tretinoin and Seminoma

Translocator protein 18 kDa (TSPO) is a high affinity cholesterol- and drug-binding protein highly expressed in steroidogenic cells, such as Leydig cells, where it plays a role in cholesterol mitochondrial transport. We have previously shown that TSPO is expressed in postnatal day 3 rat gonocytes, precursors of spermatogonial stem cells. Gonocytes undergo regulated phases of proliferation and migration, followed by retinoic acid (RA)-induced differentiation. Understanding these processes is important since their disruption may lead to the formation of carcinoma in situ, a precursor of testicular germ cell tumors (TGCTs). Previously, we showed that TSPO ligands do not regulate gonocyte proliferation. In the present study, we found that TSPO expression is downregulated in differentiating gonocytes. Similarly, in F9 embryonal carcinoma cells, a mouse TGCT cell line with embryonic stem cell properties, there is a significant decrease in TSPO expression during RA-induced differentiation. Silencing TSPO expression in gonocytes increased the stimulatory effect of RA on the expression of the differentiation marker Stra8, suggesting that TSPO exerts a repressive role on differentiation. Furthermore, in normal human testes, TSPO was located not only in Leydig cells, but also in discrete spermatogenic phases such as the forming acrosome of round spermatids. By contrast, seminomas, the most common type of TGCT, presented high levels of TSPO mRNA. TSPO protein was expressed in the cytoplasmic compartment of seminoma cells, identified by their nuclear expression of the transcription factors OCT4 and AP2G. Thus, TSPO appears to be tightly regulated during germ cell differentiation, and to be deregulated in seminomas, suggesting a role in germ cell development and pathology.

Topics: Animals; Carrier Proteins; Cell Line, Tumor; Humans; Leydig Cells; Male; Mice; Rats; Rats, Sprague-Dawley; Receptors, GABA; Receptors, GABA-A; Seminoma; Spermatogenesis; Spermatogonia; Transcription Factors; Tretinoin

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

Seminomas and embryonal carcinomas (EC) are both type II germ cell tumor (GCT) entities and develop from the same precursor lesion (carcinoma-in situ, CIS). However, they show significant differences in growth behavior, differentiation potential, and gene expression. Although ECs are prone to differentiate into all three germ layers and give rise to the non-seminomatous GCT entities teratoma, choriocarcinoma, and yolk-sac tumor, differentiation of seminomas to these entities is only rarely observed. This might reflect the ability of seminomas to actively inhibit differentiation processes evoked by environmental cues. Also, it is not known why CIS gives rise to seminoma in some patients and to non-seminoma in the others. Here, we treated the seminoma-like cell line TCam-2 with the HDAC-inhibitor Depsipeptide, the global demethylating agent 5-aza-2'-deocycytidine, all-trans retinoic acid and the monaminooxidase inhibitor Tranylcipromine and also used knock down approaches to reduce expression of the pluripotency marker NANOG and/or the inhibitor of primordial germ cell differentiation TFAP2C. We found that TCam-2 cells induce apoptosis when treated with Depsipeptide (> 10 nM) but are resistant to treatments with 5-aza-2'-deocycytidine, all-trans retinoic acid and Tranylcipromine, highlighting Depsi as a treatment option for seminomas. We show that TCam-2 cells up-regulate endoderm- and throphectoderm-associated genes after down-regulation of NANOG expression; however, morphologically no indications of differentiation could be found. Instead, we observed up-regulation of OCT3/4 and SOX17 in TCam-2-NANOG knockdown and speculate that this compensates for the loss of the NANOG protein. Hence, NANOG is not a primary target gene responsible for the inhibition of differentiation in seminomas.

