How notch signalling is regulated and how this goes wrong in breast cancer.
Notch signalling pathway is composed by transmembrane receptors and ligands and it is modulated by a number of proteins. It plays a key role in the development of an embryo and maintenance of tissues in adult life by controlling neighbouring cells communication and influencing cell fate. This literature review is focused on the regulation of notch signalling in mammalian cells and how it can cause breast cancer when it goes wrong. Peer-reviewed journals obtained from Pubmed, Scopus and Google Scholar databases were used to gather relevant information. It was understood from this research the importance of the Notch signalling pathway in all organisms and how fragile it can be seeing that a mutation in any of the Notch modulators or the components of Notch itself that results in an uncontrolled communication between adjacent cells and ultimately altering cell fate can result in a number of pathologies, including breast cancer. More precisely, Notch receptors 1, 3 and 4 were normally overexpressed in the formation or aggravation of breast tumours, while Notch2 were under expressed on breast cancer development. It was also found that potentially targeting components of Notch or its modulators by manipulating their expression can lead to design and creation of new cancer therapies.
Notch signalling is responsible for the development of an organism from embryonic stage throughout adult life by regulating cell-to-cell communication, exerting an important role in cell differentiation, proliferation and apoptosis as well as playing a role in the regulation of cell and tissue maintenance (Kumar, Juillerat-Jeanneret and Golshayan, 2016).
There is a number of proteins that are involved in the regulation and transmission of Notch signalling (Artavanis-Tsakonas, Rand and Lake, 1999). Mutations in any of these proteins or any part of the Notch signalling mechanism can result in a variety of human diseases including different types of tumours (Kumar, Juillerat-Jeanneret and Golshayan, 2016; Artavanis-Tsakonas, Rand and Lake, 1999).
Tumours are formed due to abnormalities during the cell cycle machinery which results in a cell uncontrolled proliferation and/or failing apoptosis (Kumar, Juillerat-Jeanneret and Golshayan, 2016). Since Notch pathway is seen in different tissues and it dictates cell fate, a mutation in any of its components directly affecting the cell cycle may result in oncogenesis.
The role of the Notch signalling pathway in the development and progression of cancer in mammalian cells is being studied over the past decades most commonly in mice but also in human breast cells (Kontomanolis et al., 2018).
Notch signalling mechanism
Notch gene were discovered in Drosophila melanogaster by Professor T. H. Morgan and colleagues in the early 20th century (Morgan, 1917). Notch signalling is an important pathway for cell-to-cell communication during the development process of an organism which is involved in coordinating tissue repair or regeneration and inducing differentiation of stem cells (Radtke and Raj, 2003). Moreover, Notch signalling is involved in tissue and cell maintenance and homeostasis during the adult life of an organism (Kumar, Juillerat-Jeanneret and Golshayan, 2016).
Notch is a transmembrane protein that perform its function by receiving signals from its extracellular domain and carrying the information to the nucleus of the cell (Lai, 2004). In mammals, Notch is encoded by four types of receptors (fig 1) NOTCH1, NOTCH2, NOTCH3, NOTCH4 (Uyttendaele et al., 1996) and five types of ligands (fig 1): DLL1, DLL3, DLL4 (delta-like ligands) and Jagged1 and Jagged2 (Kumar, Juillerat-Jeanneret and Golshayan, 2016). The extracellular part of its receptors and ligands are made of a repeated sequence of epidermal growth factors (EGF) which is responsible for the ligand-receptor interaction at the cell surface (Rebay et al., 1991).
The activation of Notch signalling is done by the binding of the extracellular domain of the receptor of a cell with the ligand of another neighbour cell (Feng et al., 2018). The amount and type of ligands and receptors being expressed in a cell will diffuse to the neighbouring cells and influence the fate and behaviour of all surrounding cells. In other words, a cell can acquire a signalling mode if its neighbour has an increase in ligand expression (Artavanis-Tsakonas, Rand and Lake, 1999). This amplification of signal given by the communication of adjacent cells results in the formation of biological patterns during development or tissue maintenance in adult life (Lai, 2004).
Once Notch receptor is activated by a ligand, it goes through two proteolytic cleavage: S2 and S3 (Bray, 2006). S2 forms a substrate for S3 which is cleaved by a protease complex called γ-secretase, releasing the intracellular region fragment of the Notch (Notchintra) which will move into the nucleus of the cell (Bray, 2006). In the nucleus, Notchintra interacts with the DNA-binding CLS protein (CBF-1/RBPJ-κ in Homo sapiens) forming a complex containing transcriptional co-activators of the mastermind family (Yamaguchi et al., 2008) which prompt the expression of target genes related to cell growth, differentiation, survival and maintenance (Kopan, 2002). CLS works as a repressor as well as an activator for the transcription of the target gene, the presence of Notchintra in the nuclear domain will convert the repressor form of CLS into active form (Lai, 2004) (fig. 1).
