Sage Journals: Discover world-class research

Abstract

RIZ1 displays strong tumor-suppressive activities, which has a potential histone methyltransferase activity. The objective of the study was to evaluate the level and the methylation status of RIZ1 and analyze its association with clinicopathological features and the histone in the pituitary adenomas. We found that RIZ1-positive cases were 11/50 and H-Scores 22.75 ± 11.83 in invasive pituitary adenomas and 26/53 and 66.3 ± 21.7 in non-invasive pituitary adenomas (χ² = 8.182, p = 0.004). RIZ1 and C-myc showed the opposite trend in these cases. The methylation levels of RIZ1 were more than 50% in 30.4% (7/23) CpG sites through MALDI-TOF Mass array. There was significant difference (p < 0.01) in 4 CpG sites between invasive pituitary adenoma group and non-invasive pituitary adenoma group; furthermore, the relieved methylation levels of H3K4/H3K9 and enhanced methylation levels of H3K27 in the patients’ serum were found. Furthermore, there was statistic difference of H3K4 and H3K27 methylation between invasive pituitary adenoma and non-invasive pituitary adenoma group (p < 0.01). The average progression-free survival in high RIZ1 group was 52.63 ± 7.62 months and 26.06 ± 4.23 months in low RIZ1 group (p < 0.05). Promoter region methylation of RIZ1 may play an important role in the epigenetic silencing of RIZ1 expression in pituitary adenomas, which may translate into important diagnostic and therapeutic applications.

Keywords

Pituitary adenomas RIZ1 histone methylation invasion

Introduction

Pituitary adenomas (PAs) account for 10%–15% of all intracranial neoplasms, and these tumors are invariably benign and exhibit features of differentiated pituitary cell function as well as premature proliferative arrest.¹ The mechanisms underlying pituitary tumorigenesis are not very clear. Altered expression of pituitary cell-cycle genes, activation of pituitary selective oncoproteins, or loss of pituitary suppressor factors may be associated with aberrant growth factor signaling in PAs.² Pituitary cells are among the few epithelial cell types that rarely undergo malignant transformation although several studies have been performed to identify the genetic cause of pituitary tumourigenesis.³

In recent years, emerging evidence indicates that epigenetic modifications including deoxyribonucleic acid (DNA) methylation, histone modification, nucleosome remodeling, and ribonucleic acid (RNA)-mediated targeting, are an alternative force altering the expression level of genes involved in neoplastic development, including pituitary tumorigenesis.^4,5 DNA methylation is a stable modification that leads to chromatin remodeling, resulting in transcriptional silencing without gene mutation.⁶ Aberrant methylation patterns in DNA damage repair genes may serve as predictive, diagnostic, prognostic, and chemosensitive markers of human cancer.⁷ Increasing evidence suggests, however, that most methylation changes are generated in a programmed manner and occur in a subpopulation of tissue cells during normal aging, probably predisposing them for tumorigenesis.⁸ DNA methylation–mediated gene silencing is closely linked to the deacetylation and methylation of histones at key lysine residues.⁹

Members of the PRDM family are characterized by an N terminal PR domain that is related to the SET methyltransferase domain and multiple zinc fingers that mediate sequence-specific DNA binding and protein–protein interactions.¹⁰ RIZ1, also named PRDM2, is originally isolated as a molecule that is associated with the retinoblastoma protein Rb.¹¹ RIZ1 displays methyltransferase activity toward H3K9.¹² PRDM factors either act as direct histone methyltransferases or recruit a suite of histone-modifying enzymes to target promoters.¹³

The comprehensive analysis of many histone modifications in the human genome demonstrates that the combination of several histone modified forms a modular pattern and influences transcriptional activations in a cooperative manner.^14,15 The signals of methylations of H3K9 and H4K20 are high in silent genes and repressed chromatin domains.¹⁶ H3K9 methylation can be mediated by several histone methyltransferases (HMTases).¹² Methylation of H3K27 is likely to spread over larger regions around transcription start sites of silent genes.¹⁷

In this study, we carried out series of immunohistochemistry (IHC) to assess a scatter of data on RIZ1 expression in a large cohort of PAs and analyze the promoter methylation of RIZ1 and histone methylation in PAs.

