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Small Intestinal Neuroendocrine Tumours: Genetic and Epigenetic Studies and Novel Serum Biomarkers
(Endokrinkirurgi)ORCID iD: 0000-0001-7366-6258
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Small intestinal neuroendocrine tumours (SI-NETs) are rare, hormone producing and proliferate slowly. Patients usually display metastases at time of diagnosis, the tumours are difficult to cure, and the disease course is unpredictable.

The gene expression pattern was investigated in paper I, with emphasis on aggressive disease and tumour progression. Expression microarrays were performed on 42 tumours. Unsupervised hierarchal clustering revealed three clusters that were correlated to clinical features, and expression changes from primary tumour to metastasis. Eight novel genes, ACTG2, GREM2, REG3A, TUSC2, RUNX1, TGFBR2, TPH1 and CDH6 may be of importance for tumour progression.

In paper II, expression of ACTG2 was detected in a fraction of SI-NETs, but not in normal enterochromaffin cells. Inhibition of histone methyltransferase and transfection of miR-145 induced expression and no effect was seen after DNA methylation or selective EZH2 inhibition in vitro. miR-145 expression was reduced in metastases compared to primary tumours. Overexpression of ACTG2 inhibited cell growth, and inducing ACTG2 may have therapeutic effects.

TCEB3C (Elongin A3) is located on chromosome 18 and is imprinted in some tissues. In paper III a reduced protein expression was detected. The gene was epigenetically repressed by both DNA and histone methylation in a tumour tissue specific context. The expression was also induced in primary cell cultures after DNA demethylation and pyrosequencing revealed promoter region hypermethylation. Overexpression of TCEB3C inhibited cell growth by 50%, suggesting TCEB3C to be a tumour suppressor gene.

In paper IV, 69 biomarkers were analysed in blood serum using multiplex proximity ligation assay. Nineteen markers displayed different levels between patients and controls. In an extended cohort, ELISA analysis showed elevated serum levels of Mindin, DcR3 and TFF3 in patients and protein expression in tumour cells. High levels of DcR3 and TFF3 were associated with poor survival, and DcR3 may be a marker for liver metastases. Mindin, DcR3, and TFF3 are potential novel diagnostic biomarkers for SI-NETs.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2014. , 51 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 975
Keyword [en]
SI-NET, microarray, tumour suppressor gene, epigenetic, serum biomarkers
National Category
Medical and Health Sciences Basic Medicine
Research subject
Surgery
Identifiers
URN: urn:nbn:se:rkh:diva-1470ISBN: 978-91-554-8887-1 (print)OAI: oai:DiVA.org:rkh-1470DiVA: diva2:794241
Public defence
2014-04-11, Rosénsalen, Akademiska Sjukhuset, Ing 95, 09:15 (Swedish)
Opponent
Supervisors
Available from: 2015-03-11 Created: 2015-03-10 Last updated: 2015-03-11Bibliographically approved
List of papers
1. Different gene expression profiles in metastasizing midgut carcinoid tumors
Open this publication in new window or tab >>Different gene expression profiles in metastasizing midgut carcinoid tumors
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2011 (English)In: Endocrine-Related Cancer, ISSN 1351-0088, E-ISSN 1479-6821, Vol. 18, no 4, 479-489 p.Article in journal (Refereed) Published
Abstract [en]

The genetic events leading the progression of midgut carcinoid tumors are largely unknown. The disease course varies from patient to patient, and there is a lack of reliable prognostic markers. In order to identify genes involved in tumor progression, gene expression profiling was performed on tumor specimens. Samples comprised 18 primary tumors, 17 lymph node (LN) metastases, and seven liver metastases from a total of 19 patients. Patients were grouped according to clinical data and histopathology into indolent or progressive course. RNA was subjected to a spotted oligo microarray and B-statistics were performed. Differentially expressed genes were verified using quantitative real-time PCR. Self-organizing maps demonstrated three clusters: 11 primary tumors separated in one cluster, five LN metastases in another cluster, whereas all seven liver metastases, seven primary, and 12 LN metastases formed a third cluster. There was no correlation between indolent and progressive behavior. The primary tumors with Ki67>5%, with low frequency of the carcinoid syndrome, and a tendency toward shorter survival grouped together. Primary tumors differed in expression profile from their associated LN metastases; thus, there is evidence for genetic changes from primary tumors to metastases. ACTG2, GREM2, REG3A, TUSC2, RUNX1, TPH1, TGFBR2, and CDH6 were differentially expressed between clusters and subgroups of tumors. The expression profile that assembles tumors as being genetically similar on the RNA expression level may not be concordant with the clinical disease course. This study reveals differences in gene expression profiles and novel genes that may be of importance in midgut carcinoid tumor progression.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:rkh:diva-1473 (URN)10.1530/ERC-10-0256 (DOI)21636701 (PubMedID)
Available from: 2011-10-10 Created: 2015-03-10 Last updated: 2015-03-11Bibliographically approved
2. A Plausible Role for Actin Gamma Smooth Muscle 2 (ACTG2) in Small Intestinal Neuroendocrine Tumorgenesis
Open this publication in new window or tab >>A Plausible Role for Actin Gamma Smooth Muscle 2 (ACTG2) in Small Intestinal Neuroendocrine Tumorgenesis
2016 (English)In: BMC Endocrine Disorders, ISSN 1472-6823, E-ISSN 1472-6823, Vol. 16, 19Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Small intestinal neuroendocrine tumors (SI-NETs) originate from the enterochromaffin cells in the ileum and jejunum. The knowledge about genetic and epigenetic abnormalities is limited. Low mRNA expression levels of actin gamma smooth muscle 2 (ACTG2) have been demonstrated in metastases relative to primary SI-NETs. ACTG2 and microRNA-145 (miR-145) are aberrantly expressed in other cancers and ACTG2 can be induced by miR-145. The aim of this study was to investigate the role of ACTG2 in small intestinal neuroendocrine tumorigenesis.

