Papel de la Angiogenesis en el desarrollo tumoral Pedro Sánchez Rovira Complejo Hospitalario de Jaén

Cancer Development: First Steps [slide 10] Cancer Development: First Steps1,2 To understand cancer development, there are some key differences between a cancer cell and a normal cell need to be understood. In normal cells, the growth and cell division of the cell is very highly controlled. In the lab, the growth and division of non cancerous cells is inhibited by contact- that is, cells will stop dividing when they come into contact with neighboring cells. Also, normal cells will eventually die in culture. Cancer cells, on the other hand, are not contact inhibited and will continue to grow and divide. Cancer cells are also immortal in culture. These characteristics are apparent in tumors in man-we know that tumor cells grow uncontrollably. We refer to this as uncontrolled cell proliferation. The growth, proliferation, and even death of a cancer cell are therefore dysregulated- no longer controlled by normal regulators of these cellular processes. So- what are the normal cellular processes that tell cells when to grow and divide, and what factors are responsible for the dysregulation of these processes in a cancer cell? The development of cancer- or oncogenesis- starts with the transformation of a normal cell into a cancer cell. Oncogenesis involves genetic changes in the cell that lead eventually to uncontrolled cell proliferation. Many of these genetic changes affect signal transduction pathways- or the transfer of extracellular signals that control intracellular events. Oncogenesis (tumorigenesis) Genetic changes lead to uncontrolled cell proliferation A large number of oncogenes are involved in signal transduction Kastan MB et al. In: Lippincott Williams & Wilkins; 2001:91–109 Stetler-Stevenson WG et al. In: Lippincott Williams & Wilkins; 2001:123–36

ANGIOGÉNESIS CARACTERÍSITICAS DE UNA CÉLULA CANCERÍGENA: Autoseñalización para el crecimiento Insensibilidad a señales anti-crecimiento No respuesta a señales antiapoptóticas Capacidad ilimitada de replicación Tumorigenicidad PERO ADEMÁS… ANGIOGÉNESIS ANGIOGENESIS

Cancer Development: First Steps [slide 10] Cancer Development: First Steps1,2 Initial tumor growth is supported by the surrounding tissue microenvironment, which eventually becomes rate limiting for further growth The center tumor cells become hypoxic and the tumor then initiates the recruitment of its own blood vessels – this is called the angiogenic switch One of the way the tumor cell recruits its own blood vessels is by the expression and secretion of growth factors including VEGF. Oncogenesis (tumorigenesis) Genetic changes lead to uncontrolled cell proliferation A large number of oncogenes are involved in signal transduction Angiogenesis To grow beyond 1–2 mm, the tumor needs to initiate the recruitment of its own blood vessels Kastan MB et al. In: Lippincott Williams & Wilkins; 2001:91–109 Stetler-Stevenson WG et al. In: Lippincott Williams & Wilkins; 2001:123–36 1. Kastan MB, Skapek SX. Molecular biology of cancer: the cell cycle. In: DeVita VT Jr, Hellman S, Rosenberg SA, eds. Cancer: Principles & Practice of Oncology. 6th edn. Philadelphia, PA: Lippincott Williams & Wilkins; 2001:91–109 2. Stetler-Stevenson WG, Kleiner DE Jr. Molecular biology of cancer: invasion and metastases. In: DeVita VT Jr, Hellman S, Rosenberg SA, eds. Cancer: Principles & Practice of Oncology. 6th edn. Philadelphia, PA: Lippincott Williams & Wilkins; 2001:123–36

Angiogenesis: The Formation of New Blood Vessels Tumor vascularization [slide 14] Angiogenesis: The Formation of New Blood Vessels1,2 Tumor angiogenesis is the proliferation of a network of blood vessels that penetrates into the tumor, supplying nutrients and oxygen and removing waste products Tumor angiogenesis actually starts with tumor cells releasing growth factors that send signals to surrounding vascular endothelial cells, and I’ve mentioned that the key growth factor released is VEGF. To survive and grow, solid tumors must acquire new blood vessels to provide them with nutrients and oxygen, a process known as angiogenesis Several signaling pathways are involved in regulating angiogenesis Ferrara N. Curr Top Microbiol Immunol. 1999;237:1–30 Verheul HM et al. Clin Breast Cancer. 2000;1:S80–4 1. Ferrara N. Vascular endothelial growth factor: molecular and biological aspects. Curr Top Microbiol Immunol. 1999;237:1–30 2. Verheul HMW, Pinedo HM. The role of vascular endothelial growth factor (VEGF) in tumor angiogenesis and early clinical development of VEGF-receptor kinase inhibitors. Clin Breast Cancer. 2000;1:S80–4.

