Animal models for Liver and Breast cancer

Beshoy Makram , Beshoy Adel , Christeen Tawfik , Christeen Tharwat , Ehab

Youssef , Elia Youssef , Eva Baddar , Erinee Adel , Faten William , Jhon Makram ,

Nardeen Nasser , Neveen Nady , Sousana Zaher

Faculty of pharmacy Minia University . Department of Biology .

Key words

Liver cancer models

Hepatocellular carcinoma models

Breast cancer models

Mouse models

Rabbit models

Genetically engineered mice

Rodent models

Animal models for cancer

Abbreviations

Genetically engineered mice

models (GEMMs)

Diethylnitrosamine (DEN)

Mouse mammary tumor virus

(MMTV)

Hepatocellular carcinoma (HCC)

Reactive oxygen species (ROS)

Hepatitis virus B (HBV)

Hepatitis virus C (HCV)

Phenobarbital (PB)

Severe combined immunodeficient

(SCID)

A B S T R A C T

Cancer is one of the most dangerous malignancies in

the world . In this research we discuss the animal

models for only two types of it ( Liver cancer and

Breast cancer ) . Animal models are induced to have

cancer by various ways ( oncogenes , Chemical

carcinogens , transgenic , GEMMs ) , and then they are

subjected to observation , in order to understand the

mechanism of pathogenecity of cancer , and try

anticancer drugs firstly on animals to know whether it is

effective against it or not , before moving to trials on

humans .

There are many strains of animals and that gives us clue

about the genetic differences and its effect on having

cancer or not .

Liver Cancer

1. Introduction :

Liver cancer is one of the most lethal

malignancies worldwide . The world health

organization estimates that in 2008 liver cancer

accounted for almost 700 000 deaths

worldwide , the survival rates were under 11 %

which makes it the third most frequent cause of

cancer death in men and sixth in women .

Liver cancer consists of several histologically

different primary hepatic malignancies , such as

cholangiocarcinoma , hepatoblastoma and

haemangiosarcoma , and Hepatocellular

carcinoma . HCC is the most common type

accounting for 70 % - 85 % of cases .

Risk factors for HCC are :

• Cirrhosis .

• HCV .

• HBV .

• Chronic alcohol consumption .

• Alatoxin-B1 from contaminated food .

Establishing animal models for HCC is

essential for both basic and translational studies

, and to our understanding of the mechanisms

underlying the pathogenesis of HCC .

Several animal models have been used over the

years to study HCC pathogenesis . The

laboratory mouse is one of the best

experimental systems , owing to the

physiologic , molecular and genetic similarities

to humans , its breeding capacity , short

lifespan .

The conventional strains of animals differ

greatly in their susceptibilities to spontaneous

liver tumors , and that’s good because it give us

clue to which genes are related to liver cancer

incidence .

Efforts were made to develop better models

where tumors would develop more rapidly with

high penetrance .

The pathologic alteration causing liver cancer

were originally modeled through the use of :

1) Conventional methods :

• Chemical carcinogens like

(Diethylnitrosamine ) .

• Implantation models .

• Viral infection ( HBV / HCV ) .

2) New methods :

• Genetically engineered mouse models

(GEMMs) .

2. Conventional mouse models :

2.1. Chemical Carcinogens :

The liver is the primary target site for hundreds

of chemicals including pesticides, food

additives, pharmaceuticals and industrial

intermediates . Identifying hepatocarcinogenic

compounds and understanding the cellular and

molecular processes during carcinogenic

transformation of hepatocytes is an ongoing

challenge .

The hepato carcinogens are subdivided into two

classes :

• Genotoxic : (They form DNA adducts ,

which lead to genetic changes of the

target cell , these changes can direct

normal cells to preneoplastic state

"initiation state" ) , Like : DEN .

• Non-genotoxic : ( They do not modify

DNA structure , but generally stimulate

the preneoplastic to evolve into a

malignant neoplasm by controlling cell

proliferation , apoptosis and cell

differentiation ) , Like : aflatoxin-B1 ,

alcohol , Combined oral contraceptives .

