Richard Hynes HHMI/MIT MGH Tumor Microcirculation Course Cambridge, MA June 4, 2003

1. ”Cell Adhesion in Tumor Growth, Progression and Angiogenesis" Richard Hynes HHMI/MIT MGH Tumor Microcirculation Course Cambridge, MA June 4, 2003

3. GROWTH of PRIMARY TUMOR and INITIAL INVASION Loss of cell adhesion Further loss of cell adhesion • Angiogenesis • also Lymphangiogenesis • (not shown) Gain of cell migration

4. Angiogenesis andLymphangiogenesis • Essential for growth of primary tumor • (and later of metastases) • Involves extensive migration and adhesion • of endothelial cells and pericytes • Involves organization of basement membranes

5. Metastatic Spread • Intravasation • Survival in circulation • Arrest at a distant site - selectivity?? • Intravascular Proliferation ? • Extravasation • Survival and proliferation at the new site • Angiogenesis again • All of these involve cell adhesion

6. Cell-Cell and Cell-Matrix Adhesion BASEMENT MEMBRANE (MATRIX) CELL-CELL ADHESION e.g., CADHERINS CELL-MATRIX ADHESION e.g., INTEGRINS

7. Cell-cell adhesion receptors

8. Cell-matrix adhesion receptors

9. Connections between extracellularmatrix (ECM) and the actin cytoskeleton PLAN VIEW ACTIN ECM SIDE VIEW POINTS of ATTACHMENT

10. The Molecular Linkage Between Actin and ECM via Integrins

11. Signals from Integrins Controlling Cell Behavior b

12. Functions of Cell Adhesion Receptors • Mediate adhesion to adjacent cells and to ECM • Control cell shape, polarity and migration • Control cell proliferation, survival, • gene expression and differentiation How do these functions impact tumor progression?

13. MATRIX/INTEGRINS and GROWTH CONTROL • Integrins regulate cyclin D synthesis • Integrins regulate PIP2 synthesis • Both these effects synergize with stimulation by soluble growth factors • In fact, they are necessary for growth factors to promote growth - cells will not grow with growth factors alone - they need matrix attachment through integrins. • This is “anchorage dependence of growth”

14. MATRIX/INTEGRINS and CELL SURVIVAL • Integrins regulate PI3 kinase and Akt, acting through FAK • This pathway suppresses apoptosis • So extracellular matrix, acting via integrins provides local survival signals • i.e., cells must be attached to the correct matrix in order to survive. • This is “anchorage dependence of survival”

15. ANCHORAGE DEPENDENCE • Most normal cells are dependent on anchorage for survival and proliferation • Tumor cells are not, because oncogenes provide the signals normally provided by integrins and other adhesion receptors • So tumor cells are less dependent on being attached in the correct place

16. Signals from Integrins Replaced by Oncogenes b

17. Angiogenesis Necessary for growth and survival of both primary and metastatic tumors

18. av Integrins (av and av) in Angiogenesis • upregulated on (many) angiogenic vessels • Inhibitors - some antibodies (LM609) and RGD-based peptides and peptidomimetics block angiogenesis and induce apoptosis in various model systems • MODEL:- av & avintegrins are proangiogenic and potential targets for antiangiogenesis therapy

19. Predictions from this model • Mice lacking av integrins should show defects in angiogenesis embryonic lethal - but lacks a dozen integrins   All three are viable and fertile either as single KOs or as double KOs av    av and 8 KOs show extensive angiogenesis, although they are not viable • So the simple predictions are not met

20. Conclusions from integrin knockouts • embryos of av-null mice generally show normal vascular development • the selective vascular defects in the brain are of neural/glial origin • the KO mouse has similar defects • in any event, they are not due to absence of avand/or av ( avand/or av are NOT ESSENTIAL for normal vascular development

21. What about tumor angiogenesis? • Transplantable tumors Human: • LS180: colon carcinoma • A375SM: melanoma Mouse: • CMT19T: lung carcinoma • B16FO: melanoma • Endogenous tumors • RIPTAg • MMTV-neu

22. Tumors grown in b3-null or b3/b5-null mice are BIGGER than controls B16F0 B16F0 WT WT b3-/- 3 b-/- CMT19T CMT19T

23. Tumors grown in b3-null or b3/b5-null mice are BIGGER than controls B16F0 CMT19T A375SM p< 0.02 p< 0.02 p< 0.01

24. Vessels in Tumors (A375M) Rag-null Rag-null/b3-null PECAM-1 NG-2

25. Tumors grown in b3-null or b3/b5-null mice have MORE VESSELS than controls Normal skin B16F0 tumor

26. Tumor Growth and Angiogenesis WT b3KO b3/b5DKO B16 melanoma + ++ ++ CMT19T lung carcinoma + ++ ++ LS180 adenocarcinoma + ++ ++ A375M melanoma + ++ ++ C57BL/6 Rag2 So:- tumor growth and angiogenesis are NOT dependent on vb3 or avb5. In fact, these integrins tend to inhibit them. HOW?

