Mattias K Andersson

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.

New tricks from an old oncogene: Gene fusion and copy number alterations of MYB in human cancer

MYB is a leucine zipper transcription factor that is essential for hematopoesis and for renewal of colonic crypts. There is also ample evidence showing that MYB is leukemogenic in several animal species. However, it was not until recently that clear evidence was presented showing that MYB actually is an oncogene rearranged in human cancer. In a recent study, a novel mechanism of activation of MYB involving gene fusion was identified in carcinomas of the breast and head and neck. A t(6;9) translocation was shown to generate fusions between MYB and the transcription factor gene NFIB. The fusions consistently result in loss of the 3´-end of MYB, including several highly conserved target sites for microRNAs that negatively regulate MYB expression. Deletion of these target sites may disrupt the repression of MYB, leading to overexpression of MYB-NFIB transcripts and protein and to transcriptional activation of critical MYB target genes associated with apoptosis, cell cycle control, cell growth/angiogenesis, and cell adhesion. This study, together with previous and recent data showing rearrangements and copy number alterations of the MYB locus in T-cell leukemia and certain solid tumors, will be the main focus of this review.

Diagnostic and therapeutic implications of new molecular biomarkers in salivary gland cancers.

Salivary gland carcinomas (SGCs) are uncommon tumors, constituting approximately 5% of all cancers of the head and neck. They are a heterogeneous group of diseases that pose significant diagnostic and therapeutic challenges. The treatment of patients with SGCs is mainly restricted to surgery and/or radiation therapy and there is only limited data available on the role of conventional systemic and targeted therapies in the management of patients with advanced disease. There is thus a great need to develop new molecular biomarkers to improve the diagnosis, prognostication, and therapeutic options for these patients. In this review, we will discuss the most recent developments in this field, with focus on pathognomonic gene fusions and other driver mutations of clinical significance. Comprehensive cytogenetic and molecular genetic analyses of SGCs have revealed a translocation-generated network of fusion oncogenes. The molecular targets of these fusions are transcription factors, transcriptional coactivators, and tyrosine kinase receptors. Prominent examples of clinically significant fusions are the MYB-NFIB fusion in adenoid cystic carcinoma and the CRTC1-MAML2 fusion in mucoepidermoid carcinoma. The fusions are key events in the molecular pathogenesis of these tumor types and contribute as new diagnostic, prognostic, and therapeutic biomarkers. Moreover, next-generation sequencing analysis of SGCs have revealed new druggable driver mutations, pinpointing alternative therapeutic options for subsets of patients. Continued molecular characterization of these fusions and their down-stream targets will ultimately lead to the identification of novel driver genes in SGCs and will form the basis for development of new therapeutic strategies for these patients.

The myxoid liposarcoma FUS-DDIT3 fusion oncoprotein deregulates NF-κB target genes by interaction with NFKBIZ

FUS (also called TLS), EWSR1 and TAF15 (also called TAF2N) are related genes involved in tumor type-specific fusion oncogenes in human malignancies. The FUS-DDIT3 fusion oncogene results from a t(12;16)(q13;p11) chromosome translocation and has a causative role in the initiation of myxoid/round cell liposarcomas (MLS/RCLS). The FUS-DDIT3 protein induces increased expression of the CAAT/enhancer-binding protein (C/EBP) and nuclear factor-κB (NF-κB)-controlled gene IL8, and the N-terminal FUS part is required for this activation. Chromatin immunoprecipitation analysis showed that FUS-DDIT3 binds the IL8 promoter. Expression studies of the IL8 promoter harboring a C/EBP–NF-κB composite site pinpointed the importance of NF-κB for IL8 expression in FUS-DDIT3-expressing cells. We therefore probed for possible interaction of FUS-DDIT3 with members of the NF-κB family. The nuclear factor NFKBIZ colocalizes with FUS-DDIT3 in nuclear structures, and immunoprecipitation experiments showed that FUS-DDIT3 binds the C-terminal of NFKBIZ. We also report that additional NF-κB-controlled genes are upregulated at the mRNA level in FUS-DDIT3-expressing cell lines and they can be induced by NFKBIZ. Taken together, the results indicate that FUS-DDIT3 deregulates some NF-κB-controlled genes through interactions with NFKBIZ. Similar mechanisms may be a part of the transformation process in other tumor types carrying FUS, EWSR1 and TAF15 containing fusion oncogenes.

Distinct cytoplasmic and nuclear functions of the stress induced protein DDIT3/CHOP/GADD153

