5-Hydroxymethylcytosine discriminates between parathyroid adenoma and carcinoma
© Barazeghi et al. 2016
Received: 12 October 2015
Accepted: 2 March 2016
Published: 12 March 2016
Primary hyperparathyroidism is characterized by enlarged parathyroid glands due to an adenoma (80–85 %) or multiglandular disease (~15 %) causing hypersecretion of parathyroid hormone (PTH) and generally hypercalcemia. Parathyroid cancer is rare (<1–5 %). The epigenetic mark 5-hydroxymethylcytosine (5hmC) is reduced in various cancers, and this may involve reduced expression of the ten-eleven translocation 1 (TET1) enzyme. Here, we have performed novel experiments to determine the 5hmC level and TET1 protein expression in 43 parathyroid adenomas (PAs) and 17 parathyroid carcinomas (PCs) from patients who had local invasion or metastases and to address a potential growth regulatory role of TET1.
The global 5hmC level was determined by a semi-quantitative DNA immune-dot blot assay in a smaller number of tumors. The global 5hmC level was reduced in nine PCs and 15 PAs compared to four normal tissue samples (p < 0.05), and it was most severely reduced in the PCs. By immunohistochemistry, all 17 PCs stained negatively for 5hmC and TET1 showed negative or variably heterogeneous staining for the majority. All 43 PAs displayed positive 5hmC staining, and a similar aberrant staining pattern of 5hmC and TET1 was seen in about half of the PAs. Western blotting analysis of two PCs and nine PAs showed variable TET1 protein expression levels. A significantly higher tumor weight was associated to PAs displaying a more severe aberrant staining pattern of 5hmC and TET1. Overexpression of TET1 in a colony forming assay inhibited parathyroid tumor cell growth.
5hmC can discriminate between PAs and PCs. Whether 5hmC represents a novel marker for malignancy warrants further analysis in additional parathyroid tumor cohorts. The results support a growth regulatory role of TET1 in parathyroid tissue.
Primary hyperparathyroidism (pHPT) is a common endocrine disease characterized by excessive secretion of PTH and increased level of serum calcium. Eighty to 85 % of pHPT cases are due to a benign, single adenoma and 15 % to multiglandular disease. Parathyroid carcinoma (PC) is rare, and depending on whether it is diagnosed based on pathological examination alone or biological evidence of malignant behavior, it accounts for less than 1 to <5 % of cases of pHPT [1–9]. An analysis of 286 PC cases from the USA revealed a 5-year survival rate of 86 % and a 10-year survival rate of 49 % . WHO criteria for PC include demonstration of an invasive growth pattern or distant metastasis. Pathological diagnosis of PC is a challenge also because there is no widely available and completely sensitive or specific immunohistochemical marker available. Somatic inactivating mutations of CDC73/HRPT2, encoding parafibromin, are common in PCs defined by malignant behavior (approximately 70 %) and rare (<1 %) in benign disease. Negative immunohistochemical staining for parafibromin has been suggested a marker for PC by some investigators but has been reported to be less useful by others [7, 11–14]. In PC patients who had local invasion and/or metastases at initial surgery or follow-up, negative staining for parafibromin was found in 64 % and in 10 % of patients whose diagnosis was based only on classic histological features .
The epigenetic mark 5-hydroxymethylcytosine (5hmC) was rediscovered in mammalian cells and shown to be an intermediate in DNA demethylation of 5-methylcytosine. The ten-eleven translocation (TET) family of proteins (TET1, TET2, TET3) catalyze conversion of 5-methylcytosine to 5hmC, 5-formylcytosine, and 5-carboxylcytosine and renders passive or active DNA demethylation [16, 17]. Growing evidence suggests that 5hmC not only is an intermediate in DNA demethylation but also acts as an epigenetic mark that regulates gene expression by recruiting DNA-binding proteins . 5hmC is present in many tissues and cell types and is frequently deregulated in cancer, by decreased levels of 5hmC when compared to normal tissues and some but not all tumors show changes in TET expression levels [19–28].