Some of the human carcinogens are not

carcinogenic to mouse liver .

For example , cirrhosis and liver cancer have

not been observed in mice subjected to ethanol

only . This discrepancy in response to

carcinogens can probably be explained by

species differences .

The single most frequently used chemical for

induction of HCCs in mice is DEN , agenotoxic

carcinogen .

DEN is typically administered to mice between

12 and 15 days of age by a single

intraperitoneal injection (5 µg/g body weight) .

Using this protocol , 100% of B6C3F1 male

mice developed HCCs , on average 44 weeks

after intraperitoneal injection of DEN .

Mechanism of action of DEN :

DEN is a DNA alkylating agent leading to the

formation of mutagenic DNA adducts . In

addition , DEN bioactivation by cytochrome

P450 can generate reactive oxygen species

(ROS) , which damage DNA , proteins and

lipids and lead to hepatocytes death .

Another protocol is used to initiate tumors by

using DEN/PB , which show activation of

B-catenin mutations and genomic instability in

mice .

And approximately 30 % of human HCCs

display mutations activating the B-catenin

pathway , by the same protocol .

Which is a good overlap between the DEN/PB

induced mouse tumors and their human counter

parts , further supporting the usefulness of this

chemical carcinogenesis mouse model . For this

purpose , chemicals are either administered to

newborn mice in order to determine

genotoxicity , or compounds are administered

for longer periods ( usually 2 years ) to assess

epigenetic carcinogenity .

2.2. Implantation models :

HCC is formed in these models in a way which

a HCC cell line or tissue fragment is implanted

in the desired model .

These HCC cells could be implanted in :

• Mice of the same strain from which the

implant originates ( Allograft models) .

• Immunodeficient mice ( Xenograft

models )

The first type is less frequently used , it is only

required when anticancer agents ( That works

by activation of immune system ) are tested .

In allograft models murine HCC cell lines or

tumor fragments are implanted in mice . The

HCC grafts have been derived from

spontaneously occurring HCCs in mice , from

carcinogen induced tumors or from genetically

engineered mice (GEM) .

The most widely used models for xenograft

implantation are :

• Nude mouse (nu -/-) : These mice are

hairless and have a deficiency of T

lymphocytes as well as an impaired T

and B cell function .

• Severe combined immunodeficient

(SCID) mice : are frequently used in

xenograft models of HCC . These mice

have a deficiency in number and

function of both T and B lymphocytes .

These HCC cell or tissue fragments can be

implanted into recipient mice by either :

• Ectotopic : usually occur

subcutaneously .

• Orthotopic : by subserosal injection of

HCC cells or by surgical orthotopical

implantation (SOI) of tumor fragments .

These fragments , approximately 1 mm3 in

size , are derived from surgical specimens

of human HCCs or from pieces of

subcutaneously grown HCC cells , either

from human or mouse origin .

2.3. Viral Infection :

More than 80% of HCCs in humans are

attributable to infection with either HBV or

HCV or both . So , we need to infect animal

models with HBV and HCV and study them to

know the multiple steps of genetic alterations

leading to HCC .

Only one problem in establishing such a model

is that HBV and HCV require human

hepatocytes to induce hepatitis , due to the

stringent human tropism of these viruses .

To overcome this , recently several animal

models have been developed in which human

hepatocytes or human liver tissue are

transplanted into these animals

(Immunocompromised mice or rats ) . The

transplanted hepatocytes in these animals can

be infected with HBV or HCV in vivo . These

models are promising for the evaluation of

therapeutics and prophylactics against HBV or

HCV, but are not useful to study associated

HCC . Because it may take more than 20 years

for HCC to develop in HBV or HCV infected

persons .

For that purpose , transgenic mice expressing

HBV or HCV proteins represent a better model.

