In mice, the Ter mutation causes primordial germ cell (PGC) loss in all genetic backgrounds. Ter is also a potent modifier of spontaneous testicular germ cell tumour (TGCT) susceptibility in the 129 family of inbred strains, and markedly increases TGCT incidence in 129-Ter/Ter males. In 129-Ter/Ter mice, some of the remaining PGCs transform into undifferentiated pluripotent embryonal carcinoma cells, and after birth differentiate into various cells and tissues that compose TGCTs. Here, we report the positional cloning of Ter, revealing a point mutation that introduces a termination codon in the mouse orthologue (Dnd1) of the zebrafish dead end (dnd) gene. PGC deficiency is corrected both with bacterial artificial chromosomes that contain Dnd1 and with a Dnd1-encoding transgene. Dnd1 is expressed in fetal gonads during the critical period when TGCTs originate. DND1 has an RNA recognition motif and is most similar to the apobec complementation factor, a component of the cytidine to uridine RNA-editing complex. These results suggest that Ter may adversely affect essential aspects of RNA biology during PGC development. DND1 is the first protein known to have an RNA recognition motif directly implicated as a heritable cause of spontaneous tumorigenesis. TGCT development in the 129-Ter mouse strain models paediatric TGCT in humans. This work will have important implications for our understanding of the genetic control of TGCT pathogenesis and PGC biology.

Ionizing radiation (IR) is used for patients diagnosed with unresectable non small cell lung cancer (NSCLC), however radiotherapy remains largely palliative due to radioresistance. Cancer stem cells (CSCs), as well as epithelial-mesenchymal transition (EMT), may contribute to drug and radiation resistance mechanisms in solid tumors. Here we investigated the molecular phenotype of A549 and H460 NSCLC cells that survived treatment with IR (5Gy) and are growing as floating tumor spheres and cells that are maintained in a monolayer after irradiation.Non-irradiated and irradiated cells were collected after one week, seeded onto ultra low attachment plates and propagated as tumor spheres. Bulk NSCLC cells which survived radiation and grew in spheres express cancer stem cell surface and embryonic stem cell markers and are able to self-renew, and generate differentiated progeny. These cells also have a mesenchymal phenotype. Particularly, the radiation survived sphere cells express significantly higher levels of CSC markers (CD24 and CD44), nuclear β-catenin and EMT markers (Snail1, Vimentin, and N-cadherin) than non-irradiated lung tumor sphere cells. Upregulated levels of Oct-4, Sox2 and beta-catenin were detected in H460 cells maintained in a monolayer after irradiation, but not in radiation survived adherent A459 cells.PDGFR-beta was upregulated in radiation survived sphere cells and in radiation survived adherent cells in both A549 and H460 cell lines. Combining IR treatment with axitinib or dasatinib, inhibitors with anti-PDFGR activity, potentiates the efficacy of NSCLC radiotherapy in vitro.Our findings suggest that radiation survived cells have a complex phenotype combining the properties of CSCs and EMT. CD44, SNAIL and PDGFR-beta are dramatically upregulated in radiation survived cells and might be considered as markers of radiotherapy response in NSCLC.

Inactivation of the dead-end (Dnd1) gene in the Ter mouse strain results in depletion of primordial germ cells (PGCs) so that mice become sterile. However, on the 129 mouse strain background, loss of Dnd1 also increases testicular germ cell tumor incidence in parallel to PGC depletion. We report that inactivation of Dnd1 also affects embryonic viability in the 129 strain. Mouse Dnd1 encodes two protein isoforms, DND1-isoform α (DND1-α) and DND1-isoform β (DND1-β). Using isoform specific antibodies, we determined DND1-α is expressed in embryos and embryonic gonads whereas DND1-β expression is restricted to germ cells of the adult testis. Our data implicates DND1-α isoform to be necessary for germ cell viability and therefore its loss in Ter mice results in PGC depletion, germ cell tumor development and partial embryonic lethality in the 129 strain.

