Atsushi Hosui

Inhibition of autophagy potentiates the antitumor effect of the multikinase inhibitor sorafenib in hepatocellular carcinoma

Multikinase inhibitor sorafenib inhibits proliferation and angiogenesis of tumors by suppressing the Raf/MEK/ERK signaling pathway and VEGF receptor tyrosine kinase. It significantly prolongs median survival of patients with advanced hepatocellular carcinoma (HCC) but the response is disease‐stabilizing and cytostatic rather than one of tumor regression. To examine the mechanisms underlying the relative resistance in HCC, we investigated the role of autophagy, an evolutionarily conserved self‐digestion pathway, in hepatoma cells in vitro and in vivo. Sorafenib treatment led to accumulation of autophagosomes as evidenced by conversion from LC3‐I to LC3‐II observed by immunoblot in Huh7, HLF and PLC/PRF/5 cells. This induction was due to activation of autophagic flux, as there was further increase in LC3‐II expression upon treatment with lysosomal inhibitors, clear decline of the autophagy substrate p62, and an mRFP‐GFP‐LC3 fluorescence change in sorafenib‐treated hepatoma cells. Sorafenib inhibited the mammalian target of rapamycin complex 1 and its inhibition led to accumulation of LC3‐II. Pharmacological inhibition of autophagic flux by chloroquine increased apoptosis and decreased cell viability in hepatoma cells. siRNA‐mediated knockdown of the ATG7 gene also sensitized hepatoma cells to sorafenib. Finally, sorafenib induced autophagy in Huh7 xenograft tumors in nude mice and coadministration with chloroquine significantly suppressed tumor growth compared with sorafenib alone. In conclusion, sorafenib administration induced autophagosome formation and enhanced autophagic activity, which conferred a survival advantage to hepatoma cells. Concomitant inhibition of autophagy may be an attractive strategy for unlocking the antitumor potential of sorafenib in HCC.

Increases in p53 expression induce CTGF synthesis by mouse and human hepatocytes and result in liver fibrosis in mice

The tumor suppressor p53 has been implicated in the pathogenesis of non-cancer-related conditions such as insulin resistance, cardiac failure, and early aging. In addition, accumulation of p53 has been observed in the hepatocytes of individuals with fibrotic liver diseases, but the significance of this is not known. Herein, we have mechanistically linked p53 activation in hepatocytes to liver fibrosis. Hepatocyte-specific deletion in mice of the gene encoding Mdm2, a protein that promotes p53 degradation, led to hepatocyte synthesis of connective tissue growth factor (CTGF; the hepatic fibrogenic master switch), increased hepatocyte apoptosis, and spontaneous liver fibrosis; concurrent removal of p53 completely abolished this phenotype. Compared with wild-type controls, mice with hepatocyte-specific p53 deletion exhibited similar levels of hepatocyte apoptosis but decreased liver fibrosis and hepatic CTGF expression in two models of liver fibrosis. The clinical significance of these data was highlighted by two observations. First, p53 upregulated CTGF in a human hepatocellular carcinoma cell line by repressing miR-17-92. Second, human liver samples showed a correlation between CTGF and p53-regulated gene expression, which were both increased in fibrotic livers. This study reveals that p53 induces CTGF expression and promotes liver fibrosis, suggesting that the p53/CTGF pathway may be a therapeutic target in the treatment of liver fibrosis.

The Bcl-xL inhibitor, ABT-737, efficiently induces apoptosis and suppresses growth of hepatoma cells in combination with sorafenib†

