A novel function of prolactin signaling in brain metastasis in HER2-positive breast cancer

A novel function of prolactin signaling in brain metastasis in HER2-positive breast cancer is sponsored by National Cancer Institute (NCI) - NIH. This is a research project focused on elucidating and therapeutically targeting mechanisms that enable HER2-positive breast cancer cells to colonize and thrive in the brain. The long-term goal is to understand and target prolactin signaling in brain metastasis.

Prolactin receptor signaling: A novel target for cancer treatment - Exploring anti-PRLR signaling strategies - PMC As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Front Endocrinol (Lausanne) .

2023 Jan 13;13:1112987. doi: 10. 3389/fendo.

2022.

1112987 Prolactin receptor signaling: A novel target for cancer treatment - Exploring anti-PRLR signaling strategies Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, United States Find articles by David Standing Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, United States Find articles by Prasad Dandawate Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, United States Find articles by Shrikant Anant Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, United States Edited by: Vincent Goffin, INSERM U1151 Institut Necker Enfants Malades, France Reviewed by: Dana Borcherding, Washington University in St.

Louis, United States; John Kopchick, Ohio University, United States ✉ *Correspondence: Shrikant Anant, sanant@kumc. edu This article was submitted to Translational Endocrinology, a section of the journal Frontiers in Endocrinology Received 2022 Nov 30; Accepted 2022 Dec 22; Collection date 2022. PMCID: PMC9880166 PMID: 36714582 Prolactin (PRL) is a peptide hormone mainly secreted from the anterior pituitary gland.

PRL is reported to play a role in pregnancy, mammary gland development, immune modulation, reproduction, and differentiation of islet cells. PRL binds to its receptor PRLR, which belongs to a superfamily of the class I cytokine receptor that has no intrinsic kinase activity. In canonical signaling, PRL binding to PRLR induces downstream signaling including JAK-STAT, AKT and MAPK pathways.

This leads to increased cell proliferation, stemness, migration, apoptosis inhibition, and resistance to chemotherapy. PRL-signaling is upregulated in numerous hormone-dependent cancers including breast, prostate, ovarian, and endometrial cancer. However, more recently, the pathway has been reported to play a tumor-promoting role in other cancer types such as colon, pancreas, and hepatocellular cancers.

Hence, the signaling pathway is an attractive target for drug development with blockade of the receptor being a potential therapeutic approach.

Different strategies have been developed to target this receptor including modification of PRL peptides (Del1-9-G129R-hPRL, G129R-Prl), growth hormone receptor/prolactin receptor bispecific antibody antagonist, neutralizing antibody LFA102, an antibody-drug conjugate (ABBV-176) of the humanized antibody h16f (PR-1594804) and pyrrolobenzodiazepine dimer, a bispecific antibody targeting both PRLR and CD3, an in vivo half-life extended fusion protein containing PRLR antagonist PrlRA and albumin binding domain.

There have also been attempts to discover and develop small molecular inhibitors targeting PRLR. Recently, using structure-based virtual screening, we identified a few antipsychotic drugs including penfluridol as a molecule that inhibits PRL-signaling to inhibit PDAC tumor progression.

In this review, we will summarize the recent advances in the biology of this receptor in cancer and give an account of PRLR antagonist development for the treatment of cancer.

Keywords: PrlR, antagonist, small molecule inhibitor, immunotherapy, antibody-drug conjugate Prolactin (PRL) and its cognate receptor, prolactin receptor (PRLR), have been characterized in hundreds of biological functions, especially mammary gland development and lactation. PRL is a peptide hormone that resembles the growth hormone due to a conserved helix bundle composition.

It is largely produced by the lactotrope cells of the anterior pituitary gland as a pro-hormone that undergoes proteolytic cleavage to produce a 199 amino acid active peptide ( 1 ). However, aberrant PRL levels are also observed in disease states, which may also be related to its synthesis from the affected tissues including the prostate, skin, adipose tissue, endometrium, myometrium, immune cells, brain, and breast tissues ( 2 ).

