miR-34a is upregulated in AIP mutated pituitary adenomas and induces octreotide-resistant cell proliferation and growth hormone secretion

Pituitary adenomas (PAs) are intracranial tumors associated with significant morbidity due to hormonal dysregulation, mass effects and 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 investigate the role of miRNA dysregulation in AIPmut+ versus AIPmut- PA samples by array analysis. miR-34a and miR-145 were found highly expressed in AIPmut+ vs AIPmut-somatotropinomas. Ectopic expression of AIPmut (p.R271W) in Aip-/- mouse embryonic fibroblasts 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 anti-proliferative effects. miR-34a directly targets Gnai2 encoding Gαi2, a G protein subunit inhibiting cAMP production. Accordingly, Gαi2 levels were significantly lower in AIPmut+ versus 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 downstream target of mutant AIP that promotes a cellular phenotype mirroring the aggressive clinical features of AIPmut+ acromegaly. We compared human somatotropinoma and prolactinoma with wild-type AIP to tumors with mutant AIP

垂体腺瘤（Pituitary adenomas, PAs）是一类颅内肿瘤，可因激素分泌紊乱、占位效应及沉重的治疗负担引发显著的不良临床结局。生长激素（Growth hormone, GH）分泌型垂体腺瘤（生长激素瘤，somatotropinomas）可导致肢端肥大症-巨人症。由种系AIP突变（AIPmut+）引发的遗传性生长激素瘤发病早、侵袭性强，且对包括奥曲肽在内的生长抑素类似物（somatostatin analogs, SSAs）治疗耐药。目前这类临床特征的分子机制仍不明确。本研究通过芯片分析，探究了微小RNA（microRNA, miRNA）表达失调在AIPmut+与AIPmut-垂体腺瘤样本中的作用。研究发现，相较于AIPmut-生长激素瘤，AIPmut+生长激素瘤中miR-34a与miR-145呈高表达。在Aip基因敲除小鼠胚胎成纤维细胞中异位表达突变型AIP（p.R271W）可上调miR-34a与miR-145的表达，证实了AIP突变与miRNA表达之间存在因果关联。在垂体腺瘤细胞系GH3中，过表达miR-34a可促进细胞增殖、集落形成能力与迁移能力，并抑制细胞凋亡；而miR-145仅对细胞增殖与凋亡产生轻度影响。此外，高表达miR-34a可升高细胞内环磷酸腺苷（cyclic adenosine monophosphate, cAMP）水平——这是垂体腺瘤中关键的促有丝分裂因子。至关重要的是，高表达miR-34a可显著削弱奥曲肽介导的生长激素抑制与抗增殖效应。miR-34a可直接靶向编码Gαi2的Gnai2基因，而Gαi2是一种可抑制环磷酸腺苷生成的G蛋白亚基。相应地，AIPmut+垂体腺瘤中Gαi2的表达水平显著低于AIPmut-垂体腺瘤。综上，携带AIP突变的生长激素瘤可高表达miR-34a，后者通过下调Gαi2的表达、升高环磷酸腺苷浓度，最终促进细胞增殖。miR-34a的上调还会损害垂体腺瘤细胞对奥曲肽的激素应答与抗增殖反应。因此，miR-34a是突变型AIP的下游靶标，其介导的细胞表型与AIPmut+肢端肥大症的侵袭性临床特征相符。本研究还将携带野生型AIP的人源生长激素瘤与泌乳素瘤，与携带AIP突变的肿瘤进行了对比分析。