Pediatric Mesothelioma With ALK Fusions: A Molecular and Pathologic Study of 5 Cases.

Pediatric mesotheliomas are rare and their pathogenesis remains undefined. In this study, we report 5 cases of malignant mesothelioma in children, characterized by fusions involving the anaplastic lymphoma kinase (ALK) gene. Four cases occurred in females involving the abdominal cavity and were characterized by a pure epithelioid morphology. The fifth arose in the tunica vaginalis of a 15-year-old male and displayed a biphasic epithelioid-sarcomatoid phenotype. All cases demonstrated the classic morphologic and immunohistochemical features of malignant mesothelioma, including tubulopapillary architecture and cuboidal epithelioid cells with eosinophilic cytoplasm and uniform nuclei with vesicular chromatin. Immunohistochemically, all cases showed labeling for ALK, cytokeratins, WT1, and calretinin, while lacking expression of adenocarcinoma immunomarkers. Four cases demonstrated weak-moderate labeling for PAX8 protein, which resulted in diagnostic challenges with primary peritoneal serous carcinoma. The ALK genetic abnormalities were investigated by a combination of molecular methods. Archer FusionPlex was performed in 2 cases, showing fusions between ALK with either STRN or TPM1 genes, resulting in a transcript that retained the ALK kinase domain. One case was further studied by DNA targeted sequencing, but no additional genetic alterations were observed. In 1 case, cytogenetic analysis showed the presence of a t(2;15)(p23;q22) and fluorescence in situ hybridization confirmed the ALK gene break-apart. In the remaining 2 cases, ALK gene rearrangements were demonstrated by fluorescence in situ hybridization. Unlike adult mesotheliomas, which are tightly linked to asbestos exposure, often show loss of BAP1 expression and have complex karyotypes, ALK-rearranged mesothelioma appears to be similar to other fusion-positive mesotheliomas, such as those harboring EWSR1/FUS-ATF1 fusions, sharing significant morphologic overlap, occurring in young patients and displaying a simple, translocation-driven genetic profile.

Differential expression of DNA methyltransferases and demethylases among the various testicular germ cell tumor subtypes.

Aim: Characterize DNA methyltransferases/demethylases expression in testicular germ cell tumors (TGCTs). Methods:In silico analysis of TCGA database, assessment of transcript levels of most relevant enzymes in four TGCT cell lines and validation in patient cohort (real-time quantitative polymerase chain reaction; immunohistochemistry). Results:DNMT3A, DNMT3B and TET2 were the most differentially expressed between seminomas (SEs) and nonseminomas (NSs). DNMT3B was significantly overexpressed in NS-related cell lines, and the opposite was found for TET2. Significantly higher DNMT3A/B mRNA expression was observed in NS, indicating a role for de novo methylation in reprogramming. Significantly higher TET2 protein expression was observed in SEs, suggesting active demethylation contributes for SE hypomethylated state. More differentiated histologies disclosed distinct expression patterns. Conclusion: DNA-modifying enzymes are differentially expressed between TGCT subtypes, influencing reprogramming and differentiation.

Low level expression of human telomerase reverse transcriptase predicts cancer-related death and progression in embryonal carcinoma.

INTRODUCTION: hTERT (human telomerase reverse transcriptase) is a catalytic subunit of the enzyme telomerase and has a role in cell proliferation, cellular senescence, and human aging. MATERIALS AND METHODS: The purpose of this study was to evaluate the expression and significance of hTERT protein expression as a prognostic marker in different histological subtypes of testicular germ cell tumors (TGCTs), including 46 embryonal carcinomas, 46 yolk sac tumors, 38 teratomas, 84 seminomas as well as two main subtypes of seminomas and non-seminomas using tissue microarray (TMA) technique. RESULTS: The results showed that there is a statistically significance difference between the expression of hTERT and various histological subtypes of TGCTs (P < 0.001). In embryonal carcinoma, low level expression of hTERT protein was significantly associated with advanced pT stage (P = 0.023) as well as tunica vaginalis invasion (P = 0.043). Moreover, low level expression of hTERT protein was found to be a significant predictor of worse DSS (log rank: P = 0.011) and PFS (log rank: P = 0.011) in the univariate analysis. Additionally, significant differences were observed (P =0.021, P =0.018) with 5-year survival rates for DSS and PFS of 66% and 70% for moderate as compared to 97% and 97% for high hTERT protein expression, respectively. CONCLUSION: We showed that hTERT protein expression was associated with more aggressive tumor behavior in embryonal carcinoma patients. Also, hTERT may be a novel worse prognostic indicator of DSS or PFS, if the patients are followed up for more time periods.

