The neural crest transcription factor BRN3A is essential for the proliferation and survival of melanoma cells. It is frequently expressed in melanoma but not in normal melanocytes or benign nevi. The mechanisms underlying the aberrant expression of BRN3A are unknown. Here, we investigated the epigenetic regulation of BRN3A in melanocytes and melanoma cell lines treated with DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC) inhibitors. DNMT and HAT inhibition did not significantly alter BRN3A expression levels, whereas panHDAC inhibition by trichostatin A led to increased expression. Treatment with the isoform-specific HDAC inhibitor mocetinostat, but not with PCI-34051, also increased BRN3A expression levels, suggesting that class I HDACs HDAC1, HDAC2, and HDAC3, and class IV HDAC11, were involved in the regulation of BRN3A expression. Transient silencing of HDACs 1, 2, 3, and 11 by siRNAs revealed that, specifically, HDAC2 inhibition was able to increase BRN3A expression. ChIP-Seq analysis uncovered that HDAC2 inhibition specifically increased H3K27ac levels at a distal enhancer region of the BRN3A gene. Altogether, our data suggest that HDAC2 is a key epigenetic regulator of BRN3A in melanocytes and melanoma cells. These results highlight the importance of epigenetic mechanisms in regulating melanoma oncogenes.

Melanoma cells share many biological properties with neural crest stem cells. Here we show that the homeodomain transcription factor MSX1, which is significantly correlated with melanoma disease progression, reprograms melanocytes and melanoma cells toward a neural crest precursor-like state. MSX1-reprogrammed normal human melanocytes express the neural crest marker p75 and become multipotent. MSX1 induces a phenotypic switch in melanoma, which is characterized by an oncogenic transition from an E-cadherine-high nonmigratory state toward a ZEB1-high invasive state. ZEB1 up-regulation is responsible for the MSX1-induced migratory phenotype in melanoma cells. Depletion of MSX1 significantly inhibits melanoma metastasis in vivo. These results show that neural crest-like reprogramming achieved by a single factor is a critical process for melanoma progression.

Differentiation from stem cells to lineage-specific somatic cells is not a unidirectional process, but can be reverted by genetic and/or epigenetic alterations. Here, we show that the homeodomain transcription factor Msh homeobox 1 (MSX1), which is essential for neural crest specification, reprograms melanocytes towards a neural crest precursor-like state. MSX1- expressing melanocytes express the neural crest marker p75 neurotrophin receptor and are able to differentiate into neuronal and mesenchymal lineages. Mechanistically, MSX1 binds and suppresses the proximal promoter of microphthalmia-associated transcription factor (MITF), the master transcriptional regulator of melanogenesis. Expression of MSX1 is significantly correlated with melanoma progression, as overexpression of MSX1 prompts melanoma cell motility and depletion of MSX1 significantly inhibits melanoma metastasis. Taken together, these results demonstrate that neural crest-like reprogramming in melanocytes can be achieved by a single factor and that similar dedifferentiation is a critical process for melanoma progression.

Significant advancements of mutation-based targeted therapy and immune checkpoint blockade have been achieved in melanoma. Nevertheless, acquired resistance and nonresponders to therapy require different strategies. An innovative approach is presented here that is based on the combination of innate immune system activation and simultaneous targeting of the oncogene urokinase-type plasminogen activator receptor (uPAR). We generated two triphosphate-conjugated siRNAs targeting uPAR (ppp-uPAR) by in vitro transcription. Specific uPAR knockdown and simultaneous activation of the retinoic acid-inducible gene 1 (RIG-I) was shown in different human melanoma cells, fibroblasts, and melanocytes. The compounds induced massive apoptosis in melanoma cells, whereas fibroblasts and melanocytes were less sensitive. The effects were less pronounced when the IFN receptor was blocked. Treatment with ppp-uPAR led to accumulation of p53 and induction of RIG-I-dependent proapoptotic signaling. The apoptotic effects induced by ppp-uPAR were maintained in melanoma cell lines that had acquired double resistance to B-RAF and MEK/extracellular signal-regulated kinase inhibition. Systemic intraperitoneal application of ppp-uPAR in nude mice significantly reduced growth of human melanoma xenografts and elicited a systemic innate immune response with increased serum cytokine levels. Our data suggest that ppp-uPAR represents a therapeutically attractive compound that may help overcome the strong therapy resistance of melanoma.

Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak.

