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The G0/G1 switch gene 2 (G0S2) is rapidly induced by all-trans-retinoic acid (RA)-treatment of acute promyelocytic leukemia (APL) and other cells. G0S2 regulates lipolysis via inhibition of adipose triglyceride lipase (ATGL). This study found that retinoic acid receptor (RAR), but not retinoid X receptor (RXR) agonists induced G0S2 expression in APL cells. Novel G0S2 functions were uncovered that included repression of exogenous gene expression and transcriptional activity. Transient G0S2 transfection repressed the activities of multiple reporter constructs (including the retinoid-regulated species RARβ, UBE1L and G0S2); this occurred in diverse cell contexts. This inhibition was antagonized by siRNA-mediated G0S2 knockdown. To determine the inhibitory effects were not due to transient G0S2 expression, G0S2 was stably overex-pressed in cells without appreciable basal G0S2 expression. As expected, this repressed transcriptional activities. Intriguingly, transfection of G0S2 did not affect endogenous RARβ, UBE1L or G0S2 expression. Hence, only exogenously expressed genes were affected by G0S2. The domain responsible for this repression was localized to the G0S2 hydrophobic domain (HD). This was the same region responsible for the ability of G0S2 to inhibit ATGL activity. Whether an interaction with ATGL accounted for this new G0S2 activity was studied. Mimicking the inhibition of ATGL by oleic acid treatment that increased lipid droplet size or ATGL siRNA knockdown did not recapitulate G0S2 repressive effects. Engineered gain of ATGL expression did not rescue G0S2 transcriptional repression either. Thus, transcriptional repression by G0S2 did not depend on the ability of G0S2 to inhibit ATGL. Subcellular localization studies revealed that endogenous and exogenously-expressed G0S2 proteins were localized to the cytoplasm, particularly in the perinuclear region. Expression of a mutant G0S2 species that lacked the HD domain altered cytosolic G0S2 localization. This linked G0S2 subcellular localization to G0S2 transcriptional repression. The potential mechanisms responsible for this G0S2 repression are examined.

Retinoids, vitamin A analogues that bind to retinoic acid receptor (RAR) or retinoid X receptor (RXR), play important roles in regulating cell proliferation, apoptosis, and differentiation. Recently, RXR-selective ligands, also referred to as rexinoids, have been investigated as potential chemopreventive agents for breast cancer. Our previous studies demonstrated that the rexinoid bexarotene significantly prevented ER-negative mammary tumourigenesis with less toxicity than naturally occurring retinoids in animal models. To determine whether bexarotene prevents cancer at the early stages during the multistage process of mammary carcinogenesis, we treated MMTV-erbB2 mice with bexarotene for 2 or 4 months. The development of preinvasive mammary lesions such as hyperplasias and carcinoma-in-situ was significantly inhibited. This inhibition was associated with reduced proliferation, but no induction of apoptosis. We also examined the regulation of a number of rexinoid-modulated genes including critical growth and cell cycle regulating genes using breast cell lines and mammary gland samples from mice treated with rexinoids. We showed that two of these genes (DHRS3 and DEC2) were modulated by bexarotene both in vitro and in vivo. Identification of these rexinoid-modulated genes will help us understand the mechanism by which rexinoid prevents cancer. Such rexinoid-regulated genes also represent potential biomarkers to assess the response of rexinoid treatment in clinical trials.

Purpose: To test whether a novel rexinoid, LG100268, prevents the development of preinvasive and invasive estrogen receptor–negative mammary tumorigenesis in MMTV-erbB2 mice. Experimental Design: For invasive breast cancer prevention, MMTV-erbB2 mice were treated with daily gastric gavage of vehicle, LG100268 (10 mg/kg), or LG100268 (100 mg/kg) for long term starting at 3 months of age. For preinvasive lesion study, mice were treated with daily gastric gavage of vehicle or LG100268 (100 mg/kg) for 4 months. Results: Long-term treatment with LG100268 significantly prevented invasive mammary tumor development. Median time (age) to tumor development was delayed from 217 days in vehicle group to 357 days in low-dose group. In high-dose group, only 2 of 20 mice developed tumors after 430 days of treatment. Short-term treatment of LG100268 significantly prevented the development of preinvasive mammary lesions including hyperplasia and ductal carcinoma in situ. The cancer prevention effect was associated with reduced expression of Ki67 and cyclin D1 in mammary glands by >80%. Conclusion: Rexinoid LG100268 is an effective chemopreventive agent in preventing the development of both malignant and premalignant mammary lesions in MMTV-erbB2 mice.

