Testicular Embryonal Carcinoma Cells Research Articles

Survival of Testicular Pure Embryonal Carcinoma vs. Mixed Germ Cell Tumor Patients across All Stages

Background and Objectives: The impact of pure histological subtypes in testicular non-seminoma germ cell tumors on survival, specifically regarding pure embryonal carcinoma, is not well established. Therefore, this study aimed to test for differences between pure embryonal carcinoma and mixed germ cell tumor patients within stages I, II and III in a large population-based database. Materials and Methods: We relied on the Surveillance, Epidemiology and End Results (SEER) database (2004-2019) to identify testicular pure embryonal carcinoma vs. mixed germ cell tumor patients. Cumulative incidence plots depicted cancer-specific mortality that represented the main endpoint of interest. Multivariable competing risks regression models tested for differences between pure embryonal carcinoma and mixed germ cell tumor patients in analyses addressing cancer-specific mortality and adjusted for other-cause mortality. Results: Of 11,223 patients, 2473 (22%) had pure embryonal carcinoma. Pure embryonal carcinoma patients exhibited lower cancer-specific mortality relative to their mixed germ cell tumor counterparts for both stage III (13.9 vs. 19.4%; p < 0.01) and stage II (0.5 vs. 3.4%, p < 0.01), but not in stage I (0.9 vs. 1.6%, p = 0.1). In multivariable competing risks regression models, pure embryonal carcinoma exhibited more favorable cancer-specific mortality than mixed germ cell tumor in stage III (hazard ratio 0.71, p = 0.01) and stage II (hazard ratio 0.11, p < 0.01). Conclusions: Pure embryonal carcinoma exhibits a more favorable cancer-specific mortality profile relative to mixed germ cell tumor in stage II and III testicular cancers. Consequently, the presence of mixed germ cell tumor elements may be interpreted as a risk factor for cancer-specific survival.

MP40-04 NOVEL SELECTIVE LYSINE-SPECIFIC DEMETHYLASE 1 INHIBITORS, NCL1 AND NCD38, SUPPRESS TESTICULAR TUMOR CELL PROLIFERATION

You have accessJournal of UrologyPenile & Testicular Cancer I (MP40)1 Sep 2021MP40-04 NOVEL SELECTIVE LYSINE-SPECIFIC DEMETHYLASE 1 INHIBITORS, NCL1 AND NCD38, SUPPRESS TESTICULAR TUMOR CELL PROLIFERATION Toshiki Etani, Taku Naiki, Nobuhiko Shimizu, Yusuke Noda, Takashi Nagai, Satoshi Nozaki, Keitaro Iida, Ryosuke Ando, Noriyasu Kawai, Satoru Takahashi, Takayoshi Suzuki, and Takahiro Yasui Toshiki EtaniToshiki Etani More articles by this author , Taku NaikiTaku Naiki More articles by this author , Nobuhiko ShimizuNobuhiko Shimizu More articles by this author , Yusuke NodaYusuke Noda More articles by this author , Takashi NagaiTakashi Nagai More articles by this author , Satoshi NozakiSatoshi Nozaki More articles by this author , Keitaro IidaKeitaro Iida More articles by this author , Ryosuke AndoRyosuke Ando More articles by this author , Noriyasu KawaiNoriyasu Kawai More articles by this author , Satoru TakahashiSatoru Takahashi More articles by this author , Takayoshi SuzukiTakayoshi Suzuki More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002055.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lysine-specific demethylase 1 (LSD1), the first histone demethylase to be discovered, is a novel target in prostate cancer therapy. NCL1 and NCD38, novel selective cell-active inhibitors of LSD1, were discovered at our university. We analyzed the efficacy of LSD1 inhibitors in germ cell tumors in vitro and in vivo. METHODS: Various tests were used to uncover mechanism of germ cell tumor growth and anticancer effects of NCL1 and NCD38. Cell viability was assessed using a WST-8 assay with standard vehicle, NCL1 or NCD38 in NTERA2 and Tera-1 cells (human testicular embryonal carcinoma cell lines). Flow cytometry and western blotting were performed to assess apoptosis and the cell cycle after treatment of NTERA2 cells with NCL1 or NCD38. Subcutaneous and orthotopic tumors were induced by injecting nude mice with NTERA2 cells. Thereafter, mice were injected intraperitoneally with vehicle, NCL1 or NCD38, twice a week, and their subsequent growth recorded. TUNEL staining was used to detect apoptosis in tumors. Finally, tissue arrays were prepared from tissue specimens from 35 patients with seminoma who underwent orchiectomy. RESULTS: The WST assay revealed a reduction in the number of viable cells in a dose-dependent manner after NCL1 or NCD38 treatment. In western blotting, NCL1 or NCD38 treatment induced caspase-dependent apoptosis, while Oct 3/4 and SOX2 expression was decreased. In ChIP-qPCR analysis, LSD1 inhibition increased the methylation of promoter regions of SOX2 and Oct 3/4. In flow cytometry analysis, NCL1 or NCD38 significantly induced apoptosis in a dose-dependent manner. Subcutaneous tumor volumes and serum AFP levels were significantly lower in mice treated with NCL1 or NCD38 than in controls. TUNEL analysis showed that NCL1 and NCD38 treatment induced apoptosis in NTERA2 subcutaneous tumors. Tissue array analysis showed that LSD1 expression in human seminoma specimens was significantly higher than that in noncancerous specimens. CONCLUSIONS: Germ cell tumor growth was effectively suppressed with NCL1 or NCD38 treatment in vitro and in vivo without adverse events by regulating apoptosis and suppressing SOX2 and Oct4. These results indicate the potential of NCL1 and NCD38 as therapeutic agents for germ cell tumors. Source of Funding: Grants in Aid for Scientific Research (KAKENHI), Japan Society for the Promotion of Science (JSPS), Number 17K11152 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e715-e716 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Toshiki Etani More articles by this author Taku Naiki More articles by this author Nobuhiko Shimizu More articles by this author Yusuke Noda More articles by this author Takashi Nagai More articles by this author Satoshi Nozaki More articles by this author Keitaro Iida More articles by this author Ryosuke Ando More articles by this author Noriyasu Kawai More articles by this author Satoru Takahashi More articles by this author Takayoshi Suzuki More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Loading ...

