Neuroblastoma is an embryonal malignancy of the sympathetic nervous system. Spontaneous regression and differentiation of neuroblastoma is observed in a subset of patients, and has been suggested to represent delayed activation of physiologic molecular programs of fetal neuroblasts. Homeobox genes constitute an important family of transcription factors, which play a fundamental role in morphogenesis and cell differentiation during embryogenesis. In this study, we demonstrate that expression of the majority of the human HOX class I homeobox genes is significantly associated with clinical covariates in neuroblastoma using microarray expression data of 649 primary tumors. Moreover, a HOX gene expression-based classifier predicted neuroblastoma patient outcome independently of age, stage and MYCN amplification status. Among all HOX genes, HOXC9 expression was most prominently associated with favorable prognostic markers. Most notably, elevated HOXC9 expression was significantly associated with spontaneous regression in infant neuroblastoma. Re-expression of HOXC9 in three neuroblastoma cell lines led to a significant reduction in cell viability, and abrogated tumor growth almost completely in neuroblastoma xenografts. Neuroblastoma growth arrest was related to the induction of programmed cell death, as indicated by an increase in the sub-G1 fraction and translocation of phosphatidylserine to the outer membrane. Programmed cell death was associated with the release of cytochrome c from the mitochondria into the cytosol and activation of the intrinsic cascade of caspases, indicating that HOXC9 re-expression triggers the intrinsic apoptotic pathway. Collectively, our results show a strong prognostic impact of HOX gene expression in neuroblastoma, and may point towards a role of Hox-C9 in neuroblastoma spontaneous regression.

The cell density-dependent growth inhibition of human SK-N-MC neuroblastoma cells is initiated by increased ganglioside sialidase activity leading to elevated cell surface presentation of ganglioside GM1, a ligand of galectin-1. We herein show that the extent of the cell surface expression of the galectin coincides with marked increases of the sialidase activity. Reverse transcriptase-polymerase chain reaction analysis excludes a regulation at the transcriptional level. Exposure of cells to purified galectin-1 reveals its carbohydrate-dependent activity to reduce cell proliferation. Assays to detect DNA fragmentation biochemically and cytometrically and to block caspases render it unlikely that galectin-1 acts as a classical proapoptotic factor on these cells. Because the chimeric galectin-3 shares binding sites and binding parameters with galectin-1 for these cells, we tested whether this galectin will elicit the same response as the homodimeric cross-linking galectin-1. Evidently, galectin-3 fails to affect cell growth by itself but interferes with galectin-1 upon coincubation. Its proteolytically truncated variant, the C-terminal lectin domain with impaired capacity to form aggregates when surface bound, has only weak binding properties. Thus, the way in which the galectin-1 interacts topologically with an apparently common set of ligands relative to galectin-3 is crucial for eliciting post-binding events. We conclude that galectin-1 is a probable effector in the sialidase-dependent growth control in this system. Moreover, the experiments with galectin-3 reveal functional divergence, most probably based on different topologies of presentation of homologous carbohydratebinding sites.

H epatocellular carcinoma (HCC) is the most frequent type of liver cancer and is the third leading cause of cancer-related death worldwide. 1 Various etiologies have been linked to HCC development, the most relevant being chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, chronic alcohol consumption, and, in certain geographical areas, aflatoxin B1 food contamination. 2 Approximately 10% of HCC patients show no signs of hepatitis virus infection, alcoholic history, or other defined causes, such as genetic hemochromatosis or ␣ 1 -antitrypsin deficiency. Most of these so-called cryptogenic HCCs evolve from nonalcoholic steatohepatitis (NASH). 3 Whereas generation of reactive oxygen species has been suggested to drive hepatocarcinogenesis in HCCs of alcoholic or cryptogenic etiology, viral-associated mechanisms are complex and involve both host and viral factors.Genomic instability is a common feature of human HCC. Using conventional and array-based comparative genomic hybridization (aCGH), frequent DNA copy number gains at 1q, 6p, 8q, 17q, and 20q and losses at 1p, 4q, 8p, 13q, 16q, and 17p have been identified as reviewed elsewhere. 4 Although target genes such as RB1 (13q14) and TP53 (17p13) have been identified and validated, the driving genes for most commonly altered loci remain unknown. Several CGH studies tried to identify chromosome aberrations that correlate specifically with HCC etiology; however, many failed to uncover significant differences in DNA copy number alteration or candidate genes, possibly due to underrepresentation of

To identify new tumor-suppressor gene candidates relevant for human hepatocarcinogenesis, we performed genome-wide methylation profiling and vertical integration with arraybased comparative genomic hybridization (aCGH), as well as expression data from a cohort of well-characterized human hepatocellular carcinomas (HCCs). Bisulfite-converted DNAs from 63 HCCs and 10 healthy control livers were analyzed for the methylation status of more than 14,000 genes. After defining the differentially methylated genes in HCCs, we integrated their DNA copy-number alterations as determined by aCGH data and correlated them with gene expression to identify genes potentially silenced by promoter hypermethylation. Aberrant methylation of candidates was further confirmed by pyrosequencing, and methylation dependency of silencing was determined by 5-aza-2 0 -deoxycytidine (5-aza-dC) treatment. Methylation profiling revealed 2,226 CpG sites that showed methylation differences between healthy control livers and HCCs. Of these, 537 CpG sites were hypermethylated in the tumor DNA, whereas 1,689 sites showed promoter hypomethylation. The hypermethylated set was enriched for genes known to be inactivated by the polycomb repressive complex 2, whereas the group of hypomethylated genes was enriched for imprinted genes. We identified three genes matching all of our selection criteria for a tumor-suppressor gene (period homolog 3 [PER3], insulin-like growth-factor-binding protein, acid labile subunit [IGFALS], and protein Z). PER3 was down-regulated in human HCCs, compared to peritumorous and healthy liver tissues. 5-aza-dC treatment restored PER3 expression in HCC cell lines, indicating that promoter hypermethylation was indeed responsible for gene silencing. Additionally, functional analysis supported a tumor-suppressive function for PER3 and IGFALS in vitro. Conclusion: The present study illustrates that vertical integration of methylation data with high-resolution genomic and transcriptomic data facilitates the identification of new tumor-suppressor gene candidates in human HCC. (HEPATOLOGY 2012;56:1817-1827 H epatocellular carcinoma (HCC) is the fifthmost frequent cancer worldwide and has a poor prognosis. 1 Various etiologies have been linked to HCC development, most of which cause chronic liver damage and finally lead to liver cirrhosis.The most prevalent etiological factors are chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, chronic alcohol consumption, and, in certain geographical areas, aflatoxin B1 food contamination. 2 Approximately 10% of HCC patients lack viral