NRN1 interacts with Notch to increase oncogenic STAT3 signaling in melanoma.

BACKGROUND: Melanoma is a highly heterogeneous cancer, in which frequent changes in activation of signaling pathways lead to a high adaptability to ever changing tumor microenvironments. The elucidation of cancer specific signaling pathways is of great importance, as demonstrated by the inhibitor of the common BrafV600E mutation PLX4032 in melanoma treatment. We therefore investigated signaling pathways that were influenced by neurotrophin NRN1, which has been shown to be upregulated in melanoma. METHODS: Using a cell culture model system with an NRN1 overexpression, we investigated the influence of NRN1 on melanoma cells' functionality and signaling. We employed real time cell analysis and spheroid formation assays, while for investigation of molecular mechanisms we used a kinase phosphorylation kit as well as promotor activity analysis followed by mRNA and protein analysis. RESULTS: We revealed that NRN1 interacts directly with the cleaved intracellular domain (NICD) of Notch1 and Notch3, causing a potential retention of NICD in the cytoplasm and thereby reducing the expression of its direct downstream target Hes1. This leads to decreased sequestration of JAK and STAT3 in a Hes1-driven phosphorylation complex. Consequently, our data shows less phosphorylation of STAT3 while presenting an accumulation of total protein levels of STAT3 in association with NRN1 overexpression. The potential of the STAT3 signaling pathway to act in both a tumor suppressive and oncogenic manner led us to investigate specific downstream targets - namely Vegf A, Mdr1, cMet - which were found to be upregulated under oncogenic levels of NRN1. CONCLUSIONS: In summary, we were able to show that NRN1 links oncogenic signaling events between Notch and STAT3 in melanoma. We also suggest that in future research more attention should be payed to cellular regulation of signaling molecules outside of the classically known phosphorylation events.

Loss of miR-101-3p in melanoma stabilizes genomic integrity, leading to cell death prevention.

Malignant melanoma remains the most lethal form of skin cancer, exhibiting poor prognosis after forming distant metastasis. Owing to their potential tumor-suppressive properties by regulating oncogenes and tumor suppressor genes, microRNAs are important player in melanoma development and progression. We defined the loss of miR-101-3p expression in melanoma cells compared with melanocytes and melanoblast-related cells as an early event in tumor development and aimed to understand the tumor suppressive role of miR-101-3p and its regulation of important cellular processes. Reexpression of miR-101-3p resulted in inhibition of proliferation, increase in DNA damage, and induction of apoptosis. We further determined the nuclear structure protein Lamin B1, which influences nuclear processes and heterochromatin structure, ATRX, CASP3, and PARP as an important direct target of miR-101-3p. RNA sequencing and differential gene expression analysis after miR-101-3p reexpression supported our findings and the importance of loss of mir-101-3p for melanoma progression. The validated functional effects are related to genomic instability, as recent studies suggest miRNAs plays a key role in mediating this cellular process. Therefore, we concluded that miR-101-3p reexpression increases the genomic instability, leading to irreversible DNA damage, which leads to apoptosis induction. Our findings suggest that the loss of miR-101-3p in melanoma serves as an early event in melanoma progression by influencing the genomic integrity to maintain the increased bioenergetic demand.

Nuclear AREG affects a low-proliferative phenotype and contributes to drug resistance of melanoma.

AMPHIREGULIN (AREG) is a multifaceted molecule, which acts not only as an extracellular ligand for EGF receptor (EGFR), but also as an intracellular signaling molecule. It remains elusive, however, whether AREG has a tumor suppressive or oncogenic role in melanoma. Here, we found that several melanoma cell lines express AREG, but the expression does not correlate with that of EGFR. Recombinant AREG and the neutralizing antibody experiments showed that intracellular AREG plays an important role in melanoma, implying a divergent function of AREG in addition to the role as a ligand for EGFR. Further investigation of this mechanism revealed that particularly nuclear-localized AREG regulates IGF-1R, P21 (Cip1/Waf1), TP53 and JARID1B protein accumulation in the nucleus. Furthermore, manipulation of nuclear AREG levels has influence on heterochromatin condensation (HP1beta, SETDB1) and trimethylation of histones H3K9 and H3K4. As these molecules correspond to previously identified markers for slow-cycling drug resistant cells, we speculate that nuclear AREG predisposes cells to resistance to therapy. According to the hypothesis, we detected the accumulation of AREG in the nucleus of SK-Mel-28-VR, which was cultured under Vemurafenib (VR) selection pressure, and this correlates with JARID1B expression. Here, knockdown of AREG makes the previously resistant cells more sensitive to VR treatment, resulting in inhibited proliferation. Taken together, we suggest that nuclear AREG affects a slow-cycling phenotype and increases resistance to VR, raising a possibility that AREG might be a potential therapeutic target for resistance in melanoma.