2.3.1. HBV associated HCC :

Approximately 380 million people are

chronically infected with HBV . These chronic

HBV infected people have a 100-fold greater

lifetime risk of developing HCC in comparison

with non-carriers . For this reason , HBV

infection is the leading risk factor for the

development of HCC . Worldwide , over 50%

of HCC cases are associated with chronic HBV

infection .

1st model : a mouse model in which transgenic

mice were generated that carried an integrated

HBV DNA fragment coding for the HBV large

envelope polypeptides on a C57BL/6 genetic

background . As a result , non secretable

hepatitis B surface antigen (HBsAg) particles

formed that accumulated in the endoplasmic

reticulum of the hepatocytes . In mice with

100% of the hepatocytes expressing HBsAg (

lineage 50 – 4 ) , liver injury begins at 2 - 3

months of age ; at 6 months regenerative

nodules appear and from the age of 15 months

HCCs develop .

2nd

model : Another HBV gene that has been

extensively studied is the HBx gene . Though

several research groups could not find evidence

for a hepatocarcinogenic role of HBx in HBx

transgenic mice , Kim et al did report such a

role . They produced HBx transgenic mice by

injection of HBV DNA containing the HBx

gene into single cell embryos from CD1-mice .

In these mice, liver tumors began to emerge

after 8-10 month . Both male and female

transgenic mice died early in comparison with

control CD1 mice , at the age of 11-15 month

VS 17-21 month , respectively . On autopsy ,

80% - 91% of male transgenic mice and 60% -

67% of female transgenic mice showed

one or multiple HCCs .

3rd

model : Yu et al generated transgenic HBx

mice using the same technique as Kim et al ,

but in a C57BL/6 genetic background and with

a much weaker HBx expression in the liver .

They reported an incidence of grossly

identified HCCs and small neoplastic nodules ,

without signs of cirrhosis or inflammation , in

86% of 11-18 month old HBx transgenic mice .

Possible explanations for the different outcome

in transgenic mouse models for the

hepatocarcinogenic role of HBx , may include a

difference in mouse strains that were used .

Male mice of the CD-1 strain develop

spontaneous HCC in 5.7% , an incidence that is

some-what higher than the rate in for instance

C57BL/6J mice (< 4.0%) . In addition , the

expression level of HBx-mRNA in the livers of

transgenic mice and the type of HBx used may

be different in the various studies.

Finally, the integration site of HBx in the

genome of the mice might influence the

hepatocarcinogenic effect of HBx .

Nowadays, models based on the HBsAg

transgenic mouse model of Chisari et al and

the HBx transgenic mouse model of Kim et al

are commonly used to study mechanisms

involved in hepatocarcinogenesis . These

models are also applied to study possible

synergistic relations between chemical

carcinogens ( such as aflatoxin B1 or diethyl

nitrosamine ) and HBV infection , and HBV

proteins have been shown to manipulate the

P53- , Rb- , cyclinD1 - and p21- genes .

2.3.2. HCV associated HCC :

Worldwide , approximately 30% of HCC cases

are related to chronic HCV infection , making

HCV the second most frequent cause of HCC .

Various HCV proteins have been expressed in

transgenic mice to study the pathogenesis of

HCV associated HCC , particularly the HCV

polyprotein , the core protein and the core

protein in combination with E1 and E2

envelope proteins . Interestingly , the

expression of the core protein of HCV seems

to be the major factor contributing to the

hepatocarcinogenic effect of HCV infection ,

as transgenic mice that do not express this

protein , no HCCs arise .

Moriya et al were the first to describe such a

transgenic mouse model . They generated

transgenic mice that carried the HCV core gene

. These mice showed histological features of

steatosis in the liver , without inflammation ,

from the age of 3 months and showed HCCs

with close histological resemblance of HCCs in

human chronic HCV infection , by the time

they were 16 month old .

The incidence of HCC in 16-19 month old male

transgenic mice was 26% to 31% , in contrast

to a low incidence in the female transgenic

mice , which is in accordance with the human

situation . By means of such transgenic mouse

models numerous molecular and pathogenetic

pathways have been investigated that have led

to a better understanding of HCV associated

hepatocarcinogenesis .

