Novel meriolin derivatives activate the mitochondrial apoptosis pathway in the presence of antiapoptotic Bcl-2.

Meriolin derivatives represent a new class of kinase inhibitors with a pronounced cytotoxic potential. Here, we investigated a newly synthesized meriolin derivative (termed meriolin 16) that displayed a strong apoptotic potential in Jurkat leukemia and Ramos lymphoma cells. Meriolin 16 induced apoptosis in rapid kinetics (within 2-3 h) and more potently (IC50: 50 nM) than the previously described derivatives meriolin 31 and 36 [1]. Exposure of Ramos cells to meriolin 16, 31, or 36 for 5 min was sufficient to trigger severe and irreversible cytotoxicity. Apoptosis induction by all three meriolin derivatives was independent of death receptor signaling but required caspase-9 and Apaf-1 as central mediators of the mitochondrial death pathway. Meriolin-induced mitochondrial toxicity was demonstrated by disruption of the mitochondrial membrane potential (ΔΨm), mitochondrial release of proapoptotic Smac, processing of the dynamin-like GTPase OPA1, and subsequent fragmentation of mitochondria. Remarkably, all meriolin derivatives were able to activate the mitochondrial death pathway in Jurkat cells, even in the presence of the antiapoptotic Bcl-2 protein. In addition, meriolins were capable of inducing cell death in imatinib-resistant K562 and KCL22 chronic myeloid leukemia cells as well as in cisplatin-resistant J82 urothelial carcinoma and 2102EP germ cell tumor cells. Given the frequent inactivation of the mitochondrial apoptosis pathway by tumor cells, such as through overexpression of antiapoptotic Bcl-2, meriolin derivatives emerge as promising therapeutic agents for overcoming treatment resistance.

Assessing the risk to develop a growing teratoma syndrome based on molecular and epigenetic subtyping as well as novel secreted biomarkers.

In germ cell tumors (GCT), a growing teratoma during chemotherapy with decreasing tumor markers was defined as 'growing teratoma syndrome' (GTS) by Logothetis et al. in 1982. So far, its pathogenesis and specific treatment options remain elusive. We aimed at updating the GTS definition based on molecular and epigenetic features as well as identifying circulating biomarkers. We selected 50 GTS patients for clinical characterization and subsequently 12 samples were molecularly analyzed. We further included 7 longitudinal samples of 2 GTS patients. Teratomas (TER) showing no features of GTS served as controls. GTS were stratified based on growth rates into a slow (<0.5 cm/month), medium (0.5-1.5) and rapid (>1.5) group. By analyzing DNA methylation, microRNA expression and the secretome, we identified putative epigenetic and secreted biomarkers for the GTS subgroups. We found that proteins enriched in the GTS groups compared to TER were involved in proliferation, DNA replication and the cell cycle, while proteins interacting with the immune system were depleted. Additionally, GTSrapid seem to interact more strongly with the surrounding microenvironment than GTSslow. Expression of pluripotency- and yolk-sac tumor-associated genes in GTS and formation of a yolk-sac tumor or somatic-type malignancy in the longitudinal GTS samples, pointed at an additional occult non-seminomatous component after chemotherapy. Thus, updating the Logothetis GTS definition is necessary, which we propose as follows: The GTS describes a continuously growing teratoma that might harbor occult non-seminomatous components considerably reduced during therapy but outgrowing over time again.

Molecular and histopathological characterization of seminoma patients with highly elevated human chorionic gonadotropin levels in the serum.

