Invasion Tisular2

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  • 2. Tumor Angiogenesis: Novel Targets for Anticancer Therapy Neovascularization is required to supply oxygen and nutrients to growing tumors. In fact, the onset of angiogenesis appears to permit rapid growth of tumor cells capable of such growth. 1 Research in ex vivo tumor systems shows that tumor growth beyond 1–2 mm 3 in size is dependent on angiogenesis. 1 Correlation between increased intratumoral microvessel density (a surrogate marker of angiogenesis) and poorer prognosis and/or metastatic potential has been demonstrated in a variety of cancer types, including breast cancer, 2,3 colon cancer, 4 malignant melanoma, 5 non-small cell lung cancer, 6-8 and prostate cancer. 9
  • 4. Factors Influencing Promotion of Tumor Angiogenesis A variety of normal physiologic and pathologic stimuli have been shown to induce production of angiogenic growth factors. Induction of vascular endothelial growth factor (VEGF, also known as vascular permeability factor) and other growth factors by tumor cells has been shown to occur in response to various stimuli, including Secreted proteins in the organ microenvironment, such as epidermal growth factor (EGF), 10 fibroblast growth factor (FGF), 10 insulin-like growth factor (IGF), 11 interleukin 1  (IL-1  ), 12 interleukin 6 (IL-6), 13 and platelet-derived growth factor (PDGF). 14 Metabolic stress such as hypoxia, 15 low pH, and/or hypoglycemia. 16 Mechanical stress such as pressure generated by proliferating tumor cells. 17 Inflammatory responses, such as cyclooxygenase-2 (COX-2) expression, 18 prostaglandin synthesis, 19 and mast cell activation. 20 Genetic alterations, such as activation of oncogenes such as ras and src 21,22 and inactivation of tumor suppressor genes such as p53 and von Hippel-Lindau (VHL). 23,24
  • 5. Activation of Vascular Endothelial Cells Major growth factors that have angiogenic properties include VEGF, FGF, and PDGF. VEGF has multiple effects on endothelial cells. VEGF directly initiates angiogenesis by stimulating the proliferation, 25 migration, 26 and sprouting of endothelial cells. 27 VEGF indirectly contributes to angiogenesis by inducing vascular hyperpermeability. 28,29 VEGF induces expression of matrix metalloproteinases (MMPs), enzymes that break down extracellular matrix (ECM) proteins and provide endothelial cells space to migrate. 30 VEGF induces expression of cellular adhesion molecules, such as integrins, on endothelial cells, which play an important role in cell-cell interactions and cell migration. 31 VEGF is specific for endothelial cells; its receptors are predominantly present only on endothelial cells. 32
  • 6. Signaling Pathways Activated by VEGF VEGF exerts its biologic effects by binding to its respective transmembrane receptors. Three VEGF receptors have been identified. VEGF receptor 1 (VEGFR-1), also known as Flt-1. 52 VEGF receptor 2 (VEGFR-2), also known as Flk-1/KDR. 32,53 VEGF receptor 3 (VEGFR-3), also known as Flt-4. VEGFR-2 is believed to play a central role in growth, permeability, and survival actions of VEGF and is required for development of mature endothelial cells and tumor angiogenesis. 54 VEGFR-2 is expressed almost exclusively on vascular endothelial cells and their progenitors. 52,53
  • 7. Upstream Regulators of VEGF VEGF gene expression is regulated via a variety of mechanisms, including oxygen tension, tumor suppressor genes, cytokines, and mutations in cellular oncogenes. Hypoxia rapidly and reversibly induces VEGF mRNA expression in normal and malignant cell lines. 15,57 Inactivation of tumor suppressor genes, such as p53 and the VHL gene, has been implicated in VEGF production and angiogenesis. Mutations in the p53 gene induce expression of VEGF mRNA in transfection assays. 23 Replacement of the mutant p53 gene in human colon cancer cell lines with a wild-type gene downregulates VEGF expression. 58 Absence of a wild-type VHL tumor suppressor gene results in increased expression of VEGF in renal carcinoma cells. 24 Conversely, introduction of wild-type VHL in various cell lines suppresses VEGF levels. 24,59
  • 8. Endothelial Cell Proliferation, Migration, and Differentiation Following degradation of the ECM, endothelial cells begin to proliferate in response to tumor-derived growth factors such as VEGF and migrate toward the growth factor stimulus. Cell adhesion molecules involved in cell-cell or cell-matrix interactions, such as the integrins, help mediate endothelial cell migration. Many of the integrins bind to the Arg-Gly-Asp (RGD) tripeptide sequence found on a number of matrix proteins, allowing endothelial cells to interact with a variety of ECM components. 64 The adhesion receptor integrin  v  3 is critical for the differentiation, maturation, and survival of blood vessels. 65 Present on the surface of activated endothelial cells,  v  3 enables the cells to spread, assists them in forming protrusions, and participates in lumen formation. 65 Growth factors such as FGF increase the expression of  v  3 on endothelial cells. 43
  • 9. Stabilization/Maturation of New Blood Vessels Maturation of new blood vessels involves formation of a new basement membrane and stabilization of the new vessels by recruited mural cells, which are vascular smooth muscle cells in large vessels and PCs in microvessels. 66 In vivo experiments suggest that blood vessels that do not recruit appropriate mural cells require VEGF for survival, whereas mural cell–covered vessels may survive in the absence of VEGF. 67 PDGF stimulates the development and migration of PCs. 46,66,67 Angiogenic growth factors such as VEGF 68 and PDGF 69 act as potent attractants for smooth muscle cells. The angiopoietins are a family of molecules that, like VEGF, are specific for endothelial cells. These ligands work in conjunction with VEGF in promoting angiogenesis and vascular remodeling. Angiopoietin-1 binds to the endothelial cell–specific Tie-2 receptor (also a receptor tyrosine kinase) and acts to help maintain and stabilize the mature vessels by promoting interactions between endothelial cells and smooth muscle support cells. 70
  • 10. Endothelial Cell Survival In the tumor microenvironment, proliferating endothelial cells, in the absence of survival factors, will undergo apoptosis. 71,72 A variety of factors contribute to endothelial cell survival, including growth factors such as VEGF, angiopoietin-1, and integrins. Endothelial cell survival in newly formed blood vessels is dependent on VEGF; apoptosis occurs in its absence. 71,73,74 Angiopoietin-1 has been shown to be a strong survival factor for endothelial cells. 72 Binding of integrins, such as  v  3 , to their ECM receptors, such as vitronectin, von Willebrand factor, fibronectin, and fibrinogen, provides a signal that allows continued survival of vascular cells undergoing angiogenesis, leading to differentiation and the formation of mature blood vessels. 75,76
  • 12. Strategies to Inhibit Tumor Angiogenesis Because angiogenesis is a multifactorial and multistep process, there are several potential targets for inhibiting tumor angiogenesis. Direct Inhibition: Agents can be targeted against specific tumor cell- or host-derived angiogenic growth factors involved in blood vessel formation, such as VEGF, FGF, and PDGF. Indirect Inhibition: Another strategy is to target upstream regulators of angiogenic growth factor expression, such as COX-2, tumor suppressor genes, other growth factors and cytokines, and cellular oncogenes. Additional targets include growth factors and molecules that promote endothelial cell survival, such as VEGF, angiopoietin-1, and integrins. The Pharmacia Oncology Angiogenesis Inhibitor Portfolio includes investigational agents that act against several of these angiogenesis targets.
