| 21. |
- Barazeghi, Elham, et al.
(författare)
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5-Hydroxymethylcytosine discriminates between parathyroid adenoma and carcinoma
- 2016
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Ingår i: Clinical Epigenetics. - : Springer Science and Business Media LLC. - 1868-7083 .- 1868-7075. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Background: Primary hyperparathyroidism is characterized by enlarged parathyroid glands due to an adenoma (80-85 %) or multiglandular disease (similar to 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. Results: 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. Conclusions: 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.
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| 22. |
- Barazeghi, Elham, et al.
(författare)
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A role for TET2 in parathyroid carcinoma
- 2017
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Ingår i: Endocrine-Related Cancer. - 1351-0088 .- 1479-6821. ; 24:7, s. 329-338
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Tidskriftsartikel (refereegranskat)abstract
- Primary hyperparathyroidism (pHPT) is rarely caused by parathyroid carcinoma (PC, <1-5% of pHPT cases). The TET proteins oxidize the epigenetic mark 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and inactivation by mutation or epigenetic deregulation of TET1 and TET2 play important roles in various cancers. Recently, we found that 5hmC was severely reduced in all of the analyzed PCs and with deranged expression of TET1 for the majority of PCs. Here, we have examined the expression of the TET2 protein in 15 5hmC-negative PCs from patients who had local invasion or metastases. Cell growth and cell migratory roles for TET2 as well as epigenetic deregulated expression were addressed. Immunohistochemistry revealed very low/undetectable expression of TET2 in all PCs and verified for two PCs that were available for western blotting analysis. Knockdown of TET2 in the parathyroid cell line sHPT-1 resulted in increased cell growth and increased cell migration. DNA sequencing of TET2 in PCs revealed two common variants and no obvious inactivating mutations. Quantitative bisulfite pyrosequencing analysis of the TET2 promoter CpG island revealed higher CpG methylation level in the PCs compared to that in normal tissues and treatment of a PC primary cell culture with the DNA methylation inhibitor 5-aza-2'-deoxycytidine caused increased expression of the methylated TET2 gene. Hence, the data suggest that deregulated expression of TET2 by DNA hypermethylation may contribute to the aberrantly low level of 5hmC in PCs and further that TET2 plays a cell growth and cell migratory regulatory role and may constitute a parathyroid tumor suppressor gene.
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| 23. |
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| 24. |
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| 25. |
- Barazeghi, Elham, et al.
(författare)
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Decrease of 5-hydroxymethylcytosine and TET1 with nuclear exclusion of TET2 in small intestinal neuroendocrine tumors.
- 2018
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Ingår i: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407 .- 1471-2407. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Small intestinal neuroendocrine tumors (SI-NETs) originate from enterochromaffin cells scattered in the intestinal mucosa of the ileum and jejunum. Loss of one copy of chromosome 18 is the most frequent observed aberration in primary tumors and metastases. The aim of this study was to investigate possible involvement of 5-hydroxymethylcytosine (5hmC), TET1 and TET2 in SI-NETs.METHODS: The analysis was conducted using 40 primary tumors and corresponding 47 metastases. The level of 5hmC, TET1 and TET2 was analyzed by DNA immune-dot blot assay and immunohistochemistry. Other methods included a colony forming assay, western blotting analysis, and quantitative bisulfite pyrosequencing analysis. The effect of the exportin-1 nuclear transport machinery inhibitors on cell proliferation and apoptosis was also explored using two SI-NET cell lines.RESULTS: Variable levels of 5hmC and a mosaic staining appearance with a mixture of positive and negative cell nuclei, regardless of cell number and staining strength, was observed overall both in primary tumors and metastases. Similarly aberrant staining pattern was observed for TET1 and TET2. In a number of tumors (15/32) mosaic pattern together with areas of negative staining was also observed for TET1. Abolished expression of TET1 in the tumors did not seem to involve hypermethylation of the TET1 promoter region. Overexpression of TET1 in a colony forming assay supported a function as cell growth regulator. In contrast to 5hmC and TET1, TET2 was also observed in the cytoplasm of all the analyzed SI-NETs regardless of nuclear localization. Treatment of CNDT2.5 and KRJ-I cells with the exportin-1 (XPO1/CRM1) inhibitor, leptomycin B, induced reduction in the cytoplasm and nuclear retention of TET2. Aberrant partitioning of TET2 from the nucleus to the cytoplasm seemed therefore to involve the exportin-1 nuclear transport machinery. Reduced cell proliferation and induction of apoptosis were observed after treatment of CNDT2.5 and KRJ-I cells with leptomycin B or KPT-330 (selinexor).CONCLUSIONS: SI-NETs are epigenetically dysregulated at the level of 5-hydroxymethylcytosine/ TET1/TET2. We suggest that KPT-330/selinexor or future developments should be considered and evaluated for single treatment of patients with SI-NET disease and also in combinations with somatostatin analogues, peptide receptor radiotherapy, or everolimus.
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| 26. |
- Barazeghi, Elham, et al.
