indirect immunofluorescence and Western blot assays. We found that hPRL-G129R not only inhibited PRLR-mediated intracellular signaling at the plasma membrane, but also blocked nuclear localization of the receptor in T-47D and MCF-7 cells in a time-dependent manner. Clone formation and transwell migration assays showed that hPRL-G129R inhibited PRL-driven proliferation and migration of tumor cells in vitro. Further, we found that increasing concentrations of hPRL-G129R inhibited the nuclear localization of PRLR and the levels of signal transducer and activator of transcription (STAT) 5 in tumor-bearing mice and hPRL-G129R also exerted an antiproliferative effect in vivo. These results indicate that hPRL-G129R is indeed a dual-function antagonist. This study lays a foundation for exploring and developing highly effective agents against the proliferation and progression of breast malignancies.

Structure and function of a dual antagonist of the human growth hormone and prolactin receptors with site-specific PEG conjugates.

Human growth hormone (hGH) is a pituitary-derived endocrine protein that regulates several critical postnatal physiologic processes including growth, organ development, and metabolism. Following adulthood, GH is also a regulator of multiple pathologies like fibrosis, cancer, and diabetes. Therefore, there is a significant pharmaceutical interest in developing antagonists of hGH action. Currently, there is a single FDA-approved antagonist of the hGH receptor (hGHR) prescribed for treating patients with acromegaly and discovered in our laboratory almost 3 decades ago. Here, we present the first data on the structure and function of a new set of protein antagonists with the full range of hGH actions-dual antagonists of hGH binding to the GHR as well as that of hGH binding to the prolactin receptor. We describe the site-specific PEG conjugation, purification, and subsequent characterization using MALDI-TOF, size-exclusion chromatography, thermostability, and biochemical activity in terms of ELISA-based binding affinities with GHR and prolactin receptor. Moreover, these novel hGHR antagonists display distinct antagonism of GH-induced GHR intracellular signaling in vitro and marked reduction in hepatic insulin-like growth factor 1 output in vivo. Lastly, we observed potent anticancer biological efficacies of these novel hGHR antagonists against human cancer cell lines. In conclusion, we propose that these new GHR antagonists have potential for development towards multiple clinical applications related to GH-associated pathologies.

The effects of prolactin receptor blockade in a murine endometriosis interna model.

Endometriosis in an estrogen-dependent disease that is characterized by the presence of endometrial tissue outside the uterine cavity leading to pain and infertility in many affected women. Highly efficient treatment options which create a hypo-estrogenic environment can cause side effects such as hot flushes and bone mass loss that are not favorable for premenopausal women. Previous work has demonstrated that increased local or systemic prolactin seems to be involved in the pathogenesis of endometriosis. Here we examined two prolactin receptor (PRLR) blocking antibodies in a murine endometriosis interna model which relies on the induction of systemic hyperprolactinemia in female SHN mice. The severity of the disease is determined by the degree of endometrial invasion into the myometrium. In this model, endometriosis was inhibited by clinical gold standards such as progestins and anti-estrogenic approaches. PRLR blockade completely inhibited endometriosis in this mouse model to the same extent as the anti-estrogen faslodex or the GnRH antagonist cetrorelix. In contrast to cetrorelix and faslodex, the PRLR antibodies did not decrease relative uterine weights and were thus devoid of anti-estrogenic effects. We therefore hypothesize that PRLR antibodies may present a novel and highly efficient treatment option for endometriosis with a good safety and tolerability profile. Clinical studies are on the way to test this hypothesis.

Progesterone receptor isoform-dependent cross-talk between prolactin and fatty acid synthase in breast cancer.

