Phosphorylation of retinoid X receptor alpha at serine 260 impairs its metabolism and function in human hepatocellular carcinoma

Retinoids induce apoptosis and differentiation of hepatocellular carcinoma (HCC) cells and are used clinically in the chemoprevention of HCC. We have shown previously that hepatocarcinogenesis is accompanied by accumulation of full-length retinoid X receptor alpha (RXRalpha), although the underlying mechanisms and biological implications have remained unclear. The present studies were based on the finding that the accumulated full-length RXRalpha was phosphorylated at serine/threonine residues both in all human HCC tissues examined and in human HCC-derived HuH7 cells. Phosphorylation at serine 260 of RXRalpha, a consensus site of mitogen-activated protein kinase, was closely linked to its retarded degradation, low transactivating activity, and the promotion of cancer cell growth. There was no genomic mutation in the RXRalpha gene, and abrogation of phosphorylation by mitogen-activated protein kinase-specific inhibitors restored the degradation of RXRalpha in an RXR ligand-dependent manner. These results suggest that phosphorylation of RXRalpha may interfere with its metabolism and signaling in human HCC, which could lead to growth promotion of these tumors.     

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 RXRα 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 RXRα proteins. In conclusion, the inhibition of RXRα 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 RXRα, may thus play a critical role in preventing the development of multicentric HCC. ( Cancer Sci 2009; 100: 369–374)     

Expression of interferon alpha/beta receptor in human hepatocellular carcinoma

Interferon-alpha (IFNalpha) plays a crucial role in the antiproliferation and immunoregulatory activity through the specific cell surface receptor, interferon-alpha/beta receptor (IFNalpha/betaR). We examined the immunohistochemical expression of IFNalpha/betaR in 91 hepatocellular carcinoma (HCC), HCV-related chronic hepatitis (n=38) and cirrhosis (n=53), dysplastic nodules (n=5), and normal liver (n=9). The level of IFNalpha/betaR increased in chronic hepatitis and cirrhosis compared with normal liver. All the dysplastic nodules showed moderate or high expression. In HCCs, 26% (24/91) of patients showed high IFNalpha/betaR expression while the remaining 38% (35/91) showed moderate, and 35% (32/91) no or faint expression. Clinicopathological survey demonstrated a significant correlation between IFNalpha/betaR expression and differentiation of carcinoma (P=0.0008) although there was no correlation between IFNalpha/betaR expression in HCC and survival or disease-free survival. Thus, IFNalpha/betaR was expressed not only in chronic hepatitis or liver cirrhosis but in HCC and its expression was significantly correlated with tissue differentiation of carcinoma.     

P2X3 purinergic receptor overexpression is associated with poor recurrence-free survival in hepatocellular carcinoma patients

P2 purinergic receptors are overexpressed in certain cancer tissues, but the pathophysiologic relevance of purinergic signaling in hepatocellular carcinoma (HCC) remains unknown. To examine the role of P2 purinergic signaling in the pathogenesis of HCC and characterize extracellular nucleotide effects on HCC cell proliferation, two independent HCC patient cohorts were analyzed for P2 purinergic receptor expression, and nucleotide treated HCC cell lines were evaluated for effects on proliferation and cell cycle progression. Our studies suggest that multiple P2 purinergic receptor isoforms are overexpressed in liver tumors, as compared to uninvolved liver, and dysregulation of P2 purinergic receptor expression is apparent in HCC cell lines, as compared to human primary hepatocytes. High P2X3 purinergic receptor expression is associated with poor recurrence-free survival (RFS), while high P2Y13 expression is associated with improved RFS. Extracellular nucleotide treatment alone is sufficient to induce cell cycle progression, via activation of JNK signaling, and extracellular ATP-mediated activation of P2X3 receptors promotes proliferation in HCC cells. Conclusion: Our analysis of HCC patient livers and HCC cells in vitro identifies a novel role for dysregulation of P2 purinergic signaling in the induction of hyper-proliferative HCC phenotype and identifies P2X3 purinergic receptors as potential new targets for therapy.     

