BackgroundHepatocellular carcinoma (HCC) is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be the cause of its molecular and clinicopathological heterogeneity. A better understanding of the global deregulation of methylation states and how they correlate with disease progression will aid in the design of strategies for earlier detection and better therapeutic decisions.Methods and FindingsWe characterized the changes in promoter methylation in a series of 30 HCC tumors and their respective surrounding tissue and identified methylation signatures associated with major risk factors and clinical correlates. A wide panel of cancer-related gene promoters was analyzed using Illumina bead array technology, and CpG sites were then selected according to their ability to classify clinicopathological parameters. An independent series of HCC tumors and matched surrounding tissue was used for validation of the signatures. We were able to develop and validate a signature of methylation in HCC. This signature distinguished HCC from surrounding tissue and from other tumor types, and was independent of risk factors. However, aberrant methylation of an independent subset of promoters was associated with tumor progression and etiological risk factors (HBV or HCV infection and alcohol consumption). Interestingly, distinct methylation of an independent panel of gene promoters was strongly correlated with survival after cancer therapy.ConclusionOur study shows that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression stage, with potential clinical applications in diagnosis and prognosis.

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Gastroenteropancreatic (GEP) endocrine tumors are hypervascular tumors able to synthesize and secrete high amounts of VEGF. We aimed to study the regulation of VEGF production in GEP endocrine tumors and to test whether some of the drugs currently used in their treatment, such as so- matostatin analogues and mTOR inhibitors, may interfere with VEGF secretion. We therefore analyzed the effects of the somatostatin analogue octreotide, the mTOR inhibitor rapamycin, the PI3K inhibitor LY294002, the MEK1 inhibitor PD98059 and the p38 inhibitor SB203850 on VEGF secretion, assessed by ELISA and Western blotting, in three murine endocrine cell lines, STC-1, INS-r3 and INS-r9. Octreotide and rapamycin induced a significant decrease in VEGF production by all three cell lines; LY294002 significantly inhibited VEGF production by STC-1 and INS-r3 only. We detected no effect of PD98059 whereas SB203850 significantly inhibited VEGF secretion in INS-r3 and INS-r9 cells only. By Western blotting analysis, we observed decreased intracellular levels of VEGF and HIF-1α under octreotide, rapamycin and LY294002. For rapamycin and LY294002, this effect was likely mediated by the inhibition of the mTOR/HIF-1/VEGF pathway. In addition to its well-known anti-secretory effects, octreotide may also act through the inhibition of the PI3K/Akt pathway, as suggested by the decrease in Akt phosphorylation detected in all three cell lines. In conclusion, our study points out to the complex regulation of VEGF synthesis and secretion in neoplastic GEP endocrine cells and suggests that the inhibition of VEGF production by octreotide and rapamycin may contribute to their therapeutic effects.

The TrkC neurotrophin receptor belongs to the functional dependence receptor family, members of which share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer tumor-suppressor activity. Indeed, cells that express these receptors are thought to be dependent on ligand availability for their survival, a mechanism that inhibits uncontrolled tumor cell proliferation and migration. TrkC is a classic tyrosine kinase receptor and therefore generally considered to be a protooncogene. We show here that TrkC expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, we show that TrkC silencing by promoter methylation is a selective advantage for colorectal cell lines to limit tumor cell death. Furthermore, reestablished TrkC expression in colorectal cancer cell lines is associated with tumor cell death and inhibition of in vitro characteristics of cell transformation, as well as in vivo tumor growth. Finally, we provide evidence that a mutation of TrkC detected in a sporadic cancer is a loss-ofproapoptotic function mutation. Together, these data support the conclusion that TrkC is a colorectal cancer tumor suppressor.neurotrophin-3 | caspase-3 | genetic | epigenetic T he Trk tyrosine kinase receptors and their ligands, the neurotrophins, have been studied extensively for their role in nervous system development. However, TrkA was originally cloned as an oncogene from colon carcinoma tumors in which the TrkA kinase domain was fused to the tropomyosin gene in the extracellular domain (1). This discovery motivated a great number of studies, which showed that neurotrophins (NGF, BDNF, and NT-4/5, NT-3) and their respective Trk receptors (TrkA, TrkB, and TrkC), are all involved in various malignancies (for review, see ref.2). The initial (and still generally accepted) view is that Trks, like other tyrosine kinase receptors, are oncogenic receptors, and therefore pan-Trk kinase inhibitors are currently being tested in clinical trials (3-5). Somewhat surprisingly, however, it has turned out that, at least in tumors such as neuroblastoma and medulloblastoma, TrkA, TrkB, and TrkC behave very differently, despite their close homology. TrkA and TrkC expression is associated with a good prognosis, whereas TrkB is expressed in very aggressive tumors (for review; see ref.2). The fact that the high expression of a tyrosine kinase receptor known to activate prooncogenic pathways (like the MAPK and PI3K-AKT pathways) is associated with a better outcome is counter intuitive, and suggests the possibility that TrkA and TrkC, rather than functioning solely as oncogenes, may also, in at least some cases, act as tumor suppressors. Although this notion may be ostensibly paradoxical, two recent independent studies have lent support to it, by demonstrating that both TrkA and TrkC, but not TrkB, act as dependence receptors (6, 7).Dependence receptors, which also include DCC (Deleted in Colorectal Carcinoma), UNC5H, Patched, Ne...

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The expression of ␣V integrins by neoplastic cells contributes to the promotion of local invasion and metastasis. The most characteristic extracellular ligands of ␣V integrins are vitronectin and fibronectin. Hepatocytes are the main source of vitronectin, and the capacity to synthesize and secrete vitronectin is usually retained in hepatocellular carcinoma. The aim of this study was to explore the expression, regulation, and functional role of ␣V integrins in hepatocellular carcinoma. We first analyzed the expression of ␣V integrins and their ligands fibronectin and vitronectin in 80 cases of hepatocellular carcinoma. ␣V integrin chain was detected in 44 cases and vitronectin in 50. Twenty-four of the 44 ␣V-positive tumors contained large amounts of vitronectin. These cases presented more frequently with adverse histoprognostic factors, including infiltrative growth pattern (62.5%), lack of capsule (71%), presence of capsular invasion (57%), and satellite nodules (50%). We then used HepG2 and Hep3B cell lines as in vitro models to study ␣V integrin regulation and function. HepG2 and Hep3B cells expressed ␣V integrin chain and used ␣V␤1 and ␣V␤5 for adhesion and migration on vitronectin. Tumor necrosis factor (TNF) ␣ and transforming growth factor (TGF) ␤ significantly increased the expression levels of ␣V integrins and stimulated the adhesion and migration of both HepG2 and Hep3B cell lines on vitronectin. The effects of growth factors on cell adhesion and migration were reproduced by incubation with conditioned medium from rat liver myofibroblasts. In conclusion, our results support the existence of an ␣V integrin/vitronectin connection in hepatocellular carcinoma and suggest that this connection may be an adverse prognostic factor. (HEPATOLOGY 2002;36:418-426.)

Background/Aims: While the range of therapeutic options for well-differentiated gastroenteropancreatic neuroendocrine tumors has recently increased with the emergence of targeted therapies, such as mTOR inhibitors, there is no recent progress in the treatment of poorly differentiated neuroendocrine carcinomas (PDNECs). Since PDNECs have been shown to strongly express mTOR pathway components, the aim of the present study was to assess the antitumor effect of the mTOR inhibitor everolimus in preclinical models of PDNECs. Methods: The expression of mTOR pathway components and their response to everolimus were assessed in two neuroendocrine cell lines: STC-1 and GluTag. A xenograft model of intrahepatic dissemination in the nude mouse, based on the intrasplenic injection of either STC-1 and GluTag tumor cells, was used. Animals were started on everolimus treatment 3 days after injection. The effects of treatment on tumor growth, proliferative capacities, apoptosis and in situ expression of mTOR pathway components were assessed. Results: The expression of mTOR pathway components was comparable in STC-1 and GluTag cells and in human PDNECs and could be inhibited in vitro by everolimus. In vivo, the tumor volume of STC-1 and GluTag xenografts was significantly reduced in treated animals (6.05 ± 1.84% as compared to 21.76 ± 3.88% in controls). Everolimus treatment also induced a significant decrease in Ki67 index and in the phosphorylation levels of the two major effectors of mTOR, p70S6K and 4E-BP1. Conclusion: Our experimental data suggest that mTOR inhibition could be considered a therapeutic option for high-grade gastroenteropancreatic neuroendocrine tumors.