Role of notch signalling in deveopment, cancer development and its detailed cancer cell line study for purpose of detailed targetted molecular therapeutics

1. NOTCH SIGNALLING
PATHWAY
COURSE: TARGETTED MOLECULAR THERAPEUTICS
PRESENTED BY: SAIMA BARKI
DEPARTMENT OF BIOCHEMISTRY
QUAID-e-AZAM UNIVERSITY, ISLAMABAD

2. CONTENT
 History
 Introduction
 Structure
 Function
 Signaling pathway
 Role in cancer
 invitro and invivo studies
 Therapeutic strategies
 References
2

3. NOTCH SIGNALLING
 HISTROY:
1. First discovered by T.H Morgan in 1917,, in Drosophila mellanogester, by observing
the notch phenotype in the wing of fruit fly.
2. Then identified in human also, first consideration of gene link with development
proposed by D F Pulson in 1985.
3. Spyros Artavinis-Tsakonas discovered the pathway while working on drosophilla.
Artavinis et al,. science, 1994

4. NOTCH SIGNALING: INTRODUCTION
 Mechanism: lateral inhibition and contact based signaling.
 Components:
 Both ligand and receptor bear EGF like repeats but the number vary from one to another.
 Ligand: DELTA protein, DLL 1/3/4, JAGGED 1/2
 Receptor: Notch protein, Notch 1/2/3/4,
 (ECD) contains several epidermal growth factor (EGF) like varying number of repeats and negative regulatory
region (NRR)
 TM hydrophobic
 NICD oncogenic
 Both Ligand and Receptors are membrane bound single pass glycoprotein and for functionality require proteolytic
cleavage.
 Chromosoaml lcation 9q34.3
 34 exons
 Initially equivalent level of both receptor and ligand expression, the level depend on many mechanism
expressing and of other repressing.
 Notch cleavage is of central importance that change the gene expression in the nucleus.
 The reason behind the cleavage of ligand delta like is unknown.
Al Husaini, et al.,2011

5. STRUCTURE OF LIGANDS AND RECEPTORS; CONSERVANCE
THROUGHOUT EVOLUTION
Michael et al., 2005

6. IMPORTANCE OF NOTCH PATHWAY
 Signaling through the notch pathway, considered one of the vastly used pathway from
early embryogenesis to adult life.
 Notch signaling pathway, a highly conserved pathway,
 FUNCTIONAL IN:
 1. Different developmental Processes,
 2. Homeostatic processes,
 On Notch pathway least regulatory control implication, as direct routed from
membrane to the nucleus.
 Both oncogenic and tumor suppresor under differen conditions as in some function to
promotes tissue growth and cancers while in some cell death and tumour suppression.
 Complex regulation for the functional outputs includes mechanisms:
1. the receptor–ligand landscape,
2. the tissue topology,
3. the nuclear environment
4. and the connectivity of the regulatory networks.Bray, et al., Nature (2006).

7. Functions: Physiological
NOTCH
 Stem cell maintainance
 Regulation of development
 Speciation of Cell fate
 Neuronal development
,
Shaolei Zang et al. 2010
7

8. Functions:Pathological
NOTCH
 Cell division
 Cell differentiation
 Cell survival
 Self renewal of stem cells and proginators
 Angiogenesis
 Invasion
 Migration
,
Shaolei Zang et al. 2010
8

9. DEVELOPMENTAL LOGIC
CELL TO CELL SIGNALING
 Lateral inhibition:
 Production of nerve cells in drosophila
 An epithelial cell when develop into a nerve cell, release a signal to neighboring not to do
the same.
Amina ahmed, Shaweeta chandra, et al., 2003

10. Notch Pathway: Role In Fate
Determination
A. Inductive signaling
B. Loss and gain of signaling
pathways
 Different signaling pathways result
of different interaction pattern.
 Each cell of embryo is guided along
all its one developmental pathway
or another depending on:
 Its history,
 Its position,
 Its neighboring microenvironment.
 Some of these act through relaying
signals from cell surface to interior
of cell.
Amina ahmed, Shaweeta chandra, et al., 2003

