Non-Traditional Notch Activation Promotes the Cancer Stem Cell Phenotype

Introduction to Cancer Stem Cells

Cancer stem cells (CSCs) are classically described as a tumor cell subpopulation that is multipotent, oncogenic, and capable of self-renewal.1,2 They may serve as an ongoing source for differentiating tumor cells and can exhibit characteristics that confer resistance to chemotherapy, including low proliferation rates and the expression of detoxifying enzymes such as aldehyde dehydrogenase (ALDH). These features place CSCs in a position to sustain tumors over the long-term, and understanding how these cells are regulated is crucial if they are to be targeted for therapy. Cells with characteristics of CSCs have been described in a range of tumor types. This includes colorectal cancer (CRC), the world’s third most prevalent tumor subtype accounting for almost 10% of all cancer diagnoses.3 A recent study by Lu et al. provides evidence that a non-traditional activation of the Notch pathway can drive human CRC cells toward a CSC phenotype.4

Endothelial Cells Produce a Colorectal Cancer Stem Cell-Promoting Factor

Studies have suggested that the tumor vasculature may act as more than a simple conduit that provides nutrients and O2. Vascular endothelial cells (ECs) might also act as a source for factors that promote tumor growth or the expansion of tumorigenic stem cells.5 In support of this activity, when cells derived from CRC tumors were cultured in EC-conditioned medium (EC CM), they exhibited several characteristics of CSC-like cells. These included increased expression of the marker CD133, enhanced ALDH activity, greater in vitro sphere-forming ability, and resistance to chemotherapeutic agents. These observations might result from either the expansion of existing CSC-like cells or by dedifferentiation. Interestingly, the latter appeared to be true as isolated tumor cells exhibiting characteristics of differentiation could be driven toward the CSC phenotype using EC CM. Supporting the physiological relevance of these in vitro observations, CRC cells pre-incubated with EC CM exhibited greater tumorigenicity and metastatic potential in mouse xenografts.

Jagged-1 Activation of Notch Promotes the Colorectal Cancer Stem Cell Phenotype

Wnt, Sonic Hedgehog, and Notch are well known for their morphogenic functions during early development. When dysregulated, they can have complex, context-dependent effects on tumorigenesis and many studies have linked morphogen activity to CSCs.6,7,8,9 Therefore, morphogens were investigated as a potential source of the CSC-inducing bioactivity observed in EC CM. Experiments using transcriptional reporters of Wnt, Sonic Hedgehog, and Notch indicated that only Notch activity was increased in CRC cells exposed to EC CM. In addition, tumor cells co-expressing CD133 and the active form of Notch were found adjacent to blood vessels in primary human CRC tumors and liver metastases, supporting a relationship between ECs and Notch activity in vivo. Ligands capable of inducing Notch activity include members of the Delta-like (DLL) and Jagged families, and in this model Jagged 1 appeared to be of importance. Immunoreactivity for Jagged 1 was detected in EC CM, and CSC-promoting activity was blocked by suppressing EC-derived Jagged 1 with either small interfering (si) RNA or neutralizing antibodies.

TACE/ADAM17 Shedding of a Soluble, Active Form of Jagged-1

Both Notch and its ligands are transmembrane proteins. In the canonical Notch pathway, ligand binding to receptor involves direct cell/cell interaction. Subsequently, Notch is proteolytically cleaved and its intracellular domain translocates to the nucleus, while the ligand/Notch extracellular domain complex is endocytosed by the ligand-expressing cell.10 However, the presence of a low molecular weight version of Jagged 1 in EC CM suggested that a soluble, truncated form could be the active factor responsible for promoting CSC features in CRCs. Previous studies have indicated that the membrane-anchored enzyme TACE/ADAM17 can mediate the shedding of transmembrane proteins including Jagged 1.11 Several lines of evidence indicated that the protease TACE/ADAM17 was important for generating soluble Jagged 1. Suppression of the protease, either pharmacologically or via siRNA, inhibited EC-mediated CSC induction in CRCs. In addition, co-transfection of TACE/ADAM17 and Jagged 1 enabled cultured fibroblasts to produce CSC-inducing conditioned media. Highlighting the importance of this mechanism, tumorigenicity was significantly inhibited by pharmacologically blocking TACE/ADAM17 in a mouse xenograft CRC model.


The CSC hypothesis suggests that resilient, tumorigenic stem cells that are capable of seeding new tumors exist within a cancer cell population. Many recent studies over a range of cancer subtypes suggest that the CSC population may be more heterogeneous and dynamic than originally thought. For instance, multiple genetically distinct CSC clones may exist within a tumor cell population, and certain CSCs might exhibit altered migratory capacity or the ability to fluctuate between states of differentiation depending on the microenvironment.8,12,13,14,15 This new study demonstrates a mechanism by which EC-derived Jagged 1 can drive CRC cells toward a CSC-like phenotype. It involves a soluble form of Jagged 1, and therefore, a non-traditional way of activating the Notch pathway. Notch-activated CRCs exhibited increased tumorigenicity, enhanced metastatic capability, and resistance to chemotherapy.4 All of these traits promote tumor recurrence and represent major roadblocks to conventional cancer therapy. The CSC hypothesis continues to evolve, and understanding the various mechanisms that control the context-dependent regulation of these cells will be important for the generation of targeted, multifaceted approaches to cancer treatment.


Non-traditional Notch Activation in Colorectal Cancer Cells
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Non-traditional Notch Activation in Colorectal Cancer Cells. TACE/ADAM17 mediates endothelial cell shedding of Jagged-1. Jagged-1 can bind Notch on colorectal cancer cells. This results in the sequential proteolytic cleavage of Notch, culminating in gamma-Secretase-mediated release of the Notch intracellular domain (NICD). The NICD interacts with DNA-associated proteins such as CSL to induce the transcription of Notch target genes that promote characteristics of cancer stem cells.


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