HDAC2 Is Involved in the Regulation of BRN3A in Melanocytes and Melanoma.

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.

Loss of Gene Information: Discrepancies between RNA Sequencing, cDNA Microarray, and qRT-PCR.

Molecular analyses of normal and diseased cells give insight into changes in gene expression and help in understanding the background of pathophysiological processes. Years after cDNA microarrays were established in research, RNA sequencing (RNA-seq) became a key method of quantitatively measuring the transcriptome. In this study, we compared the detection of genes by each of the transcriptome analysis methods: cDNA array, quantitative RT-PCR, and RNA-seq. As expected, we found differences in the gene expression profiles of the aforementioned techniques. Here, we present selected genes that exemplarily demonstrate the observed differences and calculations to reveal that a strong RNA secondary structure, as well as sample preparation, can affect RNA-seq. In summary, this study addresses an important issue with a strong impact on gene expression analysis in general. Therefore, we suggest that these findings need to be considered when dealing with data from transcriptome analyses.

miR-488-5p and its role in melanoma.

Due to their ability to regulate dozens to hundreds of target genes simultaneously and, therefore, influence several oncogenic pathways at the same time, microRNAs are a fascinating research object in melanoma. MicroRNAs have been identified as regulators of tumor proliferation, invasion and metastasis in melanoma. More precisely, it has been published that dysregulation of miR-488 contibutes to the progression of several cancer entities. However, the biological functions of miR-488, in special miR-488-5p in melanoma, remain unclear. This study showed the involvement of miR-488-5p in Wnt/ß-catenin pathway and the function as a tumor suppressor. Transfection of miR-488-5p mimic led to inhibition of proliferation, migration, anchorage independent growth and led to induction of apoptosis. These data indicated that miR-488-5p acts as a tumor suppressor and is lost during melanoma development. The loss of miR-488-5p was confirmed in vivo by in situ hybridization on melanoma tissue.

Aberrant expression of c-Jun in glioblastoma by internal ribosome entry site (IRES)-mediated translational activation.

Although the protooncogene c-Jun plays a critical role in cell proliferation, cell death, and malignant transformation, DNA microarray screens have identified only a few human cancer types with aberrant expression of c-Jun. Here, we show that c-Jun accumulation is robustly elevated in human glioblastoma and that this increase contributes to the malignant properties of the cells. Most importantly, the increase in c-Jun protein accumulation occurs with no corresponding increase in c-Jun mRNA or the half-life of the c-Jun protein but, rather, in the translatability of the transcript. The c-Jun 5'UTR harbors a potent internal ribosomal entry site (IRES) with a virus-like IRES domain that directs cap-independent translation in glioblastoma cells. Accumulation of c-Jun is not dependent on MAPK activity but can be stimulated by a cytoskeleton-dependent pathway. Our findings provide evidence that human c-Jun is an IRES-containing cellular transcript that contributes to cancer development through translational activation. This previously undescribed mechanism of c-Jun regulation might also be relevant to other types of human cancer and offers unique potential targets for therapy.

Loss of T-cadherin (CDH-13) regulates AKT signaling and desensitizes cells to apoptosis in melanoma.

An understanding of signaling pathways is a basic requirement for the treatment of melanoma. Currently, kinases are at the center of melanoma therapies. According to our research, additional alternative molecules are equally important for development of melanoma. In this regard, cancer progression is, among other factors, driven by an altered adhesion via cadherins. For instance, the de-regulated expression of the adhesion molecule T-cadherin is found in various cancer types, including melanoma, and influences migration and invasion. T-cadherin is thought to affect cellular function largely through its signaling and not its adhesion properties because the molecule is anchored into the cell membrane by a glycosylphosphatidylinositol (GPI) moiety. However, detailed knowledge about the consequences of the loss of T-cadherin in melanoma is currently lacking. For this reason, we were interested in assessing which signaling pathways are initiated by T-cadherin. The tumor growth of subcutaneously injected T-cadherin-positive melanoma cells was diminished compared with T-cadherin-negative cells in nude mice. The difference in tumor volume was not due to decreased proliferation but rather due to increased apoptosis. After the expression of T-cadherin was induced, we detected V-AKT murine thymoma viral oncogene homolog (AKT) and FoxO3a hypophosphorylation accompanied by the downregulation of the antiapoptotic molecules BCL-2, BCL-x and Clusterin. Furthermore, we detected a diminished transcriptional activity of CREB and AP-1. We demonstrated that T-cadherin functions as a pro-apoptotic tumor suppressor that antagonizes AKT/CREB/AP-1/FoxO3a signaling, whereas NFκB, TCF/LEF and mTOR are not part of the T-cadherin signaling pathway. Notably, we found that the restoration of T-cadherin in melanoma cells causes sensitization to apoptosis induced by CD95/Fas antibody CH-11.

