Inflammation and Cancer The Significance of COX-2

1. Inflammation and CancerThe Significance of COX-2 Dan Dixon Dept. of Biological Sciences South Carolina Cancer Center ddixon@biol.sc.edu

2. Inflammation and Cancer Inflammation is a critical component of tumor progression • Cancers can arise from sites of infection, chronic irritation and inflammation. • A vital component of the tumor microenvironment are the inflammatory cells. • Inflammation promotes the neoplastic processes involving proliferation, survival, and migration. • Tumor cells have co-opted inflammatory signaling molecules and receptors. Tumors act as wounds that fail to heal

3. Historical Perspective • In 1863, German physician Rudolf Virchow hypothesized that certain classes of irritants, together with the tissue injury and inflammation, promote enhanced cell proliferation. • In 1918, Yamagiwa and Ichikawa, who showed that repeated painting of coal tar onto rabbits' ears causes carcinomas. Rudolf Virchow the world’s first man-made cancer on the ears of a rabbit

4. Time, Feb. 23, 2004

5. Inflammation Overview • In response to tissue injury, signals start and maintain a host response to “heal” the damage. • This involves getting “activated” leukocytes (WBCs) to the proper damaged location. • Adhesion molecules and integrins with their respective receptors/ligands modulate WBC rapid adhesion, activation of gene expression, and transmigration into the tissue. • Expression of chemokines attract specific WBC populations and promote the natural progression of the inflammatory response. • The “normal” inflammatory response associated with wound healing is self-limiting. Dysregulation of key steps leads to abnormalities… pathogenesis… neoplasia.

6. eosinophil platelets neutrophil lymphocyte Monocyte Some of the Players… • Neutrophils (and eosinophils): The first cells recruited to damaged tissues and invading bactera. • Monocytes: Chemotactic factors guide monocytes to the injury migrate into the site of injury. • Macrophages: Primarily stem from differentiated monocytes; they are the main source of growth factors and cytokines. Their presence profoundly effects the surrounding microenvironment cells. • Mast cells: Release histamine, cytokines, and proteases; let the body know something is wrong. • Lymphocytes: Provide firepower for the immune response. • Platelets: They aggregate to form a fibrin clot to close the wound.

7. Wound Healing: Normal tissues have a highly organized and segregated architecture. Epithelial cells sit atop a basement membrane separated from the vascularized stromal (dermis) compartment. Upon wounding or tissue assault, platelets are activated and form a haemostatic plug where they release vasoactive mediators that regulate vascular permeability, influx of serum fibrinogen, and formation of the fibrin clot. Chemotactic factors such as transforming growth factor- and platelet-derived growth factor, derived from activated platelets, initiate granulation tissue formation, activation of fibroblasts, and induction and activation of proteolytic enzymes necessary for remodelling of the extracellular matrix (for example, matrix metalloproteinases and urokinase-type plasminogen activator). In combination, granulocytes, monocytes and fibroblasts are recruited, the venous network restored, and re-epithelialization across the wound occurs. Epithelial and stromal cell types engage in a reciprocal signalling dialogue to facilitate healing. Once the wound is healed, the reciprocal signalling subsides. Invasive Tumor Growth: Invasive carcinomas are less organized. Neoplasia-associated angiogenesis and lymphangiogenesis produces a chaotic vascular organization of blood vessels and lymphatics where neoplastic cells interact with other cell types (mesenchymal, haematopoietic and lymphoid) and a remodelled extracellular matrix. Although the vascular network is not disrupted in the same way during neoplastic progression as it is during wounding, many reciprocal interactions occur in parallel. Neoplastic cells produce an array of cytokines and chemokines that are mitogenic and/or chemoattractants for granulocytes, mast cells, monocytes/macrophages, fibroblasts and endothelial cells. In addition, activated fibroblasts and infiltrating inflammatory cells secrete proteolytic enzymes, cytokines and chemokines, which are mitogenic for neoplastic cells, as well as endothelial cells involved in neoangiogenesis and lymphangiogenesis. These factors potentiate tumour growth, stimulate angiogenesis, induce fibroblast migration and maturation, and enable metastatic spread via engagement with either the venous or lymphatic networks.

