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Studies of epigenetic deregulation in parathyroid tumors and small intestinal neuroendocrine tumors
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery. (Endocrine Surgery)
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 [en]
Epigenetics, 5hmC, TET1, TET2, parathyroid tumors, SI-NET, RRBS, PTPRM
National Category
Cancer and Oncology Endocrinology and Diabetes
Identifiers
URN: urn:nbn:se:uu:diva-330810ISBN: 978-91-513-0097-9 (print)OAI: oai:DiVA.org:uu-330810DiVA, id: diva2:1146980
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
List of papers
1. 5-Hydroxymethylcytosine discriminates between parathyroid adenoma and carcinoma
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
2. A role for TET2 in parathyroid carcinoma
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
3. Decrease of 5-hydroxymethylcytosine and TET1 with nuclear exclusion of TET2 in small intestinal neuroendocrine tumors
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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(English)In: Article in journal (Refereed) Submitted
National Category
Cancer and Oncology Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-330575 (URN)
Available from: 2017-10-03 Created: 2017-10-03 Last updated: 2017-10-04
4. Reduced representation bisulfite sequencing of small intestinal neuroendocrine tumors identifies PTPRM as a novel candidate tumor suppressor gene
Open this publication in new window or tab >>Reduced representation bisulfite sequencing of small intestinal neuroendocrine tumors identifies PTPRM as a novel candidate tumor suppressor gene
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(English)In: Article in journal (Refereed) Submitted
National Category
Cancer and Oncology Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-330794 (URN)
Available from: 2017-10-04 Created: 2017-10-04 Last updated: 2017-10-04

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