commentary

Hacker E, Hayward NK, Dumenil T et al. (2010) The

association between MC1R genotype and BRAF
mutation status in cutaneous melanoma: findings
from an Australian population. J Invest Dermatol
130:2418
Ihn H, Nakamura K, Abe M et al. (1993) Amelanotic
metastatic melanoma in a patient with
oculocutaneous albinism. J Am Acad Dermatol
28:895900
Kennedy C, ter Huurne J, Berkhout M et al.
(2001) Melanocortin 1 receptor (MC1R) gene
variants are associated with an increased risk
for cutaneous melanoma which is largely
independent of skin type and hair color.
J Invest Dermatol 117:294300
Landi MT, Bauer J, Pfeiffer RM et al. (2006) MC1R
germline variants confer risk for BRAF-mutant
melanoma. Science 313:5212
Liu W, Kelly JW, Trivett M et al. (2007) Distinct
clinical and pathological features are associated
with the BRAF(T1799A(V600E)) mutation in
primary melanoma. J Invest Dermatol 127:9005

Meyskens FL Jr, Farmer PJ, Anton-Culver H

(2004) Etiologic pathogenesis of melanoma:
a unifying hypothesis for the missing
attributable risk. Clin Cancer Res 10:25813
Palmer JS, Duffy DL, Box NF et al. (2000)
Melanocortin-1 receptor polymorphisms
and risk of melanoma: is the association
explained solely by pigmentation phenotype?
Am J Hum Genet 66:17686
Poynter JN, Elder JT, Fullen DR et al. (2006)
BRAF and NRAS mutations in melanoma and
melanocytic nevi. Melanoma Res 16:26773
Thomas NE, Edmiston SN, Alexander A et
al. (2007) Number of nevi and early-life
ambient UV exposure are associated with
BRAF-mutant melanoma. Cancer Epidemiol
Biomarkers Prev 16:9917
Van Raamsdonk CD, Barsh GS, Wakamatsu K
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and skin color by two G protein-coupled
pathways. Pigment Cell Melanoma Res
22:81926

See related article on pg 249

Xmrk in Medaka:
A New Genetic Melanoma Model
E. Elizabeth Patton1 and Rodney S. Nairn2
Melanoma is the most lethal form of skin cancer, and its incidence is rising rapidly
(Chin et al., 2006; Linos et al., 2009). Highly aggressive, metastatic melanoma is
notoriously resistant to chemotherapy, and early detection is critical for surgical
excision (Gray-Schopfer et al., 2007). A detailed knowledge of the cellular,
molecular, and genetic events during melanoma progression is highly relevant to
both diagnosis and the development of new therapies. Animal models, such as
the one described in this issue by Schartl and colleagues, are important tools for
identifying the genetic and environmental factors that contribute to melanoma
development, as well as identifying and testing new therapeutic strategies.
Journal of Investigative Dermatology (2010) 130, 1417. doi:10.1038/jid.2009.293

Xmrk, Xiphophorus, and

melanoma genetics

Fish have long been used in biomedical

research and are increasingly used as
models of human disease. Fish can
develop a wide range of cancers, including neurofibromatosis, hematological and liver cancers, and melanoma,

among others (Bunton, 1996). The

Xiphophorus hybrid melanoma was recognized in the 1920s as a genetically
controlled tumor model and has been
studied since then (Meierjohann and
Schartl, 2006). In this melanoma model,
interspecies hybrids between pigmented
platyfish (Xiphophorus maculatus) and

Institute for Genetics and Molecular Medicine, MRC Human Genetics Unit and The University of Edinburgh, Cancer Research Centre, Edinburgh, EH4 2XR. UK. 2Department of Carcinogenesis, The University
of Texas MD Anderson Cancer Center, Science Park/Research Division, Smithville, Texas 78957, USA.
1