Changes in number of alpha-adrenergic receptor subtypes in hepatocytes from rats fed 3'-methyl-4-dimethylaminoazobenzene

Changes in numbers of alpha 1- and alpha 2-adrenergic receptors in the plasma membranes of hepatocytes from female Donryu rats given feed containing 0.06% of the carcinogen 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB), were examined. alpha 1-Adrenergic receptors, measured in terms of [3H]prazosin binding, decreased to half of the control 2 weeks after the start of this diet, then gradually decreased for the next 22 weeks. alpha 2-Adrenergic receptors, measured in terms of [3H]clonidine binding, transiently increased 3-fold over the control at 2 weeks. These changes in the early period of the 3'-MeDAB diet intake may be related to hepatocarcinogenesis.     

PADI4在肝癌细胞中表达的检测

Objective  

The aim of the research was to study peptidylarginine deiminase type 4 (PAD4/PADI4) expression and its tumorigenic mechanism in hepatocellular carcinomas.     

信号调节蛋白SIRPα在肝癌内的表达及意义

目的探讨信号调节蛋白ＳＩＲＰα与肝细胞肝癌之间的关系。方法应用Ｎｏｒｔｈｅｒｎ杂交技术,检测３６对肝细胞肝癌（ＨＣＣ）和相应癌旁肝组织,以及一种正常肝细胞系、四种肝癌细胞系内ＳＩＲＰα基因表达的变化,并结合各组织标本的病理学特点进行分析。结果ＳＩＲＰα在２０例肝癌组织及两种肝癌细胞系内表达水平明显下降。在两种主要杂交信号中,肿瘤组织内３．９ｋｂ转录子表达量较癌旁组织下降２～４倍,而２．５ｋｂ转录子表达量则下降４～６倍。有９例肿瘤组织在原基因表达水平下降的同时,在３．９ｋｂ转录子的上方又出现一长度稍大的杂交信号,而相应癌旁肝组织内则为阴性。其中,５例有肝内或门静脉转移的肿瘤组织,ＳＩＲＰα表达全部下降,且均发现有这种新的基因表现形式出现。结论ＳＩＲＰα尤其是２．５ｋｂ转录子在肝癌内表达下降,与肿瘤进展程度密切相关,且肿瘤组织内存在ＳＩＲＰα相关的新的基因表现形式。ＳＩＲＰα可能为肝癌发生发展过程中一个重要的调节蛋白。     

Dissemination of hepatocellular carcinoma is mediated via chemokine receptor CXCR4

In different tumour entities, expression of the chemokine receptor 4 (CXCR4) has been linked to tumour dissemination and poor prognosis. Therefore, we evaluated, if the expression of CXCR4 exerts similar effects in human hepatocellular carcinoma (HCC). Expression analysis and functional assays were performed in vitro to elucidate the impact of CXCL12 on human hepatoma cells lines. In addition, expression of CXCR4 was evaluated in 39 patients with HCC semiquantitatively and correlated with both, tumour and patients characteristics. Human HCC and hepatoma cell lines displayed variable intensities of CXCR4 expression. Loss of p53 function did not impact on CXCR4 expression. Exposure to CXCL12 mediated a perinuclear translocation of CXCR4 in Huh7/Hep3B cells and increased the invasive potential of Huh7 cells. In HCC patients, CXCR4 expression significantly correlated with progressed local tumours (T-status; P=0.006), lymphatic metastasis (N-status; P=0.005) and distant dissemination (M-status; P=0.009), as well as with a decreased 3-year-survival rate (P=0.01). In summary, strong expression of CXCR4 is significantly associated with progressed hepatocellular cancer.     

Synergistic growth inhibition of human hepatocellular carcinoma cells by acyclic retinoid and GW4064, a farnesoid X receptor ligand

Abnormalities in the expression and function of retinoid X receptor (RXR), a master regulator of the nuclear receptor superfamily, are associated with the development of hepatocellular carcinoma (HCC). Dysfunction of farnesoid X receptor (FXR), one of the nuclear receptors that forms a heterodimer with RXR, also plays a role in liver carcinogenesis. In the present study, we examined the effects of acyclic retinoid (ACR), a synthetic retinoid targeting RXRα, plus GW4064, a ligand for FXR, on the growth of human HCC cells. We found that ACR and GW4064 preferentially inhibited the growth of HLE, HLF, and Huh7 human HCC cells in comparison with Hc normal hepatocytes. The combination of 1μM ACR plus 1μM GW4064 synergistically inhibited the growth of HLE cells by inducing apoptosis. The combined treatment with these agents acted cooperatively to induce cell cycle arrest in the G(0)/G(1) phase and inhibit the phosphorylation of RXRα, which is regarded as a critical factor for liver carcinogenesis, through inhibition of ERK and Stat3 phosphorylation. This combination also increased the expression levels of p21(CIP1) and SHP mRNA, while decreasing the levels of c-myc and cyclin D1 mRNA in HLE cells. In addition, a reporter assay indicated that the FXRE promoter activity was significantly increased by treatment with ACR plus GW4064. Our results suggest that ACR and GW4064 cooperatively inhibit RXRα phosphorylation, modulate the expression of FXR-regulated genes, thus resulting in the induction of apoptosis and the inhibition of growth in HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC.     

