RXRα Proteins Research Articles

Phosphorylation of RXRα mediates the effect of JNK to suppress hepatic FGF21 expression and promote metabolic syndrome

The cJun NH2-terminal kinase (JNK) signaling pathway in the liver promotes systemic changes in metabolism by regulating peroxisome proliferator-activated receptor α (PPARα)-dependent expression of the hepatokine fibroblast growth factor 21 (FGF21). Hepatocyte-specific gene ablation studies demonstrated that the Mapk9 gene (encoding JNK2) plays a key mechanistic role. Mutually exclusive inclusion of exons 7a and 7b yields expression of the isoforms JNK2α and JNK2β. Here we demonstrate that Fgf21 gene expression and metabolic regulation are primarily regulated by the JNK2α isoform. To identify relevant substrates of JNK2α, we performed a quantitative phosphoproteomic study of livers isolated from control mice, mice with JNK deficiency in hepatocytes, and mice that express only JNK2α or JNK2β in hepatocytes. We identified the JNK substrate retinoid X receptor α (RXRα) as a protein that exhibited JNK2α-promoted phosphorylation invivo. RXRα functions as a heterodimeric partner of PPARα and may therefore mediate the effects of JNK2α signaling on Fgf21 expression. To test this hypothesis, we established mice with hepatocyte-specific expression of wild-type or mutated RXRα proteins. We found that the RXRα phosphorylation site Ser260 was required for suppression of Fgf21 gene expression. Collectively, these data establish a JNK-mediated signaling pathway that regulates hepatic Fgf21 expression.

Oroxylin A, a natural compound, mitigates the negative effects of TNFα-treated acute myelogenous leukemia cells.

Tumor necrosis factor alpha (TNFα) is a complicated cytokine which is involved in proliferation and differentiation of acute myelogenous leukemia (AML) cells through a poorly understood mechanism. Mechanistic studies indicate that TNFα induced binding of PI3K subunit p85α to N-terminal truncated nuclear receptor RXRα (tRXRα) proteins, and activated AKT. The activated PI3K/AKT pathway negatively regulated differentiation of AML cells through the upregulation of c-Myc. In addition, TNFα also induced activation of nuclear factor κB (NF-κB), a nuclear transcription factor which was shown to promote cell proliferation. The present study demonstrates that oroxylin A, a natural compound isolated from Scutellariae radix, sensitizes leukemia cells to TNFα and markedly enhances TNFα-induced growth inhibition and differentiation of AML cell including human leukemia cell lines and primary AML cells. Activation of PI3K/AKT pathway could be inhibited by oroxylin A through inhibiting expression of tRXRα in NB4 and HL-60-resistant cells. Furthermore, we found that oroxylin A inhibited the activation of NF-κB and the DNA binding activity by TNFα proved by EMSA in these two AML cell lines. Moreover, in vivo studies showed that treatment with oroxylin A in combination with TNFα decreased AML cell population and prolonged survival in NOD/SCID mice with xenografts of primary AML cells. Overall, our results indicate that oroxylin A is able to inhibit the negative effects of TNFα for AML therapy, suggesting that combination of oroxylin A and TNFα have the potential to delay growth or eliminate the abnormal leukemic cells, thus representing a promising strategy for AML treatment.

Retinoid X receptor α downregulation is required for tail and caudal spinal cord regeneration in the adult newt.

Some adult vertebrate species, such as newts, axolotls and zebrafish, have the ability to regenerate their central nervous system (CNS). However, the factors that establish a permissive CNS environment for correct morphological and functional regeneration in these species are not well understood. Recent evidence supports a role for retinoid signaling in the intrinsic ability of neurons, in these regeneration-competent species, to regrow after CNS injury. Previously, we demonstrated that a specific retinoic acid receptor (RAR) subtype, RARβ, mediates the effects of endogenous retinoic acid (RA) on neuronal growth and guidance in the adult newt CNS after injury. Here, we now examine the expression of the retinoid X receptor RXRα (a potential heterodimeric transcriptional regulator with RARβ), in newt tail and spinal cord regeneration. We show that at 21 days post-amputation (dpa), RXRα is expressed at temporally distinct periods and in non-overlapping spatial domains compared to RARβ. Whereas RARβ protein levels increase, RXRα proteins level decrease by 21 dpa. A selective agonist for RXR, SR11237, prevents both this downregulation of RXRα and upregulation of RARβ and inhibits tail and caudal spinal cord regeneration. Moreover, treatment with a selective antagonist for RARβ, LE135, inhibits regeneration with the same morphological consequences as treatment with SR11237. Interestingly, LE135 treatment also inhibits the normal downregulation of RXRα in tail and spinal cord tissues at 21 dpa. These results reveal a previously unidentified, indirect regulatory feedback loop between these two receptor subtypes in regulating the regeneration of tail and spinal cord tissues in this regeneration-competent newt.