LRWD1 expression is regulated through DNA methylation in human testicular embryonal carcinoma cells

BackgroundSperm growth and maturation are correlated with the expression levels of Leucine-rich repeat and WD repeat-containing protein 1 (LRWD1), a widely expressed protein in the human testicles. The decrease in LRWD1 cellular level was linked to the reduction in cell growth and mitosis and the rise in cell microtubule atrophy rates. Since DNA methylation has a major regulatory role in gene expression, this study aimed at exploring the effect of the modulation of DNA methylation on LRWD1 expression levels.ResultsThe results revealed the presence of a CpG island up of 298 bps (− 253 ~ + 45) upon LRWD1 promoter in NT2/D1 cells. The hypermethylation of the LRWD1 promoter was linked to a reduction in the transcription activity in NT2/D1 cells, as indicated by luciferase reporter assay. The methylation activator, floxuridine, confirmed the decrease in the LRWD1 promoter transcriptional activity. On the other hand, 5-Aza-2′-deoxycytidine (5-Aza-dc, methylation inhibitor), significantly augmented LRWD1 promoter activity and the expression levels of mRNA and proteins. Furthermore, DNA methylation status of LRWD1 promoter in human sperm genomic DNA samples was analyzed. The results indicated that methylation of LRWD1 promoter was correlated to sperm activity.ConclusionsThus, the regulation of LRWD1 expression is correlated with the methylation status of LRWD1 promoter, which played a significant role in the modulation of spermatogenesis, sperm motility, and vitality. Based on these results, the methylation status of LRWD1 promoter may serve as a novel molecular diagnostic marker or a therapeutic target in males’ infertility.

