Publications by authors named "Helén Nilsson"

21 Publications

  • Page 1 of 1

Features of increased malignancy in eosinophilic clear cell renal cell carcinoma.

J Pathol 2020 12 24;252(4):384-397. Epub 2020 Sep 24.

Clinical Pathology, Sahlgrenska University Hospital, Gothenburg, Sweden.

Clear cell renal cell carcinoma (ccRCC) is the most common form of renal cancer. Due to inactivation of the von Hippel-Lindau tumour suppressor, the hypoxia-inducible transcription factors (HIFs) are constitutively activated in these tumours, resulting in a pseudo-hypoxic phenotype. The HIFs induce the expression of genes involved in angiogenesis and cell survival, but they also reset the cellular metabolism to protect cells from oxygen and nutrient deprivation. ccRCC tumours are highly vascularized and the cytoplasm of the cancer cells is filled with lipid droplets and glycogen, resulting in the histologically distinctive pale (clear) cytoplasm. Intratumoural heterogeneity may occur, and in some tumours, areas with granular, eosinophilic cytoplasm are found. Little is known regarding these traits and how they relate to the coexistent clear cell component, yet eosinophilic ccRCC is associated with higher grade and clinically more aggressive tumours. In this study, we have for the first time performed RNA sequencing comparing histologically verified clear cell and eosinophilic areas from ccRCC tissue, aiming to analyse the characteristics of these cell types. Findings from RNA sequencing were confirmed by immunohistochemical staining of biphasic ccRCC. We found that the eosinophilic phenotype displayed a higher proliferative drive and lower differentiation, and we confirmed a correlation to tumours of higher stage. We further identified mutations of the tumour suppressor p53 (TP53) exclusively in the eosinophilic ccRCC component, where mTORC1 activity was also elevated. Also, eosinophilic areas were less vascularized, yet harboured more abundant infiltrating immune cells. The cytoplasm of clear cell ccRCC cells was filled with lipids but had very low mitochondrial content, while the reverse was found in eosinophilic tissue. We herein suggest possible transcriptional mechanisms behind these phenomena. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756750PMC
December 2020

Localization and Regulation of Polymeric Ig Receptor in Healthy and Diseased Human Kidney.

Am J Pathol 2019 10 9;189(10):1933-1944. Epub 2019 Aug 9.

Center for Molecular Pathology, Department of Translational Medicine, Lund University, Malmö, Sweden; Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden. Electronic address:

The polymeric Ig receptor (PIgR) constitutes an important part of the immune system by mediating transcytosis of dimeric IgA into mucosal fluids. Although well studied in organs such as the intestine, the regulation and localization of PIgR in human kidney are incompletely characterized. Herein, using immunohistochemistry, we show that in healthy human kidneys, PIgR is expressed by the progenitor-like tubular scattered cells of the proximal tubules and by parietal epithelial cells of glomeruli. We further show that proximal tubular expression of PIgR becomes widespread during kidney disease, correlating to elevated levels of urinary secretory IgA. Urinary secretory IgA levels also correlated to the degree of tubular fibrosis, plasma creatinine, and urea levels. In addition, primary tubular cells were cultured to study the function and regulation of PIgR in vitro. Cellular PIgR expression was induced by conditioned medium from activated human leukocytes, as well as by inflammatory cytokines, whereas transforming growth factor-β1 caused decreased expression. Furthermore, interferon-γ increased the transcytosis of dimeric IgA in cultured tubular cells. Finally, a correlation study of mRNA data from the Genotype-Tissue Expression portal indicated that PIGR mRNA expression in kidney correlates to the expression of TNFSF13, a cytokine involved in plasma cell class switching to IgA. These results indicate that PIgR induction is an integral part of the injury phenotype of renal tubular cells.
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http://dx.doi.org/10.1016/j.ajpath.2019.06.015DOI Listing
October 2019

The GTPase domain of gamma-tubulin is required for normal mitochondrial function and spatial organization.

