Phosphorylation of Nucks1 on Serine 79: A Prognostic Marker of Recurrent Nonfunctioning Pituitary Adenoma

Introduction: Nonfunctioning pituitary adenomas (NFPA) usually present as macroadenomas. The incomplete removal of invasive NFPAs carries the increased risks of recurrence and requires adjuvant radiotherapy and surgeries. Here, we describe a comprehensive phosphoproteomics evaluation of 20 NFPAs followed by validation in a large cohort (n = 200) for biomarker of recurrence.

Material and Methods: Peptides from 20 tumors were subjected to high-throughput LC-MS/MS-Orbitrap Fusion Tribrid Mass Spectrometer. Up to five precursor ions were chosen for MS/MS analysis. Following MASCOT and SEQUEST analysis our bioinformatics pipeline (Phosphosite Plus, Gene Ontology, DAVID, and KEGG) identified 2,233 unique phosphopeptides corresponding to 1,345 phosphoproteins. Eight candidate phosphoproteins involved in cell proliferation and growth were selected for validation using immunoblotting (n = 18) and immunohistochemistry (tissue microarray containing 200 NFPA samples).

Results and Discussions: NUCKS1 phospho-Ser79 found 3.98-fold hyper phosphorylated in recurrent group in our mass spectrometry experiment. Immunohistochemistry revealed 3.2-fold upregulation (p < 0.0001) in the recurrent and 0.56 (p < 0.01) fold up change in invasive NFPA. In agreement with the mass spectrometry and IHC data, immunoblotting also revealed significantly upregulated NUCKS1 phospho-Ser79 in recurrent group. NUCKS1 phospho-Ser79 correlates with increase expression of cyclin D and hence play significant role in tumorigenesis.

Conclusion: NUCKS1 phospho-Ser79 is significantly associated with NFPA recurrence.