Retinoic Acid Receptor Target Genes Research Articles

Identification of downstream effectors of retinoic acid specifying the zebrafish pancreas by integrative genomics

Retinoic acid (RA) is a key signal for the specification of the pancreas. Still, the gene regulatory cascade triggered by RA in the endoderm remains poorly characterized. In this study, we investigated this regulatory network in zebrafish by combining RNA-seq, RAR ChIP-seq and ATAC-seq assays. By analysing the effect of RA and of the RA receptor (RAR) inverse-agonist BMS493 on the transcriptome and on the chromatin accessibility of endodermal cells, we identified a large set of genes and regulatory regions regulated by RA signalling. RAR ChIP-seq further defined the direct RAR target genes in zebrafish, including hox genes as well as several pancreatic regulators like mnx1, insm1b, hnf1ba and gata6. Comparison of zebrafish and murine RAR ChIP-seq data highlighted the conserved direct target genes and revealed that some RAR sites are under strong evolutionary constraints. Among them, a novel highly conserved RAR-induced enhancer was identified downstream of the HoxB locus and driving expression in the nervous system and in the gut in a RA-dependent manner. Finally, ATAC-seq data unveiled the role of the RAR-direct targets Hnf1ba and Gata6 in opening chromatin at many regulatory loci upon RA treatment.

A Novel Approach to Induce ATRA Mediated Differentiation in NPM1 Mutant Acute Myeloid Leukemia

▪Introduction: Retinoic acid (RA), an active metabolite of vitamin A, activates the retinoic acid receptors (RARs), ligand-controlled transcription factors that regulate expression of target genes affecting homeostasis and cellular differentiation. The success of RA in the form of all-trans retinoic acid (ATRA) to treat acute promyelocytic leukemia (APL) has stimulated considerable interest in the development of small molecules to modulate RARs in AML cells. Besides RARs, RA can also activate the pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a member of the nuclear hormone receptor superfamily of transcription factors that regulate several metabolic pathways in a tissue-selective manner. In the aqueous intracellular milieu, RA is transported by two alternative carriers, cellular retinoid-binding protein CRABP2, or fatty-acid-binding protein 5 (FABP5). These carriers preferentially deliver RA to RARs or PPARβ/δ respectively. In cells, expressing high CRABP2 and low FABP5, RA is delivered to and activates RARs, whereas in the presence of the reverse ratio, RA is delivered to and activates PPARβ/δ. Here we demonstrate that these two different modes of RA delivery leads to opposite cellular outcomes: CRABP2-mediated RA delivery to RAR prompts differentiation and growth arrest, while in cells containing high levels of FABP5, RA delivery to PPARβ/δ promotes survival and exponential proliferation. Inhibiting FABP5 carrier-function could be a useful method, therefore, to extend RA therapeutic activity to non-APL acute myeloid leukemia (AML).Methods: Genetic as well as pharmacologic in vitro and in vivo models of AML were used to validate FABP5 as a target for therapy to, increase RA stimulation of RARs instead of PPARβ/δ. Knockdown and overexpression of key regulators of these pathways were rationally used to understand pro-survival effects and proliferative mechanism of malignant myeloid cells. The efficacy of small molecule pharmacophore targeting FABP5 was evaluated in vitro in cell free and cell culture model as well as in vivo small animal model systems as a proof of therapeutic concept.Results: In public databases of AML gene expression, the majority of non-APL AMLs demonstrated upregulation of FABP5 and downregulation of CRABP2, potentially explaining limited benefit of RA to treat non-APL AMLs. On the other hand, ATRA sensitive APL samples demonstrated low FABP5, as expected from the established efficacy of RA to treat APLs. The greatest elevations of pro-survival FABP5 was in AMLs with mutated-NPM1. Consistent with the elevated FABP5 in these cells, we confirmed that RA activates pro-survival PPARβ/δ rather than pro-differentiation RAR in NPM1-mutated AML cells. We thus evaluated if inhibiting FABP5 could produce ATRA sensitization in NPM1 mutant AML cells. Genetic ablation of FABP5 using shRNA sensitized AML cells to ATRA treatment, while this effect was not seen in AML cells transfected with non-targeting scrambled shRNA. Cells stably expressing shRNA targeting FABP5 has significant growth perturbation in vivo in murine AML model. To target FABP5 using a small molecule approach, we used in silico drug-design to develop a novel small molecule FABP5 inhibitor (iFABP5): the inhibitor bound to recombinant FABP5 (Kd= 2x10 -7) with a 56-fold higher affinity than ATRA (Kd=11x10 -6 Mole), demonstrated in a cell free fluorescence reporter binding assay using 8-Anilino-1-naphthalenesulfonic acid (ANS). We then treated RA-resistant AML cells with iFABP5 alone and in combination with RA: neither iFABP5 nor RA alone had anti-proliferative activity as expected, but combination iFABP5 + RA downregulated PPARβ/δ target genes and upregulated RAR target genes, terminating AML cells growth.Conclusion: The lack of effective ATRA response in AML might be, in part, due to high expression of FABP5 and delivery of RA to pro-survival PPARβ/δ. A small molecule inhibitor of FABP5 could redirect RA to pro-terminal-differentiation RARs, and could be an approach to extending the remarkable activity of RA in APL to other AMLs. Reprogramming of the RA delivery to RAR pathway with the addition of a small molecular inhibitor of FABP5 could potentially restore therapeutic effects of ATRA in FABP5 over expressing cases of AML subtypes. DisclosuresMaciejewski: Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy. Saunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Carraway: Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Other: Independent review committee; Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astex: Other: Independent review committee.