27. a51 Integrin and Fibronectin in Angiogenesis • both are upregulated on angiogenic vessels • mice lacking a51 die with vascular defects • mice lacking  die with vascular defects • antibodies to either inhibit angiogenesis • peptides blocking their interaction inhibit angiogenesis • that is - genetics and inhibitor studies conform here • Fibronectin and a51 integrin are proangiogenic • They appear good targets for antiangiogenesis

28. A new way of thinking about av integrins in angiogenesis • The original model of their being proangiogenic does not explain all the data • Perhaps they are actually antiangiogenic or negative regulators some or all the time • The negative regulation model does a better, although not a perfect job of explaining the data

29. Possible Negative Regulation by av Integrins

30. Transdominant Inhibition Based on data showing cross- inhibition by ligation of different integrins on the same cell. Works best when the inhibitory integrin is at a high level Like avb3 and avb5 !

31. Agonists orAntagonists? • That often depends on the assay • The same agent can act as an agonist when presented on a substrate and an antagonist when presented in solution • An agent detected as an antagonist in an adhesion assay can be an agonist with respect to signaling

32. Design of anti-av integrin drugs • It is not enough just to screen for antagonists of adhesion • Figure out the (positive and negative) functions of avb3 and avb • for their ability to stimulate the negative or inhibit the positive pathways - that is, agonists or antagonists

34. Cadherins and Integrins in Tumor Invasion • Cadherins, particularly E-cadherin, are frequently lost from invasive malignant tumors • Integrins are sometimes gained by invasive tumors • This reflects the switch from sessile adherent epithelial cells to migratory, invasive mesenchymal cells • Often called the Epithelial-Mesenchymal Transition or EMT

35. EPITHELIAL- MESENCHYMAL TRANSITION CADHERINS HGF/SF Met VIMENTIN KERATINS FIBRONECTIN Common to development and tumor progression

36. RELEASE of b-CATENIN from CADHERINS ENHANCES TRANSCRIPTION

38. How do Circulating TumorCells Arrest? Mechanical trapping in small vessels? Emboli with host cells and platelets? Specific arrest via cell adhesion?

39. Could tumor cells use the same mechanisms?

40. SELECTINS and METASTASIS • Acquisition by human carcinomas of • carbohydrate ligands (S-Lex and S-Lea) for • selectins is associated with poor prognoses • Selectins are expressed by vascular cells - • platelets, leukocytes, endothelium • Could tumor cells use selectins in their • metastatic spread? S-Lex S-Lex S-Lex S-Lex

41. PLATELETS and METASTASIS • Platelets enhance metastatic spread HOW? • Provision of adhesion molecules • Adherence to tumor cells? • Bridging between tumor cells and endothelium ? • Provision of growth factors/cytokines • Protection against turbulence • Trapping of embolus • Could selectins or integrins play a role?

42. PLATELET ACTIVATION IIbb ADP Thrombin Fibrinogen von Willebrand factor Fibronectin Thrombospondin Vitronectin PSGL-1 P-selectin b a5b1 b1 b1 GPIb/V/IX Collagen von Willebrand factor IIbb a5b1 b PSGL-1 P b1 b1 PSGL-1 P GPIb/V/IX

43. SELECTINS, LIGANDS, PLATELETS and METASTASIS S-Lex S-Lex S-Lex S-Lex PLATELETS S-Lex S-Lex S-Lex S-Lex S-Lex S-Lex S-Lex S-Lex FIBRINOGEN S-Lex S-Lex S-Lex ENHANCED ADHESION and TRAPPING of TUMOR CELLS ?? S-Lex S-Lex

44. SELECTIN-DEFICIENT MICE Chr 1 All three genes ablated in all combinations P L E Stephen Robinson All strains viable and fertile

45. INTRAVENOUS INJECTION of TUMOR CELLS - SCORE LUNG METASTASES • Mice lacking one, two or all three selectins • C57BL6 background to investigate murine tumors (eg.,MC38 colon adenocarcinoma) • Rag2-/- background to investigate human tumors (eg.LS180 adenocarcinoma) • These cells express ligands for all 3 selectins • Daniela Taverna - • and collaboration with Ajit Varki/Lubor Borsig

46. SELECTIN DEPENDENCE of METASTASIS to LUNGS LS180 COLON CARCINOMA CELLS - Rag2-/- BACKGROUND Alu PCR WT

47. SELECTIN DEPENDENCE of METASTASIS to LUNGS MC38 ADENOCARCINOMA CELLS -C57BL6 BACKGROUND

48. SELECTIN DEPENDENCE of METASTASIS to LUNGS MC38 ADENOCARCINOMA CELLS - C57BL6 BACKGROUND (GFP)

49. SELECTINS and EXPERIMENTAL METASTASIS to LUNGS • P and L selectins both enhance metastasis and their effects are additive • E-selectin has rather little effect • True for injected tumor cells of either • human (LS180) or mouse (MC38) origin • Selectin ligands on the tumor cells may be contributing to metastasis

50. Activation (Biosynthesis) E E E E E E E E E E E E E Activation (Exocytosis) P P P P P P P P P P P P P P P P P P P P SELECTINS on VASCULAR CELLS Activation (Exocytosis) P P P P P P Platelets P P P P P P L Leukocytes L L L L L Activation (Shedding) L L L L L L L L L L L L L L L L L L L L L L L L L Endothelial Cells