Angiogenesis: The Role of Growth Factors and Their Receptors The major growth factors involved in angiogenesis Vascular endothelial growth factor (VEGF) Platelet-derived growth factor (PDGF) Fibroblast growth factor (FGF) All of the angiogenic factors interact with their specific receptors on blood vessels or on connective tissue In cancer, VEGF is produced by tumor cells, but acts on vascular endothelial cells to cause the growth of blood vessels [slide 15] Angiogenesis: The Role of Growth Factors and Their Receptors1–4 Vascular endothelial growth factor (VEGF) is the most potent vascular growth factor identified. VEGF binds to its receptor, VEGFR-2, on endothelial cells (which are the cells that make up the blood vessels) and initiates a signaling cascade, similar to what we just described in the tumor cell, this signaling cascade tells the endothelial cell to grow and divide leading to the formation of new blood vessels for the tumor. PDGF and its receptor is also involved in the promotion of angiogenesis; PDGFR is found predominantly on pericytes, which help support blood vessel structure and may be important in later stages of angiogenesis Ferrara N. Curr Top Microbiol Immunol. 1999;237:1–30 Clauss M. Semin Thromb Hemost. 2000;26:561–569 Ostman A et al. Adv Cancer Res. 2001;80:1–38 Bergers G et al. J Clin Invest. 2003;111:1287–95

Angiogenesis: The Role of Growth Factors and Their Receptors Receptors for VEGF (VEGFRs) and PDGF (PDGFRs) are receptor tyrosine kinases Three major types have been identified VEGFR-1 (Flt-1) VEGFR-2 (KDR, Flk-1) VEGFR-3 (Flt-4) VEGF signalling is correlated with endothelial cell migration and proliferation, resulting in angiogenesis The VEGF receptors- as well as PDGF receptors- are receptor tyrosine kinases. Three major VEGFRs have been identified: the one that is most important for angiogenesis is VEGFR-2 VEGFR-1 (Flt-1) is expressed on endothelial cells, monocytes, and other cells, including some tumor cells, and is thought to mediate cell motility actions of VEGF (endothelial migration) VEGFR-2 (KDR, FLK-1) is expressed on endothelial cells as well as other cell types. It mediates the proliferative activities of VEGF, as well as vascular permeability and endothelial cell survival VEGFR-3 (Flt-4) is expressed on lymph and tumor endothelial cells and is believed to mediate lymphangiogenesis Ferrara N. Curr Top Microbiol Immunol. 1999;237:1–30 Clauss M. Semin Thromb Hemost. 2000;26:561–569 Ostman A et al. Adv Cancer Res. 2001;80:1–38 Bergers G et al. J Clin Invest. 2003;111:1287–95

VEGF: A Key Mediator of Angiogenesis Environmental factors (hypoxia, pH) Increased VEGF levels Genes implicated in tumorigenesis (p53, p73, src, ras, vHL, bcr-abl) Growth factors, hormones (EGF, bFGF, PDGF, IGF-1, IL-1, IL-6, estrogen) bFGF, basic fibroblast growth factors; EGF, epidermal growth factor; IGF, insulin-like growth factor; IL, interleukin; PDGF, platelet-derived growth factor; VEGFR, VEGF receptor. 1. Dvorak HF. J Clin Oncol. 2002;20:4368-4380; 2. Ebos JM, et al. Mol Cancer Res. 2002;1:89-95; 3. Ferrara N, et al. Nat Med. 2003;9:669-676.

VEGF: A Key Mediator of Angiogenesis VEGFR binding and activation VEGF P P P P Endothelial cell activation KEY POINT: A number of environmental, hormonal, and genetic factors can lead to VEGF overexpression. ANGIOGENESIS Survival Proliferation Migration PLC FAK PI3-K Ras IP3 PKC AKT Paxillin MAPK ANGIOGENESIS Survival Proliferation Migration PLC FAK PI3-K Ras IP3 PKC AKT Paxillin MAPK

The VEGF Family and Its Receptors PIGF VEGF-A VEGF-B VEGF-C VEGF-D VEGFR-1 (Flt-1) VEGFR-2 (Flk-1/KDR) VEGFR-3 (Flt-4) PlGF, placenta growth factor. Angiogenesis Lymphangiogenesis Angiogenesis Lymphangiogenesis Neufeld G, et al. FASEB J. 1999;13:9-22. Ferrara et al. Nat Med. 2003;9:669.