Correspondence: E. Elizabeth Patton, Institute for Genetics and Molecular Medicine, MRC Human Genetics Unit and University of Edinburgh, Cancer Research Centre, Edinburgh EH4 2XR, United Kingdom.
E-mail: epatton@staffmail.ed.ac.uk

14

Journal of Investigative Dermatology (2010), Volume 130

nonpigmented swordtail (Xiphophorus

hellerii) parents, when backcrossed to
swordtails, produce hybrids with phenotypes that segregate in an apparently
Mendelian fashion, consistent with a
two-gene inheritance model. In 1952,
Breider hypothesized that melanomas
developed in the susceptible pigmented
Xiphophorus backcross hybrids as the
result of the loss of a regulatory locus
that inhibited genes controlling pigmentation, anticipating the oncogenetumor
suppressor gene concept by almost two
decades. Classical genetic recombination mapping subsequently established
that a sex-linked pigmentation determining locus (Mdl) and an autosomal regulatory locus (R) were indeed involved
in determining BC1 hybrid phenotypes,
including melanoma susceptibility.
In the late 1980s and early 1990s,
Schartl and colleagues published
work that associated an EGFR-related
gene, Xmrk, with the sex-linked Mdl
locus (then called Tu). The molecular
cloning and characterization of
this oncogenic melanoma receptor
kinase revolutionized the study of the
Xiphophorus melanoma model and
strengthened the concept of Xmrk as
a dominant oncogene in the model,
with R playing the role of a recessive
tumor suppressor. The true identity
of R has proven elusive, because the
cloned candidate for this susceptibility
locus, a homolog of the CDKN2 family
called CDKN2AB, does not behave as
predicted for a recessive tumor suppressor. However, a number of complexities
are revealed by studying other hybrid
crosses available in Xiphophorus, and
it is possible that R may be playing the
role of a modifier of Xmrk activity rather
than a frank suppressor, at least within
Xiphophorus interspecies hybrids (see
Butler et al., 2007, for a discussion of
this point). In any event, there is abundant evidence that Xmrk can behave
as a dominant oncogene, specifically
overexpressed in pigmented tissue
and driving oncogenesis. In this issue,
Schartl and colleagues express Xmrk as
a transgene in the pigmented tissues of
a fish other than Xiphophorus, called
medaka, and demonstrate conclusively that this gene is a powerful and
dominant oncogene driving melanoma
formation (Figure 1). Of greater interest,

commentary

Clinical Implications
Oncogenes and tumor suppressor genes are highly conserved between
fish and humans.
In medaka and zebrafish there are a wealth of mutations that give rise to
specific pigment cell phenotypes.
Transgenic medaka fish expressing the Xmrk oncogene develop nevi,
with some developing melanoma by 6 months.

in adult fish. Zebrafish and medaka

can also be modified with transgenes,
and expression of human cancer genes
driven by tissue-specific promoters has
led to the development of new models
of cancer, including leukemias, pan
creatic tumors, rhabdomyosarcoma,
and melanoma.
A new Xmrk melanoma model

however, is the identification of genetic

modifiers of the nevi and melanoma
phenotypes and the possible avenues of
further research opened by the system
described in the current study.
Using fish models to study cancer

Although the Xiphophorus hybrid melanoma system can be viewed as the

founding model for melanoma genetics, for genome-wide genetic screens
and dissecting the mechanisms of melanocyte development and melanoma
progression, the zebrafish and medaka
model fish systems offer distinct advant
ages (Patton and Zon, 2001; Kelsh et
al., 2004; White & Zon, 2008). In contrast to the live-bearing Xiphophorus,
in which the embryos are born after
30 days of in utero development, the
embryos of zebrafish and medaka are
fertilized externally. This allows for
analysis and visualization, as well as
genetic or chemical modification, from
the single-cell stage through embryogenesis to adulthood. Parents can be
bred to generate hundreds of offspring
each week. The genomic resources
available place zebrafish next to mice
and humans for comprehensive coverage of vertebrate genomes, and the
medaka genome is also available. The
ability to control gene expression with
morpholinos, RNA or DNA micro
injection, transgenes, and genetic mutations, coupled with sensitivity to small
molecules (zebrafish embryos can easily be arrayed in 96-well plates), means
that pathways relevant to cancer, such
as cell cycle and signaling pathways,
can be interrogated in the developing
embryo and tested directly in the adult.
Already
well
established
as
a powerful model organism for
studying development, the zebrafish
is gaining momentum in the cancer
field (Amatruda and Patton, 2008).

Oncogenes and tumor suppressor

genes are highly conserved between
zebrafish and humans, as are genes
involved in angiogenesis, the cell cycle,
apoptosis, and senescence. Importantly,
zebrafish can develop a wide spectrum of tumors that share histopathological features with human cancers.
The molecular pathways are also conserved; for example, liver tumors in
zebrafish share stage-specific gene
expression signatures with liver tumors
of the same histopathological grade in
humans. Both zebrafish and medaka
can be mutagenized with N-ethyl-Nnitrosourea (ENU) for genetic screens,
which allows the identification of specific phenotypes and subsequently
their associated mutant genes by positional cloning. For example, spontaneous mutations and large-scale genetic
screening in zebrafish and medaka have
generated a wealth of mutations that
give rise to specific pigment cell phenotypes (Kelsh et al., 2004; Kelsh, 2004;
White and Zon, 2008). In the cancer
field, zebrafish ENU-based genetic
screens have led to the identification of
modifier mutations that enhance cancer susceptibility and determine tumor
spectrum. In addition, insertional mutagenesis in zebrafish has generated hundreds of mutations in genetic screens,
which have been rapidly identified by
virtue of sequencing the insertion site.
This method has led to the identification of neurofibromatosis type 2 and
novel
haploinsufficient
ribosomal
mutations that confer spontaneous
neural tumors in the adult zebrafish.
In an approach called TILLING, mutations in specific genes are identified
by sequencing from ENU mutagenized
genomes, resulting in the identification of mutations in tumor suppressor
genes such as p53, PTEN, and APC, all
of which confer tumor susceptibility

Oncogenic Xmrk in the Xiphophorus

melanoma model provides a valuable
opportunity to study melanoma cancer biology (Meierjohann and Schartl,
2006), but the lack of genomic resources
and transgenic technologies has meant
that identification of genetic modifiers
has been a significant obstacle in the
Xiphophorus field. Using transgenesis,
Schartl et al. (2010, this issue) have now
built a bridge from the Xiphophorus to
the medaka system by expressing the
Xmrk oncogene in medaka and thereby
generating a tractable genetic melanoma model. In Xiphophorus, oncogenic
Xmrk is specifically expressed in the
pigment cells, and so Schartl and colleagues expressed the Xmrk gene under
the pigment cell-specific promoter, mitf,
in medaka. Hemizygous transgenic fish
expressing Xmrk develop nevi, with some
developing melanoma by 6 months.
Impressively, in homozygous transgenic
Xmrk fish, all developed melanoma
between 2 and 6 weeks of age. In this
model, the mitf promoter also appears
to express and generate tumors in the
red and yellow pigment cell types (xanthophores and erythrophores, respectively). The Xmrk melanomas are highly
invasive and appear to be metastatic.
Molecular analysis suggests that Xmrk
medaka tumor melanomas are similar to
Xmrk Xiphophorus tumors, in that both
display activation of AKT signaling and
enhanced activation of STAT5 signaling.
MITF and BCL2 are also increased in the
Xmrk medaka melanoma, providing in
vivo evidence for a role of the MitfBcl-2
axis (McGill et al., 2002).
Where the transgenic Xmrk melanoma model becomes especially useful is in the identification of genetic
modifiers of the melanoma phenotype.
Schartl and colleagues showed that the
tumor spectrum and age of onset vary
with genetic background. In the Carbio
strain, Xmrk promoted pigment cell
www.jidonline.org

15

commentary

X
Xmrk /+ male
25% WT

Xmrk /+ female
50% Xmrk /+

25% Xmrk /Xmrk

Cpd2

10 days

Cpd1

Cpd3

6 weeks

CpdA

CpdB

CpdC

6 weeks

6 months

6 months

Xmrk /Xmrk; M/+

Xmrk /+; M1/+

Xmrk /+; M2/+

Xmrk /+; M3/+

Figure 1.The Xmrk medaka model provides an opportunity to identify genetic and chemical modifiers
that suppress or enhance the nevi and melanoma phenotypes. In the mitf-Xmrk medaka melanoma model,
hemizygous Xmrk fish give rise to progeny that are 25% wild type, 50% hemizygous for Xmrk (Xmrk/+), and
25% homozygous for Xmrk (Xmrk/Xmrk; salmon-colored panels). By 10 days of development, homozygous
Xmrk/Xmrk larvae express ectopic pigmentation (fish-nevi), and by 26 weeks of age, these fish develop
melanoma. In the hemizygous Xmrk medaka, ectopic pigmentation develops by 6 weeks, and tumors
develop by 6 months. Melanomas, or other pigmented tumors, are not observed in wild-type medaka fish.
Schartl and colleagues (2010, this issue) provide evidence that genetic modifiers (M) that alter tumor biology,
such as those in background variance or ENU mutations, can be identified (red panels). In these examples,
mutations in Xmrk/Xmrk fish affect tumor progression (M; e.g., nevi, but not melanoma development); in
Xmrk/+ fish, modifiers affect tumor spectrum (M1; e.g., uveal melanoma), tumor progression (M2; e.g.,
tumor size), or tumor pathology (M3; e.g., endophytic versus exophytic melanoma). The rapid onset of fishnevi and melanoma also allows for screening for small molecules that alter tumor characteristics. Young
medaka fish can easily fit into 24- or 6-well plates, each with a different compound (Cpd) dissolved in the
water. In these examples, Cpd3 prevents the onset of Xmrk/Xmrk-induced fish nevi in 10-day-old medaka;
CpdB prevents development of melanoma in 6-week-old Xmrk/Xmrk medaka.

tumors of all three pigment cells, as well

as rare uveal melanomas. In the CabR
genetic background, melanoma was
rare and almost all tumors developed
from the red and yellow pigment cells.
These tumors were largely exophytic,
with invasion seen only in the terminal stages. In contrast, in the HB32C
genetic background, almost all tumors
were endophytic, invasive melanoma.
In another line, the albino (i-3) strain,
internal melanomas developed, in addition to a high percentage of uveal melanoma. Presuming the fish lines develop
melanocytes and other pigment cells in
a similar way, the genetic differences
between the lines appear to account for
the varied pigment cell tumor phenotypes and provide strong evidence for
how background polymorphisms can
have a profound effect on cancer biology within individuals.
Although the genetic differences in
background have yet to be identified,
16

Schartl and colleagues were able to

directly test the role of a specific gene,
p53, in tumor progression. The p53 pathway appears to play a role in melanoma
(Chin et al., 2006, Yu et al., 2009); however, mutations in p53 are relatively rare
in melanoma compared with other tumor
types. This may be in part because the
CDKN2A locus is often mutated in melanoma and encodes both p16INK4A and
p14ARF, controlling the INK4A-COK416RB and ARF-MDM2-p53 axes, respectively (Chin et al., 2006). In mice, mutations in p53 can cooperate with RAS to
promote melanoma formation (Chin et
al., 2006), and in zebrafish, BRAFV600E is
sufficient to promote nevi, but requires
loss of p53 for progression to melanoma
(Patton et al., 2005). In contrast, oncogenic Xmrk in medaka is sufficient to
promote melanoma formation, possibly
because in addition to mitogen-activated
protein kinase-pathway signaling, the
phosphoinositide 3-kinase pathway is

