Yvonne Arvidsson

The multifunctional FUS, EWS and TAF15 proto-oncoproteins show cell type-specific expression patterns and involvement in cell spreading and stress response

BackgroundFUS, EWS and TAF15 are structurally similar multifunctional proteins that were first discovered upon characterization of fusion oncogenes in human sarcomas and leukemias. The proteins belong to the FET (previously TET) family of RNA-binding proteins and are implicated in central cellular processes such as regulation of gene expression, maintenance of genomic integrity and mRNA/microRNA processing. In the present study, we investigated the expression and cellular localization of FET proteins in multiple human tissues and cell types.ResultsFUS, EWS and TAF15 were expressed in both distinct and overlapping patterns in human tissues. The three proteins showed almost ubiquitous nuclear expression and FUS and TAF15 were in addition present in the cytoplasm of most cell types. Cytoplasmic EWS was more rarely detected and seen mainly in secretory cell types. Furthermore, FET expression was downregulated in differentiating human embryonic stem cells, during induced differentiation of neuroblastoma cells and absent in terminally differentiated melanocytes and cardiac muscle cells. The FET proteins were targeted to stress granules induced by heat shock and oxidative stress and FUS required its RNA-binding domain for this translocation. Furthermore, FUS and TAF15 were detected in spreading initiation centers of adhering cells.ConclusionOur results point to cell-specific expression patterns and functions of the FET proteins rather than the housekeeping roles inferred from earlier studies. The localization of FET proteins to stress granules suggests activities in translational regulation during stress conditions. Roles in central processes such as stress response, translational control and adhesion may explain the FET proteins frequent involvement in human cancer.

ASK1 Inhibits Astroglial Development via p38 Mitogen-Activated Protein Kinase and Promotes Neuronal Differentiation in Adult Hippocampus-Derived Progenitor Cells

ABSTRACT The mechanisms controlling differentiation and lineage specification of neural stem cells are still poorly understood, and many of the molecules involved in this process and their specific functions are yet unknown. We investigated the effect of apoptosis signal-regulating kinase 1 (ASK1) on neural stem cells by infecting adult hippocampus-derived rat progenitors with an adenovirus encoding the constitutively active form of ASK1. Following ASK1 overexpression, a significantly larger number of cells differentiated into neurons and a substantial increase in Mash1 transcription was observed. Moreover, a marked depletion of glial cells was observed, persisting even after additional treatment of ASK1-infected cultures with potent glia inducers such as leukemia inhibitory factor and bone morphogenetic protein. Analysis of the promoter for glial fibrillary acidic protein revealed that ASK1 acts as a potent inhibitor of glial-specific gene transcription. However, the signal transducers and activators of transcription 3 (STAT3)-binding site in the promoter was dispensable, while the activation of p38 mitogen-activated protein kinase was crucial for this effect, suggesting the presence of a novel mechanism for the inhibition of glial differentiation.

Amyloid precursor-like protein 1 is differentially upregulated in neuroendocrine tumours of the gastrointestinal tract

We have examined the global gene expression profile of small intestinal carcinoids by microarray analysis. High expression of a number of genes was found including amyloid precursor-like protein 1 (APLP1). Quantitative real-time PCR and western blot analysis demonstrated higher expression of APLP1 in carcinoid metastases relative to primary tumours indicating a role of APLP1 in tumour dissemination. Tissue microarray analysis of gastroentero-pancreatic tumours demonstrated a high frequency of APLP1 expression and a low frequency of APLP2 expression in neuroendocrine (NE) tumours when compared with non-NE tumours at the same sites. Meta-analysis of gene expression data from a large number of tumours outside the gastrointestinal tract confirmed a correlation between APLP1 expression and NE phenotype where high expression of APLP1 was accompanied by downregulation of APLP2 in NE tumours. Cellular localization of APLP1, APLP2 and amyloid precursor protein (APP) in carcinoid cells (GOT1) by confocal microscopy demonstrated partial co-localization with synaptophysin. This suggests that the APP family of proteins is transported to the cell membrane by synaptic microvesicles and that they may influence tumour cell adhesion and invasiveness. We conclude that APLP1 is differentially upregulated in gastrointestinal NE tumours and that APLP1 may be important for the dissemination of small intestinal carcinoids. Identification of APLP1 in NE tumours offers a novel target for treatment and may also serve as a tumour-specific marker.

