Notch signaling in cancer

The evolutionarily conserved developmental pathway driven by Notch receptors and ligands has acquired multiple post-natal homeostatic functions in vertebrates. Potential roles in human physiology and pathology are being studied by an increasingly large number of investigators. While the canonical Notch signaling pathway is deceptively simple, the consequences of Notch activation on cell fate are complex and context-dependent. The manner in which other signaling pathways cross-talk with Notch signaling appears to be extraordinarily complex. Recent observations have demonstrated the importance of endocytosis, multiple ubiquitin ligases, non-visual beta-arrestins and hypoxia in modulating Notch signaling. Structural biology is shedding light on the molecular mechanisms whereby Notch interacts with its nuclear partners. Genomics is slowly unraveling the puzzle of Notch target genes in several systems. At the same time, interest in modulating Notch signaling for medical purposes has dramatically increased. Over the last few years we have learned much about Notch signaling in cancer, immune disorders, neurological disorders and most recently, stroke. The role of Notch signaling in normal and transformed stem cells is under intense investigation. Some Notch-modulating drugs are already in clinical trials, and others at various stages of development. This review will focus on the most recent findings on Notch signaling in cancer and discuss their potential clinical implications.     

Using C. elegans notch mutants in an undergraduate cancer biology laboratory course to demonstrate oncogenic effects on cell proliferation

Undergraduate laboratory exercises addressing aspects of cancer biology such as increased cell proliferation, gain-of-function signaling mutations and tumour formation often rely on tissue culture or even small mammal models. Many departments have limited or no access to these tools, and even well-equipped departments face logistical problems when incorporating these models into laboratory classes. I have developed a laboratory exercise using the microscopic worm, C. elegans, to demonstrate the effects of Notch receptor mutations on cell proliferation. Notch, which is activated by juxtacrine signaling, is mutated in many human cancers. In this exercise, students compare the germline phenotypes of worms that have a loss-of-function Notch mutation (no cells in the germline) or a gain-of-function Notch mutation (over-proliferation resulting in a germline tumour). Students also genotype the worms and perform sequence analysis to determine the effects of the mutations on the protein sequence. This laboratory exercise demonstrates oncogenic proliferation, correlates genotype to phenotype, exposes students to model organisms and introduces sequence databases and analysis. In addition to cancer biology courses, this exercise could be incorporated in courses with a focus on genetics, cell biology or developmental biology.     

Characterization of transgenic mice expressing cancer-associated variants of human NOTCH1

The Notch1 receptor plays a critical role in cell fate decisions during development. Activation of Notch signaling has been implicated in several types of cancer, particularly T-cell acute lymphoblastic leukemia (T-ALL). Consequently, several transgenic mouse strains have been made to study the role of Notch1 in T-ALL. However, the existing Notch1 transgenic lines mimic a translocation event found in only ～1% of T-ALL cases. Here we describe three novel NOTCH1 transgenic mouse strains that have Cre-inducible expression of the entire human NOTCH1 locus, each possessing a common mutation found in T-ALL. Unlike existing Notch1 transgenic strains, these NOTCH1 transgenic strains express full-length receptors from an endogenous human promoter that should be susceptible to a number of Notch antagonists that have recently been developed. These strains will allow researchers to modulate Notch signaling to study both normal development and cancer biology.     

Notch activation augments nitric oxide/soluble guanylyl cyclase signaling in immortalized ovarian surface epithelial cells and ovarian cancer cells

Nitric oxide (NO) is generated by tumor, stromal and endothelial cells and plays a multifaceted role in tumor biology. Many physiological functions of NO are mediated by soluble guanylyl cyclase (sGC) and NO/sGC signaling has been shown to promote proliferation and survival of ovarian cancer cells. However, how NO/sGC signaling is modulated in ovarian cancer cells has not been studied. The evolutionarily conserved Notch signaling pathway plays an oncogenic role in ovarian cancer. Here, we report that all three ovarian cancer cell lines we examined express a higher level of GUCY1B3 (the β subunit of sGC) compared to non-cancerous immortalized ovarian surface epithelial (IOSE) cell lines. Interestingly, the highest expression of GUCY1B3 in ovarian cancer OVCAR3 cells is concurrent with the expression of Notch3. In IOSE cells, forced activation of Notch3 increases the expression of GUCY1B3, NO-induced cGMP production, and the expression of cGMP-dependent protein kinase (PKG), thereby enhancing NO- and cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP, a direct PKG substrate protein). In contrast, inhibition of Notch by DAPT reduces GUCY1B3 expression and NO-induced cGMP production and VASP phosphorylation in OVCAR3 cells. Finally, we confirmed that inhibition of sGC by ODQ decreases growth of ovarian cancer cells. Together, our work demonstrates that Notch is a positive regulator of NO/sGC signaling in IOSE and ovarian cancer cells, providing the first evidence that Notch and NO signaling pathways interact in IOSE and ovarian cancer cells.     