Approximately 30% of seminoma (SEM) patients present with moderately elevated human chorionic gonadotropin (hCG) levels at first diagnosis. In case of high hCG serum levels, the presence of a non-SEM component, i.e. choriocarcinoma (CC), may be assumed. To characterize cases described as pure seminoma with high serum hCG levels, tissue samples and DNA were analyzed. Patient files from an international registry were screened for patients with SEM and extraordinarily high hCG serum levels. IHC and qRT-PCR analysis was performed for markers of SEM, embryonal carcinoma (EC) and CC/trophoblast cells. The cell lines TCam-2 (SEM), 2102EP, NCCIT, NT2/D1 (EC) and JAR, JEG3 and BeWo (CC) were included for comparison. Of 1031 SEM patients screened, 39 patients (3.7%) showed hCG serum levels > 1000 U/l. Of these, tumor material for IHC and RNA for qRT-PCR was available from n = 7 patients and n = 3 patients, respectively. Median pre-orchiectomy serum hCG level was 5356 U/l (range: 1224-40909 U/L). Histopathologically, all investigated samples were classified as SEM with syncytiotrophoblast sub-populations. SEM cells were SALL4+ / OCT3/4+ / D2-40+, while syncytiotrophoblast cells were hCG+ / GATA3+ / p63+ and SOX2-/CDX2-. qRT-PCR analysis detected trophoblast stem cell markers CDX2, EOMES and TFAP2C as well as the trophectoderm-specifier TEAD4, but not GATA3. Additionally, SOX17 and PRAME, but not SOX2, were detected, confirming the pure SEM-like gene expression signature of the analyzed samples. In conclusion, excessively increased hCG serum levels can appear in patients with pure SEM. To explain detectable hCG serum levels, it is important to diagnose the subtype of a SEM with syncytiotrophoblasts.

Molecular characterization of the CXCR4 / CXCR7 axis in germ cell tumors and its targetability using nanobody-drug-conjugates.

Being stimulated by the chemokine CXCL12, the CXCR4 / CXCR7 cascade is involved in tumor proliferation, migration, and metastasis. The interaction between CXCL12, secreted by cells from the microenvironment, and its receptors is complex and has been ascribed to promote chemotherapy resistance. However, the role of this signaling axis and its targetability in germ cell tumors (GCT) is not fully understood. Thus, this study investigated the therapeutic efficacy of a nanobody-drug-conjugate targeting CXCR4 (CXCR4-NDC) and functionally characterized this signaling pathway in GCT using small molecule inhibitors and nanobodies. As shown by diminished cell viability, enhanced apoptosis induction, and detection of mitotic catastrophes, we confirmed the cytotoxic efficacy of the CXCR4-NDC in CXCR4+-GCT cells (i.e. seminoma and yolk-sac tumor), while non-malignant CXCR4--fibroblasts, remained largely unaffected. Stimulation of CXCR4+ / CXCR7+-GCT cells with CXCL12 resulted in an enhanced proliferative and migratory capacity, while this effect could be reverted using CXCR4 inhibitors or a CXCR7-nanobody. Molecularly, the CXCR4 / CXCR7-signaling cascade could be activated independently of MAPK (ERK1 / 2)-phosphorylation. Although, in CXCR4- / CXCR7--embryonal carcinoma cells, CXCR7-expression was re-induced upon inhibition of ERK1 / 2-signaling. This study identified a nanobody-drug-conjugate targeting CXCR4 as a putative therapeutic option for GCT, i.e. seminoma and yolk-sac tumors. Furthermore, this study shed light on the functional role of the CXCR4 / CXCR7 / CXCL12-signaling cascade in GCT, demonstrating an important influence on proliferation and migration.

Characterizing the mutational burden, DNA methylation landscape, and proteome of germ cell tumor-related somatic-type malignancies to identify the tissue-of-origin, mechanisms of therapy resistance, and druggable targets.