Alternative Wnt-signaling axis leads to a break of oncogene-induced senescence.

Oncogene-induced senescence (OIS) is an important process that suppresses tumor development, but the molecular mechanisms of OIS are still under investigation. It is known that BRAFV600E-mutated melanocytes can overcome OIS and develop melanoma, but the underlying mechanism is largely unknown. Using an established OIS model of primary melanocytes transduced with BRAFV600E, YAP activity was shown to be induced in OIS as well as in melanoma cells compared to that in normal epidermal melanocytes. This led to the assumption that YAP activation itself is not a factor involved in the disruption of OIS. However, its role and interaction partners potentially change. As Wnt molecules are known to be important in melanoma progression, these molecules were the focus of subsequent studies. Interestingly, activation of Wnt signaling using AMBMP resulted in a disruption of OIS in BRAFV600E-transduced melanocytes. Furthermore, depletion of Wnt6, Wnt10b or ß-catenin expression in melanoma cells resulted in the induction of senescence. Given that melanoma cells do not exhibit canonical Wnt/ß-catenin activity, alternative ß-catenin signaling pathways may disrupt OIS. Here, we discovered that ß-catenin is an interaction partner of YAP on DNA in melanoma cells. Furthermore, the ß-catenin-YAP interaction changed the gene expression pattern from senescence-stabilizing genes to tumor-supportive genes. This switch is caused by transcriptional coactivation via the LEF1/TEAD interaction. The target genes with binding sites for LEF1 and TEAD are involved in rRNA processing and are associated with poor prognosis in melanoma patients. This study revealed that an alternative YAP-Wnt signaling axis is an essential molecular mechanism leading to OIS disruption in melanocytes.

The Role of T-Cadherin (CDH13) in Treatment Options with Garcinol in Melanoma.

Targeted therapies with chemotherapeutic agents and immunotherapy with checkpoint inhibitors are among the systemic therapies recommended in the guidelines for clinicians to treat melanoma. Although there have been constant improvements in the treatment of melanoma, resistance to the established therapies continues to occur. Therefore, the purpose of this study was to explore the function of garcinol with regards to specific cancer properties such as proliferation and apoptosis. Garcinol, a natural compound isolated from the plant also known as mangosteen (Garcinia mangostana), is a newly discovered option for cancer treatment. Numerous pharmaceutical substances are derived from plants. For example, the derivates of camptothecin, extracted from the bark of the Chinese tree of happiness (Camptotheca acuminate), or paclitaxel, extracted from the bark of the Western yew tree (Taxus brevifolia), are used as anti-cancer drugs. Here, we show that garcinol reduced proliferation and induced apoptosis in melanoma cell lines. In addition, we found that those cells that are positive for the expression of the cell-cell adhesion molecule T-cadherin (CDH13) respond more sensitively to treatment with garcinol. After knock-down experiments with an siRNA pool against T-cadherin, the sensitivity to garcinol decreased and proliferation and anti-apoptotic behavior of the cells was restored. We conclude that patients who are T-cadherin-positive could especially benefit from a therapy with garcinol.

Impact of LIF (leukemia inhibitory factor) expression in malignant melanoma.

