Identification of a gain-of-function mutation of the prolactin receptor in women with benign breast tumors

Abstract

There is currently no known genetic disease linked to prolactin (Prl) or its receptor (PrlR) in humans. Given the essential role of this hormonal system in breast physiology, we reasoned that genetic anomalies of Prl/PrlR genes may be related to the occurrence of breast diseases with high proliferative potential. Multiple fibroadenomas (MFA) are benign breast tumors which appear most frequently in young women, including at puberty, when Prl has well-recognized proliferative actions on the breast. In a prospective study involving 74 MFA patients and 170 control subjects, we identified four patients harboring a heterozygous single nucleotide polymorphism in exon 6 of the PrlR gene, encoding Ile(146)-->Leu substitution in its extracellular domain. This sole substitution was sufficient to confer constitutive activity to the receptor variant (PrlR(I146L)), as assessed in three reconstituted cell models (Ba/F3, HEK293 and MCF-7 cells) by Prl-independent (i) PrlR tyrosine phosphorylation, (ii) activation of signal transducer and activator of transcription 5 (STAT5) signaling, (iii) transcriptional activity toward a Prl-responsive reporter gene, and (iv) cell proliferation and protection from cell death. Constitutive activity of PrlR(I146L) in the breast sample from a patient was supported by increased STAT5 signaling. This is a unique description of a functional mutation of the PrlR associated with a human disease. Hallmarks of constitutive activity were all reversed by a specific PrlR antagonist, which opens potential therapeutic approaches for MFA, or any other disease that could be associated with this mutation in future.

Fig. 1.

MRI of a MFA patient showing several fibroadenomas (arrows) mainly located in the left breast.

Fig. 2.

Exon 6 mutation in human PrlR gene leads to I146L missense substitution. (A) Schematic representation of the 11 exons of the human PrlR gene and the corresponding protein domains. Examples of exon 6 sense sequences obtained from one homozygous patient (2 WT alleles) and one heterozygous patient harboring both WT and A-to-C mutated alleles are shown. (B) The 3D structure of the human PrlR extracellular domain (blue) complexed to growth hormone (orange) (PDB ID code 1BP3) is used to locate the I146L substitution on the folded receptor. D1 and D2 indicate cytokine receptor homology domains 1 and 2. UTR, untranslated region; TM, transmembrane.

Fig. 3.

Constitutive activity of PrlR_I146L in various in vitro assays. (A–C) HEK293, MCF-7, and Ba/F3 cells were stably transfected by using expression vectors encoding human PrlR_WT or PrlR_I146L. Each stable clone or population was serum-starved, then stimulated or not by using 40 nM human Prl (15 min) as indicated. Phosphorylated and total PrlR (Upper) and STAT5 (Lower) were analyzed by immunoblotting. Whereas tyrosine-phosphorylation of PrlR and STAT5 was observed only under Prl stimulation in cells expressing PrlR_WT, constitutive phosphorylation of both proteins (arrowheads) was observed in cells expressing PrlR_I146L. (D) Both HEK-PrlR_WT and HEK-PrlR_I146L clones also stably incorporated the Prl-responsive LHRE-luciferase gene (see SI Text). Luciferase activity was measured after 24 h treatment with (filled bars) or without (open bars) 400 nM Prl. Data normalized to basal luciferase activity of HEK-PrlR_WT show significantly higher background in HEK-PrlR_I146L cells (means ± SD, six independent experiments performed in triplicates). **, P < 0.01. (E) HEK293 cells were transiently cotransfected by using expression plasmids encoding (i) WT or mutated PrlR as indicated, (ii) LHRE-luciferase (firefly) reporter gene, and (iii) Renilla luciferase gene. Luciferase activities were measured after 24 h treatment with (filled bars) or without (open bars) 40 nM Prl. Data normalized to basal firefly/Renilla luciferase activities of cells expressing PrlR_WT show that Y587F substitution abolishes the higher background of I146L PrlR (means ± SD, three independent experiments performed in triplicates). **, P < 0.01. (F) Cell cycle distribution of Ba/F-PrlR_WT and Ba/F-PrlR_I146L cells was monitored by FACS analysis. Cells were starved by Prl depletion and stimulated with 8 nM hPrl (Right) or not (Left) for 24 h. DNA content analysis of propidium iodide-stained cells is represented. Numbers indicate the average proportion of cells ± SD (seven to nine independent series) exhibiting <2n (subG₁), 2n (G₀/G₁), and >2n (S/M). In the absence of Prl, expression of PrlR_I146L protects cells from death and stimulates division (Lower Left). (G and H) Proliferation of MCF7-PrlR_WT,WT and MCF7-PrlR_WT,I146L cells was monitored by using WST-1 reagent. This data shows that cells expressing PrlR_I146L are autonomous for growth. Error bars indicate SD (slopes differences; three independent experiments performed in triplicate). *, P < 0.05.

Fig. 4.

Immunohistochemical analysis of breast biopsies (MFA and adjacent tissue). STAT5 phosphorylation (Top) and FAS expression (Bottom) were analyzed in MFA (Right) and adjacent tissue (Left) from one homozygous patient (PrlR_WT,WT) and one heterozygous patient harboring the mutated PrlR allele (PrlR_WT,I146L). (Middle) Nonspecific staining obtained without the addition of primary antibodies. Phospho-STAT5 was predominantly found in cell nuclei (arrows). Both phospho-STAT5 and FAS were up-regulated in adjacent tissue of the patient harboring PrlR_I146L allele and in MFAs.

Fig. 5.

Inhibition of PrlR_I146L constitutive activity by a PrlR antagonist. (A) Constitutive phosphorylation of the mutated PrlR in HEK-PrlR_I146L cells was inhibited by treating cells with the JAK2 inhibitor AG490 (50 μM, 1 h), which identifies this tyrosine kinase as involved in constitutive PrlR phosphosylation. The same effect was obtained by using the PrlR antagonist Del1–9-G129R-hPrl (0.8 μM, 1 h), which demonstrates that abolition of constitutive activation can also be achieved by this PrlR-specific inhibitor. (B) Constitutive phosphorylation of STAT5 in Ba/F-PrlR_I146L cells was inhibited in a time-dependent manner by the addition of PrlR antagonist Del1–9-G129R-hPrl (0.16 μM) and remained below starting levels for at least 24 h. (C and D) Ba/F-PrlR_WT and Ba/F-PrlR_I146L cells (C) and MCF7-PrlR_WT,WT and MCF7-PrlR_WT,I146L cells (D) were treated by using the PrlR antagonist Del1–9-G129R-hPrl (0.8 μM), and cell proliferation (growth/survival ratio) was monitored after 3-day treatment using WST-1. Although the PrlR antagonist did not affect survival of cells expressing the WT PrlR (indicating absence of toxicity), it significantly reduced the growth of cells expressing the PrlR mutant (means ± SD, three independent experiments performed in triplicate). *, P < 0.05.