Making sense out of missense mutations: Mechanistic dissection of Notch receptors through structure-function studies in Drosophila

Notch signaling is involved in the development of almost all organ systems and is required post-developmentally to modulate tissue homeostasis. Rare variants in Notch signaling pathway genes are found in patients with rare Mendelian disorders, while unique or recurrent somatic mutations in a similar set of genes are identified in cancer. The human genome contains four genes that encode Notch receptors, NOTCH1-4, all of which are linked to genetic diseases and cancer. Although some mutations have been classified as clear loss- or gain-of-function alleles based on cellular or rodent based assay systems, the functional consequence of many variants/mutations in human Notch receptors remain unknown. In this review, I will first provide an overview of the domain structure of Notch receptors and discuss how each module is known to regulate Notch signaling activity in vivo using the Drosophila Notch receptor as an example. Next, I will introduce some interesting mutant alleles that have been isolated in the fly Notch gene over the past > 100 years of research and discuss how studies of these mutations have facilitated the understanding of Notch biology. By identifying unique alleles of the fly Notch gene through forward genetic screens, mapping their molecular lesions and characterizing their phenotypes in depth, one can begin to unravel new mechanistic insights into how different domains of Notch fine-tune signaling output. Such information can be useful in deciphering the functional consequences of rare variants/mutations in human Notch receptors, which in turn can influence disease management and therapy.     

Epigenetic silencing of Notch signaling in gastrointestinal cancers

The Notch signaling pathway drives proliferation, differentiation, apoptosis, cell fate choices and maintenance of stem cells during embryogenesis and in self-renewing tissues of the adult. In addition, aberrant Notch signaling has been implicated in several tumors, where Notch can function both as an oncogene or a tumor-suppressor gene, depending on the context.

This Extra View aims to review what is currently known about Notch signaling, in particular in gastrointestinal tumors, providing a summary of our data on Notch1 signaling in gastric cancer with results obtained in colorectal cancer (CRC).

We have already reported that the epigenetic regulation of the Notch ligand DLL1 controls Notch1 signaling activation in gastric cancer, and that Notch1 inhibition is associated with the diffuse type of gastric cancer. Here, we describe additional data showing that in CRC cell lines, unlike gastric cancer, DLL1 expression is not regulated by promoter methylation. Moreover, in CRC, Notch1 receptor is not affected by any mutation. These data suggest a different regulation of Notch1 signaling between gastric cancer and CRC.     

Epigenetic silencing of Notch signaling in gastrointestinal cancers

The Notch signaling pathway drives proliferation, differentiation, apoptosis, cell fate choices and maintenance of stem cells during embryogenesis and in self-renewing tissues of the adult. In addition, aberrant Notch signaling has been implicated in several tumors, where Notch can function both as an oncogene or a tumor-suppressor gene, depending on the context. This Extra View aims to review what is currently known about Notch signaling, in particular in gastrointestinal tumors, providing a summary of our data on Notch1 signaling in gastric cancer with results obtained in colorectal cancer (CRC). We have already reported that the epigenetic regulation of the Notch ligand DLL1 controls Notch1 signaling activation in gastric cancer, and that Notch1 inhibition is associated with the diffuse type of gastric cancer. Here, we describe additional data showing that in CRC cell lines, unlike gastric cancer, DLL1 expression is not regulated by promoter methylation. Moreover, in CRC, Notch1 receptor is not affected by any mutation. These data suggest a different regulation of Notch1 signaling between gastric cancer and CRC.     

