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Barazeghi, Elham
Publications (7 of 7) Show all publications
Barazeghi, E., Hellman, P., Westin, G. & Stålberg, P. (2019). PTPRM, a candidate tumor suppressor gene in small intestinal neuroendocrine tumors. Endocrine Connections, 8(8), 1126-1135
Open this publication in new window or tab >>PTPRM, a candidate tumor suppressor gene in small intestinal neuroendocrine tumors
2019 (English)In: Endocrine Connections, ISSN 2049-3614, E-ISSN 2049-3614, Vol. 8, no 8, p. 1126-1135Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
BIOSCIENTIFICA LTD, 2019
Keywords
DNA methylation, neuroendocrine tumors, epigenetic, SI-NETs, PTPRM
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-393899 (URN)10.1530/EC-19-0279 (DOI)000483142900007 ()31349215 (PubMedID)
Funder
Swedish Cancer SocietyErik, Karin och Gösta Selanders Foundation
Available from: 2019-10-18 Created: 2019-10-18 Last updated: 2019-10-18Bibliographically approved
Barazeghi, E., Prabhawa, S., Norlén, O., Hellman, P., Stålberg, P. & Westin, G. (2018). Decrease of 5-hydroxymethylcytosine and TET1 with nuclear exclusion of TET2 in small intestinal neuroendocrine tumors.. BMC Cancer, 18(1), Article ID 764.
Open this publication in new window or tab >>Decrease of 5-hydroxymethylcytosine and TET1 with nuclear exclusion of TET2 in small intestinal neuroendocrine tumors.
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2018 (English)In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 18, no 1, article id 764Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
5-hydroxymethylcytosine, Epigenetic, Neuroendocrine tumors, SI-NET, TET1, TET2
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-365219 (URN)10.1186/s12885-018-4579-z (DOI)000439700400002 ()30045709 (PubMedID)
Funder
Swedish Cancer SocietyErik, Karin och Gösta Selanders Foundation
Note

