Special approaches of tumor biology and chemotaxis Orsolya Láng 2012.

TUMOR CELLS AND MIGRATION METASTASISPRIMARY TUMOR Angiogenezis Adhesion

CELL and CELL CYCLE

Growth factors Adhesion molecules Chemokines Regulatory proteins Apoptosis

TUMOR CELL

CELL KINETICS

Doubling time of the tumor volume (Td) Time of the Cell cycle (Tc): Tc= Ts / Li Ts: S phase Li: labeling index (proportoin of cells in S phase) Growth fraction(GF): GF=P / (P+Q’) P: number of the mitotic cells Q: number of the cells in interphase Rate of the cell loss (  ):  = 1-Tpd / Td Tpd= *Ts/Li Tpd: Potential tumor volume doubling time Td: tumor volume doubling time Lymphoma 48 h Lung cancer 108 h Usually h Lymphoma 4 weeks Colon adenoma 90 weeks Usually days

Volume of the tumor tissue ~10 division =*1000 cell number increase (2 10 =1024) ~20 division= 10 6 cells = 1 mg= 1 mm 3 ~30 division= 10 9 cells = 1 g= 1 cm 3 ~40 division= cells = 1 kg 1 tumor cell ~30-33,25 division =1-10 cm 3 Time of the clinical symptomes / diagnosis ~27 division = 0,1cm 3 Earliest time of diagnosis ~40 division = cell Fatal

BIOLOGY OF THE TUMORPROGRESSION

Exogen and endogen factors Genom instability Activation of the oncogene Inactivation of tumorsuppressors Epithelial cell Hiperplastic adenoma Displatic Carcinoma in situ Invasive and metastasis carcinoma Local and systemic factors inhibitionacceleration Growth rate Ectopic survival capacity Invazivity De-Differentation Tumorigenesis

Important steps of tumor progression Transformation of the microenvironment: stromal cells, ECM components, proteolytic degradation Induction of the angiogenesis Escaping from immune-mediated rejection Formation of metastasis

MICROENVIRONMENT – STROMAL CELLS Cell types: fibroblasts, myofibroblasts, endothelial cells, lymphocytes, macrophages Function: host defence ! MALT - B cell helps to maintain lymphomas ! Growth factors are released by the stromal cells (VEGF- angiogenesis)

ANGIOGENESIS Hypoxia  formation of new vessels, proliferation of the endothelial cells Types: vessels arteriovenous shunts „dead end” /lack of smooth muscle, weak vessel wall, irregular shape(insuficient endothelial cell and basement membrane layers)/ sinuses /wall is formed by tumor cells/ Venous circulation VEGF  induces angiogensis increases permeability Lack of lymphatic vessels  OEDEMA, decresed blood flow

Strategies that tumors use to escape from immune-mediated rejection are: To decrease the antigen expression To inhibit the immune-reactive cells: degrade the chemoattractans decrease their cell adhesion inhibite their phagocytotic activity

Angiogenesis Local invasion ECM Adhesion Proteolysis Migration Intravasation Extravasationcirculation Metastasis Tumor cell Primary tumor Adhesion Proteolysis Migration Angiogenesis VEGF Angiogenin FGF spreading METASTATIC CASCADE

INVASION In situ carcinoma DECREASED CELL ADHESION, INCREASED MOTILITY ECM proteolysis

Angiogenesis Local invasion ECM Adhesion Proteolysis Migration Intravasation Extravasationcirculation Metastasis Tumor cell Primary tumor Adhesion Proteolysis Migration Angiogenesis VEGF Angiogenin FGF spreading METASTATIC CASCADE

CELL ADHESION Significant change in cell-cell and cell-ECM interactions Molecules: selectins integrins immunoglobulin superfamily cadherins catenins

SELECTINS Cell-cel junctions Types: E- endothelial cells P- trombocytes L- leukocytes Extracellular C-lectin domain Ca 2+ dependent anchorage It binds Sialyl-Le x carbohydrates „ROLLING” ! Tumor cells express increased amount of sialil-Le x or -Le a

INTEGRINS Transmembrane receptors Form cell-ECM interaction 8 , 14  subunites ~20 heterodimer Ca 2+, Mg 2+ dependent anchorage „RGD” sequence is the specific substrate Signalling: outside-in – signalling inside-out – adhesion Increased expression of integrins promotes angiogenesis and helps to bind MMPs at the cell surface EXTRAVASATION, ATTACHMENT   D G R

Integrin or celladhesion regulated signalling pathways cellproliferation PTEN RAC PI(3)K CDC42 integrin ECM ILK  -catenin Ciklin D1 BAD PKB/AKT FAK RAS RAF MEK MAPK GSK3 motilitygene expressioncellcycleapoptosis SHC GRB2/SOS