Targeting VEGF: The Bevacizumab Story KEY POINT: A number of environmental, hormonal, and genetic factors can lead to VEGF overexpression. P P P P

Targeting VEGF: The Bevacizumab Story KEY POINT: A number of environmental, hormonal, and genetic factors can lead to VEGF overexpression. P P P P VEGF Activation BLOCKED

VEGF Trap P

VEGF Trap P

VEGF Trap

VEGF Trap VEGF Trap VEGF Fc portion P P P P

VEGF Trap VEGF Trap VEGF Fc portion P P P P

VEGF Activation BLOCKED VEGF Trap VEGF Trap VEGF P P P P VEGF Activation BLOCKED

Tyrosine Kinase Inhibition and VEGF TKI (Tyrosine Kinase Inhibitor) P P P P

Tyrosine Kinase Inhibition and VEGF TKI P P P P Downstream phosphorylation BLOCKED

Tumor Angiogenesis: Bevacizumab and Multitargeted Agents Angiogenic growth factors Pericyte O2 Tumor cell Endothelial cell

Endothelial Cell VEGF-B VEGF-A VEGF-C VEGF-D VEGF-R1 (Flt-1) VEGF-R2 (KDR) VEGF-R3 (Flt-4) PROLIFERATION MIGRATION ANGIOGENESIS VASCULOGENESIS Ras Raf MEK ERK p38MAPK PI3K Akt Caspase-9 eNOS CELL ACTIVATION / SURVIVAL MIGRATION LYMPHANGIOGENESIS

Tumor Angiogenesis: Bevacizumab and Multitargeted Agents PDGF Pericyte O2 Tumor cell Endothelial cell

CELL ACTIVATION VESSEL MATURATION Pericyte PDGF-B PDGF-Rβ Ras Raf MEK ERK p38MAPK PI3K Akt Caspase-9 eNOS CELL ACTIVATION VESSEL MATURATION

Tumor Angiogenesis: Bevacizumab and Multitargeted Agents Pericyte O2 Tumor cell Endothelial cell Paracrine factors SOURCE: Angiogenesis Foundation

CELL PROLIFERATION SURVIVAL Tumor Cell KIT RET Flt-3 Multiple Growth Factors CELL PROLIFERATION SURVIVAL PI3K Akt Ras Raf MEK ERK p38MAPK Src Rac1 JNK

Tumor Angiogenesis: Cells and Pathways Tumor cell Pericyte Angiogenic growth factors O2 Endothelial cell Paracrine factors PDGF

Avastin Phase III studies in the neoadjuvant and adjuvant settings in breast cancer NSABP-B40 Docetaxel + Xeloda or gemcitabine  doxorubicin + cyclophosphamide Neoadjuvant Adjuvant E2104 / E5103 Doxorubicin + cyclophosphamide  paclitaxel HER2 negative Triple negative BEATRICE Multiple chemotherapy regimens Avastin BETH Adjuvant chemotherapy + Herceptin HER2 positive 29 29

RELACIÓN CD 105 Y RESPUESTA AL TRATAMIENTO Br J Cancer 2006; 95: 1683

CEC DURANTE EL TRATAMIENTO NEOADYUVANTE

CELULAS PROGENITORAS DURANTE EL TRATAMIENTO NEOADYUVANTE

RELACIÓN pVEGFR2 Y BEVACIZUMAB

RELACION APOPTOSIS Y TRATAMIENTO CON BEVACIZUMAB

ESTUDIO FASE II MULTICENTRICO, ABIERTO, NO ALEATORIZADO PARA EVALUAR LA EFICACIA DE UNA COMBINACIÓN DE BEVACIZUMAB Y QUIMIOTERAPIA SECUENCIAL COMO TRATAMIENTO DE PACIENTES CON CANCER DE MAMA OPERABLE HER 2 NEGATIVO ENSAYO CLÍNICO ML20382

