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Leendert H. J. Looijenga - One of the best experts on this subject based on the ideXlab platform.
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cripto expression epigenetic regulation and potential diagnostic use in testicular germ cell tumors
Molecular Oncology, 2016Co-Authors: Cassy M Spiller, Josephine Bowles, Guillaume Burnet, Hans Stoop, Peter Koopman, Ad J. M. Gillis, Leendert H. J. LooijengaAbstract:Type II germ cell tumors arise after puberty from a germ cell that was incorrectly programmed during fetal life. Failure of testicular germ cells to properly differentiate can lead to the formation of germ cell neoplasia in situ of the testis; this precursor cell invariably gives rise to germ cell cancer after puberty. The Nodal co-receptor Cripto is expressed transiently during normal germ cell development and is ectopically expressed in non-Seminomas that arise from germ cell neoplasia in situ, suggesting that its aberrant expression may underlie germ cell dysregulation and hence germ cell cancer. Here we investigated methylation of the Cripto promoter in mouse germ cells and human germ cell cancer and correlated this with the level of CRIPTO protein expression. We found hypomethylation of the CRIPTO promoter in undifferentiated fetal germ cells, embryonal carcinoma and Seminomas, but hypermethylation in differentiated fetal germ cells and the differentiated types of non-Seminomas. CRIPTO protein was strongly expressed in germ cell neoplasia in situ along with embryonal carcinoma, yolk sac tumor and Seminomas. Further, cleaved CRIPTO was detected in media from Seminoma and embryonal carcinoma cell lines, suggesting that cleaved CRIPTO may provide diagnostic indication of germ cell cancer. Accordingly, CRIPTO was detectable in serum from 6/15 patients with embryonal carcinoma, 5/15 patients with Seminoma, 4/5 patients with germ cell neoplasia in situ cells only and in 1/15 control patients. These findings suggest that CRIPTO expression may be a useful serological marker for diagnostic and/or prognostic purposes during germ cell cancer management.
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Diagnosis of testicular carcinoma in situ, (intratubular- and micro-invasive) Seminoma and embryonal carcinoma using direct enzymatic alkaline phosphatase reactivity on frozen histological sections.
Histopathology, 2011Co-Authors: Hans Stoop, A. D. Gillis, J. Wolter Oosterhuis, Katharina Biermann, Wim Kirkels, Gert Dohle, Michael Den Bakker, Leendert H. J. LooijengaAbstract:Aims: Testis-sparing surgery might benefit quality of life, but can only be applied with histological examination for Seminoma and embryonal carcinoma, and carcinoma in situ (CIS). Diagnosis is based on paraffin-embedded tissue, therefore a delay in further surgery is mostly unavoidable. Methods and Results: A total of 4,093 snap frozen samples and paraffin tissue of 1,500 patients were included. Besides standard H & e staining, the direct enzymatic alkaline phosphatase reactivity (dAP) test (duration 15 minutes). Endothelial cells served as control for the dAP test. Positive staining was found in CIS (n = 965), Seminoma (n = 1035), embryonal carcinoma (n = 584), either intratubular. Differentiated non-Seminomatas (n = 1238) showed variable staining. No staining was identified in spermatocytic Seminomas (n = 5), testicular lymphomas (n = 42), testicular rhabdomyosarcomas (n = 7), Leydig cell tumors (n = 31), Sertoli-cell-only nodules (n = 4), (epi)dermoid cyst (n = 16), normal testicular parenchyma (n = 116), testicular torsion (n = 32) and inflammation of the epididymis (n = 19). Conclusion: The dAP test is informative, reproducible, and easy tool to diagnose CIS, Seminoma and embryonal carcinoma on frozen tissue sections, being of great value in the context of sparing surgery.
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Genome-wide BAC array-CGH and expression profiling of human Seminomas, dysgerminomas, embryonal carcinomas and spermatocytic Seminomas
Cancer Research, 2005Co-Authors: Leendert H. J. Looijenga, Hans Stoop, A. D. Gillis, Remco Hersmus, Wolter OosterhuisAbstract:1650 Human germ cell tumors (GCTs) comprise various entities, which are characterized by age of the patient at clinical presentation, clinical behavior, anatomical location, histology and chromosomal constitution of the tumor. Within the testis, Seminomas and nonSeminomas (including the stem cell component embryonal carcinoma) of adolescents and adults can be identified, as well as spermatocytic Seminoma of elderly. In the ovary, the Seminoma-like tumors are known as dysgerminoma. We performed a genome wide screen for genomic imbalances using 3.7 and 32 K BAC-array CGH on testicular Seminomas (4), embryonal carcinomas (4), spermatocytic Seminomas (5), ovarian dysgerminomas (n=4) and embryonal carcinoma (1). Known changes were identified, like gain of 7, 8, 12p and X and loss of 4, 5, and 13 in Seminoma/dysgerminoma and embryonal carcinoma, as well as a consistent gain of chromosome 9 in spermatocytic Seminoma. The shortest regions of overlap (SRO) could be identified, and novel gains and losses were found, including a restricted high-level amplification of 9p in a spermatocytic Seminoma. This genomic profile was combined with data from a genome-wide expression analysis using Affymetrix U133 Plus 2.0 arrays. Hierarchical clustering (Omniviz, after K-mean analyses, 2.75 fold expression difference) based on 1354 genes dividing the subtypes of tumors correctly. SAM analyses showed no differences between Seminoma and dysgerminoma. In contrast, these tumors were significantly different from embryonal carcinomas (241 positively and 6 negatively correlated genes) and spermatocytic Seminomas (150 positively and 235 negatively correlated genes). These included previously identified diagnostic markers, like KRT8/18, BCAT1, GDF3, c-KIT, NANOG, OCT3/4 and SSX. In addition, novel differentiating genes were identified, like C6orf148, MST4, TNFRSF1A, GAGE4/8, PARD, PRSS21, and TUBA. Moreover, strong candidate genes for the SRO were identified, in particular for the high level amplification on 9p in spermatocytic Seminoma. Ingenuity analysis revealed the pathways in with these genes act in the different tumor types, like cell cycle control, cell morphology etc. This combined high through-put genomic and expression investigation on a limited set of well-defined tumors allowed identification of chromosomal regions, genes and pathways involved in the development of the cancer. This data set is informative to identify diagnostic markers, as well as determine the cell of origin of these tumors.
