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.

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;52:1310-1321 T he B cell lymphoma-2 (Bcl-2) family proteins regulate the mitochondrial pathway of apoptosis, a major form of cell death.1 They include five antiapoptotic proteins, Bcl-2, B cell lymphoma extra large (Bcl-xL), myeloid cell leukemia-1 (Mcl-1), Bcl-2-related protien A1 (Bfl-1), and Bcl-2-like 2 (Bcl-w), and two structurally related proapoptotic proteins, Bcl-2-antagonist/killer (Bak) and Bcl-2-Abbreviations: ALT, alanine aminotransferase; Bad, Bcl-2-associated agonist of cell death; Bak, Bcl-2-antagonist/killer; Bax, Bcl-2-associated X protein; Bcl-2, B cell lymphoma-2; BH3, Bcl-2 homology domain-3; Bid, BH3-interacting domain death agonist; cDNA, complementary DNA; HA, hemagglutinin; HCC, hepatocellular carcinoma; Mcl-1, myeloid cell leukemia-1; mRNA, messenger RNA; RT-PCR, reverse-transcription polymerase chain reaction; siRNA, small interfering RNA; USP9X, ubiquitin-specific peptidase 9 X-linked; WST, water-soluble tetrazolium.From the

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.Platelets are unique blood cells that do not have a nucleus but contain mitochondria and have the daily job of handling hemostasis and thrombosis (1). They are produced from megakaryocytes and once released into circulation can function for about 10 days in humans and 4 -5 days in mice (2). They are then thought to be destroyed by the reticuloendothelial system. Regarding the mechanism that controls their lifespan, several studies have observed a decrease in mitochondrial membrane potential, caspase activation, and phosphatidylserine exposure in platelets, leading to the conclusion that platelets undergo apoptotic cell death (3-5). It has been demonstrated that platelets contain several apoptosis-related proteins such as Bcl-2 family proteins and a variety of caspase family proteins (3-7). Recently, Mason et al. (8) reported that knock-out of a single allele of the bcl-x gene results in mild thrombocytopenia, which is ameliorated in a Bak knock-out background. We have also reported previously that thrombocyte-specific homozygous Bcl-xL knock-out mice show marked thrombocytopenia (9). These findings established the critical role of Bcl-2 family proteins in regulating platelet apoptosis and lifespan. Platelets may be the simplest model for the study of Bcl-2 biology with physiological relevance because they neither perform de novo protein synthesis nor undergo proliferation.The proapoptotic multidomain Bcl-2 family proteins Bak and Bax serve as ef...

BACKGROUND & AIMS: Although the tumor microenvironment plays an important role in tumor growth, it is not fully understood what role hepatic stellate cells (HSCs) play in the hepatocellular carcinoma (HCC) microenvironment. METHODS: A high-fat diet after streptozotocin was administered to HSC-specific Atg7-deficient (GFAP-Atg7 knockout [KO]) or growth differentiation factor 15 (GDF15)-deficient (GFAP-GDF15KO) mice. LX-2 cells, a human HSC cell line, were cultured with human hepatoma cells. RESULTS: In the steatohepatitis-based tumorigenesis model, GFAP-Atg7KO mice formed fewer and smaller liver tumors than their wild-type littermates. Mixed culture of LX-2 cells and hepatoma cells promoted LX-2 cell autophagy and hepatoma cell proliferation, which were attenuated by Atg7 KO in LX-2 cells. Hepatoma cell xenograft tumors grew rapidly in the presence of LX-2 cells, but Atg7 KO in LX-2 cells abolished this growth. RNA-sequencing revealed that LX-2 cells cultured with HepG2 cells highly expressed GDF15, which was abolished by Atg7 KO in LX-2 cells. GDF15 KO LX-2 cells did not show a growth-promoting effect on hepatoma cells either in vitro or in the xenograft model. GDF15 deficiency in HSCs reduced liver tumor size caused by the steatohepatitis-based tumorigenesis model. GDF15 was highly expressed and GDF15-positive nonparenchymal cells were more abundant in human HCC compared with noncancerous parts. Single-cell RNA sequencing showed that GDF15-positive rates in HSCs were higher in HCC than in background liver. Serum GDF15 levels were high in HCC patients and increased with tumor progression. CONCLUSIONS: In the HCC microenvironment, an increase of HSCs that produces GDF15 in an autophagy-dependent manner may be involved in tumor progression.

Connective tissue growth factor (CTGF) is a matricellular protein related to hepatic fibrosis. This study aims to clarify the roles of CTGF in hepatocellular carcinoma (HCC), which usually develops from fibrotic liver. CTGF was overexpressed in 93 human HCC compared with nontumorous tissues, primarily in tumor cells. Increased CTGF expression was associated with clinicopathologic malignancy of HCC. CTGF was upregulated in hepatoma cells in hepatocyte-specific Kras-mutated mice (Alb-Cre Kras). Hepatocyte-specific knockout of CTGF in these mice (Alb-Cre Kras CTGF) decreased liver tumor number and size. Hepatic stellate cells (HSC) were present in both human and murine liver tumors, and α-SMA expression, a marker of HSC activation, positively correlated with CTGF expression. Forced expression of CTGF did not affect growth of PLC/PRF/5 cells, a hepatoma cell line with little CTGF expression, but facilitated their growth in the presence of LX-2 cells, an HSC line. The growth of HepG2 cells, which express high levels of CTGF, was promoted by coculture with LX-2 cells compared with monoculture. Growth promotion by LX-2 cells was negated by an anti-CTGF antibody in both culture and xenografts. Coculturing LX-2 cells with HepG2 cells drove LX-2-derived production of IL6, which led to STAT-3 activation and proliferation of HepG2 cells. An anti-CTGF antibody reduced IL6 production in LX-2 cells and suppressed STAT-3 activation in HepG2 cells. In conclusion, our data identify tumor cell-derived CTGF as a keystone in the HCC microenvironment, activating nearby HSC that transmit progrowth signals to HCC cells, and this interaction is susceptible to inhibition by an anti-CTGF antibody. Protumor cross-talk between cancer cells and hepatic stellate cells presents an opportunity for therapeutic intervention against HCC. http://cancerres.aacrjournals.org/content/canres/78/17/4902/F1.large.jpg .