Fatty Acid Binding Protein FABP5: A Novel Therapeutic Target in Acute Myeloid Leukemia

Introduction: Retinoic acid (RA), the active metabolite of vitamin A, influences biological processes by activating the retinoic acid receptor (RAR). RARs are ligand-controlled transcription factors that function as heterodimer with retinoid X receptors (RXRs) and regulate expression of target genes affecting homeostasis of cellular differentiation and death. The success of RAR activation in the treatment of acute promyelocytic leukemia (APL), particularly by the use of all-trans retinoic acid (ATRA) has stimulated considerable interest in the development of small molecules that can modulate RAR and RXR in AML cells. RA can also activate the pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ) in a context dependent manner. In the aqueous intracellular milieu, RA is transported by the cellular retinoid-binding protein CRABP-II, or by the fatty-acid-binding protein FABP5, depending on the ratio of FABP5 to CRABP-II and cellular levels of RA. In cells, expressing high CRABP-II and low FABP5, RA activates the RAR, whereas in the presence of the reverse ratio, RA activates PPARβ/δ. These two different modes of RA delivery leads to opposite cellular outcomes. Cells harboring high level of CRABP-II, RA is delivered to RAR leading to differentiation and growth arrest. However, FABP5 high expressing cells, RA is delivered to PPARβ/δ resulting in survival and proliferation. Therefore, preventing FABP5 from such antagonism may be a novel therapeutic strategy for AML with high FABP5. Here, we report the development of a therapeutic approach using a specific FABP5 inhibitor (iFABP5) that facilitates the delivery of RA to tumor suppressor RAR. Methods: Genetic as well as pharmacologic models in AML cell lines were used to validate the therapeutic target by decoupling PPARβ/δ and RAR/RXR pathways. Knockdown and overexpression of key regulators of these pathways were rationally used to understand pro-survival effects of FABP5 overexpression. The efficacy of small molecule pharmacophore were evaluated in vitro in cell free and cell culture model systems as well as in vivo small animal model systems as a proof of therapeutic concept. Results: We analyzed AML samples from different cohorts (Beat AML and TCGA) for the expression levels of genes involved in RAR and PPARβ/δ pathways. Most of the AML patients have upregulation of pro-survival PPARβ/δ pathway genes including FABP5 and downregulation of differentiating promoting RAR pathway genes including critical RA chaperone CRABP2. On the otherhand, ATRA sensitive APL patients and cell lines have lower expression of FABP5 compared to AML. We established FABP5 as a therapeutic target for ATRA sensitization in AML cell line THP1. Genetic ablation of FABP5 using shRNA sensitizes THP1 cells to ATRA treatment compared to scramble control cells. We used in silico approach to develop a novel small molecule iFABP5 that binds to recombinant FABP5 (Kd= 2x10-7) with a 56-fold higher affinity compared to ATRA (Kd=11x10-6 Mole) in a cell free fluorescence reporter binding assay using 8-Anilino-1-naphthalenesulfonic acid (ANS). Efficacy of iFABP5 as a single agent and in combination with ATRA was evaluated in THP1, HL-60, K562, and OCI-AML5 cells. The effect of the combination was most pronounced in THP-1 cells. The iFABP5 synergizes with ATRA and induces the differentiation at 72 hours as assessed by both CD11b and CD14 levels. Treatment of cells with single agent iFABP5 leads to downregulation of PPARβ/δ target genes and upregulation of RAR target genes consistent with the genetic ablation of shFABP5. THP1 cells stably expressing shRNA targeting FABP5 has significant growth perturbation in a NSG mouse model compared to non-targeting shRNA. Pre-clinical in vivo evaluation of iFABP5 in combination with ATRA is ongoing as a therapeutic proof of concept. Conclusion: The lack of effective ATRA response in AML might be due to an aberrant activation of pro-survival PPARβ/δ pathway, which negatively affects ATRA-regulated gene expression and its antileukemic activity. Reprogramming of the RA delivery to RAR pathway with the addition of a small molecular inhibitor of FABP5 could potentially restore therapeutic effects of ATRA in FABP5 over expressing cases of AML subtypes. Disclosures Maciejewski: Novartis: Consultancy; Alexion: Consultancy.

