RESUMEN
In testicular germ cell tumor type II (TGCT), a seminoma subtype expresses an induced pluripotent stem cell (iPSC) panel with four upregulated genes, OCT4/POU5F1, SOX17, KLF4, and MYC, and embryonal carcinoma (EC) has four upregulated genes, OCT4/POU5F1, SOX2, LIN28, and NANOG. The EC panel can reprogram cells into iPSC, and both iPSC and EC can differentiate into teratoma. This review summarizes the literature on epigenetic regulation of the genes. Epigenetic mechanisms, such as methylations of cytosines on the DNA string and methylations and acetylations of histone 3 lysines, regulate expression of these driver genes between the TGCT subtypes. In TGCT, the driver genes contribute to well-known clinical characteristics and the driver genes are also important for aggressive subtypes of many other malignancies. In conclusion, epigenetic regulation of the driver genes are important for TGCT and for oncology in general.
Asunto(s)
Carcinoma Embrionario , Neoplasias de Células Germinales y Embrionarias , Neoplasias Testiculares , Masculino , Humanos , Epigénesis Genética , Neoplasias Testiculares/genética , Neoplasias de Células Germinales y Embrionarias/genética , Carcinoma Embrionario/genética , Carcinogénesis/genética , Transformación Celular Neoplásica/genéticaRESUMEN
Germ cell neoplasia in situ (GCNIS) is the precursor of both seminomatous and non-seminomatous germ cell tumors. It consists of distended tubules that may have either intratubular seminoma or intratubular embryonal carcinoma cells. Many invasive non-seminomatous tumors contain a mixture of tumor types, which are reviewed here. Morphology, aided by a panel of immunostains, can determine the presence and percent of embryonal carcinoma, yolk sac tumor, choriocarcinoma, or teratoma in such tumors. Use of immunostains, required for diagnosis in perhaps 25% of testicular neoplasms, is reviewed. Changes of classification in the AJCC (8th edition) in 2016 are discussed, including the partitioning of two tumor types: the central role of chromosome 12p amplification allows both teratoma and yolk sac tumor to be divided into prepubertal types (lacking amplification) and post-pubertal types. Occasionally, sex cord-stromal tumors, hematolymphoid tumors, or epididymal adenomatoid tumors enter the differential diagnosis of germ cell neoplasms.
Asunto(s)
Carcinoma Embrionario , Tumor del Seno Endodérmico , Neoplasias de Células Germinales y Embrionarias , Seminoma , Teratoma , Neoplasias Testiculares , Masculino , Humanos , Tumor del Seno Endodérmico/patología , Neoplasias Testiculares/genética , Neoplasias Testiculares/diagnóstico , Neoplasias Testiculares/patología , Carcinoma Embrionario/genética , Carcinoma Embrionario/metabolismo , Carcinoma Embrionario/patología , Teratoma/diagnóstico , Teratoma/genética , Teratoma/patología , Seminoma/diagnóstico , Seminoma/genética , Seminoma/patologíaRESUMEN
BACKGROUND: In recent years, several studies investigated the complex process called "reprogramming" of seminoma (S) cells. The accepted pathogenetic model is a complex network including SOX2, SOX17, OCT3/4 and PRAME, which modulates the epigenetic transcription of numerous downstream genes and drives a divergent gene expression profile resulting in the transition from pure S (P-S) to S component (S-C) of mixed germ cell tumors of the testis (M-GCTT), and finally to embryonal carcinoma (EC). Herein, we tested a large cohort of GCTT with SOX2 and PRAME to evaluate their expression in the evolutionary steps of GCTT and verify if the modulation in the expression of these two molecules could be relevant for the fate of GCTT. METHODS: We tested 43, 19 and 17 consecutive and retrospectively enrolled cases of GCTT, germ cell neoplasia in situ (GCNIS) and uninvolved background testes (UBT), respectively. SOX2 and PRAME expressions have been evaluated with H-score and compared by adopting the appropriate statistic tests (Student's t-test and Mann-Whitney U test). RESULTS: We found that SOX2 was more expressed by nonseminomatous-GCTT (NS-GCTT) (p < 0.001) and EC (p < 0.001) rather than S; by contrast, PRAME showed an opposite expression profile being expressed by S but not by NS-GCTT (p < 0.001) and EC (p < 0.001). S-C showed different expressions of SOX2 and PRAME compared to both P-S (p = 0.002 and <0.001, respectively) and EC (p < 0.001 and 0.042, respectively), with intermediate values between these latter two categories. GCNIS and UBT showed no expression of SOX2 (scattered positive Leydig cells) but high H-score levels of PRAME. CONCLUSIONS: SOX2 and PRAME are differentially expressed and specularly modulated during the "reprogramming" of S cells [P-S (high levels of PRAME, no expression/low levels of SOX2) â S-C (intermediate levels of PRAME, intermediate levels of SOX2) â EC (no expression/low levels of PRAME, high levels of SOX2)], therefore supporting a complex pathogenetic model where the interactions between these two molecules are crucial in determining the fate of GCTT.
Asunto(s)
Carcinoma Embrionario , Neoplasias de Células Germinales y Embrionarias , Seminoma , Neoplasias Testiculares , Masculino , Humanos , Seminoma/genética , Seminoma/metabolismo , Seminoma/patología , Neoplasias Testiculares/patología , Estudios Retrospectivos , Factores de Transcripción SOXB1/metabolismo , Carcinoma Embrionario/genética , Carcinoma Embrionario/metabolismo , Carcinoma Embrionario/patología , Antígenos de Neoplasias/metabolismoRESUMEN
Background: Testicular germ cell tumor (TGCT) is the most common tumor in young men, but molecular signatures, especially the alternative splicing (AS) between its subtypes have not yet been explored. Methods: To investigate the differences between TGCT subtypes, we comprehensively analyzed the data of gene expression, alternative splicing (AS), and somatic mutation in TGCT patients from the TCGA database. The gene ontology (GO) enrichment analyses were used to explore the function of differentially expressed genes and spliced genes respectively, and Spearman correlation analysis was performed to explore the correlation between differential genes and AS events. In addition, the possible patterns in which AS regulates gene expression were elaborated by the ensemble database transcript atlas. And, we identified important transcription factors that regulate gene expression and AS and functionally validated them in TGCT cell lines. Results: We found significant differences between expression and AS in embryonal carcinoma and seminoma, while mixed cell tumors were in between. GO enrichment analyses revealed that both differentially expressed and spliced genes were enriched in transcriptional regulatory pathways, and obvious correlation between expression and AS events was determined. By analyzing the transcript map and the sites where splicing occurs, we have demonstrated that AS regulates gene expression in a variety of ways. We further identified two pivot AS-related molecules (SOX2 and HDAC9) involved in AS regulation, which were validated in embryonal carcinoma and seminoma cell lines. Differences in somatic mutations between subtypes are also of concern, with our results suggesting that mutations in some genes (B3GNT8, CAPN7, FAT4, GRK1, TACC2, and TRAM1L1) occur only in embryonal carcinoma, while mutations in KIT, KARS, and NRAS are observed only in seminoma. Conclusions: In conclusion, our analysis revealed the differences in gene expression, AS and somatic mutation among TGCT subtypes, providing a molecular basis for clinical diagnosis and precise therapy of TGCT patients.
Asunto(s)
Carcinoma Embrionario , Seminoma , Neoplasias Testiculares , Masculino , Humanos , Carcinoma Embrionario/genética , Carcinoma Embrionario/metabolismo , Carcinoma Embrionario/patología , Empalme Alternativo , Seminoma/genética , Seminoma/metabolismo , Seminoma/patología , ADN Recombinante , Neoplasias Testiculares/diagnóstico , Mutación , Expresión GénicaRESUMEN
Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population-embryonal carcinoma (EC)-is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra-embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho- and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.
