Introduction
Clinically non-functioning adenomas of the pituitary gland account for about 30 % of all adenomas. About 80 % of them belong to the gonadotroph lineage. They are usually diagnosed when signs and symptoms of mass effects occur, and about 40 % of them extend to the cavernous sinus and, less commonly, invade the sellar floor, making their resection and post-operative radiotherapy challenging [
14,
34]. In addition, they are usually resistant to pharmacological treatment with somatostatin analogs and dopamine agonists. Chemotherapy has been reserved as salvage therapy in aggressive cases although the results have often been disappointing [
34]. Novel therapeutic approaches are therefore needed for these tumors.
A prerequisite to identify novel therapeutic targets for human tumors is a good understanding of the pathogenetic mechanisms. The molecular mechanisms underlying the development of PAs in general, and of gonadotroph adenomas in particular, have only been incompletely unraveled and this is in part due to the limited availability of suitable animal models and by the fact that those available are not always representative of human adenomas [
27].
We have recently identified a multiple endocrine neoplasia syndrome occurring in a Sprague–Dawley-derived rat strain (named MENX) characterized by the occurrence of spontaneous PAs with complete penetrance. MENX is caused by a biallelic loss-of-function mutation in
Cdkn1b, encoding the cell cycle inhibitor p27, which results in a highly unstable protein [
29,
33]. MENX-associated PAs are histologically and ultrastructurally remarkably similar to human gonadotroph adenomas. They also show high mitotic activity and elevated Ki67 labeling index [
26]. Primary cells derived from these tumors are a suitable model for pharmacological studies of PAs [
24].
To elucidate the mechanisms associated with the development of human gonadotroph adenomas we exploited the MENX model and performed whole genome transcriptome analysis of the rat tumors and of normal pituitary tissues of wild-type animals. With this approach, we have identified genes differentially expressed in rat PAs that had been previously found dysregulated in human adenomas by array analysis, but not yet further validated. We here show that two such genes (i.e. CYP11A1 and NUSAP1) are highly expressed at both mRNA and protein level in human gonadotropinomas and may represent novel biomarkers of these tumors. In vitro functional assays demonstrated for the first time that the high expression of Cyp11a1 promotes proliferation/survival of PA cells (gonadotroph- and somatotroph-derived), thereby supporting a role for this gene in pituitary tumorigenesis. We also determined that Cyp11a1 expression is regulated by the steroidogenic transcription factor SF-1 in gonadotroph cells. Noteworthy, in addition to NUSAP1, other genes involved in mitosis were found overexpressed in both rat and human PAs by meta-analysis. If further validated, these genes may represent novel potential targets for the therapy of gonadotropinomas.
Discussion
We recently demonstrated that PAs developing in MENX-affected rats are pathologically similar to human gonadotroph adenomas [
26]. The current study confirms this observation and shows that common genetic signatures exist between MENX-associated and human gonadotropinomas. In addition, novel putative markers of human PAs were identified, suggesting that MENX-associated neoplasms may be exploited as to gene discovery.
In silico analysis of gene expression array data of human gonadotroph adenomas identified several genes differentially expressed between tumors and normal pituitaries in both patients and MENX rats. An example of a gene down-regulated in both rats and human PAs is
Dlk1, encoding a non-canonical ligand of Notch.
DLK1 was found to be selectively silenced in NFPAs but not in other types of PA [
1,
5].
DLK1 is located in the DLK1/MEG3 imprinted locus on human chromosome 14q32.3 and
MEG3, coding for a maternally imprinted long noncoding RNA with tumor suppressive function, was also found to be specifically silenced through methylation in NFPAs, but not in other hypophyseal adenoma types [
13]. In rat PAs,
Gtl2 (
Meg3) is down-regulated (−1.95) but did not reach statistical significance.
Previous studies have compared human non-invasive and invasive/aggressive PAs with the aim to unveil the molecular mechanisms leading to an aggressive behavior and to identify markers predictive of invasiveness [
12,
19,
43]. Some genes or gene families we found up-regulated in MENX adenomas had previously been identified in human aggressive-invasive PAs, e.g.,
PTTG1,
RACGAP1,
CCNB1,
CENPE,
AURKB [
12]. PTTG1, the homologue of yeast securin, controls the faithful separation of sister chromatids [
46], while deregulated expression of centrosome proteins (CENP) and Aurora kinases has been linked to defects in the mitotic machinery [
41,
44]. Differential expression of the five genes mentioned above was observed among benign, pre-malignant and malignant tumors, with a tendency to higher expression in the more malignant ones, with
Pttg1 being expressed only in the latter group. Altogether, these findings further support our initial hypothesis that MENX-associated PAs resemble aggressive tumors [
26], and suggest that these lesions may be exploited to identify novel targets for specific therapies of aggressive PAs, currently orphan of specific molecular-targeted treatment strategies. Moreover, our data further confirm the critical role of
Pttg1 in pituitary tumorigenesis across different species and genetic backgrounds.
