Molecular Basis Of Cancer Progression1

Molecular Basis and Therapeutic Approaches of Cancer Objective: To understand to the process of tumor progression, angiogenesis, metastasis and its implication for cancer therapy

Ratchada Cressey, Ph.D 11/25/09

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Some Important Facts: General  US- 500,000 deaths per years  2nd only to heart disease as causes of motality  1 in 3 in developing country  50% die/survive  17% cured by chemotherapy  1 million new cases per year  Lung, large intestine, breast and prostate cancer = 55% of new cases and deaths in t he US 11/25/09

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Some Important Fact: Thailand  2nd only to accident as cause of motality  Distribution of the types of cancer according to the area    

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Bangkok (lung cancer) North-East (liver cancer) North (lung cancer) South (cancer of GI tract, lung cancer)

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Why do people of cancer die?

 Local Effects  Paraneoplastic Syndromes  Cancer cachexia

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I. Local effects  Tumor Impingement on nearby structures 

Pituitary adenoma on normal gland, Pancreatic carcinoma on bile duct, Esophageal carcinoma on lumen

 Ulceration/bleeding 

Colon, Gastric

 Infection (often due to obstruction) 

Pulmonary infections due to blocked bronchi (lung carcinoma), Urinary infections due to blocked ureters (cervical carcinoma)

 Rupture or Infarction 

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Ovarian 5

II.Paraneoplastic Syndromes Peptide Product PTHrP or PTH

hypercalcemia

insulin/insulin-like gastrin

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hypoglycemia

Zollinger-Ellison dis.

erythropoietin ACTH

Condition

polycythemia

Cushing’s disease

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III.Cancer Cachexia  unique form of protein-calorie malnutrition  affects 50 to 80% of human cancer patients  common cause of death  symptoms  weakness, fatigue, anorexia  physical examination abnormalities  weight loss, skeletal muscle atrophy, adipose tissue loss, myopathy  clinical pathology abnormalities  anemia, decreased serum albumin, glucose intolerance, energy

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Multi-step of carcinogenesis

The cell cycle is regulated by a number of signaling systems

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Tumors require the continuing formation of new blood vessels:  Supply oxygen and nutrients  Supply endocrine and paracrine growth-enabling factors

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Growth of tumor is dependent on angiogenesis

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What is angiogenesis?  Angiogenesis is fundamental to healing, reproduction, embryonic development.  During development, new blood vessels originate from endothelial cell precursors (angioblasts) by a process called VASCULOGENESIS or from pre-existing blood vessels by ANGIOGENESIS.  Both processes are mediated by growth factors.

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Angiogenesis: Hypoxia  Tumor cells located more than 100 μm (diffusion limit for oxygen) away from blood vessels become hypoxic.  Clones are selected in hypoxic tumors that switch to proangiogenic phenotype.  Hypoxia inducible factors (HIFs) increase transcription of angiogenic genes. 11/25/09

Carmeliet & Jain, 2000 Nature

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 The angiogenesis process begins with the degradation of the basement membrane by proteases secreted by activated endothelial cells that will migrate and proliferate, leading to the formation of solid endothelial cell sprouts into the stromal space.  Then, vascular loops are formed and capillary tubes develop with formation of tight junctions and deposition of new basement membrane. 11/25/09

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The actual process of angiogenesis involves a number of steps including:

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The release of proteases from "activated endothelial cells" The degradation of the basement membrane surrounding the existing vessel The migration of the endothelial cells into the interstitial space Endothelial cell proliferation The formation of the lumen The generation of new basement membrane with the recruitment of pericytes Fusion of the newly formed vessels

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And resuming blood flow

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I. Breaking down of the basement membrane  In order to create new capillaries, endothelial cells of existing blood vessels must degrade the basement membrane.  This process of endothelial cell invasion requires the help of 

Urokinase-plasminogen activator (uPA)

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Matrix metalloproteinases (MMPs).

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I. Breaking down of the basement membrane (2)  Plasmin degrades several extraceullar matrix (ECM) components such as fibrin, fibronectin, laminin, and the protein core of proteoglycans. Plasmin can also activate serveral MMPs such as MMP-1, MMP-3, and MMP-5.  Most types of ECM contain collagens, elastin, various glycoproteins (such as fibronectin, laminin, entactin, and nidogen), proteoglycans, and glycosaminoglycans. 

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There are at least 16 different members of MMPs that break down different components of the ECM.

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II. Endothelial cells migration and proliferation  After the degradation of the ECM, "leader" endothelial cells migrate through the broken down matrix  Proliferating endothelial cells migrate into the degraded matrix. Then they are stimulated by growth factors that were released from the degraded matrix.

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III. Cell and Matrix Interactions  The final phase of angiogenesis includes the construction of capillary loops and the determination of the polarity of the endothelial cells. 

These are required for lumen formation and involve cell-cell contact and cell-ECM interaction.

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Alterations in at least four endothelial cell functions occur during angiogenesis (1) an increase in proliferation, which provides new cells for the growing and elongating vessel, with a subsequent return to the quiescent state once the new vessel is formed (2) an initial increase and subsequent decrease in locomotion (migration), which allows the cells to translocate toward the angiogenic stimulus and to stop once they reach their destination (3) endothelial cell-to-cell interactions (4) interactions with the extracellular matrix.