Notch信号与心脏干细胞关系的研究进展

Notch信号是广泛存在于各种动物细胞中高度保守的信号途径,在干细胞生物学功能中发挥重要作用。心脏干细胞（cardiac stem cells,CSCs）是存在于心脏特殊微环境下的多潜能干细胞,其表面存在Notch受体,而与其相邻的细胞可表达Notch配体,提示心脏干细胞中的Notch信号在某些条件下可被活化。该文从Notch信号通路的组成和激活、CSCs的界定与来源、CSCs主要类型的一般生物学特征及Notch信号通路与CSCs形成、分化和增殖的关系等方面进行综述,并展望了基于CSCs在心肌再生相关转化医学研究中的前景。     

Notch signaling in blood vessels: from morphogenesis to homeostasis

Notch signaling is an evolutionarily conserved intercellular signaling pathway that plays numerous crucial roles in vascular development and physiology. Compelling evidence indicates that Notch signaling is vital for vascular morphogenesis including arterial and venous differentiation and endothelial tip and stalk cell specification during sprouting angiogenesis and also vessel maturation featured by mural cell differentiation and recruitment. Notch signaling is also required for vascular homeostasis in adults by keeping quiescent phalanx cells from re-entering cell cycle and by modulating the behavior of endothelial progenitor cells. We will summarize recent advances of Notch pathway in vascular biology with special emphasis on the underlying molecular mechanisms.     

Effects of N-[N-(3, 5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) on cell proliferation and apoptosis in Ishikawa endometrial cancer cells

Endometrial cancer is one of the most common gynecological malignancies in Japan, where the disease shows an increasing morbidity. However, surgical therapy remains the treatment of choice for endometrial cancers that tend to be insensitive to radiation therapy and chemotherapy. Therefore, novel therapeutic strategies are required. The Notch signaling pathway regulates embryogenesis and cellular development, but deregulated Notch signaling may contribute to tumorigenesis in several cancers. Moreover, γ-secretase inhibitors have been shown to be potent inhibitors of the Notch signaling pathway; they suppress cellular proliferation and induce apoptosis in several cancer cells. In the present study, we investigated the effect of N-[N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT, γ-secretase inhibitor) on the cell proliferation and apoptosis in Ishikawa endometrial cancer cells. Real-time PCR detected mRNA derived from NOTCH1 and HES1, which are target genes of the Notch signaling pathway, in Ishikawa endometrial cancer cells. After blocking Notch signaling, cellular proliferation decreased, accompanied by increased expression of p21 mRNA and decreased expression of the cyclin A protein. Furthermore, blockade of Notch signaling induced apoptosis. These results suggest that the Notch signaling pathway may be involved in cell proliferation through cell cycle regulation and apoptosis in Ishikawa endometrial cancer cells. Inhibition of the Notch signaling pathway by γ-secretase inhibitors is expected to be a potential target of novel therapeutic strategies for endometrial cancer.     

Regulation of notch signaling activity

Cell-cell signalling mediated by the receptor Notch is used widely across the metazoans to determine cell fate and regulate pattern formation. Notch signals via a conserved regulated intramembrane proteolysis. Recent analyses of the cell biology of the Notch receptor have identified several fundamental mechanisms that contribute to regulate Notch signaling activity in space and time.     

Regulation of angiogenesis via Notch signaling in breast cancer and cancer stem cells

Breast cancer angiogenesis is elicited and regulated by a number of factors including the Notch signaling. Notch receptors and ligands are expressed in breast cancer cells as well as in the stromal compartment and have been implicated in carcinogenesis. Signals exchanged between neighboring cells through the Notch pathway can amplify and consolidate molecular differences, which eventually dictate cell fates. Notch signaling and its crosstalk with many signaling pathways play an important role in breast cancer cell growth, migration, invasion, metastasis and angiogenesis, as well as cancer stem cell (CSC) self-renewal. Therefore, significant attention has been paid in recent years toward the development of clinically useful antagonists of Notch signaling. Better understanding of the structure, function and regulation of Notch intracellular signaling pathways, as well as its complex crosstalk with other oncogenic signals in breast cancer cells will be essential to ensure rational design and application of new combinatory therapeutic strategies. Novel opportunities have emerged from the discovery of Notch crosstalk with inflammatory and angiogenic cytokines and their links to CSCs. Combinatory treatments with drugs designed to prevent Notch oncogenic signal crosstalk may be advantageous over λ secretase inhibitors (GSIs) alone. In this review, we focus on the more recent advancements in our knowledge of aberrant Notch signaling contributing to breast cancer angiogenesis, as well as its crosstalk with other factors contributing to angiogenesis and CSCs.     