Material and methods

Patients and tissue specimens

The files of the Department of Neurosurgery, the First Affiliated Hospital of Zhengzhou University, from January 2010 to July 2014 were searched for 103 PAs. Histologically, the tumors were classified into 23 prolactinomas (PRL), 23 GHomas, and 57 non-functional pituitary adenoma (NFPA) according to the 2007 World Health Organization histological classification of tumors of the PAs.¹⁸ The following IPA diagnostic criteria were adopted: (1) Knosp classification grade IV tumors and Hardy classification invasive adenomas, (2) tumor cells confirmed via pathology as invading sellar bone or adjacent dura mater, and (3) tumor cells invading the sphenoid sinus cavity or peripheral vascular and nerve. Three normal pituitary specimens were from donors. The study was conducted according to an Institutional Review Board (IRB)–approved protocol (IRB of The First Affiliated Hospital of Zhengzhou University), and written informed consent was obtained from all patients for surgery and for research purposes (Table 1).

Table 1.

The clinicopathological data in 103 cases.

Type		PRL	GHomas	NFPA
Case		23	23	57
Age	Range	14–62	13–72	24–72
	Mean ± SD	39 ± 14.7	34 ± 12.2	49.6 ± 16.7
Sex	Males	13	12	29
	Females	10	11	28
Hormone	HGH (ng/mL)	2.01 ± 1.67	15.4 ± 4.73	1.14 ± 1.62
	PRL (ng/mL)	622 ± 157	23.4 ± 14.9	47 ± 21.7
	FSH (mIU/mL)	6.71 ± 3.17	5.32 ± 2.48	14.7 ± 3.6
	LH (mIU/mL)	3.51 ± 1.49	2.84 ± 1.63	5.13 ± 3.55
	TSH (µIU/mL)	1.66 ± 1.37	1.07 ± 0.93	1.84 ± 1.24
Tumor size	Volume (cm³)	8.94	6.77	19.55
	Microadenoma	2	0	0
	Macroadenoma	13	15	31
	Giant-adenoma	8	8	26
Headache	None	12	16	28
	Minor	6	4	17
	Serious	5	3	12
Invasive	Yes	11	10	29
	No	12	13	28
Visual disturbance	Yes	9	9	22
	No	14	14	35
Field-defect	One	2	3	18
	Two	4	3	16
Recurrence		2 (8.70%)	3 (13%)	12 (21.1%)

PRL: prolactinomas; NFPA: non-functional pituitary adenoma; SD: standard deviation; FSH: follicle-stimulating hormone; LH: luteinizing hormone; TSH: thyroid-stimulating hormone.

IHC

All slides were evaluated in advance using an H&E stain to assess tumor content and quality. The slides were placed in the Leica BOND-III purchased from Leica Biosystems, which is a fully automated, random, and continuous-access slide-staining system that processes IHC tests simultaneously. IHC protocol F was selected in the machine, and 3 min with ER1 (epitope retrieval) was set for heat-induced epitope retrieval (HIER) parameter. Bond^™ Polymer Refine Detection (DS9800, Leica Biosystems) was used for detection of primary antibodies. The slides were scanned into digital pictures and expression was examined using Aperio AT2 (Leica Biosystems). Primary antibodies, anti-RIZ1 (1:1000, ab3791; Abcam) and C-myc (1:500, ab17767; Abcam) was used. The optimal titer of the primary Abs for the remainder was determined based on pre-experiment results. The results were calculated using Aperio AT2 (Leica Biosystems) with digital slide-viewing software.

The staining intensity was stratified on a scale of 0–3 (0 for no staining, 1 for weak, 2 for moderate, and 3 for strong). The H-score is obtained multiplying by the intensity of its stain and a constant adjusting the mean for the strongest staining (score = 1.0× (% weak) + 2.0× (% medium) + 3.0× (%strong)).