MP11-03 NOVEL SELECTIVE LYSINE-SPECIFIC DEMETHYLASE 1 INHIBITORS SUPPRESS TESTICULAR TUMOR CELL PROLIFERATION

INTRODUCTION AND OBJECTIVE: Lysine-specific demethylase 1 (LSD1), the first histone demethylase to be discovered, is a novel target in prostate cancer therapy. NCL1 and NCD38, novel selective cell-active inhibitors of LSD1, were discovered at our university. We analyzed the efficacy of LSD1 inhibitors in germ cell tumors in vitro and in vivo. METHODS: Various tests were used to uncover mechanism of germ cell tumor growth and anticancer effects of NCL1 and NCD38. Cell viability was assessed using a WST-8 assay with standard vehicle, NCL1 or NCD38 in NTERA2 and Tera-1 cells (human testicular embryonal carcinoma cell lines). Flow cytometry and western blotting were performed to assess apoptosis and the cell cycle after treatment of NTERA2 cells with NCL1 or NCD38. Subcutaneous tumors were induced by subcutaneously injecting nude mice with NTERA2 cells. Thereafter, mice were injected intraperitoneally with vehicle, NCL1 or NCD38, twice a week, and their subsequent growth recorded. TUNEL staining was used to detect apoptosis in tumors. Finally, tissue arrays were prepared from tissue specimens from 35 patients with seminoma who underwent orchiectomy. RESULTS: The WST assay revealed a reduction in the number of viable cells in a dose-dependent manner after NCL1 or NCD38 treatment. In western blotting, NCL1 or NCD38 treatment induced caspase-dependent apoptosis, while Oct4 and SOX2 expression was decreased. In flow cytometry analysis, NCL1 or NCD38 significantly induced apoptosis in a dose-dependent manner. Subcutaneous tumor volumes and serum AFP levels were significantly lower in mice treated with NCL1 or NCD38 than in controls. TUNEL analysis showed that NCL1 and NCD38 treatment induced apoptosis in NTERA2 subcutaneous tumors. No adverse effect was observed in body weights, blood tests, and hematoxylin-eosin staining of various organs in mice. Tissue array analysis showed that LSD1 expression in human seminoma specimens was significantly higher than that in noncancerous specimens. CONCLUSIONS: Germ cell tumor growth was effectively suppressed with NCL1 or NCD38 treatment in vitro and in vivo without adverse events by regulating apoptosis and suppressing SOX2 and Oct4. These results indicate the potential of NCL1 and NCD38 as therapeutic agents for germ cell tumors.Source of Funding: Grant-in-Aid for Scientific Research 17K11152

Long noncoding RNA HOTTIP is associated with male infertility and promotes testicular embryonal carcinoma cell proliferation.

BackgroundIt has been proposed that lncRNAs, widely transcribed from genomes, play pivotal regulatory roles in a variety of biological processes, but their function in regulating spermatogenesis in human males is rarely reported.MethodsQRT‐PCR was adopted to detect HOTTIP expression level in testicular tissues from hypospermatogenesis (Hypo) patients or controls. The proliferation levels of NT2 and 293T were measured via CCK‐8 and EdU detection. Meanwhile, luciferase reporter gene assay and bioinformatics analysis were carried out to identify a target of HOTTIP. Additionally, the underlying mechanism of HOTTIP’s function was investigated using western blotting and RIP analysis.ResultsThe research results manifested that the expression of HOTTIP in testicular tissues from Hypo patients was prominently reduced in comparison with that in control testicular tissues. Interestingly, it was noted that HOTTIP exhibited a high expression in testicular embryonal carcinoma cell line NT2 compared with that in normal control cell line 293T. It was denoted in cell function evaluation that cell proliferation was impeded by downregulated HOTTIP but evidently stimulated by overexpressed HOTTIP. Moreover, HOTTIP was capable of positively modulating HOXA13 expression via the competitive binding to miR‐128‐3p.ConclusionTherefore, HOTTIP acting as ceRNAs to promote testicular embryonal carcinoma cell proliferation.