Commun Biol 2018 3;1:37. Epub 2018 May 3.

Molecular Pathology, Department of Translational Medicine, Lund University, Skåne University Hospital Malmö, 20502, Malmö, Sweden.

In the cell, γ-tubulin establishes a cellular network of threads named the γ-string meshwork. However, the functions of this meshwork remain to be determined. We investigated the traits of the meshwork and show that γ-strings have the ability to connect the cytoplasm and the mitochondrial DNA together. We also show that γ-tubulin has a role in the maintenance of the mitochondrial network and functions as reduced levels of γ-tubulin or impairment of its GTPase domain disrupts the mitochondrial network and alters both their respiratory capacity and the expression of mitochondrial-related genes. By contrast, reduced mitochondrial number or increased protein levels of γ-tubulin DNA-binding domain enhanced the association of γ-tubulin with mitochondria. Our results demonstrate that γ-tubulin is an important mitochondrial structural component that maintains the mitochondrial network, providing mitochondria with a cellular infrastructure. We propose that γ-tubulin provides a cytoskeletal element that gives form to the mitochondrial network.
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http://dx.doi.org/10.1038/s42003-018-0037-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123723PMC
May 2018

Injury induced expression of caveolar proteins in human kidney tubules - role of megakaryoblastic leukemia 1.

BMC Nephrol 2017 Oct 24;18(1):320. Epub 2017 Oct 24.

Department of Translational Medicine, Clinical Pathology, Lund University, SUS Malmö, Jan Waldenströms gata 59, SE-20502, Malmö, Sweden.

Background: Caveolae are membrane invaginations measuring 50-100 nm. These organelles, composed of caveolin and cavin proteins, are important for cellular signaling and survival. Caveolae play incompletely defined roles in human kidneys. Induction of caveolin-1/CAV1 in diseased tubules has been described previously, but the responsible mechanism remains to be defined.

Methods: Healthy and atrophying human kidneys were stained for caveolar proteins, (caveolin 1-3 and cavin 1-4) and examined by electron microscopy. Induction of caveolar proteins was studied in isolated proximal tubules and primary renal epithelial cells. These cells were challenged with hypoxia or HO. Primary tubular cells were also subjected to viral overexpression of megakaryoblastic leukemia 1 (MKL1) and MKL1 inhibition by the MKL1 inhibitor CCG-1423. Putative coregulators of MKL1 activity were investigated by Western blotting for suppressor of cancer cell invasion (SCAI) and filamin A (FLNA). Finally, correlative bioinformatic studies of mRNA expression of caveolar proteins and MKL1 were performed.

Results: In healthy kidneys, caveolar proteins were expressed by the parietal epithelial cells (PECs) of Bowman's capsule, endothelial cells and vascular smooth muscle. Electron microscopy confirmed caveolae in the PECs. No expression was seen in proximal tubules. In contrast, caveolar proteins were expressed in proximal tubules undergoing atrophy. Caveolar proteins were also induced in cultures of primary epithelial tubular cells. Expression was not enhanced by hypoxia or free radical stress (HO), but proved sensitive to inhibition of MKL1. Viral overexpression of MKL1 induced caveolin-1/CAV1, caveolin-2/CAV2 and SDPR/CAVIN2. In kidney tissue, the mRNA level of MKL1 correlated with the mRNA levels for caveolin-1/CAV1, caveolin-2/CAV2 and the archetypal MKL1 target tenascin C (TNC), as did the MKL1 coactivator FLNA. Costaining for TNC as readout for MKL1 activity demonstrated overlap with caveolin-1/CAV1 expression in PECs as well as in atrophic segments of proximal tubules.

