Affiliation of authors: Department of Retinoid Research, Ligand Pharmaceuticals Inc., San Diego, CA.
Present address: E. D. Bischoff, R. A. Heyman, X-Ceptor Therapeutics, San Diego, CA.
Correspondence to: William W. Lamph, Ph.D., Department of Retinoid Research, Ligand Pharmaceuticals Inc., 10275 Science Center Dr., San Diego, CA 92121 (e-mail: wlamph{at}ligand.com).
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During the last few years, we have focused our efforts on the design and pharmacologic characterization of both naturally occurring and synthetic compounds that interact with the retinoid X receptors (RXRs). The RXRs are members of the intracellular receptor superfamily that play a pivotal role in multiple endocrine signaling pathways. This activity stems from their ability to heterodimerize with numerous intracellular receptors, including the retinoic acid receptors (RARs), vitamin D receptor, peroxisome proliferator-activated receptors (PPARs), thyroid hormone receptors (TRs), and liver X receptors (LXRs) (5). Thus, RXR-selective ligands (rexinoids) control unique signaling pathways and offer multiple opportunities for the development of new therapies for diseases that range from cancer to metabolic disorders such as type II diabetes (6).
We have previously demonstrated that the novel rexinoid LGD1069 is highly efficacious in two different models of carcinogen-induced mammary carcinoma. LGD1069 is as potent as tamoxifen in preventing the development of mammary tumors in the N-nitrosomethylurea-induced mammary carcinoma model (7). In addition, LGD1069 was much more efficacious than traditional RAR-selective retinoids in this model (8,9). In a model of a more advanced disease state, in which classical retinoids are not active (10), LGD1069 is highly efficacious against established carcinogen-induced rat mammary tumors, causing regression in 72% of primary tumors compared with only 33% of primary tumors for tamoxifen (11). Furthermore, combination therapy with doses of LGD1069 and tamoxifen that are ineffective separately demonstrated a greater than additive antitumor effect over monotherapy. LGD1069 has been shown to be safe and tolerable in an initial clinical trial (12).
The encouraging results with LGD1069, alone or in combination therapy, led us to explore the arena of tamoxifen failure, a major hurdle in breast cancer therapy. We set out to develop a novel carcinogen-induced mammary tumor model, which closely mimics the human clinical situation, to examine the effect of LGD1069 on tumors that had stopped responding to tamoxifen therapy and may have developed resistance.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
LGD1069 (Targretin®; Ligand Pharmaceuticals Inc., San Diego, CA) was suspended in an aqueous solution composed of 10% (vol/vol) polyethylene glycol/Tween 80 (99.5 : 0.5) and 90% of 1% (wt/vol) carboxymethylcellulose (Sigma Chemical Co., St. Louis, MO). LGD1069 (100 mg/kg of body weight) was administered orally to animals in 1 mL by a 16-gauge gavage needle (Popper and Sons, New Hyde Park, NY) based on an average body weight. Tamoxifen (Sigma Chemical Co.) was formulated in purified sesame oil (Croda, Parsippany, NJ) by first dissolving in a small volume of ethanol and then evaporating the ethanol under a stream of purified nitrogen to solubilize the tamoxifen into the sesame oil. Animals were dosed with a volume of 0.1 mL (subcutaneously) at a dose of 800 µg of tamoxifen/kg of body weight.
Development of Tamoxifen-Resistant Tumors, Tumor Induction, Measurement, and Scoring
N-Nitrosomethylurea (Sigma Chemical Co.) was formulated as an aqueous solution
of 10 mg/mL by wetting N-nitrosomethylurea powder with 3% acetic acid and
dissolving it in sterile saline. Fresh solutions of N-nitrosomethylurea were injected within
30 minutes of preparation. Virgin female Sprague-Dawley rats (Harlan Sprague-Dawley, Inc.,
Indianapolis, IN), 50 days of age, received 50 mg/kg of body weight (intravenously, in the tail
vein) as previously described (7) (Fig. 1, left,
point A). Rats were housed in a U.S. Department of Agriculture (Washington, DC)-registered
facility in accordance with National Institutes of Health guidelines for the care and use of
laboratory animals. All animals received food (Teklad LM485-7012; Harlan Sprague-Dawley,
Inc.) and water ad libitum. Beginning 5 weeks after tumor induction, animals were
examined for tumors twice a week. Tumors were measured with electronic calipers (Mitutoyo
Inc., Utsunomiya, Japan), and cross-sectional areas were determined by multiplying the longest
length of the tumor by the greatest perpendicular width of the tumor.
