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The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein that plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we identified several compounds that strongly inhibit HMGA2-DNA interactions including suramin, a century-old, negatively charged antiparasitic drug. Our results show that the inhibition is likely through suramin binding to the “AT-hook” DNA-binding motifs and therefore preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. Since HMGA1 proteins also carry multiple “AT-hook” DNA-binding motifs, suramin is expected to inhibit HMGA1-DNA interactions as well. Biochemical and biophysical studies show that charge-charge interactions and hydrogen bonding between the suramin sulfonated groups and Arg/Lys residues play critical roles in the binding of suramin to the “AT-hook” DNA-binding motifs. Furthermore, our results suggest that HMGA2 may be one of suramin’s cellular targets.

Endothelins are cytokines expressed in the microenvironment of several tumors. To identify which stromal cells in the melanoma microenvironment respond to endothelin, we injected murine melanoma cell lines B16F10, YUMM1.7, and YUMMER1.7 in a transgenic mouse that overexpresses endothelin 3 (Edn3) under the control of the keratin 5 promoter in the skin (K5-Edn3). All cell lines developed larger tumors in K5-Edn3 mice than in control animals. In YUMM1.7 tumors, the Edn3 receptor, endothelin receptor B (Ednrb), was expressed in several stromal cell types including immune cells. This result was validated by the identification of Ednrb-positive stromal cells in human melanoma from previously published RNA-seq data. Regulatory T cells (Tregs) and dendritic cell numbers were significantly higher in K5-Edn3 tumors when compared to control tumors. Edn3 increased Treg proliferation in vitro and the expression of FOXP3. YUMM1.7-GFP tumors in K5-Edn3 mice were sensitive to immune checkpoint inhibitor (anti-CTLA-4) as well as to Ednrb blockage (BQ-788). Our results indicate that Ednrb signaling has an important role in the melanoma microenvironment where it mediates immunosuppression resulting in escape from tumor immunity.

Objective: Aortic valve (AV) leaflets rely on a precise extracellular matrix (ECM) microarchitecture for appropriate biomechanical performance. The ECM structure is maintained by valvular interstitial cells (VICs), which reside within the leaflets. The presence of pigment produced by a melanocytic population of VICs in mice with dark coats has been generally regarded as a nuisance, as it interferes with histological analysis of the AV leaflets. However, our previous studies have shown that the presence of pigment correlates with increased mechanical stiffness within the leaflets as measured by nanoindentation analyses. In the current study, we seek to better characterize the phenotype of understudied melanocytic VICs, explore the role of these VICs in ECM patterning, and assess the presence of these VICs in human aortic valve tissues.Approach and Results: Immunofluorescence and immunohistochemistry revealed that melanocytes within murine AV leaflets express phenotypic markers of either neuronal or glial cells. These VIC subpopulations exhibited regional patterns that corresponded to the distribution of elastin and glycosaminoglycan ECM proteins, respectively. VICs with neuronal and glial phenotypes were also found in human AV leaflets and showed ECM associations similar to those observed in murine leaflets. A subset of VICs within human AV leaflets also expressed dopachrome tautomerase, a common melanocyte marker. A spontaneous mouse mutant with no aortic valve pigmentation lacked elastic fibers and had reduced elastin gene expression within AV leaflets. A hyperpigmented transgenic mouse exhibited increased AV leaflet elastic fibers and elastin gene expression.Conclusions: Melanocytic VIC subpopulations appear critical for appropriate elastogenesis in mouse AVs, providing new insight into the regulation of AV ECM homeostasis. The identification of a similar VIC population in human AVs suggests conservation across species.

Scanning ion conductance microscopy (SICM) offers the ability to obtain nanoscale resolution images of the membranes of living cells. Here, we show that a dual-barrel nanopipette probe based potentiometric SICM (P-SICM) can simultaneously map the topography and surface potential of soft, rough and heterogeneously charged surfaces under physiological conditions. This technique was validated and tested by systematic studies on model samples, and the finite element method (FEM) based simulations confirmed its surface potential sensing capability. Using the P-SICM method, we compared both the topography and extracellular potential distributions of the membranes of normal (Mela-A) and cancerous (B16) skin cells. We further monitored the structural and electrical changes of the membranes of both types of cells after exposing them to the elevated potassium ion concentration in extracellular solution, known to depolarize and damage the cell. From surface potential imaging, we revealed the dynamic appearance of heterogeneity of the surface potential of the individual cell membrane. This P-SICM method provides new opportunities to study the structural and electrical properties of cell membrane at the nanoscale.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein which plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen high throughput screening (HTS) assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we discovered that suramin, a negatively charged antiparasitic drug potently inhibits the HMGA2-DNA interaction. Our results also show that the inhibition is through suramin binding to the AT-hooks of HMGA2, therefore blocking its DNA binding capacity. Furthermore, we demonstrate that suramin can induce brain tumor stem cells differentiation into cells with neurite-like structures, a process triggered by disrupting HMGA2-DNA interactions. Since suramin has strong antitumor and anti-metastasis activities, our discovery suggests that HMGA2 and HMGA2-like proteins may be the cellular target of this century-old drug.

