Potential Role of Selective COX-2 Inhibitors in Cancer Management

ABSTRACT: Tumorigenesis is a complex process, and understanding the mechanisms behind tumorigenesis is key to identifying effective targeted therapies. Prostaglandins are signaling lipophilic molecules derived from phospholipids that are involved in normal physiologic functions. However, overexpression of prostaglandins has been associated with tumorigenesis. Several epidemiologic studies have shown an inverse correlation between the incidence of colon cancer and the use of nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit prostaglandin synthesis. The NSAIDs target cyclooxygenases (COX), essential enzymes in prostaglandin production. Cyclooxygenase-2 (COX-2) is an inducible form of the enzyme that is usually not expressed in normal tissue. Because COX-2 is frequently overexpressed in premalignant lesions and neoplasms, specific COX-2 inhibitors have been investigated as chemoprevention and potential chemotherapeutic agents. There is now preclinical and early clinical data that suggest inhibitors of COX-2 may protect against colon, breast, lung, esophageal, and oral tumors. This paper will discuss evidence addressing the possible mechanistic contribution of COX-2 in tumorigenesis and will explore the link between COX-2 activity and carcinogenesis. The potential role of COX-2 inhibitors in the chemoprevention and treatment of various tumors will also be discussed. Clinical trials using targeted inhibitors of COX-2 will be critical in determining if COX-2 is a viable molecular target in cancer management. [ONCOLOGY 16(Suppl 4):30-36, 2002]

Prostaglandins and their derivatives aresignaling lipophilic molecules that are involved in diverse homeostatic andreactive functions, including platelet aggregation, clot formation,vasodilation, and gastric cytotoxic protection, as well as renal sodium andwater balance. The first step in the synthesis of prostaglandins is thehydrolysis of membrane phospholipids to arachidonic acid by phospholipase A₂.Arachidonic acid is then converted to an unstable product, prostaglandin G₂,which is rapidly converted to prostaglandin H₂ by the peroxidase activity of acyclooxygenase (COX). Prostaglandin H₂ is converted by tissue-specific enzymesto other prostaglandins and thromboxanes.

There are two isoforms of the COX enzyme encoded by twodifferent genes. Cyclooxygenase-1 (COX-1) is a constitutive enzyme that ispresent in most normal tissues and mediates the synthesis of prostaglandinsrequired for normal physiologic functions. The gene for COX-1 is on humanchromosome 9.[1] Cyclooxygenase-2 (COX-2) is an inducible form of the enzymethat is not normally detected in most tissues and is coded by a gene on humanchromosome 1.[2] Cyclooxygenase-2 is induced by cytokines, growth factors, tumorpromoters, and carcinogens.[3] Cyclooxygenase-2 is also induced by severaloncogenes, such as v-Src, v-Ha-ras, HER2/neu and Wnt.[4-7]

Although prostaglandins are involved in many normal physiologicfunctions, these molecules—and the COX enzymes involved in prostaglandinproduction—may also be involved in tumorigenesis. Indeed, there is evidencethat prostaglandins may contribute to tumorigenesis by inhibiting the immunesystem, stimulating cell growth, enhancing angiogenesis, increasing mutagenproduction, enhancing cell invasion, or inhibiting apoptosis. In this article,we will discuss these mechanisms and will explore the role of COX-2 intumorigenesis. We will also review clinical trials that have investigated COX-2inhibitors as chemoprotection agents and will discuss future directions in thestudy of COX-2 as a therapeutic target in cancer management.

Mechanisms of Prostaglandins in Carcinogenesis

Immunosuppressive Effects

Prostaglandins have a variety of immunosuppressive effects. Forexample, prostaglandin E₂ diminishes the cytotoxic activity of natural killercells, inhibits T-cell and B-cell growth, and decreases the production ofcytokines including tumor necrosis factor-alpha. Huang et al[8] showed thatpretreatment with a prostaglandin inhibitor prevents an increase ininterleukin-10 synthesis by peripheral blood lymphocytes. Furthermore,prostaglandins may also interfere with antigen processing by dendritic cells.[9]The ability of prostaglandins to inhibit the immune system may allow tumors togrow without surveillance and may contribute to tumorigenesis.

Mitogenic Effects

Enhanced prostaglandin synthesis may also contribute totumorigenesis by direct stimulation of cell growth. Data have shown thatprostaglandin E₂ and prostaglandin F₂-alpha can be mitogenic in BALB/c3T3fibroblasts in the presence of epidermal growth factor.[10] Furthermore,proliferation of mammary epithelial cells can be stimulated in the presence ofepidermal growth factor by prostaglandin E₁ and prostaglandin E₂.[11] In breasttissue, prostaglandins may stimulate cell growth by stimulating the aromatasegene, CYP19, and thus enhancing estrogen synthesis.[12,13] Interestingly,enhanced expression of CYP19 and COX has been found in human breast cancerspecimens.[14]

Inhibition of Apoptosis

Prolonged survival of abnormal cells favors the accumulation ofgenetic changes that may result in tumor formation. Therefore, inhibition ofapoptosis may increase the tumorigenic potential of initiated cells. Sheng etal[15] demonstrated that prostaglandin E₂ may inhibit apoptosis by inducingbcl-2. Recently it was shown that inhibition of COX-2 by celecoxib (Celebrex), aselective COX-2 inhibitor, resulted in a decrease of production of prostaglandinE₂ and TXB₂, and was associated with an increase in apoptosis in vivo.[16]

Mechanisms of Cyclooxygenase in Carcinogenesis

Effects on Metastatic Potential

Enhanced COX-2 expression may also contribute to tumorigenesisby increasing cell invasiveness. DuBois et al[17] showed that COX-2overexpression in rat intestinal epithelial cells increases cell adhesion to theextracellular matrix. Recently, Stockton and Jacobson[18] demonstrated thatCOX-2 is required for NIH3T3 cell migration in a process that appears to beregulated by the extracellular signal-regulated kinase 1/2. Furthermore, theactivity of enzymes responsible for digesting cellular basement membrane isenhanced by COX-2 overexpression in the breast cancer cell line Hs578T and coloncancer cells, which likely contributes to the increased ability of these cellsto invade through a layer of Matrigel.[19,20]

Increased Production of Mutagens

Another mechanism by which COX-2 overexpression may play a rolein carcinogenesis is an increase in the production of mutagens. One such potentmutagen is malondialdehyde, which can be produced by isomerization ofprostaglandin H₂. Malondialdehyde acts by forming adducts with deoxynucleotides,which cause frame-shifts and base-pair substitutions.[21] In addition, Wiese etal[22] showed that the peroxidase activity of cyclooxygenases can catalyze theformation of mutagens by the oxidation of aromatic amines, heterocyclic amines,and dihydrodiol derivatives of polycyclic hydrocarbons. Thus, COX-2overexpression may lead to DNA damage that may eventually lead tocarcinogenesis.

Angiogenesis

Cyclooxygenase-2 has also been implicated in enhancedangiogenesis, which plays a role in carcinogenesis. Tumor growth depends onincreased blood supply via secretion of angiogenic promoters such as vascularendothelial growth factor. Cyclooxygenase-2 overexpression in colon cancer cellsis correlated with increased production of vascular growth factors and formationof capillary networks.[23] This angiogenic effect of COX-2 can be blocked byNS398, a selective COX-2 inhibitor. Williams et al[24] demonstrated that tumorformation is markedly decreased in COX-2 knockout mice compared with wild-typemice. Furthermore, the pharmacologic inhibition of COX-2 leads to a decrease invascular endothelial growth factor production that may contribute to a decreasein tumor formation. Masferrer et al[25] showed that celecoxib, a selective COX-2inhibitor, blocks corneal blood vessel formation in a rat model. Thus, COX-2overexpression may increase tumor blood supply and may contribute to tumorgrowth.

