Proteomic profiling of cisplatin-resistant and cisplatin-sensitive germ cell tumour cell lines using quantitative mass spectrometry.

PURPOSE: Advanced testicular germ cell tumours (GCT) generally have a good prognosis owing to their unique sensitivity towards cisplatin-based chemotherapies. However, cisplatin-resistant GCT have a poor outcome. Further studies are mandatory to better understand resistance mechanisms and develop therapeutic strategies for refractory GCTs. METHODS: Protein levels in cisplatin-resistant GCT cell lines of NTERA-2, NCCIT and 2102EP were analyzed by quantitative proteomic mass spectrometry (MS) in combination with stable isotope labelling by amino acids in cell culture (SILAC). Differentially abundant protein markers of acquired cisplatin resistance were validated by Western blotting. Comprehensive bioinformatical annotation using gene set enrichment analyses (GSEA) and STRING interaction analysis were performed to identify commonly affected pathways in cisplatin resistance and the data were compared to the GCT cohort of the 'The Cancer Genome Atlas'. RESULTS: A total of 4375 proteins were quantified by MS, 144 of which were found to be differentially abundant between isogenic resistant and sensitive cell line pairs (24 proteins for NTERA-2, 60 proteins for NCCIT, 75 proteins for 2102EP). Western blotting confirmed regulation of key resistance-associated proteins (CBS, ANXA1, LDHA, CTH, FDXR). GSEA revealed a statistically significant enrichment of DNA repair-associated proteins in all three resistant cell lines and specific additional processes for individual cell lines. CONCLUSION: High resolution MS combined with SILAC is a powerful tool and 144 significantly deregulated proteins were found in cisplatin-resistant GCT cell lines. Our study provides the largest proteomic in vitro library for cisplatin resistance in GCT, yet, enabling further studies to develop new treatment options for patients with refractory GCT.

Clinical characteristics, treatment patterns and relapse in patients with clinical stage IS testicular cancer.

PURPOSE: Clinical stage I (CSI) testicular germ cell tumors (TGCT) represents disease confined to the testis without metastasis and CSIS is defined as persistently elevated tumor markers (TM) after orchiectomy, indicating subclinical metastatic disease. This study aims at assessing clinical characteristics and oncological outcome in CSIS. METHODS: Data from five tertiary referring centers in Germany were screened. We defined correct classification of CSIS according to EAU guidelines. TM levels, treatment and relapse-free survival were assessed and differences between predefined groups (chemotherapy, correct/incorrect CSIS) were analyzed with Fisher's exact and Chi-square test. RESULTS: Out of 2616 TGCT patients, 43 (1.6%) were CSIS. Thereof, 27 were correctly classified (cCSIS, 1.03%) and 16 incorrectly classified (iCSIS). TMs that defined cCSIS were in 12 (44.4%), 10 (37%), 3 (11.1%) and 2 (7.4%) patients AFP, ß-HCG, AFP plus ß-HCG and LDH, respectively. In the cCSIS group, six patients were seminoma and 21 non-seminoma. Treatment consisted of active surveillance, carboplatin-mono AUC7 and BEP (bleomycin, etoposide and cisplatin). No difference between cCSIS and iCSIS with respect to applied chemotherapy was found (p = 0.830). 5-year relapse-free survival was 88.9% and three patients (11%) in the cCSIS group relapsed. All underwent salvage treatment (3xBEP) with no documented death. CONCLUSION: Around 1% of all TGCT were classified as cCSIS patients. Identification of cCSIS is of critical importance to avoid disease progression and relapses by adequate treatment. We report a high heterogeneity of treatment patterns, associated with excellent long-term survival irrespective of the initial treatment approach.

N6-Methyladenosine detected in RNA of testicular germ cell tumors is controlled by METTL3, ALKBH5, YTHDC1/F1/F2, and HNRNPC as writers, erasers, and readers.

BACKGROUND: Type II testicular germ cell tumors (GCTs) arise from a common precursor lesion (germ cell neoplasia in situ) and are stratified into seminomas and non-seminomas, which differ considerably in morphology, gene expression, and epigenetic landscape. The N6-methyladenosine (6mA) epigenetic modification is the most abundant modification in mRNA and is also detectable in eukaryotic DNA. The functional role of 6mA is not fully understood, but 6mA residues may influence transcription by affecting splicing, miRNA processing, and mRNA stability. Additionally, the methyl group of 6mA destabilizes Watson-Crick base-pairing affecting RNA structure and protein binding. OBJECTIVES: Here, we analyzed the presence of the 6mA epigenetic modification in germ cells and GCT tissues and cell lines. MATERIALS AND METHODS: We screened for the presence of 6mA in DNA and RNA by immunohistochemistry, mass spectrometry or ELISA-based quantification assays. Additionally, expression of 6mA writer-, eraser- and reader-factors was analyzed by microarrays, qRT-PCR, western blotting and screening of public databases. RESULTS: We demonstrate that 6mA is detectable in RNA, but not DNA, of GCT cell lines and tissues, fibroblasts, and Sertoli cells as well as germ cells of different developmental stages. Based on expression analyses, our results suggest METTL3, ALKBH5, YTHDC1, YTHDF1, YTHDF2 and HNRNPC as main writers, erasers, and readers of the 6mA modification in GCTs. DISCUSSION: Owing to the lack of 6mA in DNA of GCTs, a functional role in regulating DNA transcription can be excluded. Interestingly, expression levels of 6mA regulators are comparable between tumor and normal tissues/cells, suggesting a similar mechanism of 6mA regulation in RNA. Finally, we demonstrate that 6mA levels in RNA increase upon differentiation of GCT cell lines, suggesting a role of 6mA in cell fate decisions. CONCLUSION: In summary, our data provide the starting point for further experiments deciphering the role of 6mA in the RNA of GCTs.