Melanocytic tumors in the central nervous system consist of metastatic melanoma and the more unusual primary lesions, which arise from melanocytes of the leptomeninges. Leptomeningeal melanocytic lesions include melanocytomas, diffuse melanosis and primary melanomas. Their differential diagnosis is challenging and the underlying molecular mechanisms for their generation and maintenance remain poorly characterized. The development and characterization of mouse models that parallel the human phenotype are critical for the understanding of the basic mechanisms underlying the pathology of the disease and for the pre-clinical evaluation of therapeutic strategies. We have identified glutamate signaling via the metabotropic glutamate receptor 1 (Grm1) as one key component of leptomeningeal disease. GRM1 is a member of the group I of GRMs that when bound to its ligand glutamate, couples with G alpha-q/11 (Gαq/11) to activate MEK/ERK and AKT/mTOR pathways, which are relevant downstream targets of GRM1 for melanocyte transformation. Transgenic mice TG(Grm1)EPv that express Grm1 under the regulation of the melanocyte specific promoter Dopachrome tautomerase (TG(Grm1)EPv) developed highly pigmented cutaneous melanocytic lesions located in the tail and pinnae at around 8 months of age with no apparent metastases to distant organs. Brains and spinal cords from transgenic animals of varying ages (n=15) were processed for histology and immunofluorescence. Melanosis was encountered in all animals in the leptomeninges associated to the olfactory bulb and forebrain. The other areas in which most animals displayed high levels of melanosis were the choroid plexus, cerebellopontine angle, and the lumbar spine in association with the perineurium of the nerves. Tyrosinase-related protein 1 (Trp1) positive cells were present in areas of melanosis and overlapped with high levels of Gαq/11 expression. Vimentin, known to be positive in melanocytomas but not in melanoma, was observed in the melanosis areas. Surprisingly, AKT and mTOR were not detected by immunostaining in the melanosis areas. Western blot analysis of the meninges with melanosis (n=3) showed significant elevated levels of p-ERK when compared to tissues from non-transgenic animals. A similar trend of elevated levels of expression was observed for Gαq/11, AKT, and mTOR. These observations indicate that the ectopic expression of Grm1 in melanocytes drive the formation of leptomeningeal melanocytic lesions and that the Tg(Grm1) mouse can be used for further studies aimed at understanding the mechanistic basis of leptomeningeal melanocytoma and other leptomeningeal diseases. Citation Format: Israel Castillo Gonzalez, Juliano Freitas, Xiaoshuang Li, Martina Cavallini, Lidia Kos. Characterization of a mouse model of leptomeningeal melanocytic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2916.

Cancer recurrence after therapy and long periods of remission are frequent in melanoma patients, likely due to the presence of residual disseminated tumor cells (DTCs). DTCs can enter dormancy and become refractory to targeted or conventional therapies. Extracellular matrix (ECM) proteins and basement membrane (BM) formation are critical structural components required to support the tumor microenvironment. Dystroglycan (DG) receptor complex, which is highly glycosylated in the alpha subunit, is a crucial ECM binding receptor because it functions in bridging the ECM and the cytoskeleton. DG receptor has been implicated in a variety of cancers, including melanoma, but its function in DTCs and dormancy has not been evaluated. In this study, we performed a bioinformatics analysis to study the implication of mutations in DAG1 (DG receptor gene) and its associated genes in cutaneous melanoma patients. We found that patients harboring mutations in the protein O-mannosyltransferase 1 enzyme (POMT1), which is essential for the transfer of a mannosyl residue to the alpha-DG subunit, had a significantly lower median months survival (27 months) compared to those with no POMT1 mutations (61 months). We noted missense mutations (Phe-Ser) in DAG1 occurred in the mucin-like region of the alpha subunit of DG. Also, the expression of DAG1, dystrophin (DMD), and utrophin (UTRN) at the mRNA level was significantly augmented under the presence of DAG1 mutations compared to the cohort of patients without DAG1 mutations. We evaluated the expression of alpha-DG, dystrophin, utrophin, and non-receptor tyrosine kinase (FER) to establish a potential relationship between the DAG receptor complex and FER in a metastatic mouse model. This mouse model is based on the overexpression of GPCRs (Tg(Grm1)Epv(E)/K5-tTA-Edn3) and spontaneously develops melanoma tumors on the ear, tail and dorsal skin, and presents with 90% penetrance of metastasis in the lymph nodes and lung. Using a genetic lineage tracing, we detected the presence of intravascular metastatic cells in the lung that are negative for Ki67 (proliferation marker), caspase3 (apoptosis marker), and other dormancy markers (p21, p27, p-ERK) indicating they are in a dormant state. The intravascular dormant DTC cells expressed high levels of alpha-DG and utrophin, and moderate levels of dystrophin but low levels of FER. Overall, our results suggest that alterations in alpha-DG glycosylation resulting from POMT1 mutations and subsequent FER downregulation may contribute to the dormancy status of metastatic melanoma cells in the lung vascular niche. Our findings will help us further understand the mechanisms that underlie melanoma dormancy and have the potential to contribute to the development of therapeutic strategies for recurrent disease. Citation Format: Israel Castillo Gonzalez, Mona Maharjan, Ananda Mohan Mondal, Lidia Kos. Dystroglycan receptor and FER maintain melanoma dormancy in the vascular niche [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3956.