Asunto(s)
Carcinoma Embrionario , Neoplasias de Células Germinales y Embrionarias , Neoplasias Testiculares , Antígeno CD24 , Carcinoma Embrionario/genética , Carcinoma Embrionario/patología , Humanos , Masculino , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias Testiculares/genética , Neoplasias Testiculares/patología , Microambiente TumoralRESUMEN
Oct4 is an important mammalian POU family transcription factor expressed by early human embryonic stem cells (hESCs). The precise level of Oct4 governs the pluripotency and fate determination of hESCs. Several post-translational modifications (PTMs) of Oct4 including phosphorylation, ubiquitination, and SUMOylation have been reported to regulate its critical functions in hESCs. Ubiquitination and deubiquitination of Oct4 should be well balanced to maintain the pluripotency of hESCs. The protein turnover of Oct4 is regulated by several E3 ligases through ubiquitin-mediated degradation. However, reversal of ubiquitination by deubiquitinating enzymes (DUBs) has not been reported for Oct4. In this study, we generated a ubiquitin-specific protease 3 (USP3) gene knockout using the CRISPR/Cas9 system and demonstrated that USP3 acts as a protein stabilizer of Oct4 by deubiquitinating Oct4. USP3 interacts with endogenous Oct4 and co-localizes in the nucleus of hESCs. The depletion of USP3 leads to a decrease in Oct4 protein level and loss of pluripotent morphology in hESCs. Thus, our results show that USP3 plays an important role in controlling optimum protein level of Oct4 to retain pluripotency of hESCs.
Asunto(s)
Carcinoma Embrionario/genética , Enzimas Desubicuitinizantes/metabolismo , Células Madre Embrionarias/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Sistemas CRISPR-Cas , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular Tumoral , Enzimas Desubicuitinizantes/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/enzimología , Técnicas de Inactivación de Genes , Humanos , Factor 3 de Transcripción de Unión a Octámeros/genética , Unión Proteica , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Análisis de la Célula Individual , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación/genéticaRESUMEN
The epithelium-specific ETS transcription factor-1 (ESE-1) plays multiple roles in pathogenesis and normal development of epithelial tissues. NANOG, a key mediator of stem cell self-renewal and pluripotency, is also expressed in various cancers and pluripotent cells. In this study, we investigated how ESE-1 influences NANOG expression and NANOG-induced proliferation in human germ cell-derived embryonic carcinoma NCCIT cells. Endogenous ESE-1 expression in NCCIT cells significantly increased during differentiation, whereas NANOG expression decreased. In addition, NANOG expression was downregulated by exogenous overexpression of ESE-1, and increased by shRNA-mediated knockdown of ESE-1. NANOG transcriptional activity was reduced by dose-dependent ESE-1 overexpression and a putative ESE-1 binding site (EBS) was mapped within conserved region 2. Site-directed mutagenesis of the putative EBS abrogated the repressive effect of ESE-1 on NANOG promoter activity. ESE-1 directly interacted with the putative EBS to regulate transcriptional activity of NANOG. Furthermore, NANOG-induced proliferation and colony formation of NCCIT cells were inhibited by ESE-1 overexpression and stimulated by ESE-1 shRNA-mediated knockdown. Altogether, our results suggest that ESE-1 exerts an anti-proliferative effect on NCCIT cells by acting as a novel transcriptional repressor of NANOG.
Asunto(s)
Carcinoma Embrionario/metabolismo , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteína Homeótica Nanog/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Factores de Transcripción/metabolismo , Carcinoma Embrionario/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Humanos , Proteína Homeótica Nanog/genética , Proteínas Proto-Oncogénicas c-ets/genética , Factores de Transcripción/genéticaRESUMEN
A recent study suggested a role of CHEK2 loss-of-function germ-line pathogenic variants in the predisposition to testicular cancer (TC) (AlDubayan et al. JAMA Oncol 5:514-522, 2019). We attempted to validate this finding relying on the high population frequency of recurrent CHEK2 pathogenic variants in Slavic populations. CHEK2 pathogenic alleles (c.1100delC (p.Thr367Metfs); del5395 [del ex9-10]; IVS2 + 1G > A [c.444 + 1G > A]) were detected in 7/280 (2.5%) TC patients vs. 3/424 (0.7%) healthy men and 6/1007 (0.6%) healthy women [OR 4.0 (95% CI 1.5-11), p = 0.009 for pooled control groups]. Somatic CHEK2 loss-of-heterozygosity (LOH) was detected in 4 out of 6 tumors available for analysis; strikingly all these instances of LOH involved inactivation of the wild-type allele. The CHEK2 c.470T > C (p.Ile157Thr) variant was detected in 21/280 (7.5%) affected vs. 22/424 (5.2%) non-affected men [OR 1.5 (95% CI 0.8-2.7), p = 0.3]. Somatic CHEK2 LOH was revealed only in 6 out of 21 tumors obtained from CHEK2 c.470T > C (p.Ile157Thr) carriers, with the C-allele lost in two cases and T-allele deleted in four tumors. The results of comparison of allele frequencies in TC patients versus population controls coupled with the data on CHEK2 LOH status in tumor tissues support the association of CHEK2 pathogenic variants with TC risk.