11. PROTEOLYTIC CLEAVAGE OF THE NOTCH
RECEPTOR
 Notch receptor: initially synthesized as single protein then
 3 proteolytic cleage
1. In Golgi apparatus (S1 cleavage)
 furin in its future extracellular domain converting into heterodimer and transported to the cell surface and act
as mature functional receptor, consist of 3 domains:
1. NECD
2. TM
3. NICD
 Signaling starts when ligand interact with the receptor leading to furter cleavages.
 2 .Cleavage after binding to ligand (S2 cleavage)
 Ligand binding lead to cleavage in NECD mediated by an extracellular protease ADAM.
 3. Final cleavage (S3 cleavage)
 This is done by cytoplasm gamma secretase,, letting free the NICD, very close to plasma membrane,
translocated to nucleus,
 Bind to TF CSL, replacing the repressor bound to CSL.
• Yamamoto et al., 2010;
• Bray, 2006;
• Kovall and Blacklow, 2010)

12. OVERVIEW OF NOTCH PATHWAY

13. NOTCH SIGNALLING MECHANISM: PROTEOLTIC CLEAVAGE OF BOTH
LIGANDS AND RECEPTORS
1. Context based ligand binding to receptor,,Nuclear translocation:
 Receptor activation by proteolytic cleavage, cleavage by γ-secretase proteolytic
cleavage.
 Release of intracellular cytoplasmic domain of the NOTCH receptors.
 NIC released into the cytoplasm.
 NIC translocated to the nucleus, bind to cnserved DNA binding protein
RBPJϰ/CBF-1, MASTERMIND etc like transcriptional regulatory
proteins→complex→induces→expression of several genes involve in cell divisoin,
diferentiation and survival.
2. Less characterized-direct activation of signal transduuction pathway in the
cytoplasm.
13
Artavinas, et al, 1999

14. ASSOCIATED MOLECULAR GENETIC
ALTERATIONS
Notch
• Chromosomal translocation
• Point mutation
• Chromosomal amlification
,
14
Sianov, et al., 2012

15. RESEMBLANCE BETWEEN THE PROTEOLYTIC CLEAVAGE OF
NOTCH RECEPTOR AND AMYLOID PRECURSOR PROETIN (APP)
15
Sheh lam, Wang Tl, 2007

16. Role In Different Cancers
(Major Types)
 Cervical cancer
 Breast cancer
 Prostate cancer
 Leukemia
 Tumor promoting function in breast
cancer
 Development of adenocarcinomas in the murine mammary gland
Efstratiadis et al.,2007
16

17. BREAST CANCER; THEREAPEUTIC MECHANISMS AND
RESULTS
 Most commonly diagnosed malignancy
 Leading cause of death in females in westren countries
 Mutation:
1. Activate expression or elevate the oncogenes
2. Disrupt tumor suppressor genes
3. Molecular lesions
 The notch pathway- an important role in normal breast cell development, cell fate determination and
stem cell self-renewal
 The aberrant activation of this pathway is associated with breast cancer.
 INHIBITION of Notch signaling by:
 Gamma secretase inhibitors,
 Anti-Notch1 or anti-delta-like 4 (DLL4) monoclonal antibodies
17
Guo H, et al., 2014

18. BREAST CANCER; THEREAPEUTIC MECHANISMS AND
RESULTS
 RESULTS:
1. Antitumor activity in a variety of tumors, including T-cell acute lymphoblastic leukemia (T-
ALL) and solid tumors, through multiple mechanisms:
2. Induction of cell cycle arrest or apoptosis,
3. Disruption of angiogenesis
4. Upregulation of Notch1 expression has been revealed to protect breast cancer cells from
apoptosis.
 TARGETS:
1. Hairy enhancer of split (Hes) genes,
2. p21,
3. cyclinD1,
4. c-Myc,
5. nuclear factor κB,
6. B-cell lymphoma-2 (Bcl-2)
Keyaerates, et al., 2016

19. INVITRO STUDY
1. Xanthohumol (XN)derived from hops (Humulus lupulus), inhibit cell growth,
Induce apoptosis in numerous types of human cancer, including breast, prostate, leukemia and colon
cancer cells.
2. NOW focusing on its ability to inhibit breast cancer cell proliferation, cell cycle arrest, apoptosis
induction in vitro and slowing tumor growth in vivo.
3.CELL LINES AND CUTRUES:
Human MCF-7, MDA-MB-231 and HEK-293T cells, h-TERT-BJ, MCF-10A and murine 4T1 cells
cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine
serum (FBS; maintained at 37°C in a humidified atmosphere of 5% CO2 in air.
19
Roy M, et al, 2007