  • 13. Inhibition of VEGF Activity VEGF is an attractive target for antiangiogenic strategies because it plays a pivotal role in the regulation of normal and abnormal angiogenesis. 53 Strategies involve interfering with VEGF binding to its receptor on endothelial cells such as soluble VEGF receptors and anti-VEGF monoclonal antibodies. Other strategies involve blocking VEGF receptor signaling using small molecule tyrosine kinase inhibitors (TKIs). Semaxanib (SU5416) is a synthetic, small molecule TKI that is being developed as an angiogenesis inhibitor. Semaxanib potentially targets the VEGFR-2 (Flk-1/KDR) on endothelial cells. 86 In athymic mice, semaxanib significantly inhibited the subcutaneous growth of 8 of 10 tumor lines tested including epidermal carcinoma (A431; 62% [ P = 0.0005]), fibrosarcoma (3T3Her2; 32% [ P = 0.046] and 488G2M2; 71% [ P = 0.0004]), glioma (C6; 54% [ P = 0.001]), lung carcinoma (Calu-6; 52% [ P = 0.031]), mammary carcinoma (EPH4-VEGF; 44% [ P = 0.00001]), melanoma (A375; 85% [ P = 0.0005]), and prostatic carcinoma (LNCAP; 62% [ P = 0.01). 86
  • 14. Inhibition of Multiple Growth Factor Receptors Growth factors important for tumor angiogenesis, such as VEGF, FGF, and PDGF, are ligands for receptor tyrosine kinases. The signaling pathways activated by these ligands are complex, affecting tumor angiogenesis and tumor growth via both direct and indirect mechanisms. Therefore, strategies that target multiple growth factors may demonstrate potent antiangiogenic effects. SU6668 is a synthetic, small molecule, competitive inhibitor of ATP binding in the intracellular kinase domains that targets VEGFR, PDGFR, and FGFR tyrosine kinases; enzyme kinetic experiments showed that SU6668 competitively inhibited VEGFR-2 (Flk-1) transphosphorylation (K i = 2.1 µM), FGFR-1 transphosphorylation (K i = 2.1 µM), and PDGFR autophosphorylation (K i = 0.008 µM). 90 In cell-based assays testing for tyrosine kinase activity, SU6668 inhibited tyrosine phosphorylation of VEGFR-2 (KDR) in VEGF-stimulated HUVECs in a dose-dependent manner; also, SU6668 (0.03–0.1 µM) inhibited PDGF-stimulated PDGFR tyrosine phosphorylation in NIH-3T3 cells overexpressing PDGFR, and SU6668 (  10 µM) similarly inhibited FGF-induced phosphorylation of FGFR-1 in NIH-3T3 cells.
  • 15. Inhibition of Integrins Integrins play an important role in angiogenesis and tumor cell metastases and provide an attractive therapeutic target. Specific integrins are selectively expressed in tumor-associated blood vessels and are required for angiogenesis. 31,65,93-95 Integrin expression on tumor vasculature correlates with malignant phenotype in various cancers including breast cancer. 96 Also, in some tumor models such as melanoma, the malignant phenotype is correlated with tumor cell integrin expression. 79 Integrin expression correlates with a malignant phenotype in various cancers, including breast cancer and melanoma. Various integrin antagonists that induce endothelial cell and/or tumor cell apoptosis are under development. 75,76,97,98
  • Invasion Tisular2

    2. 2. Cáncer: Características básicas
    3. 3. Características de la célula cancerosa <ul><li>Replicación auto-mantenida </li></ul><ul><li>Mayor sobrevida </li></ul><ul><li>Inestabilidad genética </li></ul><ul><li>Capacidad de inducir angiogénesis </li></ul><ul><li>Capacidad para invasión y metástasis </li></ul>
    4. 4. DEFINICION <ul><li>INVASION: Proceso mediante el cual las c é lulas migran dentro del estroma adyacente siendo por lo tanto libres de diseminarse a ó rganos distantes v í a canales vasculares o linf á ticos. </li></ul><ul><li>METASTASIS: Es la diseminaci ó n de c é lulas tumorales desde el sitio primario donde se originaron para formar tumores secundarios en otros sitios del cuerpo. </li></ul>