It can therefore participate in paracrine and autocrine signaling functions related to cell homeostasis and growth ( 3 ).

Composed of 4 parallel alpha helices, PRL, binds to PRLR via several residues, including Lys-69, Tyr-169, and H180 of Site 1, and Arg-24, Lys-124 within the Gly129 cavity and Glu-43 within the N-terminus of Site 2, stimulating dimerization of PRLR on the cell surface, leading to activation of canonical signaling via Janus kinase (JAK)-signal transducer and activator of transcription (STAT) ( Schematic of PRL : PRLR signaling.

PRL binds to PRLR, inducing JAK2 association that leads to downstream activation of multiple pathways that include STAT3, STAT5, PI3K, AKT, and ERK. Extrapituitary prolactin is thought to be regulated primarily at the transcriptional and translational level. In contrast, lactotrope cells have large vacuolar stores of PRL, which can be released by calcium-dependent exocytosis.

Transcription of PRL mRNA in tissues other than the pituitary is regulated by an alternative promoter upstream of the site utilized by lactotrope cells ( 9 ). Transcripts generated from alternative promotor driven transcription results in inclusion of an additional exon1a within the 5’untranslated region of the transcript. However, this does not alter the amino acids of the encoded protein ( 10 ).

While pituitary PRL synthesis and release is sensitive to regulation by dopamine, typically extrapituitary PRL is not ( 11 ). An exception to this is in the context of adipocytes in which PRL is dependent on dopamine ( 12 ).

The mechanisms that control expression of PRL at extrapituitary sites is poorly understood; however, the use of an alternate promoter indicates site specific regulation of PRL transcription to modulate expression, which warrants further study especially during tumorigenesis ( 13 ). PRLR is a type 1 cytokine receptor, encoded by the PRLR gene on chromosome 5.

Conserved homology permits binding by human growth hormone (GH) in addition to PRL. In humans, the PRLR gene contains 11 exons and is widely expressed throughout the body ( 14 ). PRLR can undergo alternatively splicing events resulting in the expression of several PRLR isoforms, with tissue specificity.

These isoforms have modified cytoplasmic domains, but share identical extracellular domains that bind PRL. Moreover, PRLR lacks intrinsic kinase activity, thus necessitating dependency on associated kinases such as the Janus kinases (JAKs) to further transduce signaling. PRLR is a single pass transmembrane protein that has two conserved cytoplasmic regions, Box1 and Box2, which are responsible for association with JAK2 ( 15 ).

PRLR signaling plays a major role in numerous biological functions, primarily mammary gland development and lactation. However, due to the widespread expression of PRLR within tissues, aberrant activation of this signaling has been linked to progression of prostate, breast, cervical, ovarian, and pancreatic tumors ( 16 ).

High expression of PRLR and circulating PRL can drive the expression of genes involved in proliferation, migration, and invasion of cancer cells. In breast cancer, PRL-mediated JAK/STAT signaling contributes to endocrine therapy resistance in conjunction with elevated HER2, by activating oncogenic factors such as MYC, FOS, and JUN ( 17 ).

This has been shown to be mediated, in part, by the estrogen independent activation of ERα by PRL, both in vitro and in vivo ( 18 , 19 ). In particular, PRL has been shown to activate ERα through a PAK1 mediated mechanism, circumventing the mechanism of action of anti-estrogen therapies ( 20 ).

Others have shown that PRL participates in endocrine therapy resistance through the activation of PRLR, and stimulating downstream signaling pathways that include STAT5, ERK1/2, and PI3K ( 20 – 22 ). With prostate cancer, PRL overexpression contributes to increased hyperplasia of prostatic tissues, thereby elevating the risk for developing adenocarcinomas.

Epidemiologic studies have linked PRL and STAT5 with higher grade tumors and more aggressive disease ( 23 ). Enhanced PRLR signaling in gynecological, pancreatic, and colorectal tumors promotes metastatic potential, chemoresistance, and pro-survival signaling events ( 24 – 27 ).

