Endocrine Abstracts

Background: The exact mechanism behind the hypersecretion of ACTH and lack of negative cortisol feedback on POMC regulation in functional corticotroph adenomas (FCA) is unknown. Silent corticotroph adenomas (SCA) express, but do not secrete functional ACTH and have lower POMC expression. Using RT-qPCR and immunohistochemistry, previous studies have identified some POMC-transcription factors, regulators and processing enzymes to be differentially expressed between FCA and SCA. For example, several G protein-coupled receptor (GPCR) molecules synergistically affect POMC downstream signalling and increase its expression. Also, some GTPases regulating intracellular vesicle trafficking, separately and cooperatively stimulate ACTH secretion in AtT20 cells.

Aim: To investigate differentially expressed genes (DEGs) between FCA and SCA with focus on POMC-expression regulators such as GPCR signalling and intracellular vesicle trafficking and to elucidate the mechanisms behind the SCA silence.

Material and methods: RNA sequencing was performed using Illumina high-throughput sequencing in six FCA (three women, five microadenomas) and six SCA (two women, all macroadenomas). All adenomas stained positive for ACTH. Data were analysed using the tophat 2- cufflinks-CummeRbund pipeline.

Results: We found 631 significant DEGs (fold change (FC)>1.9, q<0.05) of which 345 were up-regulated and 286 were down-regulated in SCA compared to FCA. As expected, POMC (FC=33.3) and POMC transcription factors NUR77 (FC=6.8) and TBX19 (FC=3.8) had lower expression in SCA. PCSK2 (FC=18.3), a POMC processing enzyme, was up-regulated in SCA. Reactome pathway analysis categorized 79 DEGs involved in ‘signal transduction’ including 12 up-regulated and 16 down-regulated in GPCR signalling. Among these, EDN3 (FC=190.6), RGS16 (FC=19.1), GNAS (FC=2.3), were up-regulated, GNG2 (FC=3.9), ADCY5 (FC=5.0), GRK3 (FC=2.8), MAPK1 (FC=2.3), and RASGRF2 (FC=5.9) were down-regulated in FCA compared to SCA. There were 24 DEGs found to be involved in ‘vesicle transport’. Among these, RAB8B (FC=2.7), RAB3C (FC=2.6) and RAB3GAP1 (FC=2.4) had lower expression, whereas ALS2CL (FC=4.1) and TRIP8B (FC=2.8) had higher expression in FCA compared to SCA.

Conclusion: RNA-seq. analysis showed that the FCA and SCA separate in two groups with a high number of DEGs. Lower POMC- and higher PCSK2 expression in SCA could explain the diminished ACTH production. Interestingly, the opposite regulation of RAB8B and TRIP8B suggests that they have different stimulatory effect on ACTH secretion. Several regulators of GPCR signalling and vesicle transport molecules were found to be differentially expressed and investigating their mechanism of action in further in vitro studies will increase our understanding on POMC regulation and may yield valuable knowledge for drug development.