Inflammation and CancerThe Significance of COX-2

Dan Dixon

Dept. of Biological SciencesSouth Carolina Cancer Center

ddixon@biol.sc.edu

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

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.

the world’s first man-made cancer on the ears of a rabbitRudolf Virchow

Time, Feb. 23, 2004

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.

Monocyte

lymphocyte

neutrophil

platelets

eosinophil

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.

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.

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.

• 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.

The Roles of Tumor-Associated Macrophages in Tumor Progression

Chronic Inflammation is a Risk Factor for Cancer

Ulcerative colitis (UC) AdenocarcinomaChronic

InflammationDysplasia

• 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.

Normal Colon Ulcerative colitisColon

Adenocarcinoma

Ulcerative Colitis and Increased Risk of Colorectal Cancer Over Time

Eaden JA, et al. Gut. 2001;48:526. Overall prevalence of CRC in any UC patient is 3.7%

Time from diagnosis (years)

UC

0 5 10 15 20 25 30

5

10

15

20

25

1

Cum

ulat

ive

prob

abili

ty (

%)

Inflammatory Syndromes and Their Associated Cancers

Inflammatory bowel disease Colorectal cancer

Gastric intestinal metaplasia Gastric cancer

Barrett’s esophagus Esophageal cancer

Chronic hepatitis Hepatocellular carcinoma

Chronic pancreatitis Pancreatic cancer

Oral leukoplasia Head/neck cancer

Atypical adenomatous hyperplasia Non-small cell lung cancer

Ductal carcinoma in situ Breast cancer

Prostatic intraepithelial neoplasia Prostate cancer

Bladder dysplasia Bladder cancer

Cervical dysplasia Cervical cancer

Actinic keratoses Skin cancer

Chronic Inflammation Promotes Tumor Development

mutation

initiated epitheliumepithelial cells

Promotion

benign

Inflammatory cells express factors that stimulate cell

growth and progression

carcinoma

Malignant conversion

Chronic activation promotes continued inflammation, angiogenesis, and ECM remodeling

Insulted stromal cells recruit activated

inflammatory cells

Activated monocyte/macrophages

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-TNFAntibodies: Effective in controlling IBD. Regulates TNF 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.

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.

COOH

Arachidonic Acid

Cycloxygenase Lipoxygenase Epoxygenase

Prostaglandins (PGs)Thromboxanes (TXs)

Leukotrienes (LTs)Lipoxins (LXs)

Epoxy Acids (EETs)Dihydroxy Acids

Cellular Arachidonic Acid Metabolism

ProstaglandinsLeukotrienes

Cyclooxygenase Activity is a Key

Step in Prostaglandin

Synthesis

orCOX

PGE2, PGF, and PGI2

RELAX VASCULARSMOOTH MUSCLE

PGE2 and PGI2

INCREASERENAL BLOOD FLOW

PGE2 and PGI2

RELAX BRONCHIALSMOOTH MUSCLE;

PGF CONTRACTS IT

PGE2 and PGF

CONTRACT UTERINESMOOTH MUSCLE;PGI2 RELAXES IT

PGE2 and PGI2

PROTECTGASTRIC MUCOSA

TxA2 PROMOTESPLATELET AGGREGATION;

PGI2 INHIBITS 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

Prostaglandin Signaling Mechanisms

Prostaglandins control cellular function through

G-coupled membrane receptors and nuclear

receptors

Prostaglandin Signaling Promotes Cell Growth

Prostaglandins Trigger Signal Transduction

Pathways Associated with Cancer Growth

Increased PGE2 Promotes Many Facets of Tumorigenesis

VascularAngiogenesis

ImmuneSuppression

ReducedApoptosis

ProliferationMotility

VEGF

PGE2

MetastasisInvasion

MMP-2 MMP-9 BCL-2

PI3-KActivation

IL-10 IL-12

Multiple COX Enzymes Exist in Cells

COX-1: Constitutively present

COX-2: Inducible isoform

“COX-3”: Splice variant of COX-1

Feature COX-1 COX-2

Expression Constitutive Inducible

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 withcolon, most tissues cytokines, growth factors, or tumor promoters

Gene Regulation Constitutive low-level expression Immediate-early response gene, rapidly transcribed,mRNA is rapidly degraded, translation is controlled

Differing Functions of COX-1 & COX-2

Arachidonic Acid

COX-1(constitutive)

Homeostasis• Stomach/GI protection• Platelet aggregation• Renal blood flow

COX-2(inducible)

Pathophysiology• Inflammation, Pain• Fever• Cancer• Morbus Alzheimer• Ischemia (CNS)

Evidence of a COX-2 Dependent Role in NeoplasiaEpidemiological Studies

Decreased risk of CRC-associated deaths in aspirin users. The NSAID sulindac decreases the size and number of polyps (FAP). Prostaglandin levels are increased in CR tumors. Overexpression of COX-2 detected in adenomas and adenocarcinomas.

Animal Studies Min mice and AOM-treated rats have elevated COX-2 levels. Sulindac and other NSAIDs attenuate intestinal tumor and xenografted cancer cell growth in mice.

Cellular Studies 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 Mice defective in COX-2 have a dramatic reduction (86%) in colorectal polyp formation.

Normal colon epitheliumCOX-2 protein staining in colon tumor

COX-2 is Overexpressed in Colon Cancer

COX-2 is Overexpressed

in Multiple Components of

Cancer

Increased COX-2 = Increased Prostaglandins

COX-2-derived PGs promote cell growth, proliferation, and angiogenic gene expression in cells composing a tumor

Genetic “Removal” of

COX-2 Suppresses

Tumorigenesis

COX-2Genotype

COX-2 gene dosage effect on polyp number

COX-2

COX-2COX-2

Tot

al P

olyp

Num

ber/

Mou

se

(100%)

(34%)

(14%)

Involvement of COX-2 in the Progression of Colon Cancer

NormalEpithelium

CarcinomaEarly Adenoma Late Adenoma

APCmutation

TGFß IIRhMSH

K-rasmutation

p53mutation

- COX-2 +/- COX-2 ++ COX-2 +++ COX-2

Loss of COX-2 gene regulation

COX-2 overexpression

Increased PG levels

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.

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.

Long-Term Aspirin and NSAID Use Reduces The Risk of Developing Colorectal Cancer

0 0.5 1 1.5 2

Relative Risk of Colorectal Cancer

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

Coogen 2000A

Coogen 2000B

Friedman 1998

adapted from Thun et al, JNCI, 2002

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.

Generic Name Brand Names

Made by several companies

Motrin®, Advil®, Motrin IB®

Naprosyn®, Aleve®

Relafen®

Aspirin

Ibuprofen

Naproxen

Nabumetone

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.

 Purpose-designed selective inhibitors of COX-2: the COXIBs.

Generic Name Brand Names

Celebrex®

Vioxx®

Celecoxib

Rofecoxib

Valdecoxib Bextra

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.

Grosser, T. et al. J. Clin. Invest. 2006;116:4-15

Schematic depiction of the structural differences between the substrate-binding channels of COX-1 and COX-2 that allowed

the design of selective inhibitors

COXIBs are Chemopreventive Agents

NSAIDs and COXIBs Inhibit Tumorigenesis

Study Drug Results

Arthritis StudiesVIGOR Vioxx

CLASS Celebrex

Vioxx associated w/higher risk of CV events

No difference in CV events

Colon Cancer StudiesAPPROVe

APC

PreSAP

Vioxx

Celebrex

Celebrex

2-fold increase risk of CV-events.Vioxx removed from market.

2.5-fold increase risk of CV-events.FDA recommends limited use of Celebrex and Bextra.

No difference in CV events

COXIBs are not without their problems…Summary of relevant trials examining CV events associated with extended COXIB use

The APPROVe study compared Vioxx vs. placebo in the prevention of colon cancer

Confirmed Cardiovascular Events with Extended Vioxx Use

After 18 months increases CV events were detected and led to removal of Vioxx from the market.

Where do we go from here?

You can call your lawyer…

Or dust off the bottle of aspirin…