Further Links Between COX-2 and Tumorigenesis

Cyclooxygenase-2 is upregulated in multiple human premalignantand malignant conditions, including tumors of the colon, breast, stomach, lung,pancreas, cervix, prostate, bladder, liver, skin, head and neck, and esophagus (Table1).[26-40] There are multiple lines of evidence suggesting a link betweenlevels of COX-2 and tumorigenesis. Increased levels of COX-2 are detected inpremalignant intestinal tumors in experimental animal models.[41] The knockoutof the COX-2 gene led to a marked reduction in the size and number of polyps inAPCD⁷¹⁶ mice. In addition, APCD⁷¹⁶ mice treated with a selective COX-2 inhibitorhad reduced adenoma formation.[42] Further, COX-2 knockout mice develop fewerskin papillomas than control mice.[43] Taken together, these results suggestthat inhibition of COX-2 could be important in the prevention of a variety ofepithelial tumors.

Synthetic and naturally occurring inhibitors of COX (eg,sulindac [Clinoril], ibuprofen, flurbiprofen [Ansaid], indomethacin [Indocin])have also been shown to protect against mammary, colon, oral, and esophagealcancer in experimental animals.[44-50] For example, studies have shown thatflurbiprofen, an inhibitor of COX-1 and COX-2, can inhibit the growth oftransplanted mammary tumors[51] and increase the mean survival duration inmice.[52] In addition to having therapeutic activity against established mammarytumors, flurbiprofen inhibits mammary carcinogenesis induced by a low dose ofN-methyl-N-nitrosourea in rats.[46] Other data have also demonstrated thatindomethacin, another inhibitor of both COX-1 and COX-2, has significantchemoprotective activity in rats when administered during either the early orlate stage of mammary tumorigenesis.[47] Importantly, epidemiologic studies haveshown that chronic intake of nonsteroidal anti-inflammatory drugs (NSAIDs)reduces the incidence of various human cancers, including cancers of the colon,breast, lung, esophagus, stomach, and bladder.[53-60]

Celecoxib has been evaluated as a possible chemopreventive agentfor the inhibition of tumorigenesis. In a study to evaluate the inhibitoryactivity of celecoxib against azoxymethane-induced aberrant crypt foci formationin the colon of rats, celecoxib significantly suppressed the total number ofaberrant crypt foci in rats compared with placebo.[61] In a second study ofazoxymethane-treated rats, celecoxib reduced the incidence of colon cancer by93% and tumor multiplicity by 97%.[62] These animal data provide strong evidencethat celecoxib has cancer activity in the prevention of cancer when tested in adefined model of tumorigenesis.

Recently, Harris et al[63] compared the chemoprevention effectsof celecoxib with ibuprofen and placebo in the development and growth of7,12-dimethyl-benz(a)anthracene (DMBA)-induced rat mammary tumors. Seven daysprior to receiving a single dose of 15 mg of DMBA by intubation, rats were fedeither a control diet or diets containing 1,500 ppm of celecoxib or 1,500 ppm ofibuprofen. Both celecoxib and ibuprofen significantly increased tumor latency,reduced tumor burden, and prevented tumor formation compared with placebo.However, celecoxib was more potent than ibuprofen. At 105 days after DMBAtreatment, tumor incidence was 100% in control rats compared with 32% and 60% inrats fed either celecoxib (P < .001) or ibuprofen (P < .001),respectively. The control rats had an average of 3.2 tumors compared with 0.4and 1.5 tumors in rats treated with celecoxib (P < .001) and ibuprofen(P< .001), respectively. Additionally, the average tumor volume was 1.5 cm³ incontrol rats vs 0.3 cm³ and 0.6 cm³ in rats treated with celecoxib (P < .001)or ibuprofen (P < .001), respectively. The higher potency of celecoxibsuggests that this agent may have an in vivo advantage over other NSAIDs as achemoprevention agent.[63]