Tumor cells are characterized by uncontrolled proliferation, often driven by activation of oncogenes, and apoptosis resistance. The oncogenic kinase inhibitor sorafenib can significantly prolong median survival of patients with advanced hepatocellular carcinoma (HCC), although the response is disease‐stabilizing and cytostatic rather than one of tumor regression. Bcl‐xL (B cell lymphoma extra large), an antiapoptotic member of the B cell lymphoma‐2 (Bcl‐2) family, is frequently overexpressed in HCC. Here, we present in vivo evidence that Bcl‐xL overexpression is directly linked to the rapid growth of solid tumors. We also examined whether ABT‐737, a small molecule that specifically inhibits Bcl‐xL but not myeloid cell leukemia‐1 (Mcl‐1), could control HCC progression, especially when used with sorafenib. Administration of ABT‐737, even at an in vivo effective dose, failed to suppress Huh7 xenograft tumors in mice. ABT‐737 caused the levels of Mcl‐1 expression to rapidly increase by protein stabilization. This appeared to be related to resistance to ABT‐737, because decreasing Mcl‐1 expression levels to the baseline by a small interfering RNA–mediated strategy made hepatoma cells sensitive to this agent. Importantly, administration of ABT‐737 to Mcl‐1 knockout mice induced severe liver apoptosis, suggesting that tumor‐specific inhibition of Mcl‐1 is required for therapeutic purposes. Sorafenib transcriptionally down‐regulated Mcl‐1 expression specifically in tumor cells and abolished Mcl‐1 up‐regulation induced by ABT‐737. Sorafenib, not alone but in combination with ABT‐737, efficiently induced apoptosis in hepatoma cells. This combination also led to stronger suppression of xenograft tumors than sorafenib alone. Conclusion: Bcl‐xL inactivation by ABT‐737 in combination with sorafenib was found to be safe and effective for anti‐HCC therapy in preclinical models. Direct activation of the apoptosis machinery seems to unlock the antitumor potential of oncogenic kinase inhibitors and may produce durable clinical responses against HCC. (HEPATOLOGY 2010)

Thrombocytopenia exacerbates cholestasis-induced liver fibrosis in mice.

BACKGROUND & AIMS Circulating platelet counts gradually decrease in parallel with progression of chronic liver disease. Thrombocytopenia is a common complication of advanced liver fibrosis and is thought to be a consequence of the destruction of circulating platelets that occurs during secondary portal hypertension or hypersplenism. It is not clear whether thrombocytopenia itself affects liver fibrosis. METHODS Thrombocytopenic mice were generated by disruption of Bcl-xL, which regulates platelet life span, specifically in thrombocytes. Liver fibrosis was examined in thrombocytopenic mice upon bile duct ligation. Effect of platelets on hepatic stellate cells (HSCs) was investigated in vitro. RESULTS Thrombocytopenic mice developed exacerbated liver fibrosis, with increased expression of type I collagen alpha1 and alpha2, during cholestasis. In vitro experiments revealed that, upon exposure to HSCs, platelets became activated, released hepatocyte growth factor (HGF), and then inhibited HSC expression of the type I collagen genes in a Met signal-dependent manner. In contrast to the wild-type mice, the thrombocytopenic mice did not accumulate hepatic platelets or phosphorylate Met in the liver following bile duct ligation. Administration of recombinant HGF to thrombocytopenic mice reduced liver fibrosis to the levels observed in wild-type mice and attenuated hepatic expression of the type I collagen genes. CONCLUSIONS Thrombocytopenia exacerbates liver fibrosis; platelets have a previously unrecognized, antifibrotic role in suppressing type I collagen expression via the HGF-Met signaling pathway.

Loss of STAT5 causes liver fibrosis and cancer development through increased TGF-β and STAT3 activation

The molecular mechanisms underlying the development of hepatocellular carcinoma are not fully understood. Liver-specific signal transducer and activator of transcription (STAT) 5A/B–null mice (STAT5-LKO) were treated with carbon tetrachloride (CCl4), and histological analyses revealed liver fibrosis and tumors. Transforming growth factor (TGF)–β levels and STAT3 activity were elevated in liver tissue from STAT5-LKO mice upon CCl4 treatment. To define the molecular link between STAT5 silencing and TGF-β up-regulation, as well as STAT3 activation, we examined STAT5-null mouse embryonic fibroblasts and primary hepatocytes. These cells displayed elevated TGF-β protein levels, whereas messenger RNA levels remained almost unchanged. Protease inhibitor studies revealed that STAT5 deficiency enhanced the stability of mature TGF-β. Immunoprecipitation and immunohistochemistry analyses demonstrated that STAT5, through its N-terminal sequences, could bind to TGF-β and that retroviral-mediated overexpression of STAT5 decreased TGF-β levels. To confirm the in vivo significance of the N-terminal domain of STAT5, we treated mice that expressed STAT5 lacking the N terminus (STAT5-ΔN) with CCl4. STAT5-ΔN mice developed CCl4-induced liver fibrosis but no tumors. In conclusion, loss of STAT5 results in elevated TGF-β levels and enhanced growth hormone–induced STAT3 activity. We propose that a deregulated STAT5–TGF-β–STAT3 network contributes to the development of chronic liver disease.