趋化因子CXC受体3在肝细胞癌中的表达及意义

背景与目的:最近研究揭示趋化因子及其受体网络在肿瘤侵袭转移中起重要作用。本研究旨在探讨趋化因子CXC受体3(CXCR3)在肝细胞癌(hepatocellularcarcinoma,HCC)中的表达,及其与HCC临床病理特征的关系。方法:选取7株人HCC细胞系、18例正常肝组织、64例HCC患者的癌组织和癌旁肝组织作为研究对象。采用RT-PCR和实时定量PCR方法,研究这些组织中CXCR3mRNA的表达情况。采用免疫组织化学方法,研究CXCR3蛋白的表达情况和HCC复发转移的关系。结果:在高转移细胞系MHCC97-H中,CXCR3与!2微球蛋白的mRNA拷贝均数之比为33.0×10-4,在低转移细胞系MHCC97-L中,该数值为8.7×10-4,在无转移能力的5株细胞系则未检出CXCR3的mRNA表达。在MHCC97-H和MHCC97-L中均见CXCR3蛋白强阳性染色,而在无转移能力的细胞系中只有HepG2见到微弱染色,其余4株均未见染色。CXCR3蛋白在HCC组织中的强阳性染色,与HCC的侵袭性正相关(P=0.003)。HCC组织中CXCR3蛋白强阳性的患者和CXCR3阴性或弱阳性的患者的1、2年无瘤生存率分别为66.7%、31.3%和75.0%、59.5%,差异有显著性(P=0.044)。结论:CXCR3的表达与HCC的侵袭和转移相关。     

Ubiquitin/proteasome pathway regulates levels of retinoic acid receptor gamma and retinoid X receptor alpha in human keratinocytes

Repeated exposure of human skin to solar UV radiation leads to premature aging (photoaging) and skin cancer. UV-induced skin damage can be ameliorated by all-trans retinoic acid treatment. The actions of retinoic acid in skin keratinocytes are mediated primarily by nuclear retinoic acid receptor gamma (RARgamma) and retinoid X receptor alpha (RXRalpha). We found that exposure of cultured primary human keratinocytes to UV irradiation (30 mJ/cm2) substantially reduced (50-90%) RARgamma and RXRalpha mRNA and protein within 8 h. The rates of disappearance of RARgamma and RXRalpha proteins after UV exposure or treatment with the protein synthesis inhibitor cycloheximide were similar. UV irradiation did not increase the rate of breakdown of RARgamma or RXRalpha but rather reduced their rate of synthesis. The addition of proteasome inhibitors MG132 and LLvL, but not the lysosomal inhibitor E64, prevented loss of RARgamma and RXRalpha proteins after exposure of keratinocytes to either UV radiation or cycloheximide. Soluble extracts from nonirradiated or UV-irradiated keratinocytes possessed similar levels of proteasome activity that degraded RARgamma and RXRalpha proteins in vitro. Furthermore, RARgamma and RXRalpha were polyubiquitinated in intact cells. RXRalpha was found to contain two proline, glutamate/aspartate, serine, and threonine (PEST) motifs, which confer rapid turnover of many short-lived regulatory proteins that are degraded by the ubiquitin/proteasome pathway. However, the PEST motifs in RXRalpha did not function to regulate its stability, because deletion of the PEST motifs individually or together did not alter ubiquitination or proteasome-mediated degradation of RXRalpha. These results demonstrate that loss of RARgamma and RXRalpha proteins after UV irradiation results from degradation via the ubiquitin/proteasome pathway. Taken together, the data here indicate that ubiquitin/proteasome-mediated breakdown is an important mechanism regulating the levels of nuclear retinoid receptors.     

Suppression by oestrogen of hepatocellular tumourigenesis induced in mice by 3'-methyl-4-dimethylaminoazobenzene.