Pityriasis versicolor (PV) is a common superficial fungal infection of the skin caused by Malassezia. Initially M. furfur was suggested as its main aetiological agent; however, more recent studies suggest M. globosa as the dominant species. The possibility of a variance in predominant species based on geographical basis has not been fully evaluated. The objective of this study was to identify the Malassezia species on affected and non-affected skin of students with PV who reside in a tropical environment (Abuja, Nigeria) and correlate them to clinical characteristics. In this study, the literature on prevalence of Malassezia genus in PV was also reviewed. Samples were taken from 304 PV lesions and 110 normal appearing skin. Microscopy, culture and identification of Malassezia species utilising polymerase chain reaction-restriction fragment length polymorphism analysis were performed. Three Malassezia species were detected in PV with the major species being M. furfur. On normal appearing skin, M. furfur (77.6%) and M. restricta (10.4%) were both detected. No case of M. globosa was identified in this study. There was no significant difference between species identified and clinical features of PV. M. furfur is probably still the most predominant species causing PV in the tropical environment.

Psoriasis vulgaris is a common T cell–mediated inflammatory skin disease with a suspected autoimmune pathogenesis. The human leukocyte antigen (HLA) class I allele, HLA-C*06:02, is the main psoriasis risk gene. Epidermal CD8+ T cells are essential for psoriasis development. Functional implications of HLA-C*06:02 and mechanisms of lesional T cell activation in psoriasis, however, remained elusive. Here we identify melanocytes as skin-specific target cells of an HLA-C*06:02–restricted psoriatic T cell response. We found that a Vα3S1/Vβ13S1 T cell receptor (TCR), which we had reconstituted from an epidermal CD8+ T cell clone of an HLA-C*06:02–positive psoriasis patient specifically recognizes HLA-C*06:02–positive melanocytes. Through peptide library screening, we identified ADAMTS-like protein 5 (ADAMTSL5) as an HLA-C*06:02–presented melanocytic autoantigen of the Vα3S1/Vβ13S1 TCR. Consistent with the Vα3S1/Vβ13S1-TCR reactivity, we observed numerous CD8+ T cells in psoriasis lesions attacking melanocytes, the only epidermal cells expressing ADAMTSL5. Furthermore, ADAMTSL5 stimulation induced the psoriasis signature cytokine, IL-17A, in CD8+ T cells from psoriasis patients only, supporting a role as psoriatic autoantigen. This unbiased analysis of a TCR obtained directly from tissue-infiltrating CD8+ T cells reveals that in psoriasis HLA-C*06:02 directs an autoimmune response against melanocytes through autoantigen presentation. We propose that HLA-C*06:02 may predispose to psoriasis via this newly identified autoimmune pathway.

Abstract Melanoma cells share many biological properties with neural crest cells. Here, we show that the homeodomain transcription factor Msh homeobox 1 (MSX1), which is essential for neural crest specification, reprograms melanocytes towards a neural crest precursor-like state. MSX1-reprogrammed melanocytes express the neural crest marker p75 and are able to differentiate into neuronal and mesenchymal lineages. Mechanistically, MSX1 suppresses the proximal promoter of microphthalmia-associated transcription factor (MITF), the master transcriptional regulator of melanogenesis. MSX1 expression is also significantly correlated with melanoma progression. MSX1 prompts melanoma cell motility and depletion of MSX1 significantly inhibits melanoma metastasis. These results demonstrate that not only can neural crest-like reprogramming in melanocytes be achieved by a single factor, but also that similar dedifferentiation is a critical process for melanoma progression. Citation Format: Markus V. Heppt, Wang X. Joshua, Denitsa M. Hristova, Zhi Wei, Martin Irmler, Carola Berking, Robert Besch, Johannes Beckers, Frank J. Rauscher, III, David E. Fisher, Meenhard Herlyn, Mizuho Fukunaga-Kalabis. MSX1-induced neural crest-like reprograming promotes melanoma progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A13.

Cyprinid herpesvirus-3 (CyHV-3) is an etiological agent of a notifiable disease that causes high mortality rates affecting both the common and koi carp Cyprinus carpio L. There is no current treatment strategy to save CyHV-3 infected fish. RNA mediated interference (RNAi) is an emerging strategy used for understanding gene function and is a promising method in developing novel therapeutics and antiviral medications. For this study, the possibility of activating the RNAi pathway by the use of small interfering (si)RNAs was tested to inhibit in vitro viral replication of CyHV-3 in common carp brain (CCB) cells. The siRNAs were designed to target either thymidine kinase (TK) or DNA polymerase (DP) genes, which both code for transcripts involved in DNA replication. The inhibition of viral replication caused by the siRNAs was measured by a reporter gene, termed ORF81. Treatment with siRNA targeting either TK or DP genes reduced the release of viral particles from infected CCB cells. However, siRNA targeting DP was most effective at reducing viral release as measured by qPCR.