Role of OCT4 in cisplatin treatment of testicular embryonal carcinoma

Purpose: To determine the role of embryonal transcription factor OCT4 in cisplatin treatment of testicular embryonal carcinoma.Methods: In vitro assays were employed to assess the effect of cisplatin treatment on testicular embryonal carcinoma cell lines under OCT4 silencing. Following treatment with 500 ng/μL cisplatin, MTT assay was used to examine cell proliferation of 2012-EP and 833K-E cells with or without OCT silencing, while wound healing assay was used to examine cell migration ability. Transwell assay and crystal violet staining were employed to measure cell invasive capacity, whereas the distribution pattern of cell cycle was assessed by flow cytometry. The expression levels of several critical components in tumorigenicity related pathways with or without OCT silencing were determined by Western-blot analysis.Results: Cisplatin enhanced OCT4-silenced cell viability at all concentration (p &lt; 0.01) when compared to control cells. Upon treatment with 500 ng/μL cisplatin, OCT4-silenced cells showed 2- to 3-fold enhancement in cell proliferation (p &lt; 0.001), 2-fold increase in cell migration capacity (p &lt; 0.001), and about 1.5-fold enhancement in invasive capacity (p &lt; 0.001) when compared to control cells. In addition, OCT4 silencing upregulated the expression level of the proteins involved in cell proliferation, cell mobility, cancer metastasis and cell cycle control.Conclusion: The results suggest that OCT4 may serve as a therapeutic target for testicular embryonal carcinoma treatment in combination with cisplatin by modulating OCT4 expression level. This physiological evidence indicates that OCT4 downregulation contributes to cisplatin resistance in chemotherapy and subsequent disease relapse.Keywords: OCT4, Cisplatin resistance, Testicular embryonal carcinoma, Chemotherapy

Imsnc761 and DDX6 synergistically suppress cell proliferation and promote apoptosis via p53 in testicular embryonal carcinoma cells.

Intermediate-sized non-coding RNAs (imsncRNAs) have been shown to play important regulatory roles in the development of several eukaryotic organisms. In the present research, we selected imsncRNA 761 (imsnc761) as a research target. Expression analyses in a previous study showed that imsnc761 was down-regulated in maturation-arrested testis tissues as compared with the level in normal controls. In the present study, we found that imsnc761 could interact with DEAD-box helicase 6 (DDX6) to induce NTERA-2 (NT2 (testicular embryonal carcinoma cell)) cell apoptosis and proliferation inhibition via the p53 pathway. This interaction between imsnc761 and DDX6 also inhibited mitochondrial function and specific gene transcription and translation. To facilitate further research, we used label-free quantitation method to analyze the associated differences in Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways and biological processes. This confirmed the changes in several specific pathways, which matched our molecular experimental results.

Hsa-miR-513b-5p suppresses cell proliferation and promotes P53 expression by targeting IRF2 in testicular embryonal carcinoma cells

Previous studies have reported the miR-513b is located on the X chromosome and is preferentially expressed in testis. However, the underlying mechanisms of miR-513b involved in spermatogenesis remains unknown. In this study, we found that hsa-miR-513b-5p was highly expressed in the testes of infertile males with maturation arrest compared with normal controls. Overexpression of hsa-miR-513b-5p suppressed testicular embryonal carcinoma (NT2) cell proliferation and induced apoptosis in vitro, whereas silencing of hsa-miR-513b-5p reversed these effects. In addition, we found that interferon regulatory transcription factor 2 (IRF2) was a direct and functional target of hsa-miR-513b-5p. Silencing of endogenous IRF2 enhanced hsa-miR-513b-5p-mediated effects on cell proliferation in NT2 cells, whereas overexpression of IRF2 reversed these effects. Moreover, immunoblotting showed that overexpression of hsa-miR-513b-5p or silencing of endogenous IRF2 could promote the expression of P53. Moreover, overexpression of hsa-miR-513b-5p in the absence of p53 could also induce cell apoptosis. Together, our results suggest that hsa-miR-513b-5p suppresses NT2 cell proliferation and promotes P53 protein expression by targeting IRF2, and abnormal testicular hsa-miR-513b-5p expression may contribute to maturation arrest.

Downregulation of miR-320a/383-sponge-like long non-coding RNA NLC1-C (narcolepsy candidate-region 1 genes) is associated with male infertility and promotes testicular embryonal carcinoma cell proliferation.