(författare)
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EZH2 presents a therapeutic target for neuroendocrine tumors of the small intestine
- 2021
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Small intestinal neuroendocrine tumors (SI-NETs) are slow-growing tumors that seem genetically quite stable without highly recurrent mutations, but are epigenetically dysregulated. In contrast to the undetectable expression of the enhancer of zeste homolog 2 (EZH2) histone methyltransferase in the enterochromaffin cells of the small intestine, we found high and differential expression of EZH2 in primary SI-NETs and corresponding metastases. Silencing EZH2 in the SI-NET cell line CNDT2.5 reduced cell proliferation and induced apoptosis. Furthermore, EZH2 knockout inhibited tumor progression in a CNDT2.5 SI-NET xenograft mouse model, and treatment of SI-NET cell lines CNDT2.5 and GOT1 with the EZH2-specific inhibitor CPI-1205 decreased cell viability and promoted apoptosis. Moreover, CPI-1205 treatment reduced migration capacity of CNDT2.5 cells. The EZH2 inhibitor GSK126 also repressed proliferation of CNDT2.5 cells. Recently, metformin has received wide attention as a therapeutic option in diverse cancers. In CNDT2.5 and GOT1 cells, metformin suppressed EZH2 expression, and inhibited cell proliferation. Exposure of GOT1 three-dimensional cell spheroids to CPI-1205 or metformin arrested cell proliferation and decreased spheroid size. These novel findings support a possible role of EZH2 as a candidate oncogene in SI-NETs, and suggest that CPI-1205 and metformin should be further evaluated as therapeutic options for patients with SI-NETs.
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| 27. |
- Barazeghi, Elham, et al.
(författare)
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PTPRM, a candidate tumor suppressor gene in small intestinal neuroendocrine tumors
- 2019
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Ingår i: Endocrine Connections. - : BIOSCIENTIFICA LTD. - 2049-3614. ; 8:8, s. 1126-1135
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Tidskriftsartikel (refereegranskat)abstract
- Small intestinal neuroendocrine tumors (SI-NETs) are small, slow growing neoplasms with loss of one copy of chromosome 18 as a common event. Frequently mutated genes on chromosome 18 or elsewhere have not been found so far. The aim of this study was to investigate a possible tumor suppressor role of the transmembrane receptor type tyrosine phosphatase PTP mu (PTPRM at 18p11) in SI-NETs. Immunohistochemistry, quantitative RT-PCR, colony formation assay and quantitative CpG methylation analysis by pyrosequencing were performed. Undetectable/very low levels of PTPRM or aberrant pattern of immunostaining, with both negative and positive areas, were detected in the majority of tumors (33/40), and a significantly reduced mRNA expression in metastases compared to primary tumors was observed. Both the DNA methylation inhibitor 5-aza-2'deoxycytidine and the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) induced PTPRM expression in CNDT2.5 and KRJ-I SI-NET cells. CpG methylation of upstream regulatory regions, the promoter region and the exon 1/intron 1 boundary was detected by pyrosequencing analysis of the two cell lines and not in the analyzed SI-NETs. Overexpression of PTPRM in the SI-NET cell lines reduced cell growth and cell proliferation and induced apoptosis. The tyrosine phosphatase activity of PTPRM was not involved in cell growth inhibition. The results support a role for PTPRM as a dysregulated candidate tumor suppressor gene in SI-NETs and further analyses of the involved mechanisms are warranted.
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| 28. |
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| 29. |
- Barazeghi, Elham
(författare)
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Studies of epigenetic deregulation in parathyroid tumors and small intestinal neuroendocrine tumors
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Deregulation of the epigenome is associated with the initiation and progression of various types of human cancers. Here we investigated the level of 5-hydroxymethylcytosine (5hmC), expression and function of TET1 and TET2, and DNA methylation in parathyroid tumors and small intestinal neuroendocrine tumors (SI-NETs).In Paper I, an undetectable/very low level of 5hmC in parathyroid carcinomas (PCs) compared to parathyroid adenomas with positive staining, suggested that 5hmC may represent a novel biomarker for parathyroid malignancy. Immunohistochemistry revealed that increased tumor weight in adenomas was associated with a more aberrant staining pattern of 5hmC and TET1. A growth regulatory role of TET1 was demonstrated in parathyroid tumor cells.Paper II revealed that the expression of TET2 was also deregulated in PCs, and promoter hypermethylation was detected in PCs when compared to normal parathyroid tissues. 5-aza-2′-deoxycytidine treatment of a primary PC cell culture induced TET2 expression and further supported involvement of promoter hypermethylation in TET2 gene repression. TET2 knockout demonstrated a role for TET2 in cell growth and migration, and as a candidate tumor suppressor gene.In Paper III, variable levels of 5hmC, and aberrant expression of TET1 and TET2 were observed in SI-NETs. We demonstrated a growth regulatory role for TET1, and cytoplasmic expression with absent nuclear localization for TET2 in SI-NETs. In vitro experiments supported the involvement of exportin-1 in TET2 mislocalization, and suggested that KPT-330/selinexor, an orally bioavailable selective inhibitor of exportin-1 and nuclear export, with anti-cancer effects, could be further investigated as a therapeutic option in patients with SI-NETs.In Paper IV, DNA methylation was compared between SI-NET primary tumors and metastases by reduced representation bisulfite sequencing. Three differentially methylated regions (DMR) on chromosome 18 were detected and chosen for further analyses. The PTPRM gene, at 18p11, displayed low expression in SI-NETs with high levels of methylation in the presumed CpG island shores, and in the DMR rather than the promoter region or exon 1/intron 1 boundary. PTPRM overexpression resulted in inhibition of cell growth, proliferation, and induction of apoptosis in SI-NET cells, suggesting a role for PTPRM as an epigenetically deregulated candidate tumor suppressor gene in SI-NETs.
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| 30. |
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