BackgroundDespite continuous efforts to identify genes that are pivotal regulators of advanced melanoma and closely related to it, to determine which of these genes have to be blocked in their function to keep this highly aggressive disease in check, it is far from clear which molecular pathway(s) and specific genes therein, is the Achilles’ heel of primary and metastatic melanoma. In this report, we present data, which document that the DEAD-box helicase DDX11, which is required for sister chromatid cohesion, is a crucial gatekeeper for melanoma cell survival.MethodsPerforming immunohistochemistry and immunoblot analysis, we determined expression of DDX11 in melanoma tissues and cell lines. Following transfection of melanoma cells with a DDX11-specific siRNA, we conducted a qPCR analysis to determine downregulation of DDX11 in the transfected melanoma cells. In subsequent studies, which focused upon an analysis of fluorescently labeled as well as Giesma-stained chromosome spreads, a proliferation analysis and apoptosis assays, we determined the impact of suppressing DDX11 expression on melanoma cells representing advanced melanoma.ResultThe findings of the study presented herein document that DDX11 is upregulated with progression from noninvasive to invasive melanoma, and that it is expressed at high levels in advanced melanoma. Furthermore, and equally important, we demonstrate that blocking the expression of DDX11 leads not only to inhibition of melanoma cell proliferation and severe defects in chromosome segregation, but also drives melanoma cells rapidly into massive apoptosis.ConclusionTo date, little is known as to whether helicases play a role in melanoma development and specifically, in the progression from early to advanced melanoma. In this report, we show that the helicase DDX11 is expressed at high levels in primary and metastatic melanoma, and that interfering with its expression leads to severe chromosome segregation defects, telomere shortening, and massive melanoma cell apoptosis. These findings suggest that DDX11 could be an important candidate for molecular targeted therapy for advanced melanoma.

The thymus is a vertebrate-specific organ where T lymphocytes are generated. Genetic programs that lead to thymus development are incompletely understood. We previously screened ethylnitrosourea-induced medaka mutants for recessive defects in thymus development. Here we report that one of those mutants is caused by a missense mutation in a gene encoding the previously uncharacterized protein WDR55 carrying the tryptophan-aspartate-repeat motif. We find that WDR55 is a novel nucleolar protein involved in the production of ribosomal RNA (rRNA). Defects in WDR55 cause aberrant accumulation of rRNA intermediates and cell cycle arrest. A mutation in WDR55 in zebrafish also leads to analogous defects in thymus development, whereas WDR55-null mice are lethal before implantation. These results indicate that WDR55 is a nuclear modulator of rRNA synthesis, cell cycle progression, and embryonic organogenesis including teleost thymus development.

The RNA binding protein DEAD-END (DND1) is one of the few proteins known to regulate microRNA (miRNA) activity at the level of miRNA-mRNA interaction. DND1 blocks miRNA interaction with the 3′-untranslated region (3′-UTR) of specific mRNAs and restores protein expression. Previously, we showed that the DNA cytosine deaminase, APOBEC3 (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide like 3), interacts with DND1. APOBEC3 has been primarily studied for its role in restricting and inactivating retroviruses and retroelements. In this report, we examine the significance of DND1-APOBEC3 interaction. We found that while human DND1 inhibits miRNA-mediated inhibition of P27, human APOBEC3G is able to counteract this repression and restore miRNA activity. APOBEC3G, by itself, does not affect the 3′-UTR of P27. We found that APOBEC3G also blocks DND1 function to restore miR-372 and miR-206 inhibition through the 3′-UTRs of LATS2 and CX43, respectively. In corollary experiments, we tested whether DND1 affects the viral restriction function or mutator activity of APOBEC3. We found that DND1 does not affect APOBEC3 inhibition of infectivity of exogenous retrovirus HIV (ΔVif) or retrotransposition of MusD. In addition, examination of Ter/Ter;Apobec3−/− mice, lead us to conclude that DND1 does not regulate the mutator activity of APOBEC3 in germ cells. In summary, our results show that APOBEC3 is able to modulate DND1 function to regulate miRNA mediated translational regulation in cells but DND1 does not affect known APOBEC3 function.

The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC) cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of β-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors.

BackgroundThe dead-end (Dnd1) gene is essential for maintaining the viability of germ cells. Inactivation of Dnd1 results in sterility and testicular tumors. The Dnd1 encoded protein, DND1, is able to bind to the 3′-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA.Methodology/Principal FindingsHere we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites.Conclusions/SignificanceThe 3′-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development.