Myofibroblasts acquire retinoic acid–producing ability during fibroblast-to-myofibroblast transition following kidney injury

Tubular injury and interstitial fibrosis are the hallmarks of chronic kidney disease. While recent studies have verified that proximal tubular injury triggers interstitial fibrosis, the impact of fibrosis on tubular injury and regeneration remains poorly understood. We generated a novel mouse model expressing diphtheria toxin receptor on renal fibroblasts to allow for the selective disruption of renal fibroblast function. Administration of diphtheria toxin induced upregulation of the tubular injury marker Ngal and caused tubular proliferation in healthy kidneys, whereas administration of diphtheria toxin attenuated tubular regeneration in fibrotic kidneys. Microarray analysis revealed down-regulation of the retinol biosynthesis pathway in diphtheria toxin-treated kidneys. Healthy proximal tubules expressed retinaldehyde dehydrogenase 2 (RALDH2), a rate-limiting enzyme in retinoic acid biosynthesis. After injury, proximal tubules lost RALDH2 expression, whereas renal fibroblasts acquired strong expression of RALDH2 during the transition to myofibroblasts in several models of kidney injury. The retinoic acid receptor (RAR) RARγ was expressed in proximal tubules both with and without injury, and αB-crystallin, the product of an RAR target gene, was strongly expressed in proximal tubules after injury. Furthermore, BMS493, an inverse agonist of RARs, significantly attenuated tubular proliferation invitro. In human biopsy tissue from patients with IgA nephropathy, detection of RALDH2 in the interstitium correlated with older age and lower kidney function. These results suggest a role of retinoic acid signaling and cross-talk between fibroblasts and tubular epithelial cells during tubular injury and regeneration, and may suggest a beneficial effect of fibrosis in the early response to injury.

Antagonistic Interactions between Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase and Retinoic Acid Receptor Signaling in Colorectal Cancer Cells.

Deregulated activation of RAS/extracellular signal-regulated kinase (ERK) signaling and defects in retinoic acid receptor (RAR) signaling are both implicated in many types of cancers. However, interrelationships between these alterations in regulating cancer cell fates have not been fully elucidated. Here, we show that RAS/ERK and RAR signaling pathways antagonistically interact with each other to regulate colorectal cancer (CRC) cell fates. We show that RAR signaling activation promotes spontaneous differentiation of CRC cells, while ERK activation suppresses it. Our microarray analyses identify genes whose expression levels are upregulated by RAR signaling. Notably, one of these genes, MKP4, encoding a member of dual-specificity phosphatases for mitogen-activated protein (MAP) kinases, mediates ERK inactivation upon RAR activation, thereby promoting the differentiation of CRC cells. Moreover, our results also show that RA induction of RAR target genes is suppressed by the ERK pathway activation. This suppression results from the inhibition of RAR transcriptional activity, which is shown to be mediated through an RIP140/histone deacetylase (HDAC)-mediated mechanism. These results identify antagonistic interactions between RAS/ERK and RAR signaling in the cell fate decision of CRC cells and define their underlying molecular mechanisms.

Highlights of This Issue

Mastermind-like 3 (MAML3) is a known co-activator of NOTCH signaling. Heynen and colleagues find an additional and unexpected role for MAML3 in retinoic acid signaling in neuroblastoma, which is used clinically to induce differentiation in this cancer. Elevated expression of MAML3 leads to loss of activation of a subset of retinoic acid receptor target genes and as a consequence blocks retinoic acid-induced differentiation. MAML3 also has retinoic acid independent functions in neuroblastoma through upregulation of IGF2, which activates AKT signaling downstream of IGF1R. These data shed light on why MAML3 is mutated in multiple cancers.Here, the functional and molecular consequences of cytotoxic chemotherapy treatment on pancreatic cancer-associated fibroblasts (CAFs) were investigated. Chemotherapy treated CAFs enhanced tumor cell viability, migration and invasion in vitro as well as tumor growth in vivo compared to treatment naïve CAFs. A pro-inflammatory gene expression signature mediated by stress-associated MAPK was identified in chemotherapy treated CAFs. Stress-associated MAPK inhibitors decreased expression of inflammatory mediators and reduced tumor cell viability, migration, and invasion in vitro as well as tumor growth in vivo. These results describe a novel molecular response to chemotherapy in pancreatic CAFs and represent a future therapeutic target.Increased signaling from growth-factor receptors and stress-related kinases plays a role in endocrine resistance, a major challenge in the treatment of estrogen receptor-positive breast cancer (BC) patients. An integrative in silico transcriptomic/cistromic approach implicated the AP-1 transcription complex as a signaling node in endocrine resistance. When AP-1 activity was inhibited by an inducible genetic approach in BC cells grown in vitro and in vivo, the blockade enhanced sensitivity to endocrine therapy, delayed resistance onset, and overcame the growth of tamoxifen-resistant tumors. These data demonstrate AP-1 as a driver of endocrine resistance and offer new perspectives for innovative treatment and biomarker research.The article by Kallifatidis and colleagues defines the heterotypic Src-mediated transactivation of the epidermal growth factor receptor (EGFR) by the atypical chemokine receptor, CXCR7, in prostate cancer. The highlight of the discovery is that the scaffold protein β-Arrestin 2 was found to regulate this transactivation and reduce the phosphorylation of EGFR at Tyrosyl 1110, one of the seven cytoplasmic phosphorylation domains of EGFR. Further, down-regulation of β-Arrestin 2 leads to increased mitogenic activity, decreased p21, and re-location of EGFR to the nucleus. This study reveals important tumor suppressor activity of β-Arrestin 2 in the context of EGFR signaling.