Long non-coding RNAs (lncRNAs), which are extensively transcribed from the genome, have been proposed to be key regulators of diverse biological processes. However, little is known about the role of lncRNAs in regulating spermatogenesis in human males. Here, using microarray technology, we show altered expression of lncRNAs in the testes of infertile men with maturation arrest (MA) or hypospermatogenesis (Hypo), with 757 and 2370 differentially down-regulated and 475 and 163 up-regulated lncRNAs in MA and Hypo, respectively. These findings were confirmed by quantitative real-time PCR (qRT-PCR) assays on select lncRNAs, including HOTTIP, imsrna320, imsrna292 and NLC1-C (narcolepsy candidate-region 1 genes). Interestingly, NLC1-C, also known as long intergenic non-protein-coding RNA162 (LINC00162), was down-regulated in the cytoplasm and accumulated in the nucleus of spermatogonia and primary spermatocytes in the testes of infertile men with mixed patterns of MA compared with normal control. The accumulation of NLC1-C in the nucleus repressed miR-320a and miR-383 transcript and promoted testicular embryonal carcinoma cell proliferation by binding to Nucleolin. Here, we define a novel mechanism by which lncRNAs modulate miRNA expression at the transcriptional level by binding to RNA-binding proteins to regulate human spermatogenesis.

Inhibition of testicular embryonal carcinoma cell tumorigenicity by peroxisome proliferator-activated receptor-β/δ- and retinoic acid receptor-dependent mechanisms.

Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) has important physiological functions in control of cell growth, lipid and glucose homeostasis, differentiation and inflammation. To investigate the role of PPARβ/δ in cancer, stable human testicular embryonal carcinoma cell lines were developed that constitutively express PPARβ/δ. Expression of PPARβ/δ caused enhanced activation of the receptor, and this significantly decreased proliferation, migration, invasion, anchorage-independent growth, and also reduced tumor mass and volume of ectopic xenografts derived from NT2/D1 cells compared to controls. The changes observed in xenografts were associated with decreased PPARβ/δ-dependent expression of proliferating cell nuclear antigen and octamer-binding transcription factor-3/4, suggesting suppressed tumor proliferation and induction of differentiation. Inhibition of migration and invasion was mediated by PPARβ/δ competing with formation of the retinoic acid receptor (RAR)/retinoid X receptor (RXR) complex, resulting in attenuation of RARα-dependent matrix metalloproteinase-2 expression and activity. These results demonstrate that PPARβ/δ mediates attenuation of human testicular embryonal carcinoma cell progression through a novel RAR-dependent mechanism and suggest that activation of PPARβ/δ inhibits RAR/RXR dimerization and represents a new therapeutic strategy.

Cisplatin Induces Resistance by Triggering Differentiation of Testicular Embryonal Carcinoma Cells

Although testicular germ cell tumors are generally quite responsive to treatment with cisplatin, a small fraction of them acquire resistance during therapy. Even when cisplatin treatment is successful the patient is often left with a residual teratoma at the site of the primary tumor suggesting that cisplatin may trigger differentiation in some tumors. Using the human embryonal carcinoma cell line NTera2/D1, we confirmed that exposure to the differentiating agent retinoic acid produced a reduction in pluripotency markers NANOG and POU5F1 (Oct3/4) and an acute concentration-dependent increase in resistance to both cisplatin and paclitaxel that reached as high as 18-fold for cisplatin and 61-fold for paclitaxel within four days. A two day exposure to cisplatin also produced a concentration-dependent decrease in the expression of the NANOG and POU5F1 and increased expression of three markers whose levels increase with differentiation including Nestin, SCG10 and Fibronectin. In parallel, exposure to cisplatin induced up to 6.2-fold resistance to itself and 104-fold resistance to paclitaxel. Paclitaxel did not induce differentiation or resistance to either itself or cisplatin. Neither retinoic acid nor cisplatin induced resistance in cervical or prostate cancer cell lines or other germ cell tumor lines in which they failed to alter the expression of NANOG and POU5F1. Forced expression of NANOG prevented the induction of resistance to cisplatin by retinoic acid. We conclude that cisplatin can acutely induce resistance to itself and paclitaxel by triggering a differentiation response in pluripotent germ cell tumor cells.

