Josephine Nefkens Institute

Rotterdam, Netherlands

Josephine Nefkens Institute

Rotterdam, Netherlands
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Monteiro J.,Josephine Nefkens Institute | Gaspar C.,Josephine Nefkens Institute | Gaspar C.,Instituto Medicina Molecular | Richer W.,University Pierre and Marie Curie | And 8 more authors.
Carcinogenesis | Year: 2014

Wnt signaling plays a central role in mammary stem cell (MaSC) homeostasis and in breast cancer. In particular, epigenetic alterations at different members of the Wnt pathway have been identified among triple-negative, basal-like breast cancers. Previously, we developed a mouse model for metaplastic breast adenocarcinoma, a subtype of triple-negative breast cancer, by targeting a hypomorphic mutations in the endogenous Apc gene (Apc1572T/+). Here, by employing the CD24 and CD29 cell surface antigens, we have identified a subpopulation of mammary cancer stem cells (MaCSCs) from Apc1572T/+ capable of self-renewal and differentiation both in vivo and in vitro. Moreover, immunohistochemical analysis of micro- and macrolung metastases and preliminary intravenous transplantation assays suggest that the MaCSCs underlie metastasis at distant organ sites. Expression profiling of the normal and tumor cell subpopulations encompassing MaSCs and CSCs revealed that the normal stem cell compartment is more similar to tumor cells than to their own differentiated progenies. Accordingly, Wnt signaling appears to be active in both the normal and cancer stem cell compartments, although at different levels. By comparing normal with cancer mouse mammary compartments, we identified a MaCSC gene signature able to predict outcome in breast cancer in man. Overall, our data indicate that constitutive Wnt signaling activation affects self-renewal and differentiation of MaSCs leading to metaplasia and basal-like adenocarcinomas. © The Author 2013. Published by Oxford University Press. All rights reserved.


Martens-Uzunova E.S.,Josephine Nefkens Institute | Jalava S.E.,University of Tampere | Dits N.F.,Josephine Nefkens Institute | Van Leenders G.J.L.H.,Erasmus University Rotterdam | And 6 more authors.
Oncogene | Year: 2012

Prostate cancer (PCa) is the most frequent male malignancy and the second most common cause of cancer-related death in Western countries. Current clinical and pathological methods are limited in the prediction of postoperative outcome. It is becoming increasingly evident that small non-coding RNA (ncRNA) species are associated with the development and progression of this malignancy. To assess the diversity and abundance of small ncRNAs in PCa, we analyzed the composition of the entire small transcriptome by Illumina/Solexa deep sequencing. We further analyzed the microRNA (miRNA) expression signatures of 102 fresh-frozen patient samples during PCa progression by miRNA microarrays. Both platforms were cross-validated by quantitative reverse transcriptase-PCR. Besides the altered expression of several miRNAs, our deep sequencing analyses revealed strong differential expression of small nucleolar RNAs (snoRNAs) and transfer RNAs (tRNAs). From microarray analysis, we derived a miRNA diagnostic classifier that accurately distinguishes normal from cancer samples. Furthermore, we were able to construct a PCa prognostic predictor that independently forecasts postoperative outcome. Importantly, the majority of miRNAs included in the predictor also exhibit high sequence counts and concordant differential expression in Illumina PCa samples, supported by quantitative reverse transcriptase-PCR. Our findings provide miRNA expression signatures that may serve as an accurate tool for the diagnosis and prognosis of PCa. © 2012 Macmillan Publishers Limited All rights reserved.


Ergin B.,TU Munich | Meding S.,Helmholtz Center Munich | Langer R.,TU Munich | Kap M.,Josephine Nefkens Institute | And 7 more authors.
Journal of Proteome Research | Year: 2010

Formalin fixation and paraffin embedding is the standard technique for preserving biological material for both storage and histological analysis. Although recent progress has been made in the molecular analysis of formalin-fixed, paraffin-embedded (FFPE) tissues, proteomic applications are a special challenge due to the cross-linking property of formalin. Here we present the results of a new formalin-free tissue fixative, PAXgene, and demonstrate successful extraction of nondegraded and immunoreactive protein for subsequent standard protein assays, such as Western blot analysis and reverse-phase protein arrays. High amounts of protein can be obtained from PAXgene-fixed, paraffin-embedded (PFPE) mouse liver and human spleen, breast, duodenum, and stomach tissues, similar to frozen material. By Western blot analysis, we found that the detection of membrane, cytoplasmic, nuclear, and phosphorylated protein from PAXgene-fixed human tissue samples was comparable to cryopreserved samples. Furthermore, the distribution of protein in PAXgene-fixed human tissue specimens is adequate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry for in situ proteomic analysis. Taken together, we demonstrate here that PAXgene has great potential to serve as a novel multimodal fixative for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples. © 2010 American Chemical Society.