Conclusions: Our findings support the view that MKL1 contributes to the expression of caveolar proteins in healthy kidneys and orchestrates the induction of tubular caveolar proteins in renal injury.
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http://dx.doi.org/10.1186/s12882-017-0738-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655893PMC
October 2017

Cell-Type-Specific Gene Programs of the Normal Human Nephron Define Kidney Cancer Subtypes.

Cell Rep 2017 08;20(6):1476-1489

Translational Cancer Research, Department of Laboratory Medicine, Lund University, Medicon Village, Scheelevägen 2, 223 81 Lund, Sweden. Electronic address:

Comprehensive transcriptome studies of cancers often rely on corresponding normal tissue samples to serve as a transcriptional reference. In this study, we performed in-depth analyses of normal kidney tissue transcriptomes from the TCGA and demonstrate that the histological variability in cellularity, inherent in the kidney architecture, lead to considerable transcriptional differences between samples. This should be considered when comparing expression profiles of normal and cancerous kidney tissues. We exploited these differences to define renal-cell-specific gene signatures and used these as a framework to analyze renal cell carcinoma (RCC) ontogeny. Chromophobe RCCs express FOXI1-driven genes that define collecting duct intercalated cells, whereas HNF-regulated genes, specific for proximal tubule cells, are an integral part of clear cell and papillary RCC transcriptomes. These networks may be used as a framework for understanding the interplay between genomic changes in RCC subtypes and the lineage-defining regulatory machinery of their non-neoplastic counterparts.
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http://dx.doi.org/10.1016/j.celrep.2017.07.043DOI Listing
August 2017

Papillary renal cell carcinoma-derived chemerin, IL-8, and CXCL16 promote monocyte recruitment and differentiation into foam-cell macrophages.

Lab Invest 2017 11 31;97(11):1296-1305. Epub 2017 Jul 31.

Department of Translational Medicine, Center for Molecular Pathology, Lund University, Malmö, Sweden.

Papillary renal cell carcinoma (pRCC) is the second most common type of renal cell carcinoma. The only curative treatment available for pRCC is radical surgery. If the disease becomes widespread, neither chemo- nor radiotherapy will have therapeutic effect, hence further research on pRCC is of utmost importance. Histologically, pRCC is characterized by a papillary growth pattern with focal aggregation of macrophages of the foam cell phenotype. In other forms of cancer, a clear role for tumor-associated macrophages during cancer growth and progression has been shown. Although the presence of foamy macrophages is a histological hallmark of pRCC tumors, little is known regarding their role in pRCC biology. In order to study the interaction between pRCC tumor and myeloid cells, we established primary cultures from pRCC tissue. We show that human pRCC cells secrete the chemokines IL-8, CXCL16, and chemerin, and that these factors attract primary human monocytes in vitro. RNAseq data from The Cancer Genome Atlas confirmed a high expression of these factors in pRCC tissue. Conditioned medium from pRCC cultures induced a shift in human monocytes toward the M2 macrophage phenotype. In extended cultures, these macrophages became enlarged and loaded with lipids, adopting the foam cell morphology found in pRCC tissue. These results show for the first time that pRCC primary tumor cells secrete factors that attract and differentiate monocytes into anti-inflammatory tumor-associated macrophages with foam cell histology.
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http://dx.doi.org/10.1038/labinvest.2017.78DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668481PMC
November 2017

Overexpression of Functional SLC6A3 in Clear Cell Renal Cell Carcinoma.

Clin Cancer Res 2017 04 23;23(8):2105-2115. Epub 2016 Sep 23.

Translational Cancer Research, Department of Laboratory Medicine, Lund University, Medicon Village, Lund, Sweden.

