Abstract
BackgroundSmall 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.MethodsThe 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.ResultsVariable 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).ConclusionsSI-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.
Highlights
Small intestinal neuroendocrine tumors (SI-NETs) originate from enterochromaffin cells scattered in the intestinal mucosa of the ileum and jejunum
Overall variable levels and mosaic appearance of the epigenetic mark 5hmC in SI-NETs The relative level of 5hmC in 87 primary tumors (PT) and corresponding metastases (Met) from 40 patients was determined by a semi-quantitative DNA immune-dot blot assay using a specific rabbit polyclonal antibody against 5hmC [18]
We found that regulation of TET2 localization seemed to involve the exportin-1 nuclear export machinery as treatment of SI-NET cells with the inhibitor of exportin-1, leptomycin B, caused vanished levels in the cytoplasm of the slower migrating TET2 polypeptide, and not of the more faster migrating one
Summary
Small intestinal neuroendocrine tumors (SI-NETs) originate from enterochromaffin cells scattered in the intestinal mucosa of the ileum and jejunum. Small intestinal neuroendocrine tumor (SI-NET) is the most common malignancy of the small intestine and the incidence is about one per 100,000 and is on the rise These tumors, formerly known as midgut carcinoids, are small with relatively low cellular proliferation (Ki67 proliferation index is often < 2%), the majority of patients have tumor spread to both lymph nodes and liver at the time of diagnosis with 5-year overall survival rate of around 65%. Surgical resection of the primary tumor and metastases is the only curative treatment and may alleviate symptoms caused by small intestinal obstruction or ischaemia and the carcinoid syndrome. The most common genetic aberration in these tumors is loss of one copy of chromosome 18, known to occur in > 60% of tumors [2, 3]. TCEB3C encodes elongin A3 is located at 18q21, and has been shown to be epigenetically repressed and has a growth regulatory role in SI-NET cells [11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