DDIT3, also known as GADD153 or CHOP, encodes a basic leucine zipper transcription factor of the dimer forming C/EBP family. DDIT3 is known as a key regulator of cellular stress response, but its target genes and functions are not well characterized. Here, we applied a genome wide microarray based expression analysis to identify DDIT3 target genes and functions. By analyzing cells carrying tamoxifen inducible DDIT3 expression constructs we show distinct gene expression profiles for cells with cytoplasmic and nuclear localized DDIT3. Of 175 target genes identified only 3 were regulated by DDIT3 in both cellular localizations. More than two thirds of the genes were downregulated, supporting a role for DDIT3 as a dominant negative factor that could act by either cytoplasmic or nuclear sequestration of dimer forming transcription factor partners. Functional annotation of target genes showed cell migration, proliferation and apoptosis/survival as the most affected categories. Cytoplasmic DDIT3 affected more migration associated genes, while nuclear DDIT3 regulated more cell cycle controlling genes. Cell culture experiments confirmed that cytoplasmic DDIT3 inhibited migration, while nuclear DDIT3 caused a G1 cell cycle arrest. Promoters of target genes showed no common sequence motifs, reflecting that DDIT3 forms heterodimers with several alternative transcription factors that bind to different motifs. We conclude that expression of cytoplasmic DDIT3 regulated 94 genes. Nuclear translocation of DDIT3 regulated 81 additional genes linked to functions already affected by cytoplasmic DDIT3. Characterization of DDIT3 regulated functions helps understanding its role in stress response and involvement in cancer and degenerative disorders.

Mutation profiling of adenoid cystic carcinomas from multiple anatomical sites identifies mutations in the RAS pathway, but no KIT mutations

The majority of adenoid cystic carcinomas (AdCCs), regardless of anatomical site, harbour the MYB–NFIB fusion gene. The aim of this study was to characterize the repertoire of somatic genetic events affecting known cancer genes in AdCCs.

Proliferation of Ewing sarcoma cell lines is suppressed by the receptor tyrosine kinase inhibitors gefitinib and vandetanib

BackgroundTyrosine kinase inhibitors (TKIs) have gained much attention in recent years as targeted agents for the treatment of a wide range of human cancers. We have investigated the effect of the TKIs gefitinib and vandetanib on tumor cell lines derived from Ewing sarcoma, a highly malignant tumor affecting bone and soft tissue in children and young adults. Gefitinib is an inhibitor of epidermal growth factor receptor tyrosine kinase activity (EGFR) and vandetanib selectively targets vascular endothelial growth factor receptor-2 (VEGFR-2) with additional activity against VEGFR-3, EGFR and RET kinase receptors.ResultsTwo Ewing sarcoma cell lines investigated showed high levels of nuclear EGFR expression as well as moderate expression in plasma membrane and cytoplasm. When treated with concentrations of 5 μM and more of either gefitinib or vandetanib, we observed a significant decrease in cell proliferation. However, there were no detectable changes in p44/42 MAPK and Akt-1 phosphorylation, or in the expression of cyclin D1 or c-Myc following gefitinib or vandetanib treatment.ConclusionWe conclude that Ewing sarcoma tumor cell proliferation is not highly sensitive to inhibition of EGFR signaling alone or the simultaneous inhibition of VEGFR receptors, EGFR and RET kinase. Decreased tumor cell proliferation could be achieved with gefitinib and vandetanib, but only at higher doses where non-specific effects of the compounds may be overriding. As Ewing tumor cells do not seem to depend on EGFR and VEGFR pathways for survival, other key factors in the cellular signaling of Ewing sarcoma should be targeted in order to obtain a potent therapeutic response.

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.

Gastrointestinal stromal tumors regularly express synaptic vesicle proteins: evidence of a neuroendocrine phenotype

Gastrointestinal stromal tumors (GISTs) are thought to originate from the interstitial cells of Cajal, which share many properties with neurons of the gastrointestinal tract. Recently, we demonstrated expression of the hormone ghrelin in GIST. The aim of the present study was therefore to evaluate a possible neuroendocrine phenotype of GIST. Specimens from 41 GISTs were examined for the expression of 12 different synaptic vesicle proteins. Expression of synaptic-like microvesicle proteins, e.g., Synaptic vesicle protein 2 (SV2), synaptobrevin, synapsin 1, and amphiphysin was demonstrated in a majority of GISTs by immunohistochemistry, western blotting, and quantitative reversetranscriptase PCR. One-third of the tumors also expressed the large dense core vesicle protein vesicular monoamine transporter 1. Presence of microvesicles and dense core vesicles in GIST was confirmed by electron microscopy. The expression of synaptic-like microvesicle proteins in GIST was not related to risk profile or to KIT/platelet derived growth factor alpha (PDGFRA) mutational status. Thus, GISTs regularly express a subset of synaptic-like microvesicle proteins necessary for the regulated secretion of neurotransmitters and hormones. Expression of synaptic-like micro-vesicle proteins, ghrelin and peptide hormone receptors in GIST indicate a neuroendocrine phenotype and suggest novel possibilities to treat therapy-resistant GIST.

The landscape of gene fusions and somatic mutations in salivary gland neoplasms – Implications for diagnosis and therapy

Recent studies of the genomic landscape of salivary gland tumors have provided important insights into the molecular pathogenesis of these tumors. The most consistent alterations identified include a translocation-generated gene fusion network involving transcription factors, transcriptional coactivators, tyrosine kinase receptors, and other kinases. In addition, next-generation sequencing studies of a few subtypes of salivary neoplasms have revealed hotspot mutations in individual genes and mutations clustering to specific pathways frequently altered in cancer. Although limited, these studies have opened up new avenues for improved classification and targeted therapies of salivary gland cancers. In this review, we summarize the latest developments in this field, focusing on tumor types for which clinically important molecular data are available.