Tumor cell plasticity, including transdifferentiation, is thought to be a key driver of therapy failure, tumor dormancy, and metastatic dissemination. Although melanoma cells have been shown to adopt various phenotypic features in vitro, direct in vivo evidence of metastatic cell plasticity remains sparse. Here, we combine lineage tracing in a spontaneous metastatic mouse model of melanoma, advanced imaging, and single-cell RNA sequencing approaches to search for pathophysiologically relevant melanoma cellular states. We identify melanoma cells in intravascular niches of various metastatic organs. These cells are quiescent, are negative for characteristic melanoma markers, and acquire endothelial cell features. We replicate the endothelial transdifferentiation (EndT) finding in another mouse model and provide evidence of EndT in BRAFV600E-metastatic biopsies from human lung, brain, and small intestine, thus highlighting the clinical relevance of these findings. The tumor-vasculature pattern described herein may contribute to melanoma dormancy within metastatic organs and represent a putative target for therapies. ispartof: CELL REPORTS vol:31 issue:11 ispartof: location:United States status: published

A major drawback of mechanical and prosthetic heart valves is their inability to permit somatic growth. By contrast, tissue-engineered pulmonary valves potentially have the capacity to remodel and integrate with the patient. For this purpose, adult stem cells may be suitable. Previously, human periodontal ligament cells (PDLs) have been explored as a reliable and robust progenitor cell source for cardiac muscle regeneration (Pelaez, D. Electronic Thesis and Dissertation Database, Coral Gables, FL, May 2011). Here, we investigate the potential of PDLs to support the valve lineage, specifically the concomitant differentiation to both endothelial cell (EC) and smooth muscle cell (SMC) types. We were able to successfully promote PDL differentiation to both SMC and EC phenotypes through a combination of stimulatory approaches using biochemical and mechanical flow conditioning (steady shear stress of 1 dyne/cm(2)), with flow-based mechanical conditioning having a predominant effect on PDL differentiation, particularly to ECs; in addition, strong expression of the marker FZD2 and an absence of the marker MLC1F point toward a unique manifestation of smooth muscle by PDLs after undergoing steady-flow mechanical conditioning alone, possible by only the heart valve and pericardium phenotypes. It was also determined that steady flow (which was performed using a physiologically relevant [for heart valves] magnitude of ~5-6 dynes/cm(2)) augmented the synthesis of the extracellular matrix collagen proteins. We conclude that under steady-flow dynamic culture environments, human PDLs can differentiate to heterogeneous cell populations that are relevant to heart valve tissue engineering. Further exploration of human PDLs for this purpose is thus warranted.

This work evaluates the effect of carbon nanotube (CNT) addition to plasma-sprayed hydroxyapatite (HA) coating on its tribological behavior, biocompatibility of the coating, and cytotoxicity of CNT-containing wear debris. Biological response of the CNT-containing wear debris is critical for osteoblasts, the bone-forming cells, and macrophages, the cells that clear up wear debris from blood stream. The addition of 4 wt % CNTs to HA coating reduces the volume of wear debris generation by 80% because of the improved elastic modulus and fracture toughness. CNT reinforcement has a pronounced effect on the particle size in the wear debris and subsequent biological response. There was a slight increase in the numbers and viability of osteoblasts grown on HA-CNT compared with HA alone. The cytotoxic effect of HA and HA-CNT debris to macrophages and osteoblasts was similar, demonstrating that loose CNT does not pose a problem to these cells.

Biodegradable polylactide-polycaprolactone copolymer (PLC) has been reinforced with 0, 2 and 5wt.% boron nitride nanotubes (BNNTs) for orthopedic scaffold application. Elastic modulus of the PLC-5wt.% BNNT composite, evaluated through nanoindentation technique, shows a 1370% increase. The same amount of BNNT addition to PLC enhances the tensile strength by 109%, without any adverse effect on the ductility up to 240% elongation. Interactions of the osteoblasts and macrophages with bare BNNTs prove them to be non-cytotoxic. PLC-BNNT composites displayed increased osteoblast cell viability as compared to the PLC matrix. The addition of BNNTs also resulted in an increase in the expression levels of the Runx2 gene, the main regulator of osteoblast differentiation. These results indicate that BNNT is a potential reinforcement for composites for orthopedic applications.

Endothelin (Edn) signaling via the G-coupled, endothelin receptor type B (Ednrb) is essential for the development of melanocytes from the neural crest (NC) and has been associated with melanoma progression. Edn3 plays varying roles during melanocyte development, promoting the proliferation and self-renewal of NC-derived multi- and bipotential precursors as well as the survival, proliferation, differentiation and migration of committed melanocyte precursors. Melanocyte differentiation is achieved via the interaction of Ednrb and Kit signaling, with Ednrb being specifically required in the final differentiation step, rather than in the initial specification of melanocytic fate. Ednrb has also been implicated in the de-differentiation of mature melanocytes, a process that takes place during the malignant transformation of these cells. Ednrb was found to be upregulated in melanoma metastases and was shown to alter tumor-host interactions leading to melanoma progression. Antagonists to this receptor were shown to inhibit melanoma cell growth and increase the apoptotic rate of these cells, and to lead to disease stabilization in melanoma patients. Thus, endothelin signaling inhibition may prove useful in the treatment of certain types of melanoma.

Endothelin 3 (Edn3) encodes a ligand important to developing neural crest cells and is allelic to the spontaneous mouse mutation occurring at the lethal spotting (ls) locus. Edn3(ls/ls) mutants exhibit a spotted phenotype due to reduced numbers of neural crest-derived melanocyte precursors in the skin. In this study, we show that when Edn3 is driven by the keratin 5 promoter and thereby placed proximal to melanocyte lineage cells, adult mice manifest pigmented skin harboring dermal melanocytes. Using a tetracycline inducible system, we show that the postnatal expression of Edn3 is required to maintain these dermal melanocytes, and that early expression of the Edn3 transgene is important to the onset of the hyperpigmentation phenotype. Crosses into Edn3(ls/ls) mutants demonstrate that the Edn3 transgene expression does not fully compensate for the endogenous expression pattern. Crosses into tyrosine kinase receptor Kit(Wv) mutants indicate that Edn3 can partially compensate for Kit's role in early development. Crosses into A(y) mutant mice considerably darkened their yellow coat color suggesting a previously unreported role for endothelin signaling in pigment switching. These results demonstrate that exogenous Edn3 affects both precursors and differentiated melanocytes, leading to a phenotype with characteristics similar to the human skin condition dermal melanocytosis.

Melanoma is the deadliest form of skin cancer due to its high propensity to metastasize and resistance to current therapies. We have created a spontaneous mouse model of metastatic melanoma (Dct-Grm1/K5-Edn3) where metastasis to the lungs is 80% penetrant. The primary tumors of these mice present cellular heterogeneity with cells at varying levels of differentiation. The main goal of this study is to determine the metastatic potential of the primary tumor resident Tyrosinase positive cells and evaluate the dynamic pattern of gene expression as those cells move from the primary tumors to the sites of metastasis. To accomplish this aim we crossed the Dct-Grm1/K5-Edn3 mice to CreERT2/ ROSAmT/mG mice to indelibly label Tyrosinase cell populations within the primary tumor by topical application of 4-hydroxytamoxifen at the tumor site. In vivo lineage tracing and characterization of those labeled cells was performed in the metastatic lesions. We found that Tyrosinase positive cells enter the circulation before the appearance of any noticeable tumor or nevus and establish close interactions with the vasculature as single cells. Metastatic cells in close association with the inner wall of the vasculature lose pigmentation and do not express melanocytic markers. Interestingly, those intravascular cells mimic endothelial cell properties with the expression of platelet endothelial cell adhesion molecule (PECAM-1, also known as CD31). In the lung tissue, the primary tumor derived Tyrosinase positive cells or their progeny can survive and establish successful metastases with pigmentation. This in vivo lineage tracing system in mouse will be a powerful model to evaluate and help us understand the etiology and pathogenesis of melanoma metastasis. Further characterization of those more aggressive cells in melanoma will allow for the development of new prognostic tests and novel therapeutic strategies to eliminate metastasis. Citation Format: Xiaoshuang Li, Raul Torres, Lidia Kos. Identification of the metastatic cell populations in a spontaneous mouse model of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1978. doi:10.1158/1538-7445.AM2017-1978

