Oral Oncology 73 (2017) 16–20

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Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology

Review

Current mouse models of oral squamous cell carcinoma: Genetic and

chemically induced models
Kazuhisa Ishida a,b, Hiroyuki Tomita a,⇑, Takayuki Nakashima a,b, Akihiro Hirata c, Takauji Tanaka d,
Toshiyuki Shibata b, Akira Hara a
a
Department of Tumor Pathology, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan
b
Department of Oral Maxillofacial Surgery, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan
c
Division of Animal Experiment, Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan
d
Department of Diagnostic Pathology (DDP) and Research Center of Diagnostic Pathology (RC-DiP), Gifu Municipal Hospital, 7-1 Kashima-cho, Gifu City, Gifu 500-8513, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Oral squamous cell carcinoma (OSCC) patients have a low 5-year survival rate and poor prognosis. To
Received 2 December 2016 improve survival and prognosis, the causes and processes involved in lesion development should be eval-
Received in revised form 11 July 2017 uated. For this purpose, the use of OSCC mouse models, such as chemically induced mouse models, genet-
Accepted 28 July 2017
ically modified mouse models, and transplanted (xenograft) models, is crucial. These OSCC models
exhibit both advantages and disadvantages when studying OSCC development and progression. Until a
model resembling human OSCC is developed, both the advantages and disadvantages of each model
Keywords:
should be carefully considered. In this review, we discuss OSCC mouse models and their use in cancer
Mouse model
Genetically modified mouse
research worldwide.
Oral cavity Ó 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
Squamous cell carcinoma (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction
Ninety percent of head and neck cancers are squamous cell car-
cinomas involving mucosal surfaces of the oral cavity, pharynx,
and larynx. This cancer can affect any site in the oral mucosa, typ-
ically the tongue and floor of the mouth [1]. Oral squamous cell
carcinoma (OSCC), a subset of head and neck squamous cell carci-
noma, is one of the most common human malignancies worldwide
[2,3]. Further, OSCC is the most common malignant tumor of the
head and neck, and its incidence has increased in recent years [4].
OSCC is a challenging disease to manage in the field of head and
neck cancer. The standard treatment for OSCC is a combination of
surgery, radiation, and chemotherapy. The 5-year survival rate of
OSCC is only 50%, which has remained unchanged for a decade
[5]. This poor prognosis is considered to stem from aggressive local
invasion and metastasis, leading to recurrence. Further, a lack of
applicable markers for early detection and the failure of advanced
lesions to respond to chemotherapy contribute to poor OSCC
outcomes.
OSCC lesions accumulate genetic alterations including chromo-
somal aberrations, DNA mutations, amplifications, or deletions,
and/or epigenetic alterations, such as changes in DNA methylation,
⇑ Corresponding author.
E-mail address: h_tomita@gifu-u.ac.jp (H. Tomita).
which affect genetic regulation [6]. These events are affected by
exposure to environmental agents, including tobacco smoke, alco-
hol, and viruses (e.g. human papillomavirus), which are the major
risk factors [7,8].
Insight into the mechanisms of OSCC initiation and progression
is needed, and identification of novel molecular targets to assist the
development of new therapeutic strategies is crucial [9]. New ther-
apies can be investigated both in vitro and in vivo. Animal models,
particularly mouse models, enable the study of complex biological
processes [10]. An optimal animal model would exhibit sponta-
neously occurring oral cancer; however, spontaneous OSCC is
extremely rare in laboratory animals [11].
In mouse models of OSCC, xenograft models and chemical
carcinogen-induced models are widely used. Xenograft models
with implanted human OSCC cells are frequently used to study
human tumor growth and spread as well as to develop and test
new antitumor drugs [10].
As a chemical carcinogen-induced model, the 4-nitroquinoline
1-oxide (4NQO) mouse model has been used worldwide. The
dynamics of gene expression alterations in the 4NQO mouse model
of human oral carcinogenesis that lead to the development of pre-
neoplastic histological changes such as hyperplasia and dysplasia
followed by SCC development can be characterized in this model
[12–16].