Mostert B.,Daniel Den Hoed Cancer Center | Mostert B.,Netherlands Cancer Institute | Kraan J.,Daniel Den Hoed Cancer Center | Bolt-De Vries J.,Netherlands Cancer Institute | And 10 more authors.
Breast Cancer Research and Treatment | Year: 2011

Most assays to detect circulating tumor cells (CTCs) rely on EpCAM expression on tumor cells. Recently, our group reported that in contrast to other molecular breast cancer subtypes, "normal-like" cell lines lack EpCAM expression and are thus missed when CTCs are captured with EpCAM-based technology [J Natl Cancer Inst 101(1):61-66, 2009]. Here, the use of CD146 is introduced to detect EpCAM-negative CTCs, thereby improving CTC detection. CD146 and EpCAM expression were assessed in our panel of 41 breast cancer cell lines. Cells from 14 cell lines, 9 of which normal-like, were spiked into healthy donor blood. Using CellSearch™ technology, 7.5 ml whole blood was enriched for CTCs by adding ferrofluids loaded with antibodies against EpCAM and/or CD146 followed by staining for Cytokeratin and DAPI. Hematopoietic cells and circulating endothelial cells (CECs) were counterstained with CD45 and CD34, respectively. A similar approach was applied for blood samples of 20 advanced breast cancer patients. Eight of 9 normal-like breast cancer cell lines lacked EpCAM expression but did express CD146. Five of these 8 could be adequately recovered by anti-CD146 ferrofluids. Of 20 advanced breast cancer patients whose CTCs were enumerated with anti-EpCAM and anti-CD146 ferrofluids, 9 had CD146+ CTCs. Cells from breast cancer cell lines that lack EpCAM expression frequently express CD146 and can be recovered by anti-CD146 ferrofluids. CD146+ CTCs are present in the peripheral blood of breast cancer patients with advanced disease. Combined use of anti-CD146 and anti-EpCAM is likely to improve CTC detection in breast cancer patients. © 2010 Springer Science+Business Media, LLC.


Kompier L.C.,Josephine Nefkens Institute | van Tilborg A.A.G.,Josephine Nefkens Institute | Zwarthoff E.C.,Josephine Nefkens Institute
Urologic Oncology: Seminars and Original Investigations | Year: 2010

Bladder cancer (BC) comes in two flavors: as non-muscle invasive (NMI) and as muscle invasive (MI) disease. These two subtypes differ in their genetic aberrations. In NMI-BC mutations in the FGFR3 oncogene are found with a frequency of 75%, whereas mutations in the TP53 tumor suppressor gene prevail in MI-BC. Mutations in the RAS genes occur in 15% of BC of all stages and are mutually exclusive with FGFR3 mutations. Mutations in the PIK3CA gene are found in about 13% and these almost exclusively co-occur with FGFR3 mutations. NMI-BC with FGFR3 mutations are genetically stable, but FGFR3 wild type NMI-BC and MI tumors are genetically unstable. In this paper, we discuss the use of these genetic aberrations in relation to recurrence, progression, surveillance, and therapeutic options. As of yet, there is no biomarker that can predict recurrences or the rate of recurrences when they occur. We show that FGFR3 mutations are associated with a decreased risk of progression, and a better survival both in BC and in upper urinary tract cancer. Microsatellite analysis (MA) in order to detect loss-of-heterozygosity can be used to detect recurrences in urinary cells of patients under surveillance. The results of a Dutch randomized trial show that consecutive positive MA results are a strong predictor for future recurrences. Using FGFR3 mutation analysis for those patients who have an FGFR3 mutant tumor will enhance performance of urine-based surveillance. Although FGFR3 mutations occur in only 20% of MI tumors, these tumors often have a high expression of the FGFR3 protein. This suggests that this receptor could present a target for adjuvant therapy in MI-BC. However, whether the FGFR3 pathway is active in these tumors remains to be established. © 2010 Elsevier Inc. All rights reserved.