Fig 1. Notch signalling pathway (D Bomfati, 2018)
Notch signalling is regulated positively or negatively by a variety of proteins these involve proteins that act in the extracellular domain of EGF sequence of receptors and ligands (fig. 1) to facilitate its binding, others can assist in the signal transduction in the cytoplasm after activation of Notch receptor and, proteins that can affect transcription activity of the Notch-CLS complex (Panin and Irvine, 1998). Some regulators of the Notch signalling are specific to different tissues, for instance, during bone development microRNA 34 directly affect many components of the Notch pathway, such as Notch1 and 2 receptors as well as the Jagged1 ligand (Bae et al., 2012). While transcription factor E74-like factor 5 (Elf5) is responsible for regulating the Notch pathway in mammary glands (Chakrabarti et al., 2012). Chakrabarti and colleagues (2012) reports that the absence of Elf5 highly increased the activation of Notch1 and 4 receptors while it decreased the activation of Notch2 receptor.
Notch is also regulated by other signalling pathways, such as Wtn signalling pathway (Collu, Hidalgo-Sastre and Brennan, 2014). Wtn has an opposite outcome in regard to cell fate decisions (Collu et al., 2012). They can interact with each other by three major ways: by promoting transcription of target genes that needs both pathways to be active at the same time; by activating transcription of ligands for the other pathway, this mode is often used throughout development to control their activity; and by direct molecular cross-talk (Collu, Hidalgo-Sastre and Brennan, 2014), for example, Dishevelled proteins (DVL) belonging to Wtn signalling pathway can inhibit Notch signalling by blocking the CLS activation site of Notch and consequently limiting the transcription of such (Collu et al., 2012).
What happens when it goes wrong?
A mutation in any of the components of the Notch signalling mechanism or its modulators resulting in a non-controlled communication between neighbouring cells is highly associated with the development of different types of disorders, including breast cancer (Kumar, Juillerat-Jeanneret and Golshayan, 2016). Non-regulated activation of the Notch receptors is linked to the formation of tumours in mammary glands and the stop of normal mammary glands development (O’Neill et al., 2007). More precisely, an analysis made on mice mammary epithelial cells by O’Neill and colleagues (2007) shows that abnormal activation of the receptors Notch1, 3 and 4 are associated with mammary carcinoma while there’s no reports on the activation of Notch2 resulting in tumour.
Although different studies reveal that Notch1 overexpression is highly related to T-cell acute lymphoblastic leukaemia (Weng et al., 2004; Lin et al., 2012), overexpression of Notch1 is also seen on mammary tumours and that inhibiting its expression resulted in the reduction of tumour progress (Simmons et al., 2012). Expression of Notch receptors and ligands were analysed in situ hybridization in different types of human breast cancer by Reedijk and team (2005). The team found that high expression of Notch1 and Jagged1 is highly related to a reduced patient survival. 32% of patients expressing high levels of Notch1 and Jagged1 had a 5-year survival rate (2005).
Another study done by Yamaguchi and colleagues (2008), shows that the overexpression of Notch3 is highly responsible for the proliferation of ErbB2-negative breast cancer and that the decreased expression of Notch3 considerably reduced the proliferation and induced apoptosis of this type of breast cancer.
Another analysis made by O’Neill and team (2007) suggested that Notch4 activation increases cell growth and proliferation and the overactive Notch4 formed aggressive tumours with a favourable vascular network which can lead to a malignant phenotype. They also proposed by analysing mice tumour xenograft in vivo that Notch2 contrary to the other receptors, when deactivated the decreased expression resulted in breast cancer, seeing that Notch2 controls and induce apoptosis when activated (2007). Their results suggested that activation of Notch2 reduced tumour growth by 60% and the tumours presented a high vascular network, however, small and immature vessels were seen resulting in notably smaller and necrotic tumours (2007). This imply that Notch2 can be a powerful signal inhibitor in breast cancer (O’Neill et al., 2007).
For over a century, since Notch has been discovered, it has been studied in a variety of organisms. It is notable how important the normal functioning of Notch is and how it can drastically result in disorders when something goes wrong in the pathway.