Melanoma is the most aggressive and deadliest type of skin cancer due to its high propensity to metastasize. Despite current progress in the identification of promising drugs for treating melanoma, drug resistance has emerged as a serious issue indicating that novel therapeutic strategies may be required. It is well documented that the process of converting the epithelial tumorigenic cell to a mesenchymal phenotype, the epithelial-mesenchymal transition (EMT), is responsible for conferring an invasive phenotype to cancer cells. An EMT-like process has been shown to occur during melanoma progression. High-mobility-protein AT-hook 2 (HMGA2) is a multi-function transcription factor linked to oncogenesis and EMT in a variety of cancers. HMGA2 is a small DNA-binding protein with three “AT-hook” DNA-binding motifs that specifically recognize the minor groove of AT-rich DNA sequences. HMGA2 is significantly upregulated in human primary melanoma and metastases, including those with BRAF mutations. We accessed the potential application of netropsin, a potent inhibitor of HMGA2-DNA interactions, as an EMT blocking agent of BRAF mutated melanoma cells. Three murine melanoma cells lines that carry mutations in the BRAF gene and show resistance to vemurafenib (D4M, YUMM1.1, and YUMM1.7) were used. Cells were treated with EMT Inducing Media Supplement along with Netropsin and the expression of E-cadherin as well as their migratory behavior were evaluated. Netropsin treatment of EMT induced cells was not cytotoxic, prevented changes in cell morphology, maintained high levels of E-cadherin expression and decreased migratory behavior. All three cell lines express HMGA2 which continues to be expressed in the tumors resulting from subcutaneous injections in mice. YUMM1.1 cells showed the highest levels of expression of HMGA2 amongst the three cell lines. Interestingly, in our preliminary study only YUMM1.1-derived tumors generated overt metastasis in the lung 30 days after the injections. Altogether our data showed that Netropsin is capable of blocking EMT of murine BRAF mutated melanoma cells. The results of this study will contribute to the development of strategies to prevent EMT that may result in the implementation of more effective therapies to treat melanoma. Citation Format: Juliano Freitas, Fenfei Leng, Lidia Kos. Netropsin blocks EMT of murine BRAF mutated melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 848. doi:10.1158/1538-7445.AM2017-848

In both mice and humans, mutations in the genes encoding the endothelin B receptor and its ligand endothelin 3 lead to deficiencies in neural crest-derived melanocytes and enteric neurons. The discrete steps at which endothelins exert their functions in melanocyte development were examined in mouse neural crest cell cultures. Such cultures, kept in the presence of fetal calf serum, gave rise to cells expressing the early melanoblast marker Dct even in the absence of the phorbol ester tetradecanoyl phorbol acetate (TPA) or endothelins. However, these early (Dct+) cells did not proliferate and pigmented cells never formed unless TPA or endothelins were added. In fact, endothelin 2 was as potent as TPA in promoting the generation of both Dct+melanoblasts and pigmented cells, and endothelin 1 or endothelin 3 stimulated the generation of melanoblasts and of pigmented cells to an even greater extent. The inhibition of this stimulation by the selective endothelin B receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-alpha-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine) suggested that the three endothelins all signal through the endothelin B receptor. This receptor was indeed expressed in Dct+melanoblasts, in addition to cells lacking Dct expression. The results demonstrate that endothelins are potent stimulators of melanoblast proliferation and differentiation.Key words: neural crest, melanocyte, endothelin, differentiation.

The axial structures, the notochord and the neural tube, play an essential role in the dorsoventral patterning of somites and in the differentiation of their many cell lineages. Here, we investigated the role of the axial structures in the mediolateral patterning of the somite by using a newly identified murine homeobox gene, Nkx-3.1, as a medial somitic marker in explant in vitro assays. Nkx-3.1 is dynamically expressed during somitogenesis only in the youngest, most newly-formed somites at the caudal end of the embryo. We found that the expression of Nkx-3.1 in pre-somitic tissue explants is induced by the notochord and maintained in newly-differentiated somites by the notochord and both ventral and dorsal parts of the neural tube. We showed that Sonic hedgehog (Shh) is one of the signaling molecules that can reproduce the effect of the axial structures by exposing explants to either COS cells transfected with a Shh expression construct or to recombinant SHH. Shh could induce and maintain Nkx-3.1 expression in pre-somitic mesoderm and young somites but not in more mature, differentiated ones. The effects of Shh on Nkx-3.1 expression were antagonized by a forskolin-induced increase in the activity of cyclic AMP-dependent protein kinase A. Additionally, we confirmed that the expression of the earliest expressed murine myogenic marker, myf 5, is also regulated by the axial strucutres but that Shh by itself is not capable of inducing or maintaining it. We suggest that the establishment of somitic medial and lateral compartments and the early events in myogenesis are governed by a combination of positive and inhibitory signals derived from the neighboring structures, as has previously been proposed for the dorsoventral patterning of somites.

This paper explores the nano-scratch technique for measuring the adhesion strength of a single osteoblast cell on a hydroxyapatite (HA) surface reinforced with carbon nanotubes (CNTs). This technique efficiently separates out the contribution of the environment (culture medium and substrate) from the measured adhesion force of the cell, which is a major limitation of the existing techniques. Nano-scratches were performed on plasma sprayed hydroxyapatite (HA) and HA‐CNT coatings to quantify the adhesion of the osteoblast. The presence of CNTs in HA coating promotes an increase in the adhesion of osteoblasts. The adhesion force and energy of an osteoblast on a HA‐CNT surface are 17 ± 2 μN/cell and 78 ± 14 pJ/cell respectively, as compared to 11 ± 2 μN/cell and 45 ± 10 pJ/cell on a HA surface after 1 day of incubation. The adhesion force and energy of the osteoblasts increase on both the surfaces with culture periods of up to 5 days. This increase is more pronounced for osteoblasts cultured on HA‐CNT. Staining of actin filaments revealed a higher spreading and attachment of osteoblasts on a surface containing CNTs. The affinity of CNTs to conjugate with integrin and other proteins is responsible for the enhanced attachment of osteoblasts. Our results suggest that the addition of CNTs to surfaces used in medical applications may be beneficial when stronger adhesion of osteoblasts is desired. S Online supplementary data available from stacks.iop.org/Nano/22/355703/mmedia (Some figures in this article are in colour only in the electronic version)

For several decades, tremendous advances in studying skin and hair pigmentation of mammals have been made using Mendelian genetics principles. A number of loci and their associated traits have been extensively examined, crossings performed, and phenotypes well documented. Continuously improving PCR techniques allowed the molecular cloning and sequencing of the first pigmentation genes at the end of the 20th century, a period followed by an intense effort to detect and describe polymorphisms in the coding regions and correlate allelic combinations with the observed melanogenic phenotypes. However, a number of phenotypes and biological events could not be elucidated solely by analysis of the coding regions of genes. Messenger RNA isolation, characterization and quantification techniques allowed groups to move ahead and investigate molecular mechanisms whose secrets lay within the noncoding regions of pigmentation genes transcripts such as MC1R, ASIP, or Mitf. The untranslated elements contain specific nucleotidic sequences and structures that dramatically influence the mRNA half-life and processing thus impacting protein translation and melanin production. As we are progressively uncovering the complex processes regulating melanocyte biology, unraveling complete mRNA structures and understanding mechanisms beyond coding regions has become critical.

Pigmentation of murine cardiac tricuspid valve leaflet is associated with melanocyte concentration, which affects its stiffness. Owing to its biological and viscoelastic nature, estimation of the in situ stiffness measurement becomes a challenging task. Therefore, quasi-static and nanodynamic mechanical analysis of the leaflets of the mouse tricuspid valve is performed in the current work. The mechanical properties along the leaflet vary with the degree of pigmentation. Pigmented regions of the valve leaflet that contain melanocytes displayed higher storage modulus (7–10 GPa) than non-pigmented areas (2.5–4 GPa). These results suggest that the presence of melanocytes affects the viscoelastic properties of the mouse atrioventricular valves and are important for their proper functioning in the organism.

Summary Apart from the well-studied melanocytes of the skin, eye and inner ear, another population has recently been described in the heart. In this study, we tracked cardiac melanoblasts using in situ hybridization with a dopachrome tautomerase (Dct) probe and Dct-LacZ transgenic mice. Large numbers of melanoblasts were found in the atrioventricular (AV) endocardial cushions at embryonic day (E) 14.5 and persisted in the AV valves into adulthood. The earliest time Dct-LacZ-positive cells were observed in the AV endocardial cushions was E12.5. Prior to that, between E10.5 and E11.5, small numbers of melanoblasts traveled between the post-otic area and third somite along the anterior and common cardinal veins and branchial arch arteries with other neural crest cells expressing CRABPI. Cardiac melanocytes were not found in the spotting mutants Ednrbs-l/s-l and Kitw-v/w-v, while large numbers were observed in transgenic mice that overexpress endothelin 3. These results indicate that cardiac melanocytes depend on the same signaling molecules known to be required for proper skin melanocyte development and may originate from the same precursor population. Cardiac melanocytes were not found in zebrafish or frog but were present in quail suggesting an association between cardiac melanocytes and four-chambered hearts.

The cells that form the cardiac conduction system (CCS) are recruited from embryonic cardiomyocytes. Endothelin-1 (ET-1) and Neuregulin-1 (NRG-1) have been associated with this transition in the avian and murine systems, respectively. We established murine embryonic cardiomyocyte cultures induced or not with ET-1 and/or NRG-1 to compare the expression of cardiomyocyte and CCS-specific genes. Semiquantitative reverse transcription-polymerase chain reaction analysis showed that cardiomyogenesis and CCS-specific markers, such as Nkx2.5, GATA4, Irx4, Connexin 40, Connexin 45, HF-1b, and MinK, were up-regulated in the presence of either growth factor. Additionally, immunofluorescence analysis demonstrated that ET-1 or NRG-1 increased the number of cells expressing the Purkinje fiber-specific marker Connexin 40 in induced cultures but did not selectively increase their proliferation rate. Interestingly, additive effects were not observed in ET-1 and NRG-1 combination treatments. Among other possibilities, this observation suggests that these factors may interact to promote the differentiation of the murine CCS. Developmental Dynamics 233:20–28, 2005. © 2005 Wiley-Liss, Inc.

