Conditional knockout mouse models of cancer.

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Topic Introduction

Conditional Knockout Mouse Models of Cancer Chu-Xia Deng1 Genetics of Development and Disease Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

In 2007, three scientists, Drs. Mario R. Capecchi, Martin J. Evans, and Oliver Smithies, received the Nobel Prize in Physiology or Medicine for their contributions of introducing specific gene modifications into mice. This technology, commonly referred to as gene targeting or knockout, has proven to be a powerful means for precisely manipulating the mammalian genome and has generated great impacts on virtually all phases of mammalian biology and basic biomedical research. Of note, germline mutations of many genes, especially tumor suppressors, often result in lethality during embryonic development or at developmental stages before tumor formation. This obstacle has been effectively overcome by the use of conditional knockout technology in conjunction with Cre-LoxP- or FlpFrt-mediated temporal and/or spatial systems to generate genetic switches for precise DNA recombination. Currently, numerous conditional knockout mouse models have been successfully generated and applied in studying tumor initiation, progression, and metastasis. This review summarizes some conditional mutant mouse models that are widely used in cancer research and our understanding of the possible mechanisms underlying tumorigenesis.

INTRODUCTION

Tumorigenesis is a multistep process consisting of tumor initiation, progression, and metastasis with the involvement of numerous environmental and intrinsic factors. It is general knowledge that the activation of oncogenes and inactivation of tumor-suppressor genes promote tumor formation. However, cancers are usually extremely heterogeneous on genomic and pathological levels (Cancer Genome Atlas Network 2012), which greatly limits our understanding of the role of these factors during tumorigenesis thus casting difficulties in clinical management and therapeutic treatment. Gene targeting, also called gene knockout, has widely been used to generate genetically engineered mice carrying mutations of a single gene or a combination of a number of genes for studying their functions in vivo (Capecchi 2005; Deng 2007). Of note, germline mutations created by the conventional knockout approach frequently result in embryonic or early postnatal lethality if the mutated genes are essential for mammal development (see Introduction: Analyses of Tumor-Suppressor Genes in Germline Mouse Models of Cancer [Wang and Abate-Shen 2014]). In addition, tumorigenesis in mutant mice with a whole-body gene knockout may not faithfully mimic human cancer, where the initiation of tumors is triggered by somatic mutations and the growth of tumor cells occurs in a wild-type or heterozygous environment. To overcome these difficulties, conditional knockout using the Cre-LoxP and Flp-Frt recombinase systems, mostly Cre-LoxP system, have been used successfully to generate conditional knockout mice in a spatial and temporal fashion (Le and Sauer 2000; Mortensen 2007; Nagy et al. 2009; Wang 2009; Bailey et al. 2009; Deng 2011). These animal models have provided key 1

Correspondence: [email protected]

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information regarding mechanisms underlying cancer initiation and progression, cancer cell origin, signaling pathways, and metastasis. This review will discuss several of these conditional knockout mouse models with an emphasis on a few commonly used models, such as Apc, Brca1, Brca2, p53, Pten, Rb, and Smad4, regarding their applications in cancer research and lessons learned from them. In addition, it will also summarize tumor phenotypes of many other conditional knockout stains reported in the literature. CONDITIONAL KNOCKOUT MOUSE MODELS FOR FAMILIAL BREAST CANCERS

Breast cancer is the most common cancer in women worldwide with 1,500,000 cases and 450,000 deaths each year. The majority of breast cancers are caused by sporadic genetic alterations without family history of cancer (sporadic cancers), whereas the remaining cases are inherited, which may be attributed to mutations of some tumor-suppressor genes, such as BRCA1, BRCA2, and p53 (Nathanson and Weber 2001; Oldenburg et al. 2007). BRCA1 and BRCA2 mutation carriers are estimated to have a 50%–80% and 40%–50% increased risk of developing breast cancer, respectively, by the age of 70 (Easton 1997; Chen et al. 2006). Germline mutations of p53 are found in patients with Li–Fraumeni syndrome (LFS), who develop breast cancer at a relatively early age (Li and Fraumeni 1982; Pearson et al. 1982). Besides breast cancer, LFS patients also suffer from many other types of cancers, including acute leukemia, brain tumors, and soft-tissue sarcomas (Kleihues et al. 1997). BRCA1 Conditional Mutant Mouse Models

The human BRCA1 gene contains 24 exons and encodes a multiple functional domain protein of 1863 amino acids that interacts with many important proteins (Miki et al. 1994; Deng and Brodie 2000) (Fig. 1). Alternative splicing in BRCA1 generates at least two more proteins of smaller size: BRCA1Δ11, designated BRCA1Δ672-4095 (Thakur et al. 1997), and BRCA1-IRIS, which contains 1399 amino acids encoded by an uninterrupted open-reading frame that extends from codon 1 to a termination point 34 triplets into intron 11 of BRCA1 (ElShamy and Livingston 2004). The mouse Brca1 gene shares similar genomic structure with its human counterpart and encodes a protein of 1812 amino acids (Lane et al. 1995). To study functions of Brca1 in mammary development and tumorigenesis, several distinct mutations were introduced into the Brca1 by gene targeting. These analyses have shown that Brca1 null mutation results in early postimplantation lethality, whereas a severe hypomorphic mutation of Brca1 (Brca1 Δ11/Δ11), which only deletes exon 11 and therefore disrupts only the full-length isoform of Brca1 while leaving the short form intact, causes embryonic lethality at embryonic day 12–18 (Gowen et al. 1996; Hakem et al. 1996; Liu et al. 1996; Ludwig et al. 1997; Shen E3 Ubiquitin Ligase 1314 –1863

RING BARD1

304 – 394

RB

1–109

NES E2F1 1–76

BAP1 1–100

81–99

758 – 1064

PALB2 RAD51 BRCA2 NLS 503 – 508 Coiled-coil

Phosphoprotein binding BRCT

607 – 614

cMyc

p53

175–303 433–511

224 – 500

RAD50

P P P

P

ATM CK-2 CHK-1 CDK-2

RHA 1560 –1863

341 – 748

RNA Pol II p300 CBP

RAP80 CCDC98 BACH1 CtIP 1651 –1863 p53

RbAp46 RbAp48 HDAC1 HDAC2

1760 –1863

1536 – 1863

FIGURE 1. Functional domains of BRCA1 and its interacting sites with other proteins. NES, Nuclear export sequence; NLS, nuclear localization sequence, BRCT, BRCA1 carboxyl terminus. (Modified from Deng and Brodie 2000, with permission from John Wiley and Sons.)