To study the role of HCV proteins other than

the HCV core protein in hepatocarcinogenesis ,

Lerat et al developed full length HCV

polyprotein transgenic mice and compared

them with transgenic mice encoding merely the

structural HCV proteins ( including the core

and the E1 and E2 envelope proteins ) .

HCCs occurred ( exclusively in males ) in 5 of

38 transgenic mice expressing the full HCV

polyprotein and in 1 of 43 transgenic mice

expressing the structural HCV proteins . These

findings suggest that HCV proteins, other than

the HCV core protein , may endorse

development of HCC as well , because in these

mice the HCV protein levels are much lower in

the first group .

The HCCs that develop in the mouse models

described by Moriya et al and Lerat et al show

proper ( histological ) resemblance to the

corresponding lesions in patients with HCV

associated HCC .

3. New methods :

3.1. Genetically engineered mouse models

(GEMMs) :

The introduction of transgenic mouse models in

made it possible to study the molecular features

of human malignancies in vivo. Since then ,

much progress has been made in techniques of

producing GEMMs .

In studying the molecular mechanisms involved

in hepatocarcinogenesis , GEMMs are

particularly used to explore the role of a

specific gene and to explore the interaction of

different genes ( e.g. oncogenes and tumor

suppressor genes ) in the development of HCC .

GEM is also suitable to investigate the role of

specific genes in combination with

a liver – specific carcinogen .

Table 1 :

Since the late 1980’s , transgenic SV40 T-Ag

(Simian Virus 40 T-antigen) mice have been

studied extensively . The genome of the simian

virus 40 ( a DNA tumor virus ) encodes two

oncogenic proteins, the large and small T

antigen (T-Ag and tAg , respectively , herein

together referred to as T-Ag). After infection,

large T-Ag can cause malignant transformation

of the host cell primarily by inactivating the

tumor-suppressor genes p53and Rb .

Research groups have reported the production

of transgenic mice expressing SV40 T-Ag

directed to the liver by the promoter/enhancer

antithrombin-Ⅲ( ATⅢ ) , albumin ( Alb ) and

α-1-antitrypsin ( AAT ) .

For example , Duboiset al produced transgenic

mice by putting the SV40 T-Ag under the

control of the human ATⅢ promoter . In

mouse lineages that expressed the highest level

of the transgene , by the age of 8 mo , 100% of

mice had developed HCCs and 10 % had

developed lung metastases .

Another commonly used transgenic mouse

model was described by Murakami et al . They

generated double transgenic mice over

expressing cmyc and TGF-αin the liver ( Alb-c-

myc/MT-TGF - αmice ) by crossing Alb/c-myc

mice ( transgenic mice overexpressing c-myc ,

directed by the albumin promoter ) with

MT/TGF-αmice ( transgenic mice

overexpressing TGF-α, directed by the

metallothionein 1 promoter ) .

Santoni-Rugiu et aldemonstrated that these

mice developed HCCs substantially earlier and

at a higher rate than single transgenic mice ,

overexpressing either c-myc or TGF-α . Within

8 months after birth , 100% of male and 30% of

female Alb-c-myc/MT-TGF-αmice had

developed HCCs .

Although these conventional transgenic mouse

models have been very useful to study the role

of particular genes in hepatocarcinogenesis and

to study the multistep nature of HCC

development , one limitation of these models is

the fact that the transgene is expressed in all

Hepatocytes , including the tumor

microenvironment . Furthermore , the

mutations are already present during

embryogenesis and thus, might activate

compensatory ( molecular ) pathways . To

overcome these limitations , mouse models

have recently been developed in which the

genetic alterations are induced in a tissue

specific and time controlled fashion

(conditional mouse models) .

For instance , Lewis et al used a retroviral

transduction strategy to deliver oncogenes to

hepatocytes in situ .