Asunto(s)
Alelos , Quinasa de Punto de Control 2/genética , Eliminación de Gen , Mutación de Línea Germinal/genética , Pérdida de Heterocigocidad , Neoplasias Testiculares/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Embrionario/genética , Niño , Preescolar , Tumor del Seno Endodérmico/genética , Humanos , Lactante , Masculino , Persona de Mediana Edad , Federación de Rusia , Seminoma/genética , Teratoma/genética , Adulto JovenRESUMEN
The pluripotent transcription factor NANOG is essential for maintaining embryonic stem cells and driving tumorigenesis. We previously showed that PKC activity is involved in the regulation of NANOG expression. To explore the possible involvement of microRNAs in regulating the expression of key pluripotency factors, we performed a genome-wide analysis of microRNA expression in the embryonal carcinoma cell line NT2/D1 in the presence of the PKC activator, PMA. We found that MIR630 was significantly upregulated in PMA-treated cells. Experimentally, we showed that transfection of MIR630 mimic into embryonal carcinoma cell lines directly targeted the 3'UTR of OCT4, SOX2, and NANOG and markedly suppressed their expression. RNAhybrid and RNA22 algorithms were used to predict miRNA target sites in the NANOG 3'UTR, four possible target sites of MIR630 were identified. To examine the functional interaction between MIR630 and NANOG mRNA, the predicted MIR630 target sites in the NANOG 3'UTR were deleted and the activity of the reporters were compared. After targeted mutation of the predicted MIR630 target sites, the MIR630 mimic inhibited NANOG significantly less than the wild-type reporters. It is worth noting that mutation of a single putative binding site in the 3'UTR of NANOG did not completely abolish MIR630-mediated suppression, suggesting that MIR630 in the NANOG 3'UTR may have multiple binding sites and act together to maximally repress NANOG expression. Interestingly, MIR630 mimics significantly downregulated NANOG gene transcription. Exogenous expression of OCT4, SOX2, and NANOG lacking the 3'UTR almost completely rescued the reduced transcriptional activity of MIR630. MIR630 mediated the expression of differentiation markers in NT2/D1 cells, suggesting that MIR630 leads to the differentiation of NT2/D1 cell. Our findings show that MIR630 represses NANOG through transcriptional and post-transcriptional regulation, suggesting a direct link between core pluripotency factors and MIR630.
Asunto(s)
Carcinoma Embrionario/genética , Células Madre de Carcinoma Embrionario/fisiología , MicroARNs/genética , Proteína Homeótica Nanog/genética , Interferencia de ARN/fisiología , Transcripción Genética/genética , Regiones no Traducidas 3'/genética , Sitios de Unión/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Células Madre Embrionarias/fisiología , Humanos , Mutación/genética , Regulación hacia Arriba/genéticaRESUMEN
Overactivation of the c-MET/HGF system is a feature of many cancers. We previously reported that type II testicular germ cell tumor (TGCT) cells express the c-MET receptor, forming non-seminomatous lesions that are more positive compared with seminomatous ones. Notably, we also demonstrated that NT2D1 non-seminomatous cells (derived from an embryonal carcinoma lesion) increase their proliferation, migration, and invasion in response to HGF. Herein, we report that HGF immunoreactivity is more evident in the microenvironment of embryonal carcinoma biopsies with respect to seminomatous ones, indicating a tumor-dependent modulation of the testicular niche. PI3K/AKT is one of the signaling pathways triggered by HGF through the c-MET activation cascade. Herein, we demonstrated that phospho-AKT increases in NT2D1 cells after HGF stimulation. Moreover, we found that this pathway is involved in HGF-dependent NT2D1 cell proliferation, migration, and invasion, since the co-administration of the PI3K inhibitor LY294002 together with HGF abrogates these responses. Notably, the inhibition of endogenous PI3K affects collective cell migration but does not influence proliferation or chemotactic activity. Surprisingly, LY294002 administered without the co-administration of HGF increases cell invasion at levels comparable to the HGF-administered samples. This paradoxical result highlights the role of the testicular microenvironment in the modulation of cellular responses and stimulates the study of the testicular secretome in cancer lesions.