Bexarotene, a Selective RXRα Agonist, Reverses Hypotension Associated with Inflammation and Tissue Injury in a Rat Model of Septic Shock.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that can activate or inhibit the expression of many target genes by forming a heterodimer complex with the retinoid X receptor (RXR). The aim of this study was to investigate effects of bexarotene, a selective RXRα agonist, on the changes in renal, cardiac, hepatic, and pulmonary expression/activity of inducible nitric oxide synthase (iNOS) and cytochrome P450 (CYP) 4F6 in relation to PPARα/β/γ-RXRα heterodimer formation in a rat model of septic shock. Rats were injected with dimethyl sulfoxide or bexarotene 1h after administration of saline or lipopolysaccharide (LPS). Mean arterial pressure (MAP) and heart rate (HR) were recorded from rats, which had received either saline or LPS before and after 1, 2, 3, and 4h. Serum iNOS, LTB4, myeloperoxidase (MPO), and lactate dehydrogenase (LDH) levels as well as tissue iNOS and CYP4F6 mRNA expression in addition to PPARα/β/γ and RXRα proteins were measured. LPS-induced decrease in MAP and increase in HR were associated with a decrease in PPARα/β/γ-RXRα heterodimer formation and CYP4F6 mRNA expression. LPS also caused an increase in systemic iNOS, LTB4, MPO, and LDH levels as well as iNOS mRNA expression. Bexarotene at 0.1mg/kg (i.p.) prevented the LPS-induced changes, except tachycardia. The results suggest that increased formation of PPARα/β/γ-RXRα heterodimers and CYP4F6 expression/activity in addition to decreased iNOS expression contributes to the beneficial effect of bexarotene to prevent the hypotension associated with inflammation and tissue injury during rat endotoxemia.

Targeting truncated RXRα for cancer therapy.

Retinoid X receptor-alpha (RXRα), a unique member of the nuclear receptor superfamily, is a well-established drug target, representing one of the most important targets for pharmacologic interventions and therapeutic applications for cancer. However, how RXRα regulates cancer cell growth and how RXRα modulators suppress tumorigenesis are poorly understood. Altered expression and aberrant function of RXRα are implicated in the development of cancer. Previously, several studies had demonstrated the presence of N-terminally truncated RXRα (tRXRα) proteins resulted from limited proteolysis of RXRα in tumor cells. Recently, we discovered that overexpression of tRXRα can promote tumor growth by interacting with tumor necrosis factor-alpha-induced phosphoinositide 3-kinase and NF-κB signal transduction pathways. We also identified nonsteroidal anti-inflammatory drug Sulindac and analogs as effective inhibitors of tRXRα activities via a unique binding mechanism. This review discusses the emerging roles of tRXRα and modulators in the regulation of cancer cell survival and death as well as inflammation and our recent understanding of tRXRα regulation by targeting the alternate binding sites on its surface.