MicroRNA-383 impairs phosphorylation of H2AX by targeting PNUTS and inducing cell cycle arrest in testicular embryonal carcinoma cells

Male germ cells with aberrant DNA damage are the weighted factor contributing to male infertility. Mounting evidence shows that DNA damage in male germ cells impairs spermatogenesis and lowers fecundity. MicroRNAs (miRNAs) regulating expression of multiple genes play a significant role in spermatogenesis. Our previous results have shown that microRNA-383 (miR-383) is one of the notable down-regulated microRNAs in the testes of sterile males with maturation arrest (MA) and is located predominantly in spermatogonia and primary spermatocytes. However, the role that miR-383 plays in DNA damage during spermatogenesis remains unknown. In this study, we found that miR-383 inhibited the focal formation and abundance of γH2AX, which is the major marker of sites of DNA damage, with or without ultraviolet irradiation and cisplatin in testicular embryonal carcinoma (NT-2) cells. In addition, NT-2 cells were remarkably sensitized to DNA damage reagent (cisplatin) by forcing expression of miR-383 and silencing expression of protein phosphatase 1, regulatory subunit 10 (PNUTS). By constructing Renilla luciferase reporters and co-transfecting miR-383 and reporters in NT-2 cells, we identified that PNUTS was a valid target of miR-383. Further results demonstrated that the repression of the phosphorylated form of H2AX by miR-383 was due to independent depletion of PNUTS and cell cycle arrest. In conclusion, we found a novel function of miR-383 in the DNA damage pathway. miR-383 impairs the phosphorylation of H2AX by targeting PNUTS and inducing cell cycle arrest independently, as well as sensitizing NT-2 cells to cisplatin.

MicroRNA‐302a sensitizes testicular embryonal carcinoma cells to cisplatin‐induced cell death

Cisplatin is a commonly used chemotherapeutic agent for the treatment of several human malignancies, such as testicular germ cell tumors (TGCT). The toxic effects persist and those that are present long after chemotherapy affect the overall quality of life of patients. MicroRNAs (miRNAs) play important roles in the responses of cancer cells to chemotherapy and have been shown to modulate cell sensitivity to chemotherapeutic drugs. However, the relationship between miRNA expression and cisplatin sensitivity of TGCT has not been fully explored. In this study, the effects of miR-302a on cisplatin cytotoxicity in TGCT-derived cell line NTERA-2 (NT2) were evaluated. We found that expression levels of miR-302a were increased in cisplatin-treated NT2 cells. Up-regulation of miR-302a significantly increased the sensitivity of NT2 cells to cisplatin by enhancing cisplatin-induced G2/M phase arrest and the subsequent progression to apoptosis. MiR-302a also increased the killing effects of cisplatin by lowering the apoptotic threshold; the same result was also observed in another TGCT-derived cell line, NCCIT. Furthermore, miR-302a-enhanced cisplatin sensitivity was partially mediated through the down-regulation of p21 in NT2 cells. MiR-302a induced apoptosis was further enhanced by silencing of p53 in NT2 cells. p53 levels were inversely associated with the expression of Oct4, Sox2, and Nanog in response to cisplatin. Thus, targeting miR-302a may offer new therapeutic interventions in TGCT.

The targeting and functions of miRNA-383 are mediated by FMRP during spermatogenesis

Our previous studies have shown that microRNA-383 (miR-383) expression is downregulated in the testes of infertile men with maturation arrest (MA). Abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumors. However, the mechanisms underlying the targeting and functions of miR-383 during spermatogenesis remain unknown. In this study, we found that fragile X mental retardation protein (FMRP) was associated with 88 miRNAs in mouse testis including miR-383. Knockdown of FMRP in NTERA-2 (NT2) (testicular embryonal carcinoma) cells enhanced miR-383-induced suppression of cell proliferation by decreasing the interaction between FMRP and miR-383, and then affecting miR-383 binding to the 3′-untranslated region of its target genes, including interferon regulatory factor-1 (IRF1) and Cyclin D1 both in vivo and in vitro. On the other hand, FMRP levels were also downregulated by overexpression of miR-383 in NT2 cells and GC1 (spermatogonia germ cell line). miR-383 targeted to Cyclin D1 directly, and then inhibited its downstream effectors, including phosphorylated pRb and E2F1, which ultimately resulted in decreased FMRP expression. Reduced miR-383 expression, dysregulated cyclin-dependent kinase 4 expression (one of the downstream genes of miR-383) and increased DNA damage were also observed in the testes of Fmr1 knockout mice and of MA patients with a downregulation of FMRP. A potential feedback loop between FMRP and miR-383 during spermatogenesis is proposed, and FMRP acts as a negative regulator of miR-383 functions. Our data also indicate that dysregulation of the FMRP–miR-383 pathway may partially contribute to human spermatogenic failure with MA.