Retinoids have been found to be promising chemopreventive agents that play an important role in regulating cell growth, differentiation, and apoptosis. The action of retinoids is mediated by retinoid receptors (retinoic acid receptors and retinoid X receptors), which are nuclear transcription factors that, when bound to retinoids, regulate gene expression. LGD1069 is a highly selective RXR agonist that has reduced toxicity compared with retinoids. Our previous studies have shown that RXR-selective ligands (or “rexinoids”), including LGD1069, can inhibit the growth of normal and malignant breast cells and can suppress the development of breast cancer in transgenic mice. For the current study, we attempted to identify biomarkers of the chemopreventive effect of the RXR-selective retinoid LGD1069. In these experiments, we used Affymetrix microarrays to identify target genes that were modulated by LGD1069 in normal human breast cells. Affymetrix and dChip analysis identified more than 100 genes that were up-regulated or down-regulated by LGD1069 treatment. We then tested 16 of these genes in validation experiments using quantitative reverse transcription-PCR and Western blotting of independently prepared samples, and found that 15 of 16 genes were modulated in a similar manner in these validation experiments as in the microarray experiments. Genes found to be regulated include known retinoid-regulated genes, growth regulatory genes, transcription factors, and differentiation markers. We then showed that the expression of several of these rexinoid-regulated biomarkers is modulated in vivo in mammary glands from mice treated with LGD1069. These critical growth-regulating proteins will be promising targets of future agents for the prevention and treatment of breast cancer. (Cancer Res 2006; 66(24): 12009-18)

Purpose: We tested whether a selective estrogen receptor modulator (SERM) and a rexinoid are active for prevention and treatment in the mouse mammary tumor virus-neu mouse model of estrogen receptor–negative breast cancer.Experimental Design: For prevention, mice were fed a powdered control diet, the SERM arzoxifene (Arz, 20 mg/kg diet), the rexinoid LG100268 (268, 30 mg/kg diet), or the combination for 60 weeks. In a second prevention study, mice were fed Arz (6 mg/kg diet), 268 (30 mg/kg diet), the combination of Arz and 268, the SERM acolbifene (Acol, 3 mg/kg diet), or the combination of Acol and 268 for 52 weeks. For the treatment studies, mice with tumors were fed combinations of a SERM and 268 for 4 weeks.Results: The rexinoid 268 and the SERMs Arz and Acol, as individual drugs, delayed the development of estrogen receptor–negative tumors. Moreover, the combination of a SERM and 268 was strikingly synergistic, as no tumors developed in any mouse fed the combination of 268 and a SERM. Moreover, this drug combination also induced significant tumor regression when used therapeutically. These drugs did not inhibit transgene expression in vitro or in vivo, and the combination of Arz and 268 inhibited proliferation and induced apoptosis in the tumors.Conclusion: The combination of a rexinoid and SERM should be considered for future clinical trials.

This study evaluated the anti-tumor efficacy of combining the RXR agonist, bexarotene, with the PPARgamma agonist, rosiglitazone, in colon cancer. Moser, a human colon cancer cell line, was treated with bexarotene and rosiglitazone alone or in combination and the effect on growth and differentiation were examined. The data demonstrated that the bexarotene/rosiglitazone combination produced greater efficacy in growth inhibition than either single agent. Furthermore, combination treatment acted cooperatively to decrease COX-2 expression and PGE2 synthesis while increasing expression of the differentiation marker, CEA. These findings were confirmed in vivo in a Moser xenograft tumor model. Collectively, our data suggest a potential role for utilizing a combination regimen of a RXR and PPARgamma agonist in the treatment of colon cancer.