Antitumoural Effects of the Pyridyl Cyanoguanidine CHS 828 on Three Different Types of Neuroendocrine Tumours Xenografted to Nude Mice

CHS 828, a cyanoguanidine with potent experimental antitumoural activity, inhibits activation of nuclear factor-ĸB. In the present study, marked antitumoural activity of peroral CHS 828 was shown against three different human neuroendocrine tumours, midgut carcinoid (GOT1), pancreatic carcinoid (BON), and medullary thyroid carcinoma (GOT2), transplanted in nude mice. Our results indicate that CHS 828 can be a candidate drug for treatment of neuroendocrine tumours.

Cytotoxic Effects of Valproic Acid on Neuroendocrine Tumour Cells.

Background/Aims: Histone deacetylases (HDACs) modulate lysine acetylation on histones and are frequently deregulated in cancer. HDAC inhibitors with potent anti-tumour effects have been developed and are now being tested in clinical trials. The aim of this study was to investigate the effects of valproic acid (VPA), an inhibitor of class I and class IIa HDACs, on neuroendocrine tumour (NET) cell growth. Methods: Three NET cell lines, GOT1 (small intestinal), KRJ-I (small intestinal), and BON (pancreatic), were treated with VPA and examined with respect to cell viability, cell cycle arrest, apoptosis, and global transcriptional response. Results: We found that VPA induced a dose-dependent growth inhibition of NET cells in vitro, which was mainly due to activation of extrinsic and intrinsic apoptotic pathways. VPA induced a major transcriptional response by altering the expression of 16-19% of the protein-coding genes in NET cell lines. Pathway analysis allowed the prediction of alterations in key regulatory pathways, e.g. activation of TGF-β1, FOXO3, p53 signalling, and inhibition of MYC signalling. Analysis of GOT1 xenografts showed reduced growth and reduced Ki-67 index, as well as an increase in apoptosis and necrosis after VPA treatment. Conclusions: We found that VPA treatment has a cytotoxic effect on NET cells of intestinal and pancreatic origin. There are several mechanisms by which VPA kills NET cells, which suggests the possibility of combination therapy. We propose that epigenetic therapy with HDAC inhibitors should be evaluated further in patients with NET disease.

Expression profiling of small intestinal neuroendocrine tumors identifies subgroups with clinical relevance, prognostic markers and therapeutic targets.

We wanted to define the transcriptome of small intestinal neuroendocrine tumors in order to identify clinically relevant subgroups of tumors, prognostic markers and novel targets for treatment. Genome-wide expression profiling was conducted on tumor biopsies from 33 patients with well-differentiated neuroendocrine tumors of the distal ileum and metastatic disease at the time of diagnosis. Unsupervised hierarchical clustering analysis identified three groups of tumors. The largest group, comprising half of the tumors, was characterized by longer patient survival and higher expression of neuroendocrine markers, including SSTR2. Tumors with higher grade (G2/3) or gain of chromosome 14 were associated with shorter patient survival and increased expression of cell cycle-promoting genes. Pathway analysis predicted the prostaglandin E receptor 2 (PTGER2) as the most significantly activated regulator in tumors of higher grade, whereas Forkhead box M1 (FOXM1) was the most significantly activated regulator in tumors with gain of chromosome 14. Druggable genes identified from expression profiles included clinically proven SSTR2 and also novel targets, for example, receptor tyrosine kinases (RET, FGFR1/3, PDGFRB and FLT1), epigenetic regulators, molecular chaperones and signal transduction molecules. Evaluation of candidate drug targets on neuroendocrine tumors cells (GOT1) showed significant inhibition of tumor cell growth after treatment with tyrosine kinase inhibitors or inhibitors of HDAC, HSP90 and AKT. In conclusion, we have defined the transcriptome of small intestinal neuroendocrine tumors and identified novel subgroups with clinical relevance. We found specific gene expression patterns associated with tumor grade and chromosomal alterations. Our data also suggest novel prognostic biomarkers and therapies for these patients.