Many pathologies derive from problems in the Notch signalling pathway and there are still many factors not yet understood. Nevertheless, it is becoming more evident that each of the Notch proteins (receptors and ligands) plays a different role when it comes to the normal formation of cells as well as formation and progression of cancer cells. Studies revelled that the formation of breast cancer where Notch signalling is related, there is an overexpression of either notch 1, 3 and/or 4 or under expression of Notch 2 (O’Neill et al., 2007; Yamaguchi et al., 2008; Reedijk et al., 2005).
The information available to date reveals possible therapy and treatment options by manipulating the modulators of Notch or its own components as suggested by Simmons (2012) and O’Neill (2007) teams in regards of breast cancer. Since Notch machinery is associated with the normal breast development as well as the breast cancer development, design and creation of cancer therapies by manipulating and regulating the functions of the Notch pathway requires intricate handling of such (Kumar, Juillerat-Jeanneret and Golshayan, 2016).
In the UK alone, there are more than 360.000 new cases of cancer every year (Cancer Research, 2015b), of which approximately 55.000 is breast cancer (Cancer Research, 2015a). Breast cancer accounts for 15% of all the cancers diagnosed, making it the most common one among all (2015a). In the UK, breast cancer is the second most common cause of death of women (2015a). Further comprehension and research on Notch signalling pathway can lead to more specific and efficient treatments for each subclass of breast cancer as seen by Yamaguchi and colleagues (2008) which showed that by decreasing the expression of Notch3
it reduced the development of ErbB2-negative breast cancer.
In conclusion it is possible to appreciate the importance and complexity of the Notch signalling pathway and although it has been a topic of research for decades, everything involving this pathway is not yet fully understood. Nonetheless, it is becoming clear that manipulating specific components can possibly change the outcome of cancers formed by errors in the Notch signalling pathway proving to be a potential therapeutic target.
- Artavanis-Tsakonas, S., Rand, M.D. and Lake, R.J. (1999) ‘Notch Signaling: Cell Fate Control and Signal Integration in Development’, Science, 284(5415), pp. 770-776. doi: 10.1126/science.284.5415.770.
- Bae, Y., Yang, T., Zeng, H., Campeau, P.M., Chen, Y., Bertin, T., Dawson, B.C., Munivez, E., Tao, J. and Lee, B.H. (2012) ‘miRNA-34c regulates Notch signaling during bone development’, Human molecular genetics, 21(13), pp. 2991-3000. doi: 10.1093/hmg/dds129.
- Bray, S.J. (2006) ‘Notch signalling: a simple pathway becomes complex’, Nature Reviews Molecular Cell Biology, 7(9), pp. 678-689. doi: 10.1038/nrm2009.
- Cancer Research, U.K. (2015a) Breast cancer statistics. Available at: https://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-type/breast-cancer (Accessed: Oct 31, 2018).
- Cancer Research, U.K. (2015b) Cancer Statistics for the UK. Available at: https://www.cancerresearchuk.org/health-professional/cancer-statistics-for-the-uk (Accessed: Oct 31, 2018).
- Chakrabarti, R., Wei, Y., Romano, R., DeCoste, C., Kang, Y. and Sinha, S. (2012) ‘Elf5 Regulates Mammary Gland Stem/Progenitor Cell Fate by Influencing Notch Signaling’, Stem cells, 30(7), pp. 1496-1508. doi: 10.1002/stem.1112.
- Collu, G.M., Hidalgo-Sastre, A., Acar, A., Bayston, L., Gildea, C., Leverentz, M.K., Mills, C.G., Owens, T.W., Meurette, O., Dorey, K. and Brennan, K. (2012) ‘Dishevelled limits Notch signalling through inhibition of CSL’, Development (Cambridge, England), 139(23), pp. 4405-4415. doi: 10.1242/dev.081885.
- Collu, G.M., Hidalgo-Sastre, A. and Brennan, K. (2014) ‘Wnt–Notch signalling crosstalk in development and disease’, Cellular and Molecular Life Sciences, 71(18), pp. 3553-3567. doi: 10.1007/s00018-014-1644-x.
- Feng, Y., Spezia, M., Huang, B., Huang, S., Yuan, C., Zeng, Z., Zhang, L., Ji, X., Liu, B., Liu, W., Luo, W., Lei, Y., Du, S., Vuppalapati, A., Luu, H.H., Haydon, R.C., He, T. and Ren, G. (2018) ‘Breast cancer development and progression: Risk factors, cancer stem cells, signaling pathways, genomics, and molecular pathogenesis’, Genes & Diseases, 5(2), pp. 77-106. doi: 10.1016/j.gendis.2018.05.001.
