Clin Transl Oncol (2009) 11:11-19 DOI 10.1007/s12094-009-0305-2 Abstract A new theory about the development of solid tu- mours is emerging from the idea that solid tumours, like normal adult tissues, contain stem cells (called cancer stem cells) and arise from them. Genetic mutations encoding for proteins involved in critical signalling pathways for stem cells such as BMP, Notch, Hedgehog and Wnt would allow stem cells to undergo uncontrolled proliferation and form tumours. Taking into account that cancer stem cells (CSCs) would represent the real driving force behind tumour growth and that they may be drug resistant, new agents that target the above signalling pathways could be more effec- tive than current anti-solid tumour therapies. In the present paper we will review the molecular basis of the Notch sig- nalling pathway. Additionally, we will pay attention to their role in adult stem cell self-renewal, and cell fate specifica- tion and differentiation, and we will also review evidence that supports their implication in cancer. Keywords Notch pathway �� Cancer stem cells Introduction Notch signalling is a developmental conserved sig- nalling pathway that plays a fundamental role in embryon- ic development and adulthood. During embryonic develop- ment, Notch activation allows definition of the cell fate of groups of embryonic stem cells. The Notch pathway also has a critical role in the self-renewal of adult tissues due to its involvement in the differentiation of non differentiated somatic stem cell populations into specific cell types. The Notch pathway uses both membrane-bound ligands and receptors. This fact makes fundamental the existence of intercellular contact for the signal transmission. Notch is a large membrane protein that is expressed in signal-receiving cells and, therefore, it works as a receptor. In mammals, there are four Notch receptors (Notch1���4) with a complex dimeric structure composed of transmem- brane monomer bound through non-covalent interactions to a fully extracellular monomer. On the one hand, the intra- cellular face of the transmembrane monomer contains a number of important domains which are involved in the transduction and modulation of the signal. Immediately next to the membrane there is a RAM (RBPjk Associate Notch signalling in cancer stem cells Victoria Bol��s �� Mois��s Blanco �� Vanessa Medina �� Guadalupe Aparicio �� Silvia D��az-Prado �� Enrique Grande Received: 11 November 2008 / Accepted: 26 November 2008 E D U C AT I O N A L S E R I E S G r e e n S e r i e s V. Bol��s ( ) �� E. Grande Medical Oncology Department Pfizer Spain Avda. de Europa, 20B 28108 Alcobendas, Madrid, Spain e-mail: MariaVictoria.Bolos@pfizer.com M. Blanco �� V. Medina �� S. D��az-Prado Institute for Biomedical Research (INIBIC) A Coru��a University Hospital A Coru��a, Spain V. Medina �� G. Aparicio Oncology Research Unit A Coru��a University Hospital A Coru��a, Spain S. D��az-Prado Medicine Department University of A Coru��a A Coru��a, Spain MOLECULAR TARGETS IN ONCOLOGY

12 Clin Transl Oncol (2009) 11:11-19 Molecule) domain followed by a number of motifs named Ankyrin repeats, which mediates the interaction with the Csl (CBF1 in mammals/Su(H) in Drosophila/Lag-1 in Cae norhabditis elegans) transcription factor. Additionally, there is a transactivation domain (TAD), two nuclear locali- sation signals (NLS) and a PEST region, which is the site for Notch ubiquitination. Therefore, the PEST domain neg- atively regulates protein stability. On the other hand, the extracellular face of the Notch transmembrane monomer shows a hydrophobic heterodimerisation region which me- diates the binding to the Notch extracellular monomer (Fig. 1). The extracellular Notch monomer is composed, in addi- tion to the heterodimerisation region, of a number of cys- teine-rich Lin12 repeats and, in the more distant zone, more than 36 epidermal growth factor (EGF)-like repeats that are critical for the interaction with the Notch ligand (fundamentally 11 and 12 EGF-like repeats). In mammals there are five conventional ligand mole- cules: three delta-like ligands (homologous to the delta lig- and of Drosophila), Dll1, Dll3 and Dll4, and two jagged ligands (homologous to the serrate ligand of Drosophila), Jag1 and Jag2 (Fig. 1). Both families of ligand are also transmembrane proteins with a shorter intracellular face than Notch receptors. Jagged ligands display a cysteine- rich region followed by less EGF-like repeats than Notch and, finally, a DSL (Delta, Serrate and LAG2) domain. Al- though delta-like ligands contain the entire DSL region, they lack a cysteine-rich region and have less EGF-like re- peats than jagged ligands. DSL regions are functionally important because they are responsible for interacting with the 11 and 12 EGF-like repeats of the Notch receptor. Ad- ditionally, the Notch EGF-like repeats 23���25 can also in- teract with delta-like ligands but not with the jagged lig- ands [1]. Currently, a new model of development of tumours is emerging and supports the idea that tumours, like normal adult tissues, arise from stem cells. In this model cancer stem cells (CSC) would be the real driving force behind tu- mour growth. CSCs would retain the differentiation poten- tial giving rise to non-self-renewing differentiated tumour cells that would constitute the bulk of the tumour. It seems that genetic alterations in critical signalling pathways that govern stem cells such as Notch, Wnt, BMP (Bone Mor- phogenic Protein) and Hedgehog signalling pathways would allow stem cells to become independent of growth signals or resist antigrowth signals and would allow them to undergo uncontrolled proliferation and tumorigenesis [2]. As we will review below, a growing amount of evi- dence has already shown an association between alter- ations in the Notch signalling pathway and cancer, support- Fig. 1 Notch signalling elements. NOTCH proteins are single-pass transmembrane receptors harbouring a large extracellular domain involved in ligand binding and a cytoplasmic domain involved in signal transduc- tion. The extracellular domain contains multiple EGF-like portions that are critical for ligand binding. The EGF-like portions are followed by a LIND domain (three cysteine-rich repeats) that prevents signalling in the absence of the signal. The intracellular domain is involved in protein���protein interactions and in the activation of the transcription. In fact, it contains a TAD and two NLS. The intracellular PEST sequence negatively regu- lates protein stability. Notch ligands are also bound to neighbouring cells. Ligands have an amino-terminal domain termed DSL (for Delta, Serrate and LAG-2 domain), followed by a different number of EGF-like por- tions. Adapted from [19]