Exploring epigenome-proteome interactions underlying post-surgical progression of non-functioning pituitary adenomas using hypernetwork modelling.

Non-functioning pituitary adenomas (NFPAs) present a complex clinical challenge due to their indolent and invasive growth patterns, and critical anatomical location. Post-surgical tumor progression is frequent in patients with NFPAs, which often necessitates additional therapeutic interventions. The molecular mechanisms underlying post-surgical progression remain poorly understood and there are currently no reliable methods to stratify patients according to risk of tumor progression. The aim of this study was to comprehensively characterize the molecular alterations, together with an integrated understanding of their interactions, that could potentially uncover the biological processes driving post-surgical tumor progression of NFPAs. We performed an integrated analysis of genome-wide DNA methylation and proteomics in 25 progressive and 15 indolent NFPAs using hypernetwork modelling linking CpG sites to the differentially expressed proteins to identify functional alterations associated with tumor progression. In addition, we investigated cis-regulatory relationships by examining CpG sites located within or in close proximity to the genes encoding the corresponding proteins, allowing assessment of the direct impact of DNA methylation changes on protein expression levels. Hypernetwork analysis uncovered extensive indirect and higher-order associations, capturing coordinated epigenetic influences on protein networks in indolent and progressive NFPAs. Progressive NFPAs were characterized by a compact and highly interconnected hub network with proteins primarily involved in DNA replication and transcription regulation (MCM6 and HDGFL2), chromatin organization (SAFB, HDGFL2, KDM3B, and TAF7), and cytoskeleton organization and cell structure maintenance (AJM1 and SYNE2). In contrast, indolent adenomas exhibited a broader and more diffuse network architecture with hub proteins linked to protein processing and transport (PSMD6, APMAP, B4GAT1, and COPE), extracellular matrix organization (LAMB2), and oxidative stress response (CISD2). Hub proteins in progressive NFPAs were enriched for metabolic pathways including glycolysis and tricarboxylic acid cycle, while enriched pathways for hub proteins in the indolent group were associated with genome maintenance and cellular stress responses. Hypernetwork analysis highlighted distinct epigenetic-proteomic regulatory mechanisms linked to tumor behavior that were not detected through cis-acting correlation analysis. Collectively, this integrative approach provides insight beyond direct regulation effects and offers a framework for identifying network-informed candidate markers with mechanistic relevance in tumor progression.