EP1137 - ECE_2413 - The importance of cell adhesion molecules CADM2/3 and miR-127 and miR432 for clinically non-functioning pituitary tumours

Abstract

The exact molecular mechanisms driving non-functioning-PitNETs invasiveness are still unclear, and reliable molecular biomarkers remain unknown. Although, the role of microRNAs (miRs) as promising markers of invasiveness in other PitNETs is increasingly highlighted. Our study aimed to identify novel miRs and differentially regulated genes in invasive and noninvasive NF-PitNETs. We have validated our results in a large cohort of tumour and control normal normal pituitary samples. RNA sequencing was conducted on 13 fresh-frozen human NF-PitNET samples (7 invasive and 6 noninvasive; gonadotrophs), while microRNA sequencing was performed on 12 of these samples. Validation was carried out using Fluidigm HD GEX arrays with TaqMan assays and quantitative PCR among NF-PitNETs (n = 80) and normal pituitary samples (n = 10). Confirmatory results included 23 selected mRNA genes (controls: GAPDH and SNORD61) and 38 miRs (including 35 miRs identified by microRNA sequencing, two reference genes, and three miRs previously reported in the literature). RNA sequencing revealed differential expression of 10 733 genes, of which 364 reached statistical significance (adjusted P < .05) and were involved in 783 biological processes. Parallel miRNA-Seq identified 35 downregulated miRNAs mapping to 143 Gene Ontology (GO) processes. Gene Ontology and KEGG pathway analyses highlighted dysregulation of cell adhesion and adherens junction pathways, implicating their role in tumor invasiveness. Validation in an expanded cohort demonstrated overexpression of cell adhesion molecules (CADM2, CADM3) important for cell-cell adhesion, aggregation as well as synapse organization and ephrin receptor B1 (EPHB1), whose major role includes targeted cell migration and adhesion. We also found downregulation compared to normal pituitary tissue of CRIM1 (Cysteine Rich Transmembrane BMP Regulator 1) responsible for tissue development through interactions with members of the transforming growth factor beta family, together with downregulation of BCL2L11 transcript (belonging to BCL-2 protein family) known to act as apoptotic activator. MiR-127-5p showed a weak but statistically significant positive correlation with BCL2L11 and CRIM1 and a statistically significant negative correlation with CADM2 and CADM3. Additionally, miR-432-5p was negatively correlated with CADM2 expression in NF-PitNET tissues (r = −0.4176, P = .0005). MiRs may regulate post-transcriptionally genes expression, thus our findings may suggest possible mechanisms underlying NF-PitNETs development and/or progression. Our study reveals novel genes and miRs in NF-PitNETs suggesting their potential regulatory role in cell adhesion, migration, and apoptosis therefore potentially contributing to their pathogenesis. Grants from the National Science Center, Poland PRELUDIUM UMO-2016/23/N/NZ5/02597 and SONATINA UMO-2025/56/C/NZ4/00016 received by first author (B.R.M).