The Notch signaling pathway is an architecturally simple signaling mechanism, well known for its role in cell fate regulation during organ development and in tissue homeostasis. In keeping with its importance for normal development, dysregulation of Notch signaling is increasingly associated with different types of tumors, and proteins in the Notch signaling pathway can act as oncogenes or tumor suppressors, depending on the cellular context and tumor type. In addition to a role as a driver of tumor initiation and progression in the tumor cells carrying oncogenic mutations, it is an emerging realization that Notch signaling also plays a role in non-mutated cells in the tumor microenvironment. In this review, we discuss how aberrant Notch signaling can affect three types of cells in the tumor stroma—cancer-associated fibroblasts, immune cells and vascular cells—and how this influences their interactions with the tumor cells. Insights into the roles of Notch in cells of the tumor environment and the impact on tumor-stroma interactions will lead to a deeper understanding of Notch signaling in cancer and inspire new strategies for Notch-based tumor therapy.

Breast cancer is the second leading cause of cancer deaths among women in the world. Treatment has been improved and, in combination with early detection, this has resulted in reduced mortality rates. Further improvement in therapy development is however warranted. This will be particularly important for certain sub-classes of breast cancer, such as triple-negative breast cancer, where currently no specific therapies are available. An important therapy development focus emerges from the notion that dysregulation of two major signaling pathways, Notch and Wnt signaling, are major drivers for breast cancer development. In this review, we discuss recent insights into the Notch and Wnt signaling pathways and into how they act synergistically both in normal development and cancer. We also discuss how dysregulation of the two pathways contributes to breast cancer and strategies to develop novel breast cancer therapies starting from a Notch and Wnt dysregulation perspective.

Hyperactivation of Notch signaling and the cellular hypoxic response are frequently observed in cancers, with increasing reports of connections to tumor initiation and progression. The two signaling mechanisms are known to intersect, but while it is well established that hypoxia regulates Notch signaling, less is known about whether Notch can regulate the cellular hypoxic response. We now report that Notch signaling specifically controls expression of HIF2α, a key mediator of the cellular hypoxic response. Transcriptional upregulation of HIF2α by Notch under normoxic conditions leads to elevated HIF2α protein levels in primary breast cancer cells as well as in human breast cancer, medulloblastoma, and renal cell carcinoma cell lines. The elevated level of HIF2α protein was in certain tumor cell types accompanied by downregulation of HIF1α protein levels, indicating that high Notch signaling may drive a HIF1α-to-HIF2α switch. At the transcriptome level, the presence of HIF2α was required for approximately 21% of all Notch-induced genes: among the 1062 genes that were upregulated by Notch in medulloblastoma cells during normoxia, upregulation was abrogated in 227 genes when HIF2α expression was knocked down by HIF2α siRNA. In conclusion, our data show that Notch signaling affects the hypoxic response via regulation of HIF2α, which may be important for future cancer therapies.

Epibranchial placodes are the geniculate, petrosal and nodose placodes that generate parts of cranial nerves VII, IX and X, respectively. How the three spatially separated placodes are derived from the common posterior placodal area is poorly understood. Here, we reveal that the broad posterior placode area is first patterned into a Vgll2 + /Irx5 + rostral domain and a Sox2 + /Fgf3 + / Etv5 + caudal domain relative to the first pharyngeal cleft. This initial rostral and caudal patterning is then sequentially repeated along each pharyngeal cleft for each epibranchial placode. The caudal domains give rise to the neuronal and non-neuronal cells in the placode, whereas the rostral domains are previously unrecognized structures, serving as spacers between the final placodes. Notch signalling regulates the balance between the rostral and caudal domains: high levels of Notch signalling expand the caudal domain at the expense of the rostral domain, whereas loss of Notch signalling produces the converse phenotype. Collectively, these data unravel a new patterning principle for the early phases of epibranchial placode development and a role for Notch signalling in orchestrating epibranchial placode segregation and differentiation.

Interaction of Notch receptors with Delta-and Serrate-type ligands is an evolutionarily conserved mechanism that mediates direct communication between adjacent cells and thereby regulates multiple developmental processes. Posttranslational modifications of both receptors and ligands are pivotal for normal Notch pathway function. We have identified by mass spectrometric analysis two serine and one threonine phosphorylation sites in the intracellular domain of the mouse Notch ligand DLL1. Phosphorylation requires cell membrane association of DLL1 and occurs sequentially at the two serine residues. Phosphorylation of one serine residue most likely by protein kinase B primes phosphorylation of the other serine. A DLL1 variant, in which all three identified phosphorylated serine/threonine residues are mutated to alanine and valine, was more stable than wild-type DLL1 but had reduced relative levels on the cell surface and was more effectively cleaved in the extracellular domain. In addition, the mutant variant activated Notch1 significantly less efficient than wild-type DLL1 in a coculture assay in vitro. Mice, however, whose endogenous DLL1 was replaced with the phosphorylation-deficient triple mutant developed normally, suggesting compensatory mechanisms under physiological conditions in vivo.