Peter Stålberg and Gunnar Westin contributed equally to this work

Available from: 2018-11-12 Created: 2018-11-12 Last updated: 2019-06-26Bibliographically approved
Barazeghi, E., Gill, A. J., Sidhu, S., Norlen, O., Dina, R., Palazzo, F. F., . . . Westin, G. (2017). A role for TET2 in parathyroid carcinoma. Endocrine-Related Cancer, 24(7), 329-338
Open this publication in new window or tab >>A role for TET2 in parathyroid carcinoma
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2017 (English)In: Endocrine-Related Cancer, ISSN 1351-0088, E-ISSN 1479-6821, Vol. 24, no 7, p. 329-338Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
5-hydroxymethylcytosine, TET2, primary hyperparathyroidism, parathyroid carcinoma, promoter hypermethylation, tumor suppressor
National Category
Endocrinology and Diabetes Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-330022 (URN)10.1530/ERC-17-0009 (DOI)000404978400007 ()
Funder
Swedish Cancer Society
Available from: 2017-10-09 Created: 2017-10-09 Last updated: 2018-04-03Bibliographically approved
Barazeghi, E., Prabhawa, S., Hellman, P., Norlén, O., Stålberg, P. & Westin, G. (2017). A Role of TETs and 5-Hydroxymethylcytosine in SI-NETs. Paper presented at 14th Annual ENETS Conference for the Diagnosis and Treatment of Neuroendocrine Tumor Disease, MAR 08-10, 2017, Barcelona, SPAIN. Neuroendocrinology, 105, 18-18
Open this publication in new window or tab >>A Role of TETs and 5-Hydroxymethylcytosine in SI-NETs
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2017 (English)In: Neuroendocrinology, ISSN 0028-3835, E-ISSN 1423-0194, Vol. 105, p. 18-18Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
KARGER, 2017
Keywords
si-net, 5-hydroxymethylcytosine, tets
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-346588 (URN)000413957900019 ()
Conference
14th Annual ENETS Conference for the Diagnosis and Treatment of Neuroendocrine Tumor Disease, MAR 08-10, 2017, Barcelona, SPAIN
Available from: 2018-03-20 Created: 2018-03-20 Last updated: 2018-03-20Bibliographically approved
Barazeghi, E. (2017). Studies of epigenetic deregulation in parathyroid tumors and small intestinal neuroendocrine tumors. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Studies of epigenetic deregulation in parathyroid tumors and small intestinal neuroendocrine tumors
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. p. 53
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1377
Keywords
Epigenetics, 5hmC, TET1, TET2, parathyroid tumors, SI-NET, RRBS, PTPRM
National Category
Cancer and Oncology Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-330810 (URN)978-91-513-0097-9 (ISBN)
Public defence
2017-11-24, Fåhraeussalen, Rudbecklaboratoriet Hus 5, Dag Hammarskjölds väg 20, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2017-11-02 Created: 2017-10-04 Last updated: 2018-04-03
Barazeghi, E., Gill, A. J., Sidhu, S., Norlen, O., Dina, R., Palazzo, F. F., . . . Westin, G. (2016). 5-Hydroxymethylcytosine discriminates between parathyroid adenoma and carcinoma. Clinical Epigenetics, 8, Article ID 31.
Open this publication in new window or tab >>5-Hydroxymethylcytosine discriminates between parathyroid adenoma and carcinoma
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2016 (English)In: Clinical Epigenetics, E-ISSN 1868-7083, Vol. 8, article id 31Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
5-hydroxymethylcytosine, 5hmC, Parathyroid cancer, Primary hyperparathyroidism, TET1
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-282795 (URN)10.1186/s13148-016-0197-2 (DOI)000371782000002 ()26973719 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2016-04-14 Created: 2016-04-07 Last updated: 2017-11-30Bibliographically approved
Svedlund, J., Barazeghi, E., Stålberg, P., Hellman, P., Åkerström, G., Björklund, P. & Westin, G. (2014). The histone methyltransferase EZH2, an oncogene common to benign and malignant parathyroid tumors. Endocrine-Related Cancer, 21(2), 231-239
Open this publication in new window or tab >>The histone methyltransferase EZH2, an oncogene common to benign and malignant parathyroid tumors
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2014 (English)In: Endocrine-Related Cancer, ISSN 1351-0088, E-ISSN 1479-6821, Vol. 21, no 2, p. 231-239Article in journal (Refereed) Published
Abstract [en]

Primary hyperparathyroidism (pHPT) resulting from parathyroid tumors is a common endocrine disorder with incompletely understood etiology. In renal failure, secondary hyperparathyroidism (sHPT) occurs with multiple tumor development as a result of calcium and vitamin D regulatory disturbance. The aim of the study was to investigate a potential role of the histone 3 lysine 27 methyltransferase EZH2 in parathyroid tumorigenesis. Parathyroid tumors from patients with pHPT included adenomas and carcinomas. Hyperplastic parathyroid glands from patients with HPT secondary to uremia, and normal parathyroid tissue specimens were included in the study. Quantitative RT-PCR, Western blotting, bisulfite pyrosequencing, colony formation assay, and RNA interference was used. EZH2 was overexpressed in a subset of the benign and in all malignant parathyroid tumors as determined by quantitative RT-PCR and Western blotting analysis. Overexpression was explained by EZH2 gene amplification in a large fraction of tumors. EZH2 depletion by RNA interference inhibited sHPT-1 parathyroid cell line proliferation as determined by tritium-thymidine incorporation and colony formation assay. EZH2 depletion also interfered with the Wnt/β-catenin signaling pathway by increased expression of growth-suppressive Axin 2, a negative regulator of β-catenin stability. Indeed, EZH2 contributed to the total level of aberrantly accumulated transcriptionally active (nonphosphylated) β-catenin in the parathyroid tumor cells. To our knowledge EZH2 gene amplification presents the first genetic aberration common to parathyroid adenomas, secondary hyperplastic parathyroid glands, and parathyroid carcinomas. This supports the possibility of a common pathway in parathyroid tumor development.

National Category
Surgery
Identifiers
urn:nbn:se:uu:diva-212145 (URN)10.1530/ERC-13-0497 (DOI)000344787300013 ()24292603 (PubMedID)
Available from: 2013-12-06 Created: 2013-12-06 Last updated: 2017-12-06
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