A regulatory role for the co-chaperone FKBP51s in PD-L1 expression in glioma

// Paolo D’Arrigo 1, * , Michele Russo 1, * , Anna Rea 1 , Martina Tufano 1 , Elia Guadagno 2 , Maria Laura Del Basso De Caro 2 , Roberto Pacelli 2 , Felix Hausch 3 , Stefania Staibano 2 , Gennaro Ilardi 2 , Silvia Parisi 1 , Maria Fiammetta Romano 1 and Simona Romano 1 1 Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Naples, Italy 2 Department of Advanced Biomedical Sciences, Federico II University, Naples, Italy 3 Technical University Darmstadt Institute of Organic Chemistry and Biochemistry, Darmstadt, Germany * These authors have contributed equally to this work Correspondence to: Maria Fiammetta Romano, email: mariafiammetta.romano@unina.it , romano@dbbm.unina.it Keywords: PD-L1, glioblastoma multiforme, FKBP51, ionizing radiation, SAFit Received: March 10, 2017 Accepted: June 11, 2017 Published: July 17, 2017 ABSTRACT Background: FKBP51 is a co-chaperone with isomerase activity, abundantly expressed in glioma. We previously identified a spliced isoform (FKBP51s) and highlighted a role for this protein in the upregulation of Programmed Death Ligand 1 (PD-L1) expression in melanoma. Because gliomas can express PD-L1 causing a defective host anti-tumoral immunity, we investigated whether FKBP51s was expressed in glioma and played a role in PD-L1 regulation in this tumour. Methods: We used D54 and U251 glioblastoma cell lines that constitutively expressed PD-L1. FKBP51s was measured by immunoblot, flow cytometry and microscopy. In patient tumours, IHC and qPCR were used to measure protein and mRNA levels respectively. FKBP51s depletion was achieved by siRNAs, and its enzymatic function was inhibited using selective inhibitors (SAFit). We investigated protein maturation using N-glycosidase and cell fractionation approaches. Results: FKBP51s was expressed at high levels in glioma cells. Glycosylated-PD-L1 was increased and reduced by FKBP51s overexpression or silencing, respectively. Naive PD-L1 was found in the endoplasmic reticulum (ER) of glioma cells complexed with FKBP51s, whereas the glycosylated form was measured in the Golgi apparatus. SAFit reduced PD-L1 levels (constitutively expressed and ionizing radiation-induced). SAFit reduced cell death of PBMC co-cultured with glioma. Conclusions: Here we addressed the mechanism of post-translational regulation of PD-L1 protein in glioma. FKBP51s upregulated PD-L1 expression on the plasma membrane by catalysing the protein folding required for subsequent glycosylation. Inhibition of FKBP51s isomerase activity by SAFit decreased PD-L1 levels. These findings suggest that FKBP51s is a potential target of immunomodulatory strategies for glioblastoma treatment.