transforming-growth-factor-beta and Seminoma

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-β, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-β, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-β superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline.

Topics: Active Transport, Cell Nucleus; Activins; Animals; beta Karyopherins; Cell Line; Humans; Karyopherins; Male; Mice; Neoplasms, Germ Cell and Embryonal; Seminoma; Testicular Neoplasms; Transforming Growth Factor beta

Disrupted fetal germline development underpins testicular germ cell neoplasia, which is increasing worldwide. The complex signaling milieu during normal testis development includes TGFβ superfamily ligands; this study tests the hypothesis that, activin A, a TGFβ superfamily member, can influence gonocyte development. The human seminoma-derived cell line, TCam-2, a model of fetal gonocytes, was cultured with activin A (1.25-25 ng/mL) for 48 h, or with 5 ng/mL activin A for short- (6, 24, and 48 h) and long-term (13 days) exposures, and downstream targets measured by qRT-PCR. Transcripts that exhibited significant dose-dependent responses to activin A included the early germ cell markers KIT, NODAL, and CRIPTO (NODALl co-receptor and activin inhibitor) which all increased and the differentiation marker DNMT3L which decreased. After 48 h, KIT, NODAL, and CRIPTO levels were significantly higher, while the differentiation marker NANOS2 was significantly lower. Interestingly, activin A exposure also significantly reduced both transcript and protein levels of the PIWI/piRNA pathway component DNMT3L. Because TCam-2 cells produce the activin inhibitor CRIPTO, CRIPTO was reduced using siRNA prior to activin A exposure. This selectively increased KIT in response to activin A. Other ligands present in the fetal testis (BMP4, FGF9, TGFβ1, and TGFβ2) induced distinct effects on germline marker expression. This study showed that activin A can directly modulate germline markers in this human gonocyte-like cell, promoting a less-differentiated phenotype. Additional findings indicate evidence of signaling crosstalk between activin A and NODAL, leading to target-specific effects on gonocyte differentiation.

Topics: Activins; Cell Differentiation; Gene Expression Profiling; Gene Expression Regulation; Germ Cells; Humans; Male; Nodal Protein; Seminoma; Testicular Neoplasms; Transforming Growth Factor beta

Germ cell testicular cancer is understood to arise during embryogenesis, based on the persistence of embryonic germ cell markers in carcinoma in situ and seminoma. In this study, we examine the potential of the seminoma-derived TCam-2 cell line to be used as representative in functional analyses of seminoma. We demonstrate expression of several early germ cell markers, including BLIMP1, OCT3/4, AP2γ, NANOG and KIT. Many TGF-beta superfamily receptors and downstream transcription factors are also present in these cells including the normally foetal ACTRIIA receptor, indicating potential responsiveness to TGF-beta superfamily ligands. Treatment with BMP4 or RA induces a significant increase in ACTRIA, ACTRIIA and ACTRIIB transcripts, whereas activin A decreases ACTRIB. BMP4 and RA each support TCam-2 survival and/or proliferation. In addition, despite increased KIT mRNA levels induced by BMP4, RA and activin A, activin A does not improve survival or proliferation. The capacity for BMP4 and retinoic acid to enhance foetal germ cell survival and proliferation/self-renewal has been demonstrated in mice, but not previously tested in humans. This study is the first to demonstrate a functional response in seminoma cells, using a well-characterized cell line, consistent with their foetal germ cell-like identity.

Topics: Activin Receptors, Type II; Activins; Adaptor Protein Complex 2; Biomarkers; Bone Morphogenetic Protein 4; Cell Line, Tumor; Cell Proliferation; Cell Survival; Germ Cells; Homeodomain Proteins; Humans; Ligands; Male; Nanog Homeobox Protein; Neoplasms, Germ Cell and Embryonal; Octamer Transcription Factor-3; Positive Regulatory Domain I-Binding Factor 1; Proto-Oncogene Proteins c-kit; Repressor Proteins; Seminoma; Signal Transduction; Testicular Neoplasms; Transforming Growth Factor beta; Tretinoin

Germ cell tumours (GCTs) are cancers of the testis, ovary or extragonadal sites that occur in infants, children and adults. Testicular GCT is the most common cancer in young men aged 15-40 years. Abnormalities in developmental signalling pathways such as wnt/β-catenin, TGF-β/BMP and Hedgehog have been described in many childhood tumours. To date, however, the status of BMP signalling in GCTs has not been described. Herein, we examine BMP-SMAD signalling in a set of clinically-annotated paediatric GCTs. We find that BMP signalling activity is absent in undifferentiated tumours such as seminomas and dysgerminomas, but robustly present in most yolk sac tumours, a differentiated tumour type. Gene expression profiling of TGF-β/BMP pathway genes in germinomas and yolk sac tumours reveals a set of genes that distinguish the two tumour types. There is significant intertumoural heterogeneity between tumours of the same histological subclass, implying that the BMP pathway can be differentially regulated in individual tumours. Finally, through miRNA expression profiling, we identify differential regulation of a set of miRNAs predicted to target the TGF-β/BMP pathway at multiple sites. Taken together, these results suggest that the BMP signalling pathway may represent a new therapeutical target for childhood GCTs.