Briefly, preincubation of PRL for 1 hour abrogated cisplatin-induced apoptosis of ovarian and endometrial cancer cells, as determined by Annexin V/PI staining ( 27 ). The authors demonstrate significant activation of Ras signaling, as well as STAT3, ATF-2, MEK1, CREB, and p53 within 5 minutes of PRL stimulation ( 27 ).

Interestingly, GH has been shown to induce the expression of ABC efflux transporters (ABCB1, ABCB5, ABCC1, ABCC2, ABCG1, and ABCG2), contributing to acquired drug resistance ( 28 ). Concurrently, PRL has been shown to induce the expression of ABCG2 through the activation of STAT5, leading to binding at consensus sequences upstream of the ABCG2 transcription start site ( 29 ).

Moreover, the authors further demonstrated that STAT5 was required, but insufficient for PRL induced transcription, as MAPK and PI3K inhibitors also decreased PRL induced ABCG2 expression, without affecting STAT5 DNA binding ( 29 ). In our own studies with pancreatic cancer, we observed that PRLR signaling potentiated invasive cell behavior and stemness through JAK2/STAT3 and ERK phosphorylation ( 25 ).

We had previously observed in colon cancer, that PRL enhanced stemness in a JAK2/STAT3/ERK dependent manner by modulating Notch signaling ( 26 ). Interestingly, in both pancreatic and colon cancer, we did not observe activation of STAT5 ( 25 ) As such, PRLR signaling plays an extensive role in human cancers, which has led to research directed towards developing therapeutic strategies to modulate activity.

Due to the strong evidence supporting the critical role of PRL and PRLR in human cancers, various approaches have attempted to modulate activity both by suppressing downstream signaling as well as by developing PRLR antagonists. These strategies will be discussed in more detail later. In brief, the use of a PRL antagonist peptide, G129R, was shown to block the PRL : PRLR signaling axis in ovarian cancer mouse models ( 30 ).

This resulted in greater than 90% reduction in tumor weights compared to controls, when used in combination with the standard-of-care agent paclitaxel. A preclinical study of the anti-PRLR antibody REGN2878-DM1 suggested induction of cell-cycle arrest and apoptosis in PRLR expressing breast cancer cell lines, and also exhibited synergistic activity with fulvestrant ( 31 ).

In preclinical studies with pancreatic cancer, we identified a small molecule Penfluridol to inhibit PRL induced JAK/STAT activation by competitively binding to PRLR. This resulted in suppression of cancer cell growth in vitro and in vivo ( 25 ). The efficacy of these preclinical studies demonstrates the validity of targeting PRLR while also establishing the critical role of PRL : PRLR signaling in human cancers.

In this review, we discuss the current research strategies directed towards PRL : PRLR inhibition. Due to the extensive expression of PRL and PRLR in various tissues, and the efficacy of preclinical inhibitory strategies, it is clear that the PRL : PRLR signaling axis is a critical pathway in human biology and cancers. 2.

Novel approaches to target prolactin receptor 2. 1. Competitive antagonists of the human prolactin A class of inhibitors that was first developed to target prolactin-sensitive pathologies such as dopamine-resistant prolactinomas, as well as breast, prostate and pancreatic malignancies were designed to compete with endogenous PRL for PRLR binding ( 32 ).

As such, these types of antagonists often require higher molar concentrations compared to endogenous PRL to ensure sufficient activity ( 33 ). Moreover, it is vital that any unintended agonistic properties are eliminated, particularly at high concentrations ( 33 ). As described previously, PRLR signaling is activated by the binding of PRL to a PRLR homodimer.

This interaction is ternary in nature and has 3 intermolecular interactions referred to as sites 1-3. Site 1 and 2 interactions are between prolactin and each receptor, while site 3 is the interaction between two receptor units. Once active, this ternary complex induces various downstream signaling pathways, including the JAK2-STAT3/STAT5 axis, MAP kinase, AKT, and Src kinase pathways ( 8 , 34 ).