They made use of the fact that mice do not

express the

TVA receptor , which is the receptor for the

avian leukosis sarcoma virus subgroup A

(ALSV-A) . Lewis et al generated TVA

transgenic mice , in which TVA was

specifically expressed within the liver .

Delivery of ALSV-A viruses encoding PyMT

(mouse polyoma virus middle T antigen , an

oncogene ) to these mice at the age of 2-3 d,

subsequently led to tumor formation by the age

of 4-6 months (in 17 of 26 mice) . They also

exposed TVA trans-genic mice that were

deficient for p53 to PyMT-bearing ALSV-A

viruses . Interestingly , the tumor incidence in

these mice was not increased , but 6 of 16 p53

null mice that had developed HCCs , showed

lung metastases ( in contrast with 1 of 17 p53

wild-type mice ) . Consequently , this mouse

model might be of value as a metastatic HCC

model . Moreover , this model can be easily

used to study the effect of other oncogenes in

hepatocarcinogenesis , through the delivery of

other oncogene-bearing ALSV-A viruses to

TVA transgenic mice .

In addition , Lou et al created mice with a

regulated expression of liver-specific SV40 T-

Ag . The SV40 T-Ag in these mice is preceded

by a stop signal flanked by loxP sites . Hence ,

the SV40 T-Ag is expressed upon Cre mediated

excision , either by adenoviral expression of

Cre recombinase or by administration of

tamoxifen to mice that are transgenic for a

liver-specific tamoxifen-inducible Cre . HCCs

were observed in mice 5 mo after

administration of adenoviral Cre recombinase

or tamoxifen . Several research groups

employed alternative recombi-nase-mediated

conditional gene-mutation strategies.

Colnot et al generated a mouse strain in which

exon 14 of both Apc ( adenomatous polyposis

coli ) alleles were flanked by loxP sites . The

Apc alleles become invalidated ( leading to β-

catenin signaling) upon liver-targeted

expression of Cre recombinase . Of these mice ,

67% develop HCCs 8-9 mo after Cre

recombinase administration .

Promising results have been published with

these and other conditional mouse models to

induce HCC-formation . Nonetheless , to date ,

these models are mainly used to study the

effect of genetic alterations ( mutation ,

Deletion , or overexpression of a certain gene )

on hepatocarcinogenesis and not to induce

HCCs .

Breast Cancer

1. Introduction :

Metastasis is the main cause of death in women

with breast cancer . Development of clinical

trials for tumor regression and metastasis

prevention and the elucidation of their

underlying molecular mechanisms help to

reduce the death rates of cancer patients .

Despite the accumulating knowledge of the

underlying mechanisms of metastasis and its

clinical application to breast cancer treatment ,

many patients die from relapse after the

removal of the primary tumors because of

metastasis of cancer throughout the body .

Therefore , many efforts have been made to

develop therapeutic drugs that prevent tumor

invasion and to identify diagnostic markers to

classify each stage of cancer and metastasis for

early diagnosis . For decades , an expanding

database of differentially regulated factors in

cancer system has helped researchers to

investigate how oncogenes and tumor

suppressor genes function together and to

understand their functional interactions with-in

the tumor environment . The roles of

oncogenes and tumor suppressor genes have

been validated from experimental animal

models carrying deletions or mutations of

genes initially identified in patient tissue

samples . A wealth of data generated from

animal models has provided in-sights into the

biological functions of genes and signaling

path-ways involved in cancer and has allowed

for the generation of an advanced concept of

metastasis . Especially , mouse model study

allow us to investigate the orchestrated

mechanisms of tumor incidence and metastasis

and to develop advanced mouse models

representing diverse aspects of human cancer .

In fact , although mouse model system is not

exactly matched to human in genetic

backgrounds (i.e., protein composition,

different genetic variants , and genetic

mutations ) , the growth of tumors and

metastasis in xenograft models and genetically

engineered mice mimicking human cancer

progression allow us , to some extent , to

understand the relationship between tumor

genesis or metastasis and single gene effect in

each cancer step .

