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Prolactin (PRL) and its receptor (PRLr) have been implicated in the development and progression of human breast cancer. PRL activates its receptor and induces activation of proximal Janus kinase 2 (Jak2) for signal transduction. Here, we sought to determine the role of PRLr-associated peptidyl-prolyl isomerase, cyclophilin A (CypA), in modulating structure/function relationships of the PRLr. It was demonstrated that CypA mediated PRL-induced conformational change of the CFP- and -YFP tagged forms of the PRLr cytoplasmic-tail, whereas CypA inhibition by NIM811 (N-methyl-4-isoleucine cyclosporin) or knockdown blocked the conformational change of the PRLr assessed by Fluorescence Resonance Energy Transfer (FRET) signal or efficiency. To further investigate the consequences of CypA inhibition or knockdown on the PRLr/Jak2 complex mediated signaling/functions, analyses of phospho-tyrosine residues that are believed to be important for interactions/signaling were investigated. It was found that NIM811 inhibition or CypA shRNA knockdown significantly reduced prolactin-stimulated phosphorylation of PRLr/Jak2 intermediates and their association with the PRLr in breast cancer cells. A microarray analysis revealed that NIM811 inhibited approximately 66% of the top 50 PRL-induced genes. NIM811 inhibited breast cancer cell proliferation, survival, migration and anchorage-independent growth. Subsequent NIM811 treatment of a triple negative breast cancer xenograft inhibited primary tumor growth, outgrowth of macro-metastasis and induced central tumor necrosis. Furthermore, loss of CypA in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. Overall, CypA modulates PRL-induced conformational change of the C-terminus of the PRLr through its isomerase activity, altering PRLr/Jak2 complex signaling/functions in breast/mammary tumorigenesis and metastasis.

Nous avons souhaité, dans ce travail de Thèse, préciser le rôle de l’hormone prolactine (PRL) dans la tumorigenèse des prolactinomes. Nous avons tout d’abord décrit l’histoire naturelle et moléculaire des tumeurs lactotropes développées par le modèle de souris Prlr-/-, invalidé de façon globale pour le récepteur de la PRL (PRLR). Les femelles Prlr-/- développent des prolactinomes avec 100% de pénétrance à 12 mois. Ces tumeurs sont très sécrétantes, invasives et prolifératives. L’analyse transcriptomique comparative des hypophyses de souris Prlr+/+ et Prlr-/- nous a permis de mettre en évidence de nouvelles voies de signalisation impliquées dans la survenue de ces tumeurs. Ces nouveaux gènes candidats seront à rechercher chez l’Homme. Par ailleurs, l’étude d’un autre modèle murin développé dans le cadre de ce travail, invalidé de façon spécifique dans la cellule lactotrope, a permis de démontrer pour la première fois in vivo que la PRL exerçait un rétrocontrôle autocrine sur la sécrétion et la prolifération des cellules lactotropes. Bien que nous n’ayons pas retrouvé de mutation germinale du PRLR dans une large cohorte de patients atteints de prolactinome sporadique, nos résultats suggèrent que des mutations somatiques de ce gène ne sont pas à exclure et pourraient contribuer à la survenue de la pathologie humaine. / In this work, we investigated the role of prolactin (PRL) in prolactinoma tumorigenesis. We first described the natural and molecular history of lactotroph cell tumors developed by the Prlr-/- mouse model, globally invalidated for the PRL receptor (PRLR). The Prlr-/- females develop prolactinomas with 100% penetrance at 12 months of age. These tumors are highly secreting, invasive and proliferative. The comparative transcriptomic analysis of pituitaries from Prlr+/+ and Prlr-/- mice suggested new signaling pathways involved in lactotroph adenoma developement in this mouse model. The role of these novel candidate genes remains to be demonstated in Humans. Furthermore, by studying another mouse model developed during this work, deleted for Prlr only in lactotroph cells, we demonstrated for the first time that PRL exerts an autocrine feedback on lactotroph cell secretion and proliferation in vivo. Although we did not find any germline mutation of PRLR in a large cohort of patients with sporadic prolactinoma, our results suggest that somatic mutations of this gene cannot be excluded and may contribute to the onset of the human pathology.

