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Stimulation of prolactin receptor induces STAT-5 phosphorylation and cellular invasion in glioblastoma multiforme
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0003-1616-4440
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2016 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 48, p. 79558-79569Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Impact Journals LLC , 2016. Vol. 7, no 48, p. 79558-79569
Keywords [en]
prolactin, prolactin receptor, prolactin receptor antagonist, STAT5, GBM
National Category
Cancer and Oncology Cell Biology
Identifiers
URN: urn:nbn:se:kth:diva-199535DOI: 10.18632/oncotarget.12840ISI: 000389636000109Scopus ID: 2-s2.0-84998678038OAI: oai:DiVA.org:kth-199535DiVA, id: diva2:1065683
Note

QC 20170116

Available from: 2017-01-16 Created: 2017-01-09 Last updated: 2019-08-29Bibliographically approved
In thesis
1. Exploitation of interactions with the neonatal Fc receptor to manipulate biological half-lives for therapeutic applications
Open this publication in new window or tab >>Exploitation of interactions with the neonatal Fc receptor to manipulate biological half-lives for therapeutic applications
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein engineering provides powerful tools to create useful proteins with desired properties. In this thesis, rational design principles have been used for development of fusion proteins that can interact with the neonatal Fc receptor (FcRn) for potential medical applications. 

FcRn is widely expressed in the human body. The natural ligands of FcRn are immunoglobulin G (IgG) and serum albumin (SA). FcRn can bind to both proteins in a pH dependent manner and endow them with an unusually long half-life in vivo. Protein building blocks interacting directly or indirectly with FcRn may potentially be used to either piggy-back on the FcRn-system for extension of the in vivo half-life or to saturate the system to decrease the in vivo half-life of the natural ligands. In this thesis, I have explored an FcRn binding affibody molecule (ZFcRn) and/or an albumin binding domain (ABD) for these purposes. 

In study I and II, the prolactin receptor was found to often be expressed in glioblastoma multiforme tumors from patients as well as in glioblastoma multiforme cells lines. We investigated a novel antagonist of the prolactin receptor in vitro and found that it could block signaling through the receptor as well as cellular invasiveness. An antagonist of prolactin receptor could thus potentially become a drug for treatment of glioblastoma multiforme. However, the antagonist will likely have a short plasma half-life due to its small molecular size, which limits its usability. Therefore, it was expressed as a fusion to ABD, which interacts indirectly with FcRn. The produced fusion protein was found to be able to block signaling through the prolactin receptor in vitro and also had a prolonged plasma half-life in vivo

The goal of study III was to investigate the properties of human growth hormone (hGH) when it was expressed as a protein fusion with ZFcRn, interacting directly with FcRn, and/or ABD. The fusion proteins, ZFcRn-hGH, ABD-hGH, and ZFcRn-ABD-hGH could be recombinantly expressed and successfully purified to homogeneity. They had the expected binding abilities to FcRn, SA and hGH receptor. They were all found to be able to induce signaling over the plasma membrane in a model cell line. 

Patients suffering from many autoimmune diseases produce particular IgG molecules, which are responsible for the disease symptoms. A potential treatment could be to increase the catabolism of these IgGs to relieve disease symptoms. In study IV, an FcRn interacting affibody molecule was investigated for IgG depletion by blocking the IgG/FcRn interaction. In vitro, we first found that the affibody molecule shares a common binding site with IgG on FcRn, which indicates that the affibody should be able to block IgG from binding to FcRn. In vivo, we injected large amounts of the affibody molecules in different formats in mice and found up to 39% reduction of total endogenous IgG. In a clinical setting, reduction of total IgG level would also reduce the disease causing IgGs, and potentially ameliorate the symptoms of IgG-driven autoimmune diseases. 

Taken together, I have in this thesis explored application of FcRn interacting molecules for extension of biological half-lives of therapeutically relevant proteins and reduction of total IgG level by FcRn blocking. 

Abstract [sv]

Proteinteknik tillhandahåller kraftfulla verktyg för att skapa användbara proteiner med önskade egenskaper. I denna avhandling har rationella designprinciper använts för utveckling av fusionsproteiner som kan interagera med neonatal Fc-receptorn (FcRn) för potentiella medicinska tillämpningar. 