2. Models for tumor transplantation :

Intravenous , intraperitoneal , subcutaneous or

orthotopic injection of human cancer cells into

mice is termed xenograft transplantation , and it

is a well-defined method to monitor tumor and

metastasis processes and to manipulate specific

genes related to human cancers . In an immune

compromised mouse , injected human breast

cancer cells form a tumor mass and metastasize

into other organs , as observed in cancer

patients . There are various human breast

cancer cell lines used for cancer research

(Table 1).

Unlike primary tumor growth , metastatic

properties are usually validated by colonization

of tumor cells in a secondary organ . Invasion

of tumor cells into the blood or lymphatic

vessels is a critical feature indicating the

metastatic ability of tumor cells . For this

reason , intravenous or cardiac injection of

breast cancer cells in which the target genes are

manipulated , the function of these genes in

promoting or suppressing metastasis is

monitored by comparing the number of

metastatic nodules per lung to those observed

in the control mouse . In addition to the

autonomous alteration of tumor cells , the host

system has been suggested to cooperate with

tumor cells in metastasis . Injection of breast

cancer cells mixed with bone marrow-derived

mesenchymal stem cells exhibit higher

metastatic phenotypes and allow us to examine

the importance of paracrine factors and the

tumor microenvironment . Chemokines, which

induce the migration

of leukocytes , and their receptors form

networks between the tumor and infiltrated

immune cells and influence the primary tumor

and its metastasis . Lung colony formation was

significantly decreased by treatment with a

neutralizing anti-CXCR4 antibody and

intravenously injected breast cancer cells .

Since metastasis is a multi-step process and

loss of interaction of cancer cells with

environment is important , spontaneous

metastasis from primary tumor is more

adequate to mimic human breast cancer .

Orthotopic injection is the method by which

transplanted tumor cells develop spontaneous

metastasis through primary tumor formation .

Therefore , combining different type of

injection method give rise to insight about

which step is affected by target gene

manipulation . For example, MDA-MB-231

cells including alteration of membrane-type

matrix metallo protease (MT4-MMP) does not

affect extravasation using tail vein injection but

increases intravasation step using mammary fat

pad injection . In addition, in orthotopic

injection , mastectomy of primary tumor after

mammary fat pad injection makes researchers

focus on metastatic function of genes without

side effects of primary tumor development.

Inhibition of angiopoietin-like 4 (ANGPTL4)

by perturbed TGFb signaling in tumor cells

does not change primary tumor

growth and intravasation . However , after

mastectomy of primary tumor , lung metastasis

is markedly decreased . Many cancer

cell lines can produce spontaneous metastasis

using Orthotopic injection to study entire steps

of breast cancer metastasis (Table 1) .

Furthermore , to identify new therapeutic

markers for the poor prognosis of organ

specific invasion and the efficacy of drugs , the

mechanisms by which metastatic tumor cells

choose other organs to invade have been

intensively studied . If we understand the

selection process that transformed tumor cells

use to differentially identify each target organ ,

we will be able to verify the biological

differences in each tumor cell originating from

a specific organ and the key molecules that

regulate this event . This idea has been

supported by recent researches reporting that

metastasis occurs in unusual target organ

depending presence of some of genes .

Recently, anion vivo selection method has been

proposed to dissect tumor cells and to elucidate

their organ-targeting abilities . MDA-MB-231

cells were injected into the tail veins of

immune-deficient mice , and the metastatic

cells isolated from a lung lesion were then

expanded in a culture and used again for

inoculation . When another round of

inoculation was performed, these cells showed

increased lung metastatic activity . Gene

expression profiles of these cells indicated a

specific pattern compared to those of parental

cells or intermediate populations, and revealed

key genes that determined the fate of breast

tumor cells to colonize the lung .

Interestingly , there are distinctive differences

in the gene expression profiles of populations

obtained from different target organs such as

lung , bone , and brain . This in vivo selection

experiment will allow us to identify more

efficient and specific drugs to treat metastatic

breast cancer patients .

As current research focuses on the

communication between the tumor cells and

environmental signals , the use of tumor

transplantation studies are limited to some

extent . Firstly , human breast cancer cell lines

are not a real tumor in vivo but are just cultured

Cells . Established tumor cell lines could not

represent spontaneous tumors thoroughly

because they have no more interaction with

host systems , which is a feature of tumor cells

, and are grown artificially in an aberrant

immune system . The immune-compromised

mouse , preventing immune rejection response ,

is radically different from the human system

because the immune response promotes the

primary growth of tumor cells and guides them

to secondary organs . Secondly, differences in

the genetic back-ground should be carefully

considered in order to prevent unexpected

outcomes . Although numerous targets for

cancer therapy have been elucidated using mice

model study , there are still un-solved issues

about unknown side effects . And also , there

are considerable technical issues because the

region of metastasis changes according to

certain kinds of tumor cell lines or methods of

tumor cell inoculation . These differences

might explain why transplantation models have

failed to recapitulate the complexity of the

whole cancer process .

3. Genetically engineered mouse

models :

Genetically engineered mice (GEM) for cancer

study use techniques for the genomic deletion

of tumor suppressor genes or the transgenic

insertion of oncogenes (Table 2). For breast

cancer research , a mammary gland-specific

promoter is often used to restrict the expression

of oncogenes in specific breast regions .

Transgenic mice expressing oncogenes ( PyMT

, ErbB2 , Wnt1 , or Ras ) under the control of

the MMTV ( mouse mammary tumor virus ) or

WAP (Whey Acidic Protein) promoter initiate

tumors in the mammary gland, leading to

metastasis to other organs during the latter

stages of cancer . These mice have been a

relevant tool to investigate the spontaneous

initiation of breast tumors and to follow each

step of metastasis progression. For example ,

MMTV-PyMT transgenic mice exhibit tumor

formation restricted to the mammary gland

with a short latency and pulmonary metastasis

at a high frequency . This mouse model

recapitulates human cancer stages , including

hyperplasia , adenoma , and early / late

Carcinoma . Furthermore , mammary epithelial

cell-specific deletion of tumor suppressor genes

such as Trp53 , Brca1 by conditional knock-out

strategies produces spontaneous tumors and

shows bone metastasis , loss of estrogen

receptor (ER) expression , and hormone

responsiveness as observed frequently in

human cancer patient .

Furthermore, crossing transgenic mice with

other GEM is useful to investigate the roles of

tumor-related genes and their roles in

tumorgenesis and metastasis . Delayed tumor

progression in MMTV-PyMT or MMTV-

ErbB2/neu mice crossed into an Akt or PTP1B-

deleted genetic background revealed the

importance of sig-naling pathway in breast

cancer metastasis . In contrast, the MMTV-

PyMT mouse in a background showed

increased metastatic invasion , because CD44 ,

a cell-adhesion receptor , in-creases epithelial–

stromal interactions that are involved in the

suppression of metastasis . Some reports

describing the deletion of secreted factors shed

light on the importance of the tumor

environment . Tumor-associated immune cells ,

especially macro-phages , represent a large

portion of the tumor mass and are associated

with a poor prognosis . These immune cells

release growth factors , cytokines , Chemokines

, and enzymes that promote tumor growth ,

angiogenesis , and metastasis . When CSF-1

(colony stimulating factor-1) is deleted in the

genome of an oncogenic mouse , these mice

show a delayed development of metastatic

carcinomas without affecting the progression of

the primary tumor . The selective loss of

� �CD4+T cells in PyMT/RAG1 / or IL-4 in

PyMT/IL- � �4 / mice significantly attenuates

Pulmonary metastasis through repression of IL

4 mediated epidermal growth factor receptor

(EGFR) signaling in mammary tumors . These

results suggest that innate and adaptive immune

responses coop-erate to sustain metastasis in

mammary tumors . Given that a tumor is an

acquired disease in specific cell types , the

appropriate expression or repression of certain

genes both temporally and spatially is essential

in mouse models for the successful

recapitulation of human cancer . Under the

control of the MMTV promoter , the Cre/loxP

recombinase mediated somatic deletion of

Trp53 or other tumor suppressor genes in the

mam-mary gland allows tumor progression in

specific tissues without embryonic lethality .