BACKGROUND: Germ cell tumors (GCT) might undergo transformation into a somatic-type malignancy (STM), resulting in a cell fate switch to tumors usually found in somatic tissues, such as rhabdomyosarcomas or adenocarcinomas. STM is associated with a poor prognosis, but the molecular and epigenetic mechanisms triggering STM are still enigmatic, the tissue-of-origin is under debate and biomarkers are lacking. METHODS: To address these questions, we characterized a unique cohort of STM tissues on mutational, epigenetic and protein level using modern and high-throughput methods like TSO assays, 850k DNA methylation arrays and mass spectrometry. RESULTS AND CONCLUSIONS: For the first time, we show that based on DNA methylation and proteome data carcinoma-related STM more closely resemble yolk-sac tumors, while sarcoma-related STM resemble teratoma. STM harbor mutations in FGF signaling factors (FGF6/23, FGFR1/4) highlighting the corresponding pathway as a therapeutic target. Furthermore, STM utilize signaling pathways, like AKT, FGF, MAPK, and WNT to mediate molecular functions coping with oxidative stress, toxin transport, DNA helicase activity, apoptosis and the cell cycle. Collectively, these data might explain the high therapy resistance of STM. Finally, we identified putative novel biomarkers secreted by STM, like EFEMP1, MIF, and DNA methylation at specific CpG dinucleotides.

Updating germ cell tumour pathogenesis - the ability of seminomas for FOXA2-driven extra-embryonic differentiation.

AIMS: Testicular germ cell tumours are the most common solid malignancies in young men of age 14-44 years. It is generally accepted that both seminomas and non-seminomas arise from a common precursor, the germ cell neoplasia in-situ, which itself is the result of a defective (primordial) germ cell development. The stem cell-like population of the non-seminomas, the embryonal carcinoma, is capable of the differentiation of all three germ layers (teratomas) and extra-embryonic tissues (yolk-sac tumours, choriocarcioma) into cells. In contrast, seminomas are thought to have a limited differentiation potential. Nevertheless, several studies have highlighted their ability to undergo reprogramming to an embryonal carcinoma or differentiation into other non-seminomatous entities. Here, we demonstrate that in approximately 5% of seminomas, the yolk-sac tumour driver gene FOXA2 is detectable at the protein level, indicative of an occult yolk-sac tumour subpopulation that putatively arose from seminoma cells, as the presence of other GCT entities could be excluded. The presence of these subpopulations might render the tumour more aggressive and argue for an adjustment of the therapeutic concept. We used our data to update the model of germ cell tumour pathogenesis, especially regarding the developmental potential of seminomas. Additionally, we suggest to include detection of FOXA2 into standard routine diagnosis of seminomas.

CD24 targeting with NK-CAR immunotherapy in testis, prostate, renal and (luminal-type) bladder cancer and identification of direct CD24 interaction partners.

Alternative therapeutic options targeting urologic malignancies, such as germ cell tumours, as well as urothelial, renal and prostate carcinomas, are still urgently needed. The membrane protein CD24 represents a promising immunotherapeutical approach. The present study aimed to decipher the molecular function of CD24 in vitro and evaluate the cytotoxic capacity of a third-generation natural killer (NK) cell chimeric antigen receptor (CAR) against CD24 in urologic tumour cell lines. Up to 20 urologic tumour cell lines and several non-malignant control cells were included. XTT viability assays and annexin V/propidium iodide flow cytometry analyses were performed to measure cell viability and apoptosis rates, respectively. Co-immunoprecipitation followed by mass spectrometry analyses identified direct interaction partners of CD24. Luciferase reporter assays were used to functionally validate transactivation of CD24 expression by SOX2. N- and O-glycosylation of CD24 were evaluated by enzymatic digestion and mass spectrometry. The study demonstrates that SOX2 transactivates CD24 expression in embryonal carcinoma cells. In cells of different urological origins, CD24 interacted with proteins involved in cell adhesion, ATP binding, phosphoprotein binding and post-translational modifications, such as histone acetylation and ubiquitination. Treatment of urological tumour cells with NK-CD24-CAR cells resulted in a decreased cell viability and apoptosis induction specifically in CD24+ tumour cells. Limitations of the study include the in vitro setting, which still has to be confirmed in vivo. In conclusion, we show that CD24 is a promising novel target for immune therapeutic approaches targeting urologic malignancies.

Genome-scale CRISPR screen reveals neddylation to contribute to cisplatin resistance of testicular germ cell tumours.