Oroxylin a Heightens Tumor Necrosis Factor a-Induced Differentiation Effects on Acute Myeloid Leukemia Cells Via Modulation on AKT and NF-Kb Pathways Involving RXRa

Tumor necrosis factor alpha (TNFα) is a complicated cytokine which could induce differentiation of acute myeloid leukemia (AML) cells. We have found that Oroxylin A, a natural compound isolated from Scutellariae radix, markedly enhanced TNFα-induced NBT reduction and CD11b/CD14 expression of AML cells. Besides, Giemsa staining also displayed that the combination group induced U937-MDR cells to differentiate into monocyte-like cells yet NB4 and HL-60-resistant cells to granulocytic-like cells. Further study showed that TNFα induced PI3K subunit p85α binding to N-terminal truncated nuclear receptor RXRα (tRXRα) proteins and activating AKT. On the contrary, these could be inhibited by Oroxylin A through inhibiting interaction between tRXRα and p85α in NB4 and HL-60-resistant cells. Moreover, Oroxylin A inhibited the activation of NF-κB signaling and the DNA binding activity by TNFα proved by EMSA in these two AML cells. All these suggested that Oroxylin A was able to inhibit NF-κB signaling and RXRα-dependent AKT signaling, the negative effects of TNFα for AML therapy, suggesting that combination of Oroxylin A and TNFα might be a novel candidate for differentiation therapy for AML cells and required further investigation. DisclosuresNo relevant conflicts of interest to declare.

Effects of Diisodecyl Phthalate on PPAR:RXR-Dependent Gene Expression Pathways in Sea Bream Hepatocytes.

Evidence that endocrine-disrupting chemicals (EDCs) may target metabolic disturbances, beyond interference with the functions of the endocrine systems has recently accumulated. Among EDCs, phthalate plasticizers like the diisodecyl phthalate (DiDP) are commonly found contaminants of aquatic environments and have been suggested to function as obesogens by activating peroxisome proliferator activated receptors (PPARs), a subset of nuclear receptors (NRs) that act as metabolic sensors, playing pivotal roles in lipid homeostasis. However, little is known about the modulation of PPAR signaling pathways by DiDP in fish. In this study, we have first investigated the ligand binding efficiency of DiDP to the ligand binding domains of PPARs and retinoid-X-receptor-α (RXRα) proteins in fish using a molecular docking approach. Furthermore, in silico predictions were integrated by in vitro experiments to show possible dose-relationship effects of DiDP on PPAR:RXR-dependent gene expression pathways using sea bream hepatocytes. We observed that DiDP shows high binding efficiency with piscine PPARs demonstrating a greater preference for RXRα. Our studies also demonstrated the coordinate increased expression of PPARs and RXRα, as well as their downstream target genes in vitro. Principal component analysis (PCA) showed the strength of relationship between transcription of most genes involved in fatty acid metabolism and PPAR mRNA levels. In particular, fatty acid binding protein (FABP) was highly correlated to all PPARs. The results of this study suggest that DiDP can be considered an environmental stressor that activates PPAR:RXR signaling to promote long-term changes in lipid homeostasis leading to potential deleterious physiological consequences in teleost fish.

Synergistic growth inhibition by acyclic retinoid and phosphatidylinositol 3-kinase inhibitor in human hepatoma cells

BackgroundA malfunction of RXRα due to phosphorylation is associated with liver carcinogenesis, and acyclic retinoid (ACR), which targets RXRα, can prevent the development of hepatocellular carcinoma (HCC). Activation of PI3K/Akt signaling plays a critical role in the proliferation and survival of HCC cells. The present study examined the possible combined effects of ACR and LY294002, a PI3K inhibitor, on the growth of human HCC cells.MethodsThis study examined the effects of the combination of ACR plus LY294002 on the growth of HLF human HCC cells.ResultsACR and LY294002 preferentially inhibited the growth of HLF cells in comparison with Hc normal hepatocytes. The combination of 1 μM ACR and 5 μM LY294002, in which the concentrations used are less than the IC50 values of these agents, synergistically inhibited the growth of HLF, Hep3B, and Huh7 human HCC cells. These agents when administered in combination acted cooperatively to induce apoptosis in HLF cells. The phosphorylation of RXRα, Akt, and ERK proteins in HLF cells were markedly inhibited by treatment with ACR plus LY294002. Moreover, this combination also increased RXRE promoter activity and the cellular levels of RARβ and p21CIP1, while decreasing the levels of cyclin D1.ConclusionACR and LY294002 cooperatively increase the expression of RARβ, while inhibiting the phosphorylation of RXRα, and that these effects are associated with the induction of apoptosis and the inhibition of cell growth in human HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.