Renal cell carcinoma (RCC) is derived from a tissue with a remarkable capacity for vectorial transport. We therefore performed an unbiased exploration of transporter proteins in normal kidney and kidney cancer to discover novel clinical targets. Using The Cancer Genome Atlas (TCGA) database, we investigated differences in membrane transporter expression in clear cell RCC (ccRCC) and normal kidney. We identified the dopamine transporter SLC6A3 as a specific biomarker for ccRCC. To investigate the functionality of SLC6A3, we used a [H]-dopamine uptake assay on ccRCC cells. We further explored the effect of hypoxia-inducible factor (HIF) proteins on SLC6A3 expression by introducing siRNA in ccRCC cells and by hypoxic treatment of nonmalignant cells. We show that ccRCC expresses very high transcript levels of in contrast to normal kidney tissue and other tumor types, which do not express appreciable levels of this transporter. Importantly, we demonstrate that the elevated expression of SLC6A3 in ccRCC cells is associated with specific uptake of dopamine. By targeting the expression of HIF-1α and HIF-2α, we could show that expression is primarily influenced by HIF-2α and that hypoxia can induce expression in normal renal cells. We conclude that the dopamine transporter SLC6A3 constitutes a novel biomarker that is highly specific for ccRCC. We further postulate that the protein can be exploited for diagnostic or therapeutic purposes for detection or treatment of ccRCC. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-0496DOI Listing
April 2017

Glycosaminoglycan Profiling in Patients' Plasma and Urine Predicts the Occurrence of Metastatic Clear Cell Renal Cell Carcinoma.

Cell Rep 2016 05 12;15(8):1822-36. Epub 2016 May 12.

Department of Biology and Biological Engineering, Chalmers University of Technology, 41296 Göteborg, Sweden. Electronic address:

Metabolic reprogramming is a hallmark of clear cell renal cell carcinoma (ccRCC) progression. Here, we used genome-scale metabolic modeling to elucidate metabolic reprogramming in 481 ccRCC samples and discovered strongly coordinated regulation of glycosaminoglycan (GAG) biosynthesis at the transcript and protein levels. Extracellular GAGs are implicated in metastasis, so we speculated that such regulation might translate into a non-invasive biomarker for metastatic ccRCC (mccRCC). We measured 18 GAG properties in 34 mccRCC samples versus 16 healthy plasma and/or urine samples. The GAG profiles were distinctively altered in mccRCC. We derived three GAG scores that distinguished mccRCC patients with 93.1%-100% accuracy. We validated the score accuracies in an independent cohort (up to 18 mccRCC versus nine healthy) and verified that the scores normalized in eight patients with no evidence of disease. In conclusion, coordinated regulation of GAG biosynthesis occurs in ccRCC, and non-invasive GAG profiling is suitable for mccRCC diagnosis.
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http://dx.doi.org/10.1016/j.celrep.2016.04.056DOI Listing
May 2016

Interferon-γ-induced p27KIP1 binds to and targets MYC for proteasome-mediated degradation.

Oncotarget 2016 Jan;7(3):2837-54

Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden.

The Myc oncoprotein is tightly regulated at multiple levels including ubiquitin-mediated protein turnover. We recently demonstrated that inhibition of Cdk2-mediated phosphorylation of Myc at Ser-62 pharmacologically or through interferon (IFN)-γ-induced expression of p27(Kip1) (p27) repressed Myc's activity to suppress cellular senescence and differentiation. In this study we identified an additional activity of p27 to interfere with Myc independent of Ser-62 phosphorylation. p27 is required and sufficient for IFN-γ-induced turnover of Myc. p27 interacted with Myc in the nucleus involving the C-termini of the two proteins, including Myc box 4 of Myc. The C-terminus but not the Cdk2 binding fragment of p27 was sufficient for inducing Myc degradation. Protein expression data of The Cancer Genome Atlas breast invasive carcinoma set revealed significantly lower Myc protein levels in tumors with highly expressed p27 lacking phosphorylation at Thr-157--a marker for active p27 localized in the nucleus. Further, these conditions correlated with favorable tumor stage and patient outcome. This novel regulation of Myc by IFN-γ/p27(KIP1) potentially offers new possibilities for therapeutic intervention in tumors with deregulated Myc.
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http://dx.doi.org/10.18632/oncotarget.6693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823075PMC
January 2016

High salt buffer improves integrity of RNA after fluorescence-activated cell sorting of intracellular labeled cells.