MARCADORES BIOMOLECULARES Expresión de VEGF-A, VEGF-B. Expresión de VEGFR 1,2,3 Expresión de neurofilina 1,2 Expresión de HIF-1. Expresión de PIGF. Factor 4E eucariótico de iniciación de la traslación. (eIF4E). Perfiles de expresión de la proteina y/o ARN del tumor asociados en la señalización de VEGFR/neurofilina

Estudio en ratones con doxorubicina (Naumov et al, 2003). La metástasis es un proceso dinámico en el cual las células cancerígenas latentes, las micrometástasis no angiogénicas y las macrometástasis angiogénicas pueden coexistir en cada paso del proceso. Mientras las micrometástasis no angiogénicas pueden ser vulnerables a agentes quimioterápicos citotóxicos y a anti-angiogénicos, las células latentes no proliferantes pueden no verse afectadas. Estudio en ratones con doxorubicina (Naumov et al, 2003). ANGIOGENESIS

Autopsias realizadas a individuos fallecidos en accidentes de automóvil la presencia de tumores latentes: El 39% de mujeres entre 40 - 50 años presentaban indicios de carcinoma de mama in situ (sólo un 1% fue diagnosticado de cáncer en vida en el mismo rango de edades). 46% de varones entre 60 – 70 años eran diagnosticados de cáncer de próstata (sólo 1-1,5% fueron diagnosticados de este tipo de cáncer en vida en el mismo rango de edades). Hasta un 98% de individuos con edad comprendida entre lo 50 y 70 años presentaron carcinoma microscópico de tiroides, y sólo el 0,1% fue diagnosticado en vida en el mismo rango de edades. Black and Welch, 1993 ANGIOGENESIS

ACTIVACIÓN ANGIOGÉNICA: Capacidad que tiene un tumor de pasar de un fenotipo no angiogénico a un fenotipo angiogénico. Proceso esencial para la progresión del cáncer. El fallo en el reclutamiento de nuevas células endoteliales microvasculares o en la reorganización de la vasculatura circundante conduce a tumores no angiogénicos que no superan el tamaño microscópico. ANGIOGENESIS

La hipoxia estimula la transcripción del factor celular inducible por hipoxia (HIF), dando lugar a la sobrerregulación de proteínas pro-angiogénicas (VEGF, PDGF, NOS) Temporalmente las proteínas pro-angiogénicas superan la concentración local de proteínas anti-angiogénicas, lo que provoca la angiogénesis y favorece la expansión del tumor ANGIOGENESIS

EL ESTADO OXIDATIVO MODULA LA EXPRESIÓN DE GENES IMPLICADOS EN EL CRECIMIENTO TUMORAL

ACTIVACIÓN ANGIOGÉNICA: La expansión tumoral está asociada al reclutamiento de células endoteliales, precedida de la activación angiogénica. ACTIVACIÓN ANGIOGÉNICA: Incremento en la expresión de proteínas angiogénicas (VEGF, bFGF) por células tumorales. Incremento de la expresión de proteínas angiogénicas por células del estroma como fibroblastos. Disminución en la expresión de inhibidores endógenos de la angiogénesis (trombospondina-1, TSP1) por células tumorales y células del estroma. Ocasionalmente reclutamiento de precursores endoteliales derivados de la médula ósea. ANGIOGENESIS

BIOMARCADORES DE ANGIOGÉNESIS: Permiten la detección de tumores de tamaño microscópico (≤ 1 mm diámetro) que de otra forma permanecerían indetectables. Técnica desarrollada en modelos animales. Células progenitoras endoteliales circulantes Factores angiogenicos en plaquetas (SELDI-ToF) Metaloproteinasas (MMPs) de la matriz en orina ANGIOGENESIS

Desarrollo de un panel de biomarcadores de angiogénesis capaces de: Análisis proteómico en plaquetas como biomarcadores amplificadores de angiogénesis. Proteínas asociadas con la activación angiogénica que podría ser usadas como biomarcadores de angiogénesis están siendo actualmente estudiadas. Desarrollo de un panel de biomarcadores de angiogénesis capaces de: Identificar la presencia de tumores microscópicos Predecir la activación angiogénica del tumor Guiar la terapia antiangiogénica. Uso de fármacos que aumenten la concentración endógena de proteínas anti-angiogénicas en plaquetas para retrasar o prevenir la formación de tumores en pacientes con alto riesgo de recurrencia. ANGIOGENESIS