Epigenetic treatment combinations to effectively target cisplatin-resistant germ cell tumors: past, present, and future considerations.

BACKGROUND: Type II germ cell tumors represent the most common solid malignancy in men aged 15-45 years. Despite high cure rates of >90% over all stages, 10-15% of advanced patients develop treatment resistance and potentially succumb to their disease. Treatment of refractory germ cell tumors remains unsatisfactory, and new approaches are needed to further improve outcomes. OBJECTIVES: With this narrative review, we highlight epigenetic mechanisms related to resistance to standard systemic treatment, which may act as promising targets for novel combined epigenetic treatment approaches. MATERIALS AND METHODS: A comprehensive literature search of PubMed and MEDLINE was conducted to identify original and review articles on resistance mechanisms and/or epigenetic treatment of germ cell tumors in vitro and in vivo. Review articles were hand-searched to identify additional articles. RESULTS: Distinct epigenetic phenomena have been linked to chemotherapy resistance in germ cell tumors, among which DNA hypermethylation, histone acetylation, and bromodomain proteins appear as promising targets for therapeutic exploitation. Inhibitors of key regulators, for example DNA methyltransferases (e.g. decitabine, guadecitabine), histone deacetylases (e.g. romidepsin), and bromodomain proteins (e.g. JQ1) decreased cell viability, triggered apoptosis, and growth arrest. Additionally, these epigenetic drugs induced differentiation and led to loss of pluripotency and re-sensitization towards cisplatin in cell lines and animal models. DISCUSSION: Epigenetic treatments hold promise to (i) reduce the treatment burden of and (ii) overcome resistance to standard cisplatin-based chemotherapy. Combined approaches may enhance activity, while the ideal target and treatment combination of epigenetic drugs, either with another epigenetic agent or conventional cytotoxic agents need to be defined. CONCLUSION: Epigenetic (combination) treatment for germ cell tumors should be further explored in pre-clinical and clinical research for its potential to further improve germ cell tumor treatment.

Are testicular mast cells involved in the regulation of germ cells in man?

Protease activated receptor-2 (PAR-2) is the receptor for the prototype mast cell product tryptase. PAR-2 expression by cells of the human germinal epithelium was reported, but the exact cellular sites of testicular expression remained unknown. That became of interest, because mast cells, expressing tryptase, were found in the walls of seminiferous tubules of patients suffering from sub- and infertility. This location suggested that mast cells via tryptase might be able to influence PAR-2-expressing cells in the germinal epithelium. To explore these points, we used testicular paraffin-embedded sections for immunohistochemistry. PAR-2-positive cells were mostly basally located cells of the seminiferous epithelium, namely spermatogonia. Some stained for the receptor for GDNF (GFRalpha-1), and possibly represent spermatogonial stem cells (SSCs). As true human SSCs could not be examined, we turned to TCam-2 seminoma cells, expressing PAR-2 and stem cell markers, including GFRalpha-1. TCam-2 cells robustly responded to stimulation with a specific PAR-2 agonist (SLIGKV) by increased intracellular Ca(2+) levels. Recombinant tryptase and trypsin, but not a control peptide (VKGILS) evoked this response, implying functional PAR-2. Video imaging and caspase 3/7 assays showed that SLIGKV and tryptase prevented spontaneous apoptosis and increased proliferation of TCam-2 cells. The expression of the marker of pluripotency OCT3/4 was unchanged upon activation of PAR-2, suggesting that the stem cell-like character is not changed. Furthermore, human germ cell cancers were examined. A subset of seminoma and carcinoma in situ samples expressed PAR-2, indicating that yet unknown subgroups exist. Collectively, the descriptive data obtained in human testicular sections, in germ cell cancers and the functional results in TCam-2 cells imply a trophic role of mast cell-derived tryptase for human germ cells. This may be relevant for subtypes of human germ cell cancers, and possibly SSCs. It also raises the possibility that PAR-2 agonists might be useful for the in vitro propagation of human SSCs.

TGF-ß1, EGF and FGF4 synergistically induce differentiation of the seminoma cell line TCam-2 into a cell type resembling mixed non-seminoma.

