| 1. |
- Fotouhi, Omid
(författare)
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Molecular aspects of tumor development and treatment for small intestinal neuroendocrine tumors
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Small intestinal neuroendocrine tumors (SI-NETs) may cause symptoms due to excess secretion of hormones and peptides. The molecular mechanisms behind development of SINETs are not well understood. Copy number alterations, especially loss of chromosome 18q, have been reported and recently p27 mutations were implicated in SI-NET tumorigenesis. Somatostatin analogs (SSAs) have long been used to alleviate the symptoms and have recently been shown to arrest SI-NET growth by unknown molecular mechanisms. In Study I, copy number alterations were investigated in 30 SI-NETs, using array comparative genomic hybridization. Recurrent alterations and minimal overlapping regions were observed, including losses on chromosomes 18, 16, 11 and 9 and gains on chromosome 20 and 14, 5 and 4. Using qPCR-based TaqMan assays, losses on chromosome 18, 16 and 11 were verified in an extension cohort, comprised of 43 SI-NETs, in total. Using unsupervised hierarchical clustering, a group of tumors was identified that was enriched with gains of chromosomes 20, 14, 7, 5 and 4. Gain in 20pter-p11.21 was associated with shorter survival and loss of 16q and gain of chromosome 7 were associated with metastases. In Study II, quantitative Pyrosequencing assays were used on 44 SI-NETs for promoter methylation analysis of candidate genes. Promoter hypermethylation was found for WIF1, RASSF1A, CTNNB1, CXCL14, NKX2–3, p16, LAMA1, and CDH1, but not for APC, CDH3, HIC1, P14, SMAD2, and SMAD4. Hypermethylation of WIF1 was concomitant with its mRNA downregulation in SI-NETs vs. normal intestine. Downregulation of RASSF1A and p16 was associated with a worse patient outcome. Global genome hypomethylation was demonstrated in SI-NETs. One group of tumors was identified with hypermethylation of WIF1, global hypomethylation and loss of chromosome 18 and another group with hypermethylation of RESSF1A and CTNNB1 and loss of chromosome 16. 5-azacytidine treatment of the SI-NET cell lines HC45 and CNDT2 reduced the methylation of hypermethylated genes and restored their mRNA expression. In Study III, the molecular mechanisms behind SSA treatment of NETs was examined using HiRIEF LC-MS/MS in HC45 and H727 cells treated with lanreotide at different time points. The results were confirmed for selected candidates using Western blot. The expression of Adenomatous polyposis coli (APC) was increased and survivin was decreased after 2 and 6 hours of treatment. Using shRNA against APC, the expression of survivin was elevated and siRNAs against somatostatin receptor 2 (SSTR2) suppressed APC-survivin regulation. In conclusion, lanreotide induced APC specifically through binding to SSTR2 and APC inhibited survivin. Immunohistochemistry on a tissue microarray comprised 112 NETs showed that survivin expression was associated with worse patient outcome. In Study IV, HiRIEF LC-MS/MS was used to study the mechanisms behind liver metastasis of SI-NETs. The proteome was compared between SI-NETs with and without liver metastasis at diagnosis. Higher expression of ubiquitin-like NEDD8 was seen in cases that had liver metastasis at the time of diagnosis. The NET cell lines BON-1, CNDT2, HC45 and H727 were treated with MLN4924, an inhibitor of the neddylation activating enzyme, NAE1. The proliferation of all cell lines was inhibited in a dose-dependent way. The proteome of CNDT2 and HC45 after treatment with MLN4924 was investigated using HiRIEF LCMS/MS. Neddylation seems to play a role in the progression of SI-NET and MLN4924 treatment is a promising strategy in the management of these tumors.
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| 2. |
- Fotouhi, Omid, et al.
