A cancer‐causing gene is positively correlated with male aggression in Xiphophorus cortezi

The persistence of seemingly maladaptive genes in organisms challenges evolutionary biological thought. In Xiphophorus fishes, certain melanin patterns form malignant melanomas because of a cancer‐causing gene (Xiphophorus melanoma receptor kinase; Xmrk), which arose several millions years ago from unequal meiotic recombination. Xiphophorus melanomas are male biased and induced by androgens however male behaviour and Xmrk genotype has not been investigated. This study found that male X. cortezi with the spotted caudal (Sc) pattern, from which melanomas originate, displayed increased aggression in mirror image trials. Furthermore, Xmrk males (regardless of Sc phenotype) bit and performed more agonistic displays than Xmrk deficient males. Male aggressive response decreased when males viewed their Sc image as compared with their non‐Sc image. Collectively, these results indicate that Xmrk males experience a competitive advantage over wild‐type males and that intrasexual selection could be an important component in the evolutionary maintenance of this oncogene within Xiphophorus.     

Evolution of Xmrk: an oncogene,but also a speciation gene?

Genes that exert their function when they are introduced into a foreign genetic background pose many questions to our current understanding of the forces and mechanisms that promote either the maintenance or divergence of gene functions over evolutionary time. The melanoma inducing Xmrk oncogene of the Southern platyfish (Xiphophorus maculatus) is a stable constituent of the genome of this species. It displays its tumorigenic function, however, almost exclusively only after inter‐populational or, even more severely, interspecific hybridization events. The Xiphophorus hybrid melanoma system has gained attention in biomedical research as a genetic model for studying tumor formation. From an evolutionary perspective, a prominent question is: how could this gene persist over millions of years? An attractive hypothesis is that Xmrk, acting as a detrimental gene in a hybrid genome, could be a speciation gene that shields the gene pool of its species from mixing with other closely related sympatric species. In this article, I briefly review our current knowledge of the molecular genetics and biochemical functions of the Xmrk gene and discuss aspects of its evolutionary history and presence with respect to this idea. While Xmrk as a potentially injurious oncogene has clearly survived for millions of years, its role as a speciation gene has to be questioned. BioEssays 30:822–832, 2008. © 2008 Wiley Periodicals, Inc.     

Evolutionary Origin and Molecular Biology of the Melanoma-Inducing Oncogene of Xiphophorus

Melanoma formation in platyfish/swordtail hybrids of genus Xiphophorus is due to overexpression of the receptor tyrosine kinase oncogene Xmrk. This gene is the molecular equivalent to the Tu-locus of platyfish, formerly identified by Mendelian genetics. The supposed evolutionary origin of the Xmrk oncogene is a nonhomologous recombination event in the 5’region of the corresponding Xmrk protooncogene with an anonymous sequence, D. This event led to a gene duplication of Xmrk, whereby the new copy obtained a novel promoter derived from D. Inactivity of this promoter in parental fish warrants lack of tumorigenicity of the Xmrk oncogene in wild playfish. In hybrids, however, the promoter is active. This leads to the pigment cell transforming overexpression of Xmrk.     

Proliferation and Neoplastic Transformation of Pigment Cells in Metallothionein/ret Transgenic Mice

Although melanoma is a common human disease, there were few animal models in which melanoma developed at high incidence. To date, the Xiphophorus fish has been used as a model system to study melanoma formation. Studies on this fish showed the presence of a dominant oncogene, Tu, which encodes a transmembrane, tyrosine kinase of epidermal growth factor receptor type (Wittbrodt et al., Nature, 341:415–421, 1989). Recently, we succeeded in establishing novel transgenic mouse lines in which melanosis and melanocytic tumors developed stepwise by introducing another transmembrane tyrosine kinase oncogene, ret (Iwamoto et al., EMBO J., 10:3167–3175, 1991). In our transgenic mice, high levels of expression of the ret transgene induced proliferation and neoplastic transformation of melanin-producing cells. In addition, crossbreeding experiments between transgenic mice and W^v mice showed that the ret oncogene can also induce melanogenesis and melanocyte development in W^v/W^v mice.     

Acute exposure to ultraviolet‐B radiation modulates sex steroid hormones and receptor expression in the skin and may contribute to the sex bias of melanoma in a fish model

Using the Xiphophorus fish melanoma model, we show a strong male bias for sunlight‐induced malignant melanoma, consistent with that seen in the human population. To examine underlying factors, we exposed adult X. couchianus fish to a single, sublethal dose of UVB and measured circulating sex steroid hormones and expression of associated hormone receptor genes over a 24‐h period. We found that a single exposure had profound effects on circulating levels of steroid hormones with significant decreases for all free sex steroids at 6 and 24 h and increases in conjugated 2‐estradiol and 11‐ketotestosterone at 6 and 24 h, respectively. Whereas ARα expression increased in male and female skin, neither ARβ nor either of the ERs showed significant responses to UVB in either sex. The rapid response of male androgens and their receptors in the skin after UVB irradiation implicates hormones in the male bias of skin cancer and suggests that the photoendocrine response immediately after UV exposure may be relevant to melanomagenesis.     

Genetic and environmental melanoma models in fish

Experimental animal models are extremely valuable for the study of human diseases, especially those with underlying genetic components. The exploitation of various animal models, from fruitflies to mice, has led to major advances in our understanding of the etiologies of many diseases, including cancer. Cutaneous malignant melanoma is a form of cancer for which both environmental insult (i.e., UV) and hereditary predisposition are major causative factors. Fish melanoma models have been used in studies of both spontaneous and induced melanoma formation. Genetic hybrids between platyfish and swordtails, different species of the genus Xiphophorus, have been studied since the 1920s to identify genetic determinants of pigmentation and melanoma formation. Recently, transgenesis has been used to develop zebrafish and medaka models for melanoma research. This review will provide a historical perspective on the use of fish models in melanoma research, and an updated summary of current and prospective studies using these unique experimental systems.     

Clustered organization and conservation of theXiphophorus maculatus D locus,which includes two distinct gene sequences

Genomic organization and chromosomal localization of a previously uncharacterizedD (Donor) locus inXiphophorus andPoecilia species was investigated using fluorescence in situ hybridization (FISH) and Southern blot analysis. Part of this region is thought to be involved in the recombination event leading to formation of theXmrk oncogene and it has recently been shown that this locus included two different genes, one with high homology to a zinc finger protein of the Krüppel type, and the other an unknown gene with high similarity to aCaenorhabditis elegans gene. FISH toXiphophorus chromosomes revealed that these two unrelated genes are closely linked and clustered at a unique chromosomal site. Southern blot hybridization patterns suggest that these genes exist in the genome as multiple copies. Furthermore, similar genomic organization profiles seem to prevail among other related fish. In particular, our FISH experiments reveal the existence of a conserved homologous chromosomal segment harboring the zinc finger protein sequence in several poeciliid fish.     

Assortative aggression among males in a sympatric pair of Labeotropheus from Lake Malaŵi,Africa

Female mate choice has strong experimental support as a diversifying force in the speciation of the haplochromine cichlid fishes of Lake Malaŵi, Africa. Somewhat less understood is the role that male–male aggression might have played in the evolution of new species of these fishes. In the rock-dwelling haplochromines of Lake Malaŵi, primarily territory-holding males successfully court females; by determining which males gain territories, male–male aggression could support speciation by excluding less-fit males from the breeding population. To test the hypothesis that males should direct more aggression towards conspecific rivals, the aggression directed towards conspecific and heterospecific opponents was compared in a sympatric pair of cichlids of the genus Labeotropheus Ahl 1927 (Labeotropheus fuelleborni Ahl 1927 and Labeotropheus trewavasae Fryer 1956). It was found that when presented with a pair of rivals, males of both species did direct more aggression towards the conspecific opponent, and the amount of aggression increased when the conspecific opponent was larger than the heterospecific opponent. In addition, this study found a difference in the behavioural repertoire of the species: L. fuelleborni tends to rely on displays to intimidate opponents, whereas L. trewavasae employs more physical attacks to drive away opponents. Males of both species can thus recognize conspecifics and assess an opponent's relative threat to their ability to successfully reproduce, and use species-specific strategies to intimidate opponents.     