Roth S.,Josephine Nefkens Institute | Franken P.,Josephine Nefkens Institute | Monkhorst K.,Josephine Nefkens Institute | Kong A San J.,Erasmus University Rotterdam | Fodde R.,Josephine Nefkens Institute
BMC Developmental Biology | Year: 2012

Background: To facilitate the in vivo study of esophageal (stem) cell biology in homeostasis and cancer, novel mouse models are necessary to elicit expression of candidate genes in a tissue-specific and inducible fashion. To this aim, we developed and studied a mouse model to allow labeling of esophageal cells with the histone 2B-GFP (H2B-GFP) fusion protein. Results: First, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the promoter (ED-L2) of the Epstein-Barr virus (EBV) gene encoding the latent membrane protein-1 (LMP-1). The newly generated ED-L2-rtTA2-M2 (ED-L2-rtTA) mice were then bred with the previously developed tetO-HIST1H2BJ/GFP (tetO-H2B-GFP) model to assess inducibility and tissue-specificity. Expression of the H2B-GFP fusion protein was observed upon doxycycline induction but was restricted to the terminally differentiated cells above the basal cell layer. To achieve expression in the basal compartment of the esophagus, we ubsequently employed a different transgenic model expressing the reverse transactivator rtTA2S-M2 under the control of the ubiquitous, methylation-free CpG island of the human hnRNPA2B1-CBX3 gene (hnRNP-rtTA). Upon doxycycline administration to the compound hnRNP-rtTA/tetO-H2B-GFP mice, near-complete labeling of all esophageal cells was achieved. Pulse-chase experiments confirmed that complete turnover of the esophageal epithelium in the adult mouse is achieved within 710 days. Conclusions: We show that the esophagus-specific promoter ED-L2 is expressed only in the differentiated cells above the basal layer. oreover, we confirmed that esophageal turn-over in the adult mouse does not exceed 710 days. © 2012 Roth et al.; licensee BioMed Central Ltd.


Oosterhuis J.W.,Josephine Nefkens Institute | Stoop H.,Josephine Nefkens Institute | Dohle G.,Erasmus Medical Center | Boellaard W.,Erasmus Medical Center | And 3 more authors.
International Journal of Andrology | Year: 2011

Aspects of the biopsy of the testis from the pathologist's point of view are discussed. Direct enzyme-histochemical staining for alkaline phosphatase (dAP) on frozen sections of biopsies taken during operation is a useful diagnostic tool to aid surgeons in testis-sparing surgery. Biopsy of the contralateral testis for the diagnosis of carcinoma in situ (CIS) in patients with a testicular germ cell tumour is not standard of care in most countries because of the high rate of negative biopsies. Based on risk factors for germ cell tumours, i.p. microlithiasis, a patient population is defined in which the rate of CIS in the contralateral biopsy is about 25%. It is reiterated that the diagnosis of CIS in testicular biopsies requires expertise, and should not be carried out without immunohistochemistry for markers for CIS. As OCT3/4 is increasingly used as marker, it is important to be aware that it may be false-negative in biopsies fixed in Bouin's or Stieve's fixative. Preliminary results are presented on a series of biopsies from cryptorchid testes in infants and children allowing the definition of morphological and immunohistochemical criteria for delayed maturation of gonocytes and pre-CIS. © 2011 The Authors. International Journal of Andrology © 2011 European Academy of Andrology.


Marques R.B.,Josephine Nefkens Institute | Dits N.F.,Josephine Nefkens Institute | Erkens-Schulze S.,Josephine Nefkens Institute | van IJcken W.F.J.,Erasmus Medical Center | And 2 more authors.
PLoS ONE | Year: 2011

