Heinz Arnheiter

Interferon-γ links ultraviolet radiation to melanomagenesis in mice.

Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, the incidence of which continues to rise. Epidemiological studies show that the major aetiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood. We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and aetiological criteria, but only when irradiated as neonatal pups with UVB, not UVA. However, the mechanisms underlying UVB-initiated, neonatal-specific melanomagenesis remain largely unknown. Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-γ (IFN-γ), but not type-I interferons. IFN-γ was produced by macrophages recruited to neonatal skin by UVB-induced ligands to the chemokine receptor Ccr2. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-γ blockade abolished macrophage-enhanced melanoma growth and survival. IFN-γ-producing macrophages were also identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-γ in promoting melanocytic cell survival/immunoevasion, identifying a novel candidate therapeutic target for a subset of melanoma patients.

Role of the nucleocapsid protein in regulating vesicular stomatitis virus RNA synthesis.

We describe experiments with two monoclonal antibodies to the vesicular stomatitis virus (VSV) nucleocapsid protein N with strikingly different characteristics. Antibody 1 binds to nucleocapsids and probably the pool of free (unbound) N protein; it inhibits transcription in vitro, and when microinjected into cells, protects the cells against VSV. Antibody 2 binds poorly to nucleocapsids, does not inhibit transcription, but when microinjected into cells, binds selectively to the free N and delays the appearance of progeny virus. We have confirmed these results by analyzing the effect of these antibodies on in vitro genomic RNA synthesis. The results of both the in vivo and in vitro experiments show that the replication of the VSV genome is controlled by the availability of the nucleocapsid protein, even when the polymerase has access to the host factors and multiple phosphorylated forms of the NS protein thought to be involved in genomic RNA synthesis.

Oct-3 is a maternal factor required for the first mouse embryonic division.

Oct-3 is a POU domain transcription factor that binds the octamer DNA motif and is present in mouse oocytes before and after fertilization. When fertilized oocytes were injected with antisense Oct-3 oligonucleotides or double-stranded DNA containing the octamer motif, embryonic DNA synthesis was inhibited and the embryos were arrested at the one-cell stage. In vitro synthesized Oct-3 mRNA rescued the developmental block induced by antisense Oct-3 oligonucleotide. We conclude that maternally inherited Oct-3 is required for DNA replication and division of the one-cell embryo.

Evolutionary biology. Eyes viewed from the skin.

The skin of many animals includes cells that respond to light by dispersing or aggregating granules within them. A study of such cells in frog skin now delivers the finding that they express a molecule, melanopsin, which is similar to the light-sensitive rhodopsins in eyes. In itself, that is not surprising. What is unexpected is that melanopsin is more closely related to invertebrate than to vertebrate rhodopsins, including that in the frogs own eyes. In evolutionary terms, then, this observation throws up all sorts of tantalizing questions.

Abstract 2994: Using embryonic melanoblast transcriptome analysis to identify novel mechanisms promoting metastatic melanoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Melanoma is among the most aggressive of cancers in its ability to metastasize. Although melanoma makes up <6% of all skin cancers, it contributes to over 70% of skin cancer deaths. There is no other tumor type that consistently gains metastatic potential in a matter of millimeters. However, the genes and pathways involved in this deadly manifestation remain elusive. The enhanced ability of melanoma cells to metastasize is reminiscent of the innate propensity of melanoblasts to migrate for long distances during embryonic development - from the neural crest to the eventual niche across the skin of the whole body. Once transformed, melanoma cells mimic migratory and growth capability similar to that of the embryonic melanoblasts. Therefore we hypothesize that late stage metastatic melanoma can exploit pathways employed by embryonic melanoblasts to achieve a more aggressive malignant phenotype. In the present study, we paint a novel picture of the oncological landscape based on the mouse melanoblast signature to reveal an intimate connection between tumorigenesis and developmental processes. We have, for the first time isolated and sequenced the transcriptomes of murine embryonic melanoblasts at several key representative developmental stages utilizing a newly developed genetically engineered mouse model with melanocyte-specific GFP expression. To uncover the overall classes of gene expression and to identify and characterize genesets whose expression is common and equally important to melanomagenic and developmental processes, a heat-map of the top 1000 most variable developmental genes was generated, and then shortlisted based on compared levels of expression in human and mouse metastatic melanomas and a relationship with melanoma patient survival data. A series of bioinformatics and meta-analyses led us to identify a small number of candidate genes. The resulting gene set was found to be related to early neural expression, epigenetic regulation, collagens, G-protein coupled receptors and calcium regulators. As a final step toward the identification and characterization of genes whose expression is common and equally important to both melanoma metastasis and melanoblast developmental processes, we are determining the consequences of RNAi-based knockdown on experimental metastasis potential in mouse models. Developmental genes that regulate melanoma metastatic behavior will be fully characterized. This approach should facilitate identification of novel therapeutic targets for melanoma treatment and diagnosis. Our study will attempt to provide insight into elements of melanocyte development that might prime them for metastasis in future malignancies. Pathways parallel between embryonic and metastatic melanoma cells will be identified and validated, offering both mechanistic and prognostic significance to our understanding of this fatal disease. Citation Format: Pravin J. Mishra, Theresa Guo, Raza Zaidi, Sean Davis, Heinz Arnheiter, William C. Reinhold, Paul Meltzer, Glenn Merlino. Using embryonic melanoblast transcriptome analysis to identify novel mechanisms promoting metastatic melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2994. doi:10.1158/1538-7445.AM2013-2994

Retraction: Oct-3 is a maternal factor required for the first mouse embryonic division.

preformationist chains. As the book describes, these philosophical arguments continued with the more well-known debates in developmental biology at the turn of this century-namely wholism, vitalism, and epigenesis. Thus, this is a very illuminating book for all developmental biologists. It reminds us that regeneration studies were for two hundred years the driving force in developmental biology, and the famous experiments of Roux, Dreisch, and Spemann were not performed independently but were seen as “regeneration” experiments. Indeed, all the concepts currently used in development, such as polarity, concentration gradients, thresholds, etc., arose from regeneration studies. Let us hope that the two fields will in the future become equal partners again and books such as Dinsmore’s may play some part in that.

Abstract 994: Integrated embryonic transcriptome analyses identify key melanoma metastasis regulator

The enhanced ability of melanoma cells to metastasize is reminiscent of the innate propensity of melanoblasts to migrate to distant sites during embryonic development - from the neural crest to their eventual niche in the skin. Once transformed, melanoma cells mimic migratory and growth capabilities similar to those of embryonic melanoblasts. This putative relationship between tumorigenesis and developmental processes was first suggested by Rudolf Virchow more than 150 years ago. While this theory is largely unproven, there are mechanistic links between the processes regulating development and malignancy. Here we investigate this age-old puzzle using a mouse model with melanocyte-specific GFP expression to capture and sequence embryonic melanoblasts. We have, for the first time, isolated and sequenced the transcriptomes of murine embryonic melanoblasts at several key representative developmental stages. To uncover the overall classes of gene expression and to identify and characterize genesets whose expression is common and equally important to melanomagenic and developmental processes, a heat-map of the top 1000 most variable developmental genes was generated, and then shortlisted based on compared levels of expression in human and mouse metastatic melanomas, and on the relationship with melanoma patient survival data. By integrating bioinformatics and functional data we have devised a new cross-species multi-dimensional embryonic-onco-genomics analyses (MEGA) approach and show that late stage melanomas reactivate genes used during embryonic development to achieve a more aggressive metastatic phenotype (which we refer to as metafetal genes). One such identified gene, a KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum (ER) protein retention receptor (KDELR) family member, was expressed in advanced mouse melanomas relative to normal skin or benign nevi. We confirmed this in human melanoma by showing that this metafetal gene was expressed at high levels in metastatic patient samples relative to benign lesions, and also predicted patient survival. Next, we determined the consequences of RNAi-based knockdown on experimental metastasis in mouse models. We validated the functional significance in human and mouse melanomas by showing that this KDELR plays a key role in melanoma metastasis through adaptation to chronic ER stress for survival by modulating the unfolded protein response (UPR), which can be targeted. We anticipate that this approach will identify a variety of key hardwired pathways associated with melanocyte development that can be co-opted by opportunistic metastatic melanoma cells. This approach also offers a novel perspective on melanoma therapeutics and intervention and offers both mechanistic as well as prognostic insights into our understanding of this fatal disease. Citation Format: Pravin J. Mishra, Theresa Guo, Raza Zaidi, Sean Davis, Aleksandra Michalowski, Helen Michael, William Reinhold, Heinz Arnheiter, Paul Meltzer, Glenn Merlino. Integrated embryonic transcriptome analyses identify key melanoma metastasis regulator. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 994. doi:10.1158/1538-7445.AM2014-994