Alterations in microRNA expression profile in HCV-infected hepatoma cells: involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway.

The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.

Sorafenib inhibits the shedding of major histocompatibility complex class I-related chain A on hepatocellular carcinoma cells by down-regulating a disintegrin and metalloproteinase 9.

The ectodomain of major histocompatibility complex class I–related chain A (MICA) is shed from tumor cells, and may be an important means of evading antitumor immunity. This study investigated the roles of a disintegrin and metalloproteinase 9 (ADAM9) in the shedding of MICA in human hepatocellular carcinoma (HCC). Small interfering RNA–mediated knockdown (KD) of ADAM9 resulted in up‐regulation of membrane‐bound MICA expression on the HepG2 and PLC/PRF/5 cellular surfaces and down‐regulation of soluble MICA levels in their culture supernatant. ADAM9 was cleaved at a site between Gln347 and Val348 of MICA in vitro. We constructed a plasmid of the MICA gene with mutation or deletion of the ADAM9 cleavage site to examine the detailed mechanism of MICA shedding by ADAM9 protease. The results suggested that MICA might be cleaved at the intracellular ADAM9‐recognized cleavage site and was further cleaved at the extracellular ADAM9‐independent cleavage site in HCC cells, resulting in the production of soluble MICA. Immunohistochemical analysis revealed that ADAM9 was overexpressed in human HCC compared to normal liver tissues. The cytolytic activity of natural killer (NK) cells against ADAM9KD‐HCC cells was higher than that against control cells, and the enhancement of this cytotoxicity depended on the MICA/B and NK group 2, member D pathway. Sorafenib treatment resulted in decreased expression of ADAM9, increased expression of membrane‐bound MICA expression, and decreased levels of soluble MICA in HCC cells. Adding sorafenib enhanced the NK sensitivity of HCC cells via increased expression of membrane‐bound MICA. Conclusion: ADAM9 is involved in MICA ectodomain shedding in HCC cells, and sorafenib can modulate ADAM9 expression. Sorafenib therapy may have a previously unrecognized effect on antitumor immunity in patients with HCC. (HEPATOLOGY 2010.)

Mcl‐1 and Bcl‐xL cooperatively maintain integrity of hepatocytes in developing and adult murine liver

Anti‐apoptotic members of the Bcl‐2 family, including Bcl‐2, Bcl‐xL, Mcl‐1, Bcl‐w and Bfl‐1, inhibit the mitochondrial pathway of apoptosis. Bcl‐xL and Mcl‐1 are constitutively expressed in the liver. Although previous research established Bcl‐xL as a critical apoptosis antagonist in differentiated hepatocytes, the significance of Mcl‐1 in the liver, especially in conjunction with Bcl‐xL, has not been clear. To examine this question, we generated hepatocyte‐specific Mcl‐1–deficient mice by crossing mcl‐1flox/flox mice and AlbCre mice and further crossed them with bcl‐xflox/flox mice, giving Mcl‐1/Bcl‐xL–deficient mice. The mcl‐1flox/flox AlbCre mice showed spontaneous apoptosis of hepatocytes after birth, as evidenced by elevated levels of serum alanine aminotransferase (ALT) and caspase‐3/7 activity and an increased number of terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate nick‐end labeling (TUNEL)‐positive cells in the liver; these phenotypes were very close to those previously found in hepatocyte‐specific Bcl‐xL–deficient mice. Although mcl‐1flox/+ AlbCre mice did not display apoptosis, their susceptibility to Fas‐mediated liver injury significantly increased. Further crossing of Mcl‐1 mice with Bcl‐xL mice showed that bcl‐xflox/+ mcl‐1flox/+ AlbCre mice also showed spontaneous hepatocyte apoptosis similar to Bcl‐xL–deficient or Mcl‐1–deficient mice. In contrast, bcl‐xflox/flox mcl‐1flox/+ AlbCre, bcl‐xflox/+ mcl‐1flox/flox AlbCre, and bcl‐xflox/flox mcl‐1flox/flox AlbCre mice displayed a decreased number of hepatocytes and a reduced volume of the liver on day 18.5 of embryogenesis and rapidly died within 1 day after birth, developing hepatic failure evidenced by increased levels of blood ammonia and bilirubin. Conclusion: Mcl‐1 is critical for blocking apoptosis in adult liver and, in the absence of Bcl‐xL, is essential for normal liver development. Mcl‐1 and Bcl‐xL are two major anti‐apoptotic Bcl‐2 family proteins expressed in the liver and cooperatively control hepatic integrity during liver development and in adult liver homeostasis in a gene dose‐dependent manner. (HEPATOLOGY 2009.)