Here, we have for the first time determined levels of 5hmC and TET1 in PAs and PCs and investigated whether TET1 could play a role in parathyroid tumor cell growth regulation. Our study show that negative staining for 5hmC is a frequent event in PCs compared to PAs and suggest a novel potential marker for parathyroid malignancy. Furthermore, our data suggest that TET1 has a growth regulatory role in parathyroid tissue.
Parathyroid carcinomas (n = 17) from 15 patients who had local invasion and/or metastases and single parathyroid adenomas (n = 43) were acquired from patients diagnosed and operated on in clinical routine at the Uppsala University Hospital, Uppsala, Sweden, Department of Surgery, Royal North Shore Hospital, St Leonards, Australia, and Hammersmith Hospital, London, UK. Apparent normal parathyroid tissue (n = 4) was obtained as normal parathyroid gland biopsies inadvertently removed in patients subjected to parathyroidectomy. These specimens (“normals”) that stained positive for parathyroid hormone (PTH), by immunohistochemistry of frozen tissue sections, were used for comparisons with parathyroid tumor tissues. Clinical data for patients with parathyroid adenoma or carcinoma are shown in Additional file 1: Table S1 and Additional file 2: Table S2, respectively. Informed consent and approval of the Uppsala Local Ethical Committee, the Northern Sydney Local Health District Human Research Ethics Committee, and by the Imperial College Research Ethics Committee, London, were achieved.
DNA sampling and dot blot analysis
Genomic DNA was extracted from frozen surgical specimens or cultured cells using DNeasy Blood and tissue kit (Qiagen GmbH, Hilden, Germany) and paraffin-embedded tissue sections using QIAamp DNA FFPE tissue kit (Qiagen GmbH) according to manufacturer’s instructions. 5hmC DNA standard (Zymo Research Corporation, Irvine, CA, USA) was used as a control. One microgram DNA was denatured in 0.1 M NaOH at 95 °C for 10 min, then placed on ice and neutralized with 1 M ammonium acetate. Twofold serial dilutions of the DNA samples were prepared and spotted onto Hybond-N+ nylon membrane (GE Healthcare, Piscataway, NJ, USA) in a Bio-Dot apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The spotted membrane was fixed with UV irradiation (GS Gene Linker UV chamber, Bio-Rad), blocked with 5 % skimmed milk, and incubated overnight with a rabbit polyclonal anti-5hmC antibody [19, 25, 29] (1:10K dilution, 39791; Active Motif, Carlsbad, CA, USA) and then appropriate HRP-conjugated secondary antibody. Signals were visualized with the enhanced chemiluminescence system (GE Healthcare). The same membrane was stained with 0.02 % methylene blue in 0.3 M sodium acetate to ensure equal spotting of the total DNA on the membrane. The second dot blot signal from the top for each serial dilution was used to quantify intensities by the NIH Image-J software according to the program’s instructions  and the 5hmC relative intensity was calculated by dividing the value for each sample by the value of the standard. A linear relationship was obtained for the serially diluted 5hmC standard (not shown).
Immunohistochemistry: Paraffin embedded tissue sections were deparaffinized with xylene and rehydrated through descending alcohol concentrations and distilled water. Sections were treated with 3 % hydrogen peroxide and heated in EDTA pH 8.0 (Life Technologies Corporation, Carlsbad, CA, USA ), for 40 min with microwave at 300-W power. The sections then were incubated with 2 M HCl for 2.5 min and treated with normal goat serum and the rabbit polyclonal anti-5hmC antibody (1:6000 dilution, Active Motif), rabbit polyclonal anti-TET1 (HPA019032; Prestige Antibodies, Sigma-Aldrich Sweden AB, Stockholm, Sweden) or normal horse serum and goat polyclonal anti-PTH antibody (sc-9678; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sections were washed three times with PBS, then incubated with a proper secondary antibody and ABC complex. DAB was used for visualization. Frozen tissue sections (apparent normal parathyroid tissue and parathyroid carcinoma #12 and #13) were first fixed in formalin and then stained as described above but excluding incubation in 2 M HCl. Immunofluorescence: The sections were treated and incubated with rabbit polyclonal anti-5hmC antibody (1:6000 dilution, Active Motif) as mentioned above, then washed three times with PBS (0.05 % Tween20) and incubated with proper fluorescence secondary antibody (Alexa 594, Life Technologies). Sections were washed again and mounted with Vectashield with Dapi (Vector Laboratories, Inc., Burlingame, CA, USA). Normal liver tissue was used as positive control for TET1.