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Conditional Knockout Mouse Models of Cancer

et al. 1998; Xu et al. 1999; Hohenstein et al. 2001). Although these Brca1 mutant embryos die at different stages of embryonic development, they all display developmental delay, cellular proliferation defects, widespread apoptosis, and chromosome abnormalities, highlighting an essential role of Brca1 in development and genome integrity (Deng 2002, 2006; Dine and Deng 2013). However, unlike human carriers of the BRCA1 mutation, mice heterozygous for the targeted mutations do not show obviously increased tumor formation. Thus, these early studies failed to generate mouse models for BRCA1-associated cancer. To overcome embryonic lethality, scientists used the Cre-LoxP system to generate conditional knockout Brca1 mutant mice. Cre is a 38-kDa site-specific DNA recombinase that recognizes its target site, loxP, and catalyzes recombination between two loxP sites (Sauer 1998; Deng 2012). Currently five Brca1 conditional mutant alleles that carry Cre-LoxP-mediated deletion of different exons of the Brca1 gene have been generated (Fig. 2). The first Brca1 conditional allele was generated by placing loxP sites in introns 10 and 12, respectively (Brca1 Flox11). Mammary-specific deletion of Brca1 exon 11 is achieved by crossing with mice that carry the Cre transgene under the control of the Whey acidic protein (WAP) gene promoter or the mouse mammary tumor virus (MMTV) long terminal repeats (Wagner et al. 1997). The resulting mutant (Brca1 Flox11/Flox11;Cre) mice were morphologically normal as the deletion of Brca1 primarily occurs in the mammary glands. The Brca1 Flox11/Flox11;Cre mice suffered aberrant mammary gland development with cellular proliferation defects, apoptosis, and genetic instability, which are similar to the defects observed in the Brca1 Δ11/Δ11 embryos. The mutant mice were initially tumor-free; however, after a long latency, 25% of female mutant animals developed mammary tumors with a medium time of 15 mo (Xu et al. 1999). Analysis of these tumors revealed massive chromosomal numeric and structural abnormalities including whole or partial gain of chromosome 15 centering on 15D2–D3 (orthologous to human chromosome 8q24), the map location of the c-Myc gene, loss of whole or partial chromosome 14, including 14D3, the map location of Rb1, and inactivation of p53, which is similar to human BRCA1-associated breast cancers (Weaver et al. 2002). Importantly, heterozygous deletion of p53 in Brca1 mutant tumors markedly accelerated tumorigenesis (Xu et al. 1999). Several additional Brca1 conditional null alleles were subsequently generated and knocked out using the Cre transgene driven by different promoters, including the β-lactoglobulin promoter (BLG) and keratinocyte 14 (K14) promoter (Fig. 2 and Table 1). These studies also revealed that the long latency of tumorigenesis in Brca1 mutant mice

A Xu et al. 1999 LoxP

LoxP Cre

B Poole et al. 2006 56

C

Liu et al. 2007 5

D McCarthy et al. 2007

13

22 24

E Shakya et al. 2008 2

FIGURE 2. Brca1 conditional mutant alleles generated by Cre-LoxP system. (A) Structure of a Brca1 conditional mutant allele showing exon 11 is flanked by loxP sites (Brca1Flox11). (B–E) Four additional Brca1 conditional alleles published in literature. Numbers represent exons that are flanked by loxP sites (black triangles in introns). Expression of Cre induces recombination between loxP sites leading to the deletion of sequence between the loxP sites. Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top074393

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1220 TABLE 1. Conditional knockout mouse models of breast cancer Conditional knockout alleles Flox14

Recombinase K14-Cre, WAP-Cre

APC Flox14

p53−/−

BLG-Cre

Brca1 Flox11

p53+/−, p53 Flox 5–6

MMTV-CreWAP-Cre, MMTV-CreC, WAP-CreC

Brca1 Flox5–13 Brca1 Flox22–24 Brca1 Flox2 Brca2 Flox11

p53Flox 2–10 p53+/−

K14-Cre BLG-Cre WAP-Cre K14-Cre

p53Flox 2–10

Brca2 Flox3–4

WAP-Cre

Brca2 Flox9–10

WAP-Cre

Chk1 Flox2

p53+/−

MMTV-Cre, WAP-Cre


Lfng Flox2

MMTV-Cre

Mnt Flox4–6

MMTV-Cre

p53 Flox2–10

K14

p53 Flox5–6

WAP-CreC, MMTV-Cre

Pten Flox5

MMTV-Cre

Pten Flox5

ErbB2

MMTV-NIC

Rb Flox3

p107−/−p53Flox

MMTV-Cre

Rev3L Flox two exons encoding

p53+/−

MMTV-Cre

amino acids 2776 to 2860

Smad4 Flox8

Tgfbr2 Flox1

MMTV-Cre, WAP-Cre

MMTVpolyomavirus middle T antigen (PyVmT)

MMTV-Cre

Phenotypes

Reference(s) Flox/+

About 76% Apc ;K14-Cre mice developed mammary tumors within 15 mo, whereas Apc Flox/+;WAP-Cre or Apc Flox/Flox;WAP-Cre mice were tumor-free. Apc Flox/Flox;BLG-Cre mice developed metaplasia but not neoplasia. In the absence of p53, 44.4% of lymphoma-free mice developed mammary tumor. Brca1 mutant mice develop mammary tumor after a long latency. Many tumors contained spontaneous p53 mutation. Loss of p53 markedly accelerates tumor formation. All mice develop tumors before 10 mo of age. 64% of mice developed tumors before 46 wk of age. 95% of mice developed tumors before 22 mo of age. All Brca2 F/F;p53 F/F;K14-Cre mice developed tumors within 300 d; about half were mammary tumors. No mammary tumors were observed in Brca2F/F;K14-Cre mice. 77% of mutant mice developed mammary tumors with a median tumor-free survival of 1.4 yr. The majority of mutant mice developed mammary tumors with a median tumor-free survival of 1.6 yr. Heterozygous or homozygous loss of Chk1 impairs mammary gland development. p53+/− mutation synergistically induces mammary tumor formation with a median time of 10 mo. 80% of mutant mice developed basal-like and claudin-low tumors at 15 mo of age with activation of Notch signaling, amplification of the Met/Caveolin locus, and elevated Met and Igf-1R signaling. 2/3 of mutant mice developed mammary tumor at 24 mo of age. Tumorigenesis is analogous to that caused by Myc deregulation. p53 F/F;K14-Cre female mice developed mammary tumors (20/32) and skin tumors (10/ 32) with a median latency (T50) of 288 d. All mutant mice developed ERa-positive or -negative tumors between 1 and 2 yr with high frequency of metastasis accompanied by genetic alterations commonly observed in human breast cancer. Mutant mammary epithelial cells were hyperproliferative with severely reduced apoptosis. More than 70% of mutant females developed mammary tumors within 400 d. All mutant females developed multifocal and highly metastatic mammary tumors within 120 d that was associated with an increase in angiogenesis. Rb deletion in mouse mammary progenitors collaborating with oncogenic events, such as p107 or p53 deficiency, induces luminal-B or basal-like/EMT tumors. The majority of mutant mice developed mammary tumors in p53+/+ and p53+/− backgrounds with a median time of 295 and 448 d, respectively. Squamous cell carcinoma and/or mammary abscesses occur between 5 and 16 mo of age accompanied by β-catenin accumulation at onset and throughout the process of transdifferentiation. Loss of Tgfbr2 alone in the mammary epithelium results in lobular–alveolar hyperplasia and increased apoptosis. Loss of Tgfbr2 in the context of PyVmT results in a shortened median tumor latency and an increased formation of pulmonary metastases.