BH3-only Activator Proteins Bid and Bim Are Dispensable for Bak/Bax-dependent Thrombocyte Apoptosis Induced by Bcl-xL Deficiency: MOLECULAR REQUISITES FOR THE MITOCHONDRIAL PATHWAY TO APOPTOSIS IN PLATELETS

A pivotal step in the mitochondrial pathway of apoptosis is activation of Bak and Bax, although the molecular mechanism remains controversial. To examine whether mitochondrial apoptosis can be induced by just a lack of antiapoptotic Bcl-2-like proteins or requires direct activators of the BH3-only proteins including Bid and Bim, we studied the molecular requisites for platelet apoptosis induced by Bcl-xL deficiency. Severe thrombocytopenia induced by thrombocyte-specific Bcl-xL knock-out was fully rescued in a Bak and Bax double knock-out background but not with single knock-out of either one. In sharp contrast, deficiency of either Bid, Bim, or both did not alleviate thrombocytopenia in Bcl-xL knock-out mice. An in vitro study revealed that ABT-737, a Bad mimetic, induced platelet apoptosis in association with a conformational change of the amino terminus, translocation from the cytosol to mitochondria, and homo-oligomerization of Bax. ABT-737-induced Bax activation and apoptosis were also observed in Bid/Bim-deficient platelets. Human platelets, upon storage, underwent spontaneous apoptosis with a gradual decline of Bcl-xL expression despite a decrease in Bid and Bim expression. Apoptosis was attenuated in Bak/Bax-deficient or Bcl-xL-overexpressing platelets but not in Bid/Bim-deficient platelets upon storage. In conclusion, platelet lifespan is regulated by a fine balance between anti- and proapoptotic multidomain Bcl-2 family proteins. Despite residing in platelets, BH3-only activator proteins Bid and Bim are dispensable for Bax activation and mitochondrial apoptosis.

Identification of Multiple Transcription Factors, HLF, FTF, and E4BP4, Controlling Hepatitis B Virus Enhancer II

ABSTRACT Hepatitis B virus (HBV) enhancer II (EnII) is a hepatotropiccis element which is responsible for the hepatocyte-specific gene expression of HBV. Multiple transcription factors have been demonstrated to interact with this region. In this study, the region from HBV nucleotides (nt) 1640 to 1663 in EnII was demonstrated to be essential for enhancer activity and to be another target sequence of putative transcription factors. To elucidate the factors which bind to this region, we used a yeast one-hybrid screening system and cloned three transcription factors, HLF, FTF, and E4BP4, from a human adult liver cDNA library. All of these factors had binding affinity to the sequence from nt 1640 to 1663. Investigation of the effects of these factors on transcriptional regulation revealed that HLF and FTF had stimulatory activity on nt 1640 to 1663, whereas E4BP4 had a suppressing effect. FTF coordinately activated both 3.5-kb RNA and 2.4/2.1-kb RNA transcription in a transient transfection assay with an HBV expression vector. HLF, however, activated only 3.5-kb RNA transcription, and in primer extension analysis, HLF strongly stimulated the synthesis of pregenome RNA compared to precore RNA. Thus, FTF stimulated the activity of the second enhancer, while HLF stimulated the activity of the core upstream regulatory sequence, which affects only the core promoter, and had a dominant effect on the pregenome RNA synthesis.