Prevention of lung cancer progression by bexarotene in mouse models
Y Wang1, Z Zhang1, R Yao1, D Jia1, D Wang1, RA Lubet2and M You1
1Department of Surgery and the Siteman Cancer Center, Washington University School of Medicine, St Louis, MO, USA and
2Chemoprevention Agent Development Research Group, National Cancer Institute, Rockville, MD, USA
Bexarotene (Targretins, Ligand Pharmaceuticals Inc.) is
a synthetic high-affinity RXR receptor agonist with
limited affinity for RAR receptors. Bexarotene has shown
efficacy in a phase I/II trial of non-small-cell lung cancers.
However, the chemopreventive efficacy of bexarotene has
not been determined in mouse lung cancer models. In this
study, we have investigated the ability of bexarotene to
inhibit lung tumor progression in the mutant A/J mouse
models with genetic alterations in p53 or K-ras, two of the
most commonly altered genes in human lung tumorigen-
esis. Mice were administered vinyl carbamate (VC), a
carcinogen, by a single intraperitoneal injection (i.p.) at 6
weeks of age. Bexarotene was given by gavage starting at
16 weeks after VC and was continued for 12 weeks.
Although all mice developed lung tumors, only 7% of lung
tumors were adenocarcinomas in wild-type mice, whereas
22 and 26% of lung tumors were adenocarcinomas in p53
transgenic or K-ras heterozygous deficient mice. Bexaro-
tene inhibited both tumor multiplicity and tumor volume in
mice of all three genotypes. Furthermore, bexarotene
reduced the progression of adenoma to adenocarcinoma by
B50% in both p53wt/wtK-rasko/wtand p53wt/wtK-raswt/wtmice.
Thus, bexarotene appears to be an effective preventive
agent against lung tumor growth and progression.
Oncogene (2006) 25, 1320–1329. doi:10.1038/sj.onc.1209180;
published online 24 October 2005
Keywords: chemoprevention; bexarotene; lung cancer;
Lung cancer is the most common cause of cancer
mortality in men and women in the US (Sekido et al.,
2003). The majority of lung cancer is attributable to the
use of tobacco products, tobacco smoking, and, in some
cases, other environmental risk factors (Garfinkel and
Silverberg, 1991; Sekido et al., 2003). Although the
relative risk of developing lung cancer declines in
smokers who quit, former smokers remain at risk for
the disease (Garfinkel and Silverberg, 1991). Thus, both
former and current smokers are likely to be responsive
to improved disease management, early diagnosis, and
The most frequently used mouse model of pulmonary
tumorigenesis is the A/J mouse, which is highly
susceptible to the development of both spontaneously
occurring and chemically induced lung tumors (Stoner,
1991; Malkinson, 1992). More than 50 chemicals have
been tested for chemopreventive efficacy in the A/J
mouse (Stoner, 1991; Malkinson, 1992; Herzog et al.,
1997). Generally, there are three protocols (complete,
postinitiation, and tumor progression) commonly used
for testing the effect of chemopreventive agents on
chemically induced lung tumorigenesis in A/J mice,
based on the mechanism of action of the agents and the
stages of tumor progression. As shown in Figure 1a, the
complete chemoprevention protocol involves interven-
tion beginning 1–2 weeks before carcinogenic initiation
and continuously thereafter. This model is frequently
used to examine agents that either block carcinogen
initiation and/or suppress tumor growth. However, only
limited lung tumor progression (from adenomas to
adenocarcinomas) occurs when animals are terminated
at 20–24 weeks post-carcinogen initiation. The post-
initiation chemoprevention protocol gives the agent 2–3
weeks post-carcinogen initiation and continues until
20–24 weeks for determination of the tumor suppressing
effects independent of tumor initiation. The tumor
progression chemoprevention protocol is designed to
determine the effects of preventive agents on progression
from adenomas to carcinomas by intervening at 12–20
weeks post-carcinogen initiation and continuing the
study to approximately 30 weeks post-carcinogen
initiation. The latter is perhaps the most relevant
protocol because the transition from adenomas to
carcinomas is the most frequent target for clinical
chemoprevention trials in humans.