Synthesis of a novel platinum(II) complex with 6,7-dichloro-5,8-quinolinedione and the study of its antitumor mechanism in testicular seminoma.

A new platinum(II) complex, [Pt(ClClQ)(DMSO)Cl] (1), utilizing 6,7-dichloro-5,8-quinolinedione (ClClQ) as a ligand, has been synthesized and fully characterized. Single-crystal X-ray diffraction and other spectroscopic and analytical methods revealed that the coordination geometry of Pt(II) in complex 1 can also be described as a four-coordinated square planar geometry. The aim of the study was to explore the in vitro anticancer properties of complex 1. Our studies showed that complex 1 can regulate the viability of testicular seminoma cells in vitro, including cell proliferation and apoptosis. We further observed negative regulation by complex 1 of the expression levels of the key elements in the phosphoinositide-3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3ß (GSK3ß) pathway, including phosphorylated phosphoinositide-3 kinase (p-PI3K), phosphorylated protein kinase B(p-Akt) and phosphorylated glycogen synthase kinase-3ß (p-GSK3ß). Moreover, the negative effect of complex 1 was reversed by LiCl, a GSK3ß-specific inhibitor of the PI3K signaling pathway. Meanwhile, the levels of Bcl2 associated death promoter (Bad), cytochrome c, active-caspase-3 and active-caspase-9 increased significantly. In conclusion, we observed that complex 1 can regulate the viability of testicular seminoma cells through the PI3K/Akt/GSK3ß signaling pathway and the mitochondria-mediated apoptotic pathway in vitro, and thus, complex 1 may have potential for use as a drug in the treatment of testicular germ cell tumors.

TdT expression in germ cell tumours: a possible immunohistochemical cross-reaction and diagnostic pitfall.

AIMS: Very recent papers proposed a possible role for the expression of terminal deoxynucleotidyl transferase (TdT) in the tumourigenesis of gonadal and extragonadal germ cell-derived tumours (GCTs). Our multicentric study evaluated the magnitude of the immunoreactivity for TdT in GCTs, encompassing seminoma, dysgerminoma, mature teratoma and mixed GCTs. METHODS AND RESULTS: The histological series was stained with both monoclonal and polyclonal antibodies, yielding a positivity of 80% of cases with well-defined nuclear reactivity. A significant difference in staining intensity between monoclonal and polyclonal antibodies was observed (p=0.005). However, exploiting western blot and more innovative proteomic approaches, no clear-cut evidence of the TdT protein was observed in the neoplastic tissues of the series. CONCLUSIONS: Alternatively to the pathogenetic link between TdT expression and GCTs tumourigenesis, we hypothesised the occurrence of a spurious immunohistochemical nuclear cross-reaction, a well-known phenomenon with important implications and a possible source of diagnostic pitfalls in routine practice for pathologists.

Expression of terminal deoxynucleotidyl transferase (TdT) in classical seminoma: a potential diagnostic pitfall.

Seminomas are the most frequent testicular tumors and in spite of specific markers some histological subtypes can be diagnostically challenging due to the potential overlap of morphologic features and a variant antigen expression. Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase present in hematogones, thymic T cells, lymphoblastic lymphoma/leukemia (LBL), and in some cases of acute myeloid leukemia but so far has not been described to be expressed in seminomas. After observing a reactivity of TdT in one case of seminoma, we analyzed ten additional tumors by immunohistochemistry to determine their spectrum of reactivity for TdT. In all seminoma cases investigated (10/10) as well as in two tumor-associated germ cell neoplasias in situ (2/2) the TdT staining intensity was variable but was often moderate to strong and restricted to the nucleus. We conclude that TdT expression in seminomas could represent a diagnostic pitfall in the differential diagnosis of LBL, particularly because both may lack CD45 and/or CD20 expression and-concerning relapse in long-term survivors of testicular cancer-LBL is the most frequent secondary neoplasm in the patients.

Expression patterns of HENMT1 and PIWIL1 in human testis: implications for transposon expression.

