PO-250 Phospho S82 Hsp27: a marker of invasive and recurrent non-functioning pituitary adenoma

Abstract

Introduction Introduction:Non-functioning pituitary adenomas (NFPA) usually present as macroadenomas and 42% shows invasion of the adjacent structures, such as cavernous sinus, sphenoid bone or nasopharynx and some recur locally. The incomplete surgery section of invasive non-functional pituitary adenomas (NFPAs) carries the increased risks of recurrence and requires adjuvant radiotherapy and surgeries. It is necessary to clarify the molecular mechanisms and markers of invasiveness to guide the management of NFPA patients. Here we describe a comprehensive phosphoproteomic evaluation of 20 NFPAs followed by validation in a larger set of samples. Material and methods Peptides from 20 tumours were enriched with TiO 2 beads, fractionated using bRPLC and subjected to high-throughput LC-MS/MS-Orbitrap Fusion Tribrid Mass Spectrometer. Upto 5 precursor ions were chosen for MS/MS analysis. Following MASCOT and SEQUEST analysis our bioinformatics pipeline (PhosphositePlus, Gene Ontology, DAVID, and KEGG) identified 1345 phosphoprotein groups and 2233 unique phosphopeptides. Eight candidate phosphoproteins involved in cell proliferation and growth were selected for validation using immunohistochemistry(tissue microarray containing 200 NFPA samples) and immunoblotting (n=18). Results and discussions Hsp27 phospho-Ser82 was found 2.1 fold upregulated in invasive and 7.8 fold hyperphosphorylated in recurrent group while Hsp phospho-Ser15 was found 2.1 fold upregulated in invasive and 7.8 fold hyperphosphorylated in recurrent group in our mass spectrometry experiment. Immunohistochemistry for Ser82 revealed 2.1-fold upregulation (p<0.0001) in the recurrent and 1.6 (p<0.01) fold upregulation in InvasiveNFPA. There was no significant upregulation in Ser15 was observed in invasive and recurrent groups compared to non-invasive and non-recurrent. In agreement with the mass spectrometry and IHC data, immunoblotting also relvealed significantly upregulated Hsp27 phospho-Ser 82 in invasive and recurrent group. HSPB1 serine 82 phosphorylation by VEGF activation of PKC-mediated PKD induces endothelial migration and tubulogenesis, indicating the potential importance of HSPB1 in VEGF-dependent angiogenesis and hence pituitary tumorigenesis. Conclusion Hsp27 phosphorylated at Ser82 is significantly associated with NFPA invasion and recurrence. It Provides a roadmap for patient stratification, and prognostication for recurrence and trials for targeted therapy.