BACKGROUND: Type II testicular germ cell tumours (TGCT) are the most prevalent tumours in young men. Patients suffering from cisplatin-resistant TGCTs are facing very poor prognosis demanding novel therapeutic options. Neddylation is a known posttranslational modification mediating many important biological processes, including tumorigenesis. Overactivation of the neddylation pathway promotes carcinogenesis and tumour progression in various entities by inducing proteasomal degradation of tumour suppressors (e.g., p21, p27). METHODS: We used a genome-scale CRISPR/Cas9 activation screen to identify cisplatin resistance factors. TGCT cell lines were treated with the neddylation inhibitor (MLN4924)/cisplatin/combination and investigated for changes in viability (XTT assay), apoptosis/cell cycle (flow cytometry) as well as in the transcriptome (3'mRNA sequencing). RESULTS: NAE1 overexpression was detected in cisplatin-resistant colonies from the CRISPR screen. Inhibition of neddylation using MLN4924 increased cisplatin cytotoxicity in TGCT cell lines and sensitised cisplatin-resistant cells towards cisplatin. Apoptosis, G2/M-phase cell cycle arrest, γH2A.X/P27 accumulation and mesoderm/endoderm differentiation were observed in TGCT cells, while fibroblast cells were unaffected. CONCLUSIONS: We identified overactivation of neddylation as a factor for cisplatin resistance in TGCTs and highlighted the additive effect of NAE1 inhibition by MLN4924 in combination with cisplatin as a novel treatment option for TGCTs.

Targeting CLDN6 in germ cell tumors by an antibody-drug-conjugate and studying therapy resistance of yolk-sac tumors to identify and screen specific therapeutic options.

BACKGROUND: Being the standard-of-care for four decades, cisplatin-based chemotherapy is highly efficient in treating germ cell tumors (GCT). However, often refractory patients present with a remaining (resistant) yolk-sac tumor (YST(-R)) component, resulting in poor prognosis due to lack of novel treatment options besides chemotherapy and surgery. The aim of this study was to identify novel targets for the treatment of YST by deciphering the molecular mechanisms of therapy resistance. Additionally, we screened the cytotoxic efficacy of a novel antibody-drug-conjugate targeting CLDN6 (CLDN6-ADC), as well as pharmacological inhibitors to target specifically YST. METHODS: Protein and mRNA levels of putative targets were measured by flow cytometry, immunohistochemical stainings, mass spectrometry of formalin-fixed paraffin-embedded tissues, phospho-kinase arrays, or qRT-PCR. Cell viability, apoptosis and cell cycle assays of GCT and non-cancerous cells were performed using XTT cell viability assays or Annexin V / propidium iodide flow cytometry, respectively. Druggable genomic alterations of YST(-R) tissues were identified by the TrueSight Oncology 500 assay. RESULTS: We demonstrated that treatment with a CLDN6-ADC enhanced apoptosis induction specifically in CLDN6+ GCT cells in comparison with non-cancerous controls. In a cell line-dependent manner, either an accumulation in the G2 / M cell cycle phase or a mitotic catastrophe was observed. Based on mutational and proteome profiling, this study identified drugs targeting the FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling pathways as promising approaches to target YST. Further, we identified factors relevant for MAPK signaling, translational initiation and RNA binding, extracellular matrix-related processes as well as oxidative stress and immune response to be involved in therapy resistance. CONCLUSIONS: In summary, this study offers a novel CLDN6-ADC to target GCT. Additionally, this study presents novel pharmacological inhibitors blocking FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling for the treatment of (refractory) YST patients. Finally, this study shed light on the mechanisms of therapy resistance in YST.

Establishment and Evaluation of Dual HDAC/BET Inhibitors as Therapeutic Options for Germ Cell Tumors and Other Urological Malignancies.