It is this ternary interaction between PRL and PRLR that has served as the design template for the development of competitive PRLR antagonists, such as G129R-hPRL and Del1–9-G129R-hPRL, which will be discussed in detail below. G129R-hPRL was developed in the early 1990s as the result of a mutational screen of hPRL with the purpose of identifying and characterizing binding sites in PRLR.

This was based on strategies that were utilized for growth hormone (GH) and its cognate receptor (GHR) that yielded the discovery of a potent GHR antagonist and drug, Pegvisomant ( 7 , 35 – 39 ). G129R-hPRL was tested for its inhibitory activity in the NB2 rat cell proliferation assay, because PRL induces proliferation of these cells.

Surprisingly, instead of being an antagonist, G129R-hPRL appeared to actually be a weak agonist, increasing the proliferation of NB2 cells rather than suppress it ( 7 ). Binding of G129-hPRL was confirmed by surface plasmon resonance; however, the affinity towards site 2 of PRLR was demonstrated to be decreased compared to WT hPRL ( 6 , 7 , 40 ).

Based on these findings, it was concluded that the lack of antagonistic activity was due, at least in part, to poor affinity for site 2, leading to insufficient hindrance of ligand:receptor interaction. Shortly after these initial reports, several studies determined that detection of the competitive antagonistic properties of G129-hPRL was impacted by the bioassay used and species of origin ( 41 – 43 ).

A PRL-responsive luciferase reporter assay was designed in human embryonic kidney fibroblasts (HEK293) that were transfected with a hPRLR long isoform expressing construct. Under these conditions, G129R-hPRL exerted potent antagonistic activity ( 6 ). Species specific discrepancies were also confirmed, as G129R-hPRL had reduced antagonism towards rat PRLR ( 6 ).

These findings were validated by multiple groups using various hPRLR-mediated cell bioassays and breast cancer cell lines ( 44 – 47 ). However, conflicting results were obtained when studies were performed in Ba/F03 human cells stably transduced with hPRLR (Ba/F03-hPRLR).

When stimulated with hPRL, Ba/F03-hPRLR cells exhibited increased proliferation, while G129R-hPRL failed to induce antagonistic effects, similar to results obtained previously in NB2 rat cells ( 48 ).

As such, it was hypothesized that G129R-hPRL behaves as a weak antagonist/partial agonist in sensitive bioassays, while in low sensitivity assays where the levels of PRLR activation induced by G129R-hPRL is not sufficient to produce a biological effect, it acts as an antagonist ( 48 ). Despite these contradictory findings, many studies have since been performed demonstrating antagonistic activity, which are outlined below.

In a recent report, it has been shown that G129R-hPRL blocks the activity of PRL-PRLR signaling in ovarian cancer ( 30 ). The authors demonstrate that in orthotopic mouse models G129R-hPRL inhibits tumor growth in a dose-dependent manner.

Moreover, prolonged treatment with G129R-hPRL at 100 μg/day resulted in a durable response, and reduced tumor weights by 50% compared to control, while in combination with paclitaxel produced more than a 90% inhibition ( 30 ). There was also no apparent off target toxicity with G129R-hPRL.

In in vitro studies, the authors further demonstrated that G129R-hPRL did not inhibit proliferation or migration in 2-dimensional monolayer cultures of SKOV3 cells; however, in 3-dimensional spheroid cultures of HeyA8 and SKOV3 cells, G129R-hPRL abrogated cellular growth and induced apoptosis ( 30 ).

Furthermore, G129R-hPRL attenuated PRL induced growth and activation of JAK2, STAT3, and STAT5 phosphorylation in HeyA8 cells, further supporting the antagonistic properties of G129R-hPRL ( 30 ). Several groups have studied the role of PRLR in breast cancer, and in the process have observed antagonistic activity in cells treated with the G129R-hPRL analog. Chen et al.

showed that G129R-hPRL treatment inhibited proliferation of T47D breast cancer cells and induced apoptosis within 2 hours of treatment at a dose of 50 ng/mL ( 45 ). In regard to PRLR signaling, Catalado et al. sought to determine the effect of G129R-hPRL on STAT3 activation, and identified that hPRL activated STAT3 preferentially compared to STAT5 in T47D breast cancer cells ( 47 ).

Furthermore, the authors determined that G129R-hPRL inhibited STAT3 phosphorylation ( 47 ). This was further confirmed by others, in which G129R-hPRL attenuated PRL-induced activation of JAK-STAT and MAPK pathways ( 44 ). In breast cancer xenograft models, PRL was found to induce tumor growth of T47D and MCF-7 tumors, while G129R-hPRL inhibited growth ( 49 ).

These findings provide evidence that targeting the PRL : PRLR signaling axis is feasible and that the G129-hPRL has antagonistic activity. As a result, further interest in targeting PRLR has led to several studies focused on developing G129R-hPRL fusion proteins as well as combinatorial therapeutic strategies.

A fusion protein of G129R-hPRL with Pseudomonas exotoxin A (PE40) was developed and found to competitively bind to hPRLR in T47D cells, further suppressing PRL-induced STAT5 phosphorylation and inducing caspase-independent cytotoxicity ( 50 ).

In another study, G129R-hPRL was fused to endostatin, and was shown to inhibit PRL-induced signaling in T47D breast cancer cells, while further suppressing HUVEC cell proliferation, tube formation, and tumor formation of mouse 4T1 cells in vivo ( 51 , 52 ). Tomblyn et al.

have examined the combination of three G129R-hPRL based fusion proteins, which include G129R-hPRL fusions with endostatin (an angiogenesis inhibitor), interleukin 2 (immune modulator), and PE38KDEL (a truncated cytotoxin) in allografts of a mammary carcinoma cell line (McNeuA) derived from MMTC-neu mice ( 53 ).

Treatment with these fusion proteins increased the number of cytotoxic CD8+ T cells in the tumor, while reducing recurrence and lung metastases ( 53 ). In similar studies conducted by Scotti et al, combining G129R-hPRL with Herceptin resulted in suppression of STAT3 and STAT5 phosphorylation and reduced HER2 expression in T47D and BT474 breast cancer cells ( 54 ).

The combination of G129R-hPRL with Herceptin also demonstrated an additive inhibitory effect on HER2 and MAPK activation and further suppressed tumor xenograft growth in athymic nude mice ( 54 ). Taken together, these studies demonstrate antagonistic activity of G129R-hPRL, despite previous confounding studies, and show the feasibility of inhibiting PRLR signaling to suppress cancer growth.

Due to confounding evidence of agonistic activity of G129R-hPRL, development of a second-generation PRLR antagonist was attempted, resulting in a competitive antagonist that is devoid of residual agonistic properties in cell culture and animal models ( 55 ).

The Δ1–9-G129R-hPRL is a human prolactin core protein analog that has two modifications: 1) a deletion of nine N-terminal amino acid residues and 2) a glycine substitution by arginine at residue 129 ( 55 ). This second-generation PRLR antagonist was developed following findings from G129R-hPRL.

Furthermore, crystal structures of ovine placental lactogen (PL), a polypeptide that shares high structural and functional similarities with PRL, and rat PRL binding protein (PRLBP) identified that the N-terminal region of PL is critical in site 2 binding of PRLR ( 56 ). This finding led to in depth analyses of the N-terminal domain in PRL biological activity ( 57 ).

Multiple deletion constructs were developed including deletion of amino acids 1-9 (Δ1-9-hPRL) and 1-14 (Δ1-14-hPRL) ( 58 ). Interestingly, the Δ1-9-hPRL construct increased receptor binding affinity and biological activity, while the Δ1-14-hPRL construct decreased binding affinity and activity by modulating site 2 functionality ( 55 , 58 ).

Although the effects were modest, these deletion mutations were introduced into G129R-hPRL, intending to improve upon the antagonistic properties of the parent construct. Both of the double mutant analogs, Δ1-9-G129R-hPRL and Δ1-14-G129R-hPRL, exhibited similar dose-response curves in bioassays of PRLR activity.

These new analogs failed to improve upon the antagonistic properties of the first-generation construct, G129R-hPRL; however, the authors did observe significant improvements related to agonistic activity. While G129R-hPRL displayed agonistic properties in sensitive bioassays, particularly Ba/F-LP and Nb2 cell proliferations assays, the new double mutant analogs failed to stimulate proliferation.

These data demonstrate that the absence of agonistic activity markedly improved the second-generation antagonists. Goffin et al. published a crystallographic structure of Δ1-9-G129R-hPRL to understand the structural and thermodynamic basis of PRLR antagonism ( 56 ).

The authors reported no major structural changes compared to wild type hPRL, suggesting the pure antagonistic properties of Δ1-9-G129R-hPRL are due to intrinsic mutations and deletions ( 56 ). Moreover, they compared the physiochemical, structural, and biological properties of wild type hPRL and various variants including N-terminal or Gly129 mutations, either alone or in combination.

The authors determined that human PRL activity was unaffected by N-terminal modifications; however, in the context of G129R mutants, N-terminal deletions eliminated residual agonist activity. Moreover, this was unrelated to site 1 affinity ( 56 ). Conversely, N-terminal alterations impacted biological activity only when site 2 binding was affected by G129 mutants ( 56 ).

N-terminal deletions of PRL did have measurable decreases in site 2 affinity alone, as determined by SPR; however, these modifications were insufficient to eliminate biological activity indicating the critical nature of G129 to hPRL function ( 56 ).

What this indicates is twofold: 1) that the N-terminus participated in site 2 binding and 2) that residual agonism of early PRL antagonists may be eliminated by further modifying the N-terminus interactions with site 2. Several studies have employed the second-generation antagonist to dissect PRL : PRLR biology. Ferraris et al.

studied the effects of Δ1-9-G129R-hPRL in the turnover of mouse anterior pituitary cells and PRLR expression in vivo using transgenic mice constitutively expressing the analog ( 59 ). The authors observed that the weight and proliferation index of the pituitary gland was elevated in transgenic mice expressing the antagonist compared to wild type mice ( 59 ).

Moreover, in vitro studies showed that Δ1-9-G129R-hPRL enhanced proliferation while reducing apoptosis of GH3 cells, a somatolactotrope and primary rat anterior pituitary cells ( 59 ). These data suggest that PRL acts as an antiproliferative and pro-apoptotic factor in cells of the anterior pituitary gland. Dwivedi et al.

identified hematopoietic PBX-interacting protein (HPIP) as a novel regulator of mammary epithelial cell differentiation, where Δ1-9-G129R-hPRL attenuated HPIP-mediated synthesis of PRL, activation of AKT, and synthesis of β-casein in cultured HC11 cells ( 60 ).

Recently, synthesis and purification of Δ1-9-G129R-hPRL was performed by testing different activation temperature and chromatographic techniques including nickel-affinity chromatography, size-exclusion chromatography and high-performance size-exclusion chromatography (HPSEC) ( 61 ).

Δ1-9-G129R-hPRL was extracted with more than 95% purity, enhanced solubility, correct folding, and without methionine, and has a significant potential in clinical application ( 61 ). In the context of cancer, several groups have shown anti-tumor activity and suppression of PRLR signaling following treatment with the Δ1-9-G129R-hPRL antagonist.

Treatment with Δ1-9-G129R-hPRL abolished the increase in nitric oxide production by prolactin-induced plasma membrane carboxypeptidase D in triple-negative breast cancer cell lines ( 62 ). It was further shown to inhibit prolactin-induced osteoclast differentiation and bone lysis in breast cancer cells ( 63 ). Similar inhibition of PRL-induced carboxypeptidase D was also seen in prostate cancer ( 64 ).

In addition, Hou et al. demonstrated that while PRL increased oncogenic potential in breast cancer cells by stimulating HOXA1, which in turn induced STAT5, ERK phosphorylation, and increased transcriptional activity of ELK1, SAP1A, STAT5A and B to increase cell proliferation, survival and anchorage dependent growth, following treatment with Δ1-9-G129R-hPRL ( 65 ).

