Systematic Investigation of Expression of G2/M Transition Genes Reveals CDC25 Alteration in Nonfunctioning Pituitary Adenomas

Abstract

Dysregulation of G1/S checkpoint of cell cycle has been reported in pituitary adenomas. In addition, our previous finding showing that deregulation of Wee1 kinase by microRNAs together with other studies demonstrating alteration of G2/M transition in nonfunctioning pituitary adenomas (NFPAs) suggest that G2/M transition may also be important in pituitary tumorigenesis. To systematically study the expression of members of the G2/M transition in NFPAs and to investigate potential microRNA (miRNA) involvement. Totally, 80 NFPA and 14 normal pituitary (NP) tissues were examined. Expression of 46 genes encoding members of the G2/M transition was profiled on 34 NFPA and 10 NP samples on TaqMan Low Density Array. Expression of CDC25A and two miRNAs targeting CDC25A were validated by individual quantitative real time PCR using TaqMan assays. Protein expression of CDC25A, CDC25C, CDK1 and phospho-CDK1 (Tyr-15) was investigated on tissue microarray and immunohistochemistry. Several genes' expression alteration were observed in NFPA compared to normal tissues by transcription profiling. On protein level CDC25A and both the total and the phospho-CDK1 were overexpressed in adenoma tissues. CDC25A correlated with nuclear localized CDK1 (nCDK1) and with tumor size and nCDK1 with Ki-67 index. Comparing primary vs. recurrent adenomas we found that Ki-67 proliferation index was higher and phospho-CDK1 (inactive form) was downregulated in recurrent tumors compared to primary adenomas. Investigating the potential causes behind CDC25A overexpression we could not find copy number variation at the coding region nor expression alteration of CDC25A regulating transcription factors however CDC25A targeting miRNAs were downregulated in NFPA and negatively correlated with CDC25A expression. Our results suggest that among alterations of G2/M transition of the cell cycle, overexpression of the CDK1 and CDC25A may have a role in the pathogenesis of the NFPA and that CDC25A is potentially regulated by miRNAs.

A mikro-RNS-ek szerepe az agyalapimirigy-daganatok tumorbiológiájában

“…Egy miRNS-nek a bá-zispárosodás szabályai szerint több célgénje is van, és egy célmolekula kifejeződését számos miRNS is szabályoz-hatja. A biológiai funkciók hátterében álló mechanizmusokat a sejten belüli jelátviteli útvonalakkal lehet a leginkább jellemezni, így a miRNS-ek szabályozó szerepét nemcsak egy-egy génhez (1. táblázat) [34,42,[44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59], hanem egy-egy jelátviteli útvonalhoz is lehet rendelni.…”

“…Kiegészítve és kiterjesztve korábbi vizsgálatainkat, szisztematikusan megvizsgálva a G2/M átmenetben részt vevő gének expressziójának szabályozását nem ACTH = adrenokortikotrop hormon; FSH = folliculusstimuláló hormon; GH = növekedési hormon; LH = luteinizáló hormon; NFA = nem funkcionáló, hormonálisan inaktív hypophysisadenoma; PRL = prolaktin funkcionáló agyalapimirigy-daganatokban, a 46 vizsgált gén közül számos eltérő expressziót mutatott, melyek közül a CDC25-család tagjainak miRNS-ek általi szabá-lyozására derült fény [46]. A CDC25-családba tartozó foszfatázok a CDK1-ciklin-B komplex defoszforilációja révén a G2/M ellenőrző ponton segítik a sejt mitózisba lépését.…”

“…A CDC25A emelkedett expressziót mutatott mRNS-és fehérjeszinten is a nem funkcionáló adenomákban a normál agyalapimirigy-szövethez képest, és expressziója korrelált a tumormérettel is. A CDC25A-t célzó mi-RNS-ek közül négy esetén mértek csökkent expressziót a tumoros szövetben (miR-449a, miR-449b, miR-424, miR-503), és ezekből kettő (miR-424, miR-503) negatív korrelációt mutatott a tumormérettel, valamint a CDC25A expressziójával is, ami erősíti jelentőségüket az adenoma patogenezisében (1. táblázat) [46]. Mindezen eredmények arra utalnak, hogy a G2/M át-menet aktivációja felerősödik az agyalapimirigy-adenomákban, és ebben a miRNS-ek kulcsszerepet játszanak.…”

Limitations of high throughput methods for miRNA expression profiles in non-functioning pituitary adenomas

“…Adenoma tissues were removed by transsphenoideal surgery at the Hungarian National included immunostaining for anterior pituitary hormones (GH, PRL, ACTH, LH, FSH, SF1 and TSH) and staining for Ki-67 proliferation marker. All routine immunohistochemical studies were performed at the 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary as previously described [15,16]. Normal pituitary samples were obtained by autopsy within 6 h of death from patients with no evidence of any endocrine disease (University Clinical Centre, Belgrade, Serbia) [15].…”

“…This later gene has been demonstrated that is expressed exclusively in the Rathke's pouch and adenohypophysis during the development of pituitary in both chicken and mouse embryos and not in the neuroectoderm (Proszkowiec-Weglarz et al 2011). By using these expressions it could be concluded that all pituitary samples used as normal contained all five adenohypophophysis specific cell types (data not shown) [15][16][17][18]. Tumor tissue specimens were immediately frozen in liquid nitrogen after the adenoma removal, and stored at -80 o C until use.…”