This study aimed to define the expression patterns of HENMT1 and PIWI proteins in human testis and investigate their association with transposon expression, infertility sub-type or development of testicular germ cell tumours (TGCTs). Testis biopsies showing normal spermatogenesis were used to identify normal localisation patterns of HENMT1 and PIWIL1 by immunolocalisation and RT-PCR after laser microdissection. 222 testis biopsies representing normal spermatogenesis, hypospermatogenesis, spermatogenic arrests, Sertoli cell-only (SCO) tumours and TGCTs were analysed by RT-qPCR for expression of HENMT1/PIWIL1/PIWIL2/PIWIL3/PIWIL4 and LINE-1 Additionally, HENMT1-overexpressing TCam2 seminoma cell lines were analysed for the same parameters by RT-qPCR. We found that HENMT1 and PIWIL1 are coexpressed in pachytene spermatocytes and spermatids. Expression of HENMT1, PIWIL1 and PIWIL2 was mainly dependent on germ cell content but low levels of expression were also detected in some SCO samples. Levels of HENMT1, PIWIL1 and PIWIL2 expression were low in TGCT. Samples with HENMT1, PIWIL2 and PIWIL4 expression showed significantly (P < 0.05) lower transposon expression compared to samples without expression in the same histological group. HENMT1-overexpressing TCam2 cells showed lower LINE-1 expression than empty vector-transfected control lines. Our findings support that the transposon-regulating function of the piRNA pathway found in the mouse is conserved in adult human testis. HENMT1 and PIWI proteins are expressed in a germ-cell-specific manner and required for transposon control.

An Overview on Predictive Biomarkers of Testicular Germ Cell Tumors.

Testicular germ cell tumors (TGCTs) are frequent solid malignant tumors and cause of death in men between 20-40 years of age. Genetic and environmental factors play an important role in the origin and development of TGCTs. Although the majority of TGCTs are responsive to chemotherapy, about 20% of patient presents incomplete response or tumors relapse. In addition, the current treatments cause acute toxicity and several chronic collateral effects, including sterility. The present mini-review collectively summarize the most recent findings on the new discovered molecular biomarkers such as tyrosine kinases, HMGAs, Aurora B kinase, and GPR30 receptor predictive of TGCTs and as emerging new possible molecular targets for therapeutic strategies. J. Cell. Physiol. 232: 276-280, 2017. © 2016 Wiley Periodicals, Inc.

Wilms tumor gene 1 (WT1), TP53, RAS/BRAF and KIT aberrations in testicular germ cell tumors.

PURPOSE: Wilms tumor gene 1 (WT1), a zinc-finger transcription factor essential for testis development and function, along with other genes, was investigated for their role in the pathogenesis of testicular germ cell tumors (TGCT). METHODS: In total, 284 TGCT and 100 control samples were investigated, including qPCR for WT1 expression and BRAF mutation, p53 immunohistochemistry detection, and massively parallel amplicon sequencing. RESULTS: WT1 was significantly (p < 0.0001) under-expressed in TGCT, with an increased ratio of exon 5-lacking isoforms, reaching low levels in chemo-naïve relapsed TGCT patients vs. high levels in chemotherapy-pretreated relapsed patients. BRAF V600E mutation was identified in 1% of patients only. p53 protein was lowly expressed in TGCT metastases compared to the matched primary tumors. Of 9 selected TGCT-linked genes, RAS/BRAF and WT1 mutations were frequent while significant TP53 and KIT variants were not detected (p = 0.0003). CONCLUSIONS: WT1 has been identified as a novel factor involved in TGCT pathogenesis, with a potential prognostic impact. Distinct biologic nature of the two types of relapses occurring in TGCT has been demonstrated. Differential mutation rate of the key TGCT-related genes has been documented.

DNA methyltransferase-3 like protein expression in various histological types of testicular germ cell tumor.

OBJECTIVE: DNA methyltransferase 3-like plays an important role in germ cell development. The aim of this study was to analyse the DNA methyltransferase 3-like protein expression in testicular germ cell tumors. METHODS: The immunohistochemical expression of DNA methyltransferase 3-like was examined in 86 testicular germ cell tumor specimens in various clinical settings. The association between DNA methyltransferase 3-like expression and disease stage was analyzed. RESULTS: DNA methyltransferase 3-like was strongly expressed in seven of the eight pure embryonal carcinomas (87.5%). Partial DNA methyltransferase 3-like expression was observed in 6 of 23 (26.1%) pure seminomas. Various degrees of DNA methyltransferase 3-like expression was observed in all four pure yolk sac tumors, of which three were prepubertal yolk sac tumors. In mixed germ cell tumors, DNA methyltransferase 3-like protein was expressed in various degrees in elements of the embryonal carcinoma (14/18, 77.8%), seminoma (4/11, 36.4%), teratoma (4/7, 57.1%) and choriocarcinoma (3/3, 100%) but not in the yolk sac tumors (0/4). When DNA methyltransferase 3-like expression was analyzed according to disease stages, it was significantly correlated with advanced seminoma rather than Stage I seminoma (46.2 vs. 0%, P = 0.019), whereas there was no significant difference in the DNA methyltransferase 3-like-positive proportion between Stage I and advanced disease in the mixed germ cell tumors. CONCLUSIONS: Our findings suggest that DNA methyltransferase 3-like protein may play roles not only in the development of embryonal carcinoma but also in the development of advanced pure seminoma and pure yolk sac tumor.