Topics: Apoptosis; Azacitidine; Cell Differentiation; Cell Line, Tumor; Decitabine; Depsipeptides; Down-Regulation; Drug Resistance, Neoplasm; Endoderm; Gene Knockdown Techniques; Histone Deacetylase Inhibitors; Histone Deacetylases; Homeodomain Proteins; Humans; Male; Nanog Homeobox Protein; Octamer Transcription Factor-3; Seminoma; SOXF Transcription Factors; Testicular Neoplasms; Transcription Factor AP-2; Tranylcypromine; Tretinoin; Up-Regulation

Embryonal carcinoma is a histologic subgroup of testicular germ cell tumors (TGCTs), and its cells may follow differentiation lineages in a manner similar to early embryogenesis. To acquire new knowledge about the transcriptional programs operating in this tumor development model, we used 22k oligo DNA microarrays to analyze normal and neoplastic tissue samples from human testis. Additionally, retinoic acid-induced in vitro differentiation was studied in relevant cell lines. We identified genes characterizing each of the known histologic subtypes, adding up to a total set of 687 differentially expressed genes. Among these, there was a significant overrepresentation of gene categories, such as genomic imprinting and gene transcripts associated to embryonic stem cells. Selection for genes highly expressed in the undifferentiated embryonal carcinomas resulted in the identification of 58 genes, including pluripotency markers, such as the homeobox genes NANOG and POU5F1 (OCT3/4), as well as GAL, DPPA4, and NALP7. Interestingly, abundant expression of several of the pluripotency genes was also detected in precursor lesions and seminomas. By use of tissue microarrays containing 510 clinical testicular samples, GAL and POU5F1 were up-regulated in TGCT also at the protein level and hence validated as diagnostic markers for undifferentiated tumor cells. The present study shows the unique gene expression profiles of each histologic subtype of TGCT from which we have identified deregulated components in selected processes operating in normal development, such as WNT signaling and DNA methylation.

Topics: Carcinoma in Situ; Carcinoma, Embryonal; Cell Differentiation; Cell Line, Tumor; DNA Methylation; DNA-Binding Proteins; Galanin; Gene Expression Profiling; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Genes, Homeobox; Humans; Male; Octamer Transcription Factor-3; Oligonucleotide Array Sequence Analysis; Seminoma; Testicular Neoplasms; Tissue Array Analysis; Transcription Factors; Tretinoin; Up-Regulation

Testicular germ cell tumors are the most common form of cancer in young adult males. They result from a derangement of primordial germ cells, and they grow out from a noninvasive carcinoma-in-situ precursor. Since carcinoma in situ can readily be cured by low-dose irradiation, there is a great incentive for non- or minimally invasive methods for detection of carcinoma in situ. We have recently shown that human Tera-2 embryonal carcinoma cells, obtained from a nonseminomatous testicular germ cell tumor, show alternative splicing and alternative promoter use of the platelet-derived growth factor alpha-receptor gene, giving rise to a unique 1.5-kb transcript. In this study we have set up a reverse transcriptase-polymerase chain reaction strategy for characterization of the various transcripts for this receptor. Using this technique, we show that a panel of 18 seminomas and II nonseminomatous testicular germ cell tumors all express the 1.5-kb transcript. In addition, a panel of 27 samples of testis parenchyma with established carcinoma in situ were all found to be positive for the 1.5-kb transcript, while parenchyma lacking carcinoma in situ, placenta, and control semen were all negative. These data show that the 1.5-kb platelet-derived growth factor alpha-receptor transcript can be used as a highly selective marker for detection of early stages of human testicular germ cell tumors.

Topics: Adult; Alkaline Phosphatase; Base Sequence; Biomarkers, Tumor; Carcinoma, Embryonal; Choriocarcinoma; Clone Cells; DNA Primers; Gene Expression; Germinoma; Humans; Male; Molecular Sequence Data; Polymerase Chain Reaction; Receptor, Platelet-Derived Growth Factor alpha; Receptors, Platelet-Derived Growth Factor; Seminiferous Tubules; Seminoma; Teratoma; Testicular Neoplasms; Testis; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured