The epigenetic immunomodulator, HBI-8000, enhances the response and reverses resistance to checkpoint inhibitors.

BACKGROUND: Treatment with immune checkpoint inhibitors (ICIs) targeting CTLA-4 and the PD-1/PD-L1 axis is effective against many cancer types. However, due in part to unresponsiveness or acquired resistance, not all patients experience a durable response to ICIs. HBI-8000 is a novel, orally bioavailable class I selective histone deacetylase inhibitor that directly modifies antitumor activity by inducing apoptosis, cell cycle arrest, and resensitization to apoptotic stimuli in adult T cell lymphoma patients. We hypothesized that HBI-8000 functions as an epigenetic immunomodulator to reprogram the tumor microenvironment from immunologically cold (nonresponsive) to hot (responsive). METHOD: Mice bearing syngeneic tumors (MC38 and CT26 murine colon carcinoma and A20 B-cell lymphoma were treated daily with HBI-8000 (orally), alone or in combination with PD-1, PD-1 L, or CTLA-4 antibodies. MC38 tumors were also analyzed in nanoString gene expression analysis. RESULTS: HBI-8000 augmented the activity of ICI antibodies targeting either PD-1, PD-L1 or CTLA-4, and significantly increased tumor regression (p < 0.05) in the above models. Gene expression analysis of the treated MC38 tumors revealed significant changes in mRNA expression of immune checkpoints, with enhanced dendritic cell and antigen-presenting cell functions, and modulation of MHC class I and II molecules. CONCLUSIONS: These findings suggest that HBI-8000 mediates epigenetic modifications in the tumor microenvironment, leading to improved efficacy of ICIs, and provide strong rationale for combination therapies with ICIs and HBI-8000 in the clinical setting. PRECIS: As an HDACi, HBI-8000 plays an important role in priming the immune system in the tumor microenvironment. The current preclinical data further justifies testing combination of HBI-8000 and ICIs in the clinic.

Induction of apoptosis by HBI-8000 in adult T-cell leukemia/lymphoma is associated with activation of Bim and NLRP3.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell malignancy caused by human T-cell lymphotropic virus 1. Treatment options for acute ATL patients include chemotherapy, stem cell transplantation, and recently the anti-chemokine (C-C motif) receptor 4 antibody, although most patients still have a poor prognosis and there is a clear need for additional options. HBI-8000 is a novel oral histone deacetylase inhibitor with proven efficacy for treatment of T-cell lymphomas that recently received approval in China. In the present study, we evaluated the effects of HBI-8000 on ATL-derived cell lines and primary cells obtained from Japanese ATL patients. In most cases HBI-8000 induced apoptosis in both primary ATL cells and cell lines. In addition, findings obtained with DNA microarray suggested Bim activation and, interestingly, the contribution of the NLR family, pyrin domain containing 3 (NLRP3) inflammasome pathway in HBI-8000-induced ATL cell death. Further investigations using siRNAs confirmed that Bim contributes to HBI-8000-induced apoptosis. Our results provide a rationale for a clinical investigation of the efficacy of HBI-8000 in patients with ATL. Although the role of NLRP3 inflammasome activation in ATL cell death remains to be verified, HBI-8000 may be part of a novel therapeutic strategy for cancer based on the NLRP3 pathway.

The combination of tamoxifen and the rexinoid LG100268 prevents ER-positive and ER-negative mammary tumors in p53-null mammary gland mice.

In pursuit of effective therapeutic agents for the estrogen receptor (ER)-negative breast cancer, we previously showed that bexarotene reduced mammary tumor development by 75% in ErbB2 mice. To further improve the effectiveness of breast cancer prevention, we have now investigated the effects of a combinatorial therapy consisting of two cancer preventive drugs. On the basis of the hypothesis, rexinoid LG100268 plus tamoxifen would more effectively prevent the development of both ER-positive and ER-negative breast cancer. We treated p53-null mammary gland mice with tamoxifen and LG100268, individually and in combination. By 60 weeks of age, vehicle-treated mice developed tumors in 52% of transplanted mammary glands, whereas mice treated with tamoxifen and LG100268 developed tumors in only 13% of transplanted mammary glands. To further define the mechanistic effects of this combinatorial treatment, we investigated the effects of tamoxifen and LG100268 on mammary tissue biomarkers. In mammary tissue harvested before tumor development, the proliferation markers Ki67 and cyclin D1 were significantly reduced in mice treated with the combination therapy. In addition, the rexinoid target genes ABCA1 and ABCG1 were induced in both the rexinoid and combination treatment groups, whereas expression remained constant in tamoxifen group. These results show that tamoxifen-LG100268 combinatorial treatment is more effective in preventing mammary tumors than either agent alone. In addition, these studies have identified relevant tissue biomarkers that can be used to show the effect of these agents on mammary tissue. These results support the development of clinical trials of antiestrogen and rexinoid combinatorial therapy for the prevention of patients with high-risk breast cancer.

Cancer-preventive rexinoid modulates neutral lipid contents of mammary epithelial cells through a peroxisome proliferator-activated receptor γ-dependent mechanism.

Synthetic rexinoids effectively suppress both estrogen receptor-positive and estrogen receptor-negative mammary tumors in animal models, which makes them prime candidates for a novel class of cancer-preventive agents. When used in combination with chemotherapy for non-small-cell lung cancer, the rexinoid bexarotene was most effective for patients who developed hypertriglyceridemia as a side effect. Although serum triglycerides originate from the liver, the effect of bexarotene on lipogenesis in breast epithelial cells is not known. Gene expression studies with normal mammary epithelial cells indicated that rexinoids modulate lipid metabolism, particularly enzymes involved in triglyceride synthesis. High-content analysis revealed dose-dependent accumulation of neutral lipids within adipocyte differentiation-related protein-associated cytoplasmic lipid droplets after long-term bexarotene treatment. Bexarotene also induced mRNA and protein levels for peroxisome proliferator-activated receptor (PPAR) γ, whereas selective knockdown of PPARγ attenuated the induction of both lipid droplets and adipocyte differentiation-related protein. Pharmacological activation of PPARγ, but not PPARα or retinoic acid receptors, effectively induced lipid accumulation. Furthermore, the combination of the PPARγ agonist rosiglitazone with bexarotene synergistically suppressed the growth of human mammary epithelial cells and revealed a strong, nonlinear, inverse correlation of cell growth with lipid droplet accumulation in the cell population. These findings indicate that rexinoids activate a lipogenic program in mammary epithelial cells through a retinoid X receptor/PPARγ-mediated mechanism. It is noteworthy that combining low doses of bexarotene with the PPARγ agonist rosiglitazone provides effective growth suppression of mammary epithelial cells, potentially dissociating systemic adverse effects associated with standard bexarotene treatment from the antiproliferative effects on mammary epithelium.

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.

Combination PPARgamma and RXR Agonist Treatment in Melanoma Cells: Functional Importance of S100A2.

Nuclear hormone receptors, including RXR and PPARgamma, represent novel therapeutic targets in melanoma. We have previously shown that the DRO subline of the amelanotic melanoma A375 responds to rexinoid and thiazolidinedione (TZD) treatment in vitro and in vivo. We performed microarray analysis of A375(DRO) after TZD and combination rexinoid/TZD treatment in which the calcium binding protein S100A2 had increased expression after rexinoid or TZD treatment and a synergistic increase to combination treatment. Increased S100A2 expression is dependent on an intact PPARgamma receptor, but it is not sufficient to mediate the antiproliferative effects of rexinoid/TZD treatment. Over expression of S100A2 enhanced the effect of rexinoid and TZD treatment while inhibition of S100A2 expression attenuated the response to rexinoid/TZD treatment, suggesting that S100A2 is necessary for optimal response to RXR and PPARgamma activation by respective ligands. In summary, we have identified potential downstream mediators of rexinoid and TZD treatment in a poorly differentiated melanoma and found that alterations in S100A2 expression affect RXR and PPARgamma signaling in A375(DRO) cells. These studies provide insight into potential mechanisms of tumor response or resistance to these novel therapies.

Identification of modulated genes by three classes of chemopreventive agents at preneoplastic stages in a p53-null mouse mammary tumor model.

