1. Department of Biotechnology Indian Institute of Technology Guwahati Molecular Signaling Network of Cripto-1 Biplab Bose

2. Complexity of Signaling Networks 1.Large number: molecules and their interactions 2.Non-linear architecture 3.Integration of multiple paths 4.Extensive variations: Cell specific, time specific 5.Adaptive in the face of noise

3. Human Oncofetal Protein Cripto-1 Intracellular Signaling Pathways: Nodal/ALK4/Smad-2 Glypican-1/c-Src/MAPK Glypican-1/c-Src/AKT During Development: Patterning of the Anterior/Posterior axis Specification of mesoderm and endoderm during gastrulation In Oncogenesis : Triggers proliferation Induction of cellular migration and invasion promotion of angiogenesis Stimulates EMT In Adult : Mammary gland development

4. Mitogenic Pathways of CR-1 Soluble CR-1 and membrane bound CR-1, both are functional Can signal through ErbB4 too. Can block growth inhibitory signal of TGF- 

5. ** Cell Growth Differ. 1997, 8(12):1257-66. HC-11 cells ** HUVEC cells * *J Natl Cancer Inst. 2005;97:132– 41 % Proliferation CR-1 (ng/ml) Origin of Cell-specific effect Control elements: feedback, involvement of multiple receptors Open Questions in CR-1 Signaling

6. Cripto-1 Cells with high Glypican-1 Cells with lower Glypican-1 Cell Proliferation Activation of mitogenic patwhays The Strategy to Look For Contextual Effect

7. Selecting Suitable Cellular System Cripto-1 + Glypican-1 ERK1/2 and Akt Glypican-1 has very low expression in HeLa cells in comparison to U-87 MG cells Real Time PCR was done using cyber green as the reporter dye GAPDH, β-actin and PPIA used as endogeneous control. U-87 MG: Human Glioblastoma; HeLa: Cervical Adenocarcenoma +p Proliferation

8. Expression of recombinant human Cripto-1 in E. coli C-terminal truncated CR-1 cloned in pGEX-4T2 GST pGEX-4T-2 Ptac Amp r pBR322 ori 1 169 BamHI XhoI cDNA clone of CR-1 PCR CR-1-ΔC Cloning of CR-1-ΔC SDS - PAGE Protein expressed in E. coli is purified 12% SDS-PAGE and stained by silver staining M CR1-GST 97kDa 43kDa 29 kDa 66kDa 20 kDa

9. CR-1 Induces Proliferation of U-87 MG Cell line: U-87 MG 48 hr treatment on 10 4 cells/ well. MTT assay Biphasic: Mitogenic at low concentration; inhibits at higher

10. Cell line: HeLa 48 hr treatment on 10 4 cells/ well. CR-1 Reduces Proliferation of HeLa Cell MTT assay Serum free With serum

11. Trypan Blue Exclusion Test Cell line: HeLa 72 hr treatment on 10 3 cells/ well. CR1-GST and GST: 200 ng/ml One way ANOVA with pairwise multiple comparison: *, significantly different from other treatment groups, p 0.05. CR-1 Reduces Proliferation of HeLa Cell

12. BrdU Assay Cell line: HeLa, 10 4 cells/ well. BrdU pulse: 3 hr before assay CR1-GST : 200 ng/ml CR-1 Reduces Proliferation of HeLa Cell

13. CR-1 Activates Mitogenic Pathways in U-87 MG U-87 MG Treatment : 200 ng/ml of CR1-GST and GST in serum free condition Densitometry of WB Cripto-1 + Glypican-1 ERK1/2 and Akt +p Proliferation

14. CR-1 Fails to Activate Mitogenic Pathways in HeLa Treatment : 200 ng/ml of CR1-GST and GST in serum free condition Cripto-1 + Glypican-1 ERK1/2 and Akt +p Proliferation HeLa