Leukemia inhibitory factor (LIF) signaling regulates cellular processes to maintain the self-renewal and pluripotency of embryonic stem (ES) cells. Independent of these capabilities, LIF was also identified to be responsible for cancer development and progression. However, its detailed cellular function in cancer remains unclear thus far. We found LIF to be expressed in melanoma cell lines of primary and metastatic origin and in melanoma tissue. We further elucidated stimuli that are responsible for the high expression levels of LIF. Interestingly, hypoxia, specifically through HIF-1α, is involved in regulating LIF. Furthermore, our data showed that the signaling of LIF was not mediated by the classically described pathway via STAT3, but rather through BMP4 and BMP7. We hypothesize that the co-expression of LIF and BMP is necessary for a de-differentiated cancer phenotype. Ancillary to BMP4 and BMP7, classical stem cell proteins, e.g., SOX2, NANOG, OCT3/4 and GBX2, are regulated by LIF. We therefore speculate that LIF can induce a typical "cancer stem cell"-like behavior, as the appropriate genes are regulated by LIF. Particularly, the expression of these genes has been proposed as a driving force for tumorigenesis and the initiation of metastasis. Notably, LIF has an important role not only for ES cells but also for cancer development. Melanoblast-related cells (MBrcs), which resemble the neural crest precursor cells of melanocytes, expressed LIF in minor amounts compared to normal human melanocytes. These data, along with the data that LIF is upregulated in melanoma cell lines compared to melanocytes, strongly indicate that LIF is important for the stabilization of the melanoma phenotype. To elucidate the role of LIF in cellular melanoma behavior, we analyzed proliferation, attachment, migration and colony formation after silencing LIF by siRNA, and found all four characteristics restricted. In summary, we can show that LIF is an important factor in melanoma progression.

BRN2 is a transcriptional repressor of CDH13 (T-cadherin) in melanoma cells.

T-cadherin (cadherin 13, H-cadherin, gene name CDH13) has been proposed to act as a tumor-suppressor gene as its expression is significantly diminished in several types of carcinomas, including melanomas. Allelic loss and promoter hypermethylation have been proposed as mechanisms for silencing of CDH13. However, they do not account for loss of T-cadherin expression in all carcinomas, and other genetic or epigenetic alterations can be presumed. The present study investigated transcriptional regulation of CDH13 in melanoma. Bioinformatical analysis pointed to the presence of known BRN2 (also known as POU3F2 and N-Oct-3)-binding motifs in the CDH13 promoter sequence. We found an inverse correlation between BRN2 and T-cadherin protein and transcript expression. Reporter gene analysis and electrophoretic mobility shift assays in melanoma cells demonstrated that CDH13 is a direct target of BRN2 and that BRN2 is a functional transcriptional repressor of CDH13 promoter activity. The regulatory binding element of BRN2 was located -219 bp of the CDH13 promoter proximal to the start codon and was identified as 5'-CATGCAAAA-3'. Ectopic expression of BRN2 in BRN2-negative/T-cadherin-positive melanoma cells resulted in suppression of CDH13 promoter activity, whereas BRN2 knockdown in BRN2-positive/T-cadherin-negative melanoma cells resulted in re-expression of T-cadherin transcripts and protein. Transcriptional repression of CDH13 by BRN2 may participate in malignant transformation of melanoma by increasing invasion and migration potentials of melanoma cells. The study has identified CDH13 as a novel direct BRN2 transcriptional target gene and has advanced knowledge of mechanisms underlying loss of T-cadherin expression in melanoma.

ETS-1/RhoC signaling regulates the transcription factor c-Jun in melanoma.

Recently, we discovered that the loss of E-cadherin induces c-Jun protein expression, which is a member of the AP-1 transcription factor family and a key player in the processes of cell proliferation and tumor development and also found in elevated levels in melanomas. Notably, the mRNA level of c-Jun was not affected, suggesting that c-Jun is regulated at post-transcriptional level. Here, we present data that suggest that the dynamic cytoskeletal network, linked to E-cadherin, is involved in the regulation of the c-Jun protein and transcriptional activity. In a signaling cascade, the loss of E-cadherin activates the transcriptional regulator ETS-1 and consequently leads to the induction of RhoC expression that stabilizes c-Jun in melanoma. The link between RhoC and c-Jun seems to be indirect via the cytoskeleton. We conclude that the loss of E-cadherin mediated cell-adhesion induces c-Jun protein expression in a multistep process, offering several possibilities for therapeutic intervention.

MAPK and PI3K/AKT mediated YB-1 activation promotes melanoma cell proliferation which is counteracted by an autoregulatory loop.