Topics: Adolescent; Adult; Biomarkers, Tumor; Bone Morphogenetic Proteins; Child; Child, Preschool; Dysgerminoma; Endodermal Sinus Tumor; Female; Gene Expression Profiling; Humans; Infant; Male; MicroRNAs; Ovarian Neoplasms; Seminoma; Signal Transduction; Smad Proteins; Testicular Neoplasms; Transforming Growth Factor beta

Activin is a pleiotropic growth factor belonging to the transforming growth factor-beta (TGFB) superfamily of signaling molecules. Regulated activin signaling is known to influence several steps in rodent male gamete differentiation. TGFB ligand isoforms, TGFB1-B3, also influence germ cell survival in the rodent testis at the onset of spermatogenesis and around the time of puberty. Given the importance of regulated activin and TGFB signaling in testis development and function, we sought to investigate the cellular production sites of activin/TGFB-signaling modulators in normal and dysfunctional adult human testes samples. Signaling transducers phosphorylated SMAD2/3, and signaling modulators SMAD6, MAN-1, inhibin alpha (INHA), and beta-glycan were detected in Bouins fixed, paraffin-embedded adult human testis sections using immunohistochemistry. Additional samples examined were from testicular cancer patients and from normal men subjected to gonadotropin suppression with androgen-based contraceptives. Our findings identify distinct differences between normal and gonadotropin-deprived human testis in the expression and cellular localization of activin/TGFB-signaling modulators. The presence of a nuclear phosphorylated SMAD2/3 signal in all analyzed seminoma specimens indicated active activin/TGFB signaling. Moreover, a subset of seminoma specimens exhibited selective enhanced expression of beta-glycan (4 out of 28 seminoma tumors), INHA (6 out of 28), and MAN-1 (6 out of 28), highlighting potential functional differences between individual tumors in their capacity to regulate activin/TGFB signaling. Within the heterogenous nonseminomas, expression of signaling modulators was variable and reflected the degree of somatic differentiation. Thus, synthesis of activin and TGFB-signaling modulators may be affected by spermatogenic disruption and altered hormone levels in the testis.

Topics: Activins; Adult; Health; Humans; Male; Models, Biological; Seminoma; Signal Transduction; Testicular Diseases; Testicular Neoplasms; Testis; Testosterone; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta is known as an antiproliferative factor in the majority of mammalian cells, including stem germ cells. Lack of TGF-beta-induced growth inhibition has been associated with disruptions of TGF-beta receptors and SMADs. In the present study, we performed a mutational analysis of the TGF-beta signaling system, including TGF-beta receptor type I and type II and SMADs (SMAD1-SMAD7), in 20 seminoma germ cell tumors. Using reverse transcription-PCR, single-strand conformational polymorphism, and sequencing analysis, the COOH-terminal domain of SMAD4 was found to be mutated: a single thymine was inserted between nt 1521 and 1522 in 2 of 20 tumors analyzed. This addition of a thymine creates a frameshift and a new stop signal at codon 492, which leads to premature termination of the encoded protein. Such a mutation potentially abrogates signaling from TGF-beta as well as the other TGF-beta family members, including activin and bone morphogenetic protein, which all use the SMAD pathway. Immunohistological analysis confirmed the loss of expression of SMAD4 protein in the seminoma tissues with the insertional mutation. To our knowledge, this is the first description of a novel SMAD4 insertional mutation in seminoma testicular germ cell tumors. This mutational inactivation of SMAD4/COOH-terminal domain may cause TGF-beta unresponsiveness. It could thus provide a basis for understanding the potential role of the TGF-beta system in germ cell tumorigenesis.

Topics: Amino Acid Sequence; Base Sequence; DNA-Binding Proteins; Humans; Immunohistochemistry; Molecular Sequence Data; Mutation; Polymorphism, Single-Stranded Conformational; Reverse Transcriptase Polymerase Chain Reaction; Seminoma; Smad2 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

To investigate the localization of transforming growth factors (TGF-beta 1, -beta 2 and -beta 3) and their receptors (TGF-beta RI and RII).. The study included 26 paraffin-embedded tissues from human testicular neoplasms: 15 seminomas, 2 embryonal carcinomas, 1 immature teratoma, 4 immature teratomas with embryonal carcinoma, 1 immature teratoma with seminoma, 1 seminoma with embryonal carcinoma and 2 gonadal stromal tumors (Leydig cell tumors).. TGF-beta 1 immunoreactivity was cytoplasmic and was expressed in 22 (84.6%), TGF-beta 2 in 20 (77%), TGF-beta 3 in 11 (42.3%), TGF-beta-RI in 21 (80.8%) and TGF-beta-RII in 18 (69.2%) of the 26 neoplasms. The percentage of positive immunostained cells and the intensity of staining were significantly higher in tumor than in peritumor nonneoplastic testis. In the peritumor nonneoplastic testis, Leydig, Sertoli and germ cells coexpressed both the three TGF-beta isoforms and TGF-beta-RI and RII. The myoepithelial cells of the seminiferous tubules showed immunoreactivity for TGF-beta RI and RII but not for TGF-beta s. In tumor testis areas the pattern of TGF-beta and TGF-beta receptor expression and distribution varied according to the histologic type of testicular tumor. Seminomas showed a diffuse pattern of TGF-beta immunoreactivity, whereas immature teratomas had focal and patchy distribution. In teratomas, differentiated structures contained more TGF-beta s than undifferentiated structures.

Topics: Activin Receptors, Type I; Adolescent; Adult; Aged; Carcinoma, Embryonal; Germinoma; Humans; Immunohistochemistry; Leydig Cell Tumor; Male; Middle Aged; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Seminoma; Teratoma; Testicular Neoplasms; Transforming Growth Factor beta