THE ROLE OF NOTCH IN TUMORIGENESIS: ONCOGENE OR TUMOUR SUPPRESSOR?

1. THE ROLE OF NOTCH IN TUMORIGENESIS: ONCOGENE OR TUMOUR SUPPRESSOR? Speakers: 葉恭誌 李昭鋐 組員: 林雅葶 張明俐 羅苑菁 鄭伯忻 曾昭穎 蔡志文 陳美君 江承堯 高家民 呂女秀菱 Notch

2. notch notch - In 1917,Thomas Hunt Morgan and colleagues described a strain of Drosophila with notches at the end of their wing blades. • Notch: • Genetics: Haploinsufficiency • Structure: transmembrane receptor

3. Transcription activation domain CDC10 repeats Lin12 repeats (cysteine-rich) Nuclear-localization signal Structure : transmembrane receptor • Synthesized as a single precursor protein • Cleaved in two during its transport to the cell surface. Notch receptor Drosophila Ligand (Delta) (Serrate)

4. Human Notch receptor Ligand EGF ANK RAM LN PEST TAD (Ser-like) Slight difference

5. Notch signaling • Different modification of Notch and signal crosstalk influence Notch activation. • Different cell type uses different signal. Activation leads to cleavage IC domain (Notch IC) Translocate into nucleus and bind to transcription factor CSL Notch absence: transcription repressor Notch present: Transcription activator • HES (hairy/enhancer of split) family of transcription factors • cell-cycle regulator-Waf1

6. Notch function

7. Notch function • Maintenance of an undifferentiated state • Notch signaling can maintain stem cells or precursor populations in an undifferentiated state. • Gain-of-function studies：(in the chicken) • using a dominant active Notch-IC：Notch signaling prevents progenitors from undergoing neurogenesis. • blocking the Notch pathway：excessive neurogenesis and depletion of the progenitor pool. Lewis, J. (1998) Semin. Cell Dev. Biol. 9, 583–589 Henrique, D. (1997) Curr. Biol. 7, 661–670

8. receptor ligand Equitpotent cell ? Differentiation Equitpotent cell • During the development of neuronal-precursor cells of the sensory organ in Drosophila. Participate in Cell-fate decision Epidermal cell Neuronal cell

9. Inductive cell-fate determination Notch signal Example : bipotential mouse neural-crest stem cell adopt to glial cell. Cell differentiation Mouse thymic epithelial cell T-cell Early lymphocyte precursors B-cell

10. Induction of terminal differentiation • DLL1-induced Notch signaling initiates a terminal-differentiation program in human skin. • Other possible mechanism (Jagged-mediated)：activated Notch1 causes keratinocyte growth arrest through increased p21WAF1/Cip1 expression. —Rangarajan, A. (2001) EMBO J. 20, 3427–3436

11. Notch as an oncogene

12. Notch and T-cell leukaemia • Translocation of a portion of chromosome 7 to chromosome 9, which contains T-cell receptor-b gene. • Expreesion of truncated NOTCH1 transcripts (similar to NOTCH1-IC) from TCRb promoter causes T-cell acute lymphoblastic leukaemia (T-ALL). Ellisen, L. W. et al. Cell66, 649-661 (1991).

13. Hot spots of mutations found in more than 50% of T-ALL patients HD: heterodimerization domain P: pest domain. Weng AP. et al.Science 306:269–271(2004)

14. Why does the hematopoietic oncogenic potential seem to be restricted to T cell leukemia? • Mice transplanted with Notch1-IC-expressing bone-marrow-progenitor cells from either Rag2–/– or Slp76 –/– mice failed to develop T-cell • leukaemia. • Introduction of a TCR βtransgene into Rag2–/– mice — to re-activate pre- • TCR signalling — restored the oncogenic function of Notch1-IC. Allman, D. et al. J. Exp. Med. 194, 99–106 (2001). Notch1-IC-mediated transformation is dependent on a second T-cell-specific signal that is mediated by the pre-TCR.

15. Notch and viruses • Proviral integration of the Moloney murine-leukaemia virus (MuLV) into the murine Notch1 locus causes T-ALL in mice. • Integration of the mouse mammary tumour virus (MMTV) in between the Notch/Lin12 repeats and the transmembrane domain of either the Notch1 (NI) or Notch4 (N4) gene causes mammary tumours in the mouse.

16. Notch and Epithelial tumours • Transgenic mice that express this Notch4-IC/int-3gene developed poorly differentiated mammary and salivary-gland adenocarcinomas within 7 months. • The mammary epithelial of these mice failed to branch.

17. Notch-IC (1) RAM domain : CBF binding site (2) ankyrin repeat domain (ANK) : mediates further protein- protein interactions (3) C-terminal domain : a polyglutamine region (OPA) + proline, glutamic acid, serine and threonine rich region (PEST) (4) NLS : nuclear localization sequence

18. How can Notch contribute to transformation? • CBF1 is a sequence specific DNA binding protein that functions to repress transcription of cellular genes • In some cells, Notch-assisted transformation is dependent on the ankyrin repeats of the Notch protein, not CBF1

19. E1A-immortalized baby rat kidney cell line (RKE) MOLECULAR AND CELLULAR BIOLOGY, June 2000, p. 3928–3941

20. Notch needs to partner another oncoprotein to actually cause cancer Virology 286, 23±30 (2001) AcN1: a truncated allele of Notch1 Transformation can be induced by expressiong Notch-IC with oncoproteins such as adenovirus EIA,HPV E6 and E7…

21. These oncoproteins all share the common property of being able to override the G1-S checkpoint resistance to apoptosis, anoikis or differentiation ANOIKIS: Cell death induced as a result of the absence of matrix attachment

22. The oncogenic effects of Notch include PI3-K activation and induction of ERBB2 and NF-ĸB2 expression (by NOTCH-IC) • PI3-K resistance to anoikis • ERBB2 cell proliferation and growth • NF-ĸB2 expression of genes which encode anti-apoptotic proteins

23. Notch1 as tumour suppressor NOTCH1 signaling increases expression of WAF1, which causes cell-cycle arrest in basal cells, to allow the onset of terminal differentiation

24. In Notch1-ablated skin, reactivation of Wnt and Sonic-hedgehog pathways result in the development of basal-cell-carcinoma-like tumours in the mouse. • The tumour-suppressive activity of NOTCH1 might be mediated by several routes, to induce cell-cycle arrest and differentiation • Tumour cells might counter-select against expression of Notch receptors or ligands to escape from differentiation and cell-cycle arrest

25. The two faces of Notch in cervical cancer

26. Proteins are increasingly being found to have several and possibly opposing functions

27. HPV was found in 99% cervical cancers

28. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53 1990 Cell • Telomerase activation by the E6 gene product of human papillomavirus type 16 1996 Nature =>E6 and E7 are oncoproteins in cervical cancer cells

29. BUT….. =>Notch prevent cellular proliferation in normal epithelia =>upregulate WAF1 =>cells initiation of differentiation

30. Conclusion • The outcome of Notch activation is dependent on cellular context

31. Thank you