Steven Kazianis

Mutant V600E BRAF Increases Hypoxia Inducible Factor-1α Expression in Melanoma

Mutations in the BRAF serine/threonine kinase gene are frequently found in cutaneous melanomas. Activation of hypoxia inducible factor-1alpha (HIF-1alpha) in response to both hypoxic stress and oncogenic signals has important implications in cancer development and progression. Here, we report that mutant BRAF(V600E) increases HIF-1alpha expression in melanoma cells. Our microarray profiling data in 35 melanoma and melanocyte cell lines showed that HIF-1alpha gene expression was significantly increased in melanomas harboring BRAF(V600E) mutation. Stable suppression of mutant BRAF(V600E) or both wild-type and mutant BRAF(V600E) by RNA interference in melanoma cells resulted in significantly decreased HIF-1alpha expression. Knockdown of mutant BRAF(V600E) induced significant reduction of cell survival and proliferation under hypoxic conditions, whereas knockdown of both wild-type and mutant BRAF(V600E) resulted in further reduction. The effects of BRAF knockdown can be rescued by reintroducing BRAF(V600E) into tumor cells. Transfection of BRAF(V600E) into melanoma cells with wild-type BRAF induced significantly more hypoxic tolerance. Knockdown of HIF-1alpha in melanoma cells resulted in decreased cell survival under hypoxic conditions. Pharmacologic inhibition of BRAF by BAY 43-9006 also resulted in decreased HIF-1alpha expression. Although HIF-1alpha translational rate was not changed, the protein was less stable in BRAF knockdown cells. In additional, von Hippel-Lindau protein expression was significantly increased in BRAF knockdown cells. Our data show for the first time that BRAF(V600E) mutation increases HIF-1alpha expression and melanoma cell survival under hypoxic conditions and suggest that effects of the oncogenic V600E BRAF mutation may be partially mediated through the HIF-1alpha pathway.

Dissolution of Sexual Signal Complexes in a Hybrid Zone between the Swordtails Xiphophorus birchmanni and Xiphophorus malinche (Poeciliidae)

Abstract The evolution of sexual signaling systems is influenced by natural and sexual selection acting on complex interactions among traits. Natural hybrid zones are excellent systems for assessing fitness effects on sexual phenotypes. Most documented hybrid zones, however, show little variation in sexual signals. A hybrid zone between the swordtails Xiphophorus birchmanni and Xiphophorus malinche is characterized by numerous recombinants for male sexual traits. Analyses of geographic variation in morphological and isozyme traits in the Río Calnali, Hidalgo, Mexico, reveal an upstream-to-downstream gradient from X. malinche- to X. birchmanni-type traits. A second hybrid zone, likely isolated from the R. Calnali, occurs in the nearby Arroyo Pochutla. Although the presumed female preference for swords predicts the introgression of swords from X. malinche-like populations into hybrid populations, the opposite pattern was observed. Swords are reduced in populations otherwise characterized by X. malinche traits. Sexually dimorphic traits were poorly correlated within individuals, indicating that sexual selection does not act against recombinant phenotypes. Hybrid males also exhibit trait values outside the range of parental variation. These patterns are consistent with predictions that females are permissive, preferring generally conspicuous males without attending to specific features.

Localization of a CDKN2 gene in linkage group V of Xiphophorus fishes defines it as a candidate for the DIFF tumor suppressor

The Xiphophorus hybrid melanoma model represents one of the earliest reported cases of genetically regulated tumor susceptibility. Melanoma formation in Xiphophorus hybrids may be explained by the inheritance of two genes: a sex‐linked oncogene, Xmrk, and a putative tumor suppressor locus, termed DIFF, located in Linkage Group V (LG V). Several genetic mapping procedures were used to produce a new Xiphophorus LG V map with 20 loci. All markers, particularly a recently cloned Xiphophorus CDKN2 gene family member, called CDKN2X, were tested for associations of genotype with degree of macromelanophore pigment pattern modification and susceptibility to melanoma formation in backcross hybrids of seven genetic types, involving 1,110 fish and three pigment patterns. Highly significant associations of CDKN2X genotypes with such phenotypic effects suggests that this gene is a strong candidate for the classically defined DIFF tumor suppressor gene. Because published results have documented the involvement of the CDKN2A (p16, MTS1, and INK4A) tumor suppressor gene in human melanoma formation, the possibility of CDKN2 genes acting as tumor suppressors in both man and Xiphophorus is likely. Genes Chromosomes Cancer 22:210–220, 1998.

NONMAMMALIAN MODELS FOR SUNLIGHT CARCINOGENESIS : GENETIC ANALYSIS OF MELANOMA FORMATION IN XIPHOPHORUS HYBRID FISH

Abstract— Genetic hybrids of Xiphophorus fishes have been used for decades to study heritable melanoma formation. In these models, overexpression of pigmentation patterns from melanin‐producing pigment cells can lead to genetically regulated melanoma formation in backcross hybrids. In the best studied of these models, the Gordon‐Kosswig hybrid melanoma, tumors form spontaneously in all individuals of a subset of backcross hybrids between the platyflsh Xiphophorus maculatus Jp 163 A and the swordtail species Xiphophorus helleri. Backcross hybrids susceptible to melanoma formation inherit a sex‐linked oncogene, Xmrk, associated with the spotted dorsal (Sd) pigment pattern and have lost both copies of an autosomal gene, DIFF, from the X. maculatus parent. Spontaneous melanoma formation conforms to simple, two‐gene Mendelian inheritance in which DIFF behaves as a recessive tumor suppressor gene. Recently, Xiphophorus hybrids in which melanomas can be induced by UV and near‐UV visible light exposure have been described. We report here results of genetic linkage analysis of one of these Xiphophorus light‐inducible hybrid melanoma models, in backcross hybrids between the two platyflsh species X. maculatus Jp 163 B and Xiphophorus couchianus. Our linkage results provide the first estimate of recombination between the tumor suppressor locus, DIFF, and glycerate‐2‐dehydrogenase (GLYDH) in Xiphophorus linkage group V. Also, they demonstrate that DIFF regulates hyperplasia of spotted side (Sp) pigment cells in this hybrid model, analogous to its regulation of hyperplasia of Sd pigment cells in the “classical” Gordon‐Kosswig hybrid. Joint segregation analyses of melanoma‐bearing fish indicate that segregation of DIFF is genetically linked to melanoma induction by 405 nm light in this model but that induction of melanomas by UV wavelengths apparently does not depend on segregation of the DIFF locus.

The Genus Xiphophorus in Mexico and Central America

The genus Xiphophorus is found from northeastern Mexico (Coahuila) for about 2200 Km as far as Honduras. There are 26 species, of which 21 occupy headwaters on the eastern slope of the Sierra Madre Oriental and continuing Cordillera to the southeast. Virtually all the species in the headwaters occupy limited ranges, often in rivers traversing karst country that are separated from lowland streams by underground passages. Only the three forms in the coastal plain are more widely distributed. Nineteen taxa occur within 400 Km of the Mexican Trans Volcanic Belt, suggesting that the genus may have evolved in this region. In many localities two species are sympatric, but natural hybrids are only known from three or four sites. Four monophyletic groups have been identified: the northern platyfish and the northern swordtail groups, north of the Mexican Trans Volcanic Axis, and to the south the helleri and the clemenciae swordtail groups. The status of the three southern platyfish is still not resolved and the phylogenetic relationship of the different groups to each other is still not fully understood.

Comparative structure and characterization of a CDKN2 gene in a Xiphophorus fish melanoma model

We have cloned, sequenced, and characterized the RNA expression properties of a fish CDKN2 gene from Xiphophorus helleri and X. maculatus. This gene, termed CDKN2X, shows a high degree of amino acid sequence similarity to members of the mammalian CDKN2 gene family, which includes the tumor suppressor loci CDKN2A (P16) and CDKN2B (P15). Comparative sequence analysis suggests that fish CDKN2X is similarly related to all four mammalian gene family members, and may represent a descendant of an ancestral prototypic CDKN2 gene. CDKN2X was mapped to a region on autosomal Xiphophorus linkage group V (LG V) known to contain the DIFF gene that acts as a tumor suppressor of melanoma formation in X. helleri/X. maculatus backcross hybrids. Thus, CDKN2X may be a candidate for the tumor suppressor DIFF gene. Here we have sequenced CDKN2X in both Xiphophorus species and have characterized its expression in normal and melanotic tissues within control and backcross hybrid fish. A simultaneous expressional analysis of the Xmrk-2 tyrosine kinase receptor gene, which is strongly implicated in melanomagenesis in this system, was also performed. RT – PCR analyses revealed that both genes were highly expressed in melanomas. For CDKN2X, this result contrasts numerous findings in human tumors including human melanoma in which either CDKN2A (P16) deactivation or LOH was observed.

Use of Random Amplified Polymorphic DNA (RAPD) for Identification of Largemouth Bass Subspecies and Their Intergrades

Abstract Random amplified polymorphic DNA (RAPD) analysis was investigated as an alternative to conventional electrophoresis for subspecies identification of largemouth bass Micropterus salmoides. Seventeen primers were analyzed by RAPD for their ability to identify polymorphisms, and the three primers that gave the most polymorphisms were used for the study. Fifteen markers were identified and were shown to indicate fixed differences between the two subspecies. Intergrades of unknown origin were distinguishable from pure Florida and pure northern wild and hatchery stocks, as well as first generation intergrades (F1s). The RAPD analysis was more sensitive than traditional histochemical agarose gel electrophoresis for identification of largemouth bass subspecies.

Genetic analysis of susceptibility to spontaneous and UV-induced carcinogenesis in Xiphophorus hybrid fish.

Abstract:Xiphophorus interspecies hybrids provide genetically controlled models of tumor formation. Spontaneous melanomas form in first-generation backcross (BC1) hybrids produced from backcrossing F1 hybrids derived from the platyfish X. maculatus Jp 163 A and the swordtail X. helleri to the X. helleri parental strain (the Gordon-Kosswig hybrid cross). Nodular melanomas originate in the dorsal fin from cells constituting the spotted dorsal (Sd) pigment pattern. A parallel genetic cross, with X. maculatus Jp 163 B, exhibits the spotted side (Sp) pigment pattern instead of Sd, and produces BC1 hybrids exhibiting a much lower frequency of spontaneous melanoma formation. These hybrids are susceptible to melanoma development if irradiated with UV light as fry. Other hybrids involving these two strains of X. maculatus and different swordtail and platyfish backcross parents also have been investigated as potential tumor models, and show differing susceptibilities to UV-induced and spontaneous melanomas. Genotyping of individual BC1 hybrids from several Xiphophorus crosses has implicated a locus, CDKN2X (a Xiphophorus homologue of the mammalian CDKN2 gene family, residing on Xiphophorus linkage group V), in enhancing pigmentation and the susceptibility to spontaneous and UV-induced melanoma formation in BC1 hybrids from some crosses, but not others. Homozygosity for X. helleri and X. couchianusCDKN2X alleles in BC1 hybrids can predispose individuals to melanoma, but this susceptibility is modified in other crosses depending both on the contributing sex-linked pigment pattern locus from X. maculatus (Sd or Sp), and the genetic constitution of the backcross parent. Xiphophorus BC1 hybrids constitute unique genetic models offering the potential to analyze the contributions of specific genes to spontaneous and induced tumor formation in different, but comparable genetic backgrounds.

Decreased Levels of (6-4) Photoproduct Excision Repair in Hybrid Fish of the Genus Xiphophorus¶

Abstract Selected hybridization in the fish genus Xiphophorus has been used for many years to study the genetics of malignant melanoma. Because DNA damage caused by ultraviolet radiation is implicated in the etiology of sunlight-induced melanoma, the heritability of mechanisms that mitigate DNA damage is a matter of some interest. We examined nucleotide excision repair of the two major types of DNA-damage induced by sunlight; the cyclobutane pyrimidine dimer (CPD) and the pyrimidine(6-4)pyrimidone dimer [(6-4)PD]. In most cases, removal of the (6-4)PD was more rapid than the CPD, and in many cases, the F1 hybrid showed reduced repair efficiency compared with the parental species. These data demonstrate reduced function in multienzyme hybrid systems and provide molecular support for potential reduced fitness in hybrid fish under conditions of environmental stress.

A Proposed Classification Scheme for Xiphophorus Melanomas Based on Histopathologic Analyses

Abstract: We studied the histopathologic characteristics of melanomas induced in the Xiphophorus model. This fish model has been used for several decades to study the molecular and genetic mechanisms underlying its susceptibility to melanoma induction. Numerous distinct interspecific hybrid crosses currently are being used in research on carcinogenesis. We previously reported that tumors were induced in such hybrid crosses after treatment with N-methyl-N-nitrosourea or UV radiation. In this report, we describe the histopathologic features of Xiphophorus melanomas and propose a new classification system. We suggest that melanomas in these fishes can be classified as follows: melanocytic melanomas; melanophorous–macromelanophorous polymorphic melanomas; spindle cell type melanomas; epithelioid cell melanomas; and amelanotic melanomas. The new classification of Xiphophorus melanomas should allow correlations between histopathologic characteristics and carcinogen treatment, and between histopathologic characteristics and the genetic background of the hybrid fish.