ABSTRACT: Background:Non-functional pituitary adenoma (NFPA) is highly heterogeneous with different hormone expression subtypes. Of them, follicle-stimulating hormone (FSH)-positive expression is an important subtype of NFPAs. It is well-known that FSH exerted its functions through binding its receptor. However, the expression rate of FSH receptor was significantly higher in aggressive pituitary adenomas. This study aimed to investigate the molecular characteristics of FSH-positive NFPAs for effective stratification of patient, target treatment, prognostic assessment, and personalized treatment of FSH-positive NFPAs. Methods:Tandem mass tag (TMT)-based quantitative proteomics was used to investigate differentially expressed proteins (DEPs) between FSH-positive and negative NFPAs. Gene ontology and KEGG pathway enrichment analyses were used to analyze the DEPs. Differentially expressed genes (DEGs) between invasive and non-invasive NFPAs from GEO database were analyzed with pathway enrichment analysis. Protein-protein interaction (PPI) networks were constructed based on DEPs in excetral cellular matrix (ECM)-receptor interaction, focal adhesion, and PI3K-Akt pathways. Cytoscape was used to obtain most significant modules. Western blot was used to validate the expressions of upregulated proteins (ITGA1, ITGA6, and ITGB4), the expression and phosphorylated status of Akt in PI3K-Akt pathway, and the expression of FSH receptors in FSH-positive relative to negative NFPAs. Results:A total of 594 DEPs (374 upregulated and 220 downregulated) were identified between FSH-positive and negative NFPAs. Nineteen KEGG pathway networks were identified to involve DEPs, and reveal molecular differences between FSH-positive and negative NFPAs, including three important pathways that were significantly associated with tumor invasiveness and aggressiveness: ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathways. Further, focal adhesion pathway was also confirmed with invasiveness-related NFPA DEG data that were derived from GEO database. Moreover, the significantly upregulated DEPs (ITGA1, ITGA6, and ITGB4) that were associated with tumor invasiveness and aggressiveness were confirmed by immunoaffinity analysis in FSH-positive vs. negative NFPAs. Also, the phosphorylation level but not its expression level of AKT in PI3K-AKT signaling was significantly increased, and the expression level of FSH receptor was significantly increased in FSH-positive relative to negative NFPAs. Also, overlapping analysis of 594 DEPs and 898 DEGs revealed 45 invasiveness-related DEPs, including 11 upregulated DEPs (ITGA6, FARP1, PALLD, PPBP, LIMA1, SCD, UACA, BAG3, CLU, PLEC, and GATM) that were also upregulated genes in invasive NFPAs, and 8 downregulated DEPs (ALCAM, HP, FSTL4, IL13RA2, NPTX2, DPP6, CRABP2, and SLC27A2) that were also downregulated genes in invasive NFPAs. Conclusions:FSH-positive expression was an important NFPA subtype. It was the first time for this study to reveal FSH-related proteomic variations and the corresponding molecular network alterations in FSH-positive relative to negative NFPAs. Also, three signaling pathways (ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathways) and involved upregulated proteins (ITGA1, ITGA6, ITGB4, pAKT, and FSHR) were significantly associated with tumor invasiveness and aggressiveness, and a set of invasiveness-related DEPs were identified with overlapping analysis of 594 DEPs in FSH-positive vs. negative NFPAs and 898 DEGs in invasive vs. non-invasive NFPAs. These findings offered the scientific evidence to in-depth understand molecular characteristics of FSH-positive NFPAs, and effectively stratify the post-surgery patients for personalized prognostic assessment and targeted treatment of FSH-positive NFPAs.