BACKGROUND We previously reported that a retinoid X receptor agonist bexarotene prevented and overcame acquired drug resistance in advanced breast cancer and non-small cell lung cancer. The present study was to evaluate the effect of bexarotene on the development of multidrug resistance in advanced prostate cancer. METHODS Human prostate cancer cells PC3 were repeatedly treated in culture with paclitaxel, doxorubicin, or cisplatin with or without bexarotene for 3 months. Thereafter, cells were isolated and characterized for their drug sensitivity. RESULTS Compared to parental cells, cells treated with a single therapeutic agent was resistant to the therapeutic agent, whereas cells treated with the combination remained chemosensitive. Cells with acquired drug resistance showed increased sensitivity to the cytotoxic agent when treated with the combination. Fluctuation analysis demonstrated that treatment with bexarotene decreased the rate of spontaneous development of drug resistance. These in vitro findings were further confirmed in the PC3 xenograft model. CONCLUSION Our results suggest a role of bexarotene in combination with chemotherapeutic agents in prevention and overcoming acquired drug resistance in advanced prostate cancer. © 2005 Wiley-Liss, Inc.

Acquired drug resistance represents a major challenge in the therapeutic management of breast cancer patients. We reported previously that the retinoid X receptor–selective agonist bexarotene (LGD1069, Targretin) was efficacious in treating animal models of tamoxifen-resistant breast cancer. The goal of this study was to evaluate the effect of bexarotene on development of acquired drug resistance and its role in overcoming acquired drug resistance in advanced breast cancer. Paclitaxel, doxorubicin, and cisplatin were chosen as model compounds to determine the effect of bexarotene on the development of acquired drug resistance. Human breast cancer cells MDA-MB-231 were repeatedly treated in culture with a given therapeutic agent with or without bexarotene for 3 months. Thereafter, cells were isolated and characterized for their drug sensitivity. Compared with parental cells, cells treated with a single therapeutic agent became resistant to the therapeutic agent, whereas cells treated with the bexarotene combination remained chemosensitive. Cells with acquired drug resistance, when treated with the combination, showed increased sensitivity to the cytotoxic agent. Furthermore, cells treated with the combination regimen had reduced invasiveness and angiogenic potential than their resistant counterparts. These in vitro findings were further confirmed in an in vivo MDA-MB-231 xenograft model. Our results suggest a role for bexarotene in combination with chemotherapeutic agents in prevention and overcoming acquired drug resistance in advanced breast carcinoma.

Retinoids and their derivatives can suppress the development of cancer in animals and in humans. We and others have shown that retinoid X receptor (RXR)-selective retinoids or “rexinoids” suppress the development of breast cancer in several animal models with minimal toxicity. LGD1069 (Bexarotene) is a potent RXR-selective retinoid with reduced toxicity compared with naturally occurring retinoids. In this study, we investigated the expression of LGD1069-modulated biomarkers. We previously did cDNA array analysis of LGD1069-treated breast cells using Affymetrix microarrays. These studies identified many LGD1069-regulated genes, one of which was cyclooxygenase-2 (COX-2). Because COX-2 inhibitors have been shown to prevent cancer in other model systems, we investigated whether LGD1069 inhibits the expression of COX-2 in mammary tissue and in normal human mammary epithelial cells (HMEC). In mouse mammary tumor virus-erbB2 mice treated with LGD1069, there was a marked decrease of COX-2 expression in both normal and malignant mammary tissues. The effect of LGD1069 on COX-2 expression was also investigated in normal human breast cells. COX-2 expression was markedly reduced by treatment with LGD1069 at the RNA and protein level in normal HMECs; LGD1069 suppressed COX-2 promoter activity. We also showed that LGD1069 inhibited activator protein (AP-1)-dependent transcription in these breast cells, and that suppression of COX-2 expression was due to sequestration of CBP/p300. These results from in vivo and in vitro studies suggest that LGD1069, an RXR-selective retinoid, inhibits COX-2 expression by suppression of COX-2 transcription in part through transrepression of the AP-1 transcription factor. Thus, RXR-selective retinoids that inhibit AP-1 activity and suppress COX-2 expression may be particularly promising drugs for breast cancer prevention. Furthermore, such RXR-selective retinoids may be most useful in combination with antiestrogens for more effective prevention of breast cancer in women at high risk of this disease.

We have previously shown that the retinoid X receptor (RXR) ligand bexarotene (LGD1069, Targretin) is efficacious as a chemopreventive and chemotherapeutic agent in rat N-nitroso-N-methylurea (NMU)-induced mammary carcinomas (Cancer Res 58: 479–484, 1998). To determine additional role for bexarotene in breast cancer treatment, we evaluated the effect of bexarotene on the efficacy of paclitaxel (Taxol) treatment in a rat NMU-derived mammary tumor cell line, NMU-417,in vitro and in rat NMU-induced mammary tumors in vivo. Our growth inhibition results showed that the bexarotene/paclitaxel combination produced a concentration-dependent synergy in NMU-417 tumor cell line. Synergistic growth inhibition by the combination was associated with an increase in cell death induced by both agents. In rat NMU-induced mammary tumor model in vivo, the benefit of combination therapy was observed as early as 1 week after treatment and increased as treatment continued. At the end of 6 weeks of treatment, the bexarotene/paclitaxel combination produced an overall objective response rate of 94% compared with a rate of 12% in paclitaxel-treated and 58% in bexarotene-treated animals, an effect that was more than the additive effects produced by single agents. Although both bexarotene alone and the bexarotene/paclitaxel combination reduced tumor multiplicity to similar extent, the combination regimen produced a statistically significant decrease in total tumor burden compared to single agents and untreated controls (two-tailed, p > 0.05). Combination therapy did not further alter body weight nor increase toxicity when compared to single agents. In summary, our results demonstrated the potential of using a RXR selective ligand in combination with chemotherapy for the treatment of breast cancer.

We show that the selective estrogen receptor modulator arzoxifene (Arz) and the rexinoid LG100268 (268) synergize to promote apoptosis in a rat model of estrogen receptor-positive breast carcinoma and in estrogen receptor-positive human breast cancer cells in culture. We also show that it is not necessary to administer Arz and 268 continuously during tumor progression to prevent cancer in the rat model because dosing of these drugs in combination for relatively short periods, each followed by drug-free rests, is highly effective. This new approach to chemoprevention uses high doses of drugs that are too toxic for long-term administration. However, when given for short periods, the agents are nontoxic and still induce apoptosis in breast cancer cells. We also show that the ability of the two drugs to induce apoptosis is the combined result of induction of transforming growth factor β by Arz, together with inhibition of the prosurvival nuclear factor κB and phosphatidylinositol 3′ kinase signaling pathways by 268. The new protocol we have developed for chemoprevention allows the efficacious and safe administration of 268 and Arz, and these agents now should be considered for clinical use.

Background: Retinoids, which are derivatives of vitamin A, induce differentiation of acute promyelocytic leukemia (APL) cells in vitro and in patients. However, APL cells develop resistance to retinoic acid treatment. Arsenic trioxide (As2O3) can induce clinical remission in patients with APL, including those who have relapsed after retinoic acid treatment, by inducing apoptosis (programmed cell death) of the leukemia cells. In this study, we investigated the molecular mechanisms by which As2O3 induces apoptosis in retinoic acid-sensitive NB4 APL cells, in retinoic acid-resistant derivatives of these cells, and in fresh leukemia cells from patients. Methods: Apoptosis was assessed by means of DNA fragmentation analyses, TUNEL assays (i.e., deoxyuridine triphosphate labeling of DNA nicks with terminal deoxynucleotidyl transferase), and flow cytometry. Expression of the PML/RARa fusion protein in leukemia cells was assessed by means of western blotting, ligand binding, and immunohistochemistry. Northern blotting and ribonuclease protection assays were used to evaluate changes in gene expression in response to retinoic acid and As 2 O 3 treatment. Results and Conclusions: As2O3 induces apoptosis without differentiation in retinoic acid-sensitive and retinoic acidresistant APL cells at concentrations that are achievable in patients. As 2 O 3 induces loss of the PML/RARa fusion protein in NB4 cells, in retinoic-acid resistant cells derived from them, in fresh APL cells from patients, and in non-APL cells transfected to express this protein. As2O3 and retinoic acid induce different patterns of gene regulation, and they inhibit the phenotypes induced by each other. Understanding the molecular basis of these differences in the effects of As 2O3 and retinoic acid may guide the clinical use of arsenic compounds and provide insights into the management of leukemias that do not respond to retinoic acid. [J Natl Cancer Inst 1998;90:124‐33]

9-cis retinoic acid (9-cis RA) is a retinoid receptor pan-agonist that binds with high affinity to both retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Using a variety of in vivo and in vitro cancer models, we present experimental data that 9-cis RA has activity as a potential chemotherapeutic agent. Treatment of the human promyelocytic leukemia cell line HL-60 with 9-cis RA decreases cell proliferation, increases cell differentiation, and increases apoptosis. Induction of apoptosis correlates with an increase in tissue transglutaminase (type II) activity. In vivo, 9-cis RA induces complete tumor regression of an early passage human lip squamous cell carcinoma xenograft. Finally, 9-cis RA inhibits the anchorage-independent growth of the human breast cancer cell lines MCF-7 and LY2 (an antiestrogen-resistant MCF-7 variant). Transient co-transfection assays indicate that 9-cis RA inhibits estrogen receptor transcription of an ERE-tk-LUC reporter through RAR or RXR receptors. These data suggest that retinoid receptors can antagonize estrogen-dependent transcription and provides one possible mechanism for the inhibition of cell growth by 9-cis RA in breast cancer cell lines. In summary, these findings present evidence that 9-cis RA has a wide range of activities in human cancer models.

Retinoic acid receptor (RAR) active retinoids have proven therapeutically useful for treating certain cancers and dermatological diseases. Herein, we describe the discovery of two new RAR active trienoic acid retinoids, (2E,4E,6E)-7-(3,5-di-tert-butylphenyl)-3-methylocta-2, 4,6-trienoic acid (10a, ALRT1550) and (2E,4E,6Z)-7-(3,5-di-tert-butylphenyl)-3-methylocta-2, 4,6-trienoic acid (10b, LG100567). ALRT1550 is a RAR selective retinoid which exhibits exceptional potency in both competitive binding and cotransfection assays. Moreover, it is the most potent antiproliferative retinoid described to date and thus has implications for the treatment of certain cancers. LG100567 is a potent panagonist which activates both RARs and retinoid X receptors.

Bexarotene is an RXR-selective vitamin A analog that has been shown to prevent ER-negative mammary tumorigenesis in animal models. While investigating the mechanism by which bexarotene prevents ER-negative breast cancer development, we found that the expression of cyclin D1, a critical cell cycle promoter, was repressed by bexarotene in vitro and in vivo. Time course and cycloheximide experiments show that repression of cyclin D1 is a late effect and requires new protein synthesis. Previously we discovered that DEC2 (differentially expressed in chondrocytes-2), a helix–loop–helix transcription repressor, was induced by bexarotene in human mammary epithelial cells. Therefore, we hypothesized that bexarotene represses the transcription of cyclin D1 through induction of DEC2. Luciferase reporter studies demonstrated that either bexarotene treatment or forced expression of DEC2 can repress the transcription of a cyclin D1 promoter reporter by affecting the basal transcriptional activity. Results from chromatin immunoprecipitation experiments showed that bexarotene treatment causes the recruitment of DEC2 and HDAC1 (histone deacetylase 1) to the cyclin D1 promoter. Co-immunoprecipitation confirms the interaction between DEC2 and HDAC1, suggesting that the recruitment of HDAC1 to the cyclin D1 promoter is through DEC2. Trichostatin A, a HDAC inhibitor, reverses the cyclin D1 repression by bexarotene, suggesting that repression of cyclin D1 involves histone deacetylation. Knock-down of DEC2 by siRNA abolishes the cyclin D1 repression, further supporting our hypothesis. Finally, we demonstrated that overexpression of DEC2 dramatically inhibited cell proliferation and repressed the expression of cyclin D1 in human mammary epithelial cells. These results suggest that bexarotene down-regulates cyclin D1 through induction of DEC2, followed by recruitment of HDAC1 to the cyclin D1 promoter causing transcriptional repression. By elucidating the mechanism by which rexinoids inhibit cell proliferation, it will be possible to develop more effective and less toxic drugs to prevent ER-negative breast cancers.

Purpose: To test whether the triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) and the rexinoid LG100268 (268) prevent the formation of estrogen receptor (ER)–negative mammary tumors or either arrest the growth or cause regression of established tumors in MMTV-neu mice. Experimental Design: For prevention, mice were fed control diet, CDDO-Me (60 mg/kg diet), 268 (20 mg/kg diet), or the combination for 45 weeks. For treatment, mice with established tumors at least 4 mm in diameter were fed control diet, CDDO-Me (100 mg/kg diet), 268 (60 mg/kg diet), or the combination for 4 weeks. Results: CDDO-Me and 268 significantly delayed the development of ER-negative tumors, with a 14- and 24-week delay, respectively, compared with the control group for the time required to reach 50% tumor incidence. The combination of CDDO-Me and 268 was significantly more potent than the individual drugs, as only one tumor was found in the combination group, after 45 weeks on diet, at which time all control animals had tumors. Treating established tumors with CDDO-Me arrested the growth of 86% of the tumors, and 268 induced tumor regression in 85% of tumors. CDDO-Me and 268 target different signaling pathways and cell types. CDDO-Me inhibited constitutive STAT3 phosphorylation and the degradation of IKBα in ER-negative breast cancer cells, whereas 268 blocked IKBα degradation and the release of interleukin-6 in RAW264.7 macrophage-like cells, inhibited the ability of endothelial cells to organize into networks, and blocked angiogenesis in vivo. Conclusions: CDDO-Me and 268 are useful as individual drugs to prevent ER-negative mammary tumorigenesis and to treat established tumors. They synergize when used in combination for prevention.

Female A/J mice injected with the carcinogen vinyl carbamate develop atypical adenomatous hyperplasias in lungs 4 weeks after injection with the carcinogen. The number and severity of tumors then increase over time, making these mice a useful model for evaluating potential chemopreventive agents. The rexinoid LG100268 (LG268), a selective ligand for the retinoid X receptor, and the methyl amide of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) both significantly reduced the number, size, and severity of the histopathology of lung tumors in female A/J mice when fed in diet for 14 to 20 weeks. The total tumor burden was 85% to 87% lower in mice fed LG268 and CDDO-MA than in controls, and the percentage of high-grade tumors decreased from 59% in the controls to 25% or 30% with CDDO-MA and LG268. Erlotinib, which is used to treat lung cancer patients and is an inhibitor of the epidermal growth factor receptor, was less effective in this model. Immunohistochemical staining of geminin, a marker of cell cycle progression, was higher in lung sections from control mice than in mice treated with LG268. Because rexinoids and triterpenoids signal through different biological pathways, they should be tested in combination for the prevention of lung cancer. [Mol Cancer Ther 2008;7(5):1251–7]

The synthetic rexinoid bexarotene (Targretin, LGD1069) inhibits the formation of both estrogen receptor-negative and estrogen receptor-positive breast cancer in preclinical models and controls the expression of growth-regulatory biomarkers, such as IGFBP-6 (insulin-like growth factor-binding protein 6), RARbeta, or cyclin D1. In this study, we identified a classical retinoic acid-responsive element in the first intron in the IGFBP-6 gene adjacent to a consensus AP-1 binding site, both elements essential for rexinoid-induced expression of IGFBP-6. In chromatin binding experiments, bexarotene increased the occupancy of the identified enhancer element by RXRalpha, RARbeta, cJun, cFos, and p300. In normal mammary epithelial cells and T47D breast cancer cells, small interfering RNA-mediated knockdown of all RXR isoforms or RARbeta, but not RARalpha or RARgamma alone, blocked the induction of IGFBP-6 by bexarotene. Simultaneous knockdown of RARalpha and RARgamma abrogated both the induction of RARbeta and the up-regulation and secretion of IGFBP-6. The suppression of either RARbeta or cJun by small interfering RNA blocked the recruitment of RXRalpha and cJun to the enhancer. These results demonstrate a novel cooperative interaction between retinoid receptors and AP-1 orchestrated by RARbeta and highlight a novel mechanism by which RARbeta can mediate the cancer-preventive effects of rexinoids.

Despite advances in treatment, breast cancer continues to be the second leading cause of cancer mortality in women. Statistics suggest that while focus on treatment should continue, chemopreventive approaches should also be pursued. Previous studies have demonstrated that naturally occurring retinoids such as 9-cis retinoic acid (9cRA) can prevent breast cancer in animal models. However, these studies have also shown that these compounds are too toxic for general use. Work from our laboratory showed that an RXR-selective retinoid LGD1069 prevented tumor development in animal models of cancer with reduced toxicity as compared to an RAR-selective retinoid TTNPB. In the present study, we investigated the mechanisms by which receptor-selective retinoids inhibit the growth of normal and malignant breast cells. Our results demonstrate that the synthetic retinoids tested are as effective as 9cRA in suppressing the growth of normal human mammary epithelial cells (HMECs) and estrogen receptor-positive (ER-positive) breast cancer cells. Although the receptor-selective retinoids induce minimal amounts of apoptosis in T47D breast cancer cells, the predominant factor that leads to growth arrest is G1 cell cycle blockade. Our data indicate that this blockade results from the downregulation of Cyclin D1 and Cyclin D3, which in turn causes Rb hypophosphorylation. Non-toxic retinoids that are potent inducers of cell cycle arrest may be particularly useful for the prevention of breast cancer.

This study was designed to evaluate, using preclinical models of non-small cell lung cancer (NSCLC), the growth inhibitory effects of the retinoid X receptor (RXR) agonist bexarotene (LGD1069, Targretin) in combination with cytotoxic agents currently used as standard first-line therapy in advanced disease. Although single-agent bexarotene had modest growth inhibitory effects in several cell lines, efficacy was observed only in the micromolar range (1muM), which approximates the plasma C(max) measured in pharmacokinetic studies in patients. However, when combined with paclitaxel or vinorelbine, bexarotene produced a concentration-dependent enhancement of the growth inhibitory activities of paclitaxel and vinorelbine. Formal synergy analysis using the Calu3 cell line demonstrated that the combination of bexarotene with either cytotoxic agent produced synergistic activity (combination index, CI1). The in vitro observations were confirmed in vivo in a NSCLC xenograft tumor model (Calu3), where both bexarotene/paclitaxel and bexarotene/vinorelbine combinations produced significantly greater antitumor effects than the single agents. These results demonstrate that bexarotene can cooperate with widely used cytotoxic agents to decrease the growth of NSCLC tumor cells both in vitro and in vivo, and suggest the potential benefit of adding a RXR-selective agonist in combination with chemotherapy for NSCLC treatment. Furthermore, the data support the clinical observation from phase I/IIa trials suggesting that bexarotene has beneficial effects on survival when used in combination with cytotoxic agents in advanced NSCLC.

Purpose: Paclitaxel is an important anticancer agent for the treatment of non–small cell lung cancer (NSCLC). However, its use in cancer therapy is limited by development of acquired drug resistance. The goal of this study was to determine the effect of bexarotene on development of acquired paclitaxel resistance in NSCLC. Experimental Design: Human NSCLC Calu3 cells were repeatedly treated in culture with intermittent paclitaxel alone or in combination with continuous bexarotene for 3 months. Thereafter, cells were isolated and characterized for their drug sensitivity in vitro and in vivo. Results: Repeat exposure to paclitaxel alone resulted in development of paclitaxel resistance with cross-resistance to multidrug resistance P-glycoprotein substrates, whereas the bexarotene/paclitaxel combination prevented the development of drug resistance and the cells remained chemosensitive. Furthermore, paclitaxel resistance could be overcome when the resistant cells were treated with the combination regimen. Fluctuation analysis showed that treatment with bexarotene decreased the rate of spontaneous development of paclitaxel resistance. In vivo, the bexarotene/paclitaxel combination regimen produced a statistically significant decrease in tumor growth in a Calu3 NSCLC xenograft model compared with the single agents (two-tailed, P < 0.05). In addition, paclitaxel-resistant Calu3 tumors treated with the bexarotene/paclitaxel combination showed greater delay in tumor growth compared with those treated with paclitaxel alone. Conclusions: Our results suggest that bexarotene may offer a novel approach to prevent and overcome paclitaxel resistance in patients with NSCLC.

All-trans-retinoic acid (RA) treatment of acute promyelocytic leukemia (APL) cases expressing the t(15;17) product, PML/RARalpha, is a successful example of differentiation therapy. Uncovering RA target genes is of considerable interest in APL. This study comprehensively examines in APL cells transcriptional and post-transcriptional regulation of the novel candidate RA target gene, G0S2, the G0/G1 switch gene. Reverse transcription (RT)-polymerase chain reaction (PCR) and heteronuclear PCR assays performed +/- treatment with the protein synthesis inhibitor cycloheximide (CHX) revealed G0S2 induction within 3 h of RA-treatment. Treatment with the RNA synthesis inhibitor actinomycin D did not implicate G0S2 transcript stabilization in the RA-mediated increase of G0S2 mRNA expression. Promoter elements of G0S2 were cloned into a reporter plasmid and retinoic acid receptor (RAR) co-transfection assays confirmed transcriptional activation after RA-treatment. Consistent with G0S2 being a direct RA target gene, retinoic acid response element (RARE) half-sites were found in this promoter. Mutation of these sites blocked RA-transcriptional activation of G0S2. To extend analyses to the protein expression level, a polyclonal anti-G0S2 antibody was derived and detected murine and human G0S2 species. G0S2 protein was rapidly induced in cultured NB4-S1 human APL cells and in APL transgenic mice treated with RA. An RAR pan-antagonist confirmed dependence on RARs for this induction. That these findings are clinically relevant was shown by analyses of APL cells derived directly from patients. These leukemic cells induced both a prominent increase in the cellular differentiation marker nitrotetrazolium blue (NBT) staining and marked increase in G0S2 expression. Taken together, these findings indicate G0S2 is an RA target gene. The functional role of G0S2 in retinoid response of APL warrants further study.

Background: We have previously shown that a retinoid X receptor (RXR)-selective ligand (a rexinoid), called LGD1069, is highly efficacious in both the chemoprevention and the chemotherapy for N-nitrosomethylureainduced rat mammary carcinomas. To evaluate a possible role for rexinoids in breast cancer therapy further, we have designed and characterized a novel carcinogen-induced model to mimic the clinical situation in which the tumors of patients stop responding to tamoxifen therapy and develop resistance to this drug. Methods: Rats with experimentally induced mammary tumors were treated with tamoxifen to select a population with primary tumors that failed to respond completely to the drug. Once the failure of tamoxifen therapy had been established, LGD1069 was added to the treatment regimen, and the tumors in these animals were compared with tumors in a group of animals that remained on tamoxifen alone. Results: LGD1069 in combination with tamoxifen for up to 20 weeks yielded an overall objective response rate of 94% (95% confidence interval [CI] = 86%-100%) (includes complete and partial responses) in primary tumors compared with a rate of 33% (95% CI = 11%-56%) in primary tumors treated with tamoxifen alone, a statistically significant difference (two-sided P

Jun and fos oncoproteins form a complex which regulates transcription from promoters containing AP-I binding sites. The "leucine zipper" domain of both fos and jun is necessary for the formation of the heterodimer, but the role of specific leucine residues is unclear. We have used site-specific mutagenesis to examine the contribution of individual leucine residues to the formation of a stable fos/jun protein complex and the binding of this complex to the AP-I site. Mutation of a single leucine in either fos or jun had no effect on protein complex formation. Furthermore, mutations of two consecutive leucines in jun did not interfere with heterodimer formation; however, in the case of fos, two consecutive mutations resulted in an inability to form a heterodimer. Although mutagenesis of the first leucine of the heptad repeat had no effect on protein complex formation, this mutation in either fos or jun drastically reduced the affinity of the complex for DNA. Thus, both fos and jun contribute directly to the DNA binding potential of the heterodimer.