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”Cell Adhesion in Tumor Growth, Progression and Angiogenesis"
Richard Hynes HHMI/MIT MGH Tumor Microcirculation Course Cambridge, MA June 4, 2003
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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
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Angiogenesis and Lymphangiogenesis
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
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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
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Cell-Cell and Cell-Matrix Adhesion
BASEMENT MEMBRANE (MATRIX) CELL-CELL ADHESION e.g., CADHERINS CELL-MATRIX ADHESION e.g., INTEGRINS
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Cell-cell adhesion receptors
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Cell-matrix adhesion receptors
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Connections between extracellular matrix (ECM) and the actin cytoskeleton
PLAN VIEW ACTIN ECM SIDE VIEW POINTS of ATTACHMENT
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The Molecular Linkage Between Actin and ECM via Integrins
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Signals from Integrins Controlling Cell Behavior
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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?
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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”
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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”
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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
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Signals from Integrins Replaced by Oncogenes
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Angiogenesis Necessary for growth and survival of both
primary and metastatic tumors
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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 & avintegrins are proangiogenic and potential targets for antiangiogenesis therapy
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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
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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 avand/or av ( avand/or av are NOT ESSENTIAL for normal vascular development
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What about tumor angiogenesis?
Transplantable tumors Human: LS180: colon carcinoma A375SM: melanoma Mouse: CMT19T: lung carcinoma B16FO: melanoma Endogenous tumors RIPTAg MMTV-neu
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Tumors grown in b3-null or b3/b5-null mice are BIGGER than controls
B16F0 B16F0 WT b3-/- WT 3 b-/- CMT19T CMT19T
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Tumors grown in b3-null or b3/b5-null mice are BIGGER than controls
B16F0 CMT19T A375SM p< 0.02 p< 0.01 p< 0.02
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Vessels in Tumors (A375M) Rag-null Rag-null/b3-null PECAM-1 NG-2
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Tumors grown in b3-null or b3/b5-null mice have MORE VESSELS than controls
B16F0 tumor Normal skin
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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?
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a51 Integrin and Fibronectin in Angiogenesis
both are upregulated on angiogenic vessels mice lacking a51 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 a51 integrin are proangiogenic They appear good targets for antiangiogenesis
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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
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Possible Negative Regulation by av Integrins
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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 !
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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
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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
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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
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Common to development and tumor progression
EPITHELIAL- MESENCHYMAL TRANSITION CADHERINS HGF/SF Met VIMENTIN KERATINS FIBRONECTIN Common to development and tumor progression
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RELEASE of b-CATENIN from CADHERINS ENHANCES TRANSCRIPTION
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How do Circulating Tumor Cells Arrest?
Mechanical trapping in small vessels? Emboli with host cells and platelets? Specific arrest via cell adhesion?
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Could tumor cells use the same mechanisms?
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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
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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?
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IIbb Fibrinogen IIbb P P PLATELET ACTIVATION Collagen
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 P PSGL-1 GPIb/V/IX
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TRAPPING of TUMOR CELLS ??
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
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SELECTIN-DEFICIENT MICE
Chr 1 All three genes ablated in all combinations P L E Stephen Robinson All strains viable and fertile
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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
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SELECTIN DEPENDENCE of METASTASIS to LUNGS
LS180 COLON CARCINOMA CELLS - Rag2-/- BACKGROUND Alu PCR WT
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SELECTIN DEPENDENCE of METASTASIS to LUNGS
