Keita Kanki

Retinoids and their target genes in liver functions and diseases

Retinoids have been reported to prevent several kinds of cancers, including hepatocellular carcinoma (HCC). Retinoic acid (RA) coupled with retinoic acid receptor/retinoid X receptor heterodimer exerts its functions by regulating its target genes. We previously reported that transgenic mice, in which RA signaling is suppressed in a hepatocyte-specific manner, developed liver cancer at a high rate, and that disruption of RA functions led to the increased oxidative stress via aberrant metabolisms of lipid and iron, indicating that retinoids play an important role in liver pathophysiology. These data suggest that exploring the metabolism of retinoids in liver diseases and their target genes provides us with useful information to understand the liver functions and diseases. Consequently, the altered metabolism of retinoids was observed in liver diseases, including non-alcoholic fatty liver disease. In this review, we summarize the metabolism of retinoids in the liver, highlight the functions of retinoids in HCC, non-alcoholic fatty liver disease, and alcoholic liver disease, and discuss the target genes of RA. Investigation of retinoids in the liver will likely help us identify novel therapies and diagnostic modalities for HCC.

Biological and clinical implications of retinoic acid-responsive genes in human hepatocellular carcinoma cells.

BACKGROUND & AIMSnAccumulating data from epidemiological and experimental studies have suggested that retinoids, which are vitamin A derivatives, exert antitumor activity in various organs. We performed a gene screening based on in silico analysis of retinoic acid response elements (RAREs) to identify the genes facilitating the antitumor activity of retinoic acid (RA) and investigated their clinical significance in hepatocellular carcinoma (HCC).nnnMETHODSnIn silico analysis of RAREs was performed in the 5-kb upstream region of EST clusters. Chromatin immunoprecipitation analysis of the retinoic acid receptors and gene expression analysis were performed in HuH7, HepG2, and MCF7 cells treated with all-trans RA (ATRA). mRNA expression of RA-responsive genes was investigated using tumor and non-tumor tissues of clinical HCC samples from 171 patients. The association between gene expression and survival of patients was examined by Cox regression analysis.nnnRESULTSnWe identified 201 candidate genes with promoter regions containing consensus RARE and finally selected 26 RA-responsive genes. Of these, downregulation of OTU domain-containing 7B (OTUD7B) gene, which was upregulated by ATRA, in tumor tissue was associated with a low cancer-specific survival of HCC patients. Functional analyses revealed that OTUD7B negatively regulates nuclear factor κB (NF-κB) signaling and decreases the survival of HCC cells.nnnCONCLUSIONSnWe identified RA-responsive genes which are regulated by retinoid signal and found that low-OTUD7B mRNA expression is associated with a poor prognosis for HCC patients. OTUD7B-mediated inhibition of NF-κB signaling may be an effective target for antitumor therapy for HCC.

Identification of the Genes Chemosensitizing Hepatocellular Carcinoma Cells to Interferon-α/5-Fluorouracil and Their Clinical Significance

The incidence of advanced hepatocellular carcinoma (HCC) is increasing worldwide, and its prognosis is extremely poor. Interferon-alpha (IFN-α)/5-fluorouracil (5-FU) therapy is reportedly effective in some HCC patients. In the present study, to improve HCC prognosis, we identified the genes that are sensitizing to these agents. The screening strategy was dependent on the concentration of ribozymes that rendered HepG2 cells resistant to 5-FU by the repeated transfection of ribozymes into the cells. After 10 cycles of transfection, which was initiated by 5,902,875 sequences of a ribozyme library, three genes including protein kinase, adenosine monophosphate (AMP)-activated, gamma 2 non-catalytic subunit (PRKAG2); transforming growth factor-beta receptor II (TGFBR2); and exostosin 1 (EXT1) were identified as 5-FU-sensitizing genes. Adenovirus-mediated transfer of TGFBR2 and EXT1 enhanced IFN-α/5-FU-induced cytotoxicity as well as 5-FU, although the overexpression of these genes in the absence of IFN-α/5-FU did not induce cell death. This effect was also observed in a tumor xenograft model. The mechanisms of TGFBR2 and EXT1 include activation of the TGF-β signal and induction of endoplasmic reticulum stress, resulting in apoptosis. In HCC patients treated with IFN-α/5-FU therapy, the PRKAG2 mRNA level in HCC tissues was positively correlated with survival period, suggesting that PRKAG2 enhances the effect of IFN-α/5-FU and serves as a prognostic marker for IFN-α/5-FU therapy. In conclusion, we identified three genes that chemosensitize the effects of 5-FU and IFN-α/5-FU on HCC cells and demonstrated that PRKAG2 mRNA can serve as a prognostic marker for IFN-α/5-FU therapy.

Liver Resident Macrophages (Kupffer Cells) Share Several Functional Antigens in Common with Endothelial Cells

The identification and specific functions of Kupffer cells (KCs), a liver resident macrophage subpopulation, are still unclear. We compared KCs with peritoneal macrophages using cDNA microarray analysis and found that these cells share some antigens with endothelial cells. KCs highly express VCAM‐1 and VEGF receptors (VEGF‐Rs) at transcriptional and protein levels. VCAM‐1 mediates the functional binding of KCs with lymphocytes and induces KC activation. Among the VEGF receptors, VEGF‐R2 and VEGF‐R3 were expressed on the KCs, while VEGF‐R1 was expressed on other tissue macrophage subsets. VEGF120, a ligand of both VEGF‐R1 and VEGF‐R2, transduced strong survival and chemotactic signals through the KCs, when compared to PIGF, a VEGF‐R1 ligand, indicating that VEGF‐R2 plays significant roles in regulating KC activities. Expression of the VEGF‐Rs was regulated by TLR4 signalling. These results suggest that the function of KCs is partly regulated by the common antigens shared with endothelial cells.

Impact of Preferentially Expressed Antigen of Melanoma on the Prognosis of Hepatocellular Carcinoma

Background: Retinoids, vitamin A and its derivatives, have an antitumor effect on hepatocellular carcinoma (HCC). The function of retinoids is exerted by the complex of retinoic acid (RA) with the heterodimer of retinoid X receptor and the RA receptor. The preferentially expressed antigen of melanoma (PRAME) acts as a dominant repressor of RA signaling by binding to the complex. The significance of PRAME on the prognosis of HCC remains to be clarified. Methods: PRAME mRNA expression was examined by quantitative real-time polymerase chain reaction in both tumor and non-tumor tissues of 100 HCC patients who received surgical resection. The effect of PRAME knockdown on DR5-mediated RA transcriptional activity was examined. Results: In tumor tissues, there were significant associations among PRAME expression, clinical stage, tumor markers, and tumor numbers. In non-tumor tissues, there were significant associations among PRAME expression, overall survival, and disease-free survival. The knockdown of PRAME caused no reduction in DR5-mediated transcriptional activity of RA, suggesting that PRAME acts via other mechanisms than the DR5 RA-responsive elements. Conclusion: Our findings indicate that PRAME expression is a novel prognostic marker in HCC patients.

Involvement of glucocorticoid in induction of lingual T1R3 in rodents.