METHODS: Protein expression was analyzed in SI-NETs (n = 24) and in enterochromaffin cells by immunohistochemistry. The cell line CNDT2.5 was treated with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep), the selective EZH2 inhibitor EPZ-6438, or 5-aza-2'-deoxycytidine, a DNA hypomethylating agent. Cells were transfected with ACTG2 expression plasmid or miR-145. Western blotting analysis, quantitative RT-PCR, colony formation- and viability assays were performed. miR-145 expression levels were measured in tumors.

RESULTS: Eight primary tumors and two lymph node metastases displayed variable levels of positive staining. Fourteen SI-NETs and normal enterochromaffin cells stained negatively. Overexpression of ACTG2 significantly inhibited CNDT2.5 cell growth. Treatment with DZNep or transfection with miR-145 induced ACTG2 expression (>10-fold), but no effects were detected after treatment with EPZ-6438 or 5-aza-2'-deoxycytidine. DZNep also induced miR-145 expression. SI-NETs expressed relatively low levels of miR-145, with reduced expression in metastases compared to primary tumors.

CONCLUSIONS: ACTG2 is expressed in a fraction of SI-NETs, can inhibit cell growth in vitro, and is positively regulated by miR-145. Theoretical therapeutic strategies based on these results are discussed.

Keyword
ACTG2, SI-NET, epigenetic, microRNA-145
National Category
Medical and Health Sciences Basic Medicine
Identifiers
urn:nbn:se:rkh:diva-1553 (URN)10.1186/s12902-016-0100-3 (DOI)27107594 (PubMedID)
Note

As manuscript in dissertation.

Available from: 2014-02-25 Created: 2015-03-11 Last updated: 2017-07-11Bibliographically approved
3. TCEB3C a putative tumor suppressor gene of small intestine neuroendocrine tumors
Open this publication in new window or tab >>TCEB3C a putative tumor suppressor gene of small intestine neuroendocrine tumors
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2014 (English)In: Endocrine-Related Cancer, ISSN 1351-0088, E-ISSN 1479-6821, Vol. 21, no 2, 275-284 p.Article in journal (Refereed) Published
Abstract [en]

Small intestinal neuroendocrine tumors (SI-NETs), formerly midgut carcinoids, are rare and slow-growing neoplasms. Frequent loss of one copy of chromosome 18 in primary tumors and metastases has been observed. The aim of the study was to investigate a possible role of TCEB3C (Elongin A3), currently the only imprinted gene on chromosome 18, as a tumor suppressor gene in SI-NETs, and whether its expression is epigenetically regulated. Primary tumors, metastases, the human SI-NET cell line CNDT2.5, and two other cell lines were included. Immunohistochemistry, gene copy number determination by PCR, colony formation assay, Western blotting, real-time quantitative RT-PCR, RNA interference, and quantitative CpG methylation analysis by pyrosequencing were performed. The large majority of tumors (33/43) showed very low to undetectable Elongin A3 expression and as expected 89% (40/45) displayed one TCEB3C gene copy. The DNA hypomethylating agent 5-aza-2'-deoxycytidine induced TCEB3C expression in CNDT2.5 cells, in primary SI-NET cells prepared directly after surgery, but not in two other cell lines. Also siRNA to DNMT1 and treatment with the general histone methyltransferase inhibitor 3-deazaneplanocin A induced TCEB3C expression in a cell type-specific way. CpG methylation at the TCEB3C promoter was observed in all analyzed tissues and thus not related to expression. Overexpression of TCEB3C resulted in a 50% decrease of clonogenic survival of CNDT2.5 cells, but not of control cells. The results support a putative role of TCEB3C as a tumor suppressor gene in SI-NETs. Epigenetic repression of TCEB3C seems to be tumor cell type-specific and involves both DNA and histone methylation.

National Category
Endocrinology and Diabetes Cancer and Oncology
Identifiers
urn:nbn:se:rkh:diva-1471 (URN)10.1530/ERC-13-0419 (DOI)24351681 (PubMedID)
Available from: 2014-01-17 Created: 2015-03-10 Last updated: 2015-03-11Bibliographically approved
4. Novel Serum Biomarkers in Small Intestinal Neuroendocrine Tumors
Open this publication in new window or tab >>Novel Serum Biomarkers in Small Intestinal Neuroendocrine Tumors
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(English)Manuscript (preprint) (Other academic)
Keyword
SI-NET, DcR3, TFF3, Mindin, PLA
National Category
Basic Medicine
Identifiers
urn:nbn:se:rkh:diva-1550 (URN)
Note

As manuscript in dissertation.

Available from: 2014-02-25 Created: 2015-03-11 Last updated: 2017-07-11Bibliographically approved

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