MC38 ADENOCARCINOMA CELLS -C57BL6 BACKGROUND
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SELECTIN DEPENDENCE of METASTASIS to LUNGS
MC38 ADENOCARCINOMA CELLS - C57BL6 BACKGROUND (GFP)
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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
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SELECTINS on VASCULAR CELLS
Activation (Exocytosis) P P P P P Platelets P P P P P P P Leukocytes L L 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 P P P P P P P P P P P P P P P P P Activation (Exocytosis) Endothelial Cells E E E E E E E E E E E E E Activation (Biosynthesis)
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SELECTINS, LIGANDS, PLATELETS, LEUKOCYTES and METASTASIS
S-Lex S-Lex L S-Lex S-Lex P S-Lex S-Lex S-Lex S-Lex L S-Lex S-Lex S-Lex S-Lex L L P S-Lex S-Lex S-Lex S-Lex P S-Lex P Activation (Exocytosis) P P P P P P P P P P P P P P P P P P P P
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BINDING of PLATELETS to METASTATIC CELLS
Tumor cells Platelets Lubor Borsig
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HOST CELL ENHANCEMENT of METASTASIS
Likely contributors include platelets and leukocytes binding to the tumor cells Suggests that reagents blocking selectin interactions might be useful in inhibiting metastatic spread Need to find out which are the key host cells e.g, bone marrow transplantations
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SUBCUTANEOUS INJECTION of GROWTH of PRIMARY TUMOR
TUMOR CELLS - SCORE GROWTH of PRIMARY TUMOR Mice lacking specific selectins Rag2 background to investigate human tumors (eg.LS180 adenocarcinoma)
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Subcutaneous injection of LS180 cells into selectin-deficient mice
Tumor weight 33 days WT WT WT -/- -/- -/- Daniela Taverna P p< 0.029 E p< 0.011 ELP p<
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DEPENDENCE on PRESENCE of L- SELECTIN
LS180 cells Rag-2-null background 30 days Lubor Borsig
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SELECTINS and GROWTH of
PRIMARY TUMORS Deficiencies in P, L and E-selectins all enhance tumor growth and the effects are additive True for several different tumor cell lines Suggests some anti-tumor role for leukocytes Rag-2 -/- mice lack B, T and NK-T cells Macrophages, NK cells, platelets, endothelium ???
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BONE MARROW TRANSPLANTATION FOLLOWED by TEST for TUMOR GROWTH
1. Irradiate 2. Reconstitute with Bone marrow WT or Selectin-deficient Rag-2-null mice WT or Selectin-deficient 3. After recovery Inject with tumor cells and assay Tumor growth
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Enhanced tumor growth in ELP-null mice is greatly REDUCED
by irradiation and reconstitution with Rag2-/- bone marrow Rag2BM Daniela Taverna n=3 n=5 n=7 n=5
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BONE MARROW TRANSPLANTS
CONCLUSIONS from BONE MARROW TRANSPLANTS Mice with selectin-deficient bone marrows consistently yield larger tumors Some selectin-dependent BM-derived cells suppress tumor growth Macrophages and NK cells express L-selectin and PSGL-1 Endothelium expresses P- and E-selectins Platelets express P-selectin and PSGL-1 - platelets could also recruit other cell types
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How do metastatic cells arise?
Are they all the same? Is there specificity in their arrest? Or is there specificity in their ability to grow/survive in distant sites?
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LEVEL of RhoC CONTROLS METASTASIS
A375P A375M A375P + RhoC A375M +DNRho
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Van’t Veer et al, Nature 415:530-536 (2002)
Primary breast carcinomas Can identify an expression profile that correlates with incidence of metastases Suggests bulk primary tumor already has properties that predispose to metastasis That is, not (only) rare variant metastatic cells
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Ramaswamy et al, Nature Genetics 33: 49-54 (2003)
Miscellaneous collection of 12 metastases and 64 primary tumors of same histological types - all adenocarcinomas Can identify an expression profile of 128 genes that distinguishes primaries from metastases Some primaries show the “metastasis pattern” Analyzed available data sets and found that the 128 gene set could split primaries into two sets, one of which showed the “metastasis pattern” and had poor prognosis - same result with a 17 gene set Suggests bulk primary tumors already have properties that predispose to metastasis That is, not (only) rare variant metastatic cells
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128 gene signature 17 gene signature Clustered by all genes
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17 gene signature
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Kang/Massague et al Cancer Cell (in press).
Breast cancer cell line MDA-MB-231 Select variants highly metastatic to bone They “breed true” They have a characteristic expression profile Transfection of 2 or 3 of the overexpressed genes -> increased metastasis Random isolation and screening of clones from parent line identifies clones with the “metastatic signature” These unselected clones ARE metastatic Therefore there ARE preexisting variant cells in the parent population The “metastatic signature” is overlaid on the “poor prognosis signature” of van’t Veer
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Contrasting Models for Metastatic Progression
Good prognosis Poor prognosis Metastasis a. Metastatic variants b. c. Hynes, Cell, in press 2003
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A More Elaborate Model for Metastatic Progression
Good prognosis Stromal response Metastatic variants Poor prognosis Metastasis Hynes, Cell, in press 2003
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