Resistance to apoptosis is essential for cancer growth. We previously reported that hepatic coexpression of c-Myc and E2F1, 2 key regulators of proliferation and apoptosis, enhanced hepatocellular carcinoma (HCC) development in transgenic mice. We have now investigated the molecular mechanisms underlying oncogenic cooperation between c-Myc and E2F1 in relationship to human liver cancer. Activation of pro- and antiapoptotic cascades was assessed by immunoblotting in in vivo and in vitro HCC models and in primary human HCC. Effect of antisense oligodeoxy nucleotides against c-Myc and E2F1 was studied in human HCC cell lines. Suppression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase (PIK3CA)/Akt, mammalian target of rapamycin (mTOR), and cyclooxygenase (COX)-2 pathways was achieved by pharmacologic inhibitors and specific small interfering RNA in human and mouse HCC cell lines. Our results revealed that coexpression with E2F1 did not increase proliferation triggered by c-Myc overexpression but conferred a strong resistance to c-Myc-initiated apoptosis via concomitant induction of PIK3CA/Akt/mTOR and c-Myb/COX-2 survival pathways. COX-2 was not induced in c-Myc and rarely in E2F1 tumors. In human HCC, PIK3CA/Akt/mTOR and c-Myb/COX-2 pathways were similarly activated, with levels of PIK3CA/Akt, mTOR, and c-Myb being inversely associated with patients' survival length. Knocking down c-Myc and E2F1 oncoproteins reduced PIK3CA/Akt and mTOR and completely abolished c-Myb and COX-2 expression in the human HCC cell lines. Furthermore, simultaneous inhibition of PIK3CA/Akt/mTOR and COX-2 activity in in vitro models caused massive apoptosis of neoplastic hepatocytes. From these data we conclude that E2F1 may function as a critical antiapoptotic factor both in human and in rodent liver cancer through its ability to counteract c-Myc-driven apoptosis via activation of PIK3CA/Akt/mTOR and c-Myb/COX-2 pathways. Patients with liver cancer have a highly variable clinical course, indicating that HCC may comprise several biologically distinctive subtypes reflecting a molecular heterogeneity of tumors.We have explored the potential of TGF-beta gene expression signature to refine the diagnosis and prognostic predictions of patients with HCC. Based on the fact that TGF-beta possesses both tumor-suppressive and tumor-promoting properties we hypothesized that the application of a gene expression signature specific for the TGF-beta signaling pathway to human HCC could identify more homogeneous and clinically relevant subgroups of patients with liver cancer.Applying a comparative functional genomics approach we demonstrated that a temporal TGF-beta gene expression signature established in mouse primary hepatocytes successfully discriminated distinct subgroups of HCC. The TGF-beta positive cluster included two novel homogeneous groups of HCC associated with early and late TGF-beta signatures.To evaluate the clinical significance of TGF-beta signature in the molecular classification of HCC, we then compared the distribution of several clinical and pathological variables between HCC harboring early or late TGF-beta signatures. HCC patients with a late TGF-beta signature showed a significantly compared to the patients with an early TGF-beta signature. Also, tumors expressing late TGF-beta responsive genes displayed an invasive phenotype and an increased tumor recurrence. In conclusion, we have discovered gene sets embedded in the TGF-beta signaling pathway that can be used to identify clinically relevant subgroups of HCC patients which differ greatly in term of survival. Similar data were obtained for patients with lung adenocarcinomas indicating a general applicability of the TGF-beta gene expression signature in the molecular prognostication of cancer. In the future, integration of multiple gene expression signatures specific for pathways or processes which are known to contribute to cancer development will greatly advance the molecular classification of tumors and provide a new basis for development of predictive personalized care based on targeted therapy. Epigenetics is profoundly altered in neoplasia and associated with the etiology of human tumorigenesis. In particular, aberrant promoter DNA CpG hypermethylation at the 5-methylcytosine is associated with atypical gene silencing, affecting genes involved in tumor progression and DNA repair. The epigenetic process is reversible, and inhibitors of DNA methyltransferase can induce functional re-expression of aberrantly silenced genes, causing growth arrest and apoptosis in cancer cells. Zebularine (ZEB) [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one] is a potent second generation inhibitor of DNA methyltransferase and cytidine deaminase. In contrast to analogous drugs, e.g. 5-azacytidine, ZEB is a stable hydrophilic molecule with low cellular toxicity and is currently being tested for clinical use. The epigenomics of human liver cancer is poorly defined. However, with recent advancement in array technology, large-scale studies of DNA methylation have now become feasible. We have examined the transcriptomic change following zebularine treatment of 10 liver cancer cell lines (7 HCC and 3 CCC lines), and two non-transformed hepato- and cholangiocytic cell lines using genome-wide expression analysis in order to characterize re-expression of epigenetically silenced genes. Each cell line was treated with 100 and 200M ZEB or vehicle control for 7 days prior to microarray analysis. Significant gene lists of differential expressed genes were computed at p≤ 0.001 for each individual cell line using corresponding control samples as reference. Gene signatures included 631 genes universally changed by ZEB in HCC and 451 genes in CCC cell lines. A list of 64 genes was found to be common among both groups. The comparison of these gene lists to those in the corresponding normal cell lines, also identified significantly expression changes of genes which were unique to either HCC (238 genes) or CCC cell lines (85 genes). Moreover, the ZEB gene signature applied to a data set of 54 human HCCs successfully differentiated the patients according to their survival. Following subcutaneous transplantation of the nine liver cancer cell lines into athymic mice, the four most aggressive cell lines were identified (Huh7, WRL68, KMCH, and WITT) for further investigation. The antitumor efficacy of ZEB pretreatment of these cell lines was evaluated by subcutaneous transplantation. The effects of ZEB were diverse varying from a complete inhibition (KMCH) to an acceleration of tumor growth (WRL68). Huh7 was the most responsive to ex vivo ZEB treatment among the HCC cell lines and was used for further testing ZEB therapy in a murine model of liver cancer metastases. Huh7 cells expressing luciferase were transplanted intrasplenic to immunodeficient SCID/Beige mice. One week later, mice randomly assigned to the ZEB treated group were given i.p. injections of ZEB (350 mg/kg x3 daily) for three days. Bioluminescent imaging performed within the next two weeks showed a 2-fold decrease in the bioluminescent signal in ZEB-treated animals as compared to vehicle control alone. Furthermore, the addition of a short-term pretreatment with raloxifene, known to inhibit the metabo [summary truncated at 7800 characters]
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