Kuraguchi et al. 2009 Meniel et al. 2005 Xu et al. 1999; Poole et al. 2006 Liu et al. 2007 McCarthy et al. 2007 Shakya et al. 2008 Jonkers et al. 2001 Ludwig et al. 2001 Cheung et al. 2004 Fishler et al. 2010

Xu et al. 2012

Toyo-oka et al. 2006 Liu et al. 2007 Lin et al. 2004

Li et al. 2002

Schade et al. 2009 Jiang et al. 2010 Wittschieben et al. 2010 Li et al. 2003

Forrester et al. 2005


APC

Collaborating mutant alleles



is accelerated by p53 deficiency (Table 1). Altogether these data support the theory that Brca1 deficiency does not directly cause tumor formation, but instead triggers genetic instability, which eventually results in tumorigenesis after progressively acquiring other permissive alterations, including inactivation of p53 and activation of oncogenes (Deng 2001). Human breast cancers are divided into six subtypes based on their gene-expression patterns, including luminal A, luminal B, claudin-low, basal-like, HER2-overexpressing, and normal-breastlike subtypes (Cianfrocca and Gradishar 2009; Visvader 2009). The majority of breast cancers caused by mutations in BRCA1 belong to the basal-like subtype (Rastelli et al. 2010; Nanda 2011), and >60% of them do not express detectable levels of estrogen receptor, progesterone receptor, and HER2 (triplenegative breast cancers, TNBCs). TNBC is the most aggressive type of breast cancer, characterized by advanced histological stage and nuclear grades, as well as high propensity for metastasis and poor prognosis. Because of the basal nature of BRCA1 breast cancers, it was proposed that loss of BRCA1 function in basal stem cells might be the cause for tumorigenesis with a concomitant block to luminal differentiation (Foulkes 2004). Consistently, mice carrying targeted deletion of Brca1 in mammary stem cells using a K14-Cre allele, which is expressed in the entire basal layer, generated cancers with increased expression of basal epithelial markers, reminiscent of human basal-like breast cancer (Liu et al. 2007). However, a study performed in humans detected increased luminal progenitor cells in breast tissue of BRCA1 mutation carriers as well as a correlation between the gene-expression profile of these cells and basal-like breast cancers, implicating aberrant luminal progenitors as the candidate target population for BRCA1-associated basal-like cancers (Lim et al. 2009). Of note, deletion of murine Brca1 by the BLG, which expressed in luminal epithelial cells also results in a basal phenotype (Molyneux et al. 2010). After a side-by-side comparison of gene-expression profiles, the researchers concluded that Brca1 Flox/Flox;p53 +/–;BLG-Cre mice develop mammary tumors that are resemble human BRCA1-deficient breast cancers whereas Brca1 Flox/Flox;p53 +/–;K14-Cre mice do not (Molyneux et al. 2010). Thus, it is conceivable that BRCA1-associated basal-like cancer actually originates from luminal epithelial cells, which undergo transdifferentiation during tumor progression, although the underlying mechanism remains elusive. Brca1 has also been disrupted in a number of other tissues (Table 2) and some Brca1 conditional mutant models have been used for cancer prevention and therapeutic treatment (Dine and Deng 2013). Brca2 Conditional Mutant Mouse Models

The human BRCA2 gene was cloned in 1995 (Wooster et al. 1995). Women carrying BRCA2 mutations have a significantly increased risk for developing breast cancer (Chen et al. 2006). Complete knockout of Brca2 in mice results in embryonic lethality before E8.5, whereas heterozygotes do not develop mammary tumors (Hakem et al. 1997; Sharan et al. 1997; Patel et al. 1998; Jonkers et al. 2001). So far at least three conditional mutant mice that delete exons 3–4 (Ludwig et al. 2001), 9–10 (Cheung et al. 2004), and 11 (Jonkers et al. 2001) of Brca2, respectively, have been developed to study its function in tumorigenesis. Brca2 Flox11/Flox11;K14-Cre mice showed normal mammary gland development, but showed no sign of mammary tumorigenesis until p53 was also mutated, leading to mammary tumor formation in 50% in the double-mutant mice (Jonkers et al. 2001). In contrast, although disruption of Brca2 in mammary luminal epithelium by WAP-Cre did not affect mammary gland development, it resulted in mammary tumor formation. Majority of Brca2 Flox3–4/Flox3–4;WAPCre and Brca2 Flox9–10/Flox9–10;WAP-Cre mice developed mammary tumors with the median tumor-free survival of 1.4 years (Ludwig et al. 2001) and 1.6 years (Cheung et al. 2004), respectively. The karyotypes of tumor cells had various chromosomal aberrations and ranged from diploid to hypertetraploid. Most tumors analyzed showed abnormal p53 protein expression and tumor incidence markedly increased in p53 +/− mice. Brca2 mutant tumors are histologically uniform luminal-type adenocarcinomas, showing predominantly a solid nodular tumor pattern. These features are in striking contrast to Brca1-associated mice, which are deficient in mammary gland development and develop basal-type tumors with morphological heterogeneity (Xu et al. 1999). Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top074393

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1222 TABLE 2. Conditional mutant mouse models for other organs/tissues besides mammary gland Tissues (Recombinases)

Phenotypes

Reference(s)

β-Catenin

Liver (Alb-Cre)

Brca1

Epithelial tissues (K5-Cre), ovarian granulosa cells (Fshr-Cre)

Brca1/p53

Ovarian surface epithelial cells (Adeno-Cre) Epithelial tissues (K14-Cre), thymus (Lck-Cre), ovary (Adeno-Cre)

Mutant mice developed malignant epithelial cancer, squamous cell carcinoma within 12–18 mo of age. Defective T-lymphocyte maturation, resulting in lymphatic diseases with 50% penetrance, including atypical hyperplasia and thymic lymphoma. Inactivation of Apc leads to the accumulation of nuclear β-catenin and the rapid development of renal carcinoma at 4 mo, which is accelerated by p53 deficiency, reducing the earliest onset to 2 mo. Depletion of β-catenin from mature hepatocytes promotes expansion of hepatic progenitor cells and liver tumors arose spontaneously from βcatenin-positive cells in 60% (7/12) mice at 20 mo of age. Tumor formation in the skin, the inner ear canal, and the oral epithelium after 1 yr of age; cell-nonautonomous induction of ovarian and uterine serous cystadenomas. Cre-mediated deletion of both Brca1 and p53 resulted in ovarian or reproductive tract tumor formation in 54% of mice after 17 mo. Brca2 and p53 deficiency collaboratively induced thymic lymphoma, hemangiosarcoma, osteosarcoma, fibrosarcoma, lung carcinoma, and ovary cancer with varying frequencies. The uteri of Cdh1 and p53 double-mutant mice showed histologic features of endometrial carcinomas with myometrial invasion. Fatal gastric cancer developed at 100% penetrance in double-mutant mice within a year, which frequently metastasize to lymph nodes. 2/3 of the mutant mice spontaneously developed hepatocellular carcinomas within 1 yr. Osteochondromas with 100% penetrance after induction with doxycycline up to 10 wk of age. Fbxw7 loss results in thymic lymphoma correlated with accumulation of c-Myc. More than 80% of mutant mice developed insulinomas and prolactinomas in the pancreas, but not in the pituitary gland. Deletion of Mlh1 in thymic and naïve T cells leads to a limited incidence of lymphoblastic T-cell lymphomas. Development of adenomas and adenocarcinomas carrying somatic truncation mutations of the Apc gene. 60% of NF1 mutant and 100% of NF1 and p53 double-mutant mice developed malignant astrocytoma by 40 wk. Triple-mutant mouse developed tumors that closely resembled human smallcell lung carcinoma. Loss of p16 Ink4a/p19 Arf combined with activation of Kras or inactivation of tumor-suppressor genes induces tumor formation in multiple organs/tissues studied with high frequency.

McCarty et al. 2008

APC/p53 −/−

Eyelid and conjunctiva (mGFAPCre) Hemoploritic cells (Mx-Cre induced by PolyIPolyC) Kidney (Ah-Cre)

Alpha V integrin AML


Brca2/p53

Cdh1/p53

Uterus (Pgr-Cre), Stomach (Atp4bCre)

Dicer1

Liver (Alb-Cre)

Ext1

Chondrocytes (Col2-rtTA-Cre)

Fbxw7

T cells (Lck-Cre)

Men1 Mlh1

Pituitary gland and endocrine pancreas (Rip-Cre) T cells (Lck-Cre)

Msh2

Intestinal tract (Villin-Cre)

NF1/p53

Brain (GFAP-Cre)

p130/Rb/p53

Lung (Adenoviral-Cre)

p16/Kras G12D, p16 Ink4a/ p19 Arf/Kras G12D/p53/ BRaf(VE)

Pancreas (PDX-Cre), melanocyte (Tyr-Cre-ERT2), brain (injection of lentiviral GFAP-Cre or CMV-Cre), lung (adeno-Cre)

Putz et al. 2006 Sansom et al. 2005

Wang et al. 2011

Berton et al. 2003; Chodankar et al. 2005

Quinn et al. 2009 Jonkers et al. 2001; Park and Lee 2008; Szabova et al. 2012 Reardon et al. 2012; Shimada et al. 2012

Sekine et al. 2009 Jones et al. 2010 Onoyama et al. 2007 Biondi et al. 2004; Crabtree et al. 2003 Reiss et al. 2010 Kucherlapati et al. 2010 Zhu et al. 2005; Wang et al. 2009; Chen et al. 2012b Schaffer et al. 2010 Dankort et al. 2007; Monahan et al. 2010; Qiu et al. 2011


Conditional alleles/ collaborating alleles

p53/Brca1

Pancreas (PDX-Cre)

Pten

Pten

Keratinocytes (K5-Cre), pancreas (PDX-Cre), liver (Alb-Cre), prostate (Osr1-Cre, Psa-Cre, K5Cre/ERT, K8-Cre/ERT), T-cells (Lck-Cre) Lung ((tetO)7-Cre/SP-C-rtTA)

Prkar1a

Pituitary (rGHRHR-Cre)

Rb/p130

Retina (Nes-Cre)

Rb

Prostate (PB-Cre)

Rb/p53

Cerebellum (GFAP–Cre),

Rb/p53/Brca1/Brca2

Ovary (Adeno-Cre)

Smad4

Skin (MMTV-Cre), T-cell (Lck-Cre), head-and-neck (K14- Cre.PR1 induced by treatment of RU486), odontoblats (K5-Cre) Pancreas (PDX-Cre), liver (Alb-Cre), prostate (Pb-Cre)

Smad4/Pten

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SOCS3

Gastrointestinal epithelial cells (T3b-Cre), liver (Alb-Cre)

Tgfbr2/Kras G12D/APC

Pancreas (PDX-Cre), Colon (FabplCre), Intestine (Villin-Cre), Fibroblasts (FSP-Cre)

VHL

Liver (Alb-Cre, or injection of Adenovirus-Cre), multiple tissues (Actin-Cre)

To overcome this limitation, we inserted FRT (flippase recognition target) sites flanking exons 2–6 of the endogenous p53 gene in mice to generate a p53FRT allele that can be deleted by Flp recombinase. We show that FlpOmediated deletion of p53 in mouse embryonic fibroblasts impairs the p53dependent response to genotoxic stress in vitro. In addition, using FSFKrasG12D/+; p53FRT/FRT mice, we show that an adenovirus expressing FlpO recombinase can initiate primary lung cancers and sarcomas in mice. Reduced latency of a p53 conditional knockout triggered pancreatic tumors from T50 of 68–40 d. Tumorigenesis occurs in many tissues/organs with varying frequency. Ptendeficient cells show many features including activation of Akt, enhanced tumor angiogenesis, increased cell proliferation, resistant to apoptosis, impaired differentiation, genetic instability, and/or abnormal metabolism.

Lee et al. 2012

Shakya et al. 2011 Horie et al. 2004; Korpershoek et al. 2009; Kwak et al. 2013; Lu et al. 2013; Stanger et al. 2005; Suzuki et al. 2003

All (14 of 14) mice who received doxycycline treatment at E10–16 and 87% (13 of 15) mice who received doxycycline treatment at P21–27 developed lung adenocarcinoma at 70 wk of age. Prkarla deficiency significantly increased pituitary tumorigenesis. 48% (10 of 21) mutant mice developed pituitary tumors compared with 18% (5 of 28) in the control mice at 18 mo of age. Mutant retina shows widespread apoptosis and loss of photoreceptors and bipolar cells but not tumor. All five Rb Flox/Flox;p130 −/−;Nes-Cre mice examined developed retinoblastoma. Loss of pRB-mediated cell cycle control directly caused the initiation of proliferative prostate disease but was insufficient to cause malignancy. Mutant mice deficient in Rb and p53 developed highly aggressive embryonal tumors of the cerebellum with typical features of medulloblastoma. Inactivation of RB-mediated tumor suppression induced surface epithelial proliferation with progression to stage I carcinoma. Additional biallelic inactivation and/or missense p53 mutation in the presence or absence of Brca1/2 caused progression to stage IV disease. Disruption of Smad4 alone in these organs/tissues is enough to induce cancer formation with varying frequency.

Yanagi et al. 2007

Loss of both Smad4 and Pten induces cancer formation in these organs accompanied by altered expression levels of genes that enhance tumorigenesis. All SOCS3 F/F;T3b-Cre mice developed tumors in the stomach by 2 mo of age, displaying features resembling human intestinal-type gastric tumors. Deletion of the SOCS3 gene in hepatocytes promotes the activation of STAT3, resistance to apoptosis, and an acceleration of proliferation, resulting in enhanced hepatitis-induced hepatocarcinogenesis. Loss of Tgfbr2 induces cancer formation in multiple organs, which is accelerated by oncogenic signaling such as Kras(G12D) or inactivation of tumor suppressors, such as APC. Loss of Tgfbr2 in the stroma of several organs using FSP-Cre also increases oncogenic potential of the adjacent epithelia. Mutant mice primarily developed hepatic vascular tumors. Angiectasis in multiple organs and impaired spermatogenesis were also observed.

Xu et al. 2006; Xu et al. 2010; Ding et al. 2011

Yin et al. 2008

MacPherson et al. 2004

Maddison et al. 2004 Marino et al. 2000 Szabova et al. 2012

Li et al. 2003; Yang et al. 2005; Qiao et al. 2006; Bornstein et al. 2009; Gao et al. 2009

Ogata et al. 2006; Inagaki-Ohara et al. 2012

Bhowmick et al. 2004; Biswas et al. 2004; Ijichi et al. 2006; Munoz et al. 2006

Haase et al. 2001; Ma et al. 2003


Lung (Adeno-FlpO)



p53/Kras G12D


C.-X. Deng

p53 LFS Mouse Models

p53 mutations are detected in >50% of all types of human cancers (May and May 1999; Donehower and Lozano 2009). The initial evidence for the involvement of p53 in breast cancer came from findings that germline p53 mutations cause LFS, which is associated with increased breast cancer risk (Li et al. 1991). Several point mutations in p53, including R175 and R273, are the most common hot spots for mutations found in p53-associated familial breast cancer cases and sporadic breast cancers (Lang et al. 2004; Olive et al. 2004; Wijnhoven et al. 2005; Jia et al. 2012). To study functions of p53 in LFS-associated breast cancer, mice carrying p53 R172H/+ and p53 R270H/+ mutations (corresponding to 175 and 273 in humans) were generated by using the Cre-LoxP approach. Expression of these mutant alleles in all tissues caused a broad variety of tumors (Lang et al. 2004; Olive et al. 2004). In addition, some new types of tumors were also observed in p53 R172H/– and p53 R270H/– mice in comparison with p53 −/− mice, including a variety of carcinomas and more frequent endothelial tumors. Thus, it was concluded that expression of these p53 mutant alleles under physiological conditions enhanced the oncogenic potential beyond the simple loss of p53 function, suggesting that these mutations have dominant-negative or gain-of-function properties (Lang et al. 2004; Olive et al. 2004). Because these mice do not develop mammary tumors, the Wap-Cre allele was used to express the p53 R270H allele specifically in the mammary gland. The data indicated that p53 R270H/−;Wap-Cre mice developed both spontaneous and carcinogen-induced mammary tumors at a high frequency, revealing a role for the R270H mutation of p53 in predisposing mammary tumor development in mice (Wijnhoven et al. 2005). p53 CONDITIONAL MUTANT MOUSE MODELS

Functions of p53 have also been extensively studied in mice with p53 disruption by gene targeting. p53deficient mice suffer a variety of tumors, mostly lymphoma but not mammary tumor, before they die at 6 mo of age (Donehower et al. 1992; Jacks et al. 1994). p53 +/− mice rarely develop mammary tumors unless they are in a BALB/c genetic background (Kuperwasser et al. 2000). These observations suggest that some factors or background modifiers may compensate for the loss of p53 in mammary tissues. To conduct tissue-specific disruption of p53, several conditional mutant alleles, including p53 Flox 2–10, Flox 3–4, Flox 5–6, and Flox 9–10, have been generated by using Cre-LoxP system (Tables 1 and 2). In the mammary gland, disruption of p53 in the basal layer of epithelium (and skin) by K14-Cre resulted in mammary tumors in 20 out of 32 and skin tumors in 10 out of 32 p53 Flox2–10/Flox2–10;K14-Cre female mice with a median latency (T50) of 288 d (Liu et al. 2007). Disruption of p53 in the luminal epithelium exclusively by Wap-Cre or both luminal and basal epithelia by MMTV-Cre resulted in ERα-positive or -negative tumors between 1 and 2 years with a high frequency of metastasis (Lin et al. 2004). Tumorigenesis is accompanied by genetic alterations commonly observed in human breast cancer including cMyc amplification and Her2/Neu/erbB2 activation. p53 conditional mice are also used as mouse models for many other types of tumors by crossing them with transgenic mice that express Cre in a variety of tissues, and in all cases, loss of p53 markedly accelerates tumorigenesis (Table 2). p53 is also mutated by using an Flp recombinase and its recognition target, Frt (Flp-Frt) system (Mortensen 2007). In this study, FRT sites were inserted into intron 1 and 6 to flank exons 2–6 of the p53 gene to generate a p53 FRT allele. After confirming efficient Flp-mediated deletion of p53 exons 2–6 in embryonic fibroblast cells, researchers infected p53 FRT/FRT;KrasG12D mice with adenovirus that carries Flp recombinase (Ad-FlpO). Data revealed extremity sarcomas and high-grade lung adenocarcinomas in the mutant mice as early as 8 wk after infection, which is much faster than tumorigenesis caused by activation of KrasG12D alone (Lee et al. 2012). RB CONDITIONAL MUTANT MOUSE MODELS

Retinoblastoma (RB) is the founding member of a gene family containing two other members, p107 and p130. Human RB is inactivated and results in development of RB and several other major cancers, 1224




including lung, breast, prostate, and bladder cancer (Murphree and Benedict 1984; Horowitz et al. 1990). RB interacts with the E2F family of transcription factors and controls the G1 to S phase transition of the cell cycle; therefore, RB deficiency can promote the development of cancer by deregulating the expression of many genes that are involved in the G1–S cell cycle checkpoint and DNA replication machinery (Korenjak and Brehm 2005). Because mice homozygous for Rb die at mid-gestation with defects in proliferation, neurogenesis, fetal liver erythropoiesis, and lens development (Clarke et al. 1992; Jacks et al. 1992; Vooijs and Berns 1999), it is necessary to use a conditional knockout of Rb to study its tumor-suppressor functions. Despite the fact that mutation of RB results in RB in early life of human patients, tissue-specific disruption of Rb in the mouse retina by the Cre driven a nestin promoter (Nes-Cre) induces widespread p53-dependent apoptosis, and loss of photoreceptors and bipolar cells but not RB even in a p53-mutant background. In contrast, when its family member p130 is also mutated, Rb mutation in the retina causes retinal dysplasia and RB (MacPherson et al. 2004). An earlier study also indicated that Rb and p107 double-homozygous mice developed RB at later embryonic stages (Robanus-Maandag et al. 1998). Thus, in addition to Rb deregulation, the Rb-related genes p107 and p130 are important in RB development. RB is frequently mutated in human sporadic breast tumors (Varley et al. 1989). To test whether Rb plays a role in mammary tumorigenesis, Rb was specifically disrupted in the mammary epithelium using the Cre-LoxP approach. The data indicate that inactivation of Rb in mouse mammary stem/ bipotent progenitor cells by MMTV-Cre caused acinar hyperplasia and squamous metaplasia, which is accelerated by p107 deficiency. Owing to a high expression level of p53 in those tumors, researchers subsequently deleted both Rb and p53 in the mammary epithelium. Because nearly all double-deficient mice (Rb Flox/Flox;p53 Flox/Flox;MMTV-Cre) developed lethal lymphomas within 2–6 mo, lineage marker (Lin)-negative mammary epithelial cells from these mice were purified and transplanted at 10,000 cells each into the mammary glands of 4- to 5-wk-old NOD/SCID mice to assess their ability of tumor formation. At 58–126 d after transplantation, 50% of recipient mice developed histologically uniform, aggressive, epithelial-to-mesenchymal transition (EMT)-type tumors. These results establish a causal role for Rb loss in mouse mammary tumor formation in cooperation with oncogenic events (Jiang et al. 2010). Rb was also conditionally mutated in a few other organs/tissues, such as prostate (Maddison et al. 2004) and cerebellum (Marino et al. 2000), but in most cases, Rb mutation alone is insufficient to cause malignancy unless other family members or p53 are also disrupted (Table 2). PTEN CONDITIONAL KNOCKOUT MODELS

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is most well known for its role as a lipid phosphatase that antagonizes PI3K (phosphatidylinositol-4,5-bisphosphate 3-kinase) activity. Mounting evidence indicates that PTEN is a powerful tumor suppressor. Somatic mutations of PTEN are frequently found in a variety of human cancers, whereas its germline mutations are also detected in two inherited hamartoma tumor syndromes: Cowden syndrome and Bannayan–Zonana syndrome (Hobert and Eng 2009; Hollander et al. 2011; Kurek et al. 2012). The absence of functional PTEN leads to constitutive activation of downstream components of the PI3 kinase pathway, including AKT/PKB, a survival factor that protects various cell types against apoptosis (Downward 1998). A recent study revealed that PI3K pathway is most widely mutated in cancers, highlighting an importance of PTEN in tumor suppression (Cancer Genome Atlas Network 2012). In mice, Pten homozygous mutant embryos die during gestation at E6.5–9.5, showing imbalanced overproliferation (Suzuki et al. 2008). About 50% of heterozygous mice died before 1 yr of age and many survivors developed neoplasia in multiple organ systems. Therefore, conditional knockout approaches have been performed to achieve tissue-specific knockout in a variety of organs/tissues, including the bladder, keratinocytes, liver, lung, mammary gland, pancreas, and prostate (Suzuki et al. 2003; Horie et al. 2004; Stanger et al. 2005; Xu et al. 2006, 2008; Korpershoek et al. 2009; Hill and Wu 2009, 2010; Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top074393

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Kwak et al. 2013; Lu et al. 2013). In each Pten-deficient organ or tissue examined, mutant cells always show inappropriate activation of Akt. Some other common features include enhanced tumor angiogenesis, increased cell proliferation and cell size, expansion of cancer stem cell population, resistance to apoptosis, impaired differentiation, genetic instability, and/or abnormal metabolism. In the mammary gland, disruption of Pten by the MMTV-Cre transgene caused precocious lobuloalveolar development and excessive ductal branching and delayed involution. Mutant mammary epithelial cells were hyperproliferative with severely reduced apoptosis, and >70% Pten Flox/Flox; MMTV-Cre females developed mammary tumors within 400 d of age (Li et al. 2002). When a Pten conditional allele was deleted by using an MMTV-NIC, which coexpresses activated ErbB2 and Cre from the same bicistronic transcript, all mutant females developed multifocal and highly metastatic mammary tumors within 120 d that was associated with an increase in angiogenesis (Schade et al. 2009). In the liver, disruption of Pten by Cre transgene driven by albumin promoter (Alb-Cre) resulted in hepatocyte carcinoma (HCC) starting at about 8 mo of age and affected 90% of mice when they were up to 20 mo of age (Horie et al. 2004). When a conditional mutant allele of Smad4 was introduced into Pten Flox/Flox;Alb-Cre mice to knockout both genes simultaneously, hyperplastic foci emerged exclusively from bile ducts at 2 mo of age and all mutant mice developed well-established cholangiocarcinoma (CC) at 4–7 mo of age. In humans, CC accounts for 15% of total liver cancer cases in the world. It is associated with poor prognosis and most patients die soon after diagnosis (Taylor-Robinson et al. 2001; Okuda et al. 2002; Olnes and Erlich 2004; Sirica 2005). Further analysis of CC developed in Pten Flox/Flox;Smad4 Flox/Flox;Alb-Cre mice and human CC revealed similar alterations, including p53, p16, p27, p57, SMAD4, β-catenin, cyclin D1, ERK, Ras, AKT, and c-Myc (Ito et al. 2002; Kang et al. 2002; Sugimoto et al. 2002; Wu et al. 2004; Xu et al. 2006). These findings elucidate common features between human and mouse CC formation and suggest that the Pten Flox/Flox;Smad4 Flox/Flox;Alb-Cre mice are an excellent model for mechanistic studies, drug screening, and developing new therapeutic treatments for this deadly disease. APC CONDITIONAL KNOCKOUT MODELS

Human adenomatous polyposis coli (APC) is a 312-kDa protein containing 2843 amino acids. Somatic mutations of APC have also been found in many cancers, such as lung adenocarcinoma, breast cancer, hamartomatous polyp, gastric cancer, colon, and glioblastoma. However, germline mutations of APC are detected only in colorectal polyposis and cancers (reviewed in Minde et al. 2011). In mice, homozygous disruption of Apc is embryonic lethal before E8, whereas Apc heterozygous mice suffer high frequencies of tumor formation following the loss of the wild-type allele (LOH) and the stabilization and accumulation of transcriptionally active nuclear β-catenin (Oshima et al. 1995). An Apc conditional allele was generated and bred either with BLG-Cre, K14-Cre, or WAP-Cre transgenic mice, respectively, aiming to study the role of Apc in mammary tumorigenesis. It was shown that disruption of Apc alone resulted only in metaplasia but not neoplasia. In the absence of p53, there is a rapid progression to neoplasia, with 44.4% of lymphoma-free Apc Flox/Flox;p53 −/−;BLG-Cre mice developing mammary tumors after the first pregnancy (Meniel et al. 2005). Apc Flox/Flox;K14-Cre mice showed skin abnormalities characterized by aberrant development and squamous metaplasia in many epithelial-derived tissues including teeth and thymus, and died before weaning. Of note, only Apc Flox/+;K14-Cre mice developed mammary tumors, which maintained a wild-type allele of Apc and moderately activated levels of β-catenin. On the other hand, knockout of Apc in mature luminal epithelium using WAP-Cre did not induce mammary tumors although Apc Flox/+;WAP-Cre or Apc Flox/Flox;WAP-Cre mice displayed severe squamous metaplasia, which is correlated with strong activation of the Wnt/β-catenin signaling pathway (Kuraguchi et al. 2009). This is consistent with several other cases in which Cre-mediated Apc deficiency induced Wnt/β-catenin activation, thus causing epithelial cells undergo squamous metaplasia but not neoplastic transformation. These data partially explain the colon but not mammary-specific tumor development in patients that carry germ1226




line mutations in APC, assuming different cell types might have different responses to high levels of β-catenin transcriptional activity. SMAD4 CONDITIONAL KNOCKOUT MODELS

SMAD4 is a common mediator of the TGF-β superfamily that comprises more than 40 growth and differentiation factors, playing important functions in diverse developmental processes (Weinstein et al. 2000; Weinstein and Deng 2006; Yang and Yang 2010; Lan 2011; Connolly et al. 2012; Chen et al. 2012a; Massague 2012; Pardali and Ten Dijke 2012; ). SMAD4 is also called DPC4 because its deletion was first found in 60% of pancreatic ductal adenocarcinomas (PDAC) (Hahn et al. 1996a,b). Sporadic mutations of the SMAD4 gene were subsequently detected in several other types of cancers, including stomach cancer, liver cancer, and colon cancer (Nagatake et al. 1996; Schutte et al. 1996; Maesawa et al. 1997; Friedl et al. 1999). Germline mutations of SMAD4 also contribute to familial juvenile polyposis, an autosomal-dominant disorder that may ultimately result in gastrointestinal cancer (Howe et al. 1998). As Smad4-deficient mice die at embryonic E6–7 (Sirard et al. 1998; Yang et al. 1998), conditional knockout of Smad4 becomes a logical option for studying the role of Smad4 in development and tumorigenesis. The data indicated that tissue-specific disruption of Smad4 alone is sufficient enough to induce tumor formation in a number of tissues/organs, including the mammary gland (Li et al. 2003), skin (Qiao et al. 2006), forestomach (Teng et al. 2006), and head and neck (Bornstein et al. 2009). However, despite the fact that human SMAD4 is frequently mutated in pancreas, liver, and colon cancers, deletion of mouse Smad4 in these organs alone does not result in cancer formation (Bardeesy et al. 2006; Kim et al. 2006; Izeradjene et al. 2007; Kojima et al. 2007; Xu et al. 2006, 2010). These data suggest that the tumor-suppressor functions of Smad4 are highly context-specific—that is, its loss cannot initiate tumor formation in some tissues, whereas it is sufficient to do so in many others. Regarding the context-specific tumor-suppressor function of Smad4, the following investigations might help to illustrate its nature. In the liver, as discussed earlier, when both Smad4 and Pten were deleted simultaneously, the Pten Flox/Flox;Smad4 Flox/Flox;Alb-Cre mice developed a bile duct–originated cancer, cholangiocarcinoma, which was not observed in mice carrying mutation on either genes. As Alb-Cre recombinase deletes both genes in bile ducts and hepatocytes starting from embryonic stages, these data suggest that the bile duct cells are more sensitive to tumorigenesis induced by deficiency of both Smad4 and Pten than hepatocytes (Xu et al. 2006). In the pancreas, although the absence of Smad4 alone did not trigger pancreas tumor formation, it increased the expression of an inactivated form of Pten, suggesting a role of Pten in preventing Smad4 −/− cells from undergoing malignancy. Consistent with this, disruption of both Pten and Smad4 in the pancreas using a Pdx1 promoter– driven Cre (Pdx-Cre), which is highly expressed throughout the entire pancreatic epithelium, including both endocrine and exocrine cells, induces pancreatic cancer formation. Further analysis indicated that Pten deficiency alone is enough to induce widespread premalignant lesions, and a low tumor incidence that was significantly accelerated by Smad4 deficiency. Deficiency of both genes enhanced cell proliferation and triggered transdifferentiation from acinar, centroacinar, and islet cells, accompanied by activation of Notch1 signaling. Consistently, Smad4 deficiency also synergistically induces pancreas cancer formation together with Pten mutation, suggesting the loss of Smad4 is a later event in pancreatic tumorigenesis. In another case, Kim et al. (2006) showed that tissue-specific disruption of Smad4 in T cells, but not in gastrointestinal epithelial cells, leads to spontaneous epithelial cancers throughout the gastrointestinal tract in mice (Kim et al. 2006). Tumors arising from the colon, rectum, duodenum, stomach, and oral cavity are stroma-rich with dense plasma cell infiltrates. Smad4-deficient T cells produce interleukin (IL)-5, IL-6, and IL-13, which are known mediators of plasma cell and stromal expansion. These results suggest that the gastrointestinal tumorigenesis occurs as a consequence of the loss of the normal communication between the cellular constituents of a given organ, indicating that Smad4-deficient T cells ultimately emit the wrong message to their stromal and epithelial neighbors. Disruption of TGF-β signaling by knocking out TGF-β receptor 2 Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top074393

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(Tgfbr2) in the stroma of the mammary gland, colon, and forestomach using the promoter of fibroblast-specific protein-1-driven Cre (FSP-Cre) was also found to increase the oncogenic potential of the adjacent epithelia (Bhowmick et al. 2004; Cheng et al. 2008) (Table 2), highlighting an inhibitory role of TGF-β signaling in modulating cancer–stroma interaction for tumorigenesis. In the prostate, it was shown that Smad4 serves as a barrier constraining prostate cancer growth and metastatic progression. Prostate-specific deletion of Pten results in prostate intraepithelial neoplasia, which can progress to high-grade adenocarcinoma following a long latency, albeit with minimally invasive and metastatic features (Ding et al. 2011; Kwak et al. 2013). Robust activation of the TGF-β/SMAD4 signaling was detected in these poorly progressive Pten-null prostate cancers compared with the normal prostate epithelium. It was therefore hypothesized that pathways constraining progression might be activated in Pten-deficient prostate tumors and that inactivation of such progression barriers would engender a metastasis-prone condition. To test this, Smad4 and Pten were simultaneously disrupted in the prostate by Probasin-Cre (Pb-Cre). The double-mutant mice developed invasive, metastatic, and lethal prostate cancers with 100% penetrance within 8 mo of age. Analysis of these tumors identified cell proliferation and invasion as two cardinal tumor biological features in the metastatic Smad4/Pten-null cancers, and Smad4 deficiency markedly enhanced expression of cyclin D1 and secreted phosphoprotein 1 (SPP1), which serve as key mediators of these biological processes (Ding et al. 2011). Further study revealed that cyclin D1, SPP1, PTEN, and SMAD4 comprise a four-gene signature that is prognostic for biochemical recurrence of prostatespecific antigen (PSA) and lethal metastasis in human PCA (Ding et al. 2011). Consistently, overexpression of the COUP transcription factor II (COUP-TFII), which is a key regulator to inhibit SMAD4-dependent transcription, cooperates in the mouse prostate epithelium with PTEN deletion to augment malignant progression and to produce aggressive metastatic tumors (Qin et al. 2013). Furthermore, in human prostate cancers, COUP-TFII expression or activity is tightly correlated with tumor recurrence and disease progression, which is inversely associated with TGF-β/SMAD4 signaling (Qin et al. 2013). These studies establish TGF-β/SMAD4 as a key regulator of prostate cancer progression in both mice and humans. CONCLUSIONS AND FUTURE ASPECTS

Numerous conditional knockout mouse models have been generated by gene targeting in combination with site-specific recombinases. Owing to space limitation, this review summarizes several most commonly used mouse models for cancer research in detail and outlines some others with a brief description. These models have provided valuable tools for many areas in life science and have yielded great impacts on mammalian biology and biomedicine, especially cancer research. Although the use of these models sheds light on many aspects of oncogenesis, numerous questions remain. In human familial cancers, mutation carriers are usually heterozygous, and tumorigenesis is caused by spontaneous loss of heterozygous of the wild-type allele (LOH). However, mice heterozygous for germline mutations (such as Brca1 +/− and Brca2 +/− mice) usually do not show cancer incidence that is higher than a background level. This is perhaps due to the marked, yet intrinsic difference in lifespan between the two species, highlighting a need for caution in data analysis and interpretation. In humans, cancer usually initiates from a single or a few cells that acquired permissive mutations and cancer progression occurs in a heterozygous (for familial cancers) or wild-type (for sporadic cancers) environment. In mice, however, a large number of cells are mutated at the beginning; even under the current best CreLoxP-regulated conditions, these cells underscore a necessity for the development of a precise temporal and spatial regulation system to generate mouse models that faithfully mimic human conditions. Conventional methods for making a mouse model are both time and labor consuming. To solve this problem, international consortia such as the International Knockout Mouse Consortium (IKMC) and International Mouse Phenotype Consortium (IMPC) have been organized to generate knockout and conditional knockout mice that are publically available. Recently, several new technologies, such as zinc-finger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs), and clustered 1228




regularity interspaced short palindromic repeats (CRISPRs) have been developed to precisely and efficiently edit genome of cultured mammal cells and/or zebrafish (Sung et al. 2012; Cong et al. 2013; Mali et al. 2013). These technologies and any other new related technical advances will certainly be adapted in mice to facilitate the production of mutant models with fairly higher efficiency. ACKNOWLEDGMENTS

We gratefully acknowledge the critical reading and helpful discussion of members of the Deng Laboratory. This work was supported by the Intramural Research Program of the National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health. REFERENCES Bailey JM, Creamer BA, Hollingsworth MA. 2009. What a fish can learn from a mouse: Principles and strategies for modeling human cancer in mice. Zebrafish 6: 329–337. Bardeesy N, Cheng KH, Berger JH, Chu GC, Pahler J, Olson P, Hezel AF, Horner J, Lauwers GY, Hanahan D, et al. 2006. Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer. Genes Dev 20: 3130–3146. Berton TR, Matsumoto T, Page A, Conti CJ, Deng CX, Jorcano JL, Johnson DG. 2003. Tumor formation in mice with conditional inactivation of Brca1 in epithelial tissues. Oncogene 22: 5415–5426. Bhowmick NA, Chytil A, Plieth D, Gorska AE, Dumont N, Shappell S, Washington MK, Neilson EG, Moses HL. 2004. TGF-β signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Science 303: 848–851. Biondi CA, Gartside MG, Waring P, Loffler KA, Stark MS, Magnuson MA, Kay GF, Hayward NK. 2004. Conditional inactivation of the MEN1 gene leads to pancreatic and pituitary tumorigenesis but does not affect normal development of these tissues. Mol Cell Biol 24: 3125– 3131. Biswas S, Chytil A, Washington K, Romero-Gallo J, Gorska AE, Wirth PS, Gautam S, Moses HL, Grady WM. 2004. Transforming growth factor β receptor type II inactivation promotes the establishment and progression of colon cancer. Cancer Res 64: 4687–4692. Bornstein S, White R, Malkoski S, Oka M, Han G, Cleaver T, Reh D, Andersen P, Gross N, Olson S, et al. 2009. Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation. J Clin Invest 119: 3408–3419. Cancer Genome Atlas Network. 2012. Comprehensive molecular portraits of human breast tumours. Nature 490: 61–70. Capecchi MR. 2005. Gene targeting in mice: Functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6: 507–512. Chen S, Iversen ES, Friebel T, Finkelstein D, Weber BL, Eisen A, Peterson LE, Schildkraut JM, Isaacs C, Peshkin BN, et al. 2006. Characterization of BRCA1 and BRCA2 mutations in a large United States sample. J Clin Oncol 24: 863–871. Chen G, Deng C, Li YP. 2012a. TGF-β and BMP signaling in osteoblast differentiation and bone formation. Int J Biol Sci 8: 272–288. Chen J, McKay RM, Parada LF. 2012b. Malignant glioma: Lessons from genomics, mouse models, and stem cells. Cell 149: 36–47. Cheng N, Chytil A, Shyr Y, Joly A, Moses HL. 2008. Transforming growth factor-β signaling-deficient fibroblasts enhance hepatocyte growth factor signaling in mammary carcinoma cells to promote scattering and invasion. Mol Cancer Res 6: 1521–1533. Cheung CY, Chen J, Chang TK. 2004. Evaluation of a real-time polymerase chain reaction method for the quantification of CYP1B1 gene expression in MCF-7 human breast carcinoma cells. J Pharmacol Toxicol Methods 49: 97–104. Chodankar R, Kwang S, Sangiorgi F, Hong H, Yen HY, Deng C, Pike MC, Shuler CF, Maxson R, Dubeau L. 2005. Cell-nonautonomous induction of ovarian and uterine serous cystadenomas in mice lacking a functional Brca1 in ovarian granulosa cells. Curr Biol 15: 561–565.

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