The imprinted H19 gene has riboregulatory functions. We show here that H19 transcription is up-regulated during the S-phase of growth-stimulated cells and that the H19 promoter is activated by E2F1 in breast cancer cells. H19 repression by pRb and E2F6 confirms the E2F1-dependent control of the H19 promoter. Consistently, we demonstrate by chromatin immunoprecipitation assays that endogenous E2F1 is recruited to the H19 promoter in vivo. The functionality of E2F promoter sites was further confirmed by gel shift and mutagenesis experiments, revealing that these sites are required for binding and promoter response to E2F1 exogenous expression and serum stimulation. Furthermore, we show that H19 overexpression confers a growth advantage on breast cancer cells released from growth arrest as well as in asynchronously growing cells. The H19 knockdown by small interfering RNA duplexes impedes S-phase entry in both wild-type and stably H19-transfected cells. Based on these findings, we conclude that the H19 RNA is actively linked to E2F1 to promote cell cycle progression of breast cancer cells. This clearly supports the H19 oncogenic function in breast tumor genesis.

We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140 trkA and p75 NTR . The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140 trkA as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75 NTR . In contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75 NTR as well as the activation of the transcription factor NF-kB, but neither p140 trkA nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75 NTR in NGF-mediated survival. Both the pharmacological NF-B inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75 NTR and NF-B in the activation of the survival pathway in breast cancer cells. Nerve growth factor (NGF)1 is the archetypal member of the neurotrophin superfamily, which also includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5, and NT-6 (1). NGF interacts with two classes of membrane receptor: the TrkA proto-oncogene product p140 trkA , which possesses intrinsic tyrosine kinase activity, and a secondary receptor, p75 NTR , that belongs to the tumor necrosis factor (TNF) receptor family (2). The stimulation of cell survival and cell differentiation by NGF and other neurotrophins have been described primarily in neuronal cell systems (3). Although the neurotrophic effect through p140trkA is known to involve the MAPK cascade, the role of p75 NTR is still controversial; there is evidence that it can both positively and negatively regulate neuronal cell death and differentiation, depending on the cell type examined (4). In some cases, p75NTR is an inducer of apoptosis, even without NGF stimulation (5), whereas in other cases the activation of p75 NTR by NGF results in a protection from cell death (6). In addition to its neurotrophic function, other activities of NGF have been described. For example, NGF can modulate gene expression in monocytes (7), it is chemotactic for melanocytes (8), and its inhibition on p75 NTR can block the migration of Schwann cells (9). NGF also stimulates the proliferation of chromaffin cells (10), lymphocytes (11), and keratinocytes (12). We have previously shown that NGF is mitogenic for cancerous but not normal human breast cells (13), and these data, as well as others showing a role for NGF in the stimulation of prostatic cancer cells (14 -17), implicate NGF in non-neuronal carcinogenesis.Both cellular proliferation as well as tumor c...

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