Progesterone receptor (PR) isoforms can drive unique phenotypes in luminal breast cancer (BC). Here, we hypothesized that PR-B and PR-A isoforms differentially modify the cross-talk between prolactin and fatty acid synthase (FASN) in BC. We profiled the responsiveness of the FASN gene promoter to prolactin in T47Dco BC cells constitutively expressing PR-A and PR-B, in the PR-null variant T47D-Y cell line, and in PR-null T47D-Y cells engineered to stably re-express PR-A (T47D-YA) or PR-B (T47D-YB). The capacity of prolactin to up-regulate FASN gene promoter activity in T47Dco cells was lost in T47D-Y and TD47-YA cells. Constitutively up-regulated FASN gene expression in T47-YB cells and its further stimulation by prolactin were both suppressed by the prolactin receptor antagonist hPRL-G129R. The ability of the FASN inhibitor C75 to decrease prolactin secretion was more conspicuous in T47-YB cells. In T47D-Y cells, which secreted notably less prolactin and downregulated prolactin receptor expression relative to T47Dco cells, FASN blockade resulted in an augmented secretion of prolactin and up-regulation of prolactin receptor expression. Our data reveal unforeseen PR-B isoform-specific regulatory actions in the cross-talk between prolactin and FASN signaling in BC. These findings might provide new PR-B/FASN-centered predictive and therapeutic modalities in luminal intrinsic BC subtypes.

Blockade of the Short Form of Prolactin Receptor Induces FOXO3a/EIF-4EBP1-Mediated Cell Death in Uterine Cancer.

Abnormal activity of human prolactin (PRL) and its membrane-associated receptor (PRLR) contributes to the progression of uterine carcinoma. However, the underlying mechanisms are not well understood, and current means of targeting the PRL/PRLR axis in uterine cancer are limited. Our integrated analyses using The Cancer Genome Atlas and Genotype-Tissue Expression (GTEx) databases demonstrated that a short form of PRLR (PRLR_SF) is the isoform predominantly expressed in human uterine cancers; expression of this PRLR_SF was elevated in uterine cancers in comparison with cancer-free uterine tissues. We hypothesized that the overexpression of PRLR_SF in uterine cancer cells contributes, in part, to the oncogenic activity of the PRL/PRLR axis. Next, we employed G129R, an antagonist of human PRL, to block the PRL/PRLR axis in both PTEN wt and PTEN mut orthotopic mouse models of uterine cancer. In comparison with control groups, treatment with G129R as monotherapy or in combination with paclitaxel resulted in a significant reduction of growth and progression of orthotopic uterine tumors. Results from protein profiling of uterine cancer cells and in vivo tumors revealed a set of new downstream targets for G129R. Our results showed that G129R induced sub-G0 population arrest, decreased nascent protein synthesis, and initiated FOXO3a/EIF-4EBP1-mediated cell death in both PTEN wt and PTEN mut uterine cancer cells. Collectively, our results show a unique pattern of PRLR_SF expression predominantly in uterine cancer. Moreover, FOXO3a and EIF-4EBP1 are important mediators of cell death following G129R treatment in uterine cancer models.

A first-in-human, phase 1, dose-escalation study of ABBV-176, an antibody-drug conjugate targeting the prolactin receptor, in patients with advanced solid tumors.

ABBV-176 is an antibody-drug conjugate composed of the humanized antibody h16f (PR-1594804) conjugated to a highly potent, cytotoxic cross-linking pyrrolobenzodiazepine dimer (PBD; SGD-1882) targeting the prolactin receptor (PRLR), which is overexpressed in several solid tumor types. This phase 1, dose-escalation study (NCT03145909) evaluated the safety, pharmacokinetics, and preliminary activity of ABBV-176 in patients with advanced solid tumors likely to exhibit elevated levels of PRLR. Patients received ABBV-176 once every 3 weeks. Dose escalation was by an exposure-adjusted, continual reassessment method. Dose-limiting toxicities (DLTs) were assessed from the first day of dosing until the next dose of ABBV-176 to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Nineteen patients received ABBV-176 at doses from 2.7-109.35 µg/kg. Patients enrolled had colorectal cancer (n = 11), breast cancer (n = 6), or adrenocortical carcinoma (n = 2). DLTs occurred in 4 patients and included thrombocytopenia (n = 2; both at 99.9-µg/kg dose level), neutropenia (n = 2; 78.3-µg/kg and 99.9-µg/kg dose levels), and pancytopenia (n = 1; 109.35-µg/kg dose level). The most common treatment-emergent adverse events related to ABBV-176 were thrombocytopenia, neutropenia, increased aspartate aminotransferase, nausea, fatigue, and pleural effusions. Effusions and edema were common, and timing of onset suggested possible cumulative ABBV-176 toxicity. Tumor expression of PRLR varied among patients enrolled and analyzed. No patient had an objective response. MTD was not formally determined, as identification of a tolerable dose was confounded by late-onset toxicities. ABBV-176 was associated with significant toxicity in this phase 1, dose-escalation study. Although cytopenias were often dose limiting, effusions and edema were also common and had late onset that suggested cumulative toxicity. No responses were observed, although data were available from a small number of patients with variable tumor PRLR expression. This study was terminated after the dosing of 19 patients.