The effect of Δ1-9-G129R-hPRL induced PRLR antagonism was further studied by Howell et al. in multiple breast cancer cell lines ( 66 ). As a monotherapy Δ1-9-G129R-hPRL failed to demonstrate antiproliferative effects of the cell lines, but potentiated the effects of doxorubicin and paclitaxel when used in combination ( 66 ).

Moreover, Δ1-9-G129R-hPRL inhibited the growth of colonies in soft agar and mammosphere formation supporting the rational for use in combination therapeutic strategies for breast cancer ( 66 ). Asad et al. have studied the effects of PRLR inhibition on glioblastoma multiforme (GBM) pathogenesis ( 67 ).

The authors identified that PRLR was highly expressed and was further correlated with poor survival in GBM patients ( 67 ). Moreover, Δ1-9-G129R-hPRL treatment reduced the proliferation, colony formation, chemoresistance and migration in GBM cells suggesting potential for PRLR as a therapeutic target in GBM ( 67 ).

Lastly, Δ1-9-G129R-hPRL treatment prevented early stages of prostate carcinogenesis by inhibiting STAT5 phosphorylation, proliferation, abnormal basal-cell pattern and grade of intraepithelial prostate neoplasia suggesting the application of PRLR-based therapies in prostate cancer ( 68 ).

Collectively, these studies demonstrate antagonistic activity of Δ1-9-G129R-hPRL and further provide solid evidence for targeting PRLR in human malignancies. 2. 1.

3. Improving half-life of PRLR antagonists In vivo While current PRLR antagonists have shown promise in pre-clinical applications, there remain challenges limiting their usage in clinic. PRL and current PRL antagonists have molecular weights of ~23 kDa, which are below the 60kDa cut-off values for glomerular filtration by the kidneys ( 69 ).

Hence, these are quickly cleared from the blood following intravenous delivery. Hence, the half-life of PRL in the blood is ~41 minutes ( 70 ), and speculation towards PRLR antagonists would yield similar results. As such, their application in a clinical setting is limited.

To overcome this challenge, Yu et al. have developed a PRLR antagonist fusion protein designed around Δ1-9-G129R-hPRL, and several additional mutations (C11S, S33A, Q73L, G129R and K190R). In addition, the fusion protein included an albumin binding domain (ABD) from Streptococcal protein G, also known as ABD 035 , which has 46 amino acids in a three-helix structure ( 71 ).

Surface plasmon resonance of this fusion protein, called PrlRA-ABD determined the K D to be 2. 3 ± 0. 2 vs 3.

4 ± 0. 5 nM of PrlRA alone, while PRL showed a KD value of 23 ± 4 nM ( 71 ). Furthermore, ABD-PrlRA and PrlRA both inhibited PRL-induced phosphorylation of STAT5 in U251-MG cells in a dose-dependent manner ( 71 ).

To understand the changes in pharmacokinetics both PrlRA and ABD-PrlRA were injected subcutaneously in Wistar rats at a dose of 4 mg/kg. After 24 h, serum was analyzed for PrlRA and ABD-PrlRA concentration and determined to be 150 ng/ml and 15,000 ng/ml, respectively ( 71 ). This data suggests that addition of ABD to PrlRA enhanced its in vivo half-life by 100-fold, demonstrating the feasibility of in vivo applications.

Additional strategies that have been effective for hGH may also have implications for PRL antagonists. Pegvisomant is a PEGylated G120K protein analog of hGH, and was the first drug approved as a GHR antagonist ( 39 ). Much like PRL, hGH is readily cleared by kidney filtration.

To slow clearance, polyethylene glycol (PEG) polymers were attached to hGH derivatives. The authors observed significant retention of PEG-hGH derivatives in serum compared to hGH, with measurable concentrations detected out to ~200 hours and 12 hours, respectively ( 37 ).

