Expressão dos genes GNAS e BTG2 e de um painel de microRNAs em somatotrofinomas esporádicos com e sem mutação no gene GNAS

Abstract

Introduction: Mutations in GNAS and AIP genes are present in 35% and 3%, respectively, of the sporadic somatotropinomas. Recently, increased biological importance of microRNAs (miRNAs) has been observed in pituitary tumorigenesis. However, the molecular mechanisms involved in the pathogenesis of 60% of these tumors remain to be elucidated. Objectives: To identify the prevalence of mutations in GNAS and AIP genes in a series of sporadic somatotropinomas. Compare clinical, bioquimical parametrer at diagnosis as age, tumor size and theirs aggressiveness, pre-operative growth hormone (GH), prolactin (PRL) and insulin-like growth factor-I (IGF-1) levels and treatment responsiveness between somatotropinomas with (gsp+) and without (gsp-) GNAS mutation.To analyze the expression of GNAS and BTG2 genes and a panel of miRNAs between somatotrofinomas and normal pituitaries (NP) and the association between the expression of these genes and miRNAs with aggressiveness, as well as disease control with surgery or control with all adjuvant therapeutic approaches. Material and Methods: 26 patients with acromegaly. GH basal ≤2.5μg/L or nadir after OGTT ≤1μg/L and normal IGF-I matched for age and sex were used as diagnosis and for cure criteria after transsphenoidal surgery (TS). As control after somatostatin analogues (SA), we adopted the normalization of IGF-I matched for age and sex. Tumor size was evaluated by MRI/CT and the degree of invasiveness by Hardy score (I to IV).Tumor samples (26) were obtained during TS, processed for histopathology and stored at -70°C for molecular studies. NP (07) were obtained during autopsy. Total DNA and RNA were extracted by TRIzol®. Codons 201 and 227 of the GNAS gene and the whole AIP gene were sequenced. Relative expression of BTG2 and GNAS genes and miRNAs let-7a, miR-16a, miR-21, miR-141, miR-143, miR-15a, miR-145, miR-23a, miR-23b, and miR-24-2 was measured by qPCR (TaqMan probes) using 2-ΔΔCt method. Results: Frequencies of GNAS and AIP mutations were 35% and 3.8%, respectively. There was no difference between the mean age (39.0 ± 11.5 vs 43.6 ± 9.0 years, p=0.32), basal GH (62.4±128.1 vs 39.9 ± 48.3 μg/L; p=0.39), IGF-I (435.5 ± 230.8 vs. 556.9 ± 238.3; p=0.32) and PRL (25.7 ± 29.8 vs. 30.9 ± 32.8 ng/L, p=0.69) in plasma concentration, and tumor aggressiveness (p=1.00) between (gsp+) and (gsp-) groups. We observed that 80% (04/05) of gsp+ whereas 33% (02/06) of the gsp- achieved control (p=0.07) after SA therapy adjuvant to TS. When SA, dopamine agonists and/or external radiotherapy were associated 100% (05/05) of gsp+ group and 44% (04/09) of gsp- group (p=0.08) showed disease control.There was no difference in GNAS expression between somatotropinomas and NP (1.07 ± 0.55 vs 0.98 ± 0.28, p=0.97) as well as between somatotropinomasgsp+ and gsp- (1.04 ± 0.59 vs 1.10 ± 0.55, p=0.97, respectively). Hardy I/II tumors showed higher GNAS expression than Hardy III/IV (p=0.02), but there was no association between GNAS expression and disease control with surgery alone or associated with other adjuvant therapies. We observed hypoexpression of BTG2 and miR-16a and miR-141 in somatotropinomas compared with NP (-6.6 fold, p=0.002; -10.0 fold, p=0.01; and -50.0 fold, p=0.0003, respectively) with no difference between gsp+ and gsp- somatotropinomas. There was miR-21 overexpression in somatotropinomas compared with NP (20.2 ± 18.5 vs 2.5 ± 3.6; 10.2 fold, p=0.02), with no difference between gsp+ and gsp- somatotropinomas. However, miR-145 and miR-23b were more hipoexpressed in gsp+ compared to gsp- (-4.8fold, p=0.03 and-2.7 fold, p=0.02). There was no association between the expression of BTG2 and a panel of miRNAs with aggressiveness or disease control. Conclusion: In this series of assumed sporadic somatotopinomas, the frequencies of mutations in GNAS (35%) and AIP (3.8%) were similar to the literature. There were no differences in clinical and biochemical characteristics, aggressiveness, response to therapy, and GNAS expression in patients with gsp+ and gsp- somatotropinomas. Hypoexpression of BTG2, a tumor suppressor gene related to p53 and Rb signaling pathways, low expression of tumor suppressor miRNAs and high expression of oncomirs in somatotropinomas suggest a role in the somatotrophic tumorigenesis.