D'Arrigo, Paolo (2018) A post-translational mechanism of PD-L1 regulation in glioblastoma multiforme. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Resource language: English
Title: A post-translational mechanism of PD-L1 regulation in glioblastoma multiforme
D'Arrigo, Paolopaolo.darrigo@unina.it
Date: 10 December 2018
Number of Pages: 103
Institution: Università degli Studi di Napoli Federico II
Department: Medicina Molecolare e Biotecnologie Mediche
Dottorato: Medicina molecolare e biotecnologie mediche
Ciclo di dottorato: 31
Coordinatore del Corso di dottorato:
Avvedimento, Vittorio EnricoUNSPECIFIED
Romano, Maria FiammettaUNSPECIFIED
Date: 10 December 2018
Number of Pages: 103
Keywords: Glioblastoma multiforme; PD-L1; FKBP51
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Area 06 - Scienze mediche > MED/06 - Oncologia medica
Date Deposited: 09 Jan 2019 13:28
Last Modified: 23 Jun 2020 09:57
URI: http://www.fedoa.unina.it/id/eprint/12608

Collection description

Glioblastoma Multiforme is one of the most aggressive form of malignancy of the central nervous system. To date, the treatment of this malignancy does not significantly improve the poor overall survival of the patients. The dramatic outcomes of immunotherapy in the treatment of several solid and hematologic tumors are giving new hopes for the treatment of glioblastoma. A series of clinical studies are currently ongoing, encouraged by the promising pre-clinical results achieved. PD-L1 is one of the main immunotherapy molecular targets expressed on glioma cell surface. PD-L1 interacts with the cognate receptor PD1, expressed on the lymphocytes, and conveys an immune-inhibitory signalling. Increasing evidence suggests that PD-L1, in addition to its well-known immunomodulatory function, exerts tumor intrinsic functions that sustain self-renewal and growth. Currently, the overall mechanism that regulates the expression of PD-L1 in tumor cells is still unclear. Glioma tumors express high levels of the immunophilin and co-chaperone FKBP51. Our research group have identified a splicing isoform of this immunophilin, FKBP51s, in melanoma patients and provided elements in support of a role for this isoform as a foldase that serves to PD-L1 protein maturation. The aim of my thesis work was to address the role of FKBP51s in regulation of PD-L1 expression in glioblastoma. Moreover, I also attempted to investigate whether PD-L1 could affect the tumor intrinsic pro-oncogenic properties. To analyse the role of FKBP51s on PD-L1 expression and function, I used D54 and U251 glioblastoma cell lines that constitutively express PD-L1 and FKBP51s. By immunoblot and flow cytometry, I measured the expression of FKBP51s and PD-L1. By sub-cellular fractionation and co-immunoprecipitation assays, I investigated FKBP51s localization and interaction with PD-L1. By co-cultures with PBMCs, I measured the tumor capacity to kill lymphocytes through PD-L1. By spheroids assay, immunofluorescence for Ki67 and cell counting, I measured the effect of PD-L1 and FKBP51s on glioma cell self-renewal and growth capacities. Using a murine model of glioblastoma, made by injection of GFP+luc+ U87 cells, I addressed the effect of a selective inhibitor of FKBP51, called SAFit2, on tumor growth and aggressiveness. Results obtained showed that FKBP51s is abundantly expressed in glioma cells. FKBP51s co-localized with PD-L1 in the ER. Silencing of FKBP51s expression or inhibiting its function by SAFit2 decreased glycosylated PD-L1 levels and its expression on plasma membrane. Moreover, PD-L1 silencing decreased spheroids formation especially in a subset of tumor cells which is isolated by the subventricular zone (SVZ) and has a high tumorigenic capacity. The same effect was obtained with SAFit2 that also decreased Ki67 expression and proliferation capacity of glioblastoma cells. Daily treatment with SAFit2 reduced the volumes of formed tumors, in the in vivo glioblastoma model, and reduced the vimentin expression, in comparison with tumors from mice treated with the drug vehicle. In conclusion, FKBP51s is involved in a post-transcriptional mechanism that regulates PD-L1 maturation and consequent expression on glioblastoma plasma membrane. Glioblastoma cells with a high tumorigenic capacity, identified in the SVZ, expressed higher levels of PD-L1 compared with cells from the tumor mass grown in the nigrostriatal region. Inhibiting FKBP51s function decreases PD-L1 expression and reduces self-renewal and growth capacities of glioblastoma cells in vitro and in vivo.


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