Abstract B55: Chemoprevention of obesity- and diabetes-related liver carcinogenesis by branched-chain amino acids and acyclic retinoid

Abstract Obesity and diabetes-related metabolic abnormalities, including insulin resistance, alterations in the IGF/IGF-1R axis, state of chronic inflammation, and induction of oxidative stress increase the risk of hepatocellular carcinoma (HCC). Non-alcoholic steatohepatitis, which is frequently associated with obesity and diabetes, can progress to cirrhosis and HCC. On the other hand, these findings indicate that the metabolic disorders caused by obesity and diabetes might be effective targets to prevent HCC development. For instance, branched-chain amino acids (BCAA), which improve protein energy malnutrition in patients with liver cirrhosis, improve insulin resistance and reduce the risk of HCC in obese cirrhotic patients. In an experimental study, BCAA supplementation inhibits the development of liver tumors in obese and diabetic mice by inhibiting the activation of IGF/IGF-1R axis, improving insulin resistance, reducing serum leptin levels, and attenuating hepatic steatosis and fibrosis. BCAA supplementation also inhibits the early phase of obesity-related liver tumorigenesis by attenuating chronic inflammation in both the liver and white adipose tissue. In addition, acyclic retinoid (ACR), which is a promising agent for the chemoprevention of HCC, prevents liver tumorigenesis in obese and diabetic mice by inhibiting the activation of Ras and phosphorylation of ERK and RXRα proteins. ACR administration also inhibits this tumorigenesis via improvement of insulin sensitivity and attenuation of the chronic inflammation induced by excessive fatty deposits. These findings suggest that pharmaceutical and nutraceutical approaches for targeting and restoring metabolic disorders, especially insulin resistance and chronic inflammation, may be an effective strategy for preventing the development of obesity- and diabetes-related HCC. We indicate that BCAA and ACR are considered some of the most promising agents for achieving this purpose. Citation Format: Masahito Shimizu, Hisataka Moriwaki. Chemoprevention of obesity- and diabetes-related liver carcinogenesis by branched-chain amino acids and acyclic retinoid. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B55.

50nm-Scale Localization of Single Unmodified, Isotopically Enriched, Proteins in Cells

Imaging single proteins within cells is challenging if the possibility of artefacts due to tagging or to recognition by antibodies is to be avoided. It is generally believed that the biological properties of proteins remain unaltered when 14N isotopes are replaced with 15N. 15N-enriched proteins can be localised by dynamic Secondary Ion Mass Spectrometry (D-SIMS). We describe here a novel imaging analysis algorithm to detect a few 15N-enriched proteins - and even a single protein - within a cell using D-SIMS. The algorithm distinguishes statistically between a low local increase in 15N isotopic fraction due to an enriched protein and a stochastic increase due to the background. To determine the number of enriched proteins responsible for the increase in the isotopic fraction, we use sequential D-SIMS images in which we compare the measured isotopic fractions to those expected if 1, 2 or more enriched proteins are present. The number of enriched proteins is the one that gives the best fit between the measured and the expected values. We used our method to localise 15N-enriched thymine DNA glycosylase (TDG) and retinoid X receptor α (RXRα) proteins delivered to COS-7 cells. We show that both a single TDG and a single RXRα can be detected. After 4 h incubation, both proteins were found mainly in the nucleus; RXRα as a monomer or dimer and TDG only as a monomer. After 7 h, RXRα was found in the nucleus as a monomer, dimer or tetramer, whilst TDG was no longer in the nucleus and instead formed clusters in the cytoplasm. After 24 h, RXRα formed clusters in the cytoplasm, and TDG was no longer detectable. In conclusion, single unmodified proteins in cells can be counted and localised with 50 nm resolution by combining D-SIMS with our method of analysis.

