OLADOLAPO BELLO

The study of genetically-tractable model organisms has advanced our

knowledge of the molecular control of cellular function and these

two disciplines underpin our understanding of cancer.

Background

Cancer is used to describe a large group of diseases affecting any

part of the body which are capable of enabling the rapid

proliferation of abnormal cells (malignant tumours) which exceed

their boundaries and may invade other parts of the body.

Genetically-tractable animal models of diseases have been of immense

importance in the understanding of a number of human disease

mechanisms (including cancer) through the elucidation of the genes,

molecules and pathways involved in cellular function. Some of the

models that have been employed specifically in cancer research

include the Fruit-fly (Drosophila melanogaster), Zebrafish (Danio rerio),

and the mouse (Mus musculus).

Why Genetically-tractable animal models?

In addition to characteristics such as high rates of fecundity and

ease of manipulation and storage, these models have specific

features that make them suitable for use in research. The

development of genetic techniques that allow quick and easy

identification and characterisation of genes involved in the

formation and development of tumours make Drosophila a very good model

organism for the study of cancer development and there is a high

level of conservation of genes and pathways between the fruit-fly

and humans (Potter et al., 2000). Fish are also able to develop

tumours which bear similarities with cancers, and genetic (forward &

reverse) and small molecule screens, construction of transgenic

models and disruption of specific genes can all be carried out in

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the fish, which make it an important and promising system in cancer

research (Stern & Zon, 2003). Also, it is possible to genetically

engineer mice with chemicals or retroviruses for different specific

cancer models, manipulate the levels and patterns of expression of

genes and even knockout genes in mice (Ma, 2004).

Discoveries arising from studies of genetically-tractable animal models

Shh Pathway

Patched was originally described in reports by Nüsslein-Volhard & Wieschaus in 1980, using their “saturation mutagenesis” technique, as an embryonic lethal mutant gene of Drosophila. It functions in segment polarity in the fruit-fly. The hedgehog gene was also identified as one of the several genes important for creating differences between the anterior and posterior segments of the body.Genetic studies have shown that Patched works in a pathway along withHedgehog and Smoothened, known as the SHH signalling Pathway (See figure 1). The SHH pathway and the genes involved are highly conserved between Drosophila and vertebrates (Ingham, 1998); although the pathway is implicated in tissue patterning during development and the control of proliferation of cells in vertebrates (Hahn et al.,1999).

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Fig 1: The Sonic Hedgehog Signalling Pathway (Adapted from: Sheridan, 2009)

- In the Sonic hedgehog signalling pathway, binding of Shh to patched (a 12-span

transmembrane receptor protein) prevents the normal inhibition of smoothened (a 7-

span transmembrane protein) by patched. In the absence of the Shh ligand, the

pathway is turned off by Ptch which inhibits Smo, thus inhibiting the signalling of

downstream molecules (Villavicencio et al., 2000). The Ptch gene functions as a tumour

suppressor and activating Smo mutants function as oncogenes. Ptch and Smo gene

mutations have all been detected in basal cell carcinomas and medulloblastomas.

It was initially discovered that inhibition of the Hedgehog pathway

leads to cyclopia and holoprosencephaly (Chiang et al., 1996), then

it was later found that over-activation of the Hedgehog signalling

pathway promotes Basal Cell Carcinoma and that this overexpression

is caused by either a loss of function mutation in patched or an

activated smoothened mutation. Experiments were carried out using

transgenic mice using a promoter (K5) to drive smoothened mutation

and it was observed that non-keratinised and keratohyalin granules

were absent from the stratum granulosum (Xie et al., 1998), similarly

observed in earlier Mice BCC expression experiments which drove

overexpression of Shh in the skin (Oro, 1997). It was discovered

that Cyclopamine, a plant-derived compound, inhibits the activity of

HedgeHog and prevents abnormal cell growth (Taipale et al., 2000) and

Vismodegib, a drug closely related to Cyclopamine has now been

approved by the FDA for the treatment of advanced and metastatic

forms of Basal Cell carcinoma (Dubey et al., 2013)

Wnt Signalling

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In 1982, Nusse & Varmus proposed that Mouse Mammary Tumour Virus

(MMTV) was capable of inducing tumours in mammary glands of mice by

activating the expression of a gene called Int1. Some researchers

also reported that DNA from carcinomas induced by MMTV further

induced carcinomas in other cells and that a DNA of human mammary

cancer cell line possessed a similar neoplastic transformation

potential. Nusse & Varmus hypothesized that the oncogenesis by MMTV

was dependent on the insertion of this proviral DNA in a specific

region of the host genome.

Fig 2: Insertion of MMTV into host genome leading to strong expression of

tumorigenic potential.

They tested their hypothesis by cloning integration sites from a new

provirus, isolating the uninterrupted MMTV int1 domain with the

integration site, and by using probes to detect rearrangements

within Int1 in different carcinomas. They found that although the

infection by proviral DNA of the normal mammary cells introduced the

proviral DNA into many sites in host genome, oncogenesis was more

likely to occur in cells that acquired a provirus within int1, and

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they thus concluded that the integration of MMTV DNA in target cells

showed a certain regional specificity for the int1 domain.

It was later discovered that this Int1 gene (now known as the Wnt

gene) was a homolog of an earlier described drosophila mutant found

in wingless mutant (Wg) flies. This gene was found to encode for

components of the signalling pathway known as the Wnt pathway (See

Fig). (Klaus & Birch Meier, 2008)

Fig 4: The Wnt Signalling Pathway (Source: Tsivitse, 2010) In the absence of Wnt ligands, β-catenin is taken up by the destruction complex consisting of

Adenomatous polyposis coli (APC), axins, Casein kinase 1α (Ck1α) and Glycogen

synthase kinase 3β (GSK3β), and phosphorylation of β-catenin occurs. Ubiquitylation

of the β-catenin also occurs by β-Transducin repeat-containing proteins (β-TrCP),

and the β-catenin is proteosomally degraded. The low levels of β-catenin lead to

repression of Wnt target genes, ensured by the co-repressor Groucho (not shown)

which binds to Lymphoid Enhancer Factor – T-Cell Factor (LEF-TCF) transcription

factors. When Wnt Ligands are present, LDL-related Receptor Protein (LRP) is

phosphorylated by Ck1α and GSK3β and molecules of dishevelled move to the plasma

membrane to interact receptors of Frizzled and other dishevelled molecules. The

ensuing interaction between axin and phosphorylated LRP and dishevelled leads to an

inactivation of the destruction complex and β-catenin stabilises and moves to the

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nucleus. In the nucleus, β-catenin binds to the LEF-TCF complex, displacing Groucho

and allowing transcription of Wnt target genes (Klaus & Birchmeier, 2008). Note: APC

is the unlabelled hexagonal structure next to β-catenin in the diagram.

It has now been shown that APC, a component of the Wnt pathway acts

as a tumour suppressor gene and it is commonly accepted that the

suppressor abilities of APC stem from its ability to destabilise

free B-catenin (Harada et al., 1999). It was also shown that APC loss

directly results in increased size of the proliferating crypt

compartment of the small intestine (Bienz and Clevers, 2000) and may

be implicated in about 85% of hereditary colorectal tumours (Kinzler

& Vogelstein, 1996) (See Fig). It is suggested that the

indiscriminate activation of TCF4 by stable B-catenin in the absence

of APC leads to this expansion of the crypt.

