Intact miRNAs can be isolated from the circulation in significant quantities despite the presence of extremely high levels of RNase activity. The remarkable stability of circulating miRNAs makes them excellent candidates for biomarkers in diagnostic applications as well as therapeutic targets in a variety of disease states including melanoma. Circulating RNA molecules are resistant to degradation by RNases because they are encapsulated in membrane-bound microvesicles. We describe a convenient method for the use of ExoQuick, a proprietary resin developed by Systems Biosciences (Mountain View, CA), whereby microvesicles can be purified under gentle conditions using readily available laboratory equipment. This protocol allows for isolation all microvesicles, regardless of their origin, and provides a convenient method for identifying potential cancer-specific biomarkers from biological fluids including serum and plasma.
Beginning in the last decade of the twentieth century, the fields of pigment cell research and melanoma have witnessed major breakthroughs in the understanding of the role of melanocortins in human pigmentation and the DNA damage response of human melanocytes to solar ultraviolet radiation (UV). This began with the cloning of the melanocortin 1 receptor (MC1R) gene from human melanocytes and the demonstration that the encoded receptor is functional. Subsequently, population studies found that the MC1R gene is highly polymorphic, and that some of its variants are associated with red hair phenotype, fair skin and poor tanning ability. Using human melanocytes cultured from donors with different MC1R genotypes revealed that the alleles associated with red hair color encode for a non-functional receptor. Epidemiological studies linked the MC1R red hair color variants to increased melanoma risk. Investigating the impact of different MC1R variants on the response of human melanocytes to UV led to the important discovery that the MC1R signaling activates antioxidant, DNA repair and survival pathways, in addition to stimulation of eumelanin synthesis. These effects of MC1R were absent in melanocytes expressing 2 MC1R red hair color variants that result in loss of function of the receptor. The importance of the MC1R in reducing UV-induced genotoxicity in melanocytes led us to design small peptide analogs of the physiological MC1R agonist ?-melanocortin (?-melanocyte stimulating hormone; ?-MSH) for the goal of utilizing them for melanoma chemoprevention.
The incidence of cutaneous melanoma has risen at a rate significantly higher than that for other malignancies. This increase persists despite efforts to educate the public about the dangers of excess exposure to UV radiation from both the sun and tanning beds. Melanoma affects a relatively younger population and is notorious for its propensity to metastasize and for its poor response to current therapeutic regimens. These factors make prevention an integral component to the goal of decreasing melanoma-related mortality. Transformation of melanocytes into malignant melanoma involves the interplay between genetic factors, UV exposure, and the tumor microenvironment. The roles of UV radiation in the etiology of melanoma are mediated by both direct damage of DNA through formation of photoproducts and production of reactive oxygen species (ROS). Many of the promising antioxidant agents under development for the prevention of melanoma are derived from foodstuffs. B-Raf is a member of the Raf kinase family of serine/threonine-specific protein kinases that plays a role in regulating the MAP kinase/ERKs signaling pathway. About 50 % of melanomas harbor activating BRAF mutations. BRAF mutations are found in 59 % of the melanomas arising in skin with intermittent sun exposure, such as trunk and arms, as compared with only 23 % of the acral melanomas, 11 % of mucosal melanomas, and 0 % of uveal melanomas. Two new agents, ipilimumab and vemurafenib, have been shown to improve outcome of advanced melanoma as presented at the plenary session of the 2011 annual meeting of the American Society of Clinical Oncology. Vemurafenib is the first personalized compound which demonstrated an improvement in progression-free survival (PFS) and overall survival (OS) in metastatic melanoma harboring the BRAFV600 mutation and represents the first drug of a class that exerts its anti-proliferative activity through inhibition of a highly specific molecular target. GSK2118436 (dabrafenib), the second BRAF inhibitor, in phase I and II trial obtained similar results to vemurafenib. A phase III trial is now ongoing. Taken together, the early clinical development of vemurafenib and dabrafenib clearly confirms that BRAF inhibitors can halt or reverse disease in patients with melanomas carrying this mutation, improving survival times compared with historically standard treatments (chemotherapy and interleukin-2). The clinical development of other new BRAF inhibitors such as RAF265 and LGX818 is now ongoing. Combination strategies of BRAF inhibitors with ipilimumab, an anti-CTLA-4 antibody, and/or MEK inhibitors or metformin are now under investigation in clinical trials.