J Biotechnol 2014 Dec 30;192 Pt A:62-5. Epub 2014 Sep 30.

Department of Laboratory Medicine, Center for Molecular Pathology, Lund University, Sweden.

Over the past years, massive progress has been made in the ability to collect large-scale gene expression data from a limited sample size. Combined with improvements in multiplex flow cytometry-based techniques, this has made it possible to isolate and characterize specific cellular subtypes within heterogeneous populations, with a great impact on our understanding of different biological processes. However, sorting based on intracellular markers requires fixation and permeabilization of samples, and very often the integrity of RNA molecules is compromised during this process. Many attempts have been made to improve the quality of nucleic acids from such samples, but RNA degradation still remains a limiting factor for downstream analyses. Here we present a method to isolate high quality RNA from cells that have been fixed, permeabilized, intracellularly labeled and sorted. By performing all incubation steps in the presence of a high salt buffer, RNA degradation was avoided and samples with remarkable integrity were obtained. This procedure offers a straightforward and very affordable technique to retrieve high quality RNA from isolated cell populations, which increases the possibilities to characterize gene expression profiles of subpopulations from mixed samples, a technique with implications in a broad range of research fields.
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http://dx.doi.org/10.1016/j.jbiotec.2014.09.016DOI Listing
December 2014

A perineal protection device designed to protect the perineum during labor: a multicenter randomized controlled trial.

Eur J Obstet Gynecol Reprod Biol 2014 Oct 30;181:10-4. Epub 2014 Jul 30.

Campus Helsingborg-Clinical Science Faculty of Medicine, Lund University, Södra Vallgatan 5, 251 87 Helsingborg, Sweden.

Objective: The objective of this study was to evaluate the protective effects of a new device for reducing perineal tears during vaginal childbirth.

Study Design: A multicenter open randomized controlled trial (RCT) was performed in Helsingborg, Lund and Malmö, Sweden consisting of 1148 women. Women anticipating a vaginal delivery were either randomized to the intervention group (n=574 in which the perineal protection device was used, or a control group (n=574), in which the perineal protection device was not used. The main outcome measurements were incidence of vaginal and perineal tears (1st to 4th degree tears) and adverse effects on the parturient and newborn.

Results: The incidences of first- and second-degree tears of the vagina (p=0.018) and perineum (p=0.005) were significantly reduced in the intervention group compared with the controls. In the intervention- and control group, 184 women (34.9%) and 142 (26.6%) showed no perineal tearing, respectively (p=0.034). Numbers needed to treat to avoid any perianal tearing was 12. The incidence of anal sphincter rupture (ASR) was the same in both groups (n=19; 3.4%). No negative effects on mother or child from using the device were observed.

Conclusions: The perineal protective device significantly reduced the incidence of first- and second-degree tears in the vagina and perineum during vaginal birth and also significantly increased the number of parturients with a fully intact posterior commissure. No significant reduction of ASR and no negative effects of the device were observed.
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http://dx.doi.org/10.1016/j.ejogrb.2014.07.006DOI Listing
October 2014

CDK-mediated activation of the SCF(FBXO) (28) ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer.

EMBO Mol Med 2013 Jul 14;5(7):1067-86. Epub 2013 Jun 14.

Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28) activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.
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http://dx.doi.org/10.1002/emmm.201202341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3721474PMC
July 2013

Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype.

Cancer Cell 2006 Nov;10(5):413-23

Division of Molecular Medicine, Department of Laboratory Medicine, Lund University, University Hospital MAS, SE-205 02 Malmö, Sweden.

