S179D-human PRL, a pseudophosphorylated human PRL analog, is an agonist and not an antagonist.

For many years, our group has been involved in the development of human PRL antagonists. In two recent publications, S179D-human PRL, a human PRL analog designed to mimic a putative S179-phosphorylated human PRL, was reported to be a highly potent antagonist of human PRL-induced proliferation and signaling in rat Nb2 cells. We prepared this analog with the aim of testing it in various bioassays involving the homologous, human PRL receptor. In our hands, S179D- human PRL was able to stimulate 1) the proliferation of rat Nb2 cells and of human mammary tumor epithelial cells (T-47D), 2) transcriptional activation of the lactogenic hormone response element-luciferase reporter gene, and 3) activation of the Janus kinase/signal transducer and activator of transcription and MAPK pathways. Using the previously characterized antagonist G129R-human PRL as a control, we failed to observe any evidence for antagonism of S179D-human PRL toward any of the human PRL-induced effects analyzed, including cell proliferation, transcriptional activation, and signaling. In conclusion, our data argue that S179D-human PRL is an agonist displaying slightly reduced affinity and activity due to local alteration of receptor binding site 1, and that the antagonistic properties previously attributed to S179D-human PRL cannot be confirmed in any of the assays analyzed in this study.

Human prolactin (hPRL) antagonists inhibit hPRL-activated signaling pathways involved in breast cancer cell proliferation.

The involvement of human prolactin (hPRL) in breast cancer has been recently reconsidered based on its autocrine/paracrine proliferative effect described in human mammary tumor epithelial cells. Therefore, there is growing interest in the development of potent hPRL antagonists that may inhibit this effect. We previously designed hPRL analogs displaying antagonistic properties in a human transcriptional bioassay. We now report that the most potent of those analogs, G129R-hPRL, antagonizes all hPRL-induced effects analysed in various breast cancer cell lines, including cell proliferation. The analog per se lacks intrinsic agonistic activity on PRL receptor-activated signaling cascades, cell proliferation and apoptosis, indicating that its mode of action only occurs through competitive inhibition of hPRL. We provide some molecular basis of this antagonistic effect by demonstrating that G129R-hPRL competitively inhibits hPRL-activation of the JAK-STAT and MAPK pathways, two signaling cascades involved in the mitogenic effect of hPRL in mammary epithelial cells. This competitive inhibition persists for at least 48 h, as evidenced by long term analysis of STAT5b activation or of progression through cell cycle. These results are the first demonstration at the molecular level that hPRL antagonists interfering with receptor dimerization disrupt signaling events in breast cancer cells, which prevents hPRL-induced cell proliferation.

From the molecular biology of prolactin and its receptor to the lessons learned from knockout mice models.

Prolactin (PRL), a polypeptide hormone secreted mainly by the pituitary and, to a lesser extent, by peripheral tissues, affects more physiological processes than all other pituitary hormones combined since it is involved in > 300 separate functions in vertebrates. Its main actions are related to lactation and reproduction. The initial step of PRL action is the binding to a specific membrane receptor, the PRLR, which belongs to the class 1 cytokine receptor superfamily. PRL-binding sites have been identified in a number of tissues and cell types in adult animals. Signal transduction by this receptor is mediated, at least in part, by two families of signaling molecules: Janus tyrosine kinases and signal transducers and activators of transcription (STATs). Disruption of the PRLR gene has provided a new mouse model with which to identify actions directly associated with PRL or any other PRLR ligands, such as placental lactogens. To date, several different phenotypes have been analyzed and are briefly described in this review. Coupled with the SAGE technique, this PRLR knockout model is being used to qualitatively and quantitatively evaluate the expression pattern of hepatic genes in two physiological situations: transcriptomes corresponding to livers from both wild type and PRLR KO mice are being compared, and following statistical analyses, candidate genes presenting a differential profile will be further characterized. Such a new approach will undoubtedly open future avenues of research for PRL targets. To date, no pathology linked to any mutation in the genes encoding PRL or its receptor have been identified. The development of genetic models provides new opportunities to understand how PRL can participate to the development of pathologies throughout life, as for example the initiation and progression of breast cancer.

Biological properties of human prolactin analogs depend not only on global hormone affinity, but also on the relative affinities of both receptor binding sites.