Y-box binding protein 1 (YB-1) is an oncogenic transcription and translation factor and is overexpressed in several types of cancer. Our previous data showed that YB-1 is upregulated and translocated to the nucleus during melanoma progression and that YB-1 is an important transcription factor regulating proliferation, survival, migration, invasion and chemosensitivity of melanoma cells. It has been suggested that YB-1 is activated and translocated to the nucleus after S102-phosphorylation in the DNA binding domain. In this study, we show that activation of YB-1 by S102-phosphorylation and nuclear translocation is increased during melanoma progression using a human tissue microarray with 100 melanocytic lesions. Furthermore, we analysed the mechanisms governing the expression and activity of YB-1 in melanoma cells. We show that the PI3K/AKT and p53 signalling, growth factors and chemotherapeutic agents increase YB-1 promoter activity. This, however, resulted in no or only modest increase in YB-1 protein expression. We show that the MAPK and PI3K/AKT signalling pathways, both activated in melanoma cells, as well as p53 overexpression increase YB-1 S102-phosphorylation, whereas NFκB signalling inhibits phosphorylation. Overexpression of YB-1 in melanoma cells inhibits translation efficiency and by this proliferation and survival of melanoma cells indicating that there is an autoregulatory loop restricting YB-1 protein expression. These data suggest that there is a tightly regulated feedback mechanism regulating YB-1 expression and activation, necessary for proper cell cycle progression of melanoma cells.

E-cadherin cell-cell communication in melanogenesis and during development of malignant melanoma.

Cell-cell communication is necessary for the crosstalk between cells that constitute multicellular organisms and is essential for cells to coordinate their physiological behavior to create cohesive tissues. Cellular crosstalk is not only controlled by molecules, like growth factors, hormones, ions and G-proteins, etc. but also by cell-cell contacts. These contacts are essential for intercellular communication and are involved in survival, apoptosis, proliferation, differentiation and homeostasis of entire tissues. In polarized epithelia of vertebrates, the adherent junction is part of the tripartite junctional complex that is localized at the juxtaluminal region, which includes tight junctions (including claudins, occludins, and zonula occludens proteins), desmosomal junctions (including desmogleins), and adherent junctions. In focus of the manuscript are adherent molecules of the cadherin superfamily of the skin. In the normal epidermis, melanocytes and keratinocytes are mostly connected via E-cadherin, P-cadherin and H-cadherin [1-3]. Melanocytes that reside in the basal layer of the epidermis predominantly contain E-cadherin and H-cadherin, whereas those that reside in the hair follicles are rich in P-cadherin [2]. The regulation and role of E-cadherin during melanoma development will be the focus of this review.

Alpha-Synuclein and Its Role in Melanocytes.

Pigmentation is an important process in skin physiology and skin diseases and presumably also plays a role in Parkinson's disease (PD). In PD, alpha-Synuclein (aSyn) has been shown to be involved in the pigmentation of neurons. The presynaptic protein is intensively investigated for its pathological role in PD, but its physiological function remains unknown. We hypothesized that aSyn is both involved in melanocytic differentiation and melanosome trafficking processes. We detected a strong expression of aSyn in human epidermal melanocytes (NHEMs) and observed its regulation in melanocytic differentiation via the microphthalmia-associated transcription factor (MITF), a central regulator of differentiation. Moreover, we investigated its role in pigmentation by performing siRNA experiments but found no effect on the total melanin content. We discovered a localization of aSyn to melanosomes, and further analysis of aSyn knockdown revealed an important role in melanocytic morphology and a reduction in melanosome release. Additionally, we found a reduction of transferred melanosomes in co-culture experiments of melanocytes and keratinocytes but no complete inhibition of melanosome transmission. In summary, this study highlights a novel physiological role of aSyn in melanocytic morphology and its so far unknown function in the pigment secretion in melanocytes.

Impact of CYLD on chromatin structure and histone methylation in malignant melanoma.

The tumor suppressive role of CYLD lysine 63 deubiquitinase (CYLD) is known in melanoma. To the best of our knowledge, however, the precise mechanism underlying the tumor suppressive function of CYLD has yet to be clarified. In the present study, a novel melanoma mouse model was generated, which revealed accelerated tumor growth in Cyld­knockout (Cyld­/­) compared with Cyld­wild­type (Cyld+/+) mice. To determine the underlying molecular mechanism, mutation analysis of primary tumor­derived cell lines from Cyld+/+ and Cyld­/­ mice was performed using RNA sequencing data. Variant calling revealed no common mutations in Cyld­/­ compared with Cyld+/+ cells. Thus, the epigenetic processes influencing development and progression of melanoma were investigated. Initial analysis of expression pattern of known hypermethylated genes in melanoma (suppressor of cytokine signalling, methylthioadenosine phosphorylase, cadherin 1) in the presence or absence of 5'­Aza­deoxyctidine treatment revealed that CYLD does not play a key role in DNA methylation. Chromatin accessibility and histone H3 modification assay uncovered a role of CYLD in the formation of chromatin structure. Subsequent inhibitor experiments confirmed the effect of CYLD on H3K9me2 level associated with heterochromatin. Furthermore, enhanced H3K9 dimethylation in Cyld­/­ melanoma cells was associated with upregulation of euchromatic histone lysine methyltransferase 2 (EHMT2). Moreover, the specific inhibitor of EHMT2, CM272, resulted in decreased proliferation and relaxation of compact chromatin in Cyld­deficient melanoma cells. These results reveal a novel role of CYLD in histone methylation and chromatin packaging.