http://dx.doi.org/10.1016/j.oraloncology.2017.07.028
1368-8375/Ó 2017 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 K. Ishida et al. / Oral Oncology 73 (2017) 16–20
Mice are one of the best model organisms for cancer research
because of the animals’ small size, propensity to breed in captivity,
life span of 2–3 years, many physiological and molecular similari-
ties to humans, and fully sequenced genome [17].
Here, we review mouse models used to investigate OSCC patho-
genesis and discuss both the advantages and disadvantages of their
application in cancer research.
Chemical carcinogen-induced mouse models
4-Nitroquinolone oxide (4NQO) model
The 4NQO molecule forms DNA adducts, causes substitutions of
adenosine for guanosine, and evokes intracellular oxidative stress,
resulting in genetic mutations and DNA strand breaks. All these
effects are similar to the genetic and epigenetic alterations result-
ing from exposure to tobacco carcinogens, and therefore, 4NQO
treatment serves as an alternative for tobacco exposure in animal
experiments of oral cancers [13]. 4NQO induces carcinoma in the
tongue and esophagus, and the carcinogenesis model of 4NQO
exposure imitates many situations of human oral squamous
carcinogenesis.
Historically, 4NQO, the most commonly used carcinogenic drug,
is a DNA adduct-forming agent that causes DNA damage similar to
the damage induced by cigarette smoke. In rodent models, the rat
oral cavity model was introduced in the early 1970s [18]. In this
landmark study, 4NQO dissolved in water produced palatal cancers
in all experimental rats by seven months. In addition, 75% of these
animals developed carcinomas on the dorsum of the tongue and
20% developed lesions on the gingival tissue [18]. 4NQO has also
been broadly used in mice and is useful for investigating the effects
of anti-tumor drugs.
Regarding mouse models, the 4NQO OSCC model involves a pro-
tracted multi-step process in which invasive SCC is eventually
reached after several months, and 100% of 4NQO-treated mice
exhibit precancerous lesions and tumors on the tongues and oral
mucosa [19]. Pathological analyses indicate that these lesions
and tumors are flat squamous dysplasias (from mild to severe
atypia), papillary squamous tumors (papillomas), carcinoma
Fig. 1. 4NQO-induced tongue lesions in mice. 4-NQO treatment induces tumors mimicking human oral squamous cell carcinogenesis.
17
in situ (non-invasive SCC), and invasive SCC, even after 4NQO is
withdrawn (Fig. 1) [6,20]. Multiple lesions are considered to consti-
tute the multi-step carcinogenesis of OSCC; this multi-step process
is one advantage of the 4NQO model, and the development of fully
malignant SCC is preceded by increasing grades of dysplastic
changes that mimic oral cancer development in humans. SCCs
are typically detected between 12 and 33 weeks after applying
4NQO (via drinking water) for 16 weeks [20,21].
Regarding the molecular pathology of the 4-NQO model, the
SCC tumors produced display some of the molecular changes
observed in human SCC; however, the detailed pathological and
molecular mechanisms remain unclear.
Dimethyl-1,2,benzanthracene (DMBA) model
This model involves the administration of polycyclic hydrocar-
bon 9,10 dimethyl-1,2 benzanthracene (DMBA) dissolved in ben-
zene or acetone to the cheek pouch of hamsters [22]. DMBA
induces DNA adduct formation and is mutagenic in mice [23,24].
However, the DMBA mouse model is not commonly used, and only
one study using this model has been reported [25]. In this study,
several mice treated with DMBA for 2–6 weeks developed papillo-
mas and papillomas with dysplasia.
Overall, carcinogen-induced models do not allow for the study
of specific genes in the process of oral carcinogenesis. For this pur-
pose, utilizing xenograft models or transgenic mouse models is
necessary.
Genetically modified mouse models
LSL-KrasG12D mouse
Another approach involves a novel inducible mouse model that
allows oncogene activation and/or tumor suppressor inactivation
solely in the stratified epithelia of the oral cavity (Table 1). Two
transgenic mouse models of oral cancer have been described
related to K-ras mutations. These models utilize the keratin 5
(K5) or keratin 14 (K14) promoter to overexpress the oncogene
K-rasG12D in the oral epithelium of mice [26,27]. These promoters
are optimal for targeting transgene expression to the oral cavity.
18 K. Ishida et al. / Oral Oncology 73 (2017) 16–20