Protein extracts prepared using Cytobuster Protein Extract Reagent (Merck Millipore, Billerica, MA, USA) with complete protease inhibitor cocktail (Roche Diagnostics Scandinavia AB, Bromma, Sweden). Rabbit polyclonal anti-TET1 (GTX124207; GeneTex Inc, Irvine, CA, USA) and goat polyclonal anti-Actin (sc-1616; Santa Cruz Biotechnology) were used. After incubation with the appropriate secondary antibody, bands were visualized using the enhanced chemiluminescence system (GE Healthcare).
Colony formation assay and measurement of apoptosis
sHPT-1 parathyroid tumor cells  were distributed onto 35-mm dishes (2 × 105) in DMEM/10 % fetal bovine serum (Sigma) and transfected in triplicates with 1 μg pIRES-hrGFP ll-TET1-FL  or empty vector (pIRES-hrGFP ll) using FuGENE 6 transfection reagent (Promega Biotech AB, Nacka, Sweden), according to manufacturer’s instructions. Twenty four hours after transfection, 2000 sHPT-1 cells were distributed onto six-well plates in triplicates. After additional 24 h, a fresh medium with 0.2 mg/ml Neomycin (G418, Sigma-Aldrich) was added and refreshed every 72 h. After 10-day selection in Neomycin, the cells were fixed with 10 % acetic acid/10 % methanol and stained with 0.4 % crystal violet, and visible colonies were counted. HEK293T cells (1.5 × 106) were transfected with FuGENE 6 transfection reagent (Promega Biotech AB). After 24 h, 8000 HEK293T cells were distributed onto six-well plates in triplicates and the next day 0.2 mg/ml Neomycin was added to culture medium. After 10 days in selection cells were fixed, stained and counted as described above. Successful transfection was determined by real-time quantitative RT-PCR and western blotting for sHPT-1 and HEK293T, after 72 h. The human parathyroid tumor cell line sHPT-1 was used at cell passages 18–30, and the cells were routinely tested for PTH expression by immunostaining of cultured fixed cells . The human embryonic kidney cell line HEK293T was obtained from ATCC (LGC, Promochem, Sweden) and used at cell passages 10–30. Apoptosis was measured in sHPT-1 cells 72 h after transfection and after 10 days of antibiotic selection, using the Cell Death Detection ELISA kit (Roche Molecular Biochemicals, Mannheim, Germany), according to the manufacturer’s instructions. As a positive control, cells were incubated with 0.1 μg/ml camptothecin (Sigma-Aldrich) for 72 h. Transfected cells were also incubated with FITC-labeled annexin V and propidium iodide (Sigma-Aldrich) and analyzed by flow cytometry on a FACS Canto ІІ (BD Biosciences). Annexin V-FITC binds to phosphatidylserine translocated to the external portion of the membrane as a marker of apoptosis, and propidium iodide distinguishes dead cells with ruptured membrane.
Real-time quantitative PCR
DNA-free total RNA was extracted using RNeasy Plus Mini kit (Qiagen GmbH) according to manufacturer’s instructions and successful treatment with DNase I of all RNA preparations using TURBO DNA-free™ kit (Life Technologies Corporation) was established by PCR analysis. After reverse transcription of RNA preparations with random hexamer primers using First-strand complementary DNA (cDNA) synthesis kit (GE Healthcare), real-time quantitative RT-PCR was performed on StepOnePlus RealTime PCR systems (Life Technologies Corporation) using TaqMan gene expression Master Mix and assays for TET1 transcript (Hs00286756_m1) and GAPDH (Hs02758991_g1). Each cDNA sample was analyzed in triplicate.