In neuroblastoma specimens, HIF-2alpha but not HIF-1alpha is strongly expressed in well-vascularized areas. In vitro, HIF-2alpha protein was stabilized at 5% O2 (resembling end capillary oxygen conditions) and, in contrast to the low HIF-1alpha activity at this oxygen level, actively transcribed genes like VEGF. Under hypoxia (1% O2), HIF-1alpha was transiently stabilized and primarily mediated acute responses, whereas HIF-2alpha protein gradually accumulated and governed prolonged hypoxic gene activation. Knockdown of HIF-2alpha reduced growth of neuroblastoma tumors in athymic mice. Furthermore, high HIF-2alpha protein levels were correlated with advanced clinical stage and high VEGF expression and predicted poor prognosis in a clinical neuroblastoma material. Our results demonstrate the relevance of HIF-2alpha in neuroblastoma progression and have general tumor biological implications.
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http://dx.doi.org/10.1016/j.ccr.2006.08.026DOI Listing
November 2006

The von Hippel-Lindau tumor suppressor gene expression level has prognostic value in neuroblastoma.

Int J Cancer 2006 Aug;119(3):624-9

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Deletions of the short arm of chromosome 3 are often observed in a specific subset of aggressive neuroblastomas (NBs) with loss of distal 11q and without MYCN amplification. The critical deleted region encompasses the locus of the von Hippel-Lindau gene (VHL, 3p25). Constitutional loss of function mutations in the VHL gene are responsible for the VHL syndrome, a dominantly inherited familial cancer syndrome predisposing to a variety of neoplasms, including pheochromocytoma. Pheochromocytomas are, like NB, derived from neural crest cells, but, unlike NB, consist of more mature chromaffin cells instead of immature neuroblasts. Further arguments for a putative role of VHL in NB are its function as oxygen sensitizer and the reported relation between hypoxia and dedifferentiation of NB cells, leading to a more aggressive phenotype. To test the possible involvement of VHL in NB, we did mRNA expression analysis and sought evidence for VHL gene inactivation. Although no evidence for a classic tumor suppressor role for VHL in NB could be obtained, a strong correlation was observed between reduced levels of VHL mRNA and low patient survival probability (p=0.013). Furthermore, VHL appears to have predictive power in NTRK1 (TRKA) positive tumor samples with presumed favorable prognosis, which makes it a potentially valuable marker for more accurate risk assessment in this subgroup of patients. The significance of the reduced VHL expression levels in relation to NB tumor biology remains unexplained, as functional analysis demonstrated no clear effect of the reduction in VHL mRNA expression on protein stability of its downstream target hypoxia-inducible factor alpha.
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http://dx.doi.org/10.1002/ijc.21888DOI Listing
August 2006

Translocation-excision-deletion-amplification mechanism leading to nonsyntenic coamplification of MYC and ATBF1.

Genes Chromosomes Cancer 2006 Feb;45(2):107-17

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Despite oncogene amplification being a characteristic of many tumor types, the mechanisms leading to amplicon formation have remained largely unresolved. In this study, we used a combinatorial approach of fluorescence in situ hybridization and single-nucleotide polymorphism chip gene copy number analyses to unravel the mechanism leading to nonsyntenic coamplification of MYC and ATBF1 in SJNB-12 cells. To explain our findings, we propose a complex series of events consisting of multiple double-strand breaks, accompanied (or triggered) by the formation of a reciprocal translocation t(8;16), as well as excisions and deletions near the translocation breakpoints. This study provides evidence for a translocation-excision-deletion-amplification sequence of events rather than a breakage-fusion-bridge model, which has been more frequently proposed to explain proto-oncogene amplification. Furthermore, it illustrates the power of presently available tools for detailed analysis of the complex rearrangements that accompany amplicon formation.
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http://dx.doi.org/10.1002/gcc.20272DOI Listing
February 2006

HIF-2alpha expression in human fetal paraganglia and neuroblastoma: relation to sympathetic differentiation, glucose deficiency, and hypoxia.

