Special approaches of tumor biology and chemotaxis Orsolya Láng 2014.

TUMOR CELLS AND MIGRATION METASTASISPRIMARY TUMOR Angiogenesis Adhesion

CELL and CELL CYCLE

Growth factors Adhesion molecules Chemokines Cell cyle regulatory proteins Apoptotic molecules Oncogenes

TUMOR CELL

GENERAL FEATURE OF A TUMOR CELL The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors (Hanahan és Weinberg 2000)

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 divisions =*1000 cell number increase (2 10 =1024) ~20 divisions= 10 6 cells = 1 mg= 1 mm 3 ~30 divisions= 10 9 cells = 1 g= 1 cm 3 ~40 divisions= 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 (Visualisation) ~40 division = cell Fatal Human total cell number: 3.72 ×

BIOLOGY OF THE TUMORPROGRESSION

Exogen and endogen factors Genom instability Activation of the oncogenes Inactivation of tumorsuppressors Local and systemic factors inhibitionacceleration Growth rate Ectopic survival capacity Invasivity De- d ifferentation Tumorigenesis Epithelial cell Hiperplastic adenoma Displatic Carcinoma in situ Invasive carcinoma Metastasis

Important steps of tumor progression Transformation of the microenvironment: stromal cells, ECM components, proteolytic degradation Induction of the angiogenesis (w/o max tumor size is 2mm) 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)

Macrophage Cancer cell migration is controlled by paracrine loop THE INVASIVE MICROENVIRONMENT Macrophage Blood vessel

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 angiogenesis increases permeability Lack of lymphatic vessels  OEDEMA, decresed blood flow Inhibition of the VEGF pathway is a potential therapeutical tool

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 ! E-cadherin tumorsuppressor ! Increased expression of N-cadherin  invasion ! N-cadherin cooperates with the FGF receptor  lead to the up- modulation of MMP-9 „Cadherin Switch”

CATENIN Is an intracellular molecule Fixes cadherins to F-actin !  -catenin binds to the APC gene product ! colon and liver cancer increased cytoplasmic and nuclear localised beta-catenin correlates with invasion and poor prognosis. in melanoma increased cytoplasmic and nuclear beta- catenin is currently emerging as a marker for good prognosis

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) Tissue inhibitor of metalloproteinases INVASION

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 Nature Reviews Cancer 2, (March 2002)

MMP/TIMP EXPRESSION IN BREAST CANCER Nature Reviews Cancer 2, (March 2002)

MMP – TUMORPROGRESSION ?!? Nature Reviews Cancer 2, (March 2002)

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

Blood flow patterns can predict the specific regions of metastases in approximately two-thirds of cancers TYPICAL SITE OF METASTASIS

LOCALISATION OF THE METASTASIS

CHEMOKINES – TISSUE SPECIFIC LOCALISATION

adhesion motility ?

EXTRAVASATION ? Attachment Migration