The DMBA-induction model was also used to evaluate celecoxib forefficacy against established tumors. Alshafie et al[64] examined the effect ofcelecoxib, given as a daily diet (1,500 ppm), on the growth of established DMBA-inducedtumors in rats over a 6-week treatment period. Tumors in the control groupcontinued to grow; whereas tumors in the celecoxib group markedly decreased insize. The average reduction in tumor volume relative to baseline wasapproximately 32% (P < .04). At the end of the 6-week treatment period,average tumor volume was 1.45 cm³ and 0.13 cm³ in the control and celecoxibgroups, respectively. Tumor volume regression occurred in 90% of rats treatedwith celecoxib.[64] These results suggest that celecoxib has potent antitumoractivity and chemoprevention activity in this rat mammary carcinoma model.

Clinical Trials Using COX-2 Inhibitor

Chemoprevention Trials

Familial adenomatous polyposis (FAP) is a rare disease thataccounts for approximately 1% of colorectal carcinomas annually. In individualswith FAP, numerous reports have documented the chemoprevention effects ofsulindac and other NSAIDs on existing adenomas. Waddell et al[65] was the firstto describe near-complete regression of colorectal adenomas in four patientstreated with sulindac. Subsequent studies have confirmed significant reductionsin colorectal adenomas in individuals treated with sulindac.[66]

Recently, celecoxib was evaluated in a randomized, double-blind,placebo-controlled trial in 77 FAP patients.[67] In this study, patients wererandomized to receive 100- or 400-mg celecoxib twice daily or placebo for 6months. Patients treated with 100-mg celecoxib experienced reductions in thenumber of colorectal polyps and total polyp burden, although these changes didnot reach statistical significance. Patients treated with 400-mg celecoxibachieved a 28% reduction in the number of colorectal polyps vs a 4.5% reductionin patients treated with placebo (P = .003) (Figure1).[67] Additionally, thetotal polyp burden (sum of polyp diameters) was significantly lower in thosepatients treated with 400-mg celecoxib compared with patients in the placebogroup (30.7% vs 4.9%, P = .001). Furthermore, a higher proportion of patientstreated with 400-mg celecoxib experienced at least a 25% reduction in polypnumber compared with patients treated with placebo (53% versus 7%, P = .003).Celecoxib at 400 mg twice daily also improved the endoscopic appearance of boththe colorectum and duodenum of patients with FAP.

These results in humans are consistent with previous resultsusing selective COX-2 inhibitors to prevent intestinal tumors in experimentalanimals.[42,62] Based on the results of this trial, celecoxib was approved forthe treatment of FAP.

Because of the similarities in the biology of sporadiccolorectal cancer and FAP, agents that are effective in FAP may also be usefulfor chemoprevention of sporadic colorectal adenomas. Chemoprevention of sporadiccolorectal tumors with NSAIDs has been evaluated in three small, uncontrolledstudies, with mixed results.[68-70] Only one large, placebo-controlled trial hasinvestigated NSAIDs for chemoprevention of sporadic colorectal cancer thus far.The Physicians’ Health Study investigated whether aspirin was effective, butshowed no significant difference in self-reported frequency of new colorectalmalignancies in patients treated with aspirin (325 mg every other day for 5years) compared with placebo.[71] Presently, several ongoing clinical trials areevaluating the efficacy of selective COX-2 inhibitors for the prevention ofsporadic colorectal adenomas.[72] Because the majority of colorectal cancersarise from preexisting adenomas, results from these trials are highlyanticipated.