Spring viraemia of carp virus (SVCV) is an aetiological agent of a serious disease affecting carp farms in Europe and is a member of the Rhabdoviridae family of viruses. The genome of SVCV codes for five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). RNA-mediated interference (RNAi) by small interfering RNAs (siRNAs) is a powerful tool to inhibit gene transcription and is used to study genes important for viral replication. In previous studies regarding another member of Rhabdoviridae, siRNA inhibition of the rabies virus nucleoprotein gene provided in vitro and in vivo protection against rabies. In this study, synthetic siRNA molecules were designed to target SVCV-N and SVCV-P transcripts to inhibit SVCV replication and were tested in an epithelioma papulosum cyprini (EPC) cell line. Inhibition of gene transcription was measured by real-time quantitative reverse-transcription PCR (RT-qPCR). The efficacy of using siRNA for inhibition of viral replication was analysed by RT-qPCR measurement of a reporter gene (glycoprotein) expression and by virus endpoint titration. Inhibition of nucleoprotein and phosphoprotein gene expression by siRNA reduced SVCV replication. However, use of tandem siRNAs that target phosphoprotein and nucleoprotein worked best at reducing SVCV replication.

Re-activation of molecules active in embryogenesis can play an important role in cancer, since they can provide tumor cells with malignant properties. Several members of the family of POU transcription factors are essential for the development of the nervous system and several are expressed in the neural crest, which is the same location where melanocytic cells develop from. Here, the POU transcription factor family and its role in melanocytic transformation and melanoma are reviewed.

Double-stranded RNA molecules carrying a triphosphate moiety represent a molecular structure by which the host recognizes viral infections. Such RNA molecules can be generated synthetically by chemical synthesis or by in vitro transcription (see Chapter 2, Hornung et al.). Similar to viruses, they initiate an antiviral immune response, e.g., by stimulation of the immune system. Short, double-stranded RNA in the cytosol can also trigger the RNA interference mechanism, which also has been considered as an antiviral response. Notably, synthetic RNAs that are designed to be specific for a certain host mRNA inhibit expression of the respective gene, leading to specific gene silencing. Both effects-gene silencing and immunostimulation-are interesting from a therapeutic perspective, e.g., for cancer therapy. Notably, both effects can be activated by a single molecule, an siRNA carrying a triphosphate moiety. This chapter provides information how to design such compounds with respect to the associated signaling pathways and the techniques to evaluate bifunctional RNAs in the context of tumor therapy.

The transcription factor SOX10 (SRY (sex determining region Y)-box 10) has a key role in the embryonic development of melanocytes. Recently, it has been suggested that SOX10 is highly relevant for melanoma development and survival. However, the distinct functions and downstream targets of SOX10 in melanoma remain widely unknown. In this study, we inhibited SOX10 via RNA interference in different human melanoma cell lines and found a significantly reduced invasion capacity in vitro and in the chick embryo model. At later time points, SOX10 inhibition reduced proliferation and induced cell death. We identified melanoma inhibitory activity (MIA) as a direct target gene of SOX10, which is an essential protein for melanoma cell migration and invasion. Expression levels of SOX10 and MIA strictly correlated in melanoma cell lines, and SOX10 inhibition reduced MIA expression and promoter activity. Direct binding of SOX10 to the MIA promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. Ectopic expression of MIA in SOX10-inhibited melanoma cells restored the invasion capacity, supporting the hypothesis that MIA is responsible for SOX10-mediated melanoma cell invasion. Our data provide evidence for a critical role of SOX10 in melanoma cell invasion through the regulation of MIA and highlight its role as a therapeutic target in melanoma.

Cellular heterogeneity, for example, the intratumoral coexistence of cancer cells with and without stem cell characteristics, represents a potential root of therapeutic resistance and a significant challenge for modern drug development in glioblastoma (GBM). We propose here that activation of the innate immune system by stimulation of innate immune receptors involved in antiviral and antitumor responses can similarly target different malignant populations of glioma cells. We used short-term expanded patient-specific primary human GBM cells to study the stimulation of the cytosolic nucleic acid receptors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). Specifically, we analyzed cells from the tumor core versus "residual GBM cells" derived from the tumor resection margin as well as stem cell-enriched primary cultures versus specimens without stem cell properties. A portfolio of human, nontumor neural cells was used as a control for these studies. The expression of RIG-I and MDA5 could be induced in all of these cells. Receptor stimulation with their respective ligands, p(I:C) and 3pRNA, led to in vitro evidence for an effective activation of the innate immune system. Most intriguingly, all investigated cancer cell populations additionally responded with a pronounced induction of apoptotic signaling cascades revealing a second, direct mechanism of antitumor activity. By contrast, p(I:C) and 3pRNA induced only little toxicity in human nonmalignant neural cells. Granted that the challenge of effective central nervous system (CNS) delivery can be overcome, targeting of RIG-I and MDA5 could thus become a quintessential strategy to encounter heterogeneous cancers in the sophisticated environments of the brain.