Exp Cell Res 2005 Feb;303(2):447-56

Department of Laboratory Medicine, Division of Molecular Medicine, Lund University, University Hospital MAS, Entrance 78, 205 02 Malmö, Sweden.

Solid tumors are frequently necrotic and hypoxic due to poor vascularization. Tumor cells adapt to hypoxia by modulating their phenotype. Key players in this process are the hypoxia-inducible factors (HIF-1alpha to 3alpha). HIFs are also expressed during normal development; for example, HIF-2alpha is specifically expressed and appears to be involved in the development of the murine sympathetic nervous system (SNS). Here, we demonstrate that HIF-2alpha protein is selectively present in human fetal week 8.5 SNS paraganglia. Neuroblastoma is derived from SNS precursors. In a subset of neuroblastomas, a spontaneous neuronal to neuroendocrine differentiation occurs in areas adjacent to necrotic zones. As HIF-2alpha activity has been associated not only with hypoxic but also with hypoglycemic conditions, we have investigated putative effects of hypoxia, glucose depletion, and HIF-2alpha on the neuroblastoma phenotype. HIF-2alpha was detected in hypoxic and in well-oxygenized neuroblastoma cells and tissue, presumably reflecting their embryonic features. With regard to differentiation, hypoxic cells lost their neuronal/neuroendocrine features and gained marker gene expression associated with an immature, neural crest-like phenotype. Low glucose potentiated the effect of hypoxia. These findings suggest that poorly vascularized neuroblastomas become immature and maintain a more aggressive phenotype, which possibly could involve a sustained stabilization and activation of HIF-2alpha.
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http://dx.doi.org/10.1016/j.yexcr.2004.10.003DOI Listing
February 2005

Neuroblastoma cells with overexpressed MYCN retain their capacity to undergo neuronal differentiation.

Lab Invest 2004 Apr;84(4):406-17

Department of Laboratory Medicine, Molecular Medicine, Lund University, University Hospital MAS, S-20502 Malmö, Sweden.

Amplification of MYCN in neuroblastoma strongly correlates to unfavorable outcome, but little is known of how the high MYCN expression translates into an aggressive tumor phenotype. More aggressive neuroblastomas are generally immature and overexpression of exogenous MYCN in cultured neuroblastoma cells and other neuronal cell types has been reported to inhibit induced differentiation, suggesting a link between high MYCN expression and an immature phenotype. However, we show here that MYCN is expressed in human neuroblasts of sympathetic chain ganglia at fetal week 8.5, a developmental stage at which these neuroblasts express a number of sympathetic neuronal differentiation marker genes. Analyses of 28 neuroblastoma tumor specimens and 27 cell lines for the expression of MYCN and a panel of neuronal differentiation marker genes did not reveal any correlation between MYCN and marker gene expression levels. Finally, we tested five separate differentiation protocols and show that MYCN overexpressing neuroblastoma cells with a neuronal phenotype, derived from the non-MYCN-amplified human neuroblastoma cell line SK-N-SH, retain their capacity to differentiate despite constitutive MYCN overexpression. Our results show that high MYCN expression and sympathetic differentiation are compatible, and indirectly our findings lend support to previously published MYCN neuroblastoma tumor data, which suggest that in single MYCN copy neuroblastomas there is no direct correlation between a high cellular MYCN protein content and aggressive tumor cell behavior.
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http://dx.doi.org/10.1038/labinvest.3700061DOI Listing
April 2004

Hypoxia-induced dedifferentiation in neuroblastoma cells.

Cancer Lett 2003 Jul;197(1-2):145-50

Department of Laboratory Medicine, Division of Molecular Medicine, Lund University, University Hospital MAS, S-205 02 Malmö, Sweden.