Melanomagenesis is caused by environmental factors, such as ultraviolet (UV) radiation, as well as genetic factors. The Nucleotide Excision Repair (NER) pathway repairs photoproducts induced by UV radiation. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Upon UV exposure, mice deficient in Xpc, an important protein in the NER pathway, develop skin cancer; but not melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for melanocyte precursor cell development. In humans, this pathway has also been associated with melanoma progression and its metastatic potential. The purpose of this study is the development of a UV-dependent melanoma mouse model that combines Xpc deficiency with the over-activation of the Edn3 pathway. Transgenic mice over-expressing Edn3 under the control of the keratin 5 promoter (K5-Edn3) and carrying a targeted mutation in Xpc were exposed to a single suberythemal neonatal dose of UV radiation. Immunostaining and histomorphology were used to confirm the melanocytic origin of primary skin tumors. Melanomas were only found in animals with the K5-Edn3 transgene. High penetrance was observed in Xpc null (67%, n=3) and Xpc heterozygous (67%, n=3) mice in comparison to Xpc wild type (33%, n=3) mice. RNA extracted from melanomas of Xpc null (n=2), Xpc heterozygous (n=1) and Xpc wild type (n=1) mice were screened for UV signature mutations in Hras, Kras and Nras. No mutations were found in any of these genes. These results indicate that UV radiation exposure, in conjunction with over-activation of the Edn3 pathway is sufficient to lead to melanomagenesis in mice independently of mutations in Hras, Kras or Nras. Citation Format: Ana Paula Benaduce, Deannys Batista, Gabriel Grilo, Karen Jorge, Diana Cardero, Clara Milikowski, Lidia Kos. Ultraviolet radiation induces carcinogenesis in a novel transgenic mouse model of melanoma. [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 5352. doi:10.1158/1538-7445.AM2014-5352

Genes related to the Drosophila eyes absent gene were identified in vertebrates (mouse and human), mollusks (squid), and nematodes (C. elegans). Proteins encoded by these genes consist of conserved C-terminal and variable N-terminal domains. In the conserved 271-amino acid C-terminal region, Drosophila and vertebrate proteins are 65-67% identical. A vertebrate homolog of eyes absent, designated Eya2, was mapped to Chromosome (Chr) 2 in the mouse and to Chr 20q13.1 in human. Eya2 shows a dynamic pattern of expression during development. In the mouse, expression of Eya2 was first detected in 8.5-day embryos in the region of head ectoderm fated to become the forebrain. At later stages of development, Eya2 is expressed in the olfactory placode and in a variety of neural crest derivatives. In the eye, expression of Eya2 was first detected after formation of the lens vesicle. At day 17.5, the highest level of Eya2 mRNA was observed in primary lens fibers. Low levels of Eya2 expression was detected in retina, sciera, and cornea. By postnatal day 10, Eya2 was expressed in secondary lens fibers, cornea, and retina. Although Eya2 is expressed relatively late in eye development, it belongs to the growing list of factors that may be essential for eye development across metazoan phyla. Like members of the Pax-6 gene family, eyes absent gene family members were probably first involved in functions not related to vision, with recruitment for visual system formation and function occurring later.

Melanomagenesis is influenced by the interaction of environmental (such as UV radiation) and genetic factors. The Xpa gene is important in the Nucleotide Excision Repair (NER) pathway. Similar to humans afflicted with Xeroderma Pigmentosum, which results from mutations in genes that play a role in NER, Xpa deficient mice show high sensitivity to UV light, leading to skin cancer development. However, Xpa deficient mice do not develop melanoma even upon UV exposure. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. In humans, this pathway has also been implicated in melanoma progression and its metastatic potential. The purpose of this study is to develop a UV induced melanoma mouse model that combines Xpa deficiency with the over-activation of the Edn3 pathway. To this end, transgenic mice over-expressing Edn3 under the control of the keratin 5 promoter (K5-Edn3) and carrying a targeted mutation in Xpa were exposed to a single suberythemal neonatal dose of UV radiation. A subgroup of mice was also exposed to a second dose of UV radiation at 6 weeks of age. Histomorphology and immunostaining were used to confirm the melanocytic origin of primary skin tumors and metastases. Melanoma was only found in animals with the K5-Edn3 transgene. High penetrance was observed in animals exposed to one dose of UV radiation that were Xpa null (100%, n=3) when compared to Xpa heterozygous (73%, n=11) or Xpa wild type (43%, n=7). Enlarged and hyperpigmented lymph nodes were present in animals with melanoma skin lesions and these were diagnosed as local metastases. These results indicate that UV radiation exposure, in conjunction with over-activation of the Edn3 pathway is sufficient to lead to melanomagenesis in mice. Citation Format: Ana Paula Benaduce, Deannys Batista, Gabriel Grilo, Karen Jorge, Lidia Kos. UV radiation interacts with Endothelin 3 and loss of nucleotide excision repair pathway to promote melanomagenesis. [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 1276. doi:10.1158/1538-7445.AM2013-1276

