Endocrine Abstracts

JOINT3777

Background: Histopathologically somatotropinomas are classified as sparsely granulated (SG) and densely granulated (DG). Compared to DG, SG displays aggressive clinical behaviour, including a younger presentation, a higher percentage of invasiveness, therapy resistance, and recurrence (after total removal). A quantitative proteomics-based approach can dissect the signalling pathways between SG and DG subtypes and can potentially leads to new therapeutic targets in SG subgroups.

Method: Surgically resected 8 sporadic somatotropinoma (SG (n=4) and DG (n=4)) were subjected to high-throughput label-free quantitative mass spectrometry-based (Orbitrap Exploris mass spectrometer, Thermo Scientific) proteomics analysis in triplicates. Functional enrichment was conducted to identify the biological pathways and functions of the identified proteins. Topmost significantly differentially expressed proteins (cut-off=1.5 fold-change) were considered for validation by immunofluorescence (IF) (n=10; SG=5, DG=5) and western blot (n=8 SG=4, DG=4).

Results: Mass spectrometry analysis identified 41,786 peptides corresponding to 5,163 proteins. Of these, 4,037 proteins were detected with ≥2 unique peptides. Statistical analysis showed significant differential expression of 1014 proteins, with overexpression of 44 proteins and under expression of 970 proteins. TNIP1(P=0.0001) was the most overexpressed followed by PRSS29P (P=0.001), HPN (P=0.001) and DTNP (P=0.002) while COQ6 (0.00004) was the most under expressed protein. Functional enrichment analysis showed significant enrichment of glutathione synthesis pathway (P=0.004) and integrin signalling pathway (P=0.004) for overexpressed while mRNA processing (P<0.0001) and splicing (P<0.0001) pathways were significantly enriched for under expressed proteins. TNIP1 plays important role in EGFR mediated signalling cascade and inhibition of ERK2 mediated trancription^1,2. ELK, a transcription factor regulated by ERK2, was found to be significantly enriched in our dataset (P=0.04). Notably, 122 proteins identified as targets of ELK exhibited under expression, suggesting a potential ELK-mediated transcriptional repression by TNP1. Furthermore, western blot analysis showed 5.9-fold increase (P=0.007) in TNIP1 expression in SG tumours as compared to DG tumours while there was no significant difference in the COQ6 levels between two groups. Immunofluorescence showed overexpression and nuclear localisation of TNIP1 in SG tumours as compared to DG.

Conclusion: Our findings suggest a potential role of TNIP1 in modulating ERK2-ELK signalling in SG tumours.

References: 1. Flores AM, Gurevich I, Zhang C, Ramirez VP, Devens TR, Aneskievich BJ. TNIP1 is a corepressor of agonist-bound PPARs. Archives of Biochemistry and Biophysics 2011.

2. Ramos JW. The regulation of extracellular signal-regulated kinase (ERK) in mammalian cells. The International Journal of Biochemistry & Cell Biology 2008;40:2707–19.