Genetically engineered mouse cancer models are among the most useful tools for testing the in vivo effectiveness of the various chemopreventive approaches. The p53-null mouse model of mammary carcinogenesis was previously characterized by us at the cellular, molecular, and pathologic levels. In a companion article, Medina et al. analyzed the efficacy of bexarotene, gefitinib, and celecoxib as chemopreventive agents in the same model. Here we report the global gene expression effects on mammary epithelium of such compounds, analyzing the data in light of their effectiveness as chemopreventive agents. SAGE was used to profile the transcriptome of p53-null mammary epithelium obtained from mice treated with each compound versus controls. This information was also compared with SAGE data from p53-null mouse mammary tumors. Gene expression changes induced by the chemopreventive treatments revealed a common core of 87 affected genes across treatments (P < 0.05). The effective compounds, bexarotene and gefitinib, may exert their chemopreventive activity, at least in part, by affecting a set of 34 genes related to specific cellular pathways. The gene expression signature revealed various genes previously described to be associated with breast cancer, such as the activator protein-1 complex member Fos-like antigen 2 (Fosl2), early growth response 1 (Egr1), gelsolin (Gsn), and tumor protein translationally controlled 1 (Tpt1), among others. The concerted modulation of many of these transcripts before malignant transformation seems to be conducive to predominantly decrease cell proliferation. This study has revealed candidate key pathways that can be experimentally tested in the same model system and may constitute novel targets for future translational research.

Rexinoid-induced expression of IGFBP-6 requires RARbeta-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), 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.

Transcriptomic signature of bexarotene (rexinoid LGD1069) on mammary gland from three transgenic mouse mammary cancer models.

BACKGROUND: The rexinoid bexarotene (LGD1069, Targretin) is a highly selective retinoid x receptor (RXR) agonist that inhibits the growth of pre-malignant and malignant breast cells. Bexarotene was shown to suppress the development of breast cancer in transgenic mice models without side effects. The chemopreventive effects of bexarotene are due to transcriptional modulation of cell proliferation, differentiation and apoptosis. Our goal in the present study was to obtain a profile of the genes modulated by bexarotene on mammary gland from three transgenic mouse mammary cancer models in an effort to elucidate its molecular mechanism of action and for the identification of biomarkers of effectiveness. METHODS: Serial analysis of gene expression (SAGE) was employed to profile the transcriptome of p53-null, MMTV-ErbB2, and C3(1)-SV40 mammary cells obtained from mice treated with bexarotene and their corresponding controls. RESULTS: This resulted in a dataset of approximately 360,000 transcript tags representing over 20,000 mRNAs from a total of 6 different SAGE libraries. Analysis of gene expression changes induced by bexarotene in mammary gland revealed that 89 genes were dysregulated among the three transgenic mouse mammary models. From these, 9 genes were common to the three models studied. CONCLUSION: Analysis of the indicated core of transcripts and protein-protein interactions of this commonly modulated genes indicate two functional modules significantly affected by rexinoid bexarotene related to protein biosynthesis and bioenergetics signatures, in addition to the targeting of cancer-causing genes related with cell proliferation, differentiation and apoptosis.

G0S2 is an all-trans-retinoic acid target gene.

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.

In vivo and microarray analysis of rexinoid-responsive anaplastic thyroid carcinoma.

PURPOSE: Anaplastic thyroid carcinoma is rare, yet lethal despite aggressive therapy. Molecular targeting may be beneficial using the rexinoid LGD1069, a retinoid X receptor-selective agonist, as a novel treatment. In this report, we describe the efficacy of LGD1069 in anaplastic thyroid carcinoma in vitro and assess the in vivo treatment effects on a responsive cancer. Additionally, we explore potential mediators of the rexinoid effect on a responsive anaplastic thyroid cancer using comparative microarray analysis. EXPERIMENTAL DESIGN: Anaplastic thyroid cancer cell lines DRO, ARO, and FRO were treated with LGD1069 in vitro. Responsive DRO xenograft tumors were treated with control chow or chow containing a low dose (30 mg/kg/d) or a high dose (100 mg/kg/d) of LGD1069. Comparative microarray analysis of DRO cells treated with LGD1069 compared with volume-equivalent control was assessed after 24 h of treatment to evaluate early gene expression changes. RESULTS: DRO xenograft tumor growth was inhibited by LGD1069 treatment in a dose-dependent manner. Comparative microarray analysis showed that 80 genes had a significant increase in expression and 29 genes had a decrease in expression after 24 h of treatment with LGD1069. Expression of angiopoietin-like 4 (ANGPTL4) mRNA was increased 6.5-fold. A trend towards an increase in ANGPTL4 mRNA (not statistically significant) was seen in treated tumors in vivo and this correlated with decreased tumor vascularity and increased necrosis. CONCLUSIONS: LGD1069 therapy decreases proliferation in an anaplastic thyroid cancer cell line that expresses retinoid X receptor-gamma, and this effect is confirmed with decreased tumor size in vivo in a nude mouse model. ANGPTL4 is increased in DRO in response to LGD1069 and may be a potential mediator of the effects of rexinoid treatment.