ANOVA test was used to calculate differences in relative 5hmC level between the three biological groups and to compare clinical data between the four groups of adenomas. Bonferroni test was used to adjust the p values (due to having a few number of samples, Kruskal-Wallis test was used to check the significance of the p values obtained from the ANOVA test). Differences in colony formation assay and real-time quantitative RT-PCR was evaluated using unpaired t test, and data are presented as mean ± SEM. Statistical analysis was performed using R version 3.1.1 (2014-07-10). p < 0.05 was considered significant.
Reduced global level of 5-hydroxymethylcytosine in parathyroid tumors
Immunohistochemistry of 5hmC discriminates between adenoma and carcinoma
To summarize, all 17 PCs showed negative/undetectable staining for 5hmC and all 43 PAs contained 5hmC positive cells. 5hmC may therefore be used as a marker to discriminate between benign and malignant parathyroid tumors.
TET1 expression and 5hmC level establish groups of PAs and relate to tumor weight
A growth regulatory role of TET1 in parathyroid cells
PC is difficult to diagnose; it has been reported that up to half of metastatic or recurrent PCs were first diagnosed as benign . Correct diagnosis may suggest aggressive surgery to decrease the risk of recurrence. Several immunohistochemical markers of parathyroid malignancy have been suggested (e.g., APC, galectin-3, parafibromin, PGP9.5, Rb), but none has been widely accepted as highly sensitive and specific [7, 9]. Our novel results demonstrated that all 17 analyzed PCs stained negatively for 5hmC. In contrast, all 43 adenomas showed positive staining for 5hmC. Thus, it seems that 5hmC can discriminate between PAs and PCs. We suggest that negative staining for 5hmC may present a novel potential marker for parathyroid malignancy and that this warrants investigations in additional cohorts of parathyroid tumors from patients with pHPT.
TET1 was first identified as a fusion partner of the mixed lineage leukemia (MLL) gene in acute myeloid leukemia, and it is now clear that TET1 plays an essential oncogenic role in MLL-rearranged leukemia [27, 28, 34]. On the contrary, in many solid tumors, including breast, lung, prostate, and colorectal, TET1 expression is down-regulated with reduced levels of 5hmC, and TET1 mutations are rarely observed [22, 26–28]. TET1 has been shown to play a role as tumor suppressor in breast, prostate, and colon cancers [35–37].
We found that expression of TET1 was undetectable in 7 out of the 17 PCs (41 %) and in 6 PCs the expression of TET1 was heterogeneous with areas of positive cells together with areas of negative cells. Since a clear correlation between decreased 5hmC levels and TET1 expression was observed only in 41 % of the PCs, other mechanisms must be involved. Besides indirect mechanisms, it is possible that TET2 also contributes to the observed reduced levels of 5hmC in some of the analyzed tumors [26, 28]. This will be addressed in future experiments when an appropriate antibody to TET2 is available.
A significantly higher tumor weight was found associated to PAs that displayed a more severe aberrant staining pattern of 5hmC and TET1. This strongly suggests a growth regulatory role of 5hmC and TET1. Consistently, overexpression of TET1 resulted in inhibition of parathyroid tumor cell growth. These observations support a possible role of TET1 as a tumor suppressor gene in this tissue.
Reduced expression of TET1 is expected to result in direct deregulation of parathyroid target genes, as for example described in colon cancer for the DKK3 and DKK4 inhibitors of the WNT pathway . Interestingly, impaired expression of another inhibitor of the WNT signaling pathway by DNA methylation, the tumor suppressor protein APC (adenomatous polyposis coli), has been associated with PC malignancy [38, 39].
PC is very rare, and in the absence of disseminated disease, it is sometimes difficult to distinguish PC from benign adenoma. At present, a widely available and sensitive or specific immunohistochemical marker for PC is not available. Here, we have analyzed a relatively large number of PCs and found 5hmC to present a new potential negative marker for parathyroid malignancy. Our novel findings also include an association of a more aberrant immunohistochemical staining pattern of 5hmC and TET1 to tumor weight for the PAs and a direct demonstration in vitro of a growth regulatory role for TET1 in parathyroid tumor cells.
This study has been supported by grants from The Swedish Cancer Society, Lions Fund for Cancer Research, and Erik, Karin o Gösta Selanders Stiftelse. We thank Olof Berggren for the help with the flow cytometry experiment.
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