15. Anti-Proliferative Effect in Other Cell Lines Cell lines: HT-29 and HEK 293 Expression of Glypican-1 is lowCR1-GST inhibit proliferation Real-Time PCRMTT assay

16. Understanding The Mechanism of Reduced Cell Viability In contrast to conventional wisdom, treatment with CR-1 reduces number of viable HeLa cells Cell cycle Arrest Slow cell cycle Apoptosis Necrosis

17. CR-1 Does Not Induce Apoptosis Cell line: HeLa 72 hr treatment on 10 5 cells/ well. CR1-GST and GST: 200 ng/ml; Cisplatine (3 μg/ml) as (+)ve control Flowcytometry : AnnexinV vs PI PI Annexin V Untreated Cisplatin treatedCR1-GST treated GST treated

18. Cell line: HeLa 72 hr treatment on 10 5 cells/ well. CR1-GST and GST: 200ng/ml, Cisplatine : 3μg/ml Flowcytometry : AnnexinV vs PI * * * One-way ANOVA, no significant difference among these treatment groups, p > 0.05. * CR-1 Does Not Induce Apoptosis

19. Cell line: HeLa, 10 4 cells/ well. CR1-GST and GST: 200 ng/ml Triton X-100 (0.1%) as a +Ve control CR-1 Does Not Induce Necrosis LDH Assay

20. CR-1 Does Not Induce Cell Death in HeLa HeLa cell treated with CR1-GST for 72 hr No morphological change

21. CR-1 Dose Not Arrest Cell Cycle HeLa cells were treated with CR1-GST or GST 200ng/ml 72 hr treatment Stained with propidium iodide (PI) Stained cells were analyzed by flow cytometry

22. CR-1 merely changes the cell cycle phases Each bar represent mean of four independent experiments. * and ** represent significant differences with untreated and GST treated cells, One-way ANOVA, p < 0.05.

23. CFSE (FL1-H) Count Measuring Cell Proliferation by Dye (CFSE) Dilution CFDA –SE: carboxyfluorescein diacetate succinimidyl ester CFSE: carboxyfluorescein diacetate succinimidyl CFDA-SE : Membrane permeable dye CFSE: Not membrane permeable, it can retain inside cell upto 10 successive generation Day 1Day 2Day 3

24. After CFSE (5 μM ) staining HeLa cells were treated with CR1-GST (200ng/ml), GST (200ng/ml) for 72 hr Flow cytometry was done at respective time points CR-1 Increases Doubling Time of HeLa Cells Mean doubling time, = T/log 2 ( / ); here, = geometric mean fluorescence intensity at zero hour and = geometric mean fluorescence intensity at T hour. Calculation of doubling time :

25. 27 hrs37 hrs HeLa Cell CR-1 Treated HeLa Cell Each bar represents mean of three independent experiments. *, significantly different from other treatment groups, One-way ANOVA, p < 0.05. CR-1 Increases Doubling Time of HeLa Cells

26. Cell cycle figure credit: Nature Reviews Neuroscience, 2007, 8, 438-450 Effect of CR-1 on Cyclins * Statistically significant p<0.05

27. Cell cycle figure credit: Nature Reviews Neuroscience, 2007, 8, 438-450 * Statistically significant p<0.05 Effect of CR-1 on Cdk Inhibitors

28. Anti –proliferative Pathway of CR-1

30. Status of PTEN Based Pathway in U-87 MG cells U-87 MG is a PTEN-null cell line

31. Anti-proliferative Pathway of CR-1 is Translation Dependent Cell line: HeLa 48 hr treatment: CR1-GST ( 200ng/ml) + Cyclohexamide MTT Assay

32. Two Opposing Pathways Determine The Fate

33. ≡ Incoherent Feed-Forward Deconstructing CR-1 Pathway

34. Non-adaptive System Adaptive System Incoherent Feedforward: Adaptive, Pulse generating, Ultra-sensitive switch

35. Funded by: MHRD, DBT & DST, Govt. of India