Journal of Investigative Dermatology (2010), Volume 130

also activated, among other pathways,

including the PLC-, STAT5, and FAK/
FYN pathways. When expressed in a
p53-deficient background, the Xmrkinduced tumor spectrum and age of
onset are not affected, but at later stages
there is a marked change, and the tumors
suddenly begin to grow very rapidly.
Despite the many pathways downstream
of Xmrk, loss of p53 nevertheless appears
to play an important role in melanoma
progression, supporting the notion that
the p53 pathway, despite the low rate of
mutations in melanomas, has an important role in melanoma progression.
Opportunities for genetic
and chemical screens

The power of this new Xmrk system

lies in its potential for both genetic and
chemical screens (Figure 1) (Patton and
Zon, 2001; Zon and Peterson, 2005).
Schartl and colleagues provide proof of
principle that the genetic modifiers of
the melanoma pathology can be identified in genetic screening, and although,
with the exception of the p53 mutation, the other mutations are currently
unknown, this is a significant step for
melanoma genetics. The rapid onset
of Xmrk-induced nevi at 10 days and
melanoma at 26 weeks (when the
medaka fish are only about 0.3 and 1
cm long, respectively) provides a unique
opportunity for chemotherapeutic and
chemopreventative screening. Zebrafish
are increasingly being used in the drug
discovery process, in part because the
effects of the chemical compound can
be assessed rapidly in a whole-animal
context (Zon and Peterson, 2005). The
recent development of BRAFV600E mouse
melanoma models that demonstrate
chemical and genetic control of melanoma progression provides an important
rationale for screening for genetic mutations and small molecules (or even combinations of small molecules) in animal
models (Dankort et al., 2009; Dhomen
et al., 2009; Goel et al., 2009). As the
incidence of melanoma continues to
rise and current chemotherapies remain
generally ineffective against malignant
melanoma (Gray-Schopfer et al., 2007),
animal models such as this one provide
an important means for dissecting the
genetic and environmental factors that
contribute to melanoma development

commentary

and progression, as well as identifying

the drugs and new drug-like leads that
can target this devastating disease.
CONFLICT OF INTEREST

The authors state no conflict of interest.

backgrounddependent
histopathologies.
J Invest Dermatol 130:24958
White RM, Zon LI. (2008) Melanocytes in
development, regeneration, and cancer. Cell
Stem Cell 3:24252
Yu H, McDaid R, Lee J et al. (2009) The role

of BRAF mutation and p53 inactivation

during transformation of a subpopulation of
primary human melanocytes. Am J Pathol
174:236777
Zon LI, Peterson RT (2005) In vivo drug discovery
in the zebrafish. Nat Rev Drug Discov 4:3544

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Amatruda JF, Patton EE (2008) Genetic models

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Bunton TE (1996) Experimental chemical
carcinogenesis in fish. Toxicol Pathol
24:60318
Butler AP, Trono D, Beard R et al. (2007)
Melanoma susceptibility and cell cycle
genes in Xiphophorus hybrids. Mol Carcinog
46:68591.
Chin L, Garraway LA, Fisher DE (2006) Malignant
melanoma: genetics and therapeutics in the
genomic era. Genes Dev 20:214982
Dankort D, Curley DP, Cartlidge RA et al. (2009)
Braf(V600E) cooperates with Pten loss to
induce metastatic melanoma. Nat Genet
41:54452
Dhomen N, Reis-Filho JS, da Rocha Dias S et al.
(2009) Oncogenic Braf induces melanocyte
senescence and melanoma in mice. Cancer
Cell 15:294303
Goel VK, Ibrahim N, Jiang G et al. (2009)
Melanocytic nevus-like hyperplasia and
melanoma in transgenic BRAFV600E mice.
Oncogene 28:2289-98
Gray-Schopfer V, Wellbrock C, Marais R (2007)
Melanoma biology and new targeted therapy.
Nature 445:8517
Kelsh RN (2004) Genetics and evolution of pigment
patterns in fish. Pigment Cell Res 17:32636
Kelsh RN, Inoue C, Momoi A et al. (2004) The
Tomita collection of medaka pigmentation
mutants as a resource for understanding
neural crest cell development. Mech Dev
121:84159
Linos E, Swetter SM, Cockburn MG et al. (2009)
Increasing burden of melanoma in the United
States. J Invest Dermatol 129:166674
McGill GG, Horstmann M, Widlund HR et al.
(2002) Bcl2 regulation by the melanocyte
master regulator MITF modulates lineage
survival and melanoma cell viability. Cell
109:70718
Meierjohann S, Schartl M (2006) From Mendelian
to molecular genetics: the Xiphophorus
melanoma model. Trends Genet 22:65461
Patton EE, Zon LI (2001) The art and design of
genetic screens: zebrafish. Nat Rev Genet
2:95666
Patton EE, Widlund HR, Kutok JL et al. (2005)
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Schartl M, Wilde B, Laisney JAGC et al.
(2010) A mutated EGFR is sufficient to
induce malignant melanoma with genetic

See related article on pg 270

Sun-Sensitizing Effects of PKC Shine

on Multiple Mouse Strains
Mitchell F. Denning1
Like many skin phenotypes, susceptibility to carcinogenesis is profoundly influenced by genetic background. Sand et al. (this issue) explored the sensitizing
effects of epidermal protein kinase C (PKC)expression in the hairless SKH-1
mouse strain commonly used in UV carcinogenesis studies. They reported that
PKC overexpression profoundly sensitized these mice to UV skin carcinogenesis
and activated oncogenic pathways similar to those reported in FVB/N mice.
Journal of Investigative Dermatology (2010) 130, 1719. doi:10.1038/jid.2009.354

Anyone who has witnessed the diverse

shades of sunburned skin on display at the beach can appreciate the
importance of genetic background on
the response of skin to UV radiation.
Likewise, investigators characterizing
transgenic mice are aware of the importance of genetic background on phenotype because strain-specific effects
are frequently reported. Differences in
mouse strain susceptibility to carcinogenesis, including UV skin carcinogenesis, are well known, and the genetic
loci responsible for some of the differences have been identified, including
some not related to skin pigmentation.
The study by Sand and colleagues
(2010, this issue) examines the sensitizing effects of protein kinase C (PKC)
overexpression on UV carcinogenesis in
two mouse strains, FVB/N and SKH-1.
Their findings highlight the potent effect
of PKC in enhancing skin carcinogenesis regardless of genetic background
and shed light on the potential signaling pathways involved.

PKC and skin carcinogenesis

The PKC family of serine/threonine

protein kinases occupies a central
node in numerous growth factor and
hormone receptor signal transduction
pathways. These kinases have also been
studied extensively as targets of the
mouse skin tumor promoter, TPA, which
is commonly used in two-stage mouse
skin chemical carcinogenesis studies.
Keratinocytes express at least five PKC
isoforms (, , , , and ), each with
a unique function in normal epidermal
biology. Recent studies have focused on
understanding biological functions of
the individual PKC isoforms in UV skin
carcinogenesis and identifying substrates
relevant to these functions.
Verma and co-workers at the
University of WisconsinMadison have
generated and characterized transgenic mice expressing individual PKC
isoforms in their epidermis under the
control of the K14 promoter. These studies have been instrumental in clarifying
the divergent effects of PKC isoforms on

Department of Pathology and the Cardinal Bernardin Cancer Center, Skin Cancer Research Program,
Loyola University Chicago, Maywood, Illinois, USA

Correspondence: Mitchell F. Denning, Department of Pathology and the Cardinal Bernardin Cancer
Center, Skin Cancer Research Program, Loyola University Chicago, 2160 South First Avenue, Maywood,
Illinois 60513, USA. E-mail: mdennin@lumc.edu

www.jidonline.org

17