Abstract 5341: Bifunctional integrated molecules with combined retinoic acid receptor agonist/protein deacetylase inhibitory activities as therapeutic agents for breast cancer and neuroblastoma

Abstract All-trans retinoic acid (ATRA), the oxidized form of Vitamin A, is a potent antiproliferative and differentiating agent that acts as an agonist of the retinoic acid receptors (RARs). Both 13-cis or all-trans retinoic acid are clinically used in differentiation or maintenance therapies for APL and neuroblastoma respectively. Their efficacy is however limited by resistance mechanisms. The use of small molecule inhibitors of HDACs (HDACi) for cancer treatment has been clinically validated for pan-HDAC inhibitors like SAHA (Vorinostat) or Romidepsin (Istodax) that are approved for the treatment of cutaneous T-cell lymphoma. The use of pan-HDAC inhibitors is restricted by dose-limiting toxicities at pharmacologically relevant doses, highlighting the need for alternative strategies such as use in combined therapies. Notably, synergistic activity was observed between retinoids and HDACs, which both induce cell differentiation, in a number of cancer models. Using molecular design we engineered a series of integrated hybrid small molecules that incorporates a deacetylase inhibitory activity and retinoic acid receptor (RAR) agonist. We show that these hybrid molecules inhibit the growth and survival of a wide range of cancer cell lines. In breast cancer cell lines, hybrids were shown to induce the recruitment of co-activators to RARα to the same extent as retinoic acid (RA) and to induce the release of co-repressors from RARs. Regulation of RAR-dependent transcription was shown in breast cancer and neuroblastoma cell lines using RAR reporter assays. Compounds displayed EC50 in the 10-500 nM range and an efficacy comparable to 13 cis-RA. In addition, the ability of compounds to regulate the expression of RAR target genes was confirmed in cells of both breast and neuroblastoma origin. Biochemical enzymatic assays show that HR- hybrids differentially inhibit HDAC activities with a two-log selectivity toward HDAC6 and HDAC. This inhibitory profile translated in both increased cellular acetylation of target proteins and in the down-regulation of anti-apoptotic target genes. Cytotoxicity toward breast or neuroblastoma cells correlated positively with targets’ activities. Strikingly, under conditions where 13 cis RA, SAHA or their combination had a modest or no effect on basal breast carcinoma cells viability, compounds with dual activity displayed a pronounced cytotoxic effect in the sub-uM range. Compounds were also active in the sub-micromolar range in N-Myc-amplified neuroblastoma lines IMR-32 or BE2C. In contrast compounds did not display significant cytotoxicity in non-tumorigenic primary cell lines under conditions where SAHA strongly inhibited the survival, proliferation or differentiation of these cells. Preliminary pharmacodynamic (PK) show that compounds were orally bioavailable and displayed favorable PK characteristics. Citation Format: David Cotnoir-White, Angela Miller, Bin Zhao, Isroel Weiss, James Gleason, David Bettoun, Sylvie Mader. Bifunctional integrated molecules with combined retinoic acid receptor agonist/protein deacetylase inhibitory activities as therapeutic agents for breast cancer and neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5341. doi:10.1158/1538-7445.AM2015-5341

Retinoid Acid Specifies Neuronal Identity through Graded Expression of Ascl1

SummaryCell diversity and organization in the neural tube depend on the integration of extrinsic signals acting along orthogonal axes. These are believed to specify distinct cellular identities by triggering all-or-none changes in expression of combinations of transcription factors [1]. Under the influence of a common dorsoventral signal, sonic hedgehog, and distinct anterior-posterior (A-P) inductive signals [2, 3], two topographically related progenitor pools that share a common transcriptional code produce serotonergic and V3 neurons in the hindbrain and spinal cord, respectively [4–7]. These neurons have different physiological properties, functions, and connectivity [8, 9]. Serotonergic involvement in neuropsychiatric diseases has prompted greater characterization of their postmitotic repertoire of fate determinants, which include Gata2, Lmx1b, and Pet1 [10], whereas V3 neurons express Sim1 [4]. How distinct serotonergic and V3 neuronal identities emerge from progenitors that share a common transcriptional code is not understood. Here, we show that changes in retinoid activity in these two progenitor pools determine their fates. Retinoids, via Notch signaling, control the expression level in progenitors of the transcription factor Ascl1, which selects serotonergic and V3 neuronal identities in a dose-dependent manner. Therefore, quantitative differences in the expression of a single component of a transcriptional code can select distinct cell fates.