Single-nucleotide polymorphism in the 5-α-reductase gene (SRD5A2) is associated with increased prevalence of metabolic syndrome in chemotherapy-treated testicular cancer survivors.

PURPOSE: Chemotherapy-treated testicular cancer survivors are at risk for development of the metabolic syndrome, especially in case of decreased androgen levels. Polymorphisms in the gene encoding steroid 5-α-reductase type II (SRD5A2) are involved in altered androgen metabolism. We investigated whether single-nucleotide polymorphisms (SNPs) rs523349 (V89L) and rs9282858 (A49T) in SRD5A2 are associated with cardiometabolic status in testicular cancer survivors. METHODS: In 173 chemotherapy-treated testicular cancer survivors, hormone levels and cardiometabolic status were evaluated cross-sectionally (median 5 years [range 3-20] after chemotherapy) and correlated with SNPs in SRD5A2. RESULTS: The metabolic syndrome was more prevalent in survivors who were homozygous or heterozygous variant for SRD5A2 rs523349 compared to wild type (33% versus 19%, P = 0.032). In particular, patients with lower testosterone levels (<15 nmol/l) and a variant genotype showed a high prevalence of the metabolic syndrome (66.7%). Mean intima-media thickness of the carotid artery and urinary albumin excretion, both markers of vascular damage, were higher in the group of survivors homozygous or heterozygous variant for rs523349 (0.62 versus 0.57 mm, P = 0.026; 5.6 versus 3.1 mg/24 h, P = 0.017, respectively). No association was found between cardiometabolic status and SNP rs9282858 in SRD5A2. CONCLUSION: Metabolic syndrome develops more frequently in testicular cancer survivors homozygous or heterozygous variant for SNP rs523349 in SRD5A2. Altered androgen sensitivity appears to be involved in the development of adverse metabolic and vascular changes in testicular cancer survivors and is a target for intervention.

Geranylgeraniol enhances testosterone production via the cAMP/protein kinase A pathway in testis-derived I-10 tumor cells.

Testosterone levels in men decrease with age; this decline has been linked to various diseases and can shorten life expectancy. Geranylgeraniol (GGOH) is an isoprenoid found in plants that plays an important role in several biological processes; however, its role in steroidogenesis is unknown. Here, we report that GGOH enhances the production of testosterone and its precursor progesterone in testis-derived I-10 tumor cells. GGOH induced protein kinase A (PKA) activity and increased cAMP levels and was found to regulate cAMP/PKA signaling by activating adenylate cyclase without altering phosphodiesterase activity. GGOH also stimulated mRNA and protein levels of steroidogenic acute regulatory protein, a downstream effector in the cAMP/PKA pathway. These results demonstrate that GGOH enhances steroidogenesis in testis-derived cells by modulating cAMP/PKA signaling. Our findings have potential applications for the development of therapeutics that increase testosterone levels in aging men.

Rare inactivating PDE11A variants associated with testicular germ cell tumors.

Germline inactivating mutations of isoform 4 of phosphodiesterase (PDE) 11A (coded by the PDE11A gene) have been associated with familial adrenocortical tumors and familial testicular cancer. Testicular tissue is unique in expressing all four isoforms of PDE11A. In a prior candidate gene study of 94 familial testicular germ cell tumor (TGCT) subjects, we identified a significant association between the presence of functionally abnormal variants in PDE11A and familial TGCT risk. To validate this novel observation, we sequenced the PDE11A coding region in 259 additional TGCT patients (both familial and sporadic) and 363 controls. We identified 55 PDE11A variants: 20 missense, four splice-site, two nonsense, seven synonymous, and 22 intronic. Ten missense variants were novel; nine occurred in transcript variant 4 and one in transcript variant 3. Five rare mutations (p.F258Y, p.G291R, p.V820M, p.R545X, and p.K568R) were present only in cases and were significantly more common in cases vs controls (P=0.0037). The latter two novel variants were functionally characterized and shown to be functionally inactivating, resulting in reduced PDE activity and increased cAMP levels. In further analysis of this cohort, we focused on white participants only to minimize confounding due to population stratification. This study builds upon our prior reports implicating PDE11A variants in familial TGCT, provides the first independent validation of those findings, extends that work to sporadic testicular cancer, demonstrates that these variants are uncommonly but reproducibly associated with TGCT, and refines our understanding regarding which specific inactivating PDE11A variants are most likely to be associated with TGCT risk.

