Molecular aspects of tumor development and treatment for small intestinal neuroendocrine tumors

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. LIST OF SCIENTIFIC PAPERS This thesis is based on the following papers which are referred to in the text by their Roman numerals (I-IV). I. Jamileh Hashemi, Omid Fotouhi, Luqman Sulaiman, Magnus Kjellman, Anders Höög, Jan Zedenius, Catharina Larsson: Copy number alterations in small intestinal neuroendocrine tumors determined by array comparative genomic hybridization. BMC Cancer 13:505, 2013. II. Omid Fotouhi, Maral Adel Fahmideh, Magnus Kjellman, Luqman Sulaiman, Anders Höög, Jan Zedenius, Jamileh Hashemi, Catharina Larsson: Global hypomethylation and promoter methylation in small intestinal neuroendocrine tumors – an in vivo and in vitro study. Epigenetics 9(7):987-997, 2014. III. Omid Fotouhi, Hanna Kjellin, Catharina Larsson, Jamileh Hashemi, Jorge Barriuso, C Christofer Juhlin, Ming Lu, Anders Höög, Laura G Pastrián, Angela Lamarca, Victoria Heredia Soto, Jan Zedenius, Marta Mendiola, Janne Lehtiö, Magnus Kjellman: Proteomics suggests a role for APC-survivin in response to somatostatin analog treatment of neuroendocrine tumors. J Clin Endocrinol Metab. 101(10):3616-3627, 2016. IV. Omid Fotouhi, Hanna Kjellin, Catharina Larsson, Mehran Ghaderi, Stefano Caramuta, Magnus Kjellman, C Christor Juhlin, Jan Zedenius, Lukas Orre, Janne Lehtiö: Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors. In Manuscript LIST OF RELATED PUBLICATIONS Publications by the author on related topics outside the thesis. • Luqman Sulaiman, C Christofer Juhlin, Inga-Lena Nilsson, Omid Fotouhi, Catharina Larsson, Jamileh Hashemi: Global and gene-specific promoter methylation analysis in primary hyperparathyroidism. Epigenetics 8(6): 646–655, 2013. • Na Wang, Hanna Kjellin, Anastasios Sofiadis, Omid Fotouhi, C Christofer Juhlin, Martin Bäckdahl, Jan Zedenius, Dawei Xu, Janne Lehtiö, Catharina Larsson: Genetic and epigenetic background and protein expression profiles in relation to telomerase activation in medullary thyroid carcinoma. Oncotarget 7(16):21332-21346, 2016.