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pou5f1 oct3 4 identifies cells with pluripotent potential in human germ cell tumors
Cancer Research, 2003Co-Authors: Leendert H. J. Looijenga, Hans Stoop, Ad J. M. Gillis, Hubert P J C De Leeuw, Carlos A De Gouveia Brazao, Kees Van Roozendaal, Everardus J J Van Zoelen, Rob F A Weber, Katja P Wolffenbuttel, Herman Van DekkenAbstract:Human germ cell tumors (GCTs) may have variable histology and clinical behavior, depending on factors such as sex of the patient, age at clinical diagnosis, and anatomical site of the tumor. Some types of GCT, i.e., the Seminomas/germinomas/dysgerminomas and embryonal carcinomas (the stem cell component of nonSeminomas), have pluripotent potential, which is demonstrated by their capacity to differentiate into somatic and/or extraembryonic elements. Although embryonal carcinoma cells are intrinsically pluripotent, Seminoma/germinoma/dysgerminoma cells, as well as their precursor carcinoma in situ/gonadoblastoma cells, have the phenotype of early germ cells that can be activated to pluripotency. The other types of GCT (teratomas and yolk sac tumors of infants and newborn, dermoid cyst of the ovary, and spermatocytic Seminoma of elderly) are composed of (fully) differentiated tissues and lack the appearance of undifferentiated and pluripotent stem cells. OCT3/4, a transcription factor also known as OTF3 and POU5F1, is involved in regulation of pluripotency during normal development and is detectable in embryonic stem and germ cells. We analyzed the presence of POU5F1 in GCT and other tumor types using immunohistochemistry. The protein was consistently detected in carcinoma in situ/gonadoblastoma, Seminomas/germinoma/dysgerminoma, and embryonal carcinoma but not in the various types of differentiated nonSeminomas. Multitumor tissue microarray analysis covering >100 different tumor categories and 3600 individual cancers verified that POU5F1 expression is specific for particular subtypes of GCT of adults. No protein was observed in GCT of newborn and infants, spermatocytic Seminomas, and the various tumors of nongerm cell origin. In addition, no difference in staining pattern was found in chemosensitive and chemoresistant GCT of adults. These results indicate preservation of the link between POU5F1 and pluripotency, as reported during normal development, after malignant transformation. Therefore, POU5F1 immunohistochemistry is an informative diagnostic tool for pluripotent GCT and offers new insights into the histological heterogeneity of this cancer.
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reactivity of germ cell maturation stage specific markers in spermatocytic Seminoma diagnostic and etiological implications
Laboratory Investigation, 2001Co-Authors: Hans Stoop, Wolter Oosterhuis, Ruud J.h.l.m. Van Gurp, Ronald R De Krijger, Ad Geurts Van Kessel, Beate Koberle, Leendert H. J. LooijengaAbstract:It is generally accepted that testicular Seminomas and spermatocytic Seminomas have separate pathogeneses, although the origin of these two types of germ cell tumors of the adult testis remains a matter of debate. Although an embryonic germ cell origin seems to be most likely for Seminomas, a spermatogonia-spermatocyte origin has been suggested for spermatocytic Seminoma. To shed more light on the etiology of spermatocytic Seminomas, we undertook an immunohistochemical and molecular approach using SCP1 (synaptonemal complex protein 1), SSX (synovial sarcoma on X chromosome), and XPA (xeroderma pigmentosum type A) as targets. Although a stage-specific expression pattern has been reported for SCP1 and SSX in normal spermatogenesis, we demonstrate here that it also exists for XPA. In fact, immunohistochemistry shows that the proteins of SCP1 and XPA are specifically present in the stage of primary and pachytene spermatocytes. In contrast, SSX was found in spermatogonia and primary spermatocytes, as well as in germ cells, from at least the 17th week of intrauterine development onward. Although no protein encoded by any of these genes was detected in tumor cells of a series of testicular Seminomas, all tested spermatocytic Seminomas were positive, in agreement with expression analysis. These data support the model that Seminomas originate from an embryonic germ cell, and they imply that the cell of origin of spermatocytic Seminomas is at least capable of maturing to the stage of spermatogonia-pachytene spermatocyte.
Hans Stoop - One of the best experts on this subject based on the ideXlab platform.
