Perturbation of Signal Transduction

1. - - 1 2 1 3 2 3 Perturbation of Signal Transduction Pathways for Growth Factors and Growth Inhibitors in Malignant Transformation

2. Ligandisoform Extra-cellular TM Intra-cellular Receptortype Different isoforms of PDGF form three types of receptor dimers PDGF-CC PDGF-AA PDGF-AB PDGF-BB PDGF-DD a a a b b b

3. In vivo function of PDGF Embryonal development • kidneys (mesangial cells) • blood vessels (smooth muscle cells, pericytes) • lungs (alveolar smooth muscle cells) • CNS (oligodendrocytes) Regulation of interstitial fluid pressure Stimulation of wound healing

4. Ligand-induced activation of PDGFRb causes a conformation switch in the receptor C-terminus

5. PDGFRb autoinhibition by a C-terminal motif PDGFRb 10000 No PDGF 7500 Kinase actvity 5000 Wt ct29 2500 ct46 ct75 0 0 10 20 30 40 50 60 70 Time (min) + PDGF 10000 - Y 7500 ct75- Kinase actvity ct46- 5000 2500 ct29- 0 0 10 20 30 40 50 60 70 Time (min)

6. ct46 - D850N PDGFRb activation by mutating the activation loop and the C-terminus Wt ct46 Control PDGF 80 70 60 50 Number of Foci 40 30 20 10 0 WT ct46 D850N vector ct46-D850N M-Ras Q71K

7. S S S S P P P P mitogenicity Grb2/Sos1 Ras Erk MAPK P P P P P P P P Binding of SH2 domain molecules to autophosphorylated tyrosines in the PDGF b-receptor 562 579 581 589 mitogenicity Src Shc mitogenicity Grb2/Sos1 Ras Erk MAPK P 716 740 751 763 771 775 778 P PI3’-kinase Rho, Rac, JNK MAPK actin reorganization and chemotaxis Nck P P RasGAP Akt survival P STAT P P P P 857 mitogenicity SHP-2 Grb2/Sos1 Ras Erk MAPK 966 970 1009 1021 P P mitogenicity PLC-g P

8. 1 2 3 200 PDGFRb PDGFRa RasGAP / FAK 116 Alix 97 Hsp84 Hsp A5 SHP2 66 Stam1 50 Erk1 37 Grb2 25 Identification of tyrosine phosphorylated proteins after PDGF stimulation • Serum-starved cells • PDGFRa expressing cells • stimulated with PDGF-BB • PDGFRb expressing cells • stimulated with PDGF-BB

9. Alix overexpression decreases the rate of PDGFRb removal from the cell surface PAE/PDGFRb-Alix 100 PAE/PGFRb Cell surface PDGFRb (% of initial) PAE/PDGFRb-Alix Alix 50 1.0 8.2 PAE/PDGFRb b-actin 0 0 10 30 60 PDGF-BB stimulation (min)

10. Monoubiquitylation K Ub Ub Ub Multimono- ubiquitylation K K K Ub Ub Ub Ub Ub Ub Ub Ubiquitylation of proteins Ub Ub Polyubiquitylation (via K48 and K63) K K

11. Alix overexpression reduces PDGFRb ubiquitination PAE/PDGFRb-Alix PAE/PDGFRb PDGF-BB (min) 0 30 60 120 0 30 60 120 Ip: PDGFRb Ib: HA-Ub Ub-PDGFRb Ip: PDGFRb Ib: PDGFRb PDGFRb 6 4 Ubiquitination (a.u.) PAE/PDGFRb 2 PAE/PDGFRb-Alix 0 0 60 120 Time (min)

12. P P Alix Alix Alix Alix Alix may stabilize PDGFRb by inducing Cbl degradation Ub Cbl

13. Downregulation of receptor tyrosine kinases

14. Ligand-activated PDGF b-receptor is hyperphosphorylated in TC-PTP knockout cells TC-PTP ko wt PDGF-DD - + - + IB: P-Tyr relative receptor phosphorylation IB: PDGFRb

15. Decreased rate of PDGF b-receptor degradation in TC-PTP ko MEFs

16. Direct detection of recycled PDGF b-receptors in TC-PTP ko MEFs

17. The PDGF b-receptor co-localizes with Rab4a after 10 min stimulation in TC-PTP ko MEFs PDGF b-receptor Rab4a-EGFP

18. No colocalization between Rab11EGFP and PDGFRb in TC-PTP ko MEFs after 20 min of stimulation

19. Trafficking of PDGF receptors X Y X P ESCRT? P P P PDGFRa PDGFRb P P P TC-PTP PTP? P TC-PTP? P degradation

20. P P P Structure of the hyaluronan receptor CD44 c Hyaluronan-binding domain c Link domain c c c c Basic motif Extracellular Variant exon (v1-v10) Transmembrane N-glycosylation Intracellular O-glycosylation ERM Glycosaminoglycan chain Cysteine residues c Actin Phosphorylation site

21. Co-immunoprecipitation of PDGF ß-receptor and CD44 rabbit IgG rabbit IgG rabbit IgG IP: PDGFRb PDGFRb PDGFRb PDGF-BB: - - + - - + - - + PDGFRb CD44 IB: CD44 PDGFRb PY99 rat IgG rat IgG rat IgG IP: CD-44 CD-44 CD-44 PDGF-BB: - - + - - + - - + PDGFRb CD44 IB: PDGFRb CD-44 PY99

22. Co-exposure of skin fibroblasts to PDGF-BB and hyaluronan inhibits PDGF ß-receptor activation Hyaluronan (µg/ml)0 10 25 50 100 0 10 25 50 100 PDGF-BB- - - - - + + + + + PY99 PDGFRß Monoclonal Hermes1, that block hyaluronan binding to CD44, restores PDGF ß-receptor activation Hermes 1 - - + Hyaluronan - + + PDGF-BB + + + PY99 PDGFRß

23. PTPases account for the hyaluronan-induced PDGF ß-receptor dephosphorylation Pervanadate Hyaluronan PDGF-BB PY99 PDGFRb Quantification 1 1 10 20 100220 55 225

24. Cross-talk between CD44 and the PDGF ß-receptor Hyaluronan PDGF-BB PDGF-BB CD44 CD44 -receptor -receptor PTP active P P P PTP inactive P P P P P Modulated signaling Signaling

25. Fibrotic conditions • lung fibrosis • glomerulonephritis • liver cirrhosis • myelofibrosis Atherosclerosis Malignancies • autocrine stimulation (glioblastoma, sarcoma) • paracrine stimulation (cancers) PDGF in disease

26. Dermatofibro- sarcoma protuberans Chronic mono- myclocytic leukemia Hyperoesinophilic syndrome Gastrointestinal stromal tumor Glioblastoma

27. PDGF antagonists PDGF aptamer STI571 (Glivec, imatinib) • DNA molecule whichbinds PDGF B-chainwith high affinity - LMW inhibitor of PDGFreceptors, c-Kit, Abl,and Arg - not absolutely specific - specific - expensive, cumbersometo administer - inexpensive, easy toadminister

28. Difference in STI571(Glivec/imatinib) sensitivity between different glioblastoma cultures

29. Clustering of mRNA profiles of glioblastoma cell cultures

30. 1 3 2 Effects of PDGF in tumors blood vessel tumor cells PDGF lymph vessel stromal fibroblast

34. Effect of anti-PDGFR and anti-VEGFR treatment on B16 melanoma tumor growth

35. PDGF and the interstitial fluid pressure (IFP) of tumors • PDGF b-receptor activation increases IFP in normal connective tissue • Many solid tumors display increased IFP • Increased tumor IFP prevents drug uptake into tumors by decreasing transcapillary transport • Many solid tumors express PDGF receptors on pericytes and stroma cells

36. Binding sites for signal transduction molecules on the PDGFR-b S r c m i t o g e n i c i t y m i t o g e n i c i t y S h c G r b 2 / S o s 1 R a s M A P k i n a s e m i t o g e n i c i t y G r b 2 / S o s 1 R a s M A P k i n a s e - P I 3 ' k i n a s e a c t i n r e o r g a n i z a t i o n / c h e m o t a x i s R h o , R a c / m i g r a t i o n A k t s u r v i v a l m i t o g e n i c i t y S H P - 2 G r b 2 / S o s 1 R a s M A P k i n a s e a c t i n r e o r g a n i z a t i o n / m i t o g e n i c i t y P L C - g

37. Regulation of Interstitial fluid homeostasis by the PDGFR-b via PI3'kinase injection of C48/80 subdermal injection 1 . 5 interstitial fluid pressure of PDGF-BB pif [mmHg] 1 0 . 5 time 0 [minutes] 10 40 70 20 30 50 60 - 0 . 5 - 1 mice with wild type PDGFR-b - 1 . 5 - 2 - 2 . 5 - 3 mice with PI3´K binding site mutant PDGFR-b - 3 . 5 -4

38. Forces that regulate transcapillary transport in tissues a Normal tissue b Tumour tissue Blood vessel COPIF 8 mm Hg PIF -1 to -3 mm Hg COPIF 20 mm Hg PIF 10 -30 mm Hg Net outward filtration pressure 1-3 mm Hg Net inward or outward pressure (-18-2 mm Hg)

39. a Normal tissue a Normal tissue b Tumour tissue b Tumour tissue Structural differences between normal and tumour tissues that affect interstitial fluid pressure

43. PDGF and tumor IFP • Tumor-derived PDGF contributes to increased tumor IFP Treatment with PDGF antagonist • - decreases tumor IFP - increases drug uptake in tumors - synergizes with chemotherapy of tumors

44. LICR, Uppsala Uppsala University Linköping Univ Åke Wasteson Karolinska Institute Monica Nister Arne Östman Christer Betsholtz Lund Univ. in Malmö Lars Rönnstrand Gothenburg Univ. Keiko Funa LICR, Stockholm Ulf Eriksson Acknowledgements Johan Lennartsson Piotr Wardega Lotti Rorsman Alexandra Jurek Carina Hellberg Aga Koslowska-Wardega Susann Karlsson Christian Schmees Evi Heldin Lingli Li Rainer Heuchel Monica Krampert Shioto Suzuki Aive Åhgren Bengt WestermarkLena Claesson-WelshKristofer Rubin Ken Amagasaki Gudrun Bäckström Federica Chiara Jean-Baptiste Demoulin Simon Ekman Masao Furuhashi Yuko Hasumi Daniel Hägerstrand Anders Kallin Kaska Kowanetz Ronggui Li Camilla Looman Kristian Pietras Akira Shimizu Tobias Sjöblom