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

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

An in vivo half-life extended prolactin receptor antagonist can prevent STAT5 phosphorylation.

Increasing evidence suggests that signaling through the prolactin/prolactin receptor axis is important for stimulation the growth of many cancers including glioblastoma multiforme, breast and ovarian carcinoma. Efficient inhibitors of signaling have previously been developed but their applicability as cancer drugs is limited by the short in vivo half-life. In this study, we show that a fusion protein, consisting of the prolactin receptor antagonist PrlRA and an albumin binding domain for half-life extension can be expressed as inclusion bodies in Escherichia coli and efficiently refolded and purified to homogeneity. The fusion protein was found to have strong affinity for the two intended targets: the prolactin receptor (KD = 2.3±0.2 nM) and mouse serum albumin (KD = 0.38±0.01 nM). Further investigation showed that it could efficiently prevent prolactin mediated phosphorylation of STAT5 at 100 nM concentration and above, similar to the PrlRA itself, suggesting a potential as drug for cancer therapy in the future. Complexion with HSA weakened the affinity for the receptor to 21±3 nM, however the ability to prevent phosphorylation of STAT5 was still prominent. Injection into rats showed a 100-fold higher concentration in blood after 24 h compared to PrlRA itself.

Monoclonal Antibody Against Prolactin Receptor: A Randomized Placebo-Controlled Study Evaluating Safety, Tolerability, and Pharmacokinetics of Repeated Subcutaneous Administrations in Postmenopausal Women.

BAY 1158061 is a potent monoclonal prolactin (PRL) receptor antibody, blocking PRL receptor (PRLR)-mediated signaling in a noncompetitive manner, which was tested in a randomized, placebo-controlled multiple dose study in postmenopausal women. The objective was to investigate safety, tolerability, pharmacokinetic characteristics, and effects of BAY 1158061 on serum PRL level. The study consisted of 4 parallel groups receiving up to 3 subcutaneous (sc) administrations of BAY 1158061 or placebo in 2 different dosing regimens. Twenty-nine healthy postmenopausal women were randomized and treated with BAY 1158061 or placebo: 30 mg at 14-day interval (7 participants), 60 mg at 28-day interval (8 participants), 90 mg at 14-day interval (7 participants), and placebo (7 participants). To keep the blinding, all randomized participants received sc injections biweekly (14-day interval) on 3 occasions in the lower abdomen. The PRLR antibody showed a favorable safety and tolerability profile in postmenopausal women with no distinct differences in occurrence of adverse events in BAY 1158061 or placebo-treated participants. BAY 1158061 displayed low immunogenicity with low titers of antidrug antibodies and absence of neutralizing antidrug antibodies. Pharmacokinetics were characterized by slow absorption after sc administration with median peak plasma concentrations 7 to 11 days after first dose and about 2-fold accumulation after repeated dosing every 2 weeks. The apparent mean elimination half-life was 9 to 16 days. The PRL concentration-time profiles over 24 hours showed no differences between verum- and placebo-treated participants. Based on the data obtained, BAY 1158061 is considered a good candidate for further development in endometriosis or other PRL-mediated disease conditions.

JAK2/STAT5 Pathway Mediates Prolactin-Induced Apoptosis of Lactotropes.