G-protein coupled receptors have been identified as important factors in melanoma progression including growth and proliferation of tumorigenic cells, as well as facilitating metastasis. Upregulation of the G-protein coupled receptor Endothelin receptor b (Ednrb) and its ligand Endothelin 3 (Edn3) has been implicated in melanoma progression in several ‘in vitro’ experiments. In this study we investigated the putative role of Edn3 over-expression in melanoma progression in vivo. Tg(Grm1) Epv mice over-expressing metabotropic glutamate receptor 1 (Grm1) under the Dopachrome tautomerase promoter that spontaneously produce melanocytic lesions in the ears and tails with limited metastatic capability, were crossed with transgenic mice that over-express Edn3 under the keratin 5 promoter (K5-Edn3). The Tg(Grm1)Epv/K5-Edn3 mice tumors appeared ∼ 3 months earlier and grew at a rate 3-4 times faster than the Tg(Grm1)/Epv control mice. Ninety-five percent of Dct-Grm1/ K5-Edn3 mice also displayed pigmented lesions in distant organs such as the lungs. Real Time PCR analysis showed higher expression levels of angiogenic related genes such as Hif-1α in the Dct-Grm1/K5-Edn3mice, when compared to controls. Our studies demonstrate that Edn3 over-expression enhances melanoma progression to a fully malignant state. Our preliminary data also suggests that Edn3 may be involved in tumor angiogenesis leading to faster melanoma tumor development. Citation Format: Nikeisha L. Chin, Juan Carlos Gallegos, Rosy Cruz, Ruslan Garcia, Andrea Gonzalez, Manuel Borobia, Lidia Kos. Role of endothelin 3 in melanoma progression and metastasis. [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 2819. doi:10.1158/1538-7445.AM2013-2819 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Melanocytes, the pigment-producing cells, arise from multipotent neural crest (NC) cells during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The variable spotting mouse pigmentation mutant arose spontaneously at the Jackson Laboratory. We identified a G-to-A nucleotide transition in exon 3 of the Ets1 gene in variable spotting, which results in a missense G102E mutation. Homozygous variable spotting mice exhibit sporadic white spotting. Similarly, mice carrying a targeted deletion of Ets1 exhibit hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The transcription factor Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of various NC derivatives, including melanocytes. We show that Ets1 is required early for murine NC cell and melanocyte precursor survival in vivo. Given the importance of Ets1 for Sox10 expression in the chick, we investigated a potential genetic interaction between these genes by comparing the hypopigmentation phenotypes of single and double heterozygous mice. The incidence of hypopigmentation in double heterozygotes was significantly greater than in single heterozygotes. The area of hypopigmentation in double heterozygotes was significantly larger than would be expected from the addition of the areas of hypopigmentation of single heterozygotes, suggesting that Ets1 and Sox10 interact synergistically in melanocyte development. Since Sox10 is also essential for enteric ganglia development, we examined the distal colons of Ets1 null mutants and found a significant decrease in enteric innervation, which was exacerbated by Sox10 heterozygosity. At the molecular level, Ets1 was found to activate an enhancer critical for Sox10 expression in NC-derived structures. Furthermore, enhancer activation was significantly inhibited by the variable spotting mutation. Together, these results suggest that Ets1 and Sox10 interact to promote proper melanocyte and enteric ganglia development from the NC.

Heart valves are complex structures composed of organized layers of extracellular matrix, and interstitial and overlying endothelial cells. In this article, we present the specific localization of a population of melanocytes within the murine heart valves at ages important for their post-natal development. In all stages analyzed in our study, melanocytes were found in high numbers populating the atrial aspect of the tricuspid and mitral leaflets. The pulmonary valve did not present melanocytes. To characterize a putative role for the valve melanocytes, the dynamic nanomechanical properties of tricuspid leaftets containing large numbers or no melanocytes were measured. The stiffness coefficient of hyperpigmented leaflets was higher (11.5 GPa) than the ones from wild-type (7.5 GPa) and hypopigmented (5.5 GPa) leaflets. These results suggest that melanocytes may contribute to the mechanical properties of the heart valves. The arrangement of extracellular matrix molecules such as Collagen I and Versican B is responsible for the mechanical characteristics of the leaflets. Melanocytes were found to reside primarily in areas of Versican B expression. The patterns of expression of Collagen I and Versican B were not, however, disrupted in hyper or hypopigmented leaflets. Melanocytes may affect other extracellular matrix molecules to alter the valves' microenvironment.