Truncated Notch receptors have transforming activity in vitro and in vivo. However, the role of wild-type Notch signaling in neoplastic transformation remains unclear. Ras signaling is deregulated in a large fraction of human malignancies and is a major target for the development of novel cancer treatments. We show that oncogenic Ras activates Notch signaling and that wild-type Notch-1 is necessary to maintain the neoplastic phenotype in Ras-transformed human cells in vitro and in vivo. Oncogenic Ras increases levels and activity of the intracellular form of wild-type Notch-1, and upregulates Notch ligand Delta-1 and also presenilin-1, a protein involved in Notch processing, through a p38-mediated pathway. These observations place Notch signaling among key downstream effectors of oncogenic Ras and suggest that it might be a novel therapeutic target.

High expression of Notch-1 and Jagged-1 mRNA correlates with poor prognosis in breast cancer. Elucidating the cross-talk between Notch and other major breast cancer pathways is necessary to determine which patients may benefit from Notch inhibitors, which agents should be combined with them, and which biomarkers indicate Notch activity in vivo. We explored expression of Notch receptors and ligands in clinical specimens, as well as activity, regulation, and effectors of Notch signaling using cell lines and xenografts. Ductal and lobular carcinomas commonly expressed Notch-1, Notch-4, and Jagged-1 at variable levels. However, in breast cancer cell lines, Notch-induced transcriptional activity did not correlate with Notch receptor levels and was highest in estrogen receptor α–negative (ERα–), Her2/Neu nonoverexpressing cells. In ERα+ cells, estradiol inhibited Notch activity and Notch-1IC nuclear levels and affected Notch-1 cellular distribution. Tamoxifen and raloxifene blocked this effect, reactivating Notch. Notch-1 induced Notch-4. Notch-4 expression in clinical specimens correlated with proliferation (Ki67). In MDA-MB231 (ERα–) cells, Notch-1 knockdown or γ-secretase inhibition decreased cyclins A and B1, causing G2 arrest, p53-independent induction of NOXA, and death. In T47D:A18 (ERα+) cells, the same targets were affected, and Notch inhibition potentiated the effects of tamoxifen. In vivo, γ;-secretase inhibitor treatment arrested the growth of MDA-MB231 tumors and, in combination with tamoxifen, caused regression of T47D:A18 tumors. Our data indicate that combinations of antiestrogens and Notch inhibitors may be effective in ERα+ breast cancers and that Notch signaling is a potential therapeutic target in ERα– breast cancers.

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXAdependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma. (Cancer Res 2005; 65(14): 6282-93)

The cellular heterogeneity of neoplasms has been at the center of considerable interest since the "cancer stem cell hypothesis", originally formulated for hematologic malignancies, was extended to solid tumors.

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Mesothelioma, a malignancy associated with asbestos, has been recently linked to simian virus 40 (SV40). We found that infection of human mesothelial cells by SV40 is very different from the semipermissive infection thought to be characteristic of human cells. Mesothelial cells are uniformly infected but not lysed by SV40, a mechanism related to p53, and undergo cell transformation at an extremely high rate. Exposure of mesothelial cells to asbestos complemented SV40 mutants in transformation. Our data provide a mechanistic explanation for the ability of SV40 to transform mesothelial cells preferentially and indicate that asbestos and SV40 may be cocarcinogens. M alignant mesothelioma (MM) is a tumor of the serosal lining the pleural, pericardial, and peritoneal cavities that causes about 2,500 deaths per year in the United States (1). MM arises from the malignant transformation of mesothelial cells, which are undifferentiated cells representing the adult remnants of the surface coelomic mesoderm (1). Although MM has been associated with past exposure to asbestos fibers, the mechanisms through which asbestos causes mesothelial cell transformation are unclear. The capacity of asbestos to induce autophosphorylation of the epidermal growth factor receptor, which leads to activation protein-1 activity in human mesothelial cells (HM; ref.2); the production of reactive oxygen species by cells exposed to asbestos (3); and the local and systemic immunosuppressive effects of asbestos (4) may all contribute to carcinogenesis (1). Other factors act alone or synergistically with asbestos in causing MM, because only 5-10% of individuals exposed to high levels of asbestos develop MM, and 10-20% of MM occurs in individuals with no known exposure (1).Recently, simian virus 40 (SV40) has been associated with human mesothelioma and brain and bone tumors (reviewed in refs. 1 and 5-7). SV40 (5-8) is a DNA tumor virus encoding two transforming proteins (the large tumor antigen, or Tag; and the small tumor antigen, or tag), and three capsid proteins (VP1-3). Tag is the replicase of SV40. Expression of Tag in the absence of cell lysis leads to cellular transformation through several mechanisms, including Tag-mediated inhibition of cellular p53 and Rb family proteins, induction of insulin-like growth factor-I and its receptor, and the direct mutagenic effect of Tag. SV40 tag enhances Tag functions by inhibiting protein phosphatase 2A, contributing to malignancy (1, 9). SV40 infects cells from different species, and the cell type determines the outcome of SV40 infection (5-8). Permissive monkey cells support SV40 replication, which results in cell lysis. In nonpermissive rodent cells SV40 DNA cannot be replicated, and cells are not lysed and can be transformed. Human cells are termed semipermissive because only a fraction of cells express SV40 Tag after infection, these infected cells are lysed, and cell transformation is a very rare event.SV40 is highly oncogenic in rodents (5-7). We found that intracardial injection of SV40 induced MM...

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