Combination of acyclic retinoid with branched‐chain amino acids inhibits xenograft growth of human hepatoma cells in nude mice

Combination chemoprevention is a promising strategy to improve the prognosis of hepatocellular carcinoma (HCC). A malfunction of retinoid X receptor-α (RXR-α) due to phosphorylation by Ras/mitogen-activated protein kinase is closely associated with liver carcinogenesis and acyclic retinoid (ACR) can prevent HCC development by inhibiting RXR-α phosphorylation. The present study examined the possible combined effects of ACR plus branched-chain amino acids (BCAA), which can also prevent the development of HCC in obese patients with liver cirrhosis, in human HCC xenografts in nude mice. This study examined the effects of the combination of ACR plus BCAA on the growth of Huh7 human HCC xenografts in nude mice. The effects of the combination on the phosphorylation of RXR-α, extracellular signal-regulated kinase (ERK), Akt and insulin-like growth factor-1 receptor (IGF-1R) proteins, and on the expression levels of retinoic acid receptor-β (RAR-β) and p21(CIP1) mRNA, were also examined by western blot and real-time reverse transcription polymerase chain reaction analyses, respectively. The combined treatment with ACR plus BCAA significantly inhibited the growth of Huh7 xenografts. The combination of these agents caused a marked inhibition of the phosphorylation of RXR-α, ERK, Akt and IGF-1R proteins in the xenografts. In addition, the expression levels of RAR-β and p21(CIP1) mRNA significantly increased by these agents. The combination of ACR and BCAA restores the function of RXR-α by inhibiting its phosphorylation and increasing the level of RAR-β, a heterodimeric partner for RXR-α, and thus suppresses the growth of HCC xenografts. Therefore, this combination might be an effective regimen for the treatment and, probably, chemoprevention of HCC.

Abstract 844: Acyclic retinoid inhibits diethylnitrosamine-induced liver tumorigenesis in obese and diabetic C57BL/KsJ-db/db mice

Abstract Background and aims: Obesity and the related metabolic abnormalities are associated with increased risk of hepatocellular carcinoma (HCC). Malfunctioning of retinoid X receptor (RXR)-α due to phosphorylation by Ras/MAPK also plays a critical role in liver carcinogenesis. In the present study, we examined the effects of acyclic retinoid (ACR), which targets RXRα, on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BL/KsJ-db/db (db/db) obese and diabetic mice. Methods: Male db/db mice were given tap water containing 40 ppm DEN for 2 weeks, after which they were fed a diet containing 0.03% or 0.06% of ACR throughout the experiment. Results: In mice treated with either dose of ACR for 34 weeks, the development of liver cell adenomas was significantly inhibited as compared to basal diet-fed mice. ACR markedly inhibited the activation of Ras and phosphorylation of the ERK, RXRα, and Stat3 proteins in the livers of experimental mice. It also increased the expression of RARβ and p21CIP1 mRNA, while decreasing the expression of cyclin D1, c-Fos, and c-Jun mRNA in the liver, thereby restoring RXRα function. Administration of ACR improved liver steatosis and activated the AMPK protein. The serum levels of insulin were decreased by ACR treatment, whereas the QUICKI values were increased, indicating improved insulin sensitivity. The serum levels of TNF-α and the expression levels of TNF-α, IL-6, and IL-1β mRNA in the livers of DEN-treated db/db mice were decreased by ACR treatment, suggesting attenuation of the chronic inflammation induced by excessive fatty deposits. Conclusion: ACR may be, therefore, useful in the chemoprevention of obesity-related HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 844. doi:10.1158/1538-7445.AM2011-844

C‐Jun N‐terminal kinase activation by oxidative stress suppresses retinoid signaling through proteasomal degradation of retinoic acid receptor α protein in hepatic cells