DNA extraction, purification, and bisulfite modification

Genomic DNA (gDNA) from frozen tissues was extracted by using a DNeasy kit (QIAGEN). All extracted gDNA was treated with sodium bisulfite (Sigma). Briefly, 2 µg gDNA was denatured by 5.5 µL of 3 mol fresh NaOH (final concentration 0.3 mol/L) for 10 min at 37°C; 30 µL of 10 mmol/L hydroquinone (Sigma) and 520 µL of 3 mol/L sodium bisulfite (pH 5.0) were added kept in the dark. The mixture was inverted, added to 200 µL liquid paraffin to prevent water evaporation and reagent oxidation, and then incubated at 50°C for 16 h. The modified DNA was purified using the Wizard DNA Clean-Up System (Promega). The purified DNA was treated again with NaHSO₃ and precipitated. DNA was re-dissolved in 20 µL of TAE, 2 µL of which were subjected to polymerase chain reaction (PCR) amplification.

Methylation-specific PCR and sequencing

Three CpG islands were found using the EMBOSS Cpgplot. Methylation-specific primers were designed to cover 30 CpG dinucleotides numbered 3445 upstream of the transcription start site G (forward) and 4024 upstream of the transcription start site G (reverse) according equenom^® EpiDesigner (#24). Primers specific for methylated DNA (forward 5′-ATTTAGGTTGGAGAGTAGTGGGGTA′; reverse 5′-TAATCCCAACACTTTAAAAAACCAA-3′) were added to the reaction and expected to generate 580 bp products. PCR conditions were 45 cycles of denaturation at 94°C for 20 s, annealing at 60°C for 30 s and 72°C for 1 min. Then, sequence PCR products with Sequenom^® MassARRAY and specimens’ methylation were generated by the EpiTYPER software v1.0 (Sequenom).

Histone extraction and methyltransferase assays

Specimens were cut into small pieces and added TEB buffer (PBS containing 0.5% Triton X-100, 2 mM phenylmethane sulfonyl fluoride (PMSF), and 0.02% NaN₃). The homogenized mixture was centrifuged at 3000 r/min for 5 min at 4°C. The pellet was resuspended with extraction buffer (0.5 N HCl, 10% glycerol), incubated on ice for 30 min and then centrifuged at 12,000 r/min for 5 min at 4°C. The supernatant was collected into a new vial, 8 volume of acetone was added, and incubated at −20°C overnight. Again centrifugation was carried out at 12,000 r/min for 5 min and the pellet was air-dried; the pellet was dissolved in distilled water and the protein concentration was quantified.

Histone methyltransferase activity was measured according to the Epigentek’s guide in https://www.epigentek.com/catalog/ (P-3024, P-3003, and P-3044).

Statistics

χ², log rank analysis, and Fisher’s exact tests were used to determine the significance of categorical variables. One-way analysis of variance (ANOVA) test was applied to the examination of differential expression of methylation of H3K4, H3K9, and H3K27. All p values are two sided and 0.05 was applied as the significance level.

Results

Sample characteristics of the cohort

We selected 103 consecutive patients diagnosed as PAs who had primary tumor specimens. For 103 samples, sufficient archival material was present for standard H&E staining and IHC staining using anti-RIZ1 and C-myc antibody, including 54 men and 49 women with a median age of 45.1 years (range = 13–72 years). The tumor size ranged from 1.3 to 6.1 cm in greatest dimension (median size: 4.3 cm). Additional sample characteristics are given in Table 1. According to the Knosp classification and the intraoperation, all cases were divided into invasive pituitary adenoma (IPA) group (50 cases) and non-invasive pituitary adenoma (nIPA) group (53 cases).

The headache (45.6%, 47/103) is the most initial clinic symptom, and visual disturbance (38.9%, 40/103) is a common clinic symptom. The headache of IPA was 32/56 and 15/47 in nIPA (χ² = 6.556, p = 0.01). The recurrence of IPA was 12/56 and 5/47 in nIPA (χ² = 2.80, p = 0.094).

The RIZ1 and C-myc expression in PAs

IHC was conducted to investigate the expression pattern of RIZ1 and C-myc in IPA and nIPA. Immunoreactivity was observed primarily in cytoplasm of tumor cells, and IHC staining for RIZ1 and C-myc in representative samples of PAs tissues was shown in Figure 1. In this study, the positive case was defined as more than 10% of positive cells.

Figure 1.

The expression of RIZ1 and c-myc in 103 cases: (a) Results of IHC (bar = 60 µm) and (b) results of western blot.