Asunto(s)
Carcinoma Embrionario/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Neoplasias Testiculares/metabolismo , Carcinoma Embrionario/genética , Carcinoma Embrionario/patología , Línea Celular Tumoral , Factor de Crecimiento de Hepatocito/genética , Humanos , Masculino , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Neoplasias Testiculares/genéticaRESUMEN
To elucidate the molecular pathogenesis of pediatric germ cell tumors (GCTs), we performed DNA methylation array analysis, whole transcriptome sequencing, targeted capture sequencing, and single-nucleotide polymorphism array analysis using 51 GCT samples (25 female, 26 male), including 6 germinomas, 2 embryonal carcinomas, 4 immature teratomas, 3 mature teratomas, 30 yolk sac tumors, and 6 mixed germ cell tumors. Among the 51 samples, 11 were from infants, 23 were from young children, and 17 were from those aged ≥10 years. Sixteen of the 51 samples developed in the extragonadal regions. Germinomas showed upregulation of pluripotent genes and global hypomethylation. Pluripotent genes were also highly expressed in embryonal carcinomas. These genes may play essential roles in embryonal carcinomas given that their binding sites are hypomethylated. Yolk sac tumors exhibited overexpression of endodermal genes, such as GATA6 and FOXA2, the binding sites of which were hypomethylated. Interestingly, infant yolk sac tumors had different DNA methylation patterns from those observed in older children. Teratomas had higher expression of ectodermal genes, suggesting a tridermal nature. Based on our results, we suggest that KIT, TNFRSF8, and ERBB4 may be suitable targets for the treatment of germinoma, embryonal carcinomas, and yolk sac tumors, respectively.
Asunto(s)
Neoplasias de Células Germinales y Embrionarias/genética , Carcinoma Embrionario/genética , Niño , Preescolar , Variaciones en el Número de Copia de ADN , Metilación de ADN , Tumor del Seno Endodérmico/genética , Femenino , Germinoma/genética , Humanos , Lactante , Masculino , Neoplasias de Células Germinales y Embrionarias/tratamiento farmacológico , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Teratoma/genética , Secuenciación del ExomaAsunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Carcinoma Embrionario/genética , Carcinoma Embrionario/patología , Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/terapia , Carcinoma Embrionario/terapia , Hemorragia Cerebral Intraventricular/etiología , Hemorragia Cerebral Intraventricular/mortalidad , Cisplatino/administración & dosificación , Terapia Combinada/métodos , Ciclofosfamida/administración & dosificación , Resultado Fatal , Femenino , Humanos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/cirugía , Procedimientos Neuroquirúrgicos , Fusión de Oncogenes/genética , Vincristina/administración & dosificaciónRESUMEN
An aberrant increase in pluripotency gene (PpG) expression due to enhancer reactivation could induce stemness and enhance the tumorigenicity of cancer stem cells. Silencing of PpG enhancers (PpGe) during embryonic stem cell differentiation involves Lsd1-mediated H3K4me1 demethylation and DNA methylation. Here, we observed retention of H3K4me1 and DNA hypomethylation at PpGe associated with a partial repression of PpGs in F9 embryonal carcinoma cells (ECCs) post-differentiation. H3K4me1 demethylation in F9 ECCs could not be rescued by Lsd1 overexpression. Given our observation that H3K4me1 demethylation is accompanied by strong Oct4 repression in P19 ECCs, we tested if Oct4 interaction with Lsd1 affects its catalytic activity. Our data show a dose-dependent inhibition of Lsd1 activity by Oct4 and retention of H3K4me1 at PpGe in Oct4-overexpressing P19 ECCs. These data suggest that Lsd1-Oct4 interaction in cancer stem cells could establish a "primed" enhancer state that is susceptible to reactivation, leading to aberrant PpG expression.
Asunto(s)
Elementos de Facilitación Genéticos , Histona Demetilasas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , Biocatálisis , Carcinoma Embrionario/genética , Carcinoma Embrionario/patología , Diferenciación Celular/genética , Línea Celular Tumoral , Cromatina/metabolismo , Metilación de ADN/genética , Epigénesis Genética , Histonas/metabolismo , Humanos , Masculino , Modelos Biológicos , Células Madre Pluripotentes/citologíaRESUMEN
The pluripotent mouse embryonal carcinoma cell line P19 is widely used as a model for research on all-trans-retinoid acid (RA)-induced neuronal differentiation; however, the signaling pathways involved in this process remain unclear. This study aimed to reveal the molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells. Real-time quantitative polymerase chain reaction and Western blot analysis were used to determine the expression of neuronal-specific markers, whereas flow cytometry was used to analyze cell cycle and cell apoptosis. The expression profiles of messenger RNAs (mRNAs) in RA-induced neuronal differentiation of P19 cells were analyzed using high-throughput sequencing, and the functions of differentially expressed mRNAs (DEMs) were determined by bioinformatics analysis. RA induced an increase in both class III ß-tubulin (TUBB3) and neurofilament medium (NEFM) mRNA expression, indicating that RA successfully induces neuronal differentiation of P19 cells. Cell apoptosis was not affected; however, cell proliferation decreased. We found 4117 DEMs, which were enriched in the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, Wnt signaling pathway, and cell cycle. Particularly, a few DEMs could be identified in the PI3K/Akt signaling pathway networks, such as PI3K, Akt, glycogen synthase kinase-3ß (GSK3ß), cyclin-dependent kinase 4 (CDK4), P21, and Bax. RA significantly increased the protein expression of PI3K, Akt, phosphorylated Akt, GSK3ß, phosphorylated GSK3ß, CDK4, and P21, but it reduced Bax protein expression. The Akt inhibitor affected the increase of TUBB3 and NEFM mRNA expression in RA-induced P19 cells. The molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells is potentially involved in the PI3K/Akt/GSK3ß signaling pathway. The decreased cell proliferation ability of neuronally differentiated P19 cells could be associated with the expression of cell cycle proteins.
Asunto(s)
Carcinoma Embrionario/patología , Diferenciación Celular , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Neuronas/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Tretinoina/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Embrionario/tratamiento farmacológico , Carcinoma Embrionario/genética , Carcinoma Embrionario/metabolismo , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/genética , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Células Tumorales CultivadasRESUMEN
Embryonal carcinomas (ECs) and seminomas are testicular germ cell tumors. ECs display expression of SOX2, while seminomas display expression of SOX17. In somatic differentiation, SOX17 drives endodermal cell fate. However, seminomas lack expression of endoderm markers, but show features of pluripotency. Here, we use chromatin immunoprecipitation sequencing to report and compare the binding pattern of SOX17 in seminoma-like TCam-2 cells to SOX17 in somatic cells and SOX2 in EC-like 2102EP cells. In seminoma-like cells, SOX17 was detected at canonical (SOX2/OCT4), compressed (SOX17/OCT4) and noncomposite SOX motifs. SOX17 regulates TFAP2C, PRDM1 and PRDM14, thereby maintaining latent pluripotency and suppressing somatic differentiation. In contrast, in somatic cells canonical motifs are rarely bound by SOX17. In sum, only 12% of SOX17-binding sites overlap in seminoma-like and somatic cells. This illustrates that binding site choice is highly dynamic and cell type specific. Deletion of SOX17 in seminoma-like cells resulted in loss of pluripotency, marked by a reduction of OCT4 protein level and loss of alkaline phosphatase activity. Furthermore, we found that in EC-like cells SOX2 regulates pluripotency-associated genes, most likely by partnering with OCT4. In conclusion, SOX17 (in seminomas) functionally replaces SOX2 (in ECs) to maintain expression of the pluripotency cluster.
Asunto(s)
Carcinoma Embrionario/genética , Neoplasias de Células Germinales y Embrionarias/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXF/metabolismo , Seminoma/genética , Neoplasias Testiculares/genética , Animales , Carcinoma Embrionario/patología , Diferenciación Celular/genética , Línea Celular Tumoral , Secuenciación de Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Neoplasias de Células Germinales y Embrionarias/patología , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Proteínas de Unión al ARN/genética , Seminoma/patología , Neoplasias Testiculares/patología , Factor de Transcripción AP-2/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Testicular germ cell tumour (TGCT) represents the most common malignancy in young men in large parts of the world, but the aetiology is yet unclear. Multiple TGCT susceptibility loci have been identified, and we have shown that one of these, SPRY4, may act as a TGCT oncogene. Furthermore, many of the loci are in non-coding regions of the genome. miRNAs, a class of non-coding RNAs may play a crucial role in cell proliferation, differentiation, and apoptosis, and alteration in their expression may lead to oncogenesis. Differential expression of miRNAs in TGCT and normal testis has been reported in previous studies. In this study, we used qPCR to analyse, in normal and malignant testis tissue, the expression of the ten miRNAs that we had previously identified by sequencing to be the most upregulated in TGCT. We found high expression of these miRNAs also by qPCR analysis. The levels of miR-302a-3p, miR-302b-3p, and miR-302c-3p were downregulated after treatment of the TGCT cell lines NT2-D1 and 833 K with the chemotherapy drug cisplatin. By using miRNA inhibitor-mediated transient transfection, we inhibited the expression of the three members of miR-302 family (miR-302s). Inhibition of miR-302s resulted in a decreased cell proliferation in NT2-D1 cells, but not in 833 K cells. In both cell lines, inhibition of miR-302s resulted in decreased expression of SPRY4, which we have previously shown to regulate MAPK/ERK and PI3K/Akt signalling pathways in these cells. Inhibition of miR-302b-3p and miR-302c-3p decreased phosphorylation of ERK1/2, whereas inhibition of miR-302a-3p and miR-302b-3p led to decreased expression of the apoptosis inhibitor, survivin. Our findings suggest that miR-302s act as TGCT oncogenes by inducing the expression of SPRY4 and activating MAPK/ERK pathway while inhibiting apoptosis via increased survivin expression.
Asunto(s)
Carcinogénesis , Carcinoma Embrionario/genética , MicroARNs/genética , Neoplasias de Células Germinales y Embrionarias/genética , Seminoma/genética , Neoplasias Testiculares/genética , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Cisplatino/farmacología , Resistencia a Antineoplásicos/genética , Humanos , MasculinoRESUMEN
By allelotyping analysis, we previously reported a putative progression pathway from germ cell neoplasia in situ (GCNIS) to seminoma, then to embryonal carcinoma in mixed-type testicular germ cell tumors (TGCTs), and detected that loss of heterozygosity events in seminoma components in mixed tumors were more frequent than those in pure seminomas. To elucidate a role of chromosomal instability in the progression of non-seminomatous germ cell tumor (NSGCT), we performed fluorescence in situ hybridization with centromeric probes for chromosomes 1, 7, 8, 12, 17, and X on a cohort of 52 TGCT cases with 103 histologically distinct components: 39 GCNIS lesions (16 and 23 in tumors with and without NSGCT components, respectively), 39 seminomas (27 as pure seminomas and 12 in mixed tumors), and 25 embryonal carcinomas. On a total component basis, both the mean copy number per tumor cell nucleus and the deviations from the modal number of all chromosomes examined significantly increased from GCNIS to seminoma, then to embryonal carcinoma with few exceptions. Seminoma components in mixed tumors showed a significantly greater extent of chromosomal instability in chromosomes 8 and 12 than pure seminomas, whereas no statistically significant difference was observed between GCNIS lesions with and without NSGCT components. These results suggest that not only aneuploidy, but also the cell-to-cell variation of chromosomal number is a sensitive indicator of chromosomal instability and would be implicated in the progression of NSGCT.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma Embrionario/genética , Inestabilidad Cromosómica/genética , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias Testiculares/genética , Adulto , Progresión de la Enfermedad , Humanos , Hibridación Fluorescente in Situ/métodos , Masculino , Neoplasias de Células Germinales y Embrionarias/diagnóstico , Seminoma/genética , Seminoma/metabolismo , Neoplasias Testiculares/diagnósticoRESUMEN
In 2016, the WHO classification of testicular germ cell tumors was revised considering advances in the understanding of their tumorigenesis and molecular features. This restructuring led to a division into two major groups with, on one hand, prepubertal-type tumors, not derived from germ cell neoplasia in situ (GCNIS), and on the other hand, postpubertal-type tumors, GCNIS-derived, which occur in youg men (seminoma and non seminomatous germ cell tumors - embryonal carcinoma, yolk sac tumor, teratoma and choriocarcinoma essentially). The term germ cell neoplasia in situ is consensually accepted as a new terminology for the precursor lesion. In this new classification, the term "spermatocytic seminoma" is replaced by "spermatocytic tumor", reclassified among non-GCNIS-derived tumors. The purpose of this change of nomenclature is to reflect the usually non-aggressive behaviour of this tumor and to avoid any confusion with usual seminoma. The spectrum of trophoblastic tumors continues to expand with the description of new rare entities such as the cystic trophoblastic tumor, the placental site trophoblastic tumor and the epithelioid trophoblastic tumor. This review aims to provide a focus on testicular germ cell tumors highlighting the new immunohistochemical and molecular features responsible for the restructuring of classification. The TNM staging is presented according to the AJCC 8th edition 2017 update.