We previously reported that in rats, chronic exposure to stress inhibits the induction of the common receptor (T1R3) for sweet and umami tastes. Here, we investigated whether endogenous glucocorticoids (GCs) might be responsible for this inhibition. In addition, we used mouse taste-bud cells (TB cells) expressing T1R3 to examine the effect of exogenous GC on T1R3 induction. Both adrenal glands were removed from rats [adrenalectomized (ADX) rats] and T1R3 mRNA expression in fungiform papillae was examined by real-time RT-PCR. T1R3 mRNA expression was significantly reduced in the ADX rats (versus sham-ADX rats). The reduced mRNA expression was restored to the level seen in the sham-ADX rats by administration of dexamethasone (DEX) at the smallest dose tested (0.1ng/kg, i.p.). However, with larger doses of DEX (10 and 1000ng/kg, i.p.) there was no such restoration (i.e., the expression level did not differ from that seen in ADX rats). Expression of the mRNA for the GC receptor-α was detected in mouse TB cells by RT-PCR. Significantly reduced T1R3 mRNA expression, as measured by real-time RT-PCR, was observed in TB cells at 24h after application of DEX (0.1, 1.0, or 10μM). These results suggest that in rodents: (a) a low concentration of endogenous GC is necessary and sufficient for induction of T1R3 expression, and that higher concentrations may actually inhibit such induction, and (b) this inhibitory effect may be due, at least in part, to a direct action of GC on taste cells.

Abstract 4234: Identification of molecular targets for retinoid signaling to regulate human hepatocellular carcinoma development: contribution of OTUD7B, a negative regulator of NF-κB signaling, in antitumor effect of retinoic acid

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnBACKGROUND & AIMS: The loss of retinoid content and diminished retinoid signaling and downstream gene function are closely associated with the progression of liver disease including hepatocellular carcinoma (HCC). We previously identified 26 retinoic acid (RA)-responsive genes through a gene screening based on genome-wide in silico analysis of RA response elements. To understand the clinical and biological significance of RA-responsive genes in HCC, we performed gene expression analysis using clinical HCC samples and functional analyses using HCC cell lines.nnMETHODS: mRNA expressions of RA-responsive genes were investigated in tumor and non-tumor tissues of the liver from 171 HCC patients by qRT-PCR. The association of clinicopathological parameters and gene expression with survival time was evaluated using univariate and multivariate Cox regression modeling. Potential anti-tumor activity of the candidate genes were evaluated using siRNA-mediated knockdown and adenovirus-mediated overexpression experiments in HCC cells. Functional analyses of the genes in the regulation of intracellular signal pathways were performed using luciferase-based reporter assays.nnRESULTS: Among the 9 genes that showed ATRA responsiveness in HuH7 or HepG2 cells, 3 genes, OTU domain-containing 7B (OTUD7B), kinesin family member 21B (KIF21B), and odd-skipped related 1 (OSR1) were significantly upregulated in tumor tissue compared to non-tumor tissue, and 4 genes, colony stimulating factor 3 receptor (CSF3R), thioredoxin interacting protein (TXNIP), cytochrome P450 26A1 (CYP26A1), and olfactomedin-like 1 (OLFML1) were downregulated. Frizzled 4 (FZD4) mRNA level was not different between tumor and non-tumor tissue. In tumor tissue, low expression of OTUD7B gene, which was upregulated by ATRA, was significantly associated with a low cancer-specific survival of HCC patients. Functional analyses revealed that OTUD7B negatively regulates nuclear factor κB (NF-κB) signaling and decreased the cell viability by inducing apoptosis in HCC cells. Frizzled 4 (FZD4), which was downregulated by ATRA, was shown to positively regulate Wnt/β-catenin signaling and increased the cell viability in HCC cells.nnDISCUSSIONS AND CONCLUSION: Our results suggest that the downregulation of OTUD7B expression in tumors facilitates tumor growth and enhances the cancer phenotype, leading to malignancy and poor cancer-specific survival. Therefore, retinoid signaling, which upregulates OTUD7B, prevents tumor development by suppressing aberrant NF-κB signaling in HCC cells, being beneficial for antitumor therapy for HCC. Our results also suggested that Wnt/β-catenin signaling may be a potential target for retinoid signaling to regulate HCC development.nnCitation Format: Goshi Shiota, Keita Kanki. Identification of molecular targets for retinoid signaling to regulate human hepatocellular carcinoma development: contribution of OTUD7B, a negative regulator of NF-κB signaling, in antitumor effect of retinoic acid. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4234. doi:10.1158/1538-7445.AM2014-4234