Cyclooxygenase-2 in oncogenesis

Clinica Chimica Acta 412 (2011) 671–687

Contents lists available at ScienceDirect

Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m

Invited critical review

Cyclooxygenase-2 in oncogenesis Maria Teresa Rizzo Signal Transduction Laboratory, Methodist Research Institute, Clarian Health and Department of Pharmacology, Indiana University School of Medicine, Indianapolis, IN, United States

a r t i c l e

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Article history: Received 9 November 2010 Received in revised form 20 December 2010 Accepted 21 December 2010 Available online 25 December 2010 Keywords: Cyclooxygenase-2 Tumors Prostaglandin E2 Mechanisms Signaling pathways

a b s t r a c t Compelling experimental and clinical evidence supports the notion that cyclooxygenase-2, the inducible isoform of cyclooxygenase, plays a crucial role in oncogenesis. Clinical and epidemiological data indicate that aberrant regulation of cyclooxygenase-2 in certain solid tumors and hematological malignancies is associated with adverse clinical outcome. Moreover, findings extrapolated from experimental studies in cultured tumor cells and animal tumor models indicate that cyclooxygenase-2 critically influences all stages of tumor development from tumor initiation to tumor progression. Cyclooxygenase-2 elicits cell-autonomous effects on tumor cells resulting in stimulation of growth, increased cell survival, enhanced tumor cell invasiveness, stimulation of neovascularization, and tumor evasion from the host immune system. Additionally, the oncogenic effects of cyclooxygenase-2 stem from its unique ability to impact tumor cell surroundings and create a proinflammatory environment conducive for tumor development, growth and progression. The initial enthusiasm generated by the availability of cyclooxygenase-2 selective inhibitors for cancer prevention and therapy has been lessened by the severe cardiovascular adverse side effects associated with their long-term use, as well as by the mixed results of recent clinical trials evaluating the efficacy of cyclooxygenase-2 inhibitors in adjuvant chemotherapy. Therefore, our ability to efficiently target the oncogenic effects of cyclooxygenase-2 for therapeutic and preventive purposes strictly depends on a better understanding of the spatial and temporal aspects of its activation in tumor cells along with a clearer elucidation of the signaling networks whereby cyclooxygenase-2 affects tumor cells and their interactions with the tumor microenvironment. This knowledge has the potential of leading to the identification of novel cyclooxygenase-2dependent molecular and signaling networks that can be exploited to improve cancer prevention and therapy. © 2010 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular and biochemical features of COX-2 . . . . . . . . . . . . 2.1. COX-2 gene . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. COX-2 protein. . . . . . . . . . . . . . . . . . . . . . . . 2.3. Intracellular localization . . . . . . . . . . . . . . . . . . . 2.4. Substrates. . . . . . . . . . . . . . . . . . . . . . . . . . COX-2 overexpression in solid tumors and hematological malignancies Mechanisms of COX-2 contribution to tumorigenesis . . . . . . . . . 4.1. Contribution of COX-2 to tumor initiation. . . . . . . . . . . 4.2. Contribution of COX-2 to tumor promotion . . . . . . . . . . 4.3. Contribution of COX-2 to metastatic disease. . . . . . . . . . Mechanisms regulating COX-2 expression and activity in tumor cells . 5.1. Regulation of COX-2 transcription . . . . . . . . . . . . . . 5.2. Regulation of COX-2 translation . . . . . . . . . . . . . . . 5.3. Regulation of COX-2 degradation . . . . . . . . . . . . . . . Oncogenic signaling pathways activated by COX-2 . . . . . . . . . . 6.1. PGE2-dependent signaling in tumor growth . . . . . . . . . . 6.2. PGE2-dependent signaling in angiogenesis . . . . . . . . . . 6.3. PGE2-dependent signaling in tumor invasion and metastasis . .

E-mail address: [email protected]. 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.12.026