FcRn uttrycks allmänt i människokroppen. De naturliga liganderna aför FcRn är immunglobulin G (IgG) och serumalbumin (SA). FcRn kan binda till båda proteinerna på ett pH-beroende sätt och förse dem med en ovanligt lång halveringstid in vivo. Proteinbyggstenar som interagerar direkt eller indirekt med FcRn kan potentiellt användas för att antingen åka snålskjuts på FcRn-systemet för förlängning av halveringstiden in vivo eller för att mätta systemet för att minska halveringstiden in vivo för de naturliga liganderna. I den här avhandlingen har jag utforskat en FcRn-bindande affibody molekyl (ZFcRn) och en albuminbindande domän (ABD) för dessa ändamål. 

I studie I och II befanns prolaktinreceptorn ofta uttryckt i glioblastoma multiforme tumörer från patienter såväl som i glioblastoma multiforme cellinjer. Vi undersökte en ny antagonist av prolaktinreceptorn in vitro och fann att den kunde blockera signalering genom receptorn såväl som cellulär invasivitet. En prolaktinreceptorantagonist kan således potentiellt bli ett läkemedel för behandling av glioblastoma multiforme. Men antagonisten kommer troligen att ha en kort halveringstid i blodet på grund av dess lilla storlek, vilket begränsar användbarheten. Därför uttrycktes det som en fusion till ABD, som interagerar indirekt med FcRn. Det producerade fusionsproteinet visade sig kunna blockera signalering genom prolaktinreceptorn in vitro och hade också en förlängd halveringstid i blodet in vivo

Målet med studie III var att undersöka egenskaperna hos humant tillväxthormon (hGH) när det uttrycktes som en proteinfusion med ZFcRn som interagerar direkt med FcRn och/eller ABD. Fusionsproteinerna, ZFcRn-hGH, ABD-hGH och ZFcRn-ABD-hGH kunde uttryckas rekombinant och kunde framgångsrikt renas till homogenitet. De hade de förväntade bindningsförmågorna till FcRn, SA och hGH-receptorn. De visade sig alla kunna inducera signalering över plasmamembranet i en modellcellinje. 

Patienter som lider av många autoimmuna sjukdomar producerar specifika IgG-molekyler som är ansvariga för sjukdomens symtom. En potentiell behandling kan vara att öka katabolismen av dessa IgG för att lindra sjukdomssymtomen. I studie IV undersöktes en FcRn-interagerande affibodymolekyl för IgG-minskning, detta genom att blockera IgG/FcRn-interaktionen. In vitro fann vi först att affibodymolekylerna delar ett gemensamt bindningsställe med IgG på FcRn, vilket indikerade att affibodymolekylen borde kunna blockera IgG från bindning till FcRn. In vivo injicerade vi stora mängder av affibodymolekylen i olika format i möss och fann upp till 39% reduktion av den totala mängden endogent IgG. I en klinisk kontext skulle reduktion av total IgG också minska nivån av de IgG som driver sjukdomen och potentiellt minska symtomen hos patienter som lider av IgG-drivna autoimmuna sjukdomar. 

Sammantaget har jag i den här avhandlingen undersökt tillämpningar av FcRn-interagerande molekyler för förlängning av biologiska halveringstider av terapeutiskt relevanta proteiner och dessutom reduktion av totalt IgG genom FcRn-blockering. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2019. p. 87
Series
TRITA-CBH-FOU ; 2019:41
Keywords
Glioblastoma Multiforme (GBM), Prolacin (Prl), Prolacin receptor (PrlR), Prolactin antagonist (PrlRA), Albumin binding domain (ABD), serum albumin (SA), half-life extension, FcRn, Affibody, human growth hormone (hGH), immunoglobulin G (IgG), blocking, autoimmune disease
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-256536 (URN)978-91-7873-268-5 (ISBN)
Public defence
2019-09-20, Oskar Kleins Auditorium, Roslagstullsbacken 21, Albanova University Center, Stockholm, 10:30 (English)
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Note

QC 2019-08-29

Available from: 2019-08-29 Created: 2019-08-29 Last updated: 2019-08-29Bibliographically approved

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