8. The Roles of Tumor-Associated Macrophages in Tumor Progression • Invasion: Macrophages secrete a variety of proteases to breakdown the basement membrane around areas of proliferating tumor cells thereby prompting their escape into the surrounding stromal tissue. • Angiogenesis: In areas of tumor hypoxia, macrophages cooperate with tumor cells to induce a vascular supply for the area by up-regulating a number of angiogenic growth factors. These proangiogenic factors stimulate vascular endothelial cells in neighboring areas to migrate into new vessels for the tumor. • Immunosuppression: Macrophages secrete factors that suppress the anti-tumor functions of innate immune system. • Metastasis: Macrophages associated with tumor vessels secretes factors that guide tumor cells toward blood vessels where they then escape into the circulation. In the stromal compartment, macrophages secrete growth factors to stimulate tumor cell growth and motility.

9. Chronic Inflammation is a Risk Factor for Cancer Chronic Inflammation Dysplasia Ulcerative colitis (UC) Adenocarcinoma • Patients with UC have a 5 to 7-fold greater risk of getting colon cancer. • UC persisting for 35-40 years increases the risk 20-35%. • Colon cancer associated with IBD has the worst prognosis. • Management with anti-inflammatory agents reduce incidence of cancer.

10. Colon Adenocarcinoma Ulcerative colitis Normal Colon

11. Ulcerative Colitis and Increased Risk of Colorectal Cancer Over Time 25 20 15 Cumulative probability (%) 10 5 UC 1 0 5 10 15 20 25 30 Time from diagnosis (years) • Overall prevalence of CRC in any UC patient is 3.7% Eaden JA, et al. Gut. 2001;48:526.

12. Inflammatory Syndromes and Their Associated Cancers

13. Chronic Inflammation Promotes Tumor Development Insulted stromal cells recruit activated inflammatory cells Activated monocyte/macrophages Chronic activation promotes continued inflammation, angiogenesis, and ECM remodeling Inflammatory cells express factors that stimulate cell growth and progression Malignant conversion Promotion mutation epithelial cells initiated epithelium benign carcinoma

14. Anti-Inflammatory Drugs as Chemotherapeutic Agents • Aspirin and NSAIDs: Aspirin and NSAIDs inhibit prostaglandin synthesis. Aspirin inhibits platelet aggregation. Flurbiprofin has anti-metastatic effects. NSAIDs promote apoptosis, interfere with cell-cycle progression, and stimulate immune surveillance. • Anti-TNFa Antibodies: Effective in controlling IBD. Regulates TNFa activity which promotes cytokine, chemokine, adhesions, MMPs, and pro-angiogenic activities. • Adhesion Molecule Receptor Antagonists: Cancer cells and tumors contain mucins and ligands for cell adhesion molecules. Blocking adhesion may alter metastasis. • MMP Inhibitors: MMPs can promote tumor growth and also attenuate growth.

15. COOH Arachidonic Acid Cycloxygenase Lipoxygenase Epoxygenase Prostaglandins (PGs) Thromboxanes (TXs) Leukotrienes (LTs) Lipoxins (LXs) Epoxy Acids (EETs) Dihydroxy Acids Cyclooxygenase and Arachidonic Acid Metabolism • Arachidonic acid (AA) is a 20-carbon polyunsaturated fatty acid. • AA is liberated from the membrane glycerophospholipids by phospholipases. • 3 Major pathways convert AA into biologically active eicosanoids. • Cyclooxygenase (COX) catalyses a key step in the formation of prostaglandins.

16. Cellular Arachidonic Acid Metabolism Leukotrienes Prostaglandins

17. or COX Cyclooxygenase Activity is a Key Step in Prostaglandin Synthesis

19. TxA2 PROMOTES PLATELET AGGREGATION; PGI2 INHIBITS IT PGE2, PGF, and PGI2 RELAX VASCULAR SMOOTH MUSCLE PGE2 and PGI2 RELAX BRONCHIAL SMOOTH MUSCLE; PGF CONTRACTS IT PGE2 and PGI2 INCREASE RENAL BLOOD FLOW PGE2 and PGI2 PROTECT GASTRIC MUCOSA PGE2 and PGF CONTRACT UTERINE SMOOTH MUSCLE; PGI2 RELAXES IT Prostaglandin Regulate Physiological Functions Prostaglandins are biologically active phospholipid molecules that regulate many physiological functions Proper balance of prostaglandins are critical for normal homeostasis