Combination chemoprevention of HER2/neu-induced breast cancer using a cyclooxygenase-2 inhibitor and a retinoid X receptor-selective retinoid.

The inducible prostaglandin synthase isoform cyclooxygenase-2 (COX-2) is overexpressed in approximately 40% of human breast carcinomas and in precancerous breast lesions, particularly in association with overexpression of human epidermal growth factor receptor 2 (HER2/neu). Experimental breast cancer can be suppressed by pharmacologic inhibition or genetic ablation of Cox-2, suggesting potential clinical utility of COX-2 inhibitors with respect to breast cancer. Importantly, several clinical trials have found reduced colorectal adenoma formation in individuals administered selective COX-2 inhibitors. However, such trials also identified increased cardiovascular risk associated with COX-2 inhibitor use. The goal of this research was to test whether improved chemopreventive efficacy could be achieved by combining submaximal doses of a selective COX-2 inhibitor and a retinoid X receptor-selective retinoid (rexinoid). The rate of HER2/neu-induced mammary tumor formation was substantially delayed by coadministration of the COX-2 inhibitor celecoxib (500 ppm in diet) and the rexinoid LGD1069 (10 mg/kg body weight; oral gavage) to MMTV/neu mice. Median time to tumor formation was increased from 304 to >600 days (P < 0.0001). The combination was substantially more effective than either drug individually. Similarly, potent suppression of aromatase activity was observed in mammary tissues from the combination cohort (44% of control; P < 0.001). Regulation of aromatase expression and activity by COX-derived prostaglandins is well established. Interestingly however, single agent LGD1069 significantly reduced mammary aromatase activity (71% of control; P < 0.001) without modulating eicosanoid levels. Our data show that simultaneous blockade of COX/prostaglandin signaling and retinoid X receptor-dependent transcription confers potent anticancer efficacy, suggesting a novel avenue for clinical evaluation.

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.

Synthesis and characterization of nonsteroidal glucocorticoid receptor modulators for multiple myeloma.

Structure-activity relationship studies centered around 3'-substituted (Z)-5-(2'-(thienylmethylidene))1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5H-chromeno[3,4-f]quinolines are described. A series of highly potent and efficacious selective glucocorticoid receptor modulators were identified with in vitro activity comparable to dexamethasone. In vivo evaluation of these compounds utilizing a 28 day mouse tumor xenograft model demonstrated efficacy equal to dexamethasone in the reduction of tumor volume.

Identification of biomarkers modulated by the rexinoid LGD1069 (bexarotene) in human breast cells using oligonucleotide arrays.

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.

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

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.

Differentiation and growth inhibition mediated via the RXR:PPARgamma heterodimer in colon cancer.

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.

Retinoid-induced mu opioid receptor expression by phytohemagglutinin-stimulated U937 cells.

Opioid use may be associated with an increased risk of neurological disease in human immunodeficiency virus (HIV) infection through effects on immune cell function. Studies were performed to examine the effects of specific retinoid receptor activation on mu opioid receptor (MOR) production by phytohemagglutinin (PHA)-stimulated U937 cells, a mononuclear cell line. PHA stimulation increased activation of the MOR promoter as well as levels of MOR mRNA, total receptor protein in cell lysates, and surface and cytoplasmic receptor expression. Retinoid X receptor (RXR) agonist and retinoic acid receptor (RAR) antagonist further increased MOR expression by the PHA-stimulated cells. In contrast, MOR expression was suppressed by RAR agonist and by RXR antagonist. Finally, opioid receptor binding was also increased by RXR agonist and RXR antagonist; no increase in binding occurred in the presence of RAR agonists and RXR antagonist. All together, these studies suggest that MOR expression in U937 cells can be differentially regulated by specific retinoid receptor activation. Such effects may have important clinical relevance for opioid users with HIV infection, including individuals with neurological disease.

The retinoid X receptor agonist bexarotene (Targretin) synergistically enhances the growth inhibitory activity of cytotoxic drugs in non-small cell lung cancer cells.

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, CI<1). 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.

The retinoid X receptor-selective retinoid, LGD1069, down-regulates cyclooxygenase-2 expression in human breast cells through transcription factor crosstalk: implications for molecular-based chemoprevention.

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.