We previously reported that impaired retinoid signaling causes hepatocellular carcinoma (HCC) through oxidative stress. However, the interaction between oxidative stress and retinoid signaling has not been fully understood. To address this issue, the effects of hydrogen peroxide on the transcriptional activity of RAR/RXR heterodimers, RARα and RXRα proteins and intracellular signaling pathways were examined. The transcriptional activity of RAR/RXR examined by the DR5-tk-Luc reporter assay was significantly suppressed. The RARα protein level began to decrease at 6 h after treatment and declined thereafter. However, RARα mRNA were not changed. Activation of extracellular regulated kinases (ERK), p38, c-Jun N-terminal kinase (JNK) and Akt was observed after treatment of hydrogen peroxide. SP600125, an inhibitor of JNK, reversed the RARα protein level reduced by hydrogen peroxide. Anisomycin, an activator of JNK, reduced RARα protein. Transfection of wild-type JNK-constitutive actively expressing plasmid, but not kinase-negative JNK-expressing plasmid caused reduction of RARα protein. Proteasomal degradation of RARα was observed after anisomycin treatment; however, the mutant RARα, of which phosphorylation sites are replaced with alanines, was not degradated. In hepatitis C virus (HCV)-related human liver tissues, phospho-JNK and RARα reciprocally expressed with the progression of liver disease. Finally, the staining of 8-OHdG and thioredoxin was increased with the disease progression. These data indicate that JNK activation by oxidative stress suppresses retinoid signaling through proteasomal degradation of RARα, suggesting that a vicious cycle between aberrant retinoid signaling and oxidative stress accelerates hepatocarcinogenesis.

Acyclic Retinoid Inhibits Diethylnitrosamine-Induced Liver Tumorigenesis in Obese and Diabetic C57BLKS/J- +Leprdb/+Leprdb Mice

Obesity and the related metabolic abnormalities are associated with increased risk of hepatocellular carcinoma (HCC). Malfunctioning of retinoid X receptor (RXR) α due to phosphorylation by Ras/MAPK also plays a critical role in liver carcinogenesis. In the present study, we examined the effects of acyclic retinoid (ACR), which targets RXRα, on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BLKS/J- +Lepr(db)/+Lepr(db) (db/db) obese mice. Male db/db mice were given tap water containing 40 ppm DEN for 2 weeks, after which they were fed a diet containing 0.03% or 0.06% of ACR throughout the experiment. In mice treated with either dose of ACR for 34 weeks, the development of liver cell adenomas was significantly inhibited as compared with basal diet-fed mice. ACR markedly inhibited the activation of Ras and phosphorylation of the ERK (extracellular signal-regulated kinase) and RXRα proteins in the livers of experimental mice. It also increased the expression of RAR β and p21(CIP1) mRNA while decreasing the expression of cyclin D1, c-Fos, and c-Jun mRNA in the liver, thereby restoring RXRα function. Administration of ACR improved liver steatosis and activated the AMPK protein. The serum levels of insulin decreased by ACR treatment, whereas the quantitative insulin sensitivity check index (QUICKI) values increased, indicating improved insulin sensitivity. The serum levels of TNF-α and the expression levels of TNF- α, IL-6, and IL-1 β mRNA in the livers of DEN-treated db/db mice were decreased by ACR treatment, suggesting attenuation of the chronic inflammation induced by excessive fatty deposits. ACR may be, therefore, useful in the chemoprevention of obesity-related HCC.

NSAID Sulindac and Its Analog Bind RXRα and Inhibit RXRα-Dependent AKT Signaling

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their anticancer effects through cyclooxygenase-2 (COX-2)-dependent and independent mechanisms. Here, we report that Sulindac, an NSAID, induces apoptosis by binding to retinoid X receptor-alpha (RXRalpha). We identified an N-terminally truncated RXRalpha (tRXRalpha) in several cancer cell lines and primary tumors, which interacted with the p85alpha subunit of phosphatidylinositol-3-OH kinase (PI3K). Tumor necrosis factor-alpha (TNFalpha) promoted tRXRalpha interaction with the p85alpha, activating PI3K/AKT signaling. When combined with TNFalpha, Sulindac inhibited TNFalpha-induced tRXRalpha/p85alpha interaction, leading to activation of the death receptor-mediated apoptotic pathway. We designed and synthesized a Sulindac analog K-80003, which has increased affinity to RXRalpha but lacks COX inhibitory activity. K-80003 displayed enhanced efficacy in inhibiting tRXRalpha-dependent AKT activation and tRXRalpha tumor growth in animals.