|
Previously, we published that, following 6 weeks of tamoxifen therapy at 800 µg/kg of
body weight (subcutaneously) (Fig. 1, left, point C), approximately
33% of the tumors will completely regress in response to tamoxifen therapy (11). The response in this study (n = 70 tumors) was similar, with 28.6%
of the tumors completely regressing. Animals with primary tumors that completely regressed
("Responders" in Fig. 1
) were considered to be tamoxifen
responsive and thus were removed from the study. The remaining 71.4% of the cases were
scored as progressive disease (40%), stable disease (21.4%), and partial response
(10%). These groups were designated as having failures of tamoxifen therapy and thus
were randomly assigned into two new treatment groups (Fig. 1,
left, point
C, "Tamoxifen Failures"). The animals with tumors that initially stopped
responding to tamoxifen therapy either remained on tamoxifen at 800 µg/kg of body weight
daily (Fig. 1,
left, points C to D) as a single agent or had LGD1069 daily
at 100 mg/kg of body weight (Fig. 1,
left, points C to E) added to the
tamoxifen therapy. The compounds were administered for a period of up to 20 additional weeks
of therapy. In a similar study, animals that had failed to respond to 6 weeks of tamoxifen therapy
had tamoxifen withdrawn from their therapeutic regimen before the initiation of daily LGD1069
therapy at 100 mg/kg of body weight.
At the completion of the therapy, based on the intent-to-treat principle,2 tumors were re-evaluated and again scored as progressive or stable disease or
partial or complete response on the basis of their individual therapeutic response following
combined LGD1069 and tamoxifen therapy (Fig. 1, left, point E) or
continued tamoxifen therapy alone (Fig. 1,
left, point D). Following
randomized assignment into one of the two treatment groups, there was no statistically significant
difference (Table 1
; two-sided P = .33) between the two
treatment groups in their response to the initial 6 weeks of tamoxifen therapy (Fig. 1,
left, points B to C). Second primary tumors, as defined above, did not receive the
entire initial 6-week course of tamoxifen therapy and thus are not included in the evaluation of
these data as primary lesions but are included in the total tumor burden analysis.
|
Primary tumor response rates and initial animal allocation were compared with the use of Fisher's exact test (two-tailed), with the null hypothesis of no difference between the two treatment groups. Analysis of tumor burden was performed by use of a Shapiro-Wilk test to test for normality and was followed by a t test. All statistics and quality control of data analysis were performed by Synteract, Inc., Encinitas, CA. All P values are two-sided, and values of less than .05 were considered to be statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To evaluate the antitumor efficacy of the combination of LGD1069 and
tamoxifen in a setting of tamoxifen failure, tumors that have
previously failed to respond to tamoxifen therapy were tested for their
response to rexinoid therapy. LGD1069 at a dose of 100 mg/kg per day of
body weight (orally) was added to the tamoxifen regimen (Fig. 1, left,
points C to E) for one group of animals that were compared with animals
that remained on continuous tamoxifen alone (Fig. 1,
left, points C to
D). This dose of LGD1069 was previously shown to be well tolerated and
efficacious in both long-term chemoprevention studies and therapeutic
studies on established mammary tumors (7,11). The addition of
LGD1069 to the tamoxifen treatment regimen showed statistically
significant antitumor efficacy on tumors for which tamoxifen had failed
and had potentially developed tamoxifen resistance when compared with
tumors that remained on tamoxifen therapy alone (Table
1
). After 6 weeks of additional therapy, only 3%
(95% confidence interval [CI] = 0%-9% ) of the tumors
continued
to progress following combination therapy with LGD1069 and tamoxifen,
whereas 44% (95% CI = 21%-68%) of the tumors remaining
on
tamoxifen therapy alone progressed, supporting the premise that these
tumors were tamoxifen resistant. LGD1069 in combination with tamoxifen
caused a complete response of 56% (95% CI = 39%-74%) of
primary
tumors compared with 17% (95% CI = 0%-35%) for tamoxifen
alone. In
addition, a total of 90% (95% CI = 80%-100%) of
LGD1069-treated
primary tumors showed an objective response to therapy (e.g., complete
or partial response) compared with 45% (95% CI = 21%-69%)
of the
tumors treated with tamoxifen alone (P = .0002). In a second
study, animals with tumors that failed to respond to tamoxifen therapy
had tamoxifen withdrawn before initiating LGD1069 at a dose of 100
mg/kg per day of body weight (orally) alone (Table 2)
. This
study demonstrated a 51% (95% CI = 34%-71%) objective
response rate
following an additional 6 weeks of LGD1069 therapy alone. These data suggest
that the overall response rate is enhanced by the combination of LGD1069 with
tamoxifen when compared with LGD1069 therapy alone.