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cripto expression epigenetic regulation and potential diagnostic use in testicular germ cell tumors
Molecular Oncology, 2016Co-Authors: Cassy M Spiller, Josephine Bowles, Guillaume Burnet, Hans Stoop, Peter Koopman, Ad J. M. Gillis, Leendert H. J. LooijengaAbstract:Type II germ cell tumors arise after puberty from a germ cell that was incorrectly programmed during fetal life. Failure of testicular germ cells to properly differentiate can lead to the formation of germ cell neoplasia in situ of the testis; this precursor cell invariably gives rise to germ cell cancer after puberty. The Nodal co-receptor Cripto is expressed transiently during normal germ cell development and is ectopically expressed in non-Seminomas that arise from germ cell neoplasia in situ, suggesting that its aberrant expression may underlie germ cell dysregulation and hence germ cell cancer. Here we investigated methylation of the Cripto promoter in mouse germ cells and human germ cell cancer and correlated this with the level of CRIPTO protein expression. We found hypomethylation of the CRIPTO promoter in undifferentiated fetal germ cells, embryonal carcinoma and Seminomas, but hypermethylation in differentiated fetal germ cells and the differentiated types of non-Seminomas. CRIPTO protein was strongly expressed in germ cell neoplasia in situ along with embryonal carcinoma, yolk sac tumor and Seminomas. Further, cleaved CRIPTO was detected in media from Seminoma and embryonal carcinoma cell lines, suggesting that cleaved CRIPTO may provide diagnostic indication of germ cell cancer. Accordingly, CRIPTO was detectable in serum from 6/15 patients with embryonal carcinoma, 5/15 patients with Seminoma, 4/5 patients with germ cell neoplasia in situ cells only and in 1/15 control patients. These findings suggest that CRIPTO expression may be a useful serological marker for diagnostic and/or prognostic purposes during germ cell cancer management.
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Mutations in LRRC50 predispose zebrafish and humans to Seminomas.
PLoS genetics, 2013Co-Authors: Sander G. Basten, Hans Stoop, Ad J. M. Gillis, Erica E. Davis, Ellen Van Rooijen, Nikolina Babala, Ive Logister, Zachary G. Heath, Trudy N. Jonges, Nicholas KatsanisAbstract:Seminoma is a subclass of human testicular germ cell tumors (TGCT), the most frequently observed cancer in young men with a rising incidence. Here we describe the identification of a novel gene predisposing specifically to Seminoma formation in a vertebrate model organism. Zebrafish carrying a heterozygous nonsense mutation in Leucine-Rich Repeat Containing protein 50 (lrrc50 also called dnaaf1), associated previously with ciliary function, are found to be highly susceptible to the formation of Seminomas. Genotyping of these zebrafish tumors shows loss of heterozygosity (LOH) of the wild-type lrrc50 allele in 44.4% of tumor samples, correlating with tumor progression. In humans we identified heterozygous germline LRRC50 mutations in two different pedigrees with a family history of Seminomas, resulting in a nonsense Arg488* change and a missense Thr590Met change, which show reduced expression of the wild-type allele in Seminomas. Zebrafish in vivo complementation studies indicate the Thr590Met to be a loss-of-function mutation. Moreover, we show that a pathogenic Gln307Glu change is significantly enriched in individuals with Seminoma tumors (13% of our cohort). Together, our study introduces an animal model for Seminoma and suggests LRRC50 to be a novel tumor suppressor implicated in human Seminoma pathogenesis.
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Diagnosis of testicular carcinoma in situ, (intratubular- and micro-invasive) Seminoma and embryonal carcinoma using direct enzymatic alkaline phosphatase reactivity on frozen histological sections.
Histopathology, 2011Co-Authors: Hans Stoop, A. D. Gillis, J. Wolter Oosterhuis, Katharina Biermann, Wim Kirkels, Gert Dohle, Michael Den Bakker, Leendert H. J. LooijengaAbstract:Aims: Testis-sparing surgery might benefit quality of life, but can only be applied with histological examination for Seminoma and embryonal carcinoma, and carcinoma in situ (CIS). Diagnosis is based on paraffin-embedded tissue, therefore a delay in further surgery is mostly unavoidable. Methods and Results: A total of 4,093 snap frozen samples and paraffin tissue of 1,500 patients were included. Besides standard H & e staining, the direct enzymatic alkaline phosphatase reactivity (dAP) test (duration 15 minutes). Endothelial cells served as control for the dAP test. Positive staining was found in CIS (n = 965), Seminoma (n = 1035), embryonal carcinoma (n = 584), either intratubular. Differentiated non-Seminomatas (n = 1238) showed variable staining. No staining was identified in spermatocytic Seminomas (n = 5), testicular lymphomas (n = 42), testicular rhabdomyosarcomas (n = 7), Leydig cell tumors (n = 31), Sertoli-cell-only nodules (n = 4), (epi)dermoid cyst (n = 16), normal testicular parenchyma (n = 116), testicular torsion (n = 32) and inflammation of the epididymis (n = 19). Conclusion: The dAP test is informative, reproducible, and easy tool to diagnose CIS, Seminoma and embryonal carcinoma on frozen tissue sections, being of great value in the context of sparing surgery.
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Genome-wide BAC array-CGH and expression profiling of human Seminomas, dysgerminomas, embryonal carcinomas and spermatocytic Seminomas
Cancer Research, 2005Co-Authors: Leendert H. J. Looijenga, Hans Stoop, A. D. Gillis, Remco Hersmus, Wolter OosterhuisAbstract:1650 Human germ cell tumors (GCTs) comprise various entities, which are characterized by age of the patient at clinical presentation, clinical behavior, anatomical location, histology and chromosomal constitution of the tumor. Within the testis, Seminomas and nonSeminomas (including the stem cell component embryonal carcinoma) of adolescents and adults can be identified, as well as spermatocytic Seminoma of elderly. In the ovary, the Seminoma-like tumors are known as dysgerminoma. We performed a genome wide screen for genomic imbalances using 3.7 and 32 K BAC-array CGH on testicular Seminomas (4), embryonal carcinomas (4), spermatocytic Seminomas (5), ovarian dysgerminomas (n=4) and embryonal carcinoma (1). Known changes were identified, like gain of 7, 8, 12p and X and loss of 4, 5, and 13 in Seminoma/dysgerminoma and embryonal carcinoma, as well as a consistent gain of chromosome 9 in spermatocytic Seminoma. The shortest regions of overlap (SRO) could be identified, and novel gains and losses were found, including a restricted high-level amplification of 9p in a spermatocytic Seminoma. This genomic profile was combined with data from a genome-wide expression analysis using Affymetrix U133 Plus 2.0 arrays. Hierarchical clustering (Omniviz, after K-mean analyses, 2.75 fold expression difference) based on 1354 genes dividing the subtypes of tumors correctly. SAM analyses showed no differences between Seminoma and dysgerminoma. In contrast, these tumors were significantly different from embryonal carcinomas (241 positively and 6 negatively correlated genes) and spermatocytic Seminomas (150 positively and 235 negatively correlated genes). These included previously identified diagnostic markers, like KRT8/18, BCAT1, GDF3, c-KIT, NANOG, OCT3/4 and SSX. In addition, novel differentiating genes were identified, like C6orf148, MST4, TNFRSF1A, GAGE4/8, PARD, PRSS21, and TUBA. Moreover, strong candidate genes for the SRO were identified, in particular for the high level amplification on 9p in spermatocytic Seminoma. Ingenuity analysis revealed the pathways in with these genes act in the different tumor types, like cell cycle control, cell morphology etc. This combined high through-put genomic and expression investigation on a limited set of well-defined tumors allowed identification of chromosomal regions, genes and pathways involved in the development of the cancer. This data set is informative to identify diagnostic markers, as well as determine the cell of origin of these tumors.
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pou5f1 oct3 4 identifies cells with pluripotent potential in human germ cell tumors
Cancer Research, 2003Co-Authors: Leendert H. J. Looijenga, Hans Stoop, Ad J. M. Gillis, Hubert P J C De Leeuw, Carlos A De Gouveia Brazao, Kees Van Roozendaal, Everardus J J Van Zoelen, Rob F A Weber, Katja P Wolffenbuttel, Herman Van DekkenAbstract:Human germ cell tumors (GCTs) may have variable histology and clinical behavior, depending on factors such as sex of the patient, age at clinical diagnosis, and anatomical site of the tumor. Some types of GCT, i.e., the Seminomas/germinomas/dysgerminomas and embryonal carcinomas (the stem cell component of nonSeminomas), have pluripotent potential, which is demonstrated by their capacity to differentiate into somatic and/or extraembryonic elements. Although embryonal carcinoma cells are intrinsically pluripotent, Seminoma/germinoma/dysgerminoma cells, as well as their precursor carcinoma in situ/gonadoblastoma cells, have the phenotype of early germ cells that can be activated to pluripotency. The other types of GCT (teratomas and yolk sac tumors of infants and newborn, dermoid cyst of the ovary, and spermatocytic Seminoma of elderly) are composed of (fully) differentiated tissues and lack the appearance of undifferentiated and pluripotent stem cells. OCT3/4, a transcription factor also known as OTF3 and POU5F1, is involved in regulation of pluripotency during normal development and is detectable in embryonic stem and germ cells. We analyzed the presence of POU5F1 in GCT and other tumor types using immunohistochemistry. The protein was consistently detected in carcinoma in situ/gonadoblastoma, Seminomas/germinoma/dysgerminoma, and embryonal carcinoma but not in the various types of differentiated nonSeminomas. Multitumor tissue microarray analysis covering >100 different tumor categories and 3600 individual cancers verified that POU5F1 expression is specific for particular subtypes of GCT of adults. No protein was observed in GCT of newborn and infants, spermatocytic Seminomas, and the various tumors of nongerm cell origin. In addition, no difference in staining pattern was found in chemosensitive and chemoresistant GCT of adults. These results indicate preservation of the link between POU5F1 and pluripotency, as reported during normal development, after malignant transformation. Therefore, POU5F1 immunohistochemistry is an informative diagnostic tool for pluripotent GCT and offers new insights into the histological heterogeneity of this cancer.
Hubert Schorle - One of the best experts on this subject based on the ideXlab platform.
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TCam-2 Cells Deficient for SOX2 and FOXA2 are Blocked in Differentiation and Maintain a Seminoma-Like Cell Fate In Vivo
2019Co-Authors: Daniel Nettersheim, Sina Jostes, Alena Heimsoeth, Saskia Vadder, Hubert SchorleAbstract:Testicular germ cell tumors (GCTs) are very common in young men and can be stratified into Seminomas and non-Seminomas. While Seminomas share a similar gene expression and epigenetic profile with primordial germ cells, the stem cell population of the non-Seminomas, the embryonal carcinoma (EC), resembles malignant embryonic stem cells. Thus, ECs are able to differentiate into cells of all three germ layers (teratomas) and even extra-embryonic-tissue-like cells (yolk-sac tumor, choriocarcinoma). In the last years, we demonstrated that the cellular microenvironment considerably influences the plasticity of Seminomas (TCam-2 cells). Upon a microenvironment-triggered inhibition of the BMP signaling pathway in vivo (murine flank or brain), Seminomatous TCam-2 cells reprogram to an EC-like cell fate. We identified SOX2 as a key factor activated upon BMP inhibition mediating the reprogramming process by regulating pluripotency, reprogramming and epigenetic factors. Indeed, CRISPR/Cas9 SOX2-deleted TCam-2 cells were able to maintain a Seminoma-cell fate in vivo for about six weeks, but after six weeks in vivo still small sub-populations initiated differentiation. Closer analyses of these differentiated clusters suggested that the pioneer factor FOXA2 might be the driving force behind this induction of differentiation, since many FOXA2 interacting genes and differentiation factors like AFP, EOMES,CDX1, ALB, HAND1, DKK, DLK1,MSX1and PITX2were upregulated. In this study, we generated TCam-2 cells double-deficient for SOX2and FOXA2using the CRISPR/Cas9 technique and xenografted those cells into the flank of nude mice. Upon loss of SOX2 and FOXA2, TCam-2 maintained a Seminoma cell fate for at least twelve weeks, demonstrating that both factors are key players in the reprogramming to an EC-like cell fate. Therefore, our study adds an important piece to the puzzle of GCT development and plasticity, providing interesting insights in what can be expected in a patient, when GCT cells are confronted with different microenvironments
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The plasticity of germ cell cancers and its dependence on the cellular microenvironment.
Journal of Cellular and Molecular Medicine, 2017Co-Authors: Daniel Nettersheim, Hubert SchorleAbstract:: So far, the understanding of germ cell cancer (GCC) pathogenesis is based on a model, where Seminomas and non-Seminomas represent distinct entities although originating from a common precursor termed germ cell neoplasia in situ (GCNIS). Embryonal carcinomas (ECs), the stem cell population of the non-Seminomas, is pluri- to totipotent and able to differentiate into cells of all three germ layers, giving rise to teratomas or tumours mimicking extraembryonic tissues (yolk sac tumours, choriocarcinomas). With regard to gene expression, (epi)genetics and histology, Seminomas are highly similar to GCNIS and primordial germ cells, but limited in development. It remains elusive, whether this block in differentiation is controlled by cell intrinsic mechanisms or by signals from the surrounding microenvironment. Here, we reviewed the recent literature emphasizing the plasticity of GCCs, especially of Seminomas. We propose that this plasticity is controlled by the microenvironment, allowing Seminomas to transit into an EC or mixed non-Seminoma and vice versa. We discuss several mechanisms and routes of reprogramming that might be responsible for this change in the cell fate. We finally integrate this plasticity into a new model of GCC pathogenesis, allowing for an alternative view on the dynamics of GCC development and progression.
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sox2 is essential for in vivo reprogramming of Seminoma like tcam 2 cells to an embryonal carcinoma like fate
Oncotarget, 2016Co-Authors: Daniel Nettersheim, Sina Jostes, Simon Schneider, Andrea Hofmann, Alena Heimsoeth, Martin Fellermeyer, Hubert SchorleAbstract:// Daniel Nettersheim 1 , Alena Heimsoeth 1 , Sina Jostes 1 , Simon Schneider 1 , Martin Fellermeyer 1 , Andrea Hofmann 2 , Hubert Schorle 1 1 Institute of Pathology, Department of Developmental Pathology, University of Bonn Medical School, Bonn, Germany 2 Institute of Human Genetics, University of Bonn Medical School, Bonn, Germany Correspondence to: Hubert Schorle, email: Hubert.Schorle@ukb.uni-bonn.de Keywords: TCam-2, Seminoma, embryonal carcinoma, reprogramming, SOX2/SOX17 Received: March 22, 2016 Accepted: May 19, 2016 Published: June 07, 2016 ABSTRACT Type II germ cell cancers (GCC) are divided into Seminomas, which are highly similar to primordial germ cells and embryonal carcinomas (EC), often described as malignant counterparts to embryonic stem cells. Previously, we demonstrated that the development of GCCs is a highly plastic process and strongly influenced by the microenvironment. While orthotopic transplantation into the testis promotes Seminomatous growth of the Seminoma-like cell line TCam-2, ectopic xenotransplantation into the flank initiates reprogramming into an EC-like fate. During this reprogramming, BMP signaling is inhibited, leading to induction of NODAL signaling, upregulation of pluripotency factors and downregulation of Seminoma markers, like SOX17. The pluripotency factor and EC-marker SOX2 is strongly induced. Here, we adressed the molecular role of SOX2 in this reprogramming. Using CRISPR/Cas9-mediated genome-editing, we established SOX2-deficient TCam-2 cells. Xenografting of SOX2-deficient cells into the flank of nude mice resulted in maintenance of a Seminoma-like fate, indicated by the histology and expression of OCT3/4, SOX17, TFAP2C, PRDM1 and PRAME . In SOX2-deficient cells, BMP signaling is inhibited, but NODAL signaling is not activated. Thus, SOX2 appears to be downstream of BMP signaling but upstream of NODAL activation. So, SOX2 is an essential factor in acquiring an EC-like cell fate from Seminomas. A small population of differentiated cells was identified resembling a mixed non-Seminoma. Analyses of these cells revealed downregulation of the pluripotency and Seminoma markers OCT3/4, SOX17, PRDM1 and TFAP2C. In contrast, the pioneer factor FOXA2 and its target genes were upregulated, suggesting that FOXA2 might play an important role in induction of non-Seminomatous differentiation.
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bmp inhibition in Seminomas initiates acquisition of pluripotency via nodal signaling resulting in reprogramming to an embryonal carcinoma
PLOS Genetics, 2015Co-Authors: Daniel Nettersheim, Sina Jostes, Rakesh Sharma, Simon Schneider, Andrea Hofmann, Humberto J Ferreira, Per Hoffmann, Glen Kristiansen, Manel Esteller, Hubert SchorleAbstract:Type II germ cell cancers (GCC) can be subdivided into Seminomas and non-Seminomas. Seminomas are similar to carcinoma in situ (CIS) cells, the common precursor of type II GCCs, with regard to epigenetics and expression, while embryonal carcinomas (EC) are totipotent and differentiate into teratomas, yolk-sac tumors and choriocarcinomas. GCCs can present as Seminomas with a non-Seminoma component, raising the question if a CIS gives rise to Seminomas and ECs at the same time or whether Seminomas can be reprogrammed to ECs. In this study, we utilized the Seminoma cell line TCam-2 that acquires an EC-like status after xenografting into the murine flank as a model for a Seminoma to EC transition and screened for factors initiating and driving this process. Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while Seminoma-markers were downregulated. Changes in expression level of 53 genes inversely correlated to changes in DNA methylation. Interestingly, after xenotransplantation 6 genes (GDF3, NODAL, DNMT3B, DPPA3, GAL, AK3L1) were rapidly induced, followed by demethylation of their genomic loci, suggesting that these 6 genes are poised for expression driving the reprogramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling. We propose that reprogramming of Seminomas to ECs is a multi-step process. Initially, the microenvironment causes inhibition of BMP signaling, leading to induction of NODAL signaling. During a maturation phase, a fast acting NODAL loop stimulates its own activity and temporarily inhibits BMP signaling. During the stabilization phase, a slow acting NODAL loop, involving WNTs re-establishes BMP signaling and the pluripotency circuitry. In parallel, DNMT3B-driven de novo methylation silences Seminoma-associated genes and epigenetically fixes the EC state.
