Endocrine Abstracts

Background: In our previous works the interdependence of DNA demethylation with proliferation and differentiation of pituitary neuroendocrine tumours (PitNET) and the inhibitory effect of Aspirin on pituitary cell proliferation were demonstrated. Although the role of Aspirin in epigenetic regulation was described in other malignancies, its correlation with pituitary tumorigenesis is currently unknown.

Our objective: was to investigate the genomic and epigenomic effects of Aspirin in PitNET.

Materials and Methods: DNA methylome by HPLC-MS/MS, and whole transcriptome profile were evaluated by next-generation sequencing in RC4-B/C and GH3 pituitary cell lines upon Aspirin treatment. Effects of Aspirin and demethylation agent, decitabine were further tested in vitro by RT-qPCR, western blot and functional (proliferation, viability, migration, luciferase promoter reporter) assays. DNA methylome was also correlated with PTTG1 expression in 41 human PitNET samples. Gene and protein expression data of 77 PitNET with 35 control samples were obtained from Gene Expression Omnibus and literature mining.

Results: Aspirin induced global DNA demethylation and consequential transcriptome changes in vitro including decreased global histone expression. Overexpression of Tet enzymes and their cofactor Uhrf2 was identified behind the increase of 5-hydroxymethylcytosine (5 hmC). Transcription factor regulatory relationships assessed by gene set enrichment analysis showed that Aspirin increased p53 and decreased E2f1 activities. The increased p53 activity was due to its acetylation at the K382 residue. Among p53 controlled genes, Pttg1 was also identified as downregulated upon Aspirin treatment. Aspirin reduced the expression of Pttg1 together with its interacting partners by inhibiting Pttg1 promoter activity. 5 hmC positively correlated with Tet1-3 and Tp53 expression, and negatively correlated with Pttg1 expression that was also reinforced by the effect of decitabine on Pttg1 expression. Both transcriptome data and in vitro assays proved that Aspirin inhibited the cell viability, cell proliferation and migration. Additionally, high overlap (20.15%) was found between Aspirin regulated genes and dysregulated genes in PitNET tissue samples. The 757 common genes were implicated mostly in cell proliferation, cell cycle – including p53 activity and function –, while cellular migration and genome stability were also detected.

Conclusion: We described a regulatory network where Aspirin regulated global demethylation, Tp53 activity and Pttg1 expression in pituitary cells along with decreased cell proliferation and migration. These data may suggest the potential beneficial effect of Aspirin in PitNET.

Funding: NKFI FK135065; Bolyai Research Fellowship of the Hungarian Academy of Sciences; TKP2021-EGA-44