    5. 5. INVASION TISULAR Y METASTASIS <ul><li>Causa principal de falla al tratamiento. </li></ul><ul><li>Al momento del diagn ó stico el 30% de los pacientes tienen met á stasis. </li></ul><ul><li>En otro 30% se manifestar á n posteriormente </li></ul><ul><li>Es un proceso continuo, que se inicia de forma temprana y se incrementa con el tiempo. </li></ul>
    6. 6. Los tumores benignos son frecuentes, pero tienen poco riesgo debido a que permanecen localizados y sin invasión
    7. 7. Los tumores malignos generalmente invaden tejidos vecinos y se diseminan a través del organismo Alteraciones en interaccion célula-célula y la formación de nuevos vasos sanguíneos están asociados con malignidad
    8. 8. PASOS EN EL PROCESO DE INVASIVIDAD <ul><li>Vascularizaci ó n del tumor. </li></ul><ul><li>Desprendimiento de c é lulas del tumor primario. </li></ul><ul><li>Adherencia y penetraci ó n de la membrana basal. </li></ul><ul><li>Migraci ó n a trav é s de la matriz extracelular. </li></ul>
    9. 9. DESPRENDIMIENTO DE CELULAS TUMORALES <ul><li>Disrupci ó n de interacci ó n c é lula-c é lula. </li></ul><ul><li>C é lulas epiteliales diferentes mecanismos de interacci ó n con otras ( uniones intermedias, desmosomas, CAM). </li></ul><ul><li>Alteraci ó n en la interacci ó n permite la migraci ó n celular. </li></ul><ul><li>E-cadherina: La subregulaci ó n produce desprendimiento celular, p é rdida de la arquitectura tisular y morfolog í a celular desdiferenciada. </li></ul><ul><li>Cadherinas: Ligadas al citoesqueleto por varias prote í nas (Beta cateninas). </li></ul>
    10. 10. Panorama de los diferentes tipos de moléculas que intevienen en la unión célula-célula y célula-matriz extracelular
    11. 11. Clases principales de moléculas de adhesión celular (CAMs) ‏
    12. 13. Uniones en base a cadherinas conectan células entre una y otra
    13. 14. Uniones en base a integrinas conectan células al substrato
    14. 16. MEMBRANA BASAL <ul><li>Separa a tejidos epiteliales y vasos del estroma circundante. </li></ul><ul><li>La forman prote í nas de matriz extracelular como col á gena tipo IV, laminina, proteoglicanos. </li></ul><ul><li>Provee de soporte a las c é lulas epiteliales para su anclaje, crecimiento y diferenciaci ó n. </li></ul><ul><li>Actuan como barrera al paso de c é lulas. </li></ul><ul><li>En tumores benignos siempre intacta, en c á ncer hay discontinuidad lo que permite la migraci ó n celular. </li></ul><ul><li>Actividad proteol í tica responsable de la destrucci ó n de la membrana basal. </li></ul>
    15. 21. PROTEASAS <ul><li>Enzimas proteol í ticas. Producidas tanto por c é lulas tumorales como c é lulas del hu é sped. </li></ul><ul><li>Degradaci ó n de membrana basal y matriz extracelular facilita la migraci ó n. </li></ul><ul><li>Gran n ú mero de enzimas proteol í ticas. Tienen diferente localizaci ó n. </li></ul><ul><li>- Intracelulares (catepsinas).Lisosomas. </li></ul><ul><li>- De superficie celular. uPA (activador del plasmin ó geno). Degrada MEC. Laminina, fibronectina. </li></ul><ul><li>- Extracelulares. Metaloproteasas. Las mas importantes. Son una familia numerosa. </li></ul>
    16. 22. Metaloproteasas de matriz (MMPs) ‏ <ul><li>Familia de proteasas dependientes de zinc (4) ‏ </li></ul><ul><li>Colagenasas intersticiales </li></ul><ul><li>Estromelisinas </li></ul><ul><li>Gelatinasas </li></ul><ul><li>MMPs tipo membrana </li></ul>
    17. 23. Metaloproteasas de matriz (MMPs) ‏ <ul><li>MMPs - familia de proteases zinc-dependientes </li></ul><ul><li>TIMPs - Inhibidores tisulares de MMPs </li></ul><ul><li>Balance entre MMPs yTIMPs es estricto en células normal, disregulado en CA </li></ul>MMPs Invasividad aumentada TIMPS
    18. 26. enzyme substrates collagenase-1 native collagen types I - III gelatinase-A collagen type IV, gelatin, elastin gelatinase-B collagen type IV, gelatin, elastin stromelysin-1 proteoglycans, laminin, collagen type IV, fibronectin, gelatin matrilysin proteoglycans, elastin, laminin, gelatin, fibronectin
    19. 29. FACTORES DE MOTILIDAD <ul><li>C é lulas normales y tumorales pueden secretar factores que promueven motilidad celular. </li></ul><ul><li>Factores aut ó crinos de motilidad. </li></ul><ul><li>Protrusi ó n pseudopoide. </li></ul><ul><li>Locomoci ó n : Dirigida </li></ul><ul><li>No dirigida. </li></ul>
    20. 31. Invasión: Pérdida de adhesión intercelular
    21. 32. Invasión: Unión a MEC
    22. 33. Invasión: Degradación de MEC
    23. 34. Invasión: Migración
    24. 35. PASOS EN EL PROCESO METASTASICO <ul><li>Entrada de c é lulas dentro del sistema vascular. </li></ul><ul><li>Escape de vigilancia inmunol ó gica. </li></ul><ul><li>Detenci ó n en ó rganos distales. </li></ul><ul><li>Extravasaci ó n hacia ó rganos blanco. </li></ul><ul><li>Crecimiento del dep ó sito metast á sico. </li></ul>
    25. 39. DISTRIBUCION DE METASTASIS <ul><li>Dependientes de: Tipo tumoral </li></ul><ul><li>Localizaci ó n anat ó mica. </li></ul><ul><li>Teor í a mec á nica: Determinado por patrones de flujo sangu í neo y anatomia. </li></ul><ul><li>Organo blanco: El primer lecho capilar que las c é lulas alcanzan. </li></ul><ul><li>Es un proceso no espec í fico, se relaciona al tama ñ o celular y di á metro capilar. </li></ul><ul><li>Es el tipo de distribuci ó n mas frecuente. </li></ul>
    26. 41. DISTRIBUCION DE METASTASIS <ul><li>Teor í a ó rgano-espec í fica (semilla-suelo). </li></ul><ul><li>Interacciones espec í ficas de prote í nas de superficie celular entre c é lulas tumorales y ó rganos blanco. </li></ul><ul><li>Factores de crecimiento en ó rganos blanco </li></ul><ul><li>Factores endoteliales. </li></ul><ul><li>Microambiente apropiado en ó rgano blanco da lugar al dep ó sito metast á sico. </li></ul><ul><li>Una vez detenidos en el endotelio del sitio blanco, se extravasan y realizan invasi ó n tisular. </li></ul>
    27. 44. ANGIOGENESIS <ul><li>Sin aporte adecuado de nutrientes tumores solo alcanzan 2 mm de di á metro. </li></ul><ul><li>Secreci ó n de factores angiog é nicos por c é lulas tumorales y c é lulas hu é sped normales. </li></ul><ul><li>Fundamental en aporte de nutrientes, remover productos de deshecho y formar canales vasculares para el desarrollo de met á stasis. </li></ul>
    28. 45. Angiogénesis tumoral: <ul><li>Angiogénesis </li></ul><ul><ul><li>Formación de nuevos vasos sanguíneos a partir de vasculatura preexistente </li></ul></ul><ul><ul><li>Necesaria para crecimiento tumoral y metástasis </li></ul></ul><ul><ul><li>Requiere interacciones coordinadas entre numerosas proteínas, vías de señalización y tipos celulares </li></ul></ul>
    29. 55. Estrategias para inhibir angiogénesis tumoral <ul><li>Inhibir moléculas involucradas en formación de vasos sanguíneos </li></ul><ul><ul><li>Factores angiogénicos derivados del tumor/huésped </li></ul></ul><ul><ul><li>Mediadores que incrementan la expresión de factores angiogénicos (ej, ras) ‏ </li></ul></ul><ul><li>Afectar sobrevida de células endoteliales </li></ul><ul><ul><li>VEGF </li></ul></ul><ul><ul><li>Integrinas </li></ul></ul><ul><ul><li>Angiopoietina-1 </li></ul></ul><ul><li>Inhibir MMPs </li></ul>