Gastrointestinal stromal tumors (GISTs) express somatostatin receptors and bind radiolabeled somatostatin analogs

Abstract Background. Gastrointestinal stromal tumors (GISTs) can be effectively treated with tyrosine kinase inhibitors (TKIs). However, some patients with GIST develop drug resistance, and alternative treatment strategies are therefore needed. The aim of this study was to analyze the expression of somatostatin receptors (SSTR) in GIST as a target for peptide receptor-mediated radiotherapy (PRRT). Material and methods. Expression profiling of SSTR1–5 was performed on biopsies from 34 GISTs (16 gastric tumors, 15 small intestinal tumors, and three rectal tumors). SSTR scintigraphy (111In-octreotide) and measurement of 111In activity in tumor specimens was performed in seven patients. Uptake and internalization of 177Lu- octreotate was studied in primary cell cultures from two patients. Results. Quantitative PCR analysis showed expression of SSTR1 and SSTR2 in the majority of tumors, while SSTR3–5 were expressed at low levels. Immunohistochemical analysis confirmed the presence of SSTR1 and SSTR2 proteins in all GISTs, and SSTR3–5 in a subset of tumors. Diagnostic imaging by SSTR scintigraphy, using 111In-octreotide, demonstrated tumor uptake of 111In in three of six GIST patients. Measurement of 111In activity in excised tumor specimens from five patients gave tumor-to-blood (T/B) activity ratios of between eight and 96. Tumor cells in primary culture (gastric and small intestinal GIST) specifically bound and internalized 177Lu when incubated with the therapeutic compound 177Lu-octreotate for 4–48 hours (p < 0.05). Conclusion. Peptide receptor-mediated radiotherapy via SSTR may provide a novel treatment strategy in carefully selected GIST patients with TKI-resistant tumors.

New Medical Strategies for Midgut Carcinoids

Patients with well-differentiated neuroendocrine tumours of the gastrointestinal tract often present with metastases and hormonal symptoms. These patients can be palliated by interventional tumour reduction and medical treatment with somatostatin analogues; no effective chemotherapy is available. Radionuclide therapy via somatostatin receptors is one new therapeutic alternative. The recognition that neuroendocrine tumours express specific receptors for growth factors and chemokines, which are of importance for tumour growth, vascularization, and spread, may open the way for new therapeutic approaches. The signalling pathways in carcinoid tumours are incompletely explored. This review summarizes potential new treatment strategies from clinical and experimental studies, e.g. inhibition of angiogenesis, targeting of growth factors or their receptors by tyrosine kinase inhibitors, interference with specific cellular pathways (mTOR, PI3K, RAS/RAF, Notch), and also inhibition of the proteasome and histone deacetylation. Combining targeted therapy with chemotherapy, or using drugs to sensitize for radionuclide therapy, may enhance the treatment outcome.

Neuroblastoma-specific cytotoxicity mediated by the Mash1-promoter and E. coli purine nucleoside phosphorylase

Neuroblastoma is derived from cells of neural crest origin and often expresses the transcription factor human achaete‐scute homolog 1 (HASH1). The aim of this study was to selectively kill neuroblastoma cells by expressing the suicide gene E. coli purine nucleoside phosphorylase (PNP) under the control of the Mash1 promoter, the murine homolog of HASH1.

NAMPT inhibitor GMX1778 enhances the efficacy of 177Lu-DOTATATE treatment of neuroendocrine tumors.

Neuroendocrine tumors (NETs) can be treated by peptide receptor radionuclide therapy using radiolabeled somatostatin analogs. However, the efficacy of such treatment is low and needs to be optimized. Our study evaluated the potential radiosensitizing effects of inhibition of nicotineamide phosphoribosyltransferase on 177Lu-DOTATATE treatment in a NET model. Methods: Nude mice xenografted with the human NET cell line GOT1 were treated with semiefficient doses of 177Lu-DOTATATE (7.5 MBq, intravenously) or the nicotineamide phosphoribosyltransferase inhibitor GMX1778 (100 mg/kg/wk, orally). Results: Median time to tumor progression (tumor volume larger than at day 0) was 3 d for controls, 7 d for single-dose GMX1778, 28 d for single-dose 177Lu-DOTATATE, 35 d for 3 weekly doses of GMX1778, and 98 d for combined treatment with 177Lu-DOTATATE and GMX1778 × 1. After 177Lu-DOTATATE and 3 weekly doses of GMX1778, none of the tumors progressed within 120 d. Conclusion: GMX1778 enhances the efficacy of 177Lu-DOTATATE treatment and induces a prolonged antitumor response.