Knockdown of Lamin B1 and the Corresponding Lamin B Receptor Leads to Changes in Heterochromatin State and Senescence Induction in Malignant Melanoma.

Modifications in nuclear structures of cells are implicated in several diseases including cancer. They result in changes in nuclear activity, structural dynamics and cell signalling. However, the role of the nuclear lamina and related proteins in malignant melanoma is still unknown. Its molecular characterisation might lead to a deeper understanding and the development of new therapy approaches. In this study, we analysed the functional effects of dysregulated nuclear lamin B1 (LMNB1) and its nuclear receptor (LBR). According to their cellular localisation and function, we revealed that these genes are crucially involved in nuclear processes like chromatin organisation. RNA sequencing and differential gene expression analysis after knockdown of LMNB1 and LBR revealed their implication in important cellular processes driving ER stress leading to senescence and changes in chromatin state, which were also experimentally validated. We determined that melanoma cells need both molecules independently to prevent senescence. Hence, downregulation of both molecules in a BRAFV600E melanocytic senescence model as well as in etoposide-treated melanoma cells indicates both as potential senescence markers in melanoma. Our findings suggest that LMNB1 and LBR influence senescence and affect nuclear processes like chromatin condensation and thus are functionally relevant for melanoma progression.

Melanoblasts in culture as an in vitro system to determine molecular changes in melanoma.

Many consolidated findings have revealed that cancer formation resembles events of embryonic development. In particular, the network of transcription factors and adhesion molecules is very similar when comparing neural crest-derived melanoblasts and melanoma cells. The main difference is found in the manifestation of distinct genes in melanoma, whereas in neural crest cells gene expression is tightly regulated to promote either a migratory or stationary phenotype. We established a cell culture system to generate melanoblast-related cells (MBrc) out of melanocytes as originally described by Cook et al. First, we confirmed the typical gene expression pattern of BRN-2, SOX10, PAX3 and EDNRB. Furthermore, we identified enhanced migration and proliferation similar to that of melanoma cells. Our intention of using this system was to classify the known 'melanoma-associated genes' into a subgroup of genes solely regulated by the differentiation process and a second subgroup that is unaffected by differentiation and is potentially important to the stabilization of a melanoma phenotype. The expression of melanoma-associated genes (N-cadherin, MUC-18, integrin ß3, α3, α5, αv, SLUG, TBX3, HIF1-α, BMP-4 and bFGF) was enhanced in MBrc which were de-differentiated out of melanocytes. E-cadherin, H-cadherin and ß-catenin, prevalently found to be downregulated in melanoma, were diminished in MBrc. Remarkably, the transcription factor SNAIL was unaffected by differentiation and could be one key molecule in early melanoma development that is of prevailing importance. In summary, we feel that the analysis of MBrc generated in a reproducible system will give new insight into the role and importance of melanoma-associated genes.

Amphiregulin Regulates Melanocytic Senescence.

Oncogene-induced senescence (OIS) is a decisive process to suppress tumor development, but the molecular details of OIS are still under investigation. Using an established OIS model of primary melanocytes transduced with BRAF V600E and compared to control cells, amphiregulin (AREG) was shown to be induced. In addition, AREG expression was observed in nevi, which by definition, are senescent cell clusters, compared to melanocytes. Interestingly, treatment of melanocytes with recombinant AREG did induce senescence. This led to the assumption that extracellular AREG has an important function in this process. Inhibition of the epidermal growth factor receptor (EGFR) using Gefitinib identified AREG as one of EGFR ligands responsible for senescence. Furthermore, depletion of AREG expression in senescent BRAF V600E melanocytes resulted in a significant reduction of senescent melanocytes. This study reveals AREG as an essential molecular component of signaling pathways leading to senescence in melanocytes.