Several lines of data suggest that COX-2 inhibitors may beeffective in other diseases including Barrett’s esophagus, oral leukoplakia,actinic keratoses, and bladder cancer. Cyclooxygenase-2 is overexpressed inBarrett’s esophagus and oral leukoplakia,[39,40] and NSAIDs have been shown toprevent esophageal and oral cancers in animal models.[45,73] Preclinical datahave also shown that COX-2 inhibitors protect against ultraviolet light-inducedskin cancer formation in mice,[74] suggesting that a COX-2 inhibitor may beeffective in the treatment of actinic keratoses, a premalignant skin lesion.Furthermore, there are clinical data demonstrating the benefit of the topicalNSAID diclofenac (Solaraze) in the treatment of actinic keratoses.[75]Cyclooxygenase-2 is also overexpressed in bladder cancer,[36] and selectiveCOX-2 inhibitors have been shown to protect against bladder tumorigenesis inanimal models.[76]

Based on these data, the National Cancer Institute has initiatedseveral clinical trials to further investigate the use of selective COX-2inhibitors as chemoprevention agents in these diseases (Table2). The results ofthese ongoing clinical trials will be important in determining the role of COX-2inhibitors in the prevention of certain cancers.

Chemotherapeutic Trials

Cyclooxygenase-2 inhibitors are also actively being investigatedas chemotherapeutic agents. Many preclinical studies have demonstrated thatCOX-2 inhibitors delay tumor progression but have less of an effect on tumorregression. However, COX-2 inhibitors may enhance the effects of standardanticancer therapy. Several preclinical studies have demonstrated thesynergistic antitumor efficacy of a COX-2 inhibitor when given concurrently withchemotherapy or radiation.[77,78]

Recently, Subbaramaiah et al[79] have demonstrated a linkbetween the overexpression of the HER2/neu oncogene and the upregulation ofCOX-2 in human breast cancer. In this study, increased expression of COX-2 isdetected in nearly 100% of HER2/neu-overexpressing human breast cancers. Incontrast, COX-2 is only expressed at very low levels in a minority of cases ofHER2/neu-normal breast cancer. This is the first study to demonstrate a clearlink between overexpression of HER2/neu and upregulation of COX-2 in breastcancer.

To elucidate the mechanism by which HER2/neu regulates COX-2expression, a cell culture model was used. Increased levels of prostaglandin E2production, COX-2 protein, and COX-2 mRNA were detected in HER2/neu-transformedhuman mammary epithelial cells (184B5/HER) compared with their nontransformedcounterparts (184B5).[79] Based on this study, a clinical trial is under way toassess the combined efficacy of celecoxib and trastuzumab (Herceptin) inpatients with metastatic HER2/neu-overexpressing breast cancer.

Future Directions

Significant progress has been made in our understanding of COX-2and its role in tumorigenesis. As noted, COX-2 overexpression and prostaglandinsynthesis may contribute to tumorigenesis via increased cellular proliferation,diminished immune surveillance, decreased apoptosis, enhanced cell invasiveness,increased mutagen production, and effects onangiogenesis,[6,8-11,13,14,17-25,80] although the relative importance of each ofthese effects is unknown. Clinical trials are ongoing to evaluate specific COX-2inhibitors for cancer chemoprevention in the colorectum, esophagus, skin,prostate, and bladder. Based on the available data, clinical studies should alsobe considered to evaluate COX-2 inhibitors in the prevention of cancer of thelung, pancreas, cervix, liver, stomach, and breast because COX-2 is alsooverexpressed in tumors involving these organs.[28-34,37]

Combination therapy with established chemotherapeutic agents andCOX-2 inhibitors also should be explored. Several preclinical reports have shownthat taxanes can induce COX-2 expression.[81-83] Thus, the upregulation of COX-2may decrease the antitumor efficacy of taxanes and, perhaps, the combination ofa COX-2 inhibitor with taxanes may provide additive or synergisticantineoplastic effects. Furthermore, the arthralgia/myalgia syndrome that isoften noted in patients treated with taxanes may also be ameliorated with aCOX-2 inhibitor.

Cyclooxygenase-2 inhibitors are relatively safe in humans andare an exciting new class of drugs with potential for cancer management, eitheralone or in combination with standard therapies. However, the exact role thatCOX-2 inhibitors may play in the current armamentaria of anticancer agentsremains to be fully elucidated.

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