Although some molecular changes found in mouse
lung tumors have similarities to those seen in humans,
some changes found in human lung cancer are not
observed in the more benign mouse lesions. For
example, there is a very low incidence of p53 inactiva-
tion in A/J mouse lung tumors (o5%), while p53
alterations are found in most human lung tumors (You
and Bergman, 1998; Zhang et al., 2000; Hofsteth et al.,
2004). While mutations in K-ras are commonly observed
in lung tumors from A/J mice, loss of the wt allele
Received 7 March 2005; revised 7 September 2005; accepted 9 September
2005; published online 24 October 2005
Correspondence: Professor M You, Department of Surgery and The
Alvin J Siteman Cancer Center, Washington University School of
Medicine, 660 S Euclid Avenue, Campus Box 8109, St Louis, MO
Oncogene (2006) 25, 1320–1329
& 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00
appears to be uncommon (Zhang et al., 2001). This is in
contrast to many human non-small-cell lung cancers or
lung adenocarcinomas from B6C3F1 mice where loss of
one copy of the K-ras allele, often associated with
mutation of the other allele, is relatively common
(Zhang et al., 2001; Li et al., 2003). Therefore, we have
developed models that carry a p53val135dominant-
negative mutation or a K-ras deletion on the A/J mouse
background. The mutants carrying p53 or K-ras defects
produce lung tumors with >20% adenocarcinomas at
4–6 months when treated with urethane, a lung
carcinogen (Zhang et al., 2001; Wang et al., 2003),
which allows one to determine if potential chemopre-
ventive/chemotherapeutic agents inhibit the growth and
progression of an established preneoplastic lesion to a
malignant tumor and/or cause the regression of these
lesions. This is important since individuals involved in
the testing of potential chemopreventive agents in
cancer-free current or former smokers will have
established preneoplastic lesions (Tong et al., 1996).
Thus, a successful chemopreventive agent must be able
to inhibit both the formation of new lesions and the
progression of existing lesions to tumors.
Retinoids are vitamin A analogues, which function as
regulators of cell growth, differentiation, and apoptosis
(Mangelsdorf et al., 1994; Nason-Burchenal and Dmi-
trovsky, 1999). These agents have been shown to
primarily work through specific nuclear receptors. The
first groups of receptors were designated retinoic acid
receptors (Mangelsdorf et al., 1994; Nason-Burchenal
and Dmitrovsky, 1999), while a second group of
retinoid-related receptors designated RXR (retinoid X
receptors) have been identified more recently (Heyman
et al., 1992). Both groups of receptors have been shown
to regulate gene expression (Ahuja et al., 2003). The
natural ligand for the RXR receptors is 9-cis retinoic
acid (9cRA), which interacts with high affinity both to
the RAR and RXR receptors. However, 9cRA- and
RAR-selective retinoids were found to have significant
side effects that made their use as cancer chemopreven-
tive agents problematic. Based on the isolation of these
RXR receptors, a variety of synthetic agonists have been
produced. Recent preclinical reports showed that the
RXR-selective retinoids are highly effective at inhibiting
the development of ER-positive or ER-negative mam-
mary cancer (Gottardis et al., 1996; Wu et al., 2002) and
they appear to be less toxic than the RAR-selective
retinoids. Boehm et al. (1994) developed the synthetic
RXR agonist 4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-penta-
methyl-2-naphtalenyl) propenyl] benzoic acid and it
was named bexarotene (Targretins, Ligand Pharmaceu-
Bexarotene is an RXR-selective agonist that mini-
mally binds RAR receptors (Boehm et al., 1994), and it
is the first synthetic RXR-selective agonist to enter
clinical trials for cancer therapy indications (Miller
et al., 1997). The RXR receptors form heterodimers with
a wide variety of nuclear receptors, including the
peroxisome proliferator-activated receptors (PPARa,
PPARg, and PPARd), the
(FXR), the constitutive androstane receptor (CAR)
receptor, the RAR receptors (a, b, g), the vitamin D
receptors, and the liver X receptors (LXRa and LXRb)
(Mangelsdorf and Evans, 1995; Chawla et al., 2001;