Understanding the molecular basis of phenotypic diversity is a critical challenge in biology, yet we know little about the mechanistic effects of different mutations and epistatic relationships among loci that contribute to complex traits. Pigmentation genetics offers a powerful model for identifying mutations underlying diversity and for determining how additional complexity emerges from interactions among loci. Centuries of artificial selection in domestic rock pigeons (Columba livia) have cultivated tremendous variation in plumage pigmentation through the combined effects of dozens of loci. The dominance and epistatic hierarchies of key loci governing this diversity are known through classical genetic studies, but their molecular identities and the mechanisms of their genetic interactions remain unknown. Here we identify protein-coding and cis-regulatory mutations in Tyrp1, Sox10, and Slc45a2 that underlie classical color phenotypes of pigeons and present a mechanistic explanation of their dominance and epistatic relationships. We also find unanticipated allelic heterogeneity at Tyrp1 and Sox10, indicating that color variants evolved repeatedly though mutations in the same genes. These results demonstrate how a spectrum of coding and regulatory mutations in a small number of genes can interact to generate substantial phenotypic diversity in a classic Darwinian model of evolution.
The role of selenium (Se) supplementation in cancer prevention is controversial; effects often depend on the nutritional status of the subject and on the chemical form in which Se is provided. We used a combination of in vitro and in vivo models to study two unique therapeutic windows for intervention in the process of cutaneous melanomagenisis, and to examine the utility of two different chemical forms of Se for prevention and treatment of melanoma. We studied the effects of Se in vitro on UV-induced oxidative stress in melanocytes, and on apoptosis and cell cycle progression in melanoma cells. In vivo, we used the HGF transgenic mouse model of UV-induced melanoma to demonstrate that topical treatment with l-selenomethionine results in a significant delay in the time required for UV-induced melanoma development, but also increases the rate of growth of those tumors once they appear. In a second mouse model, we found that oral administration of high dose methylseleninic acid significantly decreases the size of human melanoma xenografts. Our findings suggest that modestly elevation of selenium levels in the skin might risk acceleration of growth of incipient tumors. Additionally, certain Se compounds administered at very high doses could have utility for the treatment of fully-malignant tumors or prevention of recurrence.
Familial melanoma (FM) is a dominantly heritable cancer that is associated with mutations in the tumor suppressor CDKN2A/p16. In FM, a single inherited "hit" occurs in every somatic cell, enabling interrogation of cultured normal skin fibroblasts (SFs) from FM gene carriers as surrogates for the cell of tumor origin, namely the melanocyte. We compared the gene expression profile of SFs from FM individuals with two distinct CDKN2A/p16 mutations (V126D-p16 and R87P-p16) with the gene expression profile of SFs from age-matched individuals without p16 mutations and with no family history of melanoma. We show an altered transcriptome signature in normal SFs bearing a single-hit inherited mutation in the CDKN2A/p16 gene, wherein some of these abnormal alterations recapitulate changes observed in the corresponding cancer. Significantly, the extent of the alterations is mutation-site specific with the R87P-p16 mutation being more disruptive than the V126D-p16 mutation. We also examined changes in gene expression after exposure to ultraviolet (UV) radiation to define potential early biomarkers triggered by sun exposure. UV treatment of SFs from FM families induces distinct alterations in genes related to cell cycle regulation and DNA damage responses that are also reported to be dysregulated in melanoma. Importantly, these changes were diametrically opposed to UV-induced changes in SF from normal controls. We posit that changes identified in the transcriptome of SF from FM mutation carriers represent early events critical for melanoma development. As such, they may serve as specific biomarkers of increased risk as well as molecular targets for personalized prevention strategies in high-risk populations.
Approximately 10% of melanoma cases are familial, but only 25-40% of familial melanoma cases can be attributed to germ-line mutations in the CDKN2A - the most significant high-risk melanoma susceptibility locus identified to date. The pathogenic mutation(s) in most of the remaining familial melanoma pedigrees have not yet been identified. The most common mutations in nevi and sporadic melanoma are found in BRAF and NRAS, both of which result in constitutive activation of the MAPK pathway. However, these mutations are not found in uveal melanomas or the intradermal melanocytic proliferations known as blue nevi. Rather, multiple studies report a strong association between these lesions and somatic mutations in Guanine nucleotide-binding protein G(q) subunit alpha (GNAQ), Guanine nucleotide-binding protein G(q) subunit alpha-11 (GNA11), and BRCA1-associated protein-1 (BAP1). Recently, germ-line mutations in BAP1, the gene encoding a tumor suppressing deubiquitinating enzyme, have been associated with predisposition to a variety of cancers including uveal melanoma, but no studies have examined the association of germ-line mutations in GNAQ and GNA11 with uveal melanoma and blue nevi. We have now done so by sequencing exon 5 of both of these genes in 13 unique familial melanoma pedigrees, members of which have had either uveal or cutaneous melanoma and/or blue nevi. Germ-line DNA from a total of 22 individuals was used for sequencing; however no deleterious mutations were detected. Nevertheless, such candidate gene studies and the discovery of novel germ-line mutations associated with an increased MM susceptibility can lead to a better understanding of the pathways involved in melanocyte transformation, formulation of risk assessment, and the development of specific drug therapies.