RIZ1-positive cases were 11/50 in IPA group and 26/53 in nIPA group (Table 2; χ² = 8.182, p = 0.004). H-Scores were 22.75 ± 11.83 in IPA group and 66.3 ± 21.7 in nIPA group in Table 2 (p < 0.01). RIZ1 overexpression in malignant meningioma cells was associated with the downregulation of C-myc expression which played a role in cell-cycle progression, apoptosis, and cellular transformation.¹⁹ In this study, C-myc-positive cases were 41/50 and 42/53, respectively. H-Scores were 265 ± 27.41 and 170 ± 41.72 as shown in Table 2 (t = 2.934 p < 0.01).

Table 2.

The RIZ1 and C-myc levels in 103 cases.

Gene	RIZ1			C-myc
Classification	(−)	(+)	H-Score	(−)	(+)	H-Score
IPA	43	7	23.75	9	41	265
nIPA	34	19	66.3	11	42	170

IPA: invasive pituitary adenoma; nIPA: non-invasive pituitary adenoma.

The RIZ1 methylation level in PAs

Epigenetic activation or inactivation of genes plays a critical role in many important human diseases, especially in cancer. A major mechanism of epigenetic inactivation of genes is methylation of CpG islands in genome DNA caused by DNA methyltransferases. We investigated the RIZ1 promoter methylation status in 12 PA samples and 3 normal pituitary samples with Sequenom’s EpiTYPER assay, and 23 CpG sites per sample were analyzed (total 245 CpG sites in 15 samples, Figure 2). About 30.4% (7/23) CpG sites of RIZ1 in PAs samples were defined as having a high-degree methylation compared to normal pituitary, with mean methylation levels above 50%. Furthermore, significant difference between methylation pattern of IPA group and nIPA group was detected in 4 CpG sites (8, 11, 12, and 27; p < 0.01).

Figure 2.

The methylation CpG sites of RIZ1 promoter. Yellow clusters indicate 0% methylated, blue clusters indicate 100% methylated, color gradient between yellow and blue indicates methylation ranging from 0 to 100, red clusters indicate 0% methylated, and white clusters indicate CpG sites not analyzed. Highlight shows the hypermethylation CpG site.

The Methylation level of H3K4, H3K9 and H3K27 in PAs

Histone methyltransferases (HMTs) control or regulate DNA methylation through chromatin-dependent transcription repression or activation. Lysine methylation is associated to transcription activation or repression depending on the residue and degree of methylation.²⁰

To further understand the molecular function of RIZ1, we sought to detect the methylation of H3K4, H3K9, and H3K27 in 42 PAs samples and 3 normal pituitary samples. H3K4 mono-methylation was associated with silenced gene repression. Results of enzyme-linked immunosorbent assay (ELISA) showed the relieved mono-methylation H3K4 expression in IPA group (p < 0.01) but not in nIPA group (Figure 3). H3K9 methylation mediates heterochromatin formation by forming a binding site for HP1 and also participates in silencing gene expression at euchromatic sites. The methyltransferase activity of H3K9 in PAs was relieved compared to the normal pituitary (p < 0.01), and there was no statistic difference between IPA group and nIPA group. Increased global H3K27 methylation was also found be involved in some pathological processes such as cancer progress. There was enhanced global methylation H3K27 in IPA group (p < 0.01) compared to normal pituitary and nIPA group.

Figure 3.

The methylation level of H3K4, H3K9, and H3K27 in normal pituitary and PA samples.

RIZ1 level and PAs’ recurrence and prognostic

Follow-up periods ranged from 2 to 8 years (mean = 4.8 years). The median expression level was used as the cutoff. There was no significant correlation between RIZ1 levels and age, gender, tumor size, or hormone serum levels. However, low RIZ1 levels were more frequently observed in IPA (χ² = 9.356, p = 0.002) or recurrent tumors (χ² = 5.500, p = 0.037) in Table 3. Log rank analysis showed that the average progression-free survival (PFS) in high RIZ1 group (4/51, 52.63 months) was longer than that in low RIZ1 group (13/52, 26.06 months; p < 0.05; Figure 4).

Table 3.

Relationship between RIZ1 H-Scores in 103 patients and various clinical parameters.