Towards in vivo melanin radicals detection in melanomas by electron paramagnetic resonance (EPR) spectroscopy: a proof-of-concept study

Melanoma is the most aggressive skin tumour type. Although complete cure can be achieved when the whole tumour is resected, prognostic dramatically drops when melanoma cells reach deeper tissues and lymph nodes. Hence, there is an urgent need to develop accurate tools allowing (i) discriminating benign naevi from malignant tumours and (ii) being able to characterise melanoma infiltration. For that purpose, we exploited the paramagnetic properties of melanin by using electron paramagnetic resonance (EPR) spectroscopy to measure the melanin content in pigmented (B16F10 cancer cells) and non-pigmented melanomas (WM2664 cancer cells) inoculated intradermally in nude mice. Specifically, we took advantage of a new clinical EPR device (1?GHz), which provides sensitive measurements of radical species in vivo. Results showed that the melanin-specific EPR signal increased with tumour growth in pigmented tumours, whereas no EPR signal could be detected in achromic melanomas. These data plead for the development of new EPR spectrometers/imagers with an improved in-depth resolution for the detection of invasive melanomas.     

Receptor tyrosine kinases and their activation in melanoma

Receptor tyrosine kinases (RTKs) and their downstream signalling pathways have long been hypothesized to play key roles in melanoma development. A decade ago, evidence was derived largely from animal models, RTK expression studies and detection of activated RAS isoforms in a small fraction of melanomas. Predictions that overexpression of specific RTKs implied increased kinase activity and that some RTKs would show activating mutations in melanoma were largely untested. However, technological advances including rapid gene sequencing, siRNA methods and phospho-RTK arrays now give a more complete picture. Mutated forms of RTK genes including KIT, ERBB4, the EPH and FGFR families and others are known in melanoma. Additional over- or underexpressed RTKs and also protein tyrosine phosphatases (PTPs) have been reported, and activities measured. Complex interactions between RTKs and PTPs are implicated in the abnormal signalling driving aberrant growth and survival in malignant melanocytes, and indeed in normal melanocytic signalling including the response to ultraviolet radiation. Kinases are considered druggable targets, so characterization of global RTK activity in melanoma should assist the rational development of tyrosine kinase inhibitors for clinical use.     

Kinase gene fusions in defined subsets of melanoma

Genomic rearrangements resulting in activating kinase fusions have been increasingly described in a number of cancers including malignant melanoma, but their frequency in specific melanoma subtypes has not been reported. We used break‐apart fluorescence in situ hybridization (FISH) to identify genomic rearrangements in tissues from 59 patients with various types of malignant melanoma including acral lentiginous, mucosal, superficial spreading, and nodular. We identified four genomic rearrangements involving the genes BRAF, RET, and ROS1. Of these, three were confirmed by Immunohistochemistry (IHC) or sequencing and one was found to be an ARMC10‐BRAF fusion that has not been previously reported in melanoma. These fusions occurred in different subtypes of melanoma but all in tumors lacking known driver mutations. Our data suggest gene fusions are more common than previously thought and should be further explored particularly in melanomas lacking known driver mutations.     

Melanoma tumors frequently acquire LRP2/megalin expression,which modulates melanoma cell proliferation and survival rates

We show that the multiligand receptor megalin, known to mediate uptake and trafficking of nutrients and signaling molecules, is frequently expressed in malignant melanoma samples. Expression of megalin‐encoding mRNA was investigated in 65 samples of nevi, melanomas, and melanoma metastases and was observed in more than 60% of the malignant samples, while only in 20% of the benign counterparts. Megalin expression in nevus and melanoma samples was additionally investigated by immunohistochemistry, which confirmed our mRNA‐based observations. We furthermore show that a panel of tumor‐derived melanoma cell lines express LRP2/megalin endogenously. In these cells, megalin is internalized from the cell surface and localizes extensively to intracellular vesicles, confirming receptor activity and pointing toward association with the endocytic apparatus. Groundbreaking, our results indicate that sustained megalin expression in melanoma cells is crucial for cell maintenance, as siRNA‐mediated reduction in melanoma cell expression of LRP2/megalin significantly decreases melanoma cell proliferation and survival rates.     

Molecular pathogenesis of malignant melanoma: a different perspective from the studies of melanocytic nevus and acral melanoma

The Clark model for melanoma progression emphasizes a series of histopathological changes beginning from benign melanocytic nevus to melanoma via dysplastic nevus. Several models of the genetic basis of melanoma development and progression are based on this Clark’s multi-step model, and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia. However, our recent investigations have challenged this view, showing the polyclonality of BRAF mutations in melanocytic nevi. Furthermore, it is suggested that many melanomas, including acral and mucosal melanomas, arise de novo, not from melanocytic nevus. While mutations of the BRAF gene are frequent in melanomas on non-chronic sun damaged skin which are prevalent in Caucasians, acral and mucosal melanomas harbor mutations of the KIT gene as well as the amplifications of cyclin D1 or cyclin-dependent kinase 4 gene. Amplifications of the cyclin D1 gene are detected in normal-looking ‘field melanocytes’, which represent a latent progression phase of acral melanoma that precedes the stage of atypical melanocyte proliferation in the epidermis. Based on these observations, we propose an alternative genetic progression model for melanoma.     

Xmrk‐induced melanoma progression is affected by Sdf1 signals through Cxcr7

Chemokine signals mediated by Sdf1/Cxcl12 through the chemokine receptor Cxcr4 are thought to play an instructive role in tumor migration and organ‐specific metastasis. We have used a small aquarium fish model to contribute to a better understanding of how the course of melanoma development is influenced by Sdf1 signals in vivo. We studied oncogene‐induced skin tumor appearance and progression in the transgenic medaka (Oryzias latipes) melanoma model. Similar to humans, invasive medaka melanomas show increased levels of sdf1, cxcr4, and cxcr7 gene expression. Stable transgenic fish lines overexpressing sdf1 exclusively in pigment cells showed a reduction in melanoma appearance and progression. Remarkably, diminished levels of functional Cxcr7, but not of Cxcr4b, resulted in strongly reduced melanoma invasiveness and a repression of melanoma. Our results thereby indicate that Sdf1 signals via Cxcr7 are able to constrain melanoma growth in vivo and that these signals influence tumor outcome.     

Linkage assignment of a DNA sequence (ERCC2L1) homologous to a human DNA repair gene in Xiphophorus fishes: Implications for the evolutionary derivation of human chromosome 19

Fish gene mapping studies have identified several syntenic groups showing conservation over more than 400 million years of vertebrate evolution. In particular, Xiphophorus linkage group IV has been identified as a homolog of human chromosomes 15 and 19. During mammalian evolution, loci coding for glucosephosphate isomerase, peptidase D, muscle creatine kinase, and several DNA repair genes (ERCC1, ERCC2, and XRCC1) appear as a conserved syntenic group on human chromosome 19. When X. clemenciae and X. milleri PstI endonuclease-digested genomic DNA was used in Southern analysis with a human ERCC2 DNA repair gene probe, a strongly cross-hybridizing restriction fragment length polymorphism was observed. Backcrosses to X. clemenciae from X. milleri × X. clemenciae F₁ hybrids allowed tests for linkage of the ERCC2-like polymorphism to markers covering a large proportion of the genome. Statistically significant evidence for linkage was found only for ERCC2L1 and CKM (muscle creatine kinase), with a total of 41 parents and 2 recombinants (4.7% recombination, χ² = 35.37, P < 0.001); no evidence for linkage to GPI and PEPD in linkage group IV was detected. The human chromosome 19 synteny of ERCC2 and CKM thus appears to be conserved in Xiphophorus, while other genes located nearby on human chromosome 19 are in a separate linkage group in this fish. If Xiphophorus gene arrangements prove to be primitive, human chromosome 19 may have arisen from chromosome fusion or translocation events at some point since divergence of mammals and fishes from a common ancestor.     

MicroRNA signatures differentiate melanoma subtypes

Melanoma is an aggressive cancer that is highly resistance to therapies once metastasized. We studied microRNA (miRNA) expression in clinical melanoma subtypes and evaluated different miRNA signatures in the background of gain of function somatic and inherited mutations associated with melanoma. Total RNA from 42 patient derived primary melanoma cell lines and three independent normal primary melanocyte cell cultures was evaluated by miRNA array. MiRNA expression was then analyzed comparing subtypes and additional clinicopathologic criteria including somatic mutations. The prevalence and association of an inherited variant in a miRNA binding site in the 3’UTR of the KRAS oncogene, referred to as the KRAS-variant, was also evaluated. We show that seven miRNAs, miR-142-3p, miR-486, miR-214, miR-218, miR-362, miR-650 and miR-31, were significantly correlated with acral as compared to non-acral melanomas (p &lt; 0.04). In addition, we discovered that the KRAS-variant was enriched in non-acral melanoma (25%), and that miR-137 under expression was significantly associated with melanomas with the KRAS-variant. Our findings indicate that miRNAs are differentially expressed in melanoma subtypes and that their misregulation can be impacted by inherited gene variants, supporting the hypothesis that miRNA misregulation reflects biological differences in melanoma.     

Microsatellites in the genus Xiphophorus,developed in Xiphophorus montezumae

Species of the genus Xiphophorus (swordtails and platies) are of great interest for the study of evolution of sexually selected traits like the sword, which is an elongation of ventral fin rays of the male caudal fin, that has evolved in several species within this genus. The detection of 10 microsatellites within the genus Xiphophorus will enable studies about the correlation of this trait with sexual reproductive success of males possessing swords of different lengths. These microsatellites will also be useful in determining population structure and enable paternity analysis in these species, where sperm storage is widespread.     

Testing the phylogeny of swordtail fishes using split decomposition and spectral analysis

We examine ways of testing for the reliability of inference from biological sequence data using sequences from Xiphophorus fishes and newly implemented methodology for sequence analysis. The approach we take provides one means to examine the fit between model and data for different sequences and hence to evaluate heterogeneity between data sets. In the case of the present study we show D-loop sequences to be a better molecule for studying the phylogeny of Xiphophorus fishes than cytochrome b sequences. The results of the split decomposition and spectral analysis confirm an earlier phylogenetic hypothesis which had been based on maximum parsimony, neighbor-joining, maximum likelihood analyses. Correspondence to: A. Meyer     

Inactivation of Receptor Tyrosine Kinases Overcomes Resistance to Targeted <Emphasis Type="Italic">B-RAF</Emphasis> Inhibitors in Melanoma Cell Lines

The discovery of B-RAF activating mutations in malignant melanoma cells has led to the development of a number of targeted drugs, which block exclusively the mutant B-RAF protein. Tumor cells often acquire resistance to B-RAF inhibitors via activation of alternative signaling pathways. One of the resistance mechanisms is activation of PDGF, VEGF, c-KIT, and certain other tyrosine kinases. The possibility of overcoming the resistance to the B-RAF inhibitor Vemurafenib by inactivating receptor tyrosine kinases (RTKs) was studied in metastatic melanoma cell lines differing in B-RAF mutations and RTK activity. It was found that RTK inactivation may help to overcome resistance to B-RAF inhibitors via inhibition of tyrosine kinase phosphorylation and a subsequent blocking of the PI3K-AKT-mTOR and MEK-ERK1/2 downstream signaling pathways. The changes eventually mitigated the cell growth and enhanced the Vemurafenibdependent cell cycle arrest.