Background: Prostate epithelial cells depend on androgens for survival and function. In (early) prostate cancer (PCa) androgens also regulate tumor growth, which is exploited by hormonal therapies in metastatic disease. The aim of the present study was to characterize the androgen receptor (AR) response in hormonal therapy-resistant PC346 cells and identify potential disease markers. Methodology/Principal Findings: Human 19K oligoarrays were used to establish the androgen-regulated expression profile of androgen-responsive PC346C cells and its derivative therapy-resistant sublines: PC346DCC (vestigial AR levels), PC346Flu1 (AR overexpression) and PC346Flu2 (T877A AR mutation). In total, 107 transcripts were differentially-expressed in PC346C and derivatives after R1881 or hydroxyflutamide stimulations. The AR-regulated expression profiles reflected the AR modifications of respective therapy-resistant sublines: AR overexpression resulted in stronger and broader transcriptional response to R1881 stimulation, AR down-regulation correlated with deficient response of AR-target genes and the T877A mutation resulted in transcriptional response to both R1881 and hydroxyflutamide. This AR-target signature was linked to multiple publicly available cell line and tumor derived PCa databases, revealing that distinct functional clusters were differentially modulated during PCa progression. Differentiation and secretory functions were up-regulated in primary PCa but repressed in metastasis, whereas proliferation, cytoskeletal remodeling and adhesion were overexpressed in metastasis. Finally, the androgen-regulated genes ENDOD1, MCCC2 and ACSL3 were selected as potential disease markers for RT-PCR quantification in a distinct set of human prostate specimens. ENDOD1 and ACSL3 showed down-regulation in high-grade and metastatic PCa, while MCCC2 was overexpressed in low-grade PCa. Conclusions/Significance: AR modifications altered the transcriptional response to (anti)androgens in therapy-resistant cells. Furthermore, selective down-regulation of genes involved in differentiation and up-regulation of genes promoting proliferation and invasion suggest a disturbed balance between the growth and differentiation functions of the AR pathway during PCa progression. These findings may have implications in the current treatment and development of novel therapeutical approaches for metastatic PCa. © 2011 Marques et al.


Buxa M.K.,Justus Liebig University | Slotman J.A.,Josephine Nefkens Institute | Van Royen M.E.,Josephine Nefkens Institute | Paul M.W.,Josephine Nefkens Institute | And 2 more authors.
Biology Open | Year: 2016

Nuclear foci of chromatin binding factors are, in many cases, discussed as sites of long-range chromatin interaction in the threedimensional nuclear space. Insulator binding proteins have been shown to aggregate into insulator bodies, which are large structures not involved in insulation; however, the more diffusely distributed insulator speckles have not been analysed in this respect. Furthermore, insulator binding proteins have been shown to drive binding sites for Polycomb group proteins into Polycomb bodies. Here we find that insulator speckles, marked by the insulator binding protein dCTCF, and Polycomb bodies show differential association with the insulator protein CP190. They differ in number and threedimensional location with only 26% of the Polycomb bodies overlapping with CP190. By using fluorescence in situ hybridization (FISH) probes to identify long-range interaction (kissing) of the Hox gene clusters Antennapedia complex (ANT-C) and Bithorax complex (BX-C), we found the frequency of interaction to be very low. However, these rare kissing events were associated with insulator speckles at a significantly shorter distance and an increased speckle number. This suggests that insulator speckles are associated with long-distance interaction. © 2016. Published by The Company of Biologists Ltd.


Duijvesz D.,Erasmus Medical Center | Luider T.,Erasmus Medical Center | Bangma C.H.,Erasmus Medical Center | Jenster G.,Erasmus Medical Center | Jenster G.,Josephine Nefkens Institute
European Urology | Year: 2011

Context: Although progress has been made with regard to types of markers (protein, DNA, RNA, and metabolites) and implementation of improved technologies (mass spectrometry, arrays, and deep sequencing), the discovery of novel biomarkers for prostate cancer (PCa) in complex fluids, such as serum and urine, remains a challenge. Meanwhile, recent studies have reported that many cancer-derived proteins and RNAs are secreted through small vesicles known as exosomes. Objective: This narrative review describes recent progress in exosome research, focusing on the potential role of exosomes as novel biomarkers for PCa. The purpose of this review is to acquaint clinicians and researchers in the field of urology with the potential role of exosomes as biomarker treasure chests and with their clinical value. Evidence acquisition: Medline and Embase entries between 1966 and September 2010 were searched using the keywords exosomes, microvesicles, prostasomes, biomarkers, prostate cancer, and urology. Leading publications and articles constructively contributing to exosome research were selected for this review. Evidence synthesis: Exosomes are small vesicles (50-100 nm) secreted by almost all tissues; they represent their tissue origin. Purification of prostate- and PCa-derived exosomes will allow us to profile exosomes, providing a promising source of protein and RNA biomarkers for PCa. This profiling will contribute to the discovery of novel markers for the early diagnosis and reliable prognosis of PCa. Conclusions: Although the initial results are promising, further investigations are required to assess the clinical value of these exosomes in PCa. © 2010 European Association of Urology.

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