Feature	High^a	Low	χ²	p value
All cases	51	52
Patient’s age (years)			0.234	0.629
≥50	23	21
<50	28	31
Patient’s gender			0.470	0.493
Male	25	29
Female	26	23
Tumor diameter			3.699	0.054
≥3 cm	16	26
<3 cm	35	26
Invasion
Yes	17	33	9.356	0.002
No	34	19
Recurrence			5.500	0.037
Yes	4	13
No	47	39

The median expression level was used as the cutoff: low RIZ1 H-Scores were defined as values below the 50th percentile of the 103 patients; values at or above 50th percentile were classified as high levels.

Figure 4.

The average progression-free survival in 103 PA cases.

Discussion

Since the identification of RIZ1 as an RB-binding protein, several studies reported that the genomic region containing the RIZ1 gene (chromosome 1p36) is frequently deleted or rearranged in human tumors.^11,21,22 In this study, we examined the association of RIZ1 expression with the clinical feature in 103 PA cases through tissue microarray (TMA) and found RIZ1 was associated with an increased risk of recurrence for adenomas.

The RIZ1 gene is a member of a superfamily of histone/protein methyltransferases. Buyse et al. first found RIZ1 by retinoblastoma (Rb) probes in 1995, while performing a functional screen for Rb, and observed that it was able to interact with Rb.¹¹ The gene is commonly inactivated in human cancers. Gene knock-out study has established RIZ1 as a tumor susceptibility gene in mice. The gene has potent tumor-suppressive activities in causing apoptosis, G2/M arrest, or both.²³ RIZ1 had a direct repressor function on C-myc gene expression. RIZ1 overexpression in malignant meningioma cells was associated with the downregulation of C-myc expression.¹⁹ Moreover, C-myc, putative downstream targets of H3K9 methylation, may be involved in regulating RIZ1 tumor-suppressive effects. The reciprocal relationship between RIZ1 and C-myc was then validated in primary meningioma cells and human tumor samples.²⁴ In this study, the RIZ1-positive rate in nIPA is 22% and 49.1% in IPA group. In addition, the RIZ1 level was approximately two-fold lower in IPA group than in nIPA group through H-Scores (p < 0.01). Binary multivariate regression analysis revealed that low RIZ1 expression level was independently associated with PAs recurrence. The average PFS in high RIZ1 group was longer than that in low RIZ1 group (p < 0.05).

According to a genetic perspective, RIZ1 may be downregulated by chromosomal instability and microsatellite instability, as well as frame-shift mutations, point mutations, and heterozygote deficiency.^25
–27 From an epigenetic perspective, the deactivation of RIZ1 may occur due to promoter methylation and histone acetylation. The RIZ1 promoter has been demonstrated to have the characteristics of a CpG island, which suggests that RIZ1 is a target of inactivation by epigenetic mechanisms.²⁸ In all, 42.6% prostate cancer cases and 69% gastric adenocarcinomas cases were reported to have RIZ1 methylation, and RIZ1 methylation was more frequent in patients with a high-grade malignancy.^29,30 In this study, we explored the methylation status of RIZ1 among normal pituitary, nIPA, and IPA specimens using MALDI-TOF MS. After methylation quantification of the RIZ1, our results showed hypomethylation patterns for PAs compared with normal pituitary in 7 CpG sites; furthermore, there was statistically significant difference in 4 CpG sites between IPA group and nIPA group.

Dynamic changes in histone post-translational modifications can alter DNA-transcription factor interactions during many nuclear processes and may either accompany or precede transcriptional activation or repression. Transcription activation correlates with methylation of H3K4, together with histone acetylation (H3K9Ac).³¹ Clinical data indicate that methylation of H3K4 may be elevated in breast, kidney, and colon cancers and correlates with a poor clinical outcome.^32,33 By contrast, transcription repression often involves tri-methylation of lysine 27 of H3 (H3K27me3) and di- or tri-methylation of lysine 9 of H3 (H3K9me2/3), through the recruitment of repressive protein complexes. RIZ1 associates with >4400 promoters in G0 myoblasts, 55% of which are also marked with H3K9me2 and enriched for myogenic, cell cycle, and developmental regulators. Knockdown of RIZ1 alters histone methylation at key promoters such as Myogenin and CyclinA2 (CCNA2).³⁴ Epigenetic regulation by RIZ1 preserves key functions of the quiescent state, with implications for stem-cell self-renewal.

