Angiogenesis is essential for primary tumor growth and metastatic dissemination. E2F1, frequently upregulated in advanced cancers, was recently shown to drive malignant progression. In an attempt to decipher the molecular events underlying this behavior, we demonstrate that the tumor cell-associated vascular endothelial growth factor-C/receptor-3 (VEGF-C/VEGFR-3) axis is controlled by E2F1. Activation or forced expression of E2F1 in cancer cells leads to the upregulation of VEGFR-3 and its ligand VEGF-C, whereas E2F1 depletion prevents their expression. E2F1-dependent receptor induction is crucial for tumor cells to enhance formation of capillary tubes and neovascularization in mice. We further provide evidence for a positive feedback loop between E2F1 and VEGFR-3 signaling to stimulate pro-angiogenic platelet-derived growth factor B (PDGF-B). E2F1 or VEGFR-3 knockdown results in reduced PDGF-B levels, while the coexpression synergistically upregulates promoter activity and endogenous protein expression of PDGF-B. Our findings delineate an as yet unrecognized function of E2F1 as enhancer of angiogenesis via regulation of VEGF-C/VEGFR-3 signaling in tumors to cooperatively activate PDGF-B expression. Targeting this pathway might be reasonable to complement standard anti-angiogenic treatment of cancers with deregulated E2F1.

Chronic hepatitis B virus (HBV) infection is the major risk for hepatocellular carcinomas (HCC). HBV X protein (HBx) and p53 tumor suppressor family interactions may be crucial for HCC induction. We compared p53 and p73 interactions with HBx in normal and HCC tumor cell lines differing in their p53 status. In the latter, HBx was pro-apoptotic but exhibited opposite effects in non-tumor cells. In these normal cells, p53 and p73 were retained in the cytoplasm. In hepatoma cells, however, HBx led to nuclear translocation of p53 and p73, followed by enhanced transactivation of p53-dependent promoters. The nuclear transfer of p53, but not of p73, was abrogated by protein kinase C inhibitor Gö6976. HBx overexpression in HCC cells led to strong p53 phosphorylation at Ser15, but not in non-tumor cells. Our results define ATM kinase as mediator for HBx-induced p53 phosphorylation. While HBx promotes cell death in p53/p73-positive hepatoma cells also in presence of increased levels of the oncogenic ΔTAp73 isoform, it significantly potentiates ΔTAp73-mediated proliferation and malignant transformation of fibroblasts. Our data suggest that prevention of apoptosis in normal cells by HBx through inhibition of pro-apoptotic p53 family members via direct interaction and coaction with anti-apoptotic ΔTAp73 seems to be the key element in the decision in favor of cell survival. The complex cell context-dependent interactions between p53 family members and HBx in the regulation of apoptosis may be essential in HBV-induced HCC and anticancer therapy.

Activation of telomerase is linked to tumorigenesis and has been observed in a variety of human tumors. Previous reports demonstrated that p53 represses human telomerase reverse transcriptase (hTERT), a key component for telomerase activity. The p73 protein displays a tumor suppressor activity similar to p53. In the present study, we examined the effect of transactivation competent p73 isoforms on hTERT expression in p53-negative human H1299 cells. Overexpression of C-terminal p73 isoforms (␣, ␤, ␥, ␦) resulted in a clear down-regulation of hTERT promoter activity. The strongest inhibitory effect, comparable with p53, was observed for p73␤. Moreover, suppression of hTERT expression was also mediated by endogenous p73 after activation of E2F1 in H1299ER-E2F1 cells. Mutations in the Sp1 transcription factor-binding sites of the proximal core promoter region significantly abolished p73-induced repression, suggesting that the effect is mediated by Sp1. Finally, we demonstrate that p73 directly interacts with Sp1, suggesting that formation of a p73-Sp1 complex is the underlying mechanism for p73-triggered inhibition of hTERT expression. Our findings provide additional evidence that p73 mimics p53 in many aspects in cells lacking functional p53, thereby contributing to tumor surveillance.Telomerase is the enzyme required for the addition of telomeric repeats to the ends of linear chromosomes. It consists of an RNA subunit, a catalytic subunit (hTERT, 2 human telomerase reverse transcriptase) and telomeraseassociated proteins. The RNA component of human telomerase provides the template for telomere repeat synthesis (1). Whereas the RNA subunit of telomerase is expressed in most cells, expression of hTERT is observed at high levels in most malignant tumors and cancer cell lines but not in normal tissues (2) and was found to be closely associated with telomerase activity (3). Moreover, ectopic expression of hTERT has been shown to facilitate immortalization of human cells and to be required for the transformation of human primary cells by H-Ras and SV40 large T antigen oncoproteins (4). These findings led to the conclusion that hTERT expression is a rate-limiting step in telomerase activity and a hallmark of cancer. Although telomerase enzymatic activity can be regulated at multiple levels, several studies demonstrated that transcriptional regulation of hTERT is the major mechanism of telomerase regulation. Deletion analyses in reporter assays showed that the 200-bp proximal region of the hTERT promoter (core promoter) is responsible for most of the transcriptional activity (5). Elucidation of the mechanisms governing hTERT expression revealed two general principles for hTERT up-regulation: (i) the activation of the hTERT promoter through oncoproteins or viral integration and (ii) the derepression of the hTERT promoter through the loss of tumor suppressors. It has been shown that oncogenes such as c-myc and the human papillomavirus E6 protein activate hTERT transcription (6). Furthermore, abrogation of p53 function by th...

The E2F1 transcription factor enhances apoptosis by DNA damage in tumors lacking p53. To elucidate the mechanism of a potential cooperation between E2F1 and chemotherapy, whole-genome microarrays of chemoresistant tumor cell lines were performed focusing on the identification of cooperation response genes (CRG). This gene class is defined by a synergistic expression response upon endogenous E2F1 activation and drug treatment. Cluster analysis revealed an expression pattern of CRGs similar to E2F1 mono-therapy, suggesting that chemotherapeutics enhance E2F1-dependent gene expression at the transcriptional level. Using this approach as a tool to explore E2F1-driven gene expression in response to anticancer drugs, we identified novel apoptosis genes such as the tumor suppressor TIEG1/KLF10 as direct E2F1 targets. We show that TIEG1/KLF10 is transcriptionally activated by E2F1 and crucial for E2F1-mediated chemosensitization of cancer cells. Our results provide a broader picture of E2F1-regulated genes in conjunction with cytotoxic treatment that allows the design of more rational therapeutics.

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