Experimental Design of The Present Study – Progression Protocol
Commonly Used Chemoprevention Protocols
stages of tumor progression, three protocols are frequently used for testing the efficacies of chemopreventive agents using A/J mice. In
complete chemoprevention protocol, testing agent(s) is administrated 2 weeks before initiation of the carcinogen and continued till
the end of experiment, which is usually at 20–26 weeks. In the postinitiation chemoprevention protocol, testing agent(s) is given 2–3
weeks post-carcinogen initiation and continues to 20–24 weeks. In the tumor progression protocol, the testing agent(s) is given at 12–20
weeks post-carcinogen initiation and continues to 30–40 weeks post-carcinogen initiation. Arrow, carcinogen initiation and this time is
regarded as week 0. Arrow head, starting time of intervention which will last until the end of experiment. (b) Experimental design of the
present study. Experimental design for chemoprevention by bexarotene on VC-induced lung tumors in A/J mice carrying a dominant-
negative p53 or heterozygous deletion of K-ras is illustrated. Mice received single i.p. injection of VC at week 0 (indicated by arrow). At
16 weeks after the VC treatment (arrow head), mice were given either vehicle control or testing agent for 12 weeks. The experiment was
completed 28 weeks after VC treatment and pathology were scored. The horizontal lines indicated the time by weeks that mice were
treated with VC-control (solid line) and with VCþBexarotene (dotted line), respectively.
Experiment design. (a) Commonly used chemoprevention protocols. Based on mechanism of action of the agents and the
Prevention of lung cancer progression by bexarotene
Y Wang et al
To our knowledge, this is the first report on the potent
effect of bexarotene on lung tumor progression in
transgenic mouse models. We have shown that bexaro-
tene decreased tumor progression and growth in
genetically modified models of lung cancer. The late
intervention, 16–28 weeks post VC, which we employed
appears similar to potential phase II clinical trials where
current or former smokers with pre-existing lesions
would be examined. We believe that individuals with
precancerous lesions of the lung with a high risk of
developing lung adenocarcinomas could benefit from
effective agent like bexarotene to inhibit their progres-
sion on the basis of results from preclinical studies and
human clinical trials (Gottardis et al., 1996; Miller et al.,
1997; Bischoff et al., 1999; Khuri et al., 2001; Zhang
et al., 2004; Lubet et al., 2005). Bexarotene exhibited a
25% response rate with a better survival rates when
combined with cisplatin and vinorelbine in the treatment
of patients with non-small-cell lung cancers in a phase I/
II trial (Khuri et al., 2001). In other Phase I clinical trials
for the treatment of cancer, bexarotene was found
to suppress the growth of cutaneous lymphoma and
has now been approved for the treatment of cutaneous
T-cell lymphoma (Miller et al., 1997). In these clinical
studies, bexarotene was generally well tolerated with
hypertriglyceridemia as the most common side effect
(Miller et al., 1997; Khuri et al., 2001). Although the
incidence of hypertriglyceridemia was similar to other
studies in patients with cutaneous lymphoma and lung
cancer, hypertriglyceridemia is generally easily managed
with fenofibrate therapy (Khuri et al., 2001). Hypothy-
roidism was noted in patients treated with bexarotene at
higher doses (1300mg/m2/day) (Sherman et al., 1999).
Since p53 and K-ras appear to be the most commonly
mutated genes in human non-small-cell lung carcinomas,
agents that are effective in tumors with these mutations
are particularly appealing. Thus, bexarotene may have the
potential for use in subjects at high risk of lung cancer.
We thank Weidong Wen for his technical support. We are
grateful to Dr Roger W Wiseman for providing the original
UL53-3 mice and Dr Tyler Jacks for the original K-rasko/wt
mice. This work was supported by NIH Grants R01CA58554
Ahuja HS, Szanto A, Nagy L, Davies PJ. (2003). J Biol Regul
Homeost 17: 29–45.
Bischoff ED, Gottardis MM, Moon TE, Heyman RA, Lamph
WW. (1998). Cancer Res 58: 479–484.
