1. Notch and Cancer IPO-LISBOA CIPM Angiogenesis group Francisco Caiado Sérgio Dias

2. The Hallmarks of Cancer (Hanahan and Weinberg) Tissue Invasion and Metastasis

3. Cancer Hallmarks and Notch signaling Tissue Invasion and Metastasis Modulation of the Notch Signaling Pathway

4. Notch-Delta signaling pathway Roca, C. and Adams, R. Genes & Dev. 2007 21: 2511-2524 Regulates: establishment of patterns of gene expression; cell differentiation; regulates binary cell fate choice; maintenance of stem cell populations; Function: Embryonic Development; Adult Self-Renewing Organs; CANCER

5. Abnormal Notch signaling and cancer Oncogenic activity of Notch Tumor supressor activity of Notch Maillard,I. and Pear, W. Cancer Cell 2003

6. De la Pompa et al. Endocrine Reviews 28(3):339–363 Abnormal Notch signaling and cancer Targeting Notch signaling: γ-secretase inhibitors (GSIs) are in early clinical trials; mAbs targeting the ‘negative regulatory region’ (NRR) of notch; mAbs that against DLL4 inhibit Notch signaling in endothelial cells and cause non-functional tumor angiogenesis;

7. Cancer Hallmarks and Notch signaling Tissue Invasion and Metastasis Modulation of the Notch Signaling Pathway

8. Tumor Angiogenesis Hashizume, H.l NCR 2000 Pro-angiogenic factors: VEGF, FGF, Neuropillin, Ang-2, MMPs… Vessel stabilizing factors: Notch-Delta, PDGF- 1, Ang-1, ECMs…

9. Notch signaling and tumor angiogenesis Ridgway, J. et al Nature 2006Thuston, G. et al NCR 2006

10. Post-natal vasculogenesis Bone marrow (BM) derived progenitor cells: recruited during physiological and “malignant” angiogenesis; BM mobilization; homing to angiogenic sites (Integrins); invasion and migration; Induction of angiogenesis: Differentiation into endothelial cells; Activation of pre-existing endothelial cells; Notch signaling pathway? Rafii, S. et al NCR 2002

11. 1. Notch signaling regulates BM- progenitor endothelial differentation? Markers: CD133+CD34+KDR+ Lin- Sca-1+ Flk-1+ Igreja, C. et al Exp. Hematol. 2006Caiado, F. et al Plos One 2008

12. Notch signaling inhibition impairs adhesion and integrin expression Caiado, F. et al Plos One 2008

13. 2. BM-progenitor modulate endothelial activation via Notch signaling? Subcutaneous injection of human or mouse tumors Transplant of BM- progenitors control or with reduced Dll4 Sub – lethal irrad. NOD-SCID female Tumor growth; Tumor apoptosis; Vessel density; Vessel stability;

14. BM progenitors with reduced Dll4 decrease tumor proliferation and increase apoptosis C WT-EPC Dll4-EPC 0 2 4 6 8 10 12 014 1818 22 26 Days Tumor Volume (mm3) 0 10 20 30 40 50 60 ControlwtEPCDll4+/-EPC Apoptosis index * ** Real, C Submited 2008

15. Vessel Density : Pericyte coverage : Hypoxic index : BM progenitors with reduced Dll4 induce increased but non-functional vascularization Real, C Submited 2009

16. Notch signaling modulates BM-progenitor function during tumor angiogenesis EPC Activated endothelial cell Endothelial cell Apoptotic endothelial cell Pericyte Apoptotic pericyte Notch signaling regulates BM-progenitor cell endothelial differentiation; Dll4 expressed on BM-progenitor cells regulates endothelial stabilization during tumor angiogenesis;

17. (2008/2009) Angiogenesis Lab members (Leonor Remédio Missing); Dr.Antonio Duarte (group members), Dr.Yadgita Hideo (group members); FCT, GlaxoSmithKline, Fundação Calouste Gulbenkian; Sara Cheila A. Gomes A. Cachaco S Dias Jacinta Cristiana A. Costa Cristina Carla Tânia Francisco Acknowledgments: