William W. Lamph

Activation of retinoid X receptors induces apoptosis in HL-60 cell lines.

Retinoids induce myeloblastic leukemia (HL-60) cells to differentiate into granulocytes, which subsequently die by apoptosis. Retinoid action is mediated through at least two classes of nuclear receptors: retinoic acid receptors, which bind both all-trans retinoic acid and 9-cis retinoic acid, and retinoid X receptors, which bind only 9-cis retinoic acid. Using receptor-selective synthetic retinoids and HL-60 cell sublines with different retinoid responsiveness, we have investigated the contribution that each class of receptors makes to the processes of cellular differentiation and death. Our results demonstrate that ligand activation of retinoic acid receptors is sufficient to induce differentiation, whereas ligand activation of retinoid X receptors is essential for the induction of apoptosis in HL-60 cell lines.

The efficacy of 9-cis retinoic acid in experimental models of cancer

Summary9-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 ofin vivo andin 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.

The Selective Estrogen Receptor Modulator Arzoxifene and the Rexinoid LG100268 Cooperate to Promote Transforming Growth Factor β-Dependent Apoptosis in 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.

A Selective Retinoid X Receptor Agonist Bexarotene (Targretin) Prevents and Overcomes Acquired Paclitaxel (Taxol) Resistance in Human Non–Small Cell Lung Cancer

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.

The Rexinoid LG100268 prevents the development of preinvasive and invasive estrogen receptor negative tumors in MMTV-erbB2 mice.

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.

The combination of the rexinoid, LG100268, and a selective estrogen receptor modulator, either arzoxifene or acolbifene, synergizes in the prevention and treatment of mammary tumors in an estrogen receptor-negative model of breast cancer

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.

Receptor-selective retinoids inhibit the growth of normal and malignant breast cells by inducing G1 cell cycle blockade

SummaryDespite 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.

Synergistic effect of a retinoid X receptor-selective ligand bexarotene (LGD1069, Targretin) and paclitaxel (Taxol) in mammary carcinoma

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.

The rexinoid bexarotene represses cyclin D1 transcription by inducing the DEC2 transcriptional repressor

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.

Rexinoid-induced Expression of IGFBP-6 Requires RARβ-dependent Permissive Cooperation of Retinoid Receptors and AP-1

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), RARβ, 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 RXRα, RARβ, cJun, cFos, and p300. In normal mammary epithelial cells and T47D breast cancer cells, small interfering RNA-mediated knockdown of all RXR isoforms or RARβ, but not RARα or RARγ alone, blocked the induction of IGFBP-6 by bexarotene. Simultaneous knockdown of RARα and RARγ abrogated both the induction of RARβ and the up-regulation and secretion of IGFBP-6. The suppression of either RARβ or cJun by small interfering RNA blocked the recruitment of RXRα and cJun to the enhancer. These results demonstrate a novel cooperative interaction between retinoid receptors and AP-1 orchestrated by RARβ and highlight a novel mechanism by which RARβ can mediate the cancer-preventive effects of rexinoids.