Treatment of female C57BL/6 x DS-F1 mice with 3''-methyl-4-dimethylaminoazobenzene (3''-Me-DAB) neonatally resulted in the development of adenomatous nodules and glucose-6-phosphatase (G-6-Pase) deficient foci at 8 and 6 months of age, respectively. Ovariectomy of these mice at 1 month of age hastened the development and increased the incidences of these lesions. Subcutaneous implantation of estradiol-17 beta (E2) with ovariectomy at 1 month of age markedly decreased the incidences of adenomatous nodules and G-6-Pase deficient foci at 10 or 12 months of age, but subcutaneous implantation of progesterone did not reduce their incidences. Subcutaneous implantation of E2 into ovariectomised mice at 6 months of age resulted in significant decreases in the incidences of adenomatous nodules and G-6-Pase deficient foci at 10 months of age, but implantation of E2 into the spleen of ovariectomised mice of the same age had no effect on their incidences. The present results suggest that E2 suppresses the development of adenomatous nodules and G-6-Pase deficient foci induced in the mouse liver by 3''-Me-DAB by actions on tissues other than the liver.     

Promotion of hepatocarcinogenesis by suxibuzone in rats initiated with 3'-methyl-4-dimethylaminoazobenzene

Among phenylbutazone (PZ) and its related compounds, suxibuzone (SUX) caused the most extensive decrease in pyruvate kinase (PK) activity with lower toxicity. Therefore, we studied the effect of SUX on rat hepatocarcinogenesis to confirm our assumption that an agent which causes a prolonged decrease in PK activity in rat liver promotes hepatocarcinogenesis. For initiation rats were fed a diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) for 4 weeks. At the end of 53 weeks of the experiment the incidences of liver tumors were 14.3 and 70.0% in the rats fed basal diet and in the rats fed 0.5% SUX diet, respectively, after the initiation. No tumors were observed in rats fed the SUX diet without the initiation. The result shows that SUX promotes hepatocarcinogenesis and supports the above assumption.     

Roles of ovaries and testes in hepatocellular tumorigenesis induced in mice by 3'-methyl-4-dimethylaminoazobenzene

The roles of gonads in tumorigenesis induced in mouse liver by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) were investigated. C57BL/6 x DS-F1 mice which were 10, 12, 14, 16 and 18 days old were treated i.p. with 3'-Me-DAB. Nodular lesions induced in the liver were classified into adenomatous nodules and hepatocellular carcinomas. Adenomatous nodules were composed of a mixture of eosinophilic, basophilic, vacuolated and foamy hepatocytes in various proportions and compressed the adjacent parenchyma. These adenomatous nodules were of monoclonal origin. Carcinomas had a trabecular structure. The incidence, numbers per mouse and areas of adenomatous nodules and carcinomas in male and female mice aged 16-64 weeks were compared. Adenomatous nodules were first detected in males and females aged 24 and 52 weeks, respectively, and their incidences were much higher in males than in females of the same age. The number of adenomatous nodules per mouse and their size were also higher in males. The first carcinoma was found in a 52-week-old male, but no carcinomas were found in females even at 64 weeks of age. The effects of castration 23 days after birth on the appearance of nodular lesions in the livers of 32- to 64-week-old mice were examined. Castration of males did not affect the incidence of adenomatous nodules, but reduced the incidence of carcinomas. It also decreased the number of adenomatous nodules per mouse. Castration of females reduced the time before appearance of adenomatous nodules and increased their incidence, number per mouse, and area. A carcinoma appeared in a 64-week-old castrated female, but no similar lesion was found in intact females of the same age. These results indicate that tumorigenesis induced in mouse liver by 3'-Me-DAB is enhanced by the testes and suppressed by the ovaries.     

Prevention of recurrent hepatocellular carcinoma with retinoid]

The annual rate of recurrent hepatocellular carcinoma (HCC) after curative treatment of the initial tumor is reportedly as high as 20-25%. Such a high recurrence rate is responsible for the poor prognosis of patients with HCC. One third of the recurrent HCC arises from intrahepatic metastasis of the initial tumor, one third from multicentric carcinogenesis, and one third from unknown origin. In any type of recurrence, elimination of the transformed cell clone from the remnant liver is essential to prevent the recurrence of HCC. A retinoid analog, acyclic retinoid, induces differentiation and apoptosis of HCC cell lines in vitro. An early clinical trial showed a preventive effect of the compound on recurrent HCC. A further trial of acyclic retinoid is now under way.