- Kontomanolis, E.N., Kalagasidou, S., Pouliliou, S., Anthoulaki, X., Georgiou, N., Papamanolis, V. and Fasoulakis, Z.N. (2018) ‘The Notch Pathway in Breast Cancer Progression’, TheScientificWorldJournal, 2018, pp. 2415489. doi: 10.1155/2018/2415489.
- Kopan, R. (2002) ‘Notch: a membrane-bound transcription factor’, Journal of cell science, 115(Pt 6), pp. 1095.
- Kumar, R., Juillerat-Jeanneret, L. and Golshayan, D. (2016) ‘Notch Antagonists: Potential Modulators of Cancer and Inflammatory Diseases’, Journal of Medicinal Chemistry, 59(17), pp. 7719-7737. doi: 10.1021/acs.jmedchem.5b01516.
- Lai, E.C. (2004) ‘Notch signaling: control of cell communication and cell fate’, Development, 131(5), pp. 965-973. doi: 10.1242/dev.01074.
- Lin, C., Zheng, H., Wang, C., Yang, L., Chen, S., Li, B., Zhou, Y., Tan, H. and Li, Y. (2012) ‘Mutations increased overexpression of Notch1 in T-cell acute lymphoblastic leukemia’, Cancer cell international, 12(1), pp. 13. doi: 10.1186/1475-2867-12-13.
- Morgan, T.H. (1917) ‘The Theory of the Gene’, The American Naturalist, 51(609), pp. 513-544. doi: 10.1086/279629.
- O’Neill, C.F., Urs, S., Cinelli, C., Lincoln, A., Nadeau, R.J., León, R., Toher, J., Mouta-Bellum, C., Friesel, R.E. and Liaw, L. (2007) ‘Notch2 Signaling Induces Apoptosis and Inhibits Human MDA-MB-231 Xenograft Growth’, The American Journal of Pathology, 171(3), pp. 1023-1036. doi: 10.2353/ajpath.2007.061029.
- Panin, V.M. and Irvine, K.D. (1998) ‘Modulators of Notch signaling’, Seminars in Cell and Developmental Biology, 9(6), pp. 609-617. doi: 10.1006/scdb.1998.0263.
- Radtke, F. and Raj, K. (2003) ‘The role of Notch in tumorigenesis: oncogene or tumour suppressor?’, Nature Reviews Cancer, 3(10), pp. 756-767. doi: 10.1038/nrc1186.
- Rebay, I., Fleming, R.J., Fehon, R.G., Cherbas, L., Cherbas, P. and Artavanis-Tsakonas, S. (1991) ‘Specific EGF repeats of Notch mediate interactions with Delta and serrate: Implications for notch as a multifunctional receptor’, Cell, 67(4), pp. 687-699. doi: 10.1016/0092-8674(91)90064-6.
- Reedijk, M., Odorcic, S., Chang, L., Zhang, H., Miller, N., McCready, D.R., Lockwood, G. and Egan, S.E. (2005) ‘High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival’, Cancer Research, 65(18), pp. 8530-8537. doi: 10.1158/0008-5472.CAN-05-1069.
- Simmons, M., Serra, R., Hermance, N. and Kelliher, M. (2012) ‘NOTCH1 inhibition in vivo results in mammary tumor regression and reduced mammary tumorsphere-forming activity in vitro’, Breast Cancer Research, 14(5), pp. R126. doi: 10.1186/bcr3321.
- Uyttendaele, H., Marazzi, G., Wu, G., Yan, Q., Sassoon, D. and Kitajewski, J. (1996) ‘Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene’, Development, 122(7), pp. 2251-2259.
- Weng, A.P., Ferrando, A.A., Lee, W., Morris, J.P., Silverman, L.B., Sanchez-Irizarry, C., Blacklow, S.C., Look, A.T. and Aster, J.C. (2004) ‘Activating Mutations of NOTCH1 in Human T Cell Acute Lymphoblastic Leukemia’, Science, 306(5694), pp. 269-271. doi: 10.1126/science.1102160.
- Yamaguchi, N., Oyama, T., Ito, E., Satoh, H., Azuma, S., Hayashi, M., Shimizu, K., Honma, R., Yanagisawa, Y., Nishikawa, A., Kawamura, M., Imai, J., Ohwada, S., Tatsuta, K., Inoue, J., Semba, K. and Watanabe, S. (2008) ‘NOTCH3 signaling pathway plays crucial roles in the proliferation of ErbB2-negative human breast cancer cells.’, Cancer research, 68(6), pp. 1881-1888. doi: 10.1158/0008-5472.CAN-07-1597.