Abstract 3779: Combination chemoprevention of hepatocellular carcinoma using acyclic retinoid

Abstract Background and aims: A malfunction of retinoid X receptor-α (RXRα) due to phosphorylation by the Ras/MAPK signaling pathway is associated with the development of hepatocellular carcinoma (HCC). Acyclic retinoid (ACR: the same material as NIK-333), which has been clinically shown to reduce the incidence of a post-therapeutic recurrence of HCC, can inhibit both the Ras activity and phosphorylation of the ERK and RXRα proteins. The effects of a combination of ACR plus other agents on the inhibition of cell growth in human HCC cells were herein examined to develop a more effective strategy for the prevention of HCC. Methods and results: The combination of ACR plus vitamin K2, which inhibits the phosphorylation of the RXRα protein through the inhibition of Ras/MAPK activation, synergistically induced apoptosis and inhibited the growth of human HCC cells without affecting the growth of normal human hepatocytes. ACR and trastuzumab, the humanized anti-HER2 monoclonal antibody, also synergistically inhibited the activation of HER2, a member of receptor tyrosine kinase (RTK), and its downstream signaling pathways, thus subsequently inhibiting the phosphorylation of RXRα and the growth of HCC cells. ACR in combination with valproic acid, a histone deacetylase inhibitor, also synergistically inhibited the growth of human HCC cells and this was associated with the inhibition of phosphorylation of the RXRα and Akt proteins by valproic acid. Conclusion: The combination of ACR plus a specific agent that targets RTK and its downstream signaling pathways, including the Ras/MAPK and PI3K/Akt pathways, may be able to inhibit the phosphorylation of RXRα and it may therefore be a promising strategy to prevent the development of HCC. ACR, which targets phosphorylated RXRα, may thus play a critical role in preventing the development of HCC when it is used either alone or in combination with other agents. 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 3779.

Leukemic transformation by the APL fusion protein PRKAR1A-RARα critically depends on recruitment of RXRα

AbstractPRKAR1A (R1A)–retinoic acid receptor-α (R1A-RARα) is the sixth RARα–containing fusion protein in acute promyelocytic leukemia (APL). Using the murine bone-marrow retroviral transduction/transformation assay, we showed that R1A-RARα fusion protein could transform bone-marrow progenitor/stem cells. In gel-shift assays, R1A-RARα was able to bind to a panel of retinoic acid response elements both as a homodimer and as a heterodimer with RXRα, and demonstrated distinct DNA-binding characteristics compared with wild-type RARα/RXRα or other X-RARα chimeric proteins. The ratio of R1A-RARα to RXRα proteins affected the retinoic acid response element interaction pattern of R1A-RARα/RXRα complexes. Studies comparing R1A-RARα with R1A-RARα(ΔRIIa) demonstrated that the RIIa protein interaction domain located within R1A was responsible for R1A-RARα homodimeric DNA binding and interaction with wild-type R1A protein. However, the RIIa domain was not required for R1A-RARα–mediated transformation because its deletion in R1A-RARα(ΔRIIa) did not compromise its transformation capability. In contrast, introduction of point mutations within the RARα portion of either R1A-RARα or R1A-RARα(ΔRIIa), previously demonstrated to eliminate RXRα interaction or treatment of transduced cells with RXRα shRNA or a RXRα agonist, reduced transformation capability. Thus, leukemic transformation by APL fusion protein PRKAR1A-RARα is critically dependent on RXRα, which suggests RXRα is a promising target for APL.

Acyclic retinoid synergises with valproic acid to inhibit growth in human hepatocellular carcinoma cells

A malfunction of retinoid X receptor-α (RXRα) due to phosphorylation is associated with the development of hepatocellular carcinoma (HCC) and acyclic retinoid (ACR), which targets RXRα, can prevent the development of second primary HCC. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, induces apoptosis and cell cycle arrest in cancer cells. VPA can also enhance the sensitivity of cancer cells to retinoids. The present study examined the possible combined effects of ACR plus VPA in HepG2 human HCC cell line. The combination of 5 μM ACR and 1 mM VPA, about the IC 25 value for both compounds, synergistically inhibited the growth of HepG2 cells without affecting the growth of Hc normal human hepatocytes. The combined treatment with ACR plus VPA also acted synergistically to induce apoptosis and G 0–G 1 cell cycle arrest in HepG2 cells. This combination further exerted a synergistic inhibition of the phosphorylation of RXRα, ERK, Akt and GSK-3β proteins and caused an accumulation of acetylated histones H3 and H4 proteins. VPA enhanced the ability of ACR to raise the cellular levels of RARβ and p21 CIP1. The combination of these agents markedly increased both the RARE and RXRE promoter activities in HepG2 cells. These results suggest that ACR and VPA cooperatively increase the expression of RARβ and p21 CIP1, while inhibiting the phosphorylation of RXRα, and these effects were associated with induction of apoptosis and the inhibition of cell growth in HepG2 cells. This combination might therefore be an effective regimen for the chemoprevention and chemotherapy of HCC.

Strategy and mechanism for the prevention of hepatocellular carcinoma: Phosphorylated retinoid X receptor α is a critical target for hepatocellular carcinoma chemoprevention

Hepatocellular carcinoma (HCC) is a major health care problem worldwide. The prognosis of patients with HCC is poor because even in the early stages when surgical treatment might be expected to be curative, the incidence of recurrence in patients with underlying cirrhosis is very high due to multicentric carcinogenesis. Therefore, strategies to prevent recurrence and second primary HCC are required to improve the prognosis. One of the most practical approaches to prevent the multicentric development of HCC is 'clonal deletion' therapy, which is defined as the removal of latent (i.e. invisible) (pre)malignant clones from the liver in a hypercarcinogenic state. Retinoids, a group of structural and functional analogs of vitamin A, exert their biological function primarily through two distinct nuclear receptors, retinoic acid receptors and retinoid X receptors (RXR), and abnormalities in the expression and function of these receptors are highly associated with the development of various cancers, including HCC. In particular, a malfunction of RXRalpha due to phosphorylation by the Ras-mitogen-activated protein kinase signaling pathway is profoundly associated with the development of HCC and thus may be a critical target for HCC chemoprevention. Acyclic retinoid, which has been clinically shown to reduce the incidence of a post-therapeutic recurrence of HCC, can inhibit Ras activity and phosphorylation of the extracellular signal-regulated kinase and RXRalpha proteins. In conclusion, the inhibition of RXRalpha phosphorylation and the restoration of its physiological function as a master regulator for nuclear receptors may be a potentially effective strategy for HCC chemoprevention and clonal deletion. Acyclic retinoid, which targets phosphorylated RXRalpha, may thus play a critical role in preventing the development of multicentric HCC.

Synergistic Growth Inhibition by 9-cis-Retinoic Acid Plus Trastuzumab in Human Hepatocellular Carcinoma Cells

A malfunction of retinoid X receptor-alpha (RXRalpha) due to phosphorylation by the Ras/mitogen-activated protein kinase signaling pathway is associated with the development of hepatocellular carcinoma (HCC). The humanized anti-HER2 monoclonal antibody trastuzumab inhibits the activation of HER2 and its multiple downstream signaling pathways, including the Ras/mitogen-activated protein kinase pathway. In this study, the effects of phosphorylation of RXRalpha on the ability of RXRalpha ligand 9-cis-retinoic acid (9cRA) and trastuzumab to inhibit growth of HCC cells was examined. The effects of a combination of 9cRA plus trastuzumab on the inhibition of cell growth in HLF human HCC cells which express constitutive activation of HER2 protein were examined. The combination of 9cRA plus trastuzumab synergistically inhibited the growth of HLF cells without affecting the growth of Hc normal human hepatocytes. Combined treatment with these agents acted synergistically to induce apoptosis in HLF cells. The treatment of HLF cells with trastuzumab alone inhibited the phosphorylation of HER2, RXRalpha, ERK, Akt, and Stat3 proteins and these effects were enhanced when the cells were cotreated with 9cRA. Reporter assays indicated that the combination of 9cRA plus trastuzumab markedly increased both the retinoic acid responsive element and retinoid X responsive element promoter activities in HLF cells. 9cRA and trastuzumab cooperatively inhibit the activation of HER2 and its downstream signaling pathways, subsequently inhibiting the phosphorylation of RXRalpha and the growth of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.