Since PEGylation of hGH derivatives proved successful, we could speculate that these strategies may be useful for PRL antagonists as well; however there are challenges that must be overcome with the use of PEG based polymers. PEG chains can mask the protein binding sites, and thereby reduce affinity of biological activity ( 72 ). Therefore, design of the polymer is crucial to developing an effective PEGylated protein.

Nevertheless, this may provide additional opportunities for improving PRL antagonist half-lives, and warrant further study. 2. 2.

Antibody-based PRLR antagonists The use of antibody-based therapeutic agents has become attractive and one of the most successful strategies for the treatment of various diseases, including cancer ( 73 ). The use of monoclonal antibodies has achieved significant success in recent years, while antibody-drug conjugates have only recently been utilized for the treatment of solid tumors and lymphomas ( 73 ).

The anticancer effects of these monoclonal antibodies can be due to direct receptor blockade, immune-activated cell killing, and specific defects of antibodies on cancer vasculature and stromal components as well as drug delivery ( 74 – 77 ).

Specific examples of successful monoclonal antibody therapies targeted epidermal growth factor receptor (EGFR) ( 75 , 78 ), C-MET ( 79 ), HER2 ( 80 ), fibroblast activation protein (FAP) ( 81 ), and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) ( 82 ).

The ideal properties of monoclonal antibodies include high selectivity towards specific target antigens, activating immune cell responses, and modulating downstream signaling pathways ( 83 ). Hence, antibody design is critical for successful preclinical and clinical applications.

The successful development of monoclonal antibodies for use in a clinical setting involves identification of the physiochemical properties of the antibody, analysis of specificity, study of immune response and signaling pathways as well as in vivo antibody localization, biodistribution, toxicity, and efficacy ( 73 ).

Several monoclonal antibodies have received approval by US Food and Drug Administration in the recent decade, which have been summarized previously ( 84 – 87 ). In the context of PRLR, the presence of a defined extracellular domain structure makes it an attractive target for designing monoclonal antibody based inhibitors and therapeutics.

As such, several antibodies and antibody based constructs have been developed targeting PRLR and are being tested in preclinical and clinical studies, which will be summarized in detail in the following sections. 2. 2.

1. PRLR neutralizing antibodies Damiano et al. developed and characterized a neutralizing antibody LF102 targeting human PRLR, which was shown to inhibit the physiological functions of both autocrine and paracrine PRL ( 88 ).

The authors first generated a parental hybridoma to LFA102 in mice immunized with recombinant PRLR extracellular domain, then LFA102 was prepared by humanizing the antibody ( 88 ). Using flow activated cell sorting, the authors demonstrated that LFA102 binds to PRLR in human breast cancer cell lines, in addition to primary breast cancer cells ( 88 ).

Moreover, LFA102 was also found to bind to rat pre-T cell lymphoma cell line Nb2-11 suggesting this antibody has cross-reactivity to rat PRLR ( 88 ). To assess selectivity of LFA102, the authors used a PRLR-negative BaF3 cell line and re-expressed PRLR (BAF3-PRLR). LFA102 did not bind to PRLR-negative BaF3 cells but was found to bind to BaF3-PRLR ( 88 ).

In addition, the antibody did not interact with cells expressing murine PRLR. To determine if LFA102 acted through a competitive or non-competitive mechanism with PRLR, the authors designed a ligand competition assay using Alexa647-labeled PRL (A647-PRL).

The authors demonstrated that LFA102 did not affect A647-PRL binding to PRLR even at saturation concentrations of LFA102 ( 88 ), suggesting that LFA102 is not a ligand-competitive inhibitor. To determine whether LF102 affects PRL-mediated signaling, T47D breast cancer cells were treated with the antibody. There was significant attenuation of PRL-induced phosphorylation of STAT5, AKT, and ERK in a concentration-dependent manner ( 88 ).

However, LFA102 failed to regulate PRLR signaling when treated alone demonstrating the absence of residual agonistic activity. As proof of principle for in vivo activity, T47D-T2 xenografts were generated in NOD