A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: Effects of methylmercury, lead and aluminum evaluated by gene expression studies

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity was then evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial). The results obtained suggest that lead chloride and aluminum nitrate at low concentrations were toxic primarily to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The results obtained suggest that a neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of potential neurotoxic compounds.

Transcription Factors Sp1 and p73 Control the Expression of the Proapoptotic Protein NOXA in the Response of Testicular Embryonal Carcinoma Cells to Cisplatin

Testicular germ cell tumors (TGCTs) are highly responsive to and curable by cisplatin-based chemotherapy even in advanced stages. We have studied the molecular mechanisms involved in the induction of apoptosis in response to cisplatin, and found that proapoptotic Noxa is transcriptionally up-regulated following cisplatin exposure, even in the absence of p53, in NTERA2 cisplatin-sensitive cells but not in 1411HP-resistant cells. Blockade of Noxa reduced the apoptotic response of embryonal carcinoma (EC) NTERA2 cells to cisplatin. A detailed analysis of the Noxa promoter revealed that p73 and Sp1-like factors, Sp1 and KLF6, played key roles in the transcriptional control of this gene. Overexpression of TAp73 induced Noxa whereas the dominant negative isoform ΔNp73, reduced the levels of Noxa after cisplatin exposure in NTERA2 and 2102EP. Interestingly, down-regulation of Sp1 increased Noxa expression in response to cisplatin. However, blockade of KLF6 decreased cisplatin-induced up-regulation of Noxa in EC cell lines. In addition, tissue microarray analyses of TGCTs revealed that expression of Noxa correlates with good clinical prognosis in patients with embryonal carcinoma. Thus, our data show the transcriptional network that regulates Noxa in EC cells, which is key for their apoptotic response to cisplatin-based chemotherapy, and propose Noxa as a predictive factor of therapeutic response.

Abstract 2958: The novel protein kinase STK17A is a direct p53 target gene that mediates response to genotoxic and nutritional stress in a cancer cell context-dependent manner

Abstract STK17A (DRAK1) is a largely uncharacterized serine/threonine kinase that belongs to the death-associated protein kinase family. Prior microarray studies demonstrated that STK17A is induced with cisplatin in testicular cancer-derived human embryonal carcinoma (EC) cells. We now demonstrate the STK17A in a novel p53 target gene that is induced by a variety of DNA damaging agents in a p53-dependent manner in a number of different cell contexts. A consensus and functional p53 response element was found upstream of the STK17A promoter and endogenous p53 was shown to bind to this site in a cisplatin-dependent manner. Knockdown of STK17A conferred resistance to cisplatin-induced growth suppression and apoptotic cell death in EC cells. This was associated with up-regulation of detoxifying and antioxidant genes and reduction in reactive oxygen species (ROS), whereas overexpression of STK17A increased ROS levels in EC cells. Interestingly, we found that STK17A is highly overexpressed in clinical glioblastoma (GBM) and cell lines compared to its expression in normal brain tissue and other cancers. High STK17A expression in GBM patients correlated with poor clinical outcome and decreased survival. STK17A knockdown in GBM cells decreased clonagenic cell growth, soft agar tumorigenicity, and survival in response to genotoxic and nutritional stress. Preliminary data will be presented implicating STK17A in the regulation of autophagy and the proximal autophagy component, ULK1. These findings indicate that STK17A may be added to a growing list of direct p53 target genes involved in autophagy that includes TIGAR, Sestrins, DAPK1 and DRAM and that by impacting autophagy STK17A may modulate response to genotoxic and nutritional stress in a cell context-depend manner. This study also suggests that STK17A is a potential novel prognostic maker and therapeutic target for GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2958. doi:1538-7445.AM2012-2958

Serine/Threonine Kinase 17A Is a Novel p53 Target Gene and Modulator of Cisplatin Toxicity and Reactive Oxygen Species in Testicular Cancer Cells