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c kit is frequently mutated in bilateral germ cell tumours and down regulated during progression from intratubular germ cell neoplasia to Seminoma
The Journal of Pathology, 2007Co-Authors: Katharina Biermann, Daniel Nettersheim, Hubert Schorle, F Goke, Dawid Eckert, H Zhou, Philip Kahl, Isabella Gashaw, Reinhard ButtnerAbstract:Testicular germ cell tumours (TGCTs) are the most frequent cancer type in young men; 5% of these patients develop a second TGCT in the contralateral testis. The pathogenesis of TGCT is closely linked to primordial germ cells (PGCs) or gonocytes. The receptor tyrosine kinase (c-KIT) is necessary for migration and survival of PGCs and is expressed in intratubular neoplastic germ cells (IGCNUs) and Seminomas. We studied the frequency of c-KIT exon 11 and 17 mutations in 155 unilateral (108 Seminomas and 47 non-Seminomas) and 22 bilateral (18 Seminomas, two embryonal carcinomas, two IGCNU) cases. While no mutations were detected in exon 11, the mutation frequency in exon 17 was significantly higher in bilateral (14/22, 63.6%) compared to unilateral TGCT (10/155, 6.4%) (p < 0.001). Different activating mutations (Y823D, D816V, D816H and N822K) were detected in bilateral TGCT. Y823D mutation was identical in both testes in three cases and quantitative pyrosequencing showed that up to 76% of the cells analysed in tumour samples carried this mutation. One bilateral synchronous Seminoma revealed a S821F mutation in one testis and a Y823D mutation contralaterally. To study the role of c-KIT in TGCT progression, we compared its expression in 41 Seminomas and adjacent IGCNUs. Immunohistochemical analysis revealed that c-KIT expression was significantly reduced in Seminomas compared to IGCNUs (p < 0.006) and that there were no significant changes in c-KIT mRNA copy numbers in progressed compared to low-stage Seminomas. In summary, our study shows that patients with c-KIT mutations are more prone to develop a bilateral TGCT and suggests that in a portion of bilateral TGCTs, c-KIT mutations occur early during embryonal development, prior to the arrival of PGCs at the genital ridge. Furthermore, our findings show that c-KIT down-regulation occurs during the progression of IGCNU to Seminoma.
Michael B. Cook - One of the best experts on this subject based on the ideXlab platform.
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Significant calendar period deviations in testicular germ cell tumors indicate that postnatal exposures are etiologically relevant
Cancer Causes & Control, 2012Co-Authors: Crystal Speaks, Katherine A. Mcglynn, Michael B. CookAbstract:Purpose The current working model of type II testicular germ cell tumor (TGCT) pathogenesis states that carcinoma in situ arises during embryogenesis, is a necessary precursor, and always progresses to cancer. An implicit condition of this model is that only in utero exposures affect the development of TGCT in later life. In an age-period-cohort analysis, this working model contends an absence of calendar period deviations. We tested this contention using data from the SEER registries of the United States. Methods We assessed age-period-cohort models of TGCTs, Seminomas, and nonSeminomas for the period 1973–2008. Analyses were restricted to whites diagnosed at ages 15–74 years. We tested whether calendar period deviations were significant in TGCT incidence trends adjusted for age deviations and cohort effects. Results This analysis included 32,250 TGCTs (18,475 Seminomas and 13,775 nonSeminomas). Seminoma incidence trends have increased with an average annual percentage change in log-linear rates (net drift) of 1.25 %, relative to just 0.14 % for nonSeminoma. In more recent time periods, TGCT incidence trends have plateaued and then undergone a slight decrease. Calendar period deviations were highly statistically significant in models of TGCT ( p = 1.24^−9) and Seminoma ( p = 3.99^−14), after adjustment for age deviations and cohort effects; results for nonSeminoma ( p = 0.02) indicated that the effects of calendar period were much more muted. Conclusion Calendar period deviations play a significant role in incidence trends of TGCT, which indicates that postnatal exposures are etiologically relevant.
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Significant calendar period deviations in testicular germ cell tumors indicate that postnatal exposures are etiologically relevant
Cancer Causes & Control, 2012Co-Authors: Crystal Speaks, Katherine A. Mcglynn, Michael B. CookAbstract:Purpose The current working model of type II testicular germ cell tumor (TGCT) pathogenesis states that carcinoma in situ arises during embryogenesis, is a necessary precursor, and always progresses to cancer. An implicit condition of this model is that only in utero exposures affect the development of TGCT in later life. In an age-period-cohort analysis, this working model contends an absence of calendar period deviations. We tested this contention using data from the SEER registries of the United States. Methods We assessed age-period-cohort models of TGCTs, Seminomas, and nonSeminomas for the period 1973–2008. Analyses were restricted to whites diagnosed at ages 15–74 years. We tested whether calendar period deviations were significant in TGCT incidence trends adjusted for age deviations and cohort effects. Results This analysis included 32,250 TGCTs (18,475 Seminomas and 13,775 nonSeminomas). Seminoma incidence trends have increased with an average annual percentage change in log-linear rates (net drift) of 1.25 %, relative to just 0.14 % for nonSeminoma. In more recent time periods, TGCT incidence trends have plateaued and then undergone a slight decrease. Calendar period deviations were highly statistically significant in models of TGCT ( p = 1.24^−9) and Seminoma ( p = 3.99^−14), after adjustment for age deviations and cohort effects; results for nonSeminoma ( p = 0.02) indicated that the effects of calendar period were much more muted. Conclusion Calendar period deviations play a significant role in incidence trends of TGCT, which indicates that postnatal exposures are etiologically relevant.
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TCam-2 Cells Deficient for SOX2 and FOXA2 are Blocked in Differentiation and Maintain a Seminoma-Like Cell Fate In Vivo
2019Co-Authors: Daniel Nettersheim, Sina Jostes, Alena Heimsoeth, Saskia Vadder, Hubert SchorleAbstract:Testicular germ cell tumors (GCTs) are very common in young men and can be stratified into Seminomas and non-Seminomas. While Seminomas share a similar gene expression and epigenetic profile with primordial germ cells, the stem cell population of the non-Seminomas, the embryonal carcinoma (EC), resembles malignant embryonic stem cells. Thus, ECs are able to differentiate into cells of all three germ layers (teratomas) and even extra-embryonic-tissue-like cells (yolk-sac tumor, choriocarcinoma). In the last years, we demonstrated that the cellular microenvironment considerably influences the plasticity of Seminomas (TCam-2 cells). Upon a microenvironment-triggered inhibition of the BMP signaling pathway in vivo (murine flank or brain), Seminomatous TCam-2 cells reprogram to an EC-like cell fate. We identified SOX2 as a key factor activated upon BMP inhibition mediating the reprogramming process by regulating pluripotency, reprogramming and epigenetic factors. Indeed, CRISPR/Cas9 SOX2-deleted TCam-2 cells were able to maintain a Seminoma-cell fate in vivo for about six weeks, but after six weeks in vivo still small sub-populations initiated differentiation. Closer analyses of these differentiated clusters suggested that the pioneer factor FOXA2 might be the driving force behind this induction of differentiation, since many FOXA2 interacting genes and differentiation factors like AFP, EOMES,CDX1, ALB, HAND1, DKK, DLK1,MSX1and PITX2were upregulated. In this study, we generated TCam-2 cells double-deficient for SOX2and FOXA2using the CRISPR/Cas9 technique and xenografted those cells into the flank of nude mice. Upon loss of SOX2 and FOXA2, TCam-2 maintained a Seminoma cell fate for at least twelve weeks, demonstrating that both factors are key players in the reprogramming to an EC-like cell fate. Therefore, our study adds an important piece to the puzzle of GCT development and plasticity, providing interesting insights in what can be expected in a patient, when GCT cells are confronted with different microenvironments
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The plasticity of germ cell cancers and its dependence on the cellular microenvironment.
Journal of Cellular and Molecular Medicine, 2017Co-Authors: Daniel Nettersheim, Hubert SchorleAbstract:: So far, the understanding of germ cell cancer (GCC) pathogenesis is based on a model, where Seminomas and non-Seminomas represent distinct entities although originating from a common precursor termed germ cell neoplasia in situ (GCNIS). Embryonal carcinomas (ECs), the stem cell population of the non-Seminomas, is pluri- to totipotent and able to differentiate into cells of all three germ layers, giving rise to teratomas or tumours mimicking extraembryonic tissues (yolk sac tumours, choriocarcinomas). With regard to gene expression, (epi)genetics and histology, Seminomas are highly similar to GCNIS and primordial germ cells, but limited in development. It remains elusive, whether this block in differentiation is controlled by cell intrinsic mechanisms or by signals from the surrounding microenvironment. Here, we reviewed the recent literature emphasizing the plasticity of GCCs, especially of Seminomas. We propose that this plasticity is controlled by the microenvironment, allowing Seminomas to transit into an EC or mixed non-Seminoma and vice versa. We discuss several mechanisms and routes of reprogramming that might be responsible for this change in the cell fate. We finally integrate this plasticity into a new model of GCC pathogenesis, allowing for an alternative view on the dynamics of GCC development and progression.
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sox2 is essential for in vivo reprogramming of Seminoma like tcam 2 cells to an embryonal carcinoma like fate
Oncotarget, 2016Co-Authors: Daniel Nettersheim, Sina Jostes, Simon Schneider, Andrea Hofmann, Alena Heimsoeth, Martin Fellermeyer, Hubert SchorleAbstract:// Daniel Nettersheim 1 , Alena Heimsoeth 1 , Sina Jostes 1 , Simon Schneider 1 , Martin Fellermeyer 1 , Andrea Hofmann 2 , Hubert Schorle 1 1 Institute of Pathology, Department of Developmental Pathology, University of Bonn Medical School, Bonn, Germany 2 Institute of Human Genetics, University of Bonn Medical School, Bonn, Germany Correspondence to: Hubert Schorle, email: Hubert.Schorle@ukb.uni-bonn.de Keywords: TCam-2, Seminoma, embryonal carcinoma, reprogramming, SOX2/SOX17 Received: March 22, 2016 Accepted: May 19, 2016 Published: June 07, 2016 ABSTRACT Type II germ cell cancers (GCC) are divided into Seminomas, which are highly similar to primordial germ cells and embryonal carcinomas (EC), often described as malignant counterparts to embryonic stem cells. Previously, we demonstrated that the development of GCCs is a highly plastic process and strongly influenced by the microenvironment. While orthotopic transplantation into the testis promotes Seminomatous growth of the Seminoma-like cell line TCam-2, ectopic xenotransplantation into the flank initiates reprogramming into an EC-like fate. During this reprogramming, BMP signaling is inhibited, leading to induction of NODAL signaling, upregulation of pluripotency factors and downregulation of Seminoma markers, like SOX17. The pluripotency factor and EC-marker SOX2 is strongly induced. Here, we adressed the molecular role of SOX2 in this reprogramming. Using CRISPR/Cas9-mediated genome-editing, we established SOX2-deficient TCam-2 cells. Xenografting of SOX2-deficient cells into the flank of nude mice resulted in maintenance of a Seminoma-like fate, indicated by the histology and expression of OCT3/4, SOX17, TFAP2C, PRDM1 and PRAME . In SOX2-deficient cells, BMP signaling is inhibited, but NODAL signaling is not activated. Thus, SOX2 appears to be downstream of BMP signaling but upstream of NODAL activation. So, SOX2 is an essential factor in acquiring an EC-like cell fate from Seminomas. A small population of differentiated cells was identified resembling a mixed non-Seminoma. Analyses of these cells revealed downregulation of the pluripotency and Seminoma markers OCT3/4, SOX17, PRDM1 and TFAP2C. In contrast, the pioneer factor FOXA2 and its target genes were upregulated, suggesting that FOXA2 might play an important role in induction of non-Seminomatous differentiation.