Genes involved in cell adhesion and signaling: A new repertoire of Retinoic Acid Receptors target genes in mouse embryonic fibroblasts

Nuclear retinoic acid (RA) receptors (RARα, β and γ) are ligand-dependent transcription factors that regulate the expression of a battery of genes involved in cell differentiation and proliferation. They are also phosphoproteins and we previously showed the importance of their phosphorylation in their transcriptional activity. In the study reported here, we conducted a genome-wide analysis of the genes that are regulated by RARs in mouse embryonic fibroblasts (MEFs) by comparing wild-type MEFs to MEFs lacking the three RARs. We found that in the absence of RA, RARs control the expression of several gene transcripts associated with cell adhesion. Consequently the knockout MEFs are unable to adhere and to spread on substrates and they display a disrupted network of actin filaments, compared with the WT cells. In contrast, in the presence of the ligand, RARs control the expression of other genes involved in signaling and in RA metabolism. Taking advantage of rescue cell lines expressing the RARα or RARγ subtypes (either wild-type or mutated at the N-terminal phosphorylation sites) in the null background, we found that the expression of RA-target genes can be controlled either by a specific single RAR or by a combination of RAR isotypes, depending on the gene. We also selected genes that require the phosphorylation of the receptors for their regulation by RA. Our results increase the repertoire of genes that are regulated by RARs and highlight the complexity and diversity of the transcriptional programs regulated by RARs, depending on the gene.

Retinoic Acid Signalling Is Activated in the Postischemic Heart and May Influence Remodelling

BackgroundAll-trans retinoic acid (atRA), an active derivative of vitamin A, regulates cell differentiation, proliferation and cardiac morphogenesis via transcriptional activation of retinoic acid receptors (RARs) acting on retinoic acid response elements (RARE).We hypothesized that the retinoic acid (RA) signalling pathway is activated in myocardial ischemia and postischemic remodelling.Methods and FindingsMyocardial infarction was induced through ligating the left coronary artery in mice. In vivo cardiac activation of the RARs was measured by imaging RARE-luciferase reporter mice, and analysing expression of RAR target genes and proteins by real time RT-PCR and western blot. Endogenous retinoids in postinfarcted hearts were analysed by triple-stage liquid chromatography/tandem mass spectrometry. Cardiomyocytes (CM) and cardiofibroblasts (CF) were isolated from infarcted and sham operated RARE luciferase reporter hearts and monitored for RAR activity and expression of target genes. The effect of atRA on CF proliferation was evaluated by EdU incorporation. Myocardial infarction increased thoracic RAR activity in vivo (p<0.001), which was ascribed to the heart through ex vivo imaging (p = 0.002) with the largest signal 1 week postinfarct. This was accompanied by increased cardiac gene and protein expression of the RAR target genes retinol binding protein 1 (p = 0.01 for RNA, p = 0,006 for protein) and aldehyde dehydrogenase 1A2 (p = 0.04 for RNA, p = 0,014 for protein), while gene expression of cytochrome P450 26B1 was downregulated (p = 0.007). Concomitantly, retinol accumulated in the infarcted zone (p = 0.02). CM and CF isolated from infarcted hearts had higher luminescence than those from sham operated hearts (p = 0.02 and p = 0.008). AtRA inhibited CF proliferation in vitro (p = 0.02).ConclusionThe RA signalling pathway is activated in postischemic hearts and may play a role in regulation of damage and repair during remodelling.

Revealing a natural marine product as a novel agonist for retinoic acid receptors with a unique binding mode and inhibitory effects on cancer cells

Retinoids display anti-tumour activity on various cancer cells and therefore have been used as important therapeutic agents. However, adverse side effects and RA (retinoic acid) resistance limit further development and clinical application of retinoid-based therapeutic agents. We report in the present paper the identification of a natural marine product that activates RARs (RA receptors) with a chemical structure distinct from retinoids by high-throughput compound library screening. Luffariellolide was uncovered as a novel RAR agonist by inducing co-activator binding to these receptors in vitro, further inhibiting cell growth and regulating RAR target genes in various cancer cells. Structural and molecular studies unravelled a unique binding mode of this natural ligand to RARs with an unexpected covalent modification on the RAR. Functional characterization further revealed that luffariellolide displays chemotherapeutic potentials for overcoming RA resistance in colon cancer cells, suggesting that luffariellolide may represent a unique template for designing novel non-retinoid compounds with advantages over current RA drugs.