20. XN Regulates Notch1 Signal Pathway
Activity And Downstream Targets20
↓
Zhihong sun, et al, 2017

21. XN Inhibits Cell Growth And Migration In Breast Cancer Cell
Lines:Western Blot Analysis Of c-Myc And Survivin
21
Liaking MA, et al., 2015

22. XN Treatment Is Associated With Cell Cycle Arrest And
Apoptosis;Xn Arrests The Cell Cycle In The G0/G1 Phase
22
Zhihong sun, et al, 2017

23. XN Inhibits Breast Cancer Cells By Inducing
Apoptosis:Xn Promote Apoptosis Of Breast
Cells.
23
Zhihong sun, Qimim liu, 2017

24. XN Effects Mouse Tumor Growth And Notch 1 Protein
Expression Levels In Vivo:Xn Inhibited Tumor Growth In
Vivo.24
Zhihong sun, Qimim liu, 2017

25. Therapeutic strategies:
 Specific monoclonal antibodies.
 Gamma secretase inhibitors.
 Receptor ubiqitylation mediated strategies.
25
• Imatani et al., 2000;
• Pece et al., 2004

26. Conclusion
 Notch-1 is a transmembrane receptor protein
 Involve in many physiological functions through cell to cell communication
 2 types of ligand families can bind to Notch-1
 Majorly involve in breast cancer
 Downregulation of Notch receptors expression can act as therapeutic strategy for treatment
of cancer.
26

27. REFERENCES
1..Guo H, Wu F, Wang Y, Yan C, Su W. Overexpressed ubiquitin ligase Cullin7 in breast
cancer promotes cell proliferation and invasion via down-regulating p53. Biochem
Biophys Res Commun. 2014
2. Keyaerts M, Xavier C, Heemskerk J, Devoogdt N, Everaert H, Ackaert C, Vanhoeij M,
Duhoux FP, Gevaert T, Simon P, et al. Phase I study of 68Ga-HER2-Nanobody for
PET/CT assessment of HER2-expression in breast carcinoma. J Nucl
3. Al-Hussaini H, Subramanyam D, Reedijk M, Sridhar SS. Notch signaling pathway as a
therapeutic target in breast cancer. Mol Cancer Ther. 2011;
4. Yuan X, Zhang M, Wu H, Xu H, Han N, Chu Q, Yu S, Chen Y, Wu K. Expression of
Notch1 correlates with breast cancer progression and prognosis. PLoS One. 2015
5. Roy M, Pear WS, Aster JC. The multifaceted role of Notch in cancer. Curr Opin Genet
Dev. 2007;17:52–59. doi: 10.1016/j.gde.2006.
6. Palmer WH, Deng WM. Ligand-independent mechanisms of Notch activity. Trends
Cell Biol. 2015

28. Cont.
7. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: Cell fate control and signal integration in
development. Science. 1999
8. Kopan R, Ilagan MX. The canonical Notch signaling pathway: Unfolding the activation
mechanism. Cell. 2009
9. Radtke F, Raj K. The role of Notch in tumorigenesis: Oncogene or tumour suppressor. Nat Rev
Cancer. 2003
10. Robinson DR, Kalyana-Sundaram S, Wu YM, Shankar S, Cao X, Ateeq B, Asangani IA, Iyer M,
Maher CA, Grasso CS, et al. Functionally recurrent rearrangements of the MAST kinase and Notch gene
families in breast cancer. Nat Med. 2011
11. Michael Reedijk,1,2,3 Silvia Odorcic,1 Lynn Chang : High-level Coexpression of JAG1 and NOTCH1
Is Observed in Human Breast Cancer and Is Associated with Poor Overall Survival, Cancer Res 2005; 65:
(18). September 15, 2005
12. Efstratiadis A, Szabolcs M and Klinakis A: Notch, Myc and breast cancer. Cell Cycle 6: 418-429,
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13. Imatani A and Callahan R: Identification of a novel NOTCH-4/INT-3 RNA species encoding an
activated gene product in certain human tumor cell lines. Oncogene 19: 223-231, 2000
14. Pece S, Serresi M, Santolini E, Capra M, Hulleman E, Galimberti V, Zurrida S, Maisonneuve P, Viale
G and DiFiore PP: Loss of negative regulation by Numb over Notch is relevant to human breast

29. Thank You for Listening