Bischoff ED, Heyman RA, Lamph WW. (1999). J Natl Cancer
Inst 91: 2118–2120.
Boehm MF, Zhang L, Badea BA, White SK, Mais DE, Berger
E et al. (1994). J Med Chem 37: 2930–2941.
Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. (2001).
Science 294: 1866–1870.
Dahlquist KD, Salomonis N, Vranizan K, Lawlor SC,
Conklin BR. (2002). Nat Genet 31: 19–20.
Garfinkel L, Silverberg E. (1991). Ca Cancer J Clin 41: 137–145.
Gottardis MM, Bischoff ED, Shirley MA, Wagoner MA,
Lamph WW, Heyman RA. (1996). Cancer Res 56:
Herzog CR, Lubet RA, You MJ. (1997). Cell Biochem 28: 49–63.
Heyman RA, Mangelsdorf DJ, Dyck JA, Stein RB, Eichele G,
Evans RM et al. (1992). Cell 68: 397–406.
Hofsteth LJ, Robles AI, Yang Q. (2004). Chest 125: 83–85.
Khuri FR, Rigas JR, Figlin RA, Gralla RJ, Shin DM,
Munden R et al. (2001). J Clin Oncol 19: 2626–2637.
Konopleva M, Andreeff M. (2002). Curr Opin Hematol 9:
Lee KW, Cohen P. (2002). J Endocrinol 175: 33–40.
Li J, Zhang Z, Dai Z, Plass C, Morrison C, Wang Y et al.
(2003). Oncogene 22: 1243–1246.
Lubet RA, Christov K, Nunez NP, Hursting SD, Steele VE,
Juliana MM et al. (2005). Carcinogenesis 26: 441–448.
Malkinson AM. (1992). Cancer Res 52: 2670s–2676s.
Mangelsdorf D, Umesono K, Evans RM. (1994). The retinoid
receptors. In: Sporn MB, Roberts AB, Goodman DS (eds).
The Retinoids: Biology, Chemistry, and Medicine. Raven
Press Ltd.: New York, NY. pp 319–349.
Mangelsdorf DJ, Evans RM. (1995). Cell 83: 841–850.
Miller VA, Benedetti FM, Rigas JR, Verret AL, Pfister DG,
Straus D et al. (1997). J Clin Oncol 15: 790–795.
Nason-Burchenal K, Dmitrovsky E. (1999). The retinoids:
cancer therapy and prevention mechanisms. In: Nau H,
Blaner W (eds). Retinoids. The Biochemical and Molecular
Basis of Vitamin A and Retinoid Action (Handbook of
Experimental Pharmacology), vol. 139. Springer: Berlin. pp
Saini SP, Sonoda J, Xu L, Toma D, Uppal H, Mu Y et al.
(2004). Mol Pharmacol 65: 292–300.
Sekido Y, Fong KM, Minna JD. (2003). Annu Rev Med 54:
Sherman SI, Gopal J, Haugen BR, Chiu AC, Whaley K,
Nowlakha P et al. (1999). N Engl J Med 340: 1075–1079.
Stoner GD. (1991). Exp Lung Res 17: 405–423.
Tong L, Spitz MR, Fueger JJ, Amos CA. (1996). Cancer 78:
Wang Y, Zhang Z, Kastens E, Lubet RA, You M. (2003).
Cancer Res 63: 4389–4395.
Wu K, Zhang Y, Xu XC, Hill J, Celestino J, Kim HT et al.
(2002). Cancer Res 62: 6376–6380.
You M, Bergman G. (1998). Hematol Oncol Clin North Am 12:
Zhang Z, Liu Q, Lantry LE, Wang Y, Kelloff GJ, Anderson
MW et al. (2000). Cancer Res 60: 901–907.
Zhang Z, Wang Y, Vikis HG, Johnson L, Liu G, Li J et al.
(2001). Nat Genet 29: 25–33.
Zhang Z, Wang Y, Yao R, Li J, Yan Y, La Regina M et al.
(2004). Oncogene 23: 3841–3850.
Prevention of lung cancer progression by bexarotene
Y Wang et al