Fig 5: Adenomatous polyp formation in APC

mutant mice

A – Normal cells of the epithelium (crypt

cells in yellow; Differentiated villi

cells in blue)

B & C – Pouch outpocket formation in zone

of proliferation of crypt

D - F – Formation of microadenoma inside

a single villus G –

Microadenoma expansion into neighbouring

villus H – Formation of a

multi-villus polyp by the fusion of

multiple adenomas which are in the

process of rupturing through villus

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epithelium to break into the lumen of the gut. Adapted from: Bienz & Clevers

(2000)

Zebrafish and Cancer

Cancer is commonly seen in wild fish and assays that involve

exposure to water-borne carcinogens show that fishes can develop a

wide variety of tumours (whether benign or malignant) in almost all

organs of their bodies (Spitsbergen et al., 2000). Also, fishes can

get tumours similar to human tumours and the cell cycle genes,

tumour suppressors and oncogenes in fish and humans are similarly

conserved. An advantage of this is that forward genetic screens can

be targeted to these highly conserved pathways and carcinogenesis

assays enable us test whether a mutation causing an embryonic

phenotype may induce predisposition to cancer in adults (Amatruda et

al., 2002).

In 2005, Shepard et al., used forward genetic screens in Zebrafish

embryos to screen for cell cycle mutants using anti-PO4 Histone H3

antibodies which marked cells that were undergoing cellular

division. An increase in the number of cells was observed in a

mutant known as crb, where it was discovered that the cells were

blocked in the M-phase of mitosis. Homozygous crb mutants also

showed an increase in apoptosis. The associated gene was

positionally cloned and named b-myb. Crb mutants represented a loss of

function in the b-myb gene, which suggested that b-myb was a tumour

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suppressor. As a follow-up to these experiments, Stern et al., (2005)

developed a chemical screen using zebrafish embryos to test for

small molecules that interact with the b-myb pathway. They crossed

crb heterozygotes (since the homozygous was embryonic lethal), and

arrayed them in multiwell plates and screened them with a 16,000

compound chemical library. It was observed that a previously unknown

compound, persynthamide, could rescue the phenotype, suppressing b-

myb mitotic defects.

SETDB1 & Melanoma (Zebrafish)

BRAF (V600E) is the most common mutation in Melanoma where it

activates BRAF and causes excessive activity in the Protein Kinase

pathway (Davies et al., 2002). However, it is also found in benign

naevi of melanocytes, which suggests that there are additional

genetic occurrences leading to melanoma Tumorigenesis. In 2011, Ceol

et al carried out experiments on zebrafish to test genes that were

recurrently amplified on a region of chromosome 1 for their

associative abilities with the BRAF (v600E) mutation in the

acceleration of melanoma using DNA sequence and gene expression

analyses. They used a transgenic fish where the oncogene,

BRAF(V600E) was expressed by the melanoma-specific promoter mitfa. In

a p53-/- background, this led to tumours but when the mitfa gene was

removed, the melanocytes died and the fish were normal. Using an

expression plasmid containing mitfa and several candidate genes which

was injected in the eggs of such fish lines, they discovered that

SETDB1, an enzyme that methylates histone on lysine9 (H3K9),

increased the rate of melanoma formation and identified SETDB1 as an

oncogene in melanoma in Zebrafish. Experiments were also carried out

using chromatin immuno-precipitation in conjunction with DNA

sequencing and analyses of gene expression and they some genes (such

as the HOX genes) were also discovered which are dsyregulated

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transcriptionally, as a direct result of the increased levels of

SETDB1. These experiments show that SETDB1 is an oncogene in

melanoma formation in Zebrafish, and they also signify the

importance of chromatin factors in the regulation of Tumorigenesis.

Nodal signaling and Melanoma (Zebrafish)

Nodal ligands were initially identified by forward genetic screens

in both mice & Zebrafish. In 1986, researchers who were carrying out

a retroviral insertational mutagenesis screen in embryonic stem

cells of mice (Robertson et al., 1986). Through this screen, a mutant,

induced retrovirally, was discovered which showed defects in early

gastrulation and the locus that corresponded was discovered to be a

member of the superfamily of TGFβ ligands that often express in

mammalian nodes (Zhou et al., 1993). Ligands related to nodal were

also subsequently discovered in other species. Similarly, in 1998,

genetic screens in Zebrafish demonstrated the functions in

gastrulation of two nodal-related genes, Cyclops (cyc) and squint that

were isolated as loss-of-function alleles (Feldman et al., 1998).

In 2006, Topczewska et al., found that the nodal gene is expressed by

aggressive melanoma cells and may be highly functional in tumour

development. This was identified by studying the interactions of the

cancer cells with embryonic progenitors in zebrafish. Fluorescently-

labelled melanoma cells transplanted in zebrafish induced ectopic

structures while a control group did not. It was also observed that

the melanoma cells induced a notochord in these ectopic structures.

Again, they observed that many embryos had a duplicated embryonic

axis. Nodal protein was suggested to be implicated. The embryos were

then

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stained for markers expressed in response to high and low levels of

nodal protein and it was found that the melanoma cells induced the

expression of both goosecoid (which is only expressed with high levels

of nodal) and notail. While the melanoma cells induced ectopic

structures, only a subset of the melanoma cells expressed nodal and

it was hypothesised that these were the actual cancer cells. To test

their observations, they tested several melanoma lines for the

expression of nodal. When two cell lines derived from the same

origin were used, it was observed that one was aggressive (MUM2B)

and the other was benign producing low levels of nodal. They

observed that nodal expression had a positive correlation with

melanoma tumour progression and there was more nodal protein in the

melanoma cancer cell metastases than in the primary tumours. It was

predicted that inhibiting the nodal pathway will lead to a decrease

in cell invasion and the reduced formation of the vasculogenic

network by downregulation of Keratin expression and VE cadherin.

They inhibited nodal expression using a morpholino and observed that

it resulted in the elimination of the axis-inducing properties of

the cell and it led to melanin formation in tumours and a reduction

in the volume of melanoma formed.

Fig 6: Graph showing reduction in tumour volume as a result of inhibition

of nodal signalling (Source: Topczewska et al., 2006)

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Fig 7: The Nodal Signalling Pathway (Source: Shen, 2007) A-Nodal ligands are intracellularly expressed as homodimeric proproteins, and are externally

cleaved by Furin and Pace4, which act as proprotein convertases. B-Cleaved Nodal

ligands, in conjunction with Gdf1 and Gdf3 can then bind to an EGF-CFC co-receptor

forming a complex, with type I receptor (ALK4) and type II receptor (ActRII or

ActRIIB) dimers. C- Nodal ligand functions can be inhibited by Cerberus and Lefty,

which are soluble antagonists. Lefty can also inhibit EGF-CFC co-receptors upon

interaction with them. D-Activation of the receptor complex then leads to

phosphorylation of the type I receptor by the type II kinase, as well as the

phosphorylation of Smad2 (or Smad3). This activated Smad2/3 then interacts with

Smad4 and moves into the nucleus, while the receptor complex undergoes

internalisation and is targeted by Dpr2 for lysosomal degradation in endosomes.

E- In the nucleus, the activated Smad2-Smad4 (or Smad3-Smad4) interacts with FoxH1,

a transcription factor, or Mixer homeoproteins on promoters of target genes, leading

to transcriptional activation through interactions with the ARC105 and mediator

complex. The Nodal pathway can be inhibited when Drap1 interacts with FoxH1, or when

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Ppm1A, a Smad phosphatase, promotes the nuclear export of Smad2, possibly targeting

it for proteasomal degradation (Shen, 2007).

Limitations of genetically-tractable animal models

While there have been significant advances in cancer research as a

result of broadened understanding of cellular function through the

experimental studies of genetically-tractable model organisms,

significant genetic, immunological and molecular differences exist

between animal and human systems and results gotten from animal

systems must be translated with caution. Also, animal models may

only show distinct processes or sets of processes within a disease

and may not display the entire array of changes in physiology that

take place in the disease being modelled (Hoyte et al., 2004).

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