Genome-wide in Silico Identification of New Conserved and Functional Retinoic Acid Receptor Response Elements (Direct Repeats Separated by 5 bp)

The nuclear retinoic acid receptors interact with specific retinoic acid (RA) response elements (RAREs) located in the promoters of target genes to orchestrate transcriptional networks involved in cell growth and differentiation. Here we describe a genome-wide in silico analysis of consensus DR5 RAREs based on the recurrent RGKTSA motifs. More than 15,000 DR5 RAREs were identified and analyzed for their localization and conservation in vertebrates. We selected 138 elements located ±10 kb from transcription start sites and gene ends and conserved across more than 6 species. We also validated the functionality of these RAREs by analyzing their ability to bind retinoic acid receptors (ChIP sequencing experiments) as well as the RA regulation of the corresponding genes (RNA sequencing and quantitative real time PCR experiments). Such a strategy provided a global set of high confidence RAREs expanding the known experimentally validated RAREs repertoire associated to a series of new genes involved in cell signaling, development, and tumor suppression. Finally, the present work provides a valuable knowledge base for the analysis of a wider range of RA-target genes in different species.

Inhibition of the redox function of APE1/Ref-1 in myeloid leukemia cell lines results in a hypersensitive response to retinoic acid-induced differentiation and apoptosis

The standard of care for promyelocytic leukemia includes use of the differentiating agent all-trans retinoic acid (RA) and chemotherapy. RA induces cell differentiation through retinoic acid receptor (RAR) transcription factors. Because redox mechanisms influence how readily transcription factors bind to DNA response elements (RARE), the impact of small molecule (E3330) inhibition of the redox regulatory protein, apurinic-apyrimidinic endonuclease/redox effector factor (APE1/Ref-1) on RAR DNA binding and function in RA-induced myeloid leukemia cell differentiation and apoptosis was investigated. The redox function of APE1 was studied using the small molecule inhibitor E3330 in HL-60 and PLB acute myeloid leukemia cells. Electrophoretic mobility shift assays were employed to determine effect of inhibitor on APE1/Ref-1 redox signaling function. Trypan blue assays, Annexin-V/propidium iodide and CD11b staining, and real-time polymerase chain reaction analyses were employed to determine survival, apoptosis, and differentiation status of cells in culture. RARα binds to its RARE in a redox-dependent manner mediated by APE1/Ref-1 redox regulation. Redox-dependent RAR-RARE binding is blocked by E3330, a small molecule redox inhibitor of APE1/Ref-1. Combination treatment of RA + E3330 results in a profound hypersensitivity of myeloid leukemia cells to RA-induced differentiation and apoptosis. Additionally, redox inhibition by E3330 results in enhanced RAR target gene, BLR-1, expression in myeloid leukemia cells. The redox function of APE1/Ref-1 regulates RAR binding to its DNA RAREs influencing the response of myeloid leukemia cells to RA-induced differentiation. Targeting of APE1/Ref-1 redox function may allow manipulation of the retinoid response with therapeutic implications.

Abstract LB-335: Retinoic acid regulates TRAIL-R1 expression through a retinoic acid receptor-dependent mechanism and enhances death receptor-induced apoptosis

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 1 (TRAIL-R1) also known as death receptor 4 (DR4), is a death domain containing transmembrane receptor involved in TRAIL-mediated apoptosis. One biological function of retinoic acid (RA) among many others is to regulate apoptosis, in part through TRAIL induction. The current study reveals another novel mechanism by which RA modulates apoptosis. For the first time, we found that RA increased TRAIL-R1 expression in various types of human cancer cell lines; this effect could be abrogated by the transcriptional inhibitor actinomycin-D. Moreover, RA increased TRAIL-R1 mRNA level and transactivated its promoter. These data collectively indicate that RA induces TRAIL-R1 expression at the transcriptional level. We also found that RA-induced TRAIL-R1 expression could be abolished by some retinoic acid receptor (RAR) antagonists, whereas various RAR agonists upregulated TRAIL-R1 expression, suggesting that RA induces a RAR-dependent TRAIL-R1 expression. Importantly, we identified two putative RA responsive elements (RARE) named RARE-Pal17 (a palindrome separated by 17 bases) and RARE-DR11 (a direct repeat separated by 11 bases) in the 5′ flanking region of TRAIL-R1 gene. Deletion and mutation of RARE-DR11, but not RARE-Pal17, abrogated RA's effect on transactivation of TRAIL-R1 promoter. In agreement, ChIP assay detected only RARE-DR11 binding, but not RARE-Pal17 binding, in RA-treated cells. These data collectively indicate that the RARE-DR11 is an active one that is responsible for TRAIL-R1 gene transactivation by RA. Furthermore, RA, when combined with TRAIL or agonistic anti-TRAIL-R1 antibody, augmented apoptosis in cancer cells. Taken together, the current study provides compelling evidence that TRAIL-R1 is a RAR target gene and mediates modulation of death receptor-induced apoptosis by RA, thus highlighting a novel mechanism by which RA modulates apoptosis.(SSR, FRK and SYS are Georgia Cancer Coalition Distinguished Cancer Scholars. This study was supported by funds from the Georgia Cancer Coalition Distinguished Cancer Scholar, NIH Head and Neck SPORE P50 CA128613 and DOD VITAL awards) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-335.

DNA recognition by thyroid hormone and retinoic acid receptors: 3,4,5 rule modified

It has been proposed that retinoic acid receptors (RARs) and thyroid hormone receptors (TRs) both bind to AGGTCA "half-site" sequences, but distinguish their different target genes by recognizing different half-site spacings. We report here that artificial DNA binding sites based on these AGGTCA half-sites confer high affinity, but poor specificity, and that spacing alone does not account for the divergent DNA recognition properties of TRs and RARs. Instead, we have determined that the non-consensus half-sites that are present in naturally occurring RAR and TR target genes play a crucial role in defining receptor DNA recognition specificity, and work together with flanking sequences and half-site spacing to produce receptor-specific DNA binding in vitro. We also provide evidence that auxiliary proteins in cells generate an additional layer of receptor-specific target gene recognition, in part by destabilizing the binding of nuclear receptors to the "wrong" response elements.

Reciprocal roles of SIRT1 and SKIP in the regulation of RAR activity: implication in the retinoic acid-induced neuronal differentiation of P19 cells

Human sirtuin 1 (SIRT1) is a NAD+-dependent deacetylase that participates in cell death/survival, senescence and metabolism. Although its substrates are well characterized, no direct regulators have been defined. Here, we show that SIRT1 associates with SKI-interacting protein (SKIP) and modulates its activity as a coactivator of retinoic acid receptor (RAR). Binding assays indicated that SKIP interacts with RAR in a RA-dependent manner, through a region that overlaps the binding site for SIRT1. SKIP augmented the transcriptional activation activity of RAR by cooperating with SRC-1, and SIRT1 suppressed SKIP/SRC-1-enhanced RAR transactivation activity. The suppression was dependent on the deacetylase activity of SIRT1 and was enhanced by a SIRT1 activator, resveratrol. In contrast, the suppression was relieved by SIRT1 knockdown, overexpression of SKIP and treatment with a SIRT1 inhibitor, splitomicin. Upon SKIP overexpression, the recruitment of SIRT1 to the endogenous RARβ2 promoter was severely impaired, and SKIP was recruited to the promoter instead. Finally, resveratrol treatment inhibited RA-induced neuronal differentiation of P19 cells, accompanied by reductions in the neuronal marker nestin and a RAR target gene, RARβ2. This inhibition was relieved by either knockdown of SIRT1 or overexpression of SKIP. These data suggest that SIRT1 and SKIP play reciprocal roles in the regulation of RAR activity, which is implicated in the regulation of RA-induced neuronal differentiation of P19 cells.

UNC45A Confers Resistance to Histone Deacetylase Inhibitors and Retinoic Acid

To identify potential biomarkers of therapy response, we have previously done a large-scale gain-of-function genetic screen to identify genes whose expression confers resistance to histone deacetylase inhibitors (HDACI). This genetic screen identified two genes with a role in retinoic acid signaling, suggesting that HDACIs target retinoic acid signaling as part of their anticancer effect. We study here a third gene identified in this genetic screen, UNC45A, and assess its role in retinoic acid signaling and responses to HDACIs using cell-based proliferation and differentiation assays and transcriptional reporter gene assays. The vertebrate Unc45 genes are known for their roles in muscle development and the assembly and cochaperoning of the muscle motor protein myosin. Here, we report that human UNC45A (GCUNC45) can render transformed cells resistant to treatment with HDACIs. We show that UNC45A also inhibits signaling through the retinoic acid receptor alpha. Expression of UNC45A inhibits retinoic acid-induced proliferation arrest and differentiation of human neuroblastoma cells and inhibits the induction of endogenous retinoic acid receptor target genes. These data establish an unexpected role for UNC45A in causing resistance to both HDACI drugs and retinoic acid. Moreover, our data lend further support to the notion that HDACIs exert their anticancer effect, at least in part, through an effect on retinoic acid signaling.