Integrin or celladhesion regulated signalling pathways integrin ECM ILK  -katenin Ciklin D1 BAD PKB/AKT FAK cellproliferation RAS RAF MEK MAPK GSK3 motilitygene expressioncellcycleapoptsis SHC GRB2/SOS PTEN RAC PI(3)K CDC42

Molecular partners of the integrins Cytoskeletal components: actinin, talin,F- actin, filamin Adaptors: rack 1, ICAP-1 Calcium binding proteins: CIB, calreticulin Protein kinases: pp125FAK, p59 ILK Membrane proteins: CD9, CD16,CD47… caveolin, urokinase-plazminogen-activator receptor Ligands in ECM: collagen, laminin, fibronectin, fibrinogen, von Willebrand factor, osteopontin, elastin

IMMUNGLOBULIN SUPERFAMILY has 5 Ig-like domains at the extracellular region forms cell-cell junction interacts with integrins VCAM -  4  1, PECAM -  v  3 takes essential part in extravasation ! ! Over expression of ICAM-1, MUC18  increased inavsion ! ! Down-regulation of VCAM-1  increased metastatic potential (faster detachment)

CADHERIN Is a transmembrane glycoprotein Forms homophyl cell-cell junctions Ca 2+ dependent anchorage Classical types: E- epithelial P- placenta N- neural, Intracellular part interacts with catenins to connect aktin filaments ! Increased expression  invasion

CATENIN Is an intracellular molecule Fixes cadherins to F-actin ! Catenin expression is often decreased in carcinomas !  -catenin binds to the az APC gén termékéhez

Increases N-Cadherin B-Catenin SrC Ras Ihibits E-Cadherin aE-Catenin cMET FGFR PTEN Adhesion molecules Signal pathways Factors influencing the metastatic potential of the melanoma cells

Angiogenesis Local invasion ECM Adhesion Proteolysis Migration Intravasation ExtravasationCirculation Metastasis Tumor cell Primary tumor Adhesion Proteolysis Migration Angiogenesis VEGF Angiogenin FGF spreading METASTATIC CASCADE Integrins cadherins Selectins CAM

PROTEOLYSIS Components of the basement membrane(BM) and ECM: IV collagen, laminin, proteoglycanes Tumorcells (stromal cells) secrete proteases Cathepsin Matrix metalloproteinase (MMP) Plazmin, tPA,Urokinase (plasminogen activator inhibitor 1&2) TIMP INVASION Tissue inhibitor of metalloproteinases

MATRIX METTALLOPROTEINASES (MMP) Zn 2+ dependent endopeptidase (MMP28) ECM degradation – tissue remodelling Interstitial collagenase (MMP2) Stromalysin Gelatinase (MMP9) Membrane type MMP Produces biologically active molecules

MOLECULAR STRUCTURE OF THE MATRIX METTALLOPROTEINASES SUBSTRATE OF TIMP

MMP/TIMP EXPRESSION IN BREAST CANCER

MMP – TUMORPROGRESSION ?!?

Angiogenesis Local invasion ECM Adhesion Proteolysis Migration Intravasation ExtravasationCirculation Metastasis Tumor cell Primary tumor Adhesion Proteolysis Migration Angiogenesis VEGF Angiogenin FGF spreading METASTATIC CASCADE Integrins cadherins Selectins CAM MMP/TIMP Cathepsin Plasminogen

MIGRATORY MECHANISMS IN TUMOR Small-cell lung cancer

FORMS OF MIGRATORY ADAPTATION

2D –3D MIGRATIONS

STEPS OF 3D MIGRATION 1. Pseudopod protrusion 2. Formation of focal contact 3. Focal ECM proteolysis 4. Actomyosin contraction 5. Detachment

Cell-cell interactions visualized in tumorigenesis

Angiogenesis Local invasion ECM Adhesion Proteolysis Migration Intravasation ExtravasationCirculation Metastasis Tumor cell Primary tumor Adhesion Proteolysis Migration Angiogenesis VEGF Angiogenin FGF spreading METASTATIC CASCADE Integrins cadherins Selectins CAM MMP/TIMP Cathepsin Plasminogen AMF/gp78 Autotaxin HGF/c-MET

!! Tumor markers e.g. cytokeratin, mucin HEMATOGENIC DISSEMINATION

EXTRAVASATION ? Attachment Migration

LOCALISATION OF THE METASTASIS

CHEMOKINES – TISSUE SPECIFIC LOCALISATION

adhesion motility ?