Zinc increases the affinity of human growth hormone (hGH) for the human prolactin receptor (hPRLR) due to the coordination of one zinc ion involving Glu-174(hGH) and His-18(hGH). In contrast, binding of hPRL to the hPRLR is zinc-independent. We engineered in binding site 1 of hPRL a hGH-like zinc coordination site, by mutating Asp-183(hPRL) (homologous to Glu-174(hGH)) into Glu (D183E mutation). This mutation was also introduced into G129R hPRL, a binding site 2 mutant (Goffin, V., Kinet, S., Ferrag, F., Binart, N., Martial, J. A. , and Kelly, P. A. (1996) J. Biol. Chem. 271, 16573-16579). These analogs were characterized using a stable clone expressing both the hPRLR and a PRLR-responsive reporter gene. The D183E mutation per se decreases the binding affinity and transcriptional activity of hPRL. However, this loss is partially rescued by the addition of zinc and the effect is much more marked on bioactivity than on binding affinity. These data indicate that the D183E mutation confers zinc sensitivity to hPRL biological properties. Due to an impaired site 2, the agonistic activity of G129R analog is almost nil. Although the double mutant D183E/G129R displays lower affinity ( approximately 1 log) compared with G129R hPRL, it unexpectedly recovers partial agonistic activity in the absence of zinc. Moreover, whereas zinc increases the affinity of D183E/G129R, it paradoxically abolishes its agonistic activity. Our results demonstrate that the biological properties of hPRL analogs do not necessarily parallel their overall affinity. Rather, the relative affinities of the individual binding sites 1 and 2 may play an even more important role.

The human growth hormone antagonist B2036 does not interact with the prolactin receptor.

The human growth hormone (hGH) antagonist B2036 combines a single amino acid substitution impairing receptor binding site 2 (G120K) with eight additional amino acid substitutions that improve binding site 1 affinity. This hGH antagonist is being tested for treating pathologies linked to excess hGH levels. B2036-PEG is a polyethylene glycol (PEG) conjugated form of B2036 that has an increased half-life due to reduced renal clearance. It is currently in phase III trials for acromegaly. Human GH is also able to bind to the receptor of prolactin (PRLR). Since activation of PRLR can promote an array of pathological states (reproduction disorders, breast cancer), the ability of B2036-PEG to interact with the PRLR had to be determined. In this study, we compared four hGH antagonists (G120K, G120K-PEG, B2036 and B2036-PEG) in three bioassays: proliferation of rat Nb2 cells, binding to the human PRLR and activation of human PRLR-mediated signaling in a cell line stably expressing this receptor and a luciferase reporter gene. Agonistic and antagonistic properties were characterized. Our data show that B2036-PEG does not bind, activate or antagonize PRLRs, either from rat or human origin. These observations further demonstrate that the eight amino acid substitutions within binding site 1 provide binding specificity directed towards the human GH receptor.

Should prolactin be reconsidered as a therapeutic target in human breast cancer?

Although prolactin (PRL) has been long suspected to be involved in the progression of human breast cancer, the failure of clinical improvement by treatment with dopamine agonists, which lower circulating levels of PRL, rapidly reduced the interest of oncologists concerning a potential role of this pituitary hormone in the development of breast cancer. Within the last few years, however, several studies reported first, that PRL is also synthesized in the mammary gland, and second that it exerts its proliferative action in an autocrine/paracrine manner. These observations have led to a reconsideration of the role of PRL as an active participant in breast cancer and are an impetus to search for alternative strategies aimed at inhibiting the proliferative effects of PRL on tumor mammary cells. In this report, we discuss the three possible levels that can be targeted for this purpose: the mammary synthesis of PRL, the interaction of the hormone with its receptor at the surface of mammary cells, and the intracellular signaling cascades triggered by the activated receptor. For each of these steps, we discuss the molecular event(s) that can be targeted, our understanding of the mechanisms involving these putative targets as well as the tools currently available for their inhibition. Besides its proliferative effect, PRL is also involved in the control of angiogenesis through one of its cleaved fragments, named PRL 16K, which has been shown to inhibit the angiogenic process. In view of this biological activity, we discuss first the cleavage of PRL with respect to the human mammary gland and, second, the hypothesis speculating that a balance between the proliferative effect of intact PRL and the anti-angiogenic activity of its 16K-like fragments might be physiologically relevant in the evolution of mammary tumors. If true, our hypothesis would suggest that the enzymatic cleavage of PRL could represent a new molecular target in the search for alternative strategies in the treatment of breast cancer.