Testicular cancer is highly curable with cisplatin-based therapy, and testicular cancer-derived human embryonal carcinoma (EC) cells undergo a p53-dominant transcriptional response to cisplatin. In this study, we have discovered that a poorly characterized member of the death-associated protein family of serine/threonine kinases, STK17A (also called DRAK1), is a novel p53 target gene. Cisplatin-mediated induction of STK17A in the EC cell line NT2/D1 was prevented with p53 siRNA. Furthermore, STK17A was induced with cisplatin in HCT116 and MCF10A cells but to a much lesser extent in isogenic p53-suppressed cells. A functional p53 response element that binds endogenous p53 in a cisplatin-dependent manner was identified 5 kb upstream of the first coding exon of STK17A. STK17A is not present in the mouse genome, but the closely related gene STK17B is induced with cisplatin in mouse NIH3T3 cells, although this induction is p53-independent. Interestingly, in human cells containing both STK17A and STK17B, only STK17A is induced with cisplatin. Knockdown of STK17A conferred resistance to cisplatin-induced growth suppression and apoptotic cell death in EC cells. This was associated with the up-regulation of detoxifying and antioxidant genes, including metallothioneins MT1H, MT1M, and MT1X that have previously been implicated in cisplatin resistance. In addition, knockdown of STK17A resulted in decreased cellular reactive oxygen species, whereas STK17A overexpression increased reactive oxygen species. In summary, we have identified STK17A as a novel direct target of p53 and a modulator of cisplatin toxicity and reactive oxygen species in testicular cancer cells.

Downregulation of microRNA-383 is associated with male infertility and promotes testicular embryonal carcinoma cell proliferation by targeting IRF1

Our previous studies have shown that microRNA-383 (miR-383) expression is downregulated in the testes of infertile men with maturation arrest (MA). However, the underlying mechanisms of miR-383 involved in the pathogenesis of MA remain unknown. In this study, we showed that downregulation of miR-383 was associated with hyperactive proliferation of germ cells in patients with mixed patterns of MA. Overexpression of miR-383 in NT2 (testicular embryonal carcinoma) cells resulted in suppression of proliferation, G1-phase arrest and induction of apoptosis, whereas silencing of miR-383 reversed these effects. The effects of miR-383 were mediated through targeting a tumor suppressor, interferon regulatory factor-1 (IRF1), and miR-383 was negatively correlated with IRF1 protein expression in vivo. miR-383 inhibited IRF1 by affecting its mRNA stability, which subsequently reduced the levels of the targets of IRF1, namely cyclin D1, CDK2 and p21. Downregulation of IRF1 or cyclin D1, but not that of CDK2, enhanced miR-383-mediated effects, whereas silencing of p21 partially inhibited the effects of miR-383. Moreover, miR-383 downregulated CDK4 by increasing proteasome-dependent degradation of CDK4, which in turn resulted in an inhibition of phosphorylated retinoblastoma protein (pRb) phosphorylation. These results suggest that miR-383 functions as a negative regulator of proliferation by targeting IRF1, in part, through inactivation of the pRb pathway. Abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumor.

MdmX is a substrate for the deubiquitinating enzyme USP2a

It has previously been shown that ubiquitin-specific protease 2a (USP2a) is a regulator of the Mdm2/p53 pathway. USP2a binds to Mdm2 and can deubiquitinate Mdm2 without reversing Mdm2-mediated p53 ubiquitination. Overexpression of USP2a causes accumulation of Mdm2 and promotes p53 degradation. We now show that MdmX is also a substrate for USP2a. MdmX associates with USP2a independently of Mdm2. Ectopic expression of wild-type USP2a but not a catalytic mutant prevents Mdm2-mediated degradation of MdmX. This correlates with the ability of wild-type USP2a to deubiquitinate MdmX. siRNA-mediated knockdown of USP2a in NTERA-2 testicular embryonal carcinoma cells and MCF7 breast cancer cells causes destabilization of MdmX and results in a decrease in MdmX protein levels, showing that endogenous USP2a participates in the regulation of MdmX stability. The therapeutic drug, cisplatin decreases MdmX protein expression. USP2a mRNA and protein levels were also reduced after cisplatin exposure. The magnitude and time course of USP2a downregulation suggests that the reduction in USP2a levels could contribute to the decrease in MdmX expression following treatment with cisplatin. Knockdown of USP2a increases the sensitivity of NTERA-2 cells to cisplatin, raising the possibility that suppression of USP2a in combination with cisplatin may be an approach for cancer therapy.