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bmp inhibition in Seminomas initiates acquisition of pluripotency via nodal signaling resulting in reprogramming to an embryonal carcinoma
PLOS Genetics, 2015Co-Authors: Daniel Nettersheim, Sina Jostes, Rakesh Sharma, Simon Schneider, Andrea Hofmann, Humberto J Ferreira, Per Hoffmann, Glen Kristiansen, Manel Esteller, Hubert SchorleAbstract:Type II germ cell cancers (GCC) can be subdivided into Seminomas and non-Seminomas. Seminomas are similar to carcinoma in situ (CIS) cells, the common precursor of type II GCCs, with regard to epigenetics and expression, while embryonal carcinomas (EC) are totipotent and differentiate into teratomas, yolk-sac tumors and choriocarcinomas. GCCs can present as Seminomas with a non-Seminoma component, raising the question if a CIS gives rise to Seminomas and ECs at the same time or whether Seminomas can be reprogrammed to ECs. In this study, we utilized the Seminoma cell line TCam-2 that acquires an EC-like status after xenografting into the murine flank as a model for a Seminoma to EC transition and screened for factors initiating and driving this process. Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while Seminoma-markers were downregulated. Changes in expression level of 53 genes inversely correlated to changes in DNA methylation. Interestingly, after xenotransplantation 6 genes (GDF3, NODAL, DNMT3B, DPPA3, GAL, AK3L1) were rapidly induced, followed by demethylation of their genomic loci, suggesting that these 6 genes are poised for expression driving the reprogramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogramming, resulting in activation of the pluripotency-associated genes and NODAL signaling. We propose that reprogramming of Seminomas to ECs is a multi-step process. Initially, the microenvironment causes inhibition of BMP signaling, leading to induction of NODAL signaling. During a maturation phase, a fast acting NODAL loop stimulates its own activity and temporarily inhibits BMP signaling. During the stabilization phase, a slow acting NODAL loop, involving WNTs re-establishes BMP signaling and the pluripotency circuitry. In parallel, DNMT3B-driven de novo methylation silences Seminoma-associated genes and epigenetically fixes the EC state.
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c kit is frequently mutated in bilateral germ cell tumours and down regulated during progression from intratubular germ cell neoplasia to Seminoma
The Journal of Pathology, 2007Co-Authors: Katharina Biermann, Daniel Nettersheim, Hubert Schorle, F Goke, Dawid Eckert, H Zhou, Philip Kahl, Isabella Gashaw, Reinhard ButtnerAbstract:Testicular germ cell tumours (TGCTs) are the most frequent cancer type in young men; 5% of these patients develop a second TGCT in the contralateral testis. The pathogenesis of TGCT is closely linked to primordial germ cells (PGCs) or gonocytes. The receptor tyrosine kinase (c-KIT) is necessary for migration and survival of PGCs and is expressed in intratubular neoplastic germ cells (IGCNUs) and Seminomas. We studied the frequency of c-KIT exon 11 and 17 mutations in 155 unilateral (108 Seminomas and 47 non-Seminomas) and 22 bilateral (18 Seminomas, two embryonal carcinomas, two IGCNU) cases. While no mutations were detected in exon 11, the mutation frequency in exon 17 was significantly higher in bilateral (14/22, 63.6%) compared to unilateral TGCT (10/155, 6.4%) (p < 0.001). Different activating mutations (Y823D, D816V, D816H and N822K) were detected in bilateral TGCT. Y823D mutation was identical in both testes in three cases and quantitative pyrosequencing showed that up to 76% of the cells analysed in tumour samples carried this mutation. One bilateral synchronous Seminoma revealed a S821F mutation in one testis and a Y823D mutation contralaterally. To study the role of c-KIT in TGCT progression, we compared its expression in 41 Seminomas and adjacent IGCNUs. Immunohistochemical analysis revealed that c-KIT expression was significantly reduced in Seminomas compared to IGCNUs (p < 0.006) and that there were no significant changes in c-KIT mRNA copy numbers in progressed compared to low-stage Seminomas. In summary, our study shows that patients with c-KIT mutations are more prone to develop a bilateral TGCT and suggests that in a portion of bilateral TGCTs, c-KIT mutations occur early during embryonal development, prior to the arrival of PGCs at the genital ridge. Furthermore, our findings show that c-KIT down-regulation occurs during the progression of IGCNU to Seminoma.