In this study, relieved methylation of H3K4 and enriched methylation of H3K27 were observed in IPA group and relieved methylation of H3K9 was observed between IPA group and nIPA group. Changes in expression of key histone modifications are found in PAs cases, which would be expected because tumor cells require instant changes in gene expression and chromatin structure in order to promote cell proliferation and tumor cell invasion. The results of the survival analyses of the individual markers reflect our expected results based on the cellular functions of RIZ1 and the respective histone modifications. We also found that nuclear expression of methylation on H3K4 and H4K27 has prognostic value in IPA cases. Methylation of H3K4 and H3K27 involves modifications found on gene promoter regions and is associated with activation of gene transcription, and lower expression of H3K4 and higher expression of H3K27 in tumors could lead to aberrant gene transcription, including genes required for cell survival, proliferation, and migration. For methylation of H3K9, literature shows conflicting results with respect to patient survival and prognosis, depending on the type of cancer.³⁵ Histone modification of H3K9 is associated with silencing of gene transcription.³⁶ In this study, there is no statistic difference between IPA group and nIPA group although the lower methylation of H3K9 in PAs.

Somatic gene mutations are uncommon in sporadic PAs, and recent studies lend support to epigenetic modification as a potential cause of tumorigenesis and tumor progression. We found that RIZ1 was silenced through DNA methylation and showed particular susceptibility to epigenetic modification, with abnormal DNA methylation in >50% of PA samples. And H3K4 and H3K27 methylation may play an important role regulating the tumor invasion.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by First Affiliated Hospital of Zhengzhou University Research and Development Funds.

References

Ezzat

Asa

Couldwell

. The prevalence of pituitary adenomas: a systematic review. Cancer 2004; 101(3): 613–619.

Melmed

Acromegaly pathogenesis and treatment. J Clin Invest 2009; 119: 3189–3202.

Lecoq

Kamenický

Guiochon-Mantel

. Genetic mutations in sporadic pituitary adenomas—what to screen for? Nat Rev Endocrinol 2015; 11: 43–54.

Sapochnik

Nieto

Fuertes

. Molecular mechanisms underlying pituitary pathogenesis. Biochem Genet 2016; 54(2): 107–119.

Chen

Zhao

TC.

Histone deacetylases and mechanisms of regulation of gene expression. Crit Rev Oncog 2015; 20(1–2): 35–47.

Jones

Baylin

SB.

The epigenomics of cancer. Cell 2007; 128: 683–692.

Gao

Herman

Guo

The clinical value of aberrant epigenetic changes of DNA damage repair genes in human cancer. Oncotarget 2016; 7: 37331–37346.

Klutstein

Nejman

Greenfield

. DNA methylation in cancer and aging. Cancer Res 2016; 76(12): 3446–3450.

Xiong

Dowdy

Podratz

. Histone deacetylase inhibitors decrease DNA methyltransferase-3B messenger RNA stability and down-regulate de novo DNA methyltransferase activity in human endometrial cells. Cancer Res 2005; 65: 2684–2689.

10.

Steele-Perkins

Fang

Yang

. Tumor formation and inactivation of RIZ1, an Rb-binding member of a nuclear protein-methyltransferase superfamily. Genes Dev 2001; 15(17): 2250–2262.

11.

Buyse

Shao

Huang

The retinoblastoma protein binds to RIZ, a zinc-finger protein that shares an epitope with the adenovirus E1A protein. Proc Natl Acad Sci U S A 1995; 92: 4467–4471.

12.

Zhang

Wen

Shi

Lysine methylation: beyond histones. Acta Biochim Biophys Sin 2012; 44: 14–27.

13.

Hohenauer

Moore

AW.

The Prdm family: expanding roles in stem cells and development. Development 2012; 139: 2267–2282.

14.

Wang

Zang

Rosenfeld

. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat Genet 2008; 40: 897–903.

15.

Kurdistani

Tavazoie

Grunstein

Mapping global histone acetylation patterns to gene expression. Cell 2004; 117: 721–733.

16.

Choe

Hong

Park

. Functional elements demarcated by histone modifications in breast cancer cells. Biochem Biophys Res Commun 2012; 418(3): 475–482.

17.

Nichol

Dupéré-Richer

Ezponda

. H3K27 methylation: a focal point of epigenetic deregulation in cancer. Adv Cancer Res 2016; 131: 59–95.

18.

Roncaroli

Scheithauer

BW.

Papillary tumor of the pineal region and spindle cell oncocytoma of the pituitary: new tumor entities in the 2007 WHO Classification. Brain Pathol 2007; 17(3): 314–318.

19.

Liu

Wang

Liu

. Retinoblastoma protein-interacting zinc-finger gene 1 (RIZ1) dysregulation in human malignant meningiomas. Oncogene 2013; 32(10): 1216–1222.

20.

Varier

Timmers

HT.

Histone lysine methylation and demethylation pathways in cancer. Biochim Biophys Acta 2011; 1815(1): 75–89.

21.

Lal

Padmanabha

Smith

. RIZ1 is epigenetically inactivated by promoter hypermethylation in thyroid carcinoma. Cancer 2006; 107(12): 2752–2759.

22.

Mzoughi

Tan

Low

. The role of PRDMs in cancer: one family, two sides. Curr Opin Genet Dev 2016; 36: 83–91.

23.

Canote

Carling

. The tumor suppressor gene RIZ in cancer gene therapy. Oncol Rep 2002; 9(1): 57–60.

24.

Ding

Wang

Jiang

. The transducible TAT-RIZ1-PR protein exerts histone methyltransferase activity and tumor-suppressive functions in human malignant meningiomas. Biomaterials 2015; 56: 165–178.

25.

Chadwick

Jiang

Bennington

. Candidate tumor suppressor RIZ is frequently involved in colorectal carcinogenesis. Proc Natl Acad Sci U S A 2000; 97(6): 2662–2667.

26.

Sun

Geyer

Yang

Cloning, expression, purification and crystallization of the PR domain of human retinoblastoma protein-binding zinc finger protein 1 (RIZ1). Int J Mol Sci 2008; 9(6): 943–950.

27.

Gao

Wang

Lan

. Lower PRDM2 expression is associated with dopamine-agonist resistance and tumor recurrence in prolactinomas. BMC Cancer 2015; 12(15): 272.

28.

Carling

Fang

. Hypermethylation in human cancers of the RIZ1 tumor suppressor gene, a member of a histone/protein methyltransferase superfamily. Cancer Res 2001; 61: 8094–8099.

29.

Hasegawa

Matsubara

Teishima

. DNA methylation of the RIZ1 gene is associated with nuclear accumulation of p53 in prostate cancer. Cancer Sci 2007; 98: 32–36.

30.

Oshimo

Oue

Mitani

. Frequent epigenetic inactivation of RIZ1 by promoter hypermethylation in human gastric carcinoma. Int J Cancer 2004; 110: 212–218.

31.

Ramakrishnan

Pokhrel

Palani

. Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription. Nat Commun 2016; 7: 11949.

32.

Benard

Goossens-Beumer

Van Hoesel

. Histone trimethylation at H3K4, H3K9 and H4K20 correlates with patient survival and tumor recurrence in early-stage colon cancer. BMC Cancer 2014; 14: 531.

33.

Mungamuri

Murk

Grumolato

. Chromatin modifications sequentially enhance ErbB2 expression in ErbB2-positive breast cancers. Cell Rep 2013; 5(2): 302–313.

34.

Cheedipudi

Puri

Saleh

. A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene. Nucleic Acids Res 2015; 43(13): 6236–6256.

35.

Yokoyama

Hieda

Nishioka

. Cancer-associated upregulation of histone H3 lysine 9 trimethylation promotes cell motility in vitro and drives tumor formation in vivo. Cancer Sci 2013; 104: 889–895.

36.

Kimura

Histone modifications for human epigenome analysis. J Hum Genet 2013; 58: 439–445.

RIZ1 and histone methylation status in pituitary adenomas

Abstract

Keywords

Introduction

Material and methods

Patients and tissue specimens

IHC

DNA extraction, purification, and bisulfite modification

Methylation-specific PCR and sequencing

Histone extraction and methyltransferase assays

Statistics

Results

Sample characteristics of the cohort

The RIZ1 and C-myc expression in PAs

The RIZ1 methylation level in PAs

The Methylation level of H3K4, H3K9 and H3K27 in PAs

RIZ1 level and PAs’ recurrence and prognostic

Discussion

Footnotes

Declaration of conflicting interests

Funding

References