Asunto(s)
Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de Células Germinales y Embrionarias/patología , Neoplasias Testiculares/genética , Neoplasias Testiculares/patología , Factores de Edad , Carcinoma Embrionario/clasificación , Carcinoma Embrionario/genética , Carcinoma Embrionario/patología , Coriocarcinoma/clasificación , Coriocarcinoma/genética , Coriocarcinoma/patología , Coriocarcinoma no Gestacional/clasificación , Coriocarcinoma no Gestacional/genética , Coriocarcinoma no Gestacional/patología , Eliminación de Gen , Humanos , Inmunohistoquímica , Masculino , Mutación/genética , Neoplasias de Células Germinales y Embrionarias/clasificación , Seminoma/clasificación , Seminoma/genética , Seminoma/patología , Teratoma/clasificación , Teratoma/genética , Teratoma/patología , Terminología como Asunto , Neoplasias Testiculares/clasificaciónRESUMEN
Testicular germ cell tumors (TGCTs) are generally rare but represent the most common solid tumors in young men. They are classified broadly into seminoma, which resemble primordial germ cells (PGCs), and non-seminoma, which are either undifferentiated (embryonic carcinoma) or differentiated (teratoma, yolk sac tumor, choriocarcinomas) patterning. A widespread role for microRNAs (miRNAs), in diverse molecular processes driving initiation and progression of various types of TGCTs has been recently studied. We discuss the involvement of different miRNAs in the development and progression of different types of TGCTs. Moreover, we highlight the aberrant expression of miRNAs in TGCTs and several targets, which may define miRNAs as oncomiRs or tumor suppressors. A better understanding of miRNA biology may ultimately yield further insight into the molecular mechanisms of tumorigenesis and new therapeutic strategies against TGCTs.
Asunto(s)
MicroARNs/genética , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias Testiculares/genética , Animales , Biomarcadores de Tumor/genética , Carcinoma Embrionario/diagnóstico , Carcinoma Embrionario/genética , Coriocarcinoma/diagnóstico , Coriocarcinoma/genética , Progresión de la Enfermedad , Tumor del Seno Endodérmico/diagnóstico , Tumor del Seno Endodérmico/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Masculino , Pronóstico , Seminoma/diagnóstico , Seminoma/genética , Teratoma/diagnóstico , Teratoma/genética , Resultado del TratamientoRESUMEN
Seminoma and embryonal carcinoma (EC), two typical types of testicular germ cell tumors (TGCTs), present significant differences in growth behavior, expression characteristics, differentiation potential, clinical features, therapy, and prognosis. The purpose of this study was to compare the distinctive or preference metabolic pathways between seminoma and EC. The Cancer Genome Atlas revealed that many genes encoding metabolic enzymes could distinguish between seminoma and EC. Using well-characterized cell line models for seminoma (Tcam-2 cells) and EC (NT2 cells), we characterized their metabolite profiles using ultraperformance liquid chromatography coupled to Q-TOF mass spectrometry (UPLC/Q-TOF MS). In general, the integrated results from transcriptome and metabolite profiling revealed that seminoma and EC exhibited distinctive characteristics in the metabolisms of amino acids, glucose, fatty acids, sphingolipids, nucleotides, and drugs. Notably, an attenuation of citric acid cycle/mitochondrial oxidative phosphorylation and sphingolipid biosynthesis as well as an increase in arachidonic acid metabolism and (very) long-chain fatty acid abundance occurred in seminoma as compared with EC. Our study suggests histologic subtype-dependent metabolic reprogramming in TGCTs and will lead to a better understanding of the metabolic signatures and biology of TGCT subtypes.