The gene transcript profile responses to metal oxide nanoparticles was studied using human cell lines derived from the colon and skin tumors. Much of the research on nanoparticle toxicology has focused on models of inhalation and intact skin exposure, and effects of ingestion exposure and application to diseased skin are relatively unknown. Powders of nominally nanosized SiO2, TiO2, ZnO and Fe2O3 were chosen because these substances are widely used in consumer products. The four oxides were evaluated using colon-derived cell lines, RKO and CaCo-2, and ZnO and TiO2 were evaluated further using skin-derived cell lines HaCaT and SK Mel-28. ZnO induced the most notable gene transcription changes, even though this material was applied at the lowest concentration. Nano-sized and conventional ZnO induced similar responses suggesting common mechanisms of action. The results showed neither a non-specific response pattern common to all substances nor synergy of the particles with TNF-? cotreatment. The response to ZnO was not consistent with a pronounced proinflammatory signature, but involved changes in metal metabolism, chaperonin proteins, and protein folding genes. This response was observed in all cell lines when ZnO was in contact with the human cells. When the cells were exposed to soluble Zn, the genes involved in metal metabolism were induced but the genes involved in protein refoldling were unaffected. This provides some of the first data on the effects of commercial metal oxide nanoparticles on human colon-derived and skin-derived cells.
A role for dopamine (DA) in the regulation of clock genes in the mammalian brain is suggested by evidence that manipulations of DA receptors can alter the expression of some clock genes outside the suprachiasmatic nucleus (SCN), the master circadian clock. The role of endogenous DA in the regulation of clock gene expression is unknown. Here, we demonstrate a direct relationship between extracellular DA levels and the rhythm of expression of the clock protein PERIOD2 (PER2) in the dorsal striatum of the male Wistar rat. Specifically, we show that the peak of the daily rhythm of extracellular DA in the dorsal striatum precedes the peak of PER2 by ?6 h and that depletion of striatal DA by 6-hydroxydopamine or ?-methyl-para-tyrosine or blockade of D(2) DA receptors by raclopride blunts the rhythm of striatal PER2. Furthermore, timed daily activation of D(2) DA receptors, but not D(1) DA receptors, restores and entrains the PER2 rhythm in the DA-depleted striatum. None of these manipulations had any effect on the PER2 rhythm in the SCN. Our findings are consistent with the idea that the rhythm of expression of PER2 in the dorsal striatum depends on daily dopaminergic activation of D(2) DA receptors. These observations may have implications for circadian abnormalities seen in Parkinsons disease.
The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to ?-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to ?-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively. These variants had different heterozygous effects on receptor function. Microarray data confirmed the observed differences in responses of melanocytes with functional vs. nonfunctional receptor to ?-melanocortin and ultraviolet radiation, and identified DNA repair and antioxidant genes that are modulated by ?-melanocortin. Our findings highlight the molecular mechanisms by which the melanocortin 1 receptor genotype controls genomic stability of and the mutagenic effect of ultraviolet radiation on human melanocytes.
Proteomic approaches have identified cancer specific biomarker proteins in the nuclear matrix fraction of cancer cells. We wanted to determine whether a similar approach could be used to investigate melanoma biomarkers.
The selenoenzyme thioredoxin reductase 1 has a complex role relating to cell growth. It is induced as a component of the cellular response to potentially mutagenic oxidants, but also appears to provide growth advantages to transformed cells by inhibiting apoptosis. In addition, selenocysteine-deficient or alkylated forms of thioredoxin reductase 1 have also demonstrated oxidative, pro-apoptotic activity. Therefore, a greater understanding of the role of thioredoxin reductase in redox initiated apoptotic processes is warranted.
Induction of oxidative stress has been implicated in UV-induced melanoma. We sought to determine whether the antioxidant N-acetylcysteine (NAC) could be safely administered to protect melanocytic nevi from the oxidative stress resulting from acute UV exposure.
Oculocutaneous albinism type 2 (OCA2) is caused by mutations of the OCA2 gene. Individuals affected by OCA2 as well as other types of albinism are at a significantly increased risk for sun-induced skin-cancers, including malignant melanoma (MM).
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