Malignant germ-cell tumours arise from a neoplastic precursor, the carcinoma in situ, and develop into seminomas and/or non-seminomas (embryonal carcinomas, teratomas, yolk-sac tumours and choriocarcinomas). Based on histological and clinical findings, it has been postulated that seminomas can eventually transform into non-seminomas. Here, we used the cell line TCam-2 as model for seminomas and interrogated their differentiation potential. We demonstrate that TCam-2 cells are able to differentiate into mixed non-seminomatous lineages after supplementing the media with TGF-ß1, EGF and FGF4. On a molecular level, the differentiation is initiated by repression of BMP/SMAD signalling. As a consequence, BLIMP1, a molecule known to inhibit the differentiation of murine primordial germ cells, is down-regulated and differentiation-inhibiting histone modifications are lost. The appearance of multinucleated giant cells and the expression of marker genes indicate that cells differentiate predominantly into extra-embryonic choriocarcinoma-like cells. This is most likely due to the presence of components of the Hippo pathway, TEAD4 and YAP1. These molecules have been described to trigger extra-embryonic fate determination in the murine system. This study supports the model that seminomas indeed have an intrinsic ability to transform into a non-seminoma. In addition, the data suggest that the transformation does not require an additional mutation, but can be triggered by changes in the tumour microenvironment.

The role of BLIMP1 and its putative downstream target TFAP2C in germ cell development and germ cell tumours.

During the past years, much information has been gathered regarding the genetic and epigenetic programmes leading to the specification and maintenance of primordial germ cells. Expression of the transcriptional regulator BLIMP1 (PRDM1) is regarded as the main event in germ cell specification. BLIMP1 induces a set of target genes, one of them being transcription factor TFAP2C (AP-2γ, Tcfap2c). In murine loss of function models Blimp1 and Tcfap2c share an identical phenotype, strengthening the assumption that they act in the same pathway. One major role of this pathway is the inhibition of somatic differentiation in germ cells. BLIMP1 and TFAP2C are also expressed in carcinoma in situ (CIS, IGCNU, TIN) and in seminoma. As pointed out herein, the presence of both proteins helps to explain the undifferentiated nature of these germ cell tumours. In addition, we performed a meta-analysis of high-throughput datasets searching for TFAP2C/Tcfap2c target genes. This analysis leads us to suggest Nanos3, Dmrt1 and Dnmt3b as potential TFAP2C/Tcfap2c target genes with relevance to germ cell development and germ cell tumours.

TCam-2 but not JKT-1 cells resemble seminoma in cell culture.

Of all malignancies diagnosed in men between 17 and 45 years of age, 60% are germ cell tumors (GCT). GCT arise from carcinoma in situ cells, which are thought to originate from a transformed fetal germ cell, the gonocyte. Seminoma together with embryonal carcinoma represent the most frequent subtypes of GCT. However, the nature of the molecular pathways involved in seminoma formation remains elusive. Therefore, analysis of appropriate cell culture systems is an important prerequisite for further understanding of the etiology of this tumor entity. Although several cell lines for embryonal carcinoma have been established and analyzed, so far only two cell lines from seminoma patients have been reported. In the present study, we have analyzed these seminoma cell lines (TCam-2 and JKT-1) and compared the gene-expression profiles with those of normal tissue and of seminoma and embryonal carcinoma by using DNA Array technology. We have found that TCam-2 clusters with the group of classical seminoma, whereas JKT-1 clusters with the group of embryonal carcinoma. Using reverse transcription/polymerase chain reaction, Western blot, and immunohistochemistry, we have confirmed the seminoma-like nature of TCam-2, whereas JKT-1 lacks expression for most of the genes detectable in GCTs, thus making doubtful the germ cell nature of this cell line. The data represent the first genome-wide expression analysis of the two cell lines and comparison/clustering with subgroups of germ cell tumors. Only TCam-2 seems to represent a suitable in vitro model for seminoma.

c-KIT is frequently mutated in bilateral germ cell tumours and down-regulated during progression from intratubular germ cell neoplasia to seminoma.

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.

[Embryonal germ cells and germ cell tumors]. / Embryonale Keimzellen und Keimzelltumoren.

Testicular germ cell tumors comprise of group of pluripotent tumors including seminomas and nonseminomas, arise from intratubular germ cell neoplasia and originate from the primordial germ cells/ gonocytes. Many well characterized markers of embryonic stem cells including CD9, PODXL and centromere-specific histone-H3-like protein CENPA are consistently expressed in TGCTs. In embryonic stem cells, pluripotency and self renewal capacities are provided by a network of OCT3/4, NANOG and SOX2. In testicular germ cell tumors, pluripotency genes OCT3/4 und NANOG are upregulated both, in seminomas and non-seminomas, while SOX2 is differentially upregulated in embryonal carcinomas only. Similar to embryonic stem cells, most histological elements of type II GCTs are sensitive to chemotherapy and irradiation. Furthermore, all invasive TGCTs show a consistent gain of the short arm of chromosome 12, as found in ES cells upon extensive in vitro culturing. Moreover, the genetic constitution of testicular germ cell tumors can also be linked to characteristics of embryonic stem cells, likely related to their specific inability to repair DNA damage and their high sensitivity to apoptotic cell death. In conclusion, testicular germ cell tumors represent embryonic cancers found in adults. Both the seminomas and nonseminomas have their specific population of stem cells representative of the primordial germ cells/gonocytes and for embryonic stem cells, respectively.