Table 1 PI3K/Akt pathway. PTEN/PI3K/Akt signaling is thought to play a

Genetically engineerd mouse models of oral squamous cell carcinoma. pivotal role in HNSCC carcinogenesis [31]. TGF-b signaling plays
Model Description Features References both tumor-suppressing and tumor-promoting roles in the car-
LSL-Kras G12D; K5 tet-on-inducible This model develops [26] cinogenesis of various cancer types. In the carcinogenesis of the
K5-rTA form of an papillomas in the Tgfbr1/Pten 2cKO model, PTEN loss leads to hyperproliferation of
oncogenic Kras skin, oral mucosa, the epithelium, and loss of Tgfbr1 contributes to increased inflam-
knock-in tongue, esophagus, or mation, enhanced angiogenesis, and immune suppression. Thus,
forestomach.
LSL-Kras G12D; K5 or K14 Cre- These models [27]
the Tgfbr1/Pten 2cKO model indicates that HNSCC carcinogenesis
K5 or K14 inducible form of develop papillomas, is affected by not only epithelial oncogenic changes, but also the
CrePR1 an oncogenic Kras hyperplasia, andd tumor microenvironment.
knock-in SCCs in the skin, oral
mucosa, tongue,
esophagus, or p53R172H; K5.CrePR1 and p53flox/flox; K5 CrePR1 mice
forestomach. p53 R172H; K5.CrePR1 (16%) and p53 flox/flox; K5 CrePR1 (25%) mice
L2D1+;p53+/ Human CyclinD1 Both models [28,29]
develop oral SCCs [32]. In these models, skin SCCs developed more
and L2D1+; (D1) expression developed invasive
p53 / under the ED-L2 oral-esophageal SCC often than oral SCCs, and metastases were observed. Deleting the
(L2) promoter and Cdkn2a gene enhances skin tumor formation and promotes metas-
p53 homzygous/ tasis in both models; yet, the role of this gene in oral tumors is
heterozygous unclear. p53 R172H; K5.CrePR1 and p53 flox/flox; K5 CrePR1 mouse
knockout
Tgfbr1/Pten K14 Cre-inducible Visible tumors in the [30]
models suggest that both gain and loss of p53 function is associ-
2cKO (Tgfbr1 Tgfbr1 and Pten oral cavity developed ated with HNSCC carcinogenesis.
flox/flox; Pten knockout and the tumors Taken together, the tumor microenvironment in transgenic
flox/flox; K14- histopathologically mice is quite different from that in humans, because mouse stro-
CreER tam) were well
mal cells also contain the transgene. Although the use of oral
differentiated scc
p53 R172H; K5. K5 Cre-inducible In both models, skin [32] mucosa-specific promoters, such as the K5 or K14 promoter, min-
CrePR1 and p53 expression and SCCs developed more imizes the leakage of transgene expression, tissue specificity is not
p53 flox/flox; p53 knockout than oral SCCs, and absolute. Further, a single gene never predominates the natural
K5 CrePR1 observed metastases. process of oral cancer carcinogenesis. The use of one or two more
specific genes such as K-ras or Akt to drive tumor formation in
transgenic mice may not necessarily reflect the carcinogenic pro-
K5 is expressed within the basal epithelium of the tongue and cess in humans. In fact, the frequency of K-ras mutations is low
forestomach, and K14 is mainly expressed in the basal layer of in human head and neck cancer [33,34].
the oral mucosa and tongue [27].
In one model, the K-rasG12D oncogene driven by the K5 or K14
promoter is distributed under the control of Cre recombinase fused Mouse models that combine genetic modification with chemical
to a mutant of the human progesterone receptor [27]. Administra- carcinogen treatment
tion of RU486 induces the overexpression of oncogenic K-rasG12D in
the oral epithelium of mice. Administration of RU486 to mice XPA / ; p53+/ mouse + 4NQO
resulted in the development of squamous papillomas in the oral An XPA / mouse strain carrying mutant alleles for p53 was
cavity. generated to explore the effects of the interaction between nucleo-
In another similar model, expression of the K-rasG12D oncogene tide excision repair machinery with p53 in oral tumorigenesis
driven by the K5 promoter is under the control of tet-responsive (Table 2). In the 4NQO mouse model, despite the same tumor fre-
elements. Oncogenic K-rasG12D overexpression is induced by quency, XPA / p53+/ mice reached 100% SCC incidence at
administering doxycycline to the mice [26]. The doxycycline- 25 weeks compared to 50 weeks for XPA / p53+/+ littermates [35].
inducible mice develop dysplasia and SCC in the skin, oral mucosa,
tongue, esophagus, or forestomach. L2D1+ mouse + 4NQO
The L2D1+ mouse is characterized by human cyclin D1 overex-
L2D1+/p53+/ and L2D1+/p53 / mice pression controlled by the ED2-L2 promoter of the Epstein-Barr
In the head and neck region, the first genetic mouse model virus [28]. Upon 4NQO treatment, this mouse model exhibits accel-
specifically designed to develop SCC was generated by Opitz erated susceptibility to oral dysplasia and SCC [36].
et al. [28]. They used the Epstein-Barr virus ED-L2 promoter (L2)
to target genes specifically in the oral- esophageal squamous PIK3CA-GEMM mouse + 4NQO
epithelium [29] and generated the L2-cyclin D1 (L2D1+) mouse, PIK3CA-GEMM mice are also known as K5. GLp65/tataPIK3CA
which fuses L2 to human cyclin D1 cDNA. They crossed L2D1+ mice mice [37]. This model overexpresses PIK3CA specifically in the buc-
with p53+/ and p53 / mice, resulting in compound mice that cal and tongue epithelium upon RU486 induction. PIK3CA gene
developed invasive oral-esophageal SCC. Further, cell lines from amplification or gain-of-function mutations have been reported
the oral epithelia of L2D1+/p53+/ and L2D1+/p53 / mice induced in human HNSCCs [38–40]. In the PIK3CA-GEMM mouse model,
tumor formation in athymic nu/nu mice [28]. PIK3CA overexpression alone was insufficient for SCC develop-
ment. Upon 4NQO administration, 39% of mice developed SCCs at
Tgfbr1/Pten 2cKO (Tgfbr1flox/flox; Ptenflox/flox; K14-CreERtam) mice 5–6 months and 100% of mice developed SCCs at 10–12 months.
Tgfbr1/Pten 2cKO mice develop hyperplastic changes in the oral This mouse model exhibits enhanced SCC development compared
epithelium 4 weeks after tamoxifen (tam) induction [30]. Ten with wild-type control mice treated with 4NQO. Further, this
weeks after tam induction, visible tumors in the oral cavity mouse model promotes tumor invasion and metastasis into lymph
develop, and histopathology shows that the tumors were well- nodes and lungs. The mechanisms of carcinogenesis in this model
differentiated SCC, mimicking human head and neck SCC (HNSCC). are related to PI3K/PDK1 and TGF-b signaling, similar to the Tgfbr1/
PTEN is a tumor suppressor gene and a negative regulator of the Pten 2cKO mouse model [30].
 K. Ishida et al. / Oral Oncology 73 (2017) 16–20 19

Table 2 model of head and neck cancer established from fresh surgical
Mouse models that combine genetic modification with chemical carcinogen specimens of 130 human head and neck cancers implanted subcu-
treatment.
taneously into nude mice.
Model Description Features References One advantage of xenograft models is that several mice injected
XPA / ; p53+/ XPA / knockout 4NQO-treated [5] with oral SCC cells developed lymphatic and pulmonary metasta-
mouse + 4NQO and carrying XPA / p53+/ sis, as observed in human oral SCCs [46,47].
mutant alleles for mice reached 100% One disadvantage of xenograft models is that human cell lines
p53 SCC incidence at
25 weeks
can only be implanted into the tissues of athymic or severe com-
L2D1++ 4NQO Human CyclinD1 By 4NQO, this model [36] bined immunodeficiency mice. The use of immune-deficient mice
(D1) expression accelerated prevents the study of interactions between the tumor and
under the ED-L2 susceptibility to oral microenvironment related to the host immune system.
(L2) promoter dysplasia and SCC
PIK3CA-GEMM PIK3CA-GEMM With 4NQO [37]
mouse + 4NQO mouse is also administration, the
shown as K5. mouse model
GLp65/tataPIK3CA developed SCCs in
Conclusions and future perspectives
mouse. This model 39% mice at 5–
can overexpress 6 months and 100% Mouse models have enabled the use of new approaches to
PIK3CA mice at 10– develop cancer prevention and treatment; identification of early
specifically in the 12 months. Further,
diagnostic markers and novel therapeutic targets; and understand-
buccal and tongue the mouse model
epithelium by promoted tumor ing of the in vivo biology and genetics of tumor initiation, promo-
RU486 induction. invasion and tion, progression, and metastasis. No mouse model is perfectly
metastasis of lymph suitable for studying any type of human cancer. Genetically mod-
node and lung. ified mouse models have only been recently developed and are
K14-GFP-miR-211 K14-GFP-miR-211 By 4NQO [42]
transgenic transgenic mouse administration, this
promising; however, their low tumor penetrance causes uncer-
mouse + 4NQO expresses EGFP mouse model tainty in aspects of experimental design such as endpoint timing
and miR-211 developed SCCs in and actual number of mice available for study [6]. However,
constitutively the tongue, early-stage models would enable the further study of early-stage
driven by K14 esophagus, buccal
indicators (precancerous lesions) and biomarkers, as well as the
promoter in mucosa.
squamous investigation of more appropriate treatments [48].
epithelium Chemical 4NQO carcinogenesis models better reflect the clinical
Dusp1 / mouse Dusp1 / Dusp1 / mice [43] setting because of the diversity and heterogeneous nature of the
+ 4NQO knockout with 4NQO resulting lesions. Chemoprevention has been widely studied using
administration
enhanced oral SCC
4NQO carcinogenesis models. The 4NQO model is attractive for
and tumor- chemoprevention studies because of its easily observable features
associated and tissue sampling for pathologic and molecular analyses.
inflammation with Currently, few neck metastasis models are available for drug tri-
IL1 b upregulation.
als and clinical research on OSCC. In xenograft model, feasible
mouse models of the neck metastasis bone destruction in the jaw
K14-GFP-miR-211 transgenic mouse + 4NQO are developed, and useful for elucidating the metastatic mecha-
MicroRNA-211 (miR-211) promotes HNSCC formation and is nism of OSCC [49,50]. However, the future development of the
implicated in decreased patient survival [41]. K14-GFP-miR-211 genetically modified mouse models with the metastasis could
transgenic mice express EGFP and miR-211 constitutively driven advance the discovery of new therapies for OSCC.
by the K14 promoter in the squamous epithelium [42]. Hyperpro- Animal models in which the early stage of OSCC carcinogenesis
liferation and wall thickness, but not tumor formation, were can be replicated remain to be developed. Such a model will allow
observed in the oral epithelium of these mice. Upon 4NQO admin- for the identification of predictive and correlative biomarkers for
istration, these mice develop SCCs in the tongue, esophagus, and evaluating specific therapeutic approaches.
buccal mucosa. The incidence, number, and diameter of tumors An ideal mouse OSCC model would be a combination of a
in this mouse cohort significantly increases upon 4NQO treatment carcinogen-induced model and transgenic model in which the
compared with tumors in wild type control mice treated with application of carcinogenic agents to transgenic mice leads to early
4NQO. Further, the histology of these tumors mimics the histology tumor formation. This model would be analogous to chronic expo-
of human HNSCCs. sure to tobacco and alcohol in an individual with genetic or epige-
netic predispositions for developing cancers in the oral cavity.
Dusp1 / mouse + 4NQO Until a model resembling human OSCC is developed, both the
Dusp1 / mice exhibit enhanced oral cancer development advantages and disadvantages of each model should be carefully
upon 4NQO administration compared with wild type control mice considered.
[43]. This mouse model exhibits accelerated tumor-associated In conclusion, taking both their advantages and disadvantages
inflammation, including IL1b upregulation. DUSP1 is a dual- into account, OSCC mouse models exhibit clear benefits and appear
specificity phosphatase that controls the mitogen-activated pro- suitable for application in therapeutic research. Clinically relevant
tein kinase (MAPK) pathway [44]. The characteristics of this mouse mouse models that overcome current limitations are required for
model indicate that DUSP1 plays an important role in the tumor studying OSCC chemoprevention and treatment.
microenvironment of oral carcinogenesis.

Transplanted (xenograft) models Acknowledgements

Xenograft models of head and neck cancers have been reported We thank all the members of our laboratory and our collabora-
since the early 1980s. Braakhuis et al. [45] reported a xenograft tors for their research work and helpful discussions.
20 K. Ishida et al. / Oral Oncology 73 (2017) 16–20
This work was partially supported by grants from the Ministry
of Education, Culture, Sports, Science, and Technology of Japan
(#15K11289 and #26430111).
Conflicting interests
The authors declare no conflicts of interest.
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