Prolactinomas are increasingly viewed as a "problem of signal transduction." Consequently, the identification of factors and signaling pathways that control lactotrope cell turnover is needed in order to encourage new therapeutic developments. We have previously shown that prolactin (PRL) acts as a proapoptotic and antiproliferative factor on lactotropes, maintaining anterior pituitary cell homeostasis, which contrasts with the classical antiapoptotic and/or proliferative actions exerted by PRL in most other target tissues. We aimed to investigate the PRLR-triggered signaling pathways mediating these nonclassical effects of PRL in the pituitary. Our results suggest that (i) the PRLR/Jak2/STAT5 pathway is constitutively active in GH3 cells and contributes to PRL-induced apoptosis by increasing the Bax/Bcl-2 ratio, (ii) PRL inhibits ERK1/2 and Akt phosphorylation, thereby contributing to its proapoptotic effect, and (iii) the PI3K/Akt pathway participates in the PRL-mediated control of lactotrope proliferation. We hypothesize that the alteration of PRL actions in lactotrope homeostasis due to the dysregulation of any of the mechanisms of actions described above may contribute to the pathogenesis of prolactinomas.

Aberrantly high expression of the CUB and zona pellucida-like domain-containing protein 1 (CUZD1) in mammary epithelium leads to breast tumorigenesis.

The peptide hormone prolactin (PRL) and certain members of the epidermal growth factor (EGF) family play central roles in mammary gland development and physiology, and their dysregulation has been implicated in mammary tumorigenesis. Our recent studies have revealed that the CUB and zona pellucida-like domain-containing protein 1 (CUZD1) is a critical factor for PRL-mediated activation of the transcription factor STAT5 in mouse mammary epithelium. Of note, CUZD1 controls production of a specific subset of the EGF family growth factors and consequent activation of their receptors. Here, we found that consistent with this finding, CUZD1 overexpression in non-transformed mammary epithelial HC11 cells increases their proliferation and induces tumorigenic characteristics in these cells. When introduced orthotopically in mouse mammary glands, these cells formed adenocarcinomas, exhibiting elevated levels of STAT5 phosphorylation and activation of the EGF signaling pathway. Selective blockade of STAT5 phosphorylation by pimozide, a small-molecule inhibitor, markedly reduced the production of the EGF family growth factors and inhibited PRL-induced tumor cell proliferation in vitro Pimozide administration to mice also suppressed CUZD1-driven mammary tumorigenesis in vivo Analysis of human MCF7 breast cancer cells indicated that CUZD1 controls the production of the same subset of EGF family members in these cells as in the mouse. Moreover, pimozide treatment reduced the proliferation of these cancer cells. Collectively, these findings indicate that overexpression of CUZD1, a regulator of growth factor pathways controlled by PRL and STAT5, promotes mammary tumorigenesis. Blockade of the STAT5 signaling pathway downstream of CUZD1 may offer a therapeutic strategy for managing these breast tumors.

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.

Internal image anti-idiotypic antibody: A new strategy for the development a new category of prolactin receptor (PRLR) antagonist.

Over the past decades, a number of prolactin receptor (PRLR) antagonists have been developed, which can be divided into two categories, PRLR analogue and anti-PRLR antibody. However, until now, there have been no commercially available PRLR antagonists. Here, we described a new approach for the preparation of PRLR antagonist, namely internal image anti-idiotypic antibody strategy. The hybridoma technique was used to generate anti-idiotypic antibodies to PRL. Competitive ELISA, competitive receptor-binding analysis and immunofluorescence assay (IFA) were then used to screen and characterize anti-idiotypic antibodies to PRL. One internal image anti-idiotypic antibody, termed MG7, was obtained. A series of experiments demonstrated that MG7 behaved as a typical internal image anti-idiotypic antibody (Ab2ß). MG7 exhibited effective antagonistic activity, which not only inhibited PRL binding to PRLR in a dose-dependent manner but also inhibited PRLR-mediated intracellular signalling. Furthermore, MG7 also blocked Nb2 cell proliferation induced by PRL. The current study suggests that MG7 has the potential application in the PRL/PRLR-related studies in future. In addition, this work also suggests that the internal image anti-idiotypic antibody may represent a novel strategy for the development of PRLR antagonist.

Bispecific Antibodies and Antibody-Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs.

