Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors arising from chromaffin cells of adrenal medulla or sympathetic or parasympathetic paraganglia, respectively. To identify new therapeutic targets, we performed a detailed membrane-focused proteomic analysis of five human paraganglioma (PGL) samples. Using the Pitchfork strategy, which combines specific enrichments of glycopeptides, hydrophobic transmembrane segments, and non-glycosylated extra-membrane peptides, we identified over 1800 integral membrane proteins (IMPs). We found 45 "tumor enriched" proteins, i.e., proteins identified in all five PGLs but not found in control chromaffin tissue. Among them, 18 IMPs were predicted to be localized on the cell surface, a preferred drug targeting site, including prostate-specific membrane antigen (PSMA), a well-established target for nuclear imaging and therapy of advanced prostate cancer. Using specific antibodies, we verified PSMA expression in 22 well-characterized human PPGL samples. Compared to control chromaffin tissue, PSMA was markedly overexpressed in high-risk PPGLs belonging to the established Cluster 1, which is characterized by worse clinical outcomes, pseudohypoxia, multiplicity, recurrence, and metastasis, specifically including SDHB, VHL, and EPAS1 mutations. Using immunohistochemistry, we localized PSMA expression to tumor vasculature. Our study provides the first direct evidence of PSMA overexpression in PPGLs which could translate to therapeutic and diagnostic applications of anti-PSMA radio-conjugates in high-risk PPGLs.

BIOCEV 1st Faculty of Medicine Charles University 25250 Vestec Czech Republic

Department of Internal Medicine 3 University Hospital Olomouc and Faculty of Medicine and Dentistry Palacky University 77900 Olomouc Czech Republic

Department of Urology University Hospital Olomouc and Faculty of Medicine and Dentistry Palacky University 77900 Olomouc Czech Republic

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University and General University Hospital 12800 Prague Czech Republic

Laboratory of Pathology National Cancer Institute NIH Bethesda MD 20892 USA

Section on Medical Neuroendocrinology Eunice Kennedy Shriver National Institute of Child Health and Human Development NIH Bethesda MD 20892 USA

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Identification of potential molecular targets for the treatment of cluster 1 human pheochromocytoma and paraganglioma via comprehensive proteomic characterization