Proteomic analysis of 3-MCPD and 3-MCPD dipalmitate toxicity in rat testis.

Thermal treatment of foodstuff containing fats and salt promotes the formation of 3-chloropropane-1,2-diol (3-MCPD) and its fatty acid esters. 3-MCPD-exposed rats develop testicular lesions and Leydig cell tumors. 3-MCPD and 3-MCPD ester toxicity is thought to be caused by 3-MCPD and its metabolites, since 3-MCPD esters are hydrolyzed in the gut. Inhibition of glycolysis is one of the few known molecular mechanisms of 3-MCPD toxicity. To obtain deeper insight into this process, a comparative proteomic approach was chosen, based on a 28-days repeated-dose feeding study with male Wistar rats. Animals received equimolar doses of 3-MCPD or 3-MCPD dipalmitate. A lower dose of 3-MCPD dipalmitate was also administered. Absence of histopathological changes supported an analysis of early cellular disturbance. Testes were analyzed by two-dimensional gel electrophoresis followed by mass-spectrometric protein identification. Data provide a comprehensive overview of proteomic changes induced by 3-MCPD and 3-MCPD dipalmitate in rat testis in an early phase of organ impairment. Results are compatible with known 3-MCPD effects on reproductive function, substantially extend our knowledge about cellular responses to 3-MCPD and support the hypothesis that toxicity of 3-MCPD and 3-MCPD esters is mediated via common effectors. DJ-1 was identified as a candidate marker for 3-MCPD exposure.

Serum metallothionein in patients with testicular cancer.

PURPOSE: Metallothioneins (MTs) have been disclosed as a useful diagnostic factor for tumour progression and drug resistance in a variety of malignancies. Increased levels of MT in blood serum have been found in patients with several types of cancer, but there is no available information on serum MT levels in patients with testicular germ cell tumour (TGCT). The aim of the study was to determine MT levels in serum of patients with TGCT and to evaluate the portion of platinum (Pt) that binds to MT after cisplatin administration since MTs could be involved in drug resistance. METHODS: Concentration of total MT was determined in serum of 25 men with newly diagnosed TGCT by differential pulse voltammetry. The fractionation of serum was carried out by size exclusion high-performance liquid chromatography (SE-HPLC), while concentration of Pt in collected fractions was determined by inductively coupled plasma mass spectrometry. RESULTS: Concentration of serum MT was significantly higher in TGCT patients than in healthy volunteers. The results of SE-HPLC analysis showed that only a small amount of Pt was bound to proteins in the area of MT elution. CONCLUSIONS: Significant increase in MT levels in individuals with TGCT indicates certain health problem and, in combination with other commonly used diagnostic tools, could improve early diagnosis.

EMMPRIN/CD147-encriched membrane vesicles released from malignant human testicular germ cells increase MMP production through tumor-stroma interaction.

BACKGROUND: Elevated levels of EMMPRIN/CD147 in cancer tissues have been correlated with tumor progression but the regulation of its expression is not yet understood. Here, the regulation of EMMPRIN expression was investigated in testicular germ cell tumor (TGCTs) cell lines. METHODS: EMMPRIN expression in seminoma JKT-1 and embryonal carcinoma NT2/D1 cell lines was determined by Western blot, immunofluorescence and qRT-PCR. Membrane vesicles (MVs) secreted from these cells, treated or not with EMMPRIN siRNA, were isolated by differential centrifugations of their conditioned medium. MMP-2 was analyzed by zymography and qRT-PCR. RESULTS: The more aggressive embryonic carcinoma NT2/D1 cells expressed more EMMPRIN mRNA than the seminoma JKT-1 cells, but surprisingly contained less EMMPRIN protein, as determined by immunoblotting and immunostaining. The protein/mRNA discrepancy was not due to accelerated protein degradation in NT2/D1 cells, but by the secretion of EMMPRIN within MVs, as the vesicles released from NT2/D1 contained considerably more EMMPRIN than those released from JKT-1. EMMPRIN-containing MVs obtained from NT2/D1, but not from EMMPRIN-siRNA treated NT2/D1, increased MMP-2 production in fibroblasts to a greater extent than those from JKT-1 cells. CONCLUSION AND GENERAL SIGNIFICANCE: The data presented show that the more aggressive embryonic carcinoma cells synthesize more EMMPRIN than seminoma cells, but which they preferentially target to secreted MVs, unlike seminoma cells which retain EMMPRIN within the cell membrane. This cellular event points to a mechanism by which EMMPRIN expressed by malignant testicular cells can exert its MMP inducing effect on distant cells within the tumor microenvironment to promote tumor invasion. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Identification of ZDHHC14 as a novel human tumour suppressor gene.

Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase-dependent apoptotic pathway and heterozygous knockout of ZDHHC14 increased [CORRECTED] cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down-regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer.

Fatty acid synthase overexpression in adult testicular germ cell tumors: potential role in the progression of non-seminomatous germ cell tumors.

Overexpression of fatty acid synthase (FASN), which is a key enzyme responsible for the endogenous synthesis of fatty acids, and its association with multistep progression have been demonstrated in various human malignant tumors. We aimed to clarify the potential role of FASN overexpression in the development and progression of adult testicular germ cell tumors (TGCTs). From the primary sites of a cohort of 113 TGCT cases, we obtained 221 histological components: 53 intratubular germ cell neoplasias, unclassified (IGCNUs), 84 seminomas, 32 embryonal carcinomas, seven choriocarcinomas, 21 yolk sac tumors, and 24 teratomas. Samples were analyzed for overexpression of FASN by immunohistochemistry. Intensities of immunoreactivity and the fraction of positive cells were classified into each four categories (intensity, 0 to 3; fraction, 0-10 % = 1, 11-50 % = 2, 51-80 % = 3, and >80 % = 4). The overall score was determined by multiplication of both scores and overall scores greater than 6 were considered FASN overexpression. On a component basis, FASN overexpression was detected in 8 % of seminomas but not in IGCNUs (0 %) and was detected frequently in non-seminomatous germ cell tumors (NSGCTs) (88 % of embryonal carcinomas, all choriocarcinomas, 81 % of yolk sac tumors, and 54 % of teratomas). There were no cases of a mixed tumor (i.e., a tumor with multiple histological components) that overexpressed FASN in seminoma components but not in co-existing NSGCT components, suggesting sequential progression. Our immunohistochemical data suggest that FASN overexpression occurs as a late event during the progression from IGCNUs/seminomas to NSGCTs.

Tumor-suppressive function of protein-tyrosine phosphatase non-receptor type 23 in testicular germ cell tumors is lost upon overexpression of miR142-3p microRNA.

Protein-tyrosine phosphatase non-receptor type 23 (PTPN23) is a candidate tumor suppressor involved in the tumorigenesis of various organs. However, its physiological role(s) and detailed expression profile(s) have not yet been elucidated. We investigated the function and regulation of PTPN23 in the formation of testicular germ cell tumors (TGCTs). Expression of PTPN23 in human TGCT cell lines was significantly lower than that in spermatogonial stem cells in mice. Overexpression of PTPN23 in NEC8, a human TGCT cell line, suppressed soft agar colony formation in vitro and tumor formation in nude mice in vivo. These data indicate that PTPN23 functions as a tumor suppressor in TGCTs. Multiple computational algorithms predicted that the 3' UTR of human PTPN23 is a target for miR-142-3p. A luciferase reporter assay confirmed that miR-142-3p bound directly to the 3' UTR of PTPN23. Introduction of pre-miR-142 in the PTPN23 transfectant of NEC8 led to suppressed expression of PTPN23 and increased soft agar colony formation. Quantitative RT-PCR data revealed a significantly higher expression of miR-142-3p in human seminomas compared with normal testes. No difference in mRNA expression between seminoma and non-seminoma samples was detected by in situ hybridization. Both quantitative RT-PCR and immunohistochemical analyses revealed that PTPN23 expression was significantly lower in TGCTs than in normal testicular tissues. Finally, a lack of PTPN23 protein expression in human TGCTs correlated with a relatively higher miR-142-3p expression. These data suggest that PTPN23 is a tumor suppressor and that repression of PTPN23 expression by miR-142-3p plays an important role in the pathogenesis of TGCTs.