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

Application of a Home Energy Management System for Incentive-Based Demand Response Program Implementation

This paper presents an experimental real-time implementation of an incentive-based demand response program with hardware demonstration of a home energy management system. This system controls the electricity consumption of a residential electricity customer. For this purpose, the real consumption and generation profiles of a typical Portuguese household equipped with a home-scale photovoltaic system are employed. These profiles are simulated by the real-time digital simulator using real hardware resources. In the case studies, three different scenarios are simulated for a period of 24 hours with the consideration of the demand response programs and a 2 kW photovoltaic system. Different pricing scenarios are considered and the performance of the home energy management system is evaluated under each scenario. The focus is given to demonstrate how a home-scale photovoltaic system, and demand response programs, especially load-shifting scenario, can be cost-effective in the daily electricity costs of the residential customers.This work has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 641794 (project DREAM-GO) and from FEDER Funds through COMPETE program and from National Funds through FCT under the project UID/EEA/00760/2013info:eu-repo/semantics/publishedVersio

Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors.

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

Regional differences in somatostatin receptor 2 (SSTR2) immunoreactivity is coupled to level of bowel invasion in small intestinal neuroendocrine tumors

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.Funding Agencies|Swedish Cancer Society; Swedish Society for Medical Research; Radiumhemmets Forskningsfonder</p

Regional differences in somatostatin receptor 2 (SSTR2) immunoreactivity is coupled to level of bowel invasion in small intestinal neuroendocrine tumors

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.Funding Agencies|Swedish Cancer Society; Swedish Society for Medical Research; Radiumhemmets Forskningsfonder</p

Regional differences in somatostatin receptor 2 (SSTR2) immunoreactivity is coupled to level of bowel invasion in small intestinal neuroendocrine tumors

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.Funding Agencies|Swedish Cancer Society; Swedish Society for Medical Research; Radiumhemmets Forskningsfonder</p

Demand response implementation in smart households

Home energy management system (HEMS) is essential for residential electricity consumers to participate actively in demand response (DR) programs. Dynamic pricing schemes are not sufficiently effective for end-users without utilizing a HEMS for consumption management. In this paper, an intelligent HEMS algorithm is proposed to schedule the consumption of controllable appliances in a smart household. Electric vehicle (EV) and electric water heater (EWH) are incorporated in the HEMS. They are controllable appliances with storage capability. EVs are flexible energy-intensive loads, which can provide advantages of a dispatchable source. It is expected that the penetration of EVs will grow considerably in future. This algorithm is designed for a smart household with a rooftop photovoltaic (PV) system integrated with an energy storage system (ESS). Simulation results are presented under different pricing and DR programs to demonstrate the application of the HEMS and to verify its’ effectiveness. Case studies are conducted using real measurements. They consider the household load, the rooftop PV generation forecast and the built-in parameters of controllable appliances as inputs. The results exhibit that the daily household energy cost reduces 29.5%–31.5% by using the proposed optimization-based algorithm in the HEMS instead of a simple rule-based algorithm under different pricing schemes.info:eu-repo/semantics/publishedVersio

Telomerase activation in small intestinal neuroendocrine tumours is associated with aberrant TERT promoter methylation, but not hot-spot mutations

Telomere maintenance is a critical requirement for enabling replicative immortality and tumour development. Here, telomerase expression and activity, telomere length (TL) and potential regulatory factors that can underlie telomerase machinery alterations in small intestinal neuroendocrine tumours (SI-NETs) were analyzed. Telomerase activity assessed by TRAP assay was increased in SI-NETs compared to normal ileum (P < 0.001). The telomerase reverse transcriptase gene (TERT) was over-expressed in SI-NETs vs. normal ileal samples (P = 0.01). Furthermore, relative TL assessed by qPCR was found shorter in tumours compared with normal ileum (P = 0.02) and in distant metastasis samples compared to primary tumours and local metastases (P= 0.02). TERT promoter hotspot mutations were not present and TERT copy number gain was only observed in 3/70 tumour samples. TERT or chromosome 18 copy number alterations were not associated with telomerase expression and activity or TL. However, hypermethylation of TERT promoter in Region B – in the proximity of the transcription start site – was inversely correlated with TERT expression and telomerase activity and positively correlated with TL. Global LINE1 methylation was positively correlated with TERT promoter Region B methylation and was inversely correlated with telomerase activity, TERT expression and the upstream Region A methylation. The results show that telomerase activation, TERT expression and shorter telomeres are commonly found in SI-NETs. Aberrant DNA methylation of TERT promoter and of LINE1 can be implicated in abnormal regulation of TERT in SI-NETs

Alternative mRNA Splicing Controls the Functions of the Histone H3K27 Demethylase UTX/KDM6A

The UTX/KDM6A histone H3K27 demethylase plays an important role in development and is frequently mutated in cancers such as urothelial cancer. Despite many studies on UTX proteins, variations in mRNA splicing have been overlooked. Using Nanopore sequencing, we present a comprehensive analysis of UTX/KDM6A splicing events in human cell lines and in tissue samples from bladder cancer cases and normal epithelia. We found that the central region of UTX mRNAs encoded by exons 12 to 17 undergoes extensive alternative splicing. Up to half of all stable mRNAs (8–48% in bladder tissues and 18–58% in cell lines) are represented by the UTX canonical isoform lacking exon 14 encoding a nuclear localization sequence, and hence exon 14-containing UTX isoforms exclusively localize to the nucleus, unlike the cytonuclear localization of the canonical isoform. Chromatin association was also higher for exon-14-containing isoforms compared to the canonical UTX. Using quantitative mass spectrometry, we found that all UTX isoforms integrated into the MLL3 and MLL4, PR-DUB and MiDAC complexes. Interestingly, one of the novel UTX isoforms, which lacks exons 14 and 16, fails to interact with PR-DUB and MiDAC complex members. In conclusion, UTX mRNAs undergo extensive alternative splicing, which controls the subcellular localization of UTX and its interactions with other chromatin regulatory complexes

Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors.

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