Urological malignancies represent major challenges for clinicians, with annually rising incidences. In addition, cisplatin treatment induced long-term toxicities and the development of therapy resistance emphasize the need for novel therapeutics. In this study, we analyzed the effects of novel histone deacetylase (HDAC) and bromodomain and extraterminal domain-containing (BET) inhibitors to combine them into a potent HDAC-BET-fusion molecule and to understand their molecular mode-of-action. Treatment of (cisplatin-resistant) germ cell tumors (GCT), urothelial, renal, and prostate carcinoma cells with the HDAC, BET, and dual inhibitors decreased cell viability, induced apoptosis, and affected the cell cycle. Furthermore, a dual inhibitor considerably decreased tumor burden in GCT xenograft models. On a molecular level, correlating RNA- to ATAC-sequencing data indicated a considerable induction of gene expression, accompanied by site-specific changes of chromatin accessibility after HDAC inhibitor application. Upregulated genes could be linked to intra- and extra-cellular trafficking, cellular organization, and neuronal processes, including neuroendocrine differentiation. Regarding chromatin accessibility on a global level, an equal distribution of active or repressed DNA accessibility has been detected after HDAC inhibitor treatment, questioning the current understanding of HDAC inhibitor function. In summary, our HDAC, BET, and dual inhibitors represent a new treatment alternative for urological malignancies. Furthermore, we shed light on new molecular and epigenetic mechanisms of the tested epi-drugs, allowing for a better understanding of the underlying modes-of-action and risk assessment for the patient.

[Current view on testicular tumors from a developmental biological perspective : Important biomarkers and molecular pathological investigations]. / Aktuelle Betrachtung der Hodentumoren aus entwicklungsbiologischer Sicht : Wichtige Biomarker und molekularpathologische Untersuchungen.

BACKGROUND: Germ cell tumors (GCTs) are the most common type of cancer in Germany in young men between 15 and 44 years of age. The routinely performed diagnostic procedures are essential for the patient's treatment, but can be difficult due to heterogenous histologies. Additionally, the molecular mechanisms of the development of the special forms growing teratoma syndrome (GTS) and testicular tumors with malignant somatic transformation (MST) as well as of therapy resistance are not fully understood. OBJECTIVES: Updated understanding of the molecular processes underlying GCT development and their special forms as well as recommendations for new and useful biomarkers. RESULTS: The development of GCTs is a dynamic process largely influenced by the microenvironment. Seminomas (SEs) in particular seem to posses a higher cellular plasticity than previously assumed, allowing SEs to be reprogrammed into an embryonal carcinoma (EC) or differentiate into extra-embryonal tissues (yolk sac tumors [YSTs], trophoblastic differentiation). Novel serological (mi371a-3p) and pathological (FOXA2) biomarkers are well suited to early detect GCTs and YSTs, respectively. For more aggressive tumors and special cases (GTS, MST), there are still no reliable diagnostics or specific/tailored therapies available. CONCLUSION: The ability of SEs to transit into EC or YSTs should be considered during therapy. Future research should focus on deciphering the special forms GTS and MST as well as the early recognition of YSTs, since their development seems to be an escape mechanism to chemotherapy.

Profiling the 3D interaction between germ cell tumors and microenvironmental cells at the transcriptome and secretome level.

The tumor microenvironment (TM), consisting of the extracellular matrix (ECM), fibroblasts, endothelial cells, and immune cells, might affect tumor invasiveness and the outcome of standard chemotherapy. This study investigated the cross talk between germ cell tumors (GCT) and surrounding TM cells (macrophages, T-lymphocytes, endothelial cells, and fibroblasts) at the transcriptome and secretome level. Using high-throughput approaches of three-dimensional (3D) co-cultured cellular aggregates, this study offers newly identified pathways to be studied with regard to sensitivity toward cisplatin-based chemotherapy or tumor invasiveness as a consequence of the cross talk between tumor cells and TM components. Mass-spectrometry-based secretome analyses revealed that TM cells secreted factors involved in ECM organization, cell adhesion, angiogenesis, and regulation of insulin-like growth factor (IGF) transport. To evaluate direct cell-cell contacts, green fluorescent protein (GFP)-expressing GCT cells and mCherry-expressing TM cells were co-cultured in 3D. Afterward, cell populations were separated by flow cytometry and analyzed by RNA sequencing. Correlating the secretome with transcriptome data indicated molecular processes such as cell adhesion and components of the ECM being enriched in most cell populations. Re-analyses of secretome data with regard to lysine- and proline-hydroxylated peptides revealed a gain in proteins, such as collagens and fibronectin. Cultivation of GCT cells on collagen I/IV- or fibronectin-coated plates significantly elevated adhesive and migratory capacity, while decreasing cisplatin sensitivity of GCT cells. Correspondingly, cisplatin sensitivity was significantly reduced in GCT cells under the influence of conditioned medium from fibroblasts and endothelial cells. This study sheds light on the cross talk between GCT cells and their circumjacent TM, which results in deposition of the ECM and eventually promotes a pro-tumorigenic environment through enhanced migratory and adhesive capacity, as well as decreased cisplatin sensitivity. Hence, our observations indicate that targeting the ECM and its cellular components might be a novel therapeutic option in combination with cisplatin-based chemotherapy for GCT patients.

Therapeutical interference with the epigenetic landscape of germ cell tumors: a comparative drug study and new mechanistical insights.

BACKGROUND: Type II germ cell tumors (GCT) are the most common solid cancers in males of age 15 to 35 years. Treatment of these tumors includes cisplatin-based therapy achieving high cure rates, but also leading to late toxicities. As mainly young men are suffering from GCTs, late toxicities play a major role regarding life expectancy, and the development of therapy resistance emphasizes the need for alternative therapeutic options. GCTs are highly susceptible to interference with the epigenetic landscape; therefore, this study focuses on screening of drugs against epigenetic factors as a treatment option for GCTs. RESULTS: We present seven different epigenetic inhibitors efficiently decreasing cell viability in GCT cell lines including cisplatin-resistant subclones at low concentrations by targeting epigenetic modifiers and interactors, like histone deacetylases (Quisinostat), histone demethylases (JIB-04), histone methyltransferases (Chaetocin), epigenetic readers (MZ-1, LP99) and polycomb-repressive complexes (PRT4165, GSK343). Mass spectrometry-based analyses of the histone modification landscape revealed effects beyond the expected mode-of-action of each drug, suggesting a wider spectrum of activity than initially assumed. Moreover, we characterized the effects of each drug on the transcriptome of GCT cells by RNA sequencing and found common deregulations in gene expression of ion transporters and DNA-binding factors. A kinase array revealed deregulations of signaling pathways, like cAMP, JAK-STAT and WNT. CONCLUSION: Our study identified seven drugs against epigenetic modifiers to treat cisplatin-resistant GCTs. Further, we extensively analyzed off-target effects and modes-of-action, which are important for risk assessment of the individual drugs.

Primary mediastinal germ cell tumours: an immunohistochemical and molecular diagnostic approach.