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Angiogenic factors  Cytokines, chemokines and angiogenic enzymes are direct-acting molecules that activate a broad range of target cells  VEGF family and angiopoietins which act on endothelial cells specifically  Indirect-acting factors whose effect results from direct-acting factors from macrophages, endothelial cells, or tumor cells.

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1. Cytokines  The most studied are tumor necrosis factor-α (TNFα ) and transforming growth factor-β (TGF-β ).  both inhibit endothelial cell proliferation in vitro. 

in vivo TGF-β induces angiogenesis and stimulates the expression of TNF-α , FGF-2, platelet derived growth factor (PDGF) and VEGF by attracted inflammatory cells.

 TNF-α has been shown to increase the expression of VEGF and its receptors.

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2. VEGF

(Vascular endothelial growth factor)  Expression of VEGF is required during embryonic development for the formation of normal blood vessels,  Loss of even a single VEGF allele is lethal, suggesting that normal VEGF levels are critical for the regulation of vessel development.

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VEGF has been studied both in vivo and in vitro and both have given similar results:  In vivo - VEGF has been shown to regulate vascular permeability. This is important because it is one of the initiation steps of angiogenesis.  In vitro - VEGF has been shown to stimulate ECM breakdown, migration, proliferation and with other enzymes.

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VEGF mediates angiogenesis through its tyrosine kinase receptor

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VEGF and Oxygen  VEGF levels are regulated by tissue oxygen tension 

Exposure to hypoxia induces VEGF expression rapidly and reversibly, through both increased transcription and stabilization of the mRNA.

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Hypoxic upregulation of VEGF thus provides a compensatory mechanism by which tissues (or tumors) can increase their oxygenation through induction of blood vessel growth.

 Hypoxia also regulates the VEGF receptor gene expression 11/25/09

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• Under conditions of hypoxia, ECs upregulate VEGF, which, once secreted, may then interact with its receptor. Such an autocrine loop provides the basis for amplification of any given VEGF secreted or administered into an ischemic territory. ECs stimulated to proliferate in response to VEGF may then serve as additional sources of VEGF synthesis, thus amplifying the effect of the initial dose of VEGF.

• Factors secreted by hypoxic myocytes up-regulate VEGF receptor expression on ECs within the hypoxic milieu. Such localized receptor expression may explain the finding that angiogenesis does not occur indiscriminately, but rather at sites of tissue ischemia. (From Horowitz et al )

Survival: Correlation With Blood Vessel Number and VEGF Levels

Takahashi et al. Arch Surg. 1997;132:541.

Role of Angiogenesis in Primary and Metastatic Tumors

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Metastasis: Correlation With Blood Vessel Number and VEGF Levels

Takahashi et al. Cancer Res. 1995;55:3964. 11/25/09

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ANTIANGIOGENIC THERAPY ~

200 biotech and big pharma companies are pursuing angiogenesis research

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Anti-VEGF drugs

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Anti-VEGF drug

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Inhibit Tumor Angiogenesis

Fidler et al. In DeVita et al. Cancer: Principles and Practice of Oncology. 6th ed. 2001:137.

Response of a solid tumor to treatment with angiogenesis inhibitors. Tumor is not ameliorated!

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Angiogenesis Inhibitors – Phase III Clinical Trials Product

Description

Disease Target

Avastatin (Genentech)

Monoclonal antibody that Breast and colorectal disables vascular endothelial cancer growth factor (VEGF), a promoter of angiogeneis

BMS275291 Synthetic compound having Non-small cell lung cancer (Bristol-Myers Squibb) multiple effects Interferon α

Protein that inhibits release Various tumors of growth factors such as VEGF Jain & Carmeleit, Scientific American (December, 2001) 39-45

Angiogenesis Inhibitors–Phase III Clinical Trials Product

Description

Disease Target

Marimastat (British Biotech)

Synthetic compound having Breast and prostate cancer multiple effects

Neovastat (Aeterna)

Naturally occurring inhibitor Non-small cell lung with a range of properties cancer and renal cancer

SU5416 (Sugen)

Synthetic compound that blocks the receptor for VEGF

Colorectal cancer

Thalidomide (Celgene)

Organic molecule whose specific mode of action is unknown

Renal cancer and multiple myeloma

Jain & Carmeleit, Scientific American (December, 2001) 39-45

Angiogenesis Inhibition  Difficult to completely inhibit angiogenesis  

Range of factors inducing angiogenesis Difficulty in eliminating all activity for particular factor

 Angiogenesis is required for normal processes – possible side effects  Suppress growth of metastases in combination with other treatments

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Angiogenesis Inhibition  Paradox: antiangiogenic therapy increases effectiveness of conventional therapies 

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Would expect that reduced blood supply to tumor would make chemotherapy less effective

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Angiogenesis Inhibition 

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For some antiangiogenic therapies, delivery of chemotherapeutic agents, nutrients and oxygen improves Antiangiogenic factors “normalize” tumor vasculature  Tumor

blood vessels structurally disorganized, dilated, leaky  Angiogenesis inhibitors can reduce diameter, make vessels less leaky

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