Furthermore , the use of the inducible Cre

system with the reverse tetracycline

transactivator (rtTA) regulates the expression

of target genes in a time-dependent manner. In

this system , the tetracycline-mediated gene

deletion has no effect on embryonic

development , and the effects of the deleted

gene only arise during cancer development . In

a tumor model , the deletion of GATA3

(MMTV-PyMT;Wap-rtTA-Cre;GATA3 fl/fl)

attenuated tumor burden by alteration in tumor

differentiation and dissemination . An

advanced inducible system is useful for the

study of the cooperation between oncogenes .

Bi-transgenic mice with MMTV-rtTA and

tetO-myc develop invasive mammary

adenocarcinoma only in the presence of

tetracycline. After tumor formation , repression

of myc expression by the withdrawal of

tetracycline resulted in almost complete

regression of the tumor , but half of tumor cells

resumed growth . These results indicate that

inducible mouse models have made enabled to

conduct experiments using multiple genetic

mutations of oncogenes involved in the

initiation and maintenance of tumors . However

, most tumors from GEM fail to fully

recapitulate hu-man breast cancer. There are

several reasons for this observation .

First, different results can be obtained from

GEM depending on the method used to

generate the model; these have not made the

interpretation of data generated from GEM

straightforward .

For example , tumor latency and metastatic

ability are different according to the type of cre

mouse strain used. The percentage of

ER/progesterone receptor (PR)-positive tumors

are high in Wap-Cre/Trp53 � �/

mice but are low in MMTV- � �Cre/Trp53 /

mice .

Second , although the molecular profiles of

mammary tumors from GEM show similar

patterns compared to subtypes of human breast

cancers (Luminal A, Luminal B, ERBB+,

Basal-like, and Normal breast-like), but not a

single mouse model shows all the expression

patterns and characteristics of human cancer .

These limitations of GEM are much more

evident in drug development . As the majority

of GEM have hormone-independent

phenotypes , GEM cannot be directly applied

for the study of breast cancer patients with an

estrogen positive status . Furthermore ,

therapeutic compounds designed on the results

obtained with GEM could result in unexpected

outcomes due to the absence of homologous

target molecules in mice ( for example , IL-8.

Cytogenetic differences also introduce further

problems, i.e., BRCA1 and p53 are on the same

chromosome in mice , and thus, deletion of

both genes is common ; however , this does not

happen in humans .

4. Conclusion :

The study of mouse models has recapitulated

the behaviors and properties of human cancer

cells and revealed the complex interactions

between tumor cells and their environment by

which tumor progression and metastasis is

promoted and modulated. Indeed , the roles of

various tumor suppressor genes and oncogenes

were functionally studied with mouse models ,

and this has enabled to address new concepts

and the underlying disease mechanisms .

Although many genetically modified mice have

been generated , the limitations of mouse

models including the differences in molecular

components between species , differential gene

expression profiles , and genetic backgrounds

make it difficult to apply the knowledge gained

from these models directly to patient treatment

and drug development . The accumulating

knowledge from the study of mouse models ,

however , has allowed us to overcome

hurdles and to make progress in a wide range of

technical methods .

Humanized mice projects have been planned

with an aim to generate partial human immune

systems in mice , and in the near future , mice

possessing intact populations of human cells

might be produced . Finally , advanced mouse

models will provide more powerful methods to

elucidate the underlying mechanisms of tumor

progression and metastasis and to introduce

drugs targeted to individual cancer patients .

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