AIMS: Primary mediastinal germ cell tumours (PMGCTs) are rare mediastinal neoplasms, and their diagnosis can be challenging, owing to small biopsy samples. The aim of this study was to develop a diagnostic algorithm using immunohistochemical staining, with a focus on novel markers, and molecular analysis of isochromosome 12p [i(12p)]. METHODS AND RESULTS: Paraffin-embedded tissues of 32 mediastinal tumours were analysed with immunohistochemical staining for sal-like transcription factor 4 (SALL4), Lin-28 homologue A (LIN28), octamer-binding transcription factor 3/4 (OCT3/4), D2-40, cluster of differentiation 117 (CD117), sex-determining region Y-box 17, sex-determining region Y-box 2 (SOX2), cluster of differentiation 30, the ß-subunit of human chorionic gonadotropin (ß-hCG), GATA-binding protein 3 (GATA3), forkhead box protein A2 (FOXA2), glypican-3 (GPC3), α-fetoprotein (AFP), terminal deoxynucleotidyl transferase (TdT), nuclear protein of the testis (NUT), and pan-cytokeratin. Quantitative real-time polymerase chain reaction was performed to investigate the i(12p) status. Fifteen seminomas, seven teratomas, one yolk sac tumour, one choriocarcinoma and seven mixed PMGCTs were diagnosed. Each entity had different immunohistochemical staining patterns, which helped to distinguish them: OCT3/4, D2-40, CD117 and TdT for seminoma; OCT3/4 and SOX2 for embryonal carcinoma; FOXA2, GPC3 and AFP for yolk sac tumour; and ß-hCG and GATA3 for choriocarcinoma. Mature teratomas stained positively for pan-cytokeratin in epithelial components and focally for SALL4, SOX2, GATA3, D2-40, and FOXA2. Furthermore, a NUT carcinoma mimicking a PMGCT was diagnosed, showing strong nuclear SOX2 staining and speckled nuclear NUT staining. i(12p) was detected in 24 of 27 PMGCTs (89%). CONCLUSION: A diagnostic algorithm is of great importance for a reliable diagnosis of PMGCT in, usually small, tissue biopsy samples. Therefore, a combination of three to four antibodies to identify the correct histological subtype is usually necessary, in addition to morphological features. The i(12p) status serves as an additional option to indicate a germ cell origin in selected cases.

The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas.

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population-embryonal carcinoma (EC)-is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra-embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho- and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.

The component of the m6A writer complex VIRMA is implicated in aggressive tumor phenotype, DNA damage response and cisplatin resistance in germ cell tumors.

BACKGROUND: Germ cell tumors (GCTs) are developmental cancers, tightly linked to embryogenesis and germ cell development. The recent and expanding field of RNA modifications is being increasingly implicated in such molecular events, as well as in tumor progression and resistance to therapy, but still rarely explored in GCTs. In this work, and as a follow-up of our recent study on this topic in TGCT tissue samples, we aim to investigate the role of N6-methyladenosine (m6A), the most abundant of such modifications in mRNA, in in vitro and in vivo models representative of such tumors. METHODS: Four cell lines representative of GCTs (three testicular and one mediastinal), including an isogenic cisplatin resistant subline, were used. CRISPR/Cas9-mediated knockdown of VIRMA was established and the chorioallantoic membrane assay was used to study its phenotypic effect in vivo. RESULTS: We demonstrated the differential expression of the various m6A writers, readers and erasers in GCT cell lines representative of the major classes of these tumors, seminomas and non-seminomas, and we evidenced changes occurring upon differentiation with all-trans retinoic acid treatment. We showed differential expression also among cells sensitive and resistant to cisplatin treatment, implicating these players in acquisition of cisplatin resistant phenotype. Knockdown of VIRMA led to disruption of the remaining methyltransferase complex and decrease in m6A abundance, as well as overall reduced tumor aggressiveness (with decreased cell viability, tumor cell proliferation, migration, and invasion) and increased sensitivity to cisplatin treatment, both in vitro and confirmed in vivo. Enhanced response to cisplatin after VIRMA knockdown was related to significant increase in DNA damage (with higher γH2AX and GADD45B levels) and downregulation of XLF and MRE11. CONCLUSIONS: VIRMA has an oncogenic role in GCTs confirming our previous tissue-based study and is further involved in response to cisplatin by interfering with DNA repair. These data contribute to our better understanding of the emergence of cisplatin resistance in GCTs and support recent attempts to therapeutically target elements of the m6A writer complex.

The developmental origin of cancers defines basic principles of cisplatin resistance.

Cisplatin-based chemotherapy has been used for more than four decades as a standard therapeutic option in several tumor entities. However, being a multifaceted and heterogeneous phenomenon, inherent or acquired resistance to cisplatin remains a major obstacle during the treatment of several solid malignancies and inevitably results in disease progression. Hence, we felt there was an urgent need to evaluate common mechanisms between multifarious cancer entities to identify patient-specific therapeutic strategies. We found joint molecular and (epi)genetic resistance mechanisms and specific cisplatin-induced mutational signatures that depended on the developmental origin (endo-, meso-, ectoderm) of the tumor tissue. Based on the findings of thirteen tumor entities, we identified three resistance groups, where Group 1 (endodermal origin) prominently indicates NRF2-pathway activation, Group 2 (mesodermal origin, primordial germ cells) shares elevated DNA repair mechanisms and decreased apoptosis induction, and Group 3 (ectodermal and paraxial mesodermal origin) commonly presents deregulated apoptosis induction and alternating pathways as the main cisplatin-induced resistance mechanisms. This review further proposes potential and novel therapeutic strategies to improve the outcome of cisplatin-based chemotherapy.

On the origin of germ cell neoplasia in situ: Dedifferentiation of human adult Sertoli cells in cross talk with seminoma cells in vitro.

Germ cell neoplasia in situ (GCNIS) is the noninvasive precursor of testicular germ cell tumors type II, the most common cancer in young men, which originates from embryonic germ cells blocked in their maturation. GCNIS is associated with impaired Sertoli cells (SCs) that express fetal keratin 18 (KRT18) and the pluripotency factor SRY-Box transcription factor 2 (SOX2). According to the current theory concerning the origin of GCNIS, these SCs are prepubertal cells arrested in their maturation due to (epi)genetic anomalies and/or environmental antiandrogens. Thus, they are unable to support the development of germ cells, which leads to their maturational block and further progresses into GCNIS. Alternatively, these SCs are hypothesized to be adult cells dedifferentiating secondarily under the influence of GCNIS. To examine whether tumor cells can dedifferentiate SCs, we established a coculture model of adult human SCs (FS1) and a seminoma cell line similar to GCNIS (TCam-2). After 2 wk of coculture, FS1 cells showed progressive expression of KRT18 and SOX2, mimicking the in vivo changes. TCam-2 cells showed SOX2 expression and upregulation of further pluripotency- and reprogramming-associated genes, suggesting a seminoma to embryonal carcinoma transition. Thus, our FS1/TCam-2 coculture model is a valuable tool for investigating interactions between SCs and seminoma cells. Our immunohistochemical and ultrastructural studies of human testicular biopsies with varying degrees of GCNIS compared to biopsies from fetuses, patients with androgen insensitivity syndrome, and patients showing normal spermatogenesis further suggest that GCNIS-associated SCs represent adult cells undergoing progressive dedifferentiation.

Molecular and epigenetic pathogenesis of germ cell tumors.

The development of germ cell tumors (GCTs) is a unique pathogenesis occurring at an early developmental stage during specification, migration or colonization of primordial germ cells (PGCs) in the genital ridge. Since driver mutations could not be identified so far, the involvement of the epigenetic machinery during the pathogenesis seems to play a crucial role. Currently, it is investigated whether epigenetic modifications occurring between the omnipotent two-cell stage and the pluripotent implanting PGCs might result in disturbances eventually leading to GCTs. Although progress in understanding epigenetic mechanisms during PGC development is ongoing, little is known about the complete picture of its involvement during GCT development and eventual classification into clinical subtypes. This review will shed light into the current knowledge of the complex epigenetic and molecular contribution during pathogenesis of GCTs by emphasizing on early developmental stages until arrival of late PGCs in the gonads. We questioned how misguided migrating and/or colonizing PGCs develop to either type I or type II GCTs. Additionally, we asked how pluripotency can be regulated during PGC development and which epigenetic changes contribute to GCT pathogenesis. We propose that SOX2 and SOX17 determine either embryonic stem cell-like (embryonal carcinoma) or PGC-like cell fate (seminoma). Finally, we suggest that factors secreted by the microenvironment, i.e. BMPs and BMP inhibiting molecules, dictate the fate decision of germ cell neoplasia in situ (into seminoma and embryonal carcinoma) and seminomas (into embryonal carcinoma or extraembryonic lineage), indicating an important role of the microenvironment on GCT plasticity.