<p>Functioning as a “powerhouse”, the liver adapts to the metabolic needs of the body by maintaining a homeostatic balance. Prolactin receptor (PRLR) has been found to have a copious existence in the liver. Having established a well-defined role in both reproductive and endocrine systems, the role of this transmembrane protein in hepatocytes is yet to be elucidated. Due to its abundant nature, we hypothesized that PRLR is required for maintaining hepatic homeostasis and plays a role in liver diseases. To test this hypothesis, we defined two specific aims. The first was to explore whether PRLR loss-of-function affects liver structure and function in physiological conditions. The second was to determine whether PRLR is associated with liver pathology. We deleted the <i>Prlr</i> gene specifically in hepatocytes using a virus-based approach and evaluated liver function, transcriptome, and activities of downstream signaling molecules. Due to the absence of PRLR, we found that the urea cycle was disrupted, concomitant with excessive accumulation of urea in the blood; 133 genes exhibited differential expression, largely associated with hepatocyte structure, metabolism, and inflammation; and the activities of STAT3 and 5 were reduced. The results signify that PRLR indeed plays a homeostatic role in the liver. We also used <i>Prlr</i>^+/- mice to assess whether the loss of one allele of the <i>Prlr</i> gene alters maternal hepatic adaptations to pregnancy. As a result, in the pre-pregnancy state and during the first half of gestation, the expression of maternal hepatic PRLR protein was reduced approximately by half owing to <i>Prlr</i> insufficiency. However, during the second half of pregnancy, we observed compensatory upregulation of this molecule, leading to minimal interference in STAT 3 and 5 signaling and liver size. Contrary to a previous study in the breast and ovary, our results suggest that one allele of <i>Prlr</i> may be sufficient for the maternal liver to respond to this physiological stimulus (pregnancy). Furthermore, we examined the expression and activity of PRLR in fatty as well as cholestatic livers. Using a high fat diet, we induced non-alcoholic fatty liver disease (NAFLD). Strikingly and for the first time, we discovered that the short isoform of PRLR (PRLR-S) was completely inactivated in response to NAFLD, whereas the long isoform remained unchanged. This finding strongly suggests the involvement of PRLR-S in lipid metabolism. We also postulate that PRLR-L may be the major regulator of STAT signaling in the liver, consistent with other reports. Lastly, we induced extrahepatic cholestasis via bile duct ligation (BDL) in mice. As this liver disease progressed, the expression of both isoforms of PRLR generally declined and was surprisingly accompanied by increased STAT 3 and 5 activity. The data suggests that PRLR participates in this disease progression, with a disconnection between PRLR signaling and STAT proteins. Collectively, our preliminary studies suggest that PRLR signaling is required to maintain liver homeostasis and more prominently, is involved in liver diseases, especially NAFLD. These findings lay a foundation for our future studies.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Functioning as a “powerhouse”, the liver adapts to the metabolic needs of the body by maintaining a homeostatic balance. Prolactin receptor (PRLR) has been found to have a copious existence in the liver. Having established a well-defined role in both reproductive and endocrine systems, the role of this transmembrane protein in hepatocytes is yet to be elucidated. Due to its abundant nature, we hypothesized that PRLR is required for maintaining hepatic homeostasis and plays a role in liver diseases. To test this hypothesis, we defined two specific aims. The first was to explore whether PRLR loss-of-function affects liver structure and function in physiological conditions. The second was to determine whether PRLR is associated with liver pathology. We deleted the Prlr gene specifically in hepatocytes using a virus-based approach and evaluated liver function, transcriptome, and activities of downstream signaling molecules. Due to the absence of PRLR, we found that the urea cycle was disrupted, concomitant with excessive accumulation of urea in the blood; 133 genes exhibited differential expression, largely associated with hepatocyte structure, metabolism, and inflammation; and the activities of STAT3 and 5 were reduced. The results signify that PRLR indeed plays a homeostatic role in the liver. We also used Prlr+/- mice to assess whether the loss of one allele of the Prlr gene alters maternal hepatic adaptations to pregnancy. As a result, in the pre-pregnancy state and during the first half of gestation, the expression of maternal hepatic PRLR protein was reduced approximately by half owing to Prlr insufficiency. However, during the second half of pregnancy, we observed compensatory upregulation of this molecule, leading to minimal interference in STAT 3 and 5 signaling and liver size. Contrary to a previous study in the breast and ovary, our results suggest that one allele of Prlr may be sufficient for the maternal liver to respond to this physiological stimulus (pregnancy). Furthermore, we examined the expression and activity of PRLR in fatty as well as cholestatic livers. Using a high fat diet, we induced non-alcoholic fatty liver disease (NAFLD). Strikingly and for the first time, we discovered that the short isoform of PRLR (PRLR-S) was completely inactivated in response to NAFLD, whereas the long isoform remained unchanged. This finding strongly suggests the involvement of PRLR-S in lipid metabolism. We also postulate that PRLR-L may be the major regulator of STAT signaling in the liver, consistent with other reports. Lastly, we induced extrahepatic cholestasis via bile duct ligation (BDL) in mice. As this liver disease progressed, the expression of both isoforms of PRLR generally declined and was surprisingly accompanied by increased STAT 3 and 5 activity. The data suggests that PRLR participates in this disease progression, with a disconnection between PRLR signaling and STAT proteins. Collectively, our preliminary studies suggest that PRLR signaling is required to maintain liver homeostasis and more prominently, is involved in liver diseases, especially NAFLD. These findings lay a foundation for our future studies.

One of the oldest, extant, lineages of vertebrates, the sea lamprey, was used to clarify the evolutionary origin and divergence of the growth hormone receptor (GHR) family. A single, full-length, cDNA, and a second, partial, cDNA were identified and shown to encode proteins that share amino acid identity with GHRs and prolactin receptors (PRLR s) previously identified. The complexity of the dynamic signaling system, with special emphasis on this system in fish and in the context of the evolution of this system, is discussed in the first chapter. The second chapter integrates the new insights gained by these studies. Included is a newly proposed phylogenetic analysis and revised nomenclature-system for vertebrate GHRs that better represents the evolutionary history of the receptor family. The molecular evolution of the receptors is, furthermore, highlighted as the backdrop for the continued discussion regarding how the GH-family of hormones exhibit such coordinated and pleiotropic actions.

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