The properties of cell surface proteins targeted by antibody-drug conjugates (ADCs) have not been fully exploited; of particular importance are the rate of internalization and the route of intracellular trafficking. In this study, we compared the trafficking of HER2, which is the target of the clinically approved ADC ado-trastuzumab emtansine (T-DM1), with that of prolactin receptor (PRLR), another potential target in breast cancer. In contrast to HER2, we found that PRLR is rapidly and constitutively internalized, and traffics efficiently to lysosomes, where it is degraded. The PRLR cytoplasmic domain is necessary to promote rapid internalization and degradation, and when transferred to HER2, enhances HER2 degradation. In accordance with these findings, low levels of cell surface PRLR (∼30,000 surface receptors per cell) are sufficient to mediate effective killing by PRLR ADC, whereas cell killing by HER2 ADC requires higher levels of cell surface HER2 (∼106 surface receptors per cell). Noncovalently cross-linking HER2 to PRLR at the cell surface, using a bispecific antibody that binds to both receptors, dramatically enhances the degradation of HER2 as well as the cell killing activity of a noncompeting HER2 ADC. Furthermore, in breast cancer cells that coexpress HER2 and PRLR, a HER2xPRLR bispecific ADC kills more effectively than HER2 ADC. These results emphasize that intracellular trafficking of ADC targets is a key property for their activity and, further, that coupling an ADC target to a rapidly internalizing protein may be a useful approach to enhance internalization and cell killing activity of ADCs. Mol Cancer Ther; 16(4); 681-93. ©2017 AACR.

Development of a new anti-prolactin receptor (PRLR) antibody, F56, which can serve as a PRLR antagonist.

In this work, we developed a new prolactin receptor (PRLR) antagonist using the hybridoma technique. A series of monoclonal antibodies against prolactin receptor (PRLR) was prepared, from which we characterized and selected one anti-PRLR antibody, F56. Epitome mapping showed that F56 and prolactin (PRL) share a common binding epitope on PRLR, and therefore, F56 could compete with prolactin (PRL) for binding to PRLR. Subsequent experiments indicated that F56 could effectively neutralize PRLR-mediated intracellular signalling molecules, such as signal transducer and activator of transcription (STAT) and extracellular signal-regulated kinase-1 and kinase 2 (ERK1/2), either by endogenously expressed PRLR or in a cell model transfected with PRLR. In addition, further experiments showed that F56 could effectively inhibit PRL-induced cell proliferation. The current study suggests that F56 has potential applications in PRLR-related studies.

Stimulation of prolactin receptor induces STAT-5 phosphorylation and cellular invasion in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in humans and is characterized with poor outcome. In this study, we investigated components of prolactin (Prl) system in cell models of GBM and in histological tissue sections obtained from GBM patients. Expression of Prolactin receptor (PrlR) was detected at high levels in U251-MG, at low levels in U87-MG and barely detectable in U373 cell lines and in 66% of brain tumor tissues from 32 GBM patients by immunohistochemical technique. In addition, stimulation of U251-MG and U87-MG cells but not U373 with Prl resulted in increased STAT5 phosphorylation and only in U251-MG cells with increased cellular invasion. Furthermore, STAT5 phosphorylation and cellular invasion induced in Prl stimulated cells were significantly reduced by using a Prl receptor antagonist that consists of Prl with four amino acid replacements. We conclude that Prl receptor is expressed at different levels in the majority of GBM tumors and that blocking of PrlR in U251-MG cells significantly reduce cellular invasion.

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.

Phase I Study of the Prolactin Receptor Antagonist LFA102 in Metastatic Breast and Castration-Resistant Prostate Cancer.

