MiR-34a is upregulated in AIP-mutated somatotropinomas and promotes octreotide resistance.

Eva‐Maria Bogner,Martin Irmler,Sebastian Gulde,Auli Karhu,Albert Beckers,Johannes Beckers,Hermine Mohr,Natalia S Pellegata,Adrian F Daly

doi:10.1002/ijc.33268

Abstract

Pituitary adenomas (PAs) are intracranial tumors associated with significant morbidity due to hormonal dysregulation, mass effects and have a heavy treatment burden. Growth hormone (GH)-secreting PAs (somatotropinomas) cause acromegaly-gigantism. Genetic forms of somatotropinomas due to germline AIP mutations (AIPmut+) have an early onset and are aggressive and resistant to treatment with somatostatin analogs (SSAs), including octreotide. The molecular underpinnings of these clinical features remain unclear. We investigated the role of miRNA dysregulation in AIPmut+ vs AIPmut- PA samples by array analysis. miR-34a and miR-145 were highly expressed in AIPmut+ vs AIPmut- somatotropinomas. Ectopic expression of AIPmut (p.R271W) in Aip-/- mouse embryonic fibroblasts (MEFs) upregulated miR-34a and miR-145, establishing a causal link between AIPmut and miRNA expression. In PA cells (GH3), miR-34a overexpression promoted proliferation, clonogenicity, migration and suppressed apoptosis, whereas miR-145 moderately affected proliferation and apoptosis. Moreover, high miR-34a expression increased intracellular cAMP, a critical mitogenic factor in PAs. Crucially, high miR-34a expression significantly blunted octreotide-mediated GH inhibition and antiproliferative effects. miR-34a directly targets Gnai2 encoding Gαi2, a G protein subunit inhibiting cAMP production. Accordingly, Gαi2 levels were significantly lower in AIPmut+ vs AIPmut- PA. Taken together, somatotropinomas with AIP mutations overexpress miR-34a, which in turn downregulates Gαi2 expression, increases cAMP concentration and ultimately promotes cell growth. Upregulation of miR-34a also impairs the hormonal and antiproliferative response of PA cells to octreotide. Thus, miR-34a is a novel downstream target of mutant AIP that promotes a cellular phenotype mirroring the aggressive clinical features of AIPmut+ acromegaly.

Highlights

Pituitary adenomas (PAs) comprise up to 15% of intracranial tumors and clinically relevant pituitary adenoma (PA) occur in approximately 1:1000 of the general population.[1,2] They represent a challenging health burden due to disordered hormonal function and tumor growth; PA treatments are invasive, costly and require significant expertise.[3]
We show that miR-34a has pro-oncogenic functions in PAs, likely mediated by increased cyclic adenosine monophosphate signaling, and that G-protein inhibitory alpha subunit (Gαi)[2] is a direct target of miR-34a and is differentially expressed in human PAs depending on AIPmut status
AIPmut in acromegaly leads to such somatostatin analogs (SSA) resistance, which has a major clinical impact on patients as AIPmut are associated with young-onset, large and invasive PAs.[10]

Summary

| INTRODUCTION

Pituitary adenomas (PAs) comprise up to 15% of intracranial tumors and clinically relevant PAs occur in approximately 1:1000 of the general population.[1,2] They represent a challenging health burden due to disordered hormonal function and tumor growth; PA treatments are invasive, costly and require significant expertise.[3] PAs usually occur sporadically but about 5% of cases present as part of hereditary syndromes, like familial isolated pituitary adenomas (FIPA), multiple endocrine neoplasia type 1 and 4 (MEN1, MEN4), Carney complex, McCune-Albright syndrome and X-linked acrogigantism.[4,5,6,7] Mutations of the aryl hydrocarbon receptor-interacting protein (AIP) gene (AIPmut) account for 15%-20% of FIPA kindreds and are important in the pathogenesis of pediatric-onset PAs.[8,9]. We show that miR-34a has pro-oncogenic functions in PAs, likely mediated by increased cyclic adenosine monophosphate (cAMP) signaling, and that Gαi[2] is a direct target of miR-34a and is differentially expressed in human PAs depending on AIPmut status. We report for the first time that miR-34a upregulation leads to increased cell proliferation and GH secretion in vitro, and induces resistance to the antiproliferative and hormonal effects of the first-generation somatostatin analog, octreotide

| MATERIALS AND METHODS

| RESULTS

Findings

| DISCUSSION