20. Prostaglandin Signaling Mechanisms Prostaglandins control cellular function through G-coupled membrane receptors and nuclear receptors

21. Prostaglandin Signaling Promotes Cell Growth

22. Prostaglandins Trigger Signal Transduction Pathways Associated with Cancer Growth

23. Increased PGE2 Promotes Many Facets of Tumorigenesis Metastasis Invasion Vascular Angiogenesis Reduced Apoptosis MMP-2  MMP-9  VEGF  BCL-2  PGE2 IL-10  IL-12  PI3-K Activation Immune Suppression Proliferation Motility

24. Multiple COX Enzymes Exist in Cells COX-1: Constitutively present COX-2: Inducible isoform “COX-3”: Splice variant of COX-1 Feature COX-1COX-2 Expression ConstitutiveInducible Protein Size 72 kDa 72/74 kDa Gene Size 22 kb 8.3 kb mRNA size 2.7 kb 4.5 kb; contains multiple AU-rich elements in 3’UTR Localization ER, nuclear envelope ER, nuclear envelope Cell & Tissue Expression Platelets, stomach, kidney, Expressed in most cells or tissues after stimulation with colon, most tissues cytokines, growth factors, or tumor promoters Gene Regulation Constitutive low-level expressionImmediate-early response gene, rapidly transcribed, mRNA is rapidly degraded, translation is controlled

25. Differing Functions of COX-1 & COX-2 Arachidonic Acid COX-2 (inducible) COX-1 (constitutive) • Pathophysiology • Inflammation, Pain • Fever • Cancer • Morbus Alzheimer • Ischemia (CNS) • Homeostasis • Stomach/GI protection • Platelet aggregation • Renal blood flow

26. Evidence of a COX-2 Dependent Role in Neoplasia Epidemiological Studies w Decreased risk of CRC-associated deaths in aspirin users. w The NSAID sulindac decreases the size and number of polyps (FAP). w Prostaglandin levels are increased in CR tumors. w Overexpression of COX-2 detected in adenomas and adenocarcinomas. Animal Studies wMin mice and AOM-treated rats have elevated COX-2 levels. w Sulindac and other NSAIDs attenuate intestinal tumor and xenografted cancer cell growth in mice. Cellular Studies w Overexpression of COX-2 in epithelial cells results in: Decreased apoptosis Angiogenesis (increased VEFG, FGF, PDGF… expression) Metastatic potential (increased adhesion and MMP expression) Genetic Model w Mice defective in COX-2 have a dramatic reduction (86%) in colorectal polyp formation.

27. COX-2 is Overexpressed in Colon Cancer COX-2 protein staining in colon tumor Normal colon epithelium

28. COX-2 is Overexpressed in Multiple Components of Cancer

29. Increased COX-2 = Increased Prostaglandins COX-2-derived PGs promote cell growth, proliferation, and angiogenic gene expression in cells composing a tumor

30. COX-2 gene dosage effect on polyp number (100%) COX-2 COX-2 Total Polyp Number/Mouse COX-2 (34%) (14%) COX-2 Genotype Genetic “Removal” of COX-2 Suppresses Tumorigenesis

31. APC mutation TGFß IIR hMSH K-ras mutation p53 mutation Normal Epithelium Carcinoma Late Adenoma Early Adenoma Involvement of COX-2 in the Progression of Colon Cancer - COX-2 +/- COX-2 ++ COX-2 +++ COX-2 Loss of COX-2 gene regulation COX-2 overexpression Increased PG levels

32. NSAIDs: A History of COX Inhibition • BC: Ancient Greeks and Romans used salicylate extracts derived from willow leaves as analgesics and antipyretics. • Middle Ages: Medicinal herb gardens featured salicylate containing wintergreen and meadowsweet plants. • 1763: Edward Stone reported on use of willow bark powder as an anti-inflammatory. • 1853: Von Gerhardt synthesizes a crude form of aspirin (acetylsalicyclic acid). • 1860: Felix Hoffman working for Bayer synthesizes pure aspirin. • 1949: The NSAID Phenylbutazone introduced. • 1963: Indomethacin introduced. • 1971: Vane and Piper demonstrated NSAIDs inhibit prostaglandin production. • 1974: Ibuprofen introduced. • 1976: Miyamoto et al purified the COX-1 enzyme. • 1989: Simmons et al (and others) identified the COX-2 enzyme. • 1999: The COXIBs Celebrex (celecoxib) and Vioxx (rofecoxib) introduced.

33. Aspirin and NSAIDs • NSAIDs have pain-relieving (analgesic) effects as well as effects of reducing inflammation and fever. • NSAIDs are non-selective COX-1 and COX-2 enzymatic inhibitors. • Aspirin is an irreversible inhibitor of COX; Other NSAIDs are reversible. • Aspirin protects against CV disease; NSAIDs are chemopreventive. • Chronic NSAID use can lead to GI ulcers and renal problems.

34. Kune 1988 Rosenberg 1991 Suh 1993, men Suh 1993, women Peleg 1994 Muscat 1994, men Muscat 1994, women Muller 1994 Reeves 1996 Bansal 1996 LaVecchia 1997 Rosenberg 1998 Friedman 1998 Coogen 2000A Coogen 2000B 0 0.5 1 1.5 2 Relative Risk of Colorectal Cancer Long-Term Aspirin and NSAID Use Reduces The Risk of Developing Colorectal Cancer adapted from Thun et al, JNCI, 2002

35. Generic Name Brand Names Aspirin Made by several companies Ibuprofen Motrin®, Advil®, Motrin IB® Naproxen Naprosyn®, Aleve® Nabumetone Relafen® NSAIDs • Non Steroidal Anti-Inflammatory Drugs (NSAIDs) are the oldest and most widely used drugs in human history. • NSAIDs have pain-relieving (analgesic) effects as well as effects of reducing inflammation and fever. • NSAIDs are non-selective COX-1 and COX-2 enzymatic inhibitors. • Chronic NSAID use reduces CRC risk ~50% and reduce polyp size and number. • Extended NSAID use can lead to GI ulcers and renal problems.

36. COXIBs • COX-2 inhibitors (COXIBs) are a special category of NSAIDs that target only COX-2 enzyme. • Because they do not block the actions of the COX-1 enzyme, COXIBs generally have the side-effects of traditional NSAIDs. • COXIBs reduce CRC polyp burden by 31% in colon cancer patients. • COX-2 inhibitors also don’t offer the same kind of protection against heart disease.

37. Generic Name Brand Names Celecoxib Celebrex® Rofecoxib Vioxx® Valdecoxib Bextra  Purpose-designed selective inhibitors of COX-2: the COXIBs. Although rofecoxib (Vioxx) has been withdrawn, celecoxib (Celebrex) and valdecoxib (Bextra) remain on the US market. Lumiracoxib and etoricoxib remain under consideration by the FDA.

38. Schematic depiction of the structural differences between the substrate-binding channels of COX-1 and COX-2 that allowed the design of selective inhibitors Grosser, T. et al. J. Clin. Invest. 2006;116:4-15

39. COXIBs are Chemopreventive Agents

40. NSAIDs and COXIBs Inhibit Tumorigenesis

41. Arthritis Studies VIGOR Vioxx Vioxx associated w/higher risk of CV events CLASS Celebrex No difference in CV events Colon Cancer Studies APPROVe Vioxx 2-fold increase risk of CV-events. Vioxx removed from market. APC Celebrex 2.5-fold increase risk of CV-events. FDA recommends limited use of Celebrex and Bextra. PreSAP Celebrex No difference in CV events COXIBs are not without their problems… Summary of relevant trials examining CV events associated with extended COXIB use Study Drug Results

42. After 18 months increases CV events were detected and led to removal of Vioxx from the market. Confirmed Cardiovascular Events with Extended Vioxx Use The APPROVe study compared Vioxx vs. placebo in the prevention of colon cancer

43. You can call your lawyer… Or dust off the bottle of aspirin… Where do we go from here?