Hypoxia in solid tumors is associated with aggressive behavior and poor outcome. We recently discovered that hypoxia alters the expression of differentiation marker genes in neuroblastoma cells, in that the tumor cells adjust to the hypoxic environment by down-regulating genes associated with a neuronal and upregulating genes associated with a neural crest-like phenotype. As there is a correlation in neuroblastoma between low stage of differentiation and high (aggressive) clinical stage, we propose that dedifferentiation of neuroblastoma cells in hypoxic tumor regions contribute to the malignancy of the tumor.
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http://dx.doi.org/10.1016/s0304-3835(03)00095-8DOI Listing
July 2003

Expression of trkB in human neuroblastoma in relation to MYCN expression and retinoic acid treatment.

Lab Invest 2003 Jun;83(6):813-23

Department of Laboratory Medicine, Lund University, University Hospital MAS, Malmö, Sweden.

Expression of full-length trkB can be found in some highly malignant neuroblastoma tumors with an amplified MYCN gene. This contrasts sympathetic neuroblasts, from which neuroblastomas are thought to arise, which neither express trkB nor are dependent on the p145(trkB) ligands, brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5, for their normal development. In this study we show that trkB was expressed in two out of five neuroblastoma tumors with amplified MYCN, while no trkB expression was observed when the MYCN gene was overexpressed in a non-MYCN-amplified neuroblastoma cell line. This shows that MYCN overexpression per se is not sufficient to induce trkB expression. trkB expression and BDNF responsiveness in neuroblastoma cells can be induced by all-trans-retinoic acid (RA). When SH-SY5Y cells were stimulated with a combination of RA and BDNF, norepinephrine and tyrosine hydroxylase levels were unaltered, showing that the cells did not change toward a more catecholaminergic sympathetic phenotype. However, expression of growth-associated protein 43, indicative of a neuronal phenotype, was elevated. Vesicular acetylcholine transporter, choline acetyl transferase, and neuropeptide tyrosine mRNA levels also increased in RA-BDNF-treated cells, which could suggest that these cells develop into a sympathetic cholinergic phenotype. In addition, treatment with RA-induced expression of the platelet-derived growth factor receptor-alpha. As previously shown for BDNF, platelet-derived growth factor stimulated growth of the RA-treated cells, findings that could have clinical relevance. If these receptors mediate a mitogenic signal in vivo also, this might limit the effect of RA treatment on neuroblastoma patients.
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http://dx.doi.org/10.1097/01.lab.0000074895.48776.d8DOI Listing
June 2003

Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest-like phenotype.

Proc Natl Acad Sci U S A 2002 May;99(10):7021-6

Department of Laboratory Medicine, Division of Molecular Medicine, Lund University, University Hospital MAS, S-205 02 Malmö, Sweden.

Insufficient oxygen and nutrient supply often restrain solid tumor growth, and the hypoxia-inducible factors (HIF) 1 alpha and HIF-2 alpha are key transcription regulators of phenotypic adaptation to low oxygen levels. Moreover, mouse gene disruption studies have implicated HIF-2 alpha in embryonic regulation of tyrosine hydroxylase, a hallmark gene of the sympathetic nervous system. Neuroblastoma tumors originate from immature sympathetic cells, and therefore we investigated the effect of hypoxia on the differentiation status of human neuroblastoma cells. Hypoxia stabilized HIF-1 alpha and HIF-2 alpha proteins and activated the expression of known hypoxia-induced genes, such as vascular endothelial growth factor and tyrosine hydroxylase. These changes in gene expression also occurred in hypoxic regions of experimental neuroblastoma xenografts grown in mice. In contrast, hypoxia decreased the expression of several neuronal/neuroendocrine marker genes but induced genes expressed in neural crest sympathetic progenitors, for instance c-kit and Notch-1. Thus, hypoxia apparently causes dedifferentiation both in vitro and in vivo. These findings suggest a novel mechanism for selection of highly malignant tumor cells with stem-cell characteristics.
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http://dx.doi.org/10.1073/pnas.102660199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124521PMC
May 2002