|
|
Total Tumor Burden Following Combination LGD1069 and Tamoxifen Therapy
The carcinogen N-nitrosomethylurea puts animals at risk for
developing multiple mammary tumors. These tumors were not derivatives
or metastases of the primary tumor but developed as independent clones
as a result of the carcinogen initiation. Tumors were allowed to
develop in naive, untreated animals until at least one tumor reached 75
mm2 in the cross-sectional surface area, defined as a primary
tumor. Most animals developed additional tumors, defined as second
primary tumors, which arose following the initiation of tamoxifen
therapy (Fig. 1, point B). This model allows one to study the effect of
therapeutic agents on both primary and second primary tumors.
To examine the effect of LGD1069 and tamoxifen combination on all tumors, the total tumor
burden was evaluated. Tumor burden was defined as the mean of the sum of the cross-sectional
surface area for all tumors, primary and/or second primary, on an animal. This analysis compared
the change in total tumor burden from the initiation of LGD1069 and tamoxifen combination
therapy with the final analysis (Fig. 1, left, points C to D or C to E). The
LGD1069 and tamoxifen combination therapy statistically significantly decreased the primary
tumor burden by 68% (95% CI = -86% to
-50%), from 304 mm2 to 132 mm2 (Fig. 2,
B; P = .03; Student's t test). In contrast, the primary
tumor burden increased in the tamoxifen-alone group by 15% (95% CI =
-57% to 88%), from 341 mm2 to 439 mm2. When
both the primary and second primary tumor burdens are combined, LGD1069 decreased the total
tumor burden by 34% (95% CI = -65% to -3%)
compared with an increase of 12% (95% CI = -29% to
53%) for tamoxifen treatment alone. Therefore, LGD1069 in combination with tamoxifen
decreased the growth of all tumors more effectively than did tamoxifen alone.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The N-nitrosomethylurea-induced mammary carcinoma model in the rat is a hormone-dependent model that has been historically employed to identify tamoxifen and other antihormonal agents or retinoids as potential therapeutic agents for both the prevention and treatment of human breast cancer (8,14,15). Our previous work (7,11) demonstrated that LGD1069, a rexinoid, not only was a highly effective chemopreventive agent in this model but also was more efficacious than tamoxifen at causing complete response of established rat mammary carcinomas. Furthermore, we demonstrated that the combination of ineffective doses of LGD1069 and tamoxifen led to a statistically significant number of complete responses of established mammary tumors. To further explore the activity of rexinoids in a carcinogen-induced mammary carcinoma model, we have developed a novel model that escapes antihormonal tamoxifen therapy yetof intereststill retains sensitivity to rexinoid therapy. The current data clearly demonstrate that LGD1069 can overcome tamoxifen failure and provide a rationale for combining LGD1069 with tamoxifen in the clinic to increase the antitumor efficacy of tamoxifen, as well as providing a rationale to use LGD1069 as a novel therapy in patients for whom tamoxifen therapy has failed. Additionally, in an adjuvant setting, LGD1069, in combination with tamoxifen, may delay the time to disease progression and/or enhance therapeutic efficacy.
We have shown that LGD1069 is an effective chemopreventive and chemotherapeutic agent
in the N-nitrosomethylurea-induced mammary tumor model (7,11); thus, LGD1069 can influence both the development and the maintenance of the
malignant phenotype. The efficacy of tamoxifen therapy is dependent on the expression of
estrogen receptors; thus, its utility is limited to tumors that express these receptors. Similarly, the
recently approved breast cancer therapeutic agent Herceptin is directed toward tumors that
express the HER-2/neu proto-oncogene. RXR is expressed ubiquitously in adult tissue,
which is different than many receptor subtypes. Thus, unlike tamoxifen or Herceptin that are
targeted therapies, LGD1069 does not depend on the restricted expression pattern of a
sex-steroid hormone receptor or a specific growth factor receptor; therefore, the agent may have
effects on a variety of breast tumors as well as other cancers. The potential mechanisms by which
LGD1069 functions in these cancer models may include interference with the estrogen signaling
pathway, modulation of breast cancer epithelial cell differentiation, or an antiproliferative effect.
Although these potential mechanisms for the action of LGD1069 may imply that the compound
would have wide-ranging activities in many cells and tissues, LGD1069 has been shown to be safe
and tolerable in a phase I clinical trial (12).
To further elucidate the mechanism by which rexinoids exert their anticancer activity, we are
trying to identify the key heterodimeric partners for RXR in mammary carcinoma models. RXR
forms heterodimers with a number of nonsteroid receptors, such as RAR, LXR, PPAR, and TR.
Although the heterodimeric partner involved in mediating the action of LGD1069 on mammary
carcinoma is not known, one likely family of partners are the PPARs. RXR-ligand activation of
the RXR : PPAR heterodimer has been shown to play a critical role in adipocyte
differentiation (16). Consistent with that observation is the preliminary
evidence from our laboratory that suggests that the mechanism of action of RXR-ligands in breast
cancer involves altering the genetic program of differentiation in mammary epithelial cells by
inducing the modification of lipid metabolism leading to terminal cell division (17). This hypothesis is further supported by the observation that activation of the RXR :
PPAR
transcriptional pathway can induce terminal differentiation of malignant breast
epithelial cells as well as other human tumor cells with diverse origins, including liposarcoma and
prostate and colon carcinomas (18-22). Thus, activation by LGD1069 of
the RXR : PPAR heterodimer (23) or other receptors (such as the orphan
receptor LXR) may play a major role in cellular metabolism or differentiation by reprogramming
gene expression. This modification of gene expression may lead to a novel therapy for breast
cancer, allowing LGD1069 to interfere with mammary carcinoma at three distinct phases of the
disease: 1) during carcinogenesis as a chemopreventive, 2) as a therapeutic agent acting on
established disease, and 3) as an effective therapeutic agent in the presence of antiestrogen failure.
![]() |
NOTES |
---|
2 A succinct definition and justification for this principle are given at http://www.pharminfo.com/conference/clintrial/cln_itt.html, Response 4.
Editor's note: E. D. Bischoff and W. W. Lamph hold stock in Ligand Pharmaceuticals Inc., the manufacturer of LGD1069.
We thank Marco Gottardis for his instrumental role on initiating work with rexinoids and breast cancer; Michael Sporn for his insightful comments and critical review of this manuscript; Diane Crombie, Peter Davies, and Marcus Boehm for their scientific contributions and discussions; and Kathy Conrad and Rene Prudente for their constant diligence and the highest caliber of work.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Early Breast Cancer Trialists' Collaborative Group. Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune therapy. 133 randomised trials involving 31,000 recurrences and 24,000 deaths among 75,000 women. Lancet 1992;339:71-85.[Medline]
2 Early Breast Cancer Trialists' Collaborative Group. Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune therapy. 133 randomised trials involving 31,000 recurrences and 24,000 deaths among 75,000 women. Lancet 1992;339: 1-15.[Medline]
3 Saez RA, Osborne CK. Hormonal treatment of advanced breast cancer. In: Kennedy BJ, editor. Breast cancer (current clinical oncology). New York (NY): Alan R. Liss; 1989. p. 163-72.
4 Coombes RC, Haynes BP, Dowsett M, Quigley M, English J, Judson IR, et al. Idoxifene: report of a phase I study in patients with metastatic breast cancer. Cancer Res 1995;55:1070-4.[Abstract]
5 Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell 1995;83:841-50.[Medline]
6 Mukherjee R, Davies PJ, Crombie DL, Bischoff ED, Cesario RM, Jow L, et al. Sensitization of diabetic and obese mice to insulin by retinoid X receptor agonists. Nature 1997;386:407-10.[Medline]
7 Gottardis MM, Bischoff ED, Shirley MA, Wagoner MA, Lamph WW, Heyman RA. Chemoprevention of mammary carcinoma by LGD1069 (Targretin): an RXR-selective ligand. Cancer Res 1996;56:5566-70.[Abstract]
8 Moon RC, Mehta RG, Rao KV. Retinoids and cancer in experimental animals. In: Sporn MB, Roberts AB, Goodman DS, editors. The retinoids: biology, chemistry, and medicine. New York (NY): Raven Press; 1994. p. 573-95.
9 Moon RC, Mehta RG, Detrisac CJ. Retinoids as chemopreventive agents for breast cancer. Cancer Detect Prev 1992;16:73-9.[Medline]
10 Teelmann K, Tsukaguchi T, Klaus M, Eliason JF. Comparison of the therapeutic effects of a new arotinoid, Ro 40-8757, and all-trans- and 13-cis-retinoic acids on rat breast cancer. Cancer Res 1993;53:2319-25.[Abstract]
11 Bischoff ED, Gottardis MM, Moon TE, Heyman RA, Lamph WW. Beyond tamoxifen: the retinoid X receptor-selective ligand LGD1069 (TARGRETIN) causes complete regression of mammary carcinoma. Cancer Res 1998;58:479-84.[Abstract]
12 Miller VA, Benedetti FM, Rigas JR, Verret AL, Pfister DG, Straus D, et al. Initial clinical trial of a selective retinoid X receptor ligand, LGD1069. J Clin Oncol 1997;15:790-5.[Abstract]
13 Rose DP, Mountjoy KG. Influence of thyroidectomy and prolactin suppression on the growth of N-nitrosomethylurea-induced rat mammary carcinomas. Cancer Res 1983;43:2588-91.[Abstract]
14 Gottardis MM, Jordan VC. Antitumor actions of keoxifene and tamoxifen in the N-nitrosomethylurea-induced rat mammary carcinoma model. Cancer Res 1987;47:4020-4.[Abstract]
15 Anzano MA, Byers SW, Smith JM, Peer CW, Mullen LT, Brown CC, et al. Prevention of breast cancer in the rat with 9-cis-retinoic acid as a single agent and in combination with tamoxifen. Cancer Res 1994;54:4614-7.[Abstract]
16
Schulman IG, Shao G, Heyman RA. Transactivation by retinoid
X receptor-peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimers:
intermolecular synergy requires only the PPARgamma hormone-dependent activation function. Mol Cell Biol 1998;18:3483-94.
17 Agarwal VR, Bischoff ED, Cooke TA, Love DL, Heyman RA, Lamph WW. Targretin causes complete regression of mammary carcinoma by modulating differentiation in mammary glands. In: Proceedings of the 90th Annual Meeting of the American Association for Cancer Research. Vol. 40. Philadelphia (PA): American Association for Cancer Research, Inc.; 1999. p. A23.
18 Mueller E, Sarraf P, Tontonoz P, Evans RM, Martin KJ, Zhang M, et al. Terminal differentiation of human breast cancer through PPAR gamma. Mol Cell 1998;1:465-70.[Medline]
19 Sarraf P, Mueller E, Jones D, King FJ, DeAngelo DJ, Partridge JB, et al. Differentiation and reversal of malignant changes in colon cancer through PPARgamma. Nat Med 1998;4:1046-52.[Medline]
20 Kubota T, Koshizuka K, Williamson EA, Asou H, Said JW, Holden S, et al. Ligand for peroxisome proliferator-activated receptor gamma (troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo. Cancer Res 1998;58:3344-52.[Abstract]
21
Demetri GD, Fletcher CD, Mueller E, Sarraf P, Naujoks R,
Campbell N, et al. Induction of solid tumor differentiation by the peroxisome
proliferator-activated receptor-gamma ligand troglitazone in patients with liposarcoma. Proc Natl Acad Sci U S A 1999;96:3951-6.
22
Tontonoz P, Singer S, Forman BM, Sarraf P, Fletcher JA,
Fletcher CD, et al. Terminal differentiation of human liposarcoma cells induced by ligands for
peroxisome proliferator-activated receptor gamma and the retinoid X receptor. Proc Natl
Acad Sci U S A 1997;94:237-41.
23
Mukherjee R, Strasser J, Jow L, Hoener P, Paterniti JR, Jr,
Heyman RA. RXR agonists activate PPARalpha-inducible genes, lower triglycerides, and raise
HDL levels in vivo. Arterioscler Thromb Vasc Biol 1998;18:272-6.
Manuscript received July 2, 1999; revised October 3, 1999; accepted October 13, 1999.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |