Endocrine Abstracts

JOINT1570

Background: Somatotropinomas are histopathologically divided into sparsely (SG) and densely granulated (DG). SG exhibits aggressive clinical behaviour (presentation at a younger age, high proportion of invasiveness, resistance to treatments and recurrence (inspite of complete removal) as compared to DG. Dysregulation of LKB1-AMPK pathway can support tumour survival and growth by promoting autophagy and providing an adaptive response to a nutrient-poor microenvironment.

Method: High-throughput label-free quantitative mass spectrometry-based (Orbitrap Exploris mass spectrometer, Thermo Scientific) phosphoproteomics analysis was performed on SG (n = 4) and DG (n = 4) tumours in triplicates. Candidates of significantly enriched pathways (cut-off=1.5fold-change) were considered for validation by immunohistochemistry (n = 32; SG=24, DG=8) on tissue microarray in separate group of samples.

Results and Discussion: In our data (total=10418) 835 over and 4246 under phosphorylated proteins were detected. There was significant over-phosphorylation of 85 proteins in the LKB1-AMPK pathway (fold enrichment = 1.8, P = 2.2E-09) in SG tumours. Commercially available antibody guided the selection of four candidates TSC1 Ser1080, AMPKβ Ser108, MAPT Thr548, and ULK1 Ser638 for IHC. Over-phosphorylation of AMPKβ Ser108 (6.0-fold, P = 0.01) suggests metabolic adaptation to energy stress. Increase in TSC1 Ser1080 phosphorylation (9.5-fold, P = 0.01) implies inhibition of the TSC complex, potentially activating mTOR signalling and promoting anabolic processes in SG tumours. Over-phosphorylation of MAPT at Thr548 (3.8-fold, P = 0.02) suggests enhanced cytoskeletal dynamics, which can potentially facilitate invasion. ULK1 Ser638 phosphorylation showed increase (1.8-fold) phosphorylation. Ratio of Phosphorylated ULK1 Ser638 to Total ULK1 was significantly increased in SG tumours (P = 0.01). This trend may indicate autophagic adaptation to metabolic stress. Upstream analysis identified PRKAA1 and GSK3β as regulators of AMPKβ Ser108 and MAPT Thr548, respectively, mTOR phosphorylates TSC1 Ser1080 and ULK1 Ser638. IHC confirmed increase in mTOR expression in SG tumours (2.66-fold P = 0.003) compared to DG, underscoring mTOR’s role in SG tumours. We observed significant correlation of AMPKβ Ser108 phosphorylation with MAPT Thr548 (r = 0.7, P = 0.0001) and TSC1 Ser1080 (r = 0.59, P = 0.002) phosphorylation. ULK1 Ser638 phosphorylation levels significantly correlated (r = 0.5, P = 0.01) with mTOR levels. Age was found to be inversely correlated with MAPT Thr548 (r=-0.5, P = 0.01) and ULK1 Ser638 (r=-0.4, P = 0.02). Maximum tumour diameter showed significant positive correlation with ULK1 Ser638 (r = 0.5, P = 0.01) and mTOR expression (0.4, P = 0.03) levels while tumour volume was found to be correlated with mTOR expression only (r = 0.6, P = 0.02).

Conclusions: Our results reveal a coordinated network of mTOR signalling complemented by PRKAA1-mediated AMPK activation and GSK3β-driven cytoskeletal remodelling in SG tumours.