NUSAP1 is among the genes overexpressed in both rat and human PAs but has not so far been investigated in these tumors. We here report that 90 % of human gonadotroph adenomas express the
NUSAP1 gene at high level. Concordantly, the number of cells immunoreactive for NuSAP was higher in tumors compared with normal pituitaries. NuSAP is a microtubule-associated protein that bundles and stabilizes microtubules thereby linking chromosomes to the mitotic spindle [
35]. Recently, overexpression of
NUSAP1 has been found in various human cancers by array analysis, including glioblastomas, hepatocellular carcinomas, pancreatic adenocarcinoma (reviewed in [
21]). The high expression of the
NUSAP1 gene has been associated with poor prognosis in melanoma patients [
3], with a malignancy-risk genetic signature in breast cancer [
4] and with recurrence in prostate cancer [
15]. The study on prostate cancer has validated gene up-regulation seen by array analysis with immunostaining for the NuSAP protein: tumors with Gleason grading scores ≥7 had more NuSAP-positive cells than tumors with lower scores [
15]. We estimated the percentage of NuSAP-positive cells in human gonadotroph tumors (average 0.5 %, range 0.2–1 %) and found a correlation with that of Ki-67-positive cells (average 1.2 %, range 0.5–2 %). Further studies are required to determine whether NuSAP labeling index correlates with the outcome of patients with PA and whether it provides a more accurate estimate of aggressive behavior than Ki67. Noteworthy, in addition to
NUSAP1, our meta-analysis of human PA array data has identified the up-regulation of genes encoding other proteins playing a critical role in mitosis, mitotic spindle checkpoint and dynamics, or cytokinesis such as
BUB1,
CCNB1,
CDC2,
KIF4,
KIF11, PRC1, in gonadotroph adenomas (Supplementary Table 2). These genes, if experimentally verified to be involved in PAs, may represent novel therapeutic targets for these tumors. While classical antimitotic compounds have limited clinical applications because of severe side effects, a new generation of drugs has been developed that targets kinesins (KIF proteins) and kinases with unique function in mitosis. These antitumor agents could be tested for the treatment of gonadotroph adenomas.
We observed that SF-1 and P450scc are co-expressed in PA cells of MENX-affected animals, and both siRNA-mediated silencing of the
Nr5a1 gene and treatment with the SF-1 inhibitor IsoQ resulted in the down-regulation of
Cyp11a1 in rat primary tumor cells and in LβT2 gonadotroph cells. These results indicate that SF-1 regulates
Cyp11a1 expression in gonadotroph cells. The transcriptional regulation of
Cyp11a1 in steroidogenic tissues is complex and involves the concerted action of several tissue-specific trans-regulators such as SF-1, DAX1, TReP-132, LBP, and GATA together with transcription factors having a more widespread expression, including AP-1, Sp1, and AP-2 [
16]. Further studies are needed to identify the additional factors involved in the transcriptional regulation of
Cyp11a1 in PA cells.
CYP11A1 was up-regulated in 75 % of human gonadotropinomas and P450scc protein expression was high in these tumors, while barely detectable in normal pituitaries. P450scc has been detected in the developing mouse at embryonic day 9.5 in Rathke’s pouch and at day 18.5 in the pituitary primordium [
17]. However, its potential role in pituitary development or in the adult pituitary has not been addressed. Here we show that
Cyp11a1 plays a pro-proliferative role in Y1 adrenocortical cancer cells, but also in GH3 somatotroph adenoma cells and in rat primary pituitary gonadotroph cells. To the best of our knowledge, only two studies have so far addressed the putative role of
Cyp11a1 in regulating cell proliferation or viability. The first one showed that overexpression of
CYP11A1 in tumorigenic and non-tumorigenic mammalian cell lines can either inhibit or enhance cell viability in a cell type-specific manner [
7]. In cells sensitive to
CYP11A1, gene overexpression led to decreased cell proliferation through increase of p21 expression and induction of apoptosis [
7]. The authors did not investigate the molecular processes associated with
CYP11A1-mediated increase in cell proliferation. Based on our results, when the reduction of
CYP11A1 level decreases cell proliferation (as seen in PA cells), both apoptosis and p21 expression are also involved, indicating that these are common pathways downstream of
CYP11A1 signaling. In a more recent study, a truncated isoform of P450scc was found to suppress osteoblast proliferation in physiological conditions [
38]. Unlike the full length P450scc isoform, which is located in the mitochondria, this small isoform localizes to cytoplasm and nucleus, and might therefore be involved in still unidentified molecular pathways. Our studies show that
Cyp11a1 plays a pro-proliferative role in adrenocortical cancer and PA cells, thereby providing additional evidence supporting a role for this gene in tumorigenesis.
In conclusion, we here provide experimental evidence that the MENX animal model can be exploited as experimental tool to shed light into the pathomechanisms involved in human pituitary tumorigenesis. Several of the dysregulated genes here reported have never been associated with PAs, and may represent novel biomarkers for future clinical applications.