LESSONS LEARNED: Despite evidence for a role for prolactin signaling in breast and prostate tumorigenesis, a prolactin receptor-binding monoclonal antibody has not produced clinical efficacy.Increased serum prolactin levels may be a biomarker for prolactin receptor inhibition.Results from the pharmacokinetic and pharmacodynamics (PD) studies suggest that inappropriately long dosing intervals and insufficient exposure to LFA102 may have resulted in lack of antitumor efficacy.Based on preclinical data, combination therapy of LFA102 with those novel agents targeting hormonal pathways in metastatic castration-resistant prostate cancer and metastatic breast cancer is promising.Given the PD evidence of prolactin receptor blockade by LFA102, this drug has the potential to be used in conditions such as hyperprolactinemia that are associated with high prolactin levels. BACKGROUND: Prolactin receptor (PRLR) signaling is implicated in breast and prostate cancer. LFA102, a humanized monoclonal antibody (mAb) that binds to and inhibits the PRLR, has exhibited promising preclinical antitumor activity. METHODS: Patients with PRLR-positive metastatic breast cancer (MBC) or metastatic castration-resistant prostate cancer (mCRPC) received doses of LFA102 at 3-60 mg/kg intravenously once every 4 weeks. Objectives were to determine the maximum tolerated dose (MTD) and/or recommended dose for expansion (RDE) to investigate the safety/tolerability of LFA102 and to assess pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity. RESULTS: A total of 73 patients were enrolled at 5 dose levels. The MTD was not reached because of lack of dose-limiting toxicities. The RDE was established at 60 mg/kg based on PK and PD analysis and safety data. The most common all-cause adverse events (AEs) were fatigue (44%) and nausea (33%) regardless of relationship. Grade 3/4 AEs reported to be related to LFA102 occurred in 4% of patients. LFA102 exposure increased approximately dose proportionally across the doses tested. Serum prolactin levels increased in response to LFA102 administration, suggesting its potential as a biomarker for PRLR inhibition. No antitumor activity was detected. CONCLUSION: Treatment with LFA102 was safe and well tolerated, but did not show antitumor activity as monotherapy at the doses tested.

Different intracellular signalling pathways triggered by an anti-prolactin receptor (PRLR) antibody: Implication for a signal-specific PRLR agonist.

In this work, we prepared a panel of monoclonal antibodies directed against prolactin receptor (PRLR) using the hybridoma technique. Of these monoclonal antibodies (Mabs), the Mab designated B6 was chosen for further characterization based on its biological activity. We first demonstrated that B6 can specifically bind to the prolactin receptor (PRLR) expressed on target cells by immunoprecipitation and Western blotting analysis. Subsequently, epitope mapping studies using a competitive receptor-binding assay indicated that B6 epitopes partially overlapped with those of prolactin (PRL). We then examined the resulting signal transduction pathways activated by this antibody in T-47D and CHO-PRLR cells and found that B6 induced different intracellular signalling compared with prolactin, which activates serine-threonine kinase (AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription1 (STAT1) and STAT3 but not STAT5. The present study suggests that: (a) B6 may be a signal-specific prolactin receptor (PRLR) agonist; (b) B6 may be a biological reagent that can be used to explore the mechanism of PRLR-mediated intracellular signalling. In addition, this work also implies a strategy for preparing signal-specific cytokine agonists.

Prolactin potentiates the activity of acid-sensing ion channels in female rat primary sensory neurons.

Prolactin (PRL) is a polypeptide hormone produced and released from the pituitary and extrapituitary tissues. It regulates activity of nociceptors and causes hyperalgesia in pain conditions, but little is known the molecular mechanism. We report here that PRL can exert a potentiating effect on the functional activity of acid-sensing ion channels (ASICs), key sensors for extracellular protons. First, PRL dose-dependently increased the amplitude of ASIC currents with an EC50 of (5.89 ± 0.28) × 10(-8) M. PRL potentiation of ASIC currents was also pH dependent. Second, PRL potentiation of ASIC currents was blocked by Δ1-9-G129R-hPRL, a PRL receptor antagonist, and removed by intracellular dialysis of either protein kinase C inhibitor GF109203X, protein interacting with C-kinase 1(PICK1) inhibitor FSC-231, or PI3K inhibitor AS605240. Third, PRL altered acidosis-evoked membrane excitability of DRG neurons and caused a significant increase in the amplitude of the depolarization and the number of spikes induced by acid stimuli. Four, PRL exacerbated nociceptive responses to injection of acetic acid in female rats. Finally, PRL displayed a stronger effect on ASIC mediated-currents and nociceptive behavior in intact female rats than OVX female and male rats and thus modulation of PRL may be gender-dependent. These results suggest that PRL up-regulates the activity of ASICs and enhances ASIC mediated nociceptive responses in female rats, which reveal a novel peripheral mechanism underlying PRL involvement in hyperalgesia.