(författare)
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Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors
- 2019
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Ingår i: Oncogene. - : NATURE PUBLISHING GROUP. - 0950-9232 .- 1476-5594. ; 38:43, s. 6881-6897
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Tidskriftsartikel (refereegranskat)abstract
- Patients with small intestinal neuroendocrine tumors (SI-NETs) frequently develop spread disease; however, the underlying molecular mechanisms of disease progression are not known and effective preventive treatment strategies are lacking. Here, protein expression profiling was performed by HiRIEF-LC-MS in 14 primary SI-NETs from patients with and without liver metastases detected at the time of surgery and initial treatment. Among differentially expressed proteins, overexpression of the ubiquitin-like protein NEDD8 was identified in samples from patients with liver metastasis. Further, NEDD8 correlation analysis indicated co-expression with RBX1, a key component in cullin-RING ubiquitin ligases (CRLs). In vitro inhibition of neddylation with the therapeutic agent pevonedistat (MLN4924) resulted in a dramatic decrease of proliferation in SI-NET cell lines. Subsequent mass spectrometry-based proteomics analysis of pevonedistat effects and effects of the proteasome inhibitor bortezomib revealed stabilization of multiple targets of CRLs including p27, an established tumor suppressor in SI-NET. Silencing of NEDD8 and RBX1 using siRNA resulted in a stabilization of p27, suggesting that the cellular levels of NEDD8 and RBX1 affect CRL activity. Inhibition of CRL activity, by either NEDD8/RBX1 silencing or pevonedistat treatment of cells resulted in induction of apoptosis that could be partially rescued by siRNA-based silencing of p27. Differential expression of both p27 and NEDD8 was confirmed in a second cohort of SI-NET using immunohistochemistry. Collectively, these findings suggest a role for CRLs and the ubiquitin proteasome system in suppression of p27 in SI-NET, and inhibition of neddylation as a putative therapeutic strategy in SI-NET.
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| 3. |
- Fotouhi, Omid, et al.
(författare)
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Regional differences in somatostatin receptor 2 (SSTR2) immunoreactivity is coupled to level of bowel invasion in small intestinal neuroendocrine tumors
- 2018
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Ingår i: Neuro - endocrinology letters. - Stockholm, Sweden : Maghira & Maas Publications. - 0172-780X. ; 39:4, s. 305-309
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Somatostatin receptor (SSTR) expression constitutes a pivotal cornerstone for accurate radiological detection and medical treatment of small intestinal neuroendocrine tumors (SI-NETs), and the development of somatostatin analogues for these purposes have revolutionized the clinical work-up. Previous assessments of SSTR isoform expression in SI-NETs have found correlations to overall prognosis and treatment response, however these analyses usually report overall tumoral immunoreactivity, and little is reported regarding histo-regional differences in expressional patterns.METHODS: Thirty-seven primary SI-NETs (WHO grade I, n=32 and WHO grade II, n=5) were collected and assessed for SSTR2 immunohistochemistry. Samples were stratified with regards to histological level of bowel infiltration and spread (mucosal region, muscularis propria region, subserosal region) and each of these tumoral regions was separately scored by SSTR2 staining localization (membrane, cytoplasmic), overall staining intensity and local staining differences within each region.RESULTS: SSTR2 immunoreactivity was progressively weaker as the tumor cells advanced through the small intestinal layers. This was exemplified by a reduction in the amount of tumor samples with strong SSTR2 expression in the deeper histological levels of the section; 56% of tumors displayed strong SSTR2 expression in the mucosal region, as compared to 29% and 30% of tumors within muscularis propria and subserosal layers, respectively.CONCLUSIONS: This observation indicates a down-regulation of SSTR2 expression as the tumors progress through the intestinal wall, which might signify underlying biological processes of importance for SI-NET invasion behavior.
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| 4. |
- Kharaziha, Pedram, et al.
(författare)
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Functional characterization of novel germline TP53 variants in Swedish families
- 2019
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Ingår i: Clinical Genetics. - : Wiley. - 0009-9163 .- 1399-0004. ; 96:3, s. 216-225
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Tidskriftsartikel (refereegranskat)abstract
- Pathogenic germline TP53 variants predispose to a wide range of early onset cancers, often recognized as the Li-Fraumeni syndrome (LFS). They are also identified in 1% of families with hereditary breast cancer (HrBC) that do not fulfill the criteria for LFS. In this study, we present a total of 24 different TP53 variants identified in 31 Swedish families with LFS or HrBC. Ten of these variants, nine exonic and one splice, have previously not been described as germline pathogenic variants. The nine exonic variants were functionally characterized and demonstrated partial transactivation activity compared to wild-type p53. Some show nuclear localization similar to wild-type p53 while others possess cytoplasmic or perinuclear localization. The four frameshift variants (W91Gfs*32, L111 Wfs*12, S227 Lfs*20 and S240Kfs*25) had negligible, while F134 L and T231del had low level of p53 activity. The L111 Wfs*12 and T231del variants are also deficient for induction of apoptosis. The missense variant R110C retain p53 effects and the nonsense E349* shows at least partial transcription factor activity but has reduced ability to trigger apoptosis. This is the first functional characterization of novel germline TP53 pathogenic or likely pathogenic variants in the Swedish cohort as an attempt to understand its association with LFS and HrBC, respectively.
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