All-trans-Retinoic Acid Represses Obesity and Insulin Resistance by Activating both Peroxisome Proliferation-Activated Receptor β/δ and Retinoic Acid Receptor

Many biological activities of all-trans-retinoic acid (RA) are mediated by the ligand-activated transcription factors termed retinoic acid receptors (RARs), but this hormone can also activate the nuclear receptor peroxisome proliferation-activated receptor beta/delta (PPARbeta/delta). We show here that adipocyte differentiation is accompanied by a shift in RA signaling which, in mature adipocytes, allows RA to activate both RARs and PPARbeta/delta, thereby enhancing lipolysis and depleting lipid stores. In vivo studies using a dietary-induced mouse model of obesity indicated that onset of obesity is accompanied by downregulation of adipose PPARbeta/delta expression and activity. RA treatment of obese mice induced expression of PPARbeta/delta and RAR target genes involved in regulation of lipid homeostasis, leading to weight loss and improved insulin responsiveness. RA treatment also restored adipose PPARbeta/delta expression. The data indicate that suppression of obesity and insulin resistance by RA is largely mediated by PPARbeta/delta and is further enhanced by activation of RARs. By targeting two nuclear receptors, RA may be a uniquely efficacious agent in the therapy and prevention of the metabolic syndrome.

The endogenous retinoid metabolite S‐4‐oxo‐9‐cis‐13,14‐dihydro‐retinoic acid activates retinoic acid receptor signalling both in vitro and in vivo

Retinoic acid receptor (RAR) and retinoid X receptor are ligand-induced transcription factors that belong to the nuclear receptor family. The receptors are activated by small hydrophobic compounds, such as all-trans-retinoic acid and 9-cis-retinoic acid, respectively. Interestingly, these receptors are also targets for a number of exogenous compounds. In this study, we characterized the biological activity of the 9-cis-substituted retinoic acid metabolite, S-4-oxo-9-cis-13,14-dihydro-retinoic acid (S-4o9cDH-RA). The endogenous levels of this metabolite in wild-type mice and rats were found to be higher than those of all-trans-retinoic acid, especially in the liver. Using cell-based luciferase reporter systems, we showed that S-4o9cDH-RA activates the transcription of retinoic acid response element-containing genes in several cell types, both from a simple 2xDR5 element and from the promoter of the natural retinoid target gene RARbeta2. In addition, quantitative RT-PCR analysis demonstrated that S-4o9cDH-RA treatment significantly increases the endogenous mRNA levels of the RAR target gene RARbeta2. Utilizing a limited proteolytic digestion assay, we showed that S-4o9cDH-RA induces conformational changes to both RARalpha and RARbeta in the same manner as does all-trans-retinoic acid, suggesting that S-4o9cDH-RA is indeed an endogenous ligand for these receptors. These in vitro results were corroborated in an in vivo system, where S-4o9cDH-RA induced morphological changes similar to those of all-trans-retinoic acid in the developing chicken wing bud. When locally applied to the wing bud, S-4o9cDH-RA induced digit pattern duplications in a dose-dependent fashion. The results illustrate that S-4o9cDH-RA closely mimics all-trans-retinoic acid with regard to pattern respecification. Finally, using quantitative RT-PCR analysis, we showed that S-4o9cDH-RA induces the transcription of several retinoic acid-regulated genes in chick wing buds, including Hoxb8, RARbeta2, shh, Cyp26 and bmp2. Although S-4o9cDH-RA was less potent when compared with all-trans-retinoic acid, the findings clearly demonstrate that S-4o9cDH-RA has the capacity to bind and activate nuclear retinoid receptors and regulate gene transcription both in vitro and in vivo.

A2E, a Pigment of the Lipofuscin of Retinal Pigment Epithelial Cells, Is an Endogenous Ligand for Retinoic Acid Receptor

Lipofuscin contains fluorophores, which represent a biomarker for cellular aging. Although it remains unsubstantiated clinically, experimental results support that the accumulation of lipofuscin is related to an increased risk of choroidal neovascularization due to age-related macular degeneration, a leading cause of legal blindness. Here, we report that a major lipofuscin component, A2E, activates the retinoic acid receptor (RAR). In vitro experiments using luciferase reporter assay, competitional binding assay, analysis of target genes, and chromatin immunoprecipitation (ChIP) assay strongly suggest that A2E is a bona fide ligand for RAR and induces sustained activation of RAR target genes. A2E-induced vascular endothelial growth factor (VEGF) expression in a human retinal pigment epithelial cell line (ARPE-19) and RAR antagonist blocked the up-regulation of VEGF. The conditioned medium of A2E-treated ARPE-19 cells induced tube formation in human umbilical vascular endothelial cells, which was blocked by the RAR antagonist and anti-VEGF antibody. These results suggest that A2E accumulation results in the phenotypic alteration of retinal pigment epithelial cells, predisposing the environment to choroidal neovascularization development. This is mediated through the agonistic function of A2E, at least in part. The results of this study provide a novel potential therapeutic target for this incurable condition.