Background
Pituitary adenoma (PA) is a common intracranial neoplasm with reported estimated prevalence rates to be 14.4% to 22.5% in pooled autopsy and radiological series, respectively [
1]. PAs are generally benign but can behave clinically in different ways. Some of pituitary tumors are hormonally inactive, others secrete hormones in excess, and some of PAs can cause morbidity because of dysregulation of hormone production and/or symptoms of mass effect [
2,
3]. The pituitary gland is localized in a dual bag attached to the inferior aspect of the diaphragm of the sella and surrounded by venous spaces that correspond laterally to the cavernous sinuses [
4]. Cavernous sinus invasion has an influence on the management and prognosis of PA [
5], because dual wall invasion usually implies partial surgical removal of the tumor [
6]. Early prediction of which pituitary tumor will recur and/or exhibit an invasive phenotype remains difficult despite the introduction of several tissue-based molecular markers [
7].
Associations between tumors (including glioblastoma, gastric cancer, colorectal cancer, breast and liver cancers, meningioma, bladder and thyroid cancers) and N-myc downstream-regulated gene 2 (
NDRG2) have been reported in numerous studies [
8‐
16].
NDRG2 is a member of the NDRG family, which consists of
NDRG1, NDRG2, NDRG3 and
NDRG4 [
17] and is located at chromosome 14q11.2, a region that has been reported to harbor a tumor suppressor gene [
18].
NDRG2 is highly expressed in the brain and skeletal muscle, while it is marginally expressed or almost undetectable in the several human cancer cell lines [
17,
19]. NDRG proteins are reported to be involved in cell proliferation, differentiation, migration, invasion and stress response [
19]. It was shown that NDRG2 reduce tumor cell proliferation in glioblastomas [
20]. Also, NDRG2 upregulation was associated with Alzheimer's disease or cerebral ischemia [
21,
22]. Several studies have shown
NDRG2 promoter CpG island methylation and down-regulation in liver [
13], gastric [
10], colorectal cancers (CRC) [
23,
24], glioblastomas [
8,
9] and anaplastic meningioma [
25]. However,
NDRG2 promoter methylation and mRNA expression levels in PAs has not been investigated.
The aim of this study was to determinate aberrant promoter methylation and mRNA expression of NDRG2 in PAs and to evaluate the associations between the methylation profile of gene, mRNA expression, patients’ clinical characteristics and tumor invasiveness and recurrence.
Methods
Description of subject
One hundred forty one pituitary adenoma tissues and clinical patient data were collected at the Department of Neurosurgery of Hospital of Lithuanian University of Health Sciences between 2010 and 2016. Tumor tissues were frozen in liquid nitrogen immediately after their surgical resection. The age at the time of the operation, gender, relapse, size and diagnoses of Cushing syndrome, acromegaly or prolactinoma were collected for each patient. The endocrinological features were: 73 functioning and 68 nonfunctioning adenomas. According to the clinical findings functioning adenomas were: 7 growth hormone (GH) - secreting adenomas, 2 insulin-like grow factor 1 (IGF-1) - secreting adenomas, 1 cortisol (COR) - secreting adenoma, 44 prolactin (PRL) - secreting adenomas, 1 adrenocorticotropic hormone (ACTH) - secreting adenoma and 18 adenomas secreting more than one hormone. According to tumor size all PAs were macroadenomas (greater than 10 mm).
Invasion of pituitary adenomas were analyzed using MR imaging findings and classified according to Hardy classification, modified by Wilson [
5]. The Knosp classification system was used to quantify the invasion of the cavernous sinus [
6]. Invasiveness was established in 71 patients with pituitary adenoma. From them, 51 invasive and 20 non-invasive PAs were found.
Tissue specimens were pulverized and stored at -80 °C until DNA and RNA was obtained. Genomic DNA was extracted from 119 PA specimens by SDS/proteinase K treatment, followed by phenol–chloroform extraction and ethanol precipitation. The remaining 22 samples were missing because of containing too small an amount of tumor tissue. All the samples were stored at -20 °C until DNA was modified with sodium bisulfite.
Total RNA was extracted from 141 PAs using Trizol reagent, according to the manufacturer’s protocol (Ambion, Life Technologies) and stored at -80 °C until cDNA synthesis. However, 10 mRNA samples were lost because the concentrations for cDNA synthesis were too small. The genomic DNA and RNA concentrations and purity was determined using Nanodrop spectrophotometer (Eppendorf). For pure DNA, A260/280 is ~1.8 and for pure RNA A260/280 is ~2.
Bisulfite Modification and MS-PCR
Extracted genomic DNA of 119 PA samples was modified with EZ DNA methylation kit™ (Zymo Research), according to the manufacturer’s instructions. The sodium bisulfite treated DNA was eluted in 40 μL of nuclease-free water.
After bisulfite modification, the methylation-specific polymerase chain reactions (MS-PCR) were performed in 15 μl of 7.5 μL Maxima® Hot Start PCR Master Mix (ThermoFisher Scientific) with Hot Start Taq DNA polymerase, 10 pmol of each primer (Metabion International AG) and nuclease-free water. Primers for methylated
NDRG2 allele were: 5'-AGAGGTATTAGGATTTTGGGTACG-3' (forward) and 5'-GCTAAAAAAACGAAAATCTCGC-3' (reverse) and for unmethylated allele: 5'-AGAGGTATTAGGATTTT GGGTATGA-3' (forward) and 5'-CCACTAAAAAAACAAAAATCTCACC-3' (reverse), according to the published data [
23]. The reaction was hotstart at 95 °C for 5 min. The amplifications were carried out in a thermal cycler (Eppendorf) for 38 cycles, each of which consisted of denaturation at 92 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 15 s, followed by a final 5 min extension at 72 °C. For each set of methylation-specific PCR reactions methylated (Bisulfite-Converted Universal Methylated Human DNA Standard (Zymo Research, USA)), unmethylated (human blood lymphocyte DNA, treated with bisulfite) and negative (nuclease-free water) controls were included in all reactions.
The MS-PCR products were analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized under UV illumination.
cDNA synthesis and qRT-PCR
First-strand cDNA was produced from total RNA by using RevertAid H Minus M-MuLV Reverse Transcriptase (ThermoFisher Scientific) and random hexamer primers (ThermoFisher Scientific), according to the manufacturer’s protocol. Negative controls were prepared as above, but without Reverse Transcriptase.
For the
NDRG2 gene mRNA expression, Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green chemistry in a Real-Time PCR System “Applied Biosystems 7500 Fast” (Applied Biosystems, USA). The 12 μl reaction mixture contained of 6 μl Maxima SYBR Green/ROX qPCR Master Mix (2×) (ThermoFisher Scientific), 15 ng of the cDNA, nuclease-free water and gene-specific primers:
NDRG2 forward 5`-AGAGCTACGACCTGAC-3`, reverse 5`-AGCACTGTGTGTACAG-3` resulting in a 128 bp PCR amplicon to a total concentration of 0.6 μM. The housekeeping gene
β-actin was used as an internal control with primers: forward 5`-CATTACACATCCAACC-3`, reverse 5`-GGAGTCAGCCTGAGGA-3`, resulting in a 184 bp PCR amplicon to a total concentration of 0.1 μM. The
NDRG2 and
β-actin primers were designed according to the published data [
26]. The PCR amplification was performed after denaturation step at 95 °C for 10 min followed by 40 cycles, each of which consisted of denaturation at 92 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s, and a final step for the generation of a melting curve to distinguish between the main PCR product and primer-dimers. All measurements were performed in triplicate.
The comparative 2
-ΔΔCt method was used for the calculations of
NDRG2 gene mRNA expression. The comparison was carried out between PA normalized threshold cycle (Ct) values and healthy human brain (RHB) normalized Ct values: ΔΔCt = (Ct,
NDRG2
- Ct,
β-actin
)
PA sample x - (Ct,
NDRG2
- Ct,
β-actin
)
RHB [
27]. The final result was given as log2(2
-ΔΔCt) calculation.
For standard curve design, RHB “FirstChoice Human Brain Reference RNA” (Ambion, cat. No. AM6050) was used. Standard curve parameters for NDRG2 were: efficiency 99.71%, R2 0.994, slope −3.33; for β-actin were: efficiency 100.08%, R2 0.997, slope −3.32.
Statistical analysis
The SPSS Statistics 19 (SPSS Inc., Chicago, IL) software package was used for statistical analysis. Chi-square test was used to evaluate associations among NDRG2 gene promoter methylation, mRNA expression levels and clinical characteristics (age, gender, relapse, Cushing syndrome, acromegaly, prolactinoma, invasiveness, secreting and non-secreting pituitary adenomas and hormone groups). The correlation between NDRG2 gene expression and methylation and the other clinical factors were evaluated by use of the Mann-Whitney test. Kruskal–Wallis test was used to reveal the difference across medians of NDRG2 mRNA expression in all hormone groups. The significance level was defined as p value less than 0.05.
Discussion
Pituitary adenoma is a common benign monoclonal neoplasm [
28]. Early prediction of which pituitary tumors will recur and/or exhibit an invasive phenotype remains difficult [
7].