Development and cancer: lessons learned in the pancreas

Cancer progression and organ development are similar phenomena. Both involve rapid bursts of proliferation, angiogenesis, tissue remodeling, and cell migration. Therefore, it is not surprising that both processes utilize similar signaling machinery. In fact, many recent studies have suggested that cancer is a disease triggered by the erroneous re-activation of signaling pathways that are typically downregulated after the completion of embryonic development. This link between embryonic development and cancer is particularly exciting because it suggests that we might be able to exploit the knowledge gained in studies of Developmental Biology to obtain novel insights into tumor biology. Our evolving understanding of pancreatic adenocarcinoma is an excellent example of this relationship between development and cancer. Here we discuss recent studies have indicated important roles for two major developmental signaling pathways in pancreatic cancer: Notch and Hedgehog (Hh).     

Convergence of Wnt and Notch signaling controls ovarian cancer cell survival

In the last 40 years ovarian cancer mortality rates have slightly declined and, consequently, it continues to be the fifth cause of cancer death in women. In the present study, we showed that β-catenin signaling is involved in the functions of ovarian cancer cells and interacts with the Notch system. Wnt and Notch systems showed to be prosurvival for ovarian cancer cells and their inhibition impaired cell proliferation and migration. We also demonstrated that the inhibition of β-catenin by means of two molecules, XAV939 and ICG-001, decreased the proliferation of the IGROV1 and SKOV3 ovarian cancer cell lines and that ICG-001 increased the percentage of IGROV1 cells undergoing apoptosis. The simultaneous inhibition of β-catenin and Notch signaling, by using the DAPT inhibitor, decreased ovarian cancer cell proliferation to the same extent as targeting only the Wnt/β-catenin pathway. A similar effect was observed in IGROV1 cell migration with ICG-001 and DAPT. ICG-001 increased the Notch target genes Hes-1 and Hey-1 and increased Jagged1 expression. However, no changes were observed in Dll4 or Notch 1 and 4 expressions. Our results suggest that Notch and β-catenin signaling co-operate in ovarian cancer to ensure the proliferation and migration of cells and that this could be achieved, at least partly, by the upregulation of Notch Jagged1 ligand in the absence of Wnt signaling. We showed that the Wnt pathway crosstalks with Notch in ovarian cancer cell functions, which may have implications in ovarian cancer therapeutics.     

Notch signaling contributes to lung cancer clonogenic capacity in vitro but may be circumvented in tumorigenesis in vivo

The Notch signaling pathway is a critical embryonic developmental regulatory pathway that has been implicated in oncogenesis. In non-small cell lung cancer (NSCLC), recent evidence suggests that Notch signaling may contribute to maintenance of a cancer stem or progenitor cell compartment required for tumorigenesis. We explored whether intact Notch signaling is required for NSCLC clonogenic and tumorigenic potential in vitro and in vivo using a series of genetically modified model systems. In keeping with previous observations, we find that Notch3 in particular is upregulated in human lung cancer lines and that downregulation of Notch signaling using a selective γ-secretase inhibitor (MRK-003) is associated with decreased proliferation and clonogenic capacity in vitro. We show that this phenotype is rescued with the expression of NICD3, a constitutively active cleaved form of Notch3 not affected by γ-secretase inhibition. Using an inducible LSL-KRAS(G12D) model of lung cancer in vivo, we show a transient upregulation of Notch pathway activity in early tumor precursor lesions. However, a more rigorous test of the requirement for Notch signaling in lung oncogenesis, crossing the LSL-KRAS(G12D) mouse model with a transgenic with a similarly inducible global dominant-negative suppressor of Notch activity, LSL-DNMAML (dominant-negative mastermind-like), reveals no evidence of Notch pathway requirement for lung tumor initiation or growth in vivo. Distinct Notch family members may have different and potentially opposing activities in oncogenesis, and targeted inhibition of individual Notch family members may be a more effective anticancer strategy than global pathway suppression.