During the past decade, cancer stem cells (CSCs) have been increasingly identified in many malignancies. Although the origin and plasticity of these cells remain controversial, tumour heterogeneity and the presence of small populations of cells with stem-like characteristics is established in most malignancies. CSCs display many features of embryonic or tissue stem cells, and typically demonstrate persistent activation of one or more highly conserved signal transduction pathways involved in development and tissue homeostasis, including the Notch, Hedgehog (HH), and Wnt pathways. CSCs generally have slow growth rates and are resistant to chemotherapy and/or radiotherapy. Thus, new treatment strategies targeting these pathways to control stem-cell replication, survival and differentiation are under development. Herein, we provide an update on the latest advances in the clinical development of such approaches, and discuss strategies for overcoming CSC-associated primary or acquired resistance to cancer treatment. Given the crosstalk between the different embryonic developmental signalling pathways, as well as other pathways, designing clinical trials that target CSCs with rational combinations of agents to inhibit possible compensatory escape mechanisms could be of particular importance. We also share our views on the future directions for targeting CSCs to advance the clinical development of these classes of agents.

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)