NDRG2 gene may be a promising target for cancer, because
NDRG2 down-regulation is associated with cancer development and progression, including such features as malignant clinical manifestations and increased pathological grade. Moreover, this gene is a relevant biomarker for predicting aggressive behavior, tumor recurrence and overall patient survival [
29]. Therefore, should be further studies to show that NDRG2 up-regulation may be a promising therapeutic strategy for the treatment of cancer and that might be associated with PA development, as well.
We began our study by defining
NDRG2 promoter methylation status in PA samples, including all the clinically functioning and hormonally inactive types. We have determined 22.69% (27/119)
NDRG2 methylation frequency. These results indicate that
NDRG2 gene has low methylation status in PAs. A number of studies using various techniques have shown that epigenetic silencing of the
NDRG2 promoter has been found in the majority of primary tumors, and different cancer cell lines and other tumor tissues such as glioma (46.3 - 62%), primary gastric (54%) and colorectal carcinoma (64.28%) cancers [
8‐
11].
NDRG2 promoter methylation was observed to be associated with the invasiveness in gastric and colorectal cancer and with the aggressiveness of glioma tumor [
8‐
10]. However, our analysis has hown no significant association between
NDRG2 gene methylation and pituitary adenoma invasiveness. Meanwhile, we have demonstrated that hypersecretion of PRL, IGF-1, GH, ACTH hormones appear with a methylated
NDRG2 gene more often than with an unmethylated gene. Also, in most cases nonfunctioning PAs have an unmethylated
NDRG2 gene. However, the mechanisms related to this are still unknown.
Moreover, previous studies have shown that
NDRG2 mRNA expression is low in numerous types of tumor tissues and cancer cell lines, and is a novel tumor suppressor candidate gene [
8,
12‐
16]. It was observed that
NDRG2 expression loss is significantly correlated with aggressive tumor behaviors such as late tumor-node-metastasis stage, differentiation grade, portal vein thrombi, infiltrative growth pattern, nodal/distant metastasis, as well as shorter patient survival rates in liver cancer [
13]. Also,
NDRG2 overexpression can inhibit tumor growth and invasion in vitro in bladder and breast cancer [
12,
15]. Meanwhile, our results have showed no correlation between
NDRG2 gene mRNA expression and pituitary adenoma invasiveness. The mechanism of
NDRG2 expression in pituitary adenoma proliferation and invasion has not yet been reported, making it necessary to further elucidate the role of
NDRG2 gene in pathogenesis of PA.
In addition, we have analyzed the associations of
NDRG2 gene mRNA expression with clinical features of PAs. Our study has revealed that in the case of acromegaly,
NDRG2 gene mRNA expression is significantly lower than in other diagnoses of PAs. It is known that acromegaly is an insidious disorder characterized by excess secretion of growth hormone and elevated circulating levels of insulin-like growth factor-I [
30], and in our examination, in most cases the hypersecretion of GH and IGF-1 hormones were determined with low
NDRG2 gene expression level as well. Moreover,
NDRG2 gene mRNA expression is significantly higher with diagnoses of prolactinoma than in other diagnoses of PAs, and in most cases the hypersecretion of PRL hormone that causes prolactinoma have been detected with medium
NDRG2 expression level. These results are consistent with previous reports that the expression of
NDRG2 is regulated by many hormones, including adrenal steroids [
31], dexamethasone [
32], insulin [
33], androgen [
34] and aldosterone [
35]. It was also shown that hormone estrogen can enhance the expression of
NDRG2 and may influence Na+/K + -ATPase activation as well as ion transport in salivary glands, brain, heart, skeletal muscle, and kidney where Na+/K + -ATPases were enriched [
36]. Also, in human and in animal models estrogen stimulates PRL secretion in vitro and induces PRL adenomas in vivo [
37]. However, more studies for signal pathway are needed to show the mechanism underlying and the significant results we showed in this study.
Conclusion
This is the first study that has demonstrated the NDRG2 gene promoter methylation and mRNA expression in patients with diagnoses of pituitary adenoma and analyzed the relationships between NDRG2 epigenetic changes and the association with PA clinical features including patient age, gender, relapse, hormone groups, invasiveness, diagnoses of prolactinoma, acromegaly and Cushing syndrome. Our data have revealed that in the case of acromegaly, NDRG2 gene mRNA expression is significantly lower than in other diagnosis of PAs and PA that secretes hormones GH and IGF-1 hormones have low NDRG2 gene expression level as well. Moreover, NDRG2 gene expression is significantly higher with diagnoses of prolactinoma than in other diagnosis of PAs. Therefore, there is need for intensive research to confirm our findings and justify the hypothesis that NDRG2 could be a diagnostic marker for diagnosis of prolactinoma and acromegaly in PAs.
Acknowledgements
The authors thank Silvija Jakstiene for radiological evaluation.