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6.4. PGE2-dependent signaling in tumor survival . . . . . . . . . . 6.5. PGE2-dependent signaling pathways in tumor immune tolerance 7. Mechanism of action and antitumoral effects of COX-2 inhibitors . . . 8. Clinical use of COX-2 inhibitors . . . . . . . . . . . . . . . . . . . 9. Concluding remarks and future perspectives . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Cyclooxygenase-2 (COX-2), or prostaglandin endoperoxide synthase-2 (PGES-2), is the inducible isoform of cyclooxygenase (COX), the rate-limiting enzyme that catalyzes the initial step of arachidonic acid metabolic transformation into prostanoids. Conversely, cyclooxygenase-1 (COX-1), or prostaglandin endoperoxide synthase-1 (PGES-1), represents the constitutive isoform of COX [1]. Both isoforms belong to the myeloperoxidase family and are distantly related to other fatty acid oxygenases present in lower organisms and plants [2,3]. The presence of an inducible isoform of cyclooxygenase was initially suggested by the work of Levine et al. who reported a transient increase in the production of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) in canine kidney cells following stimulation with mitogens, tumor promoters, and carcinogens [4–6]. Importantly, the production of these newly synthesized prostaglandins was blunted by protein synthesis and transcription inhibitors, suggesting the requirement for the de novo synthesis of a new cyclooxygenase activity distinct from the constitutive activity of COX1 [5,6]. In 1991, two independent groups reported the discovery of an inducible immediate response gene that shared high homology with COX. Xie et al. identified and characterized a pp60-vsrc-inducible early response gene from chicken embryo fibroblasts [7]. This gene was homologous to a 12-O-tetradecanoylphorbol-13-acetate (TPA)sensitive gene called TPA-inducible-sequence clone 10 (TIS10), which was isolated and cloned by Herschman's laboratory from mitogenstimulated Swiss 3T3 fibroblasts [8]. Both genes encoded a protein that shared 60% homology with COX-1. Moreover, cells transfected with the TIS10 expression vector displayed higher levels of COX activity and produced elevated amount of PGE2 [9]. Hla et al. later reported the cloning, sequencing and expression of the inducible cyclooxygenase gene in human cells, which was named COX-2 [10]. Subsequent work demonstrated that COX-1 and COX-2 are differentially regulated [11]. In striking contrast to the constitutive expression of COX-1, expression levels of COX-2 transiently increased in cells stimulated by proinflammatory cytokines [12]. Together, these observations led to the notion that while COX-1 contributes to homeostatic cellular functions [1], COX-2 contributes to disease processes, including hyperalgesia, inflammation, and cancer [13,14]. The roles and cellular outcomes of COX-1 and COX-2 are, however, more complex than what initially postulated. For example, COX-2 is constitutively expressed in certain organs and tissues where regulates physiological responses [15–17], while COX-1 can be upregulated in response to cell injury and cell differentiation [18,19]. Results from studies carried out in COX-1 or COX-2 knockout mice further underscore the complex role of COX-2 and COX-1 in cellular functions [20]. For example, despite its role in induction of inflammation, COX-2 is also required for the resolution of the inflammatory response [20]. The discovery of COX-2 opened a new frontier in cancer research and spurred intensive investigations that led to our current understanding of its contribution to tumorigenesis. Following the initial reports of COX-2 overexpression in colorectal cancer [21], several other epithelial tumors were found to constitutively express COX-2 [22–26]. Moreover, recent evidence indicates that COX-2 is overexpressed in several hematological malignancies [27], suggesting, therefore, a broader involvement of COX-2 in tumorigenesis. This

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review summarizes the salient molecular and biochemical features of COX-2 and its contribution to oncogenesis. Emphasis is placed on reviewing recent information pertaining to the mechanisms and signaling pathways whereby COX-2 contributes to tumorigenesis. The implication of COX-2 inhibition for cancer chemoprevention and therapy is discussed. 2. Molecular and biochemical features of COX-2 2.1. COX-2 gene The gene encoding for human COX-2 is located on chromosome 1 (1q25.2–q25.3) and shares about 60% homology with COX-1 [28,29]. The COX-2 gene is approximately 8.3 kb long, consists of 10 exons and 9 introns, and is transcribed into three mRNA polyadenylated variants of 2.8, 3.2, and 4.6 kb, which are expressed in a tissue- and stimulus specific manner [29]. Additional COX-2 gene variants are produced by mRNA alternative splicing and single nucleotide polymorphism [30,31]. At the present time, the significance of these variants is poorly understood, although there is evidence that they might have important pathophysiological roles [31]. In contrast to the COX-1 promoter, the promoter region of COX-2 possesses features of an immediate early response gene. Accordingly, the 5'-untranslated region (UTR) contains a TATA box, a CCATT/enhancer binding protein (C/EBP), and putative binding sites for several transcription factors, including, among others, the cAMP response element binding protein (CREB), the nuclear factor kB (NF-kB), the activating protein-1 (AP-1), the nuclear factor for interleukin-6 (NF-IL6), and the nuclear factor for activated T-lymphocytes (NFAT) [32]. The 3'-UTR contains several AUrich elements (AREs) that are responsible for the rapid degradation and short half-life of COX-2 mRNA [33]. 2.2. COX-2 protein The COX-2 gene encodes for a homodimeric protein of 604 amino acids in size, which is highly similar in structure and enzymatic activity to COX-1 [34]. X-ray crystallography analyses revealed the presence of several functional domains within the N-terminus and C-terminus of COX-2 monomers, which regulate COX-2 cellular localization, membrane anchoring, and catalytic activity (Fig. 1) [34]. Starting from the N-terminus, the hydrophobic signal peptide domain directs nascent COX-2 polypeptides into the lumen of the endoplasmic reticulum (ER). Notably, the signal peptide domain of COX-2 is shorter of 17 amino acids compared to the signal peptide domain of COX-1. The dimerization domain contains an epidermal growth factor (EGF)-like module and is responsible for the formation of COX-2 homodimers. The membrane-binding domain is composed of four short amphipathic α helices that anchor COX-2 to only one leaflet of the lipid bilayer in the luminal side of the ER and in the outer and inner membranes of the nuclear envelope. The globular catalytic domain, which constitutes the bulk of the COX-2 protein, is formed by two distinct interconnecting lobes that contain the cyclooxygenase active site and the peroxidase active site, which are separated by the heme prosthetic group. The cyclooxygenase site is a long, narrow and dead-end hydrophobic channel that extends into the globular

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Fig. 1. Schematic representation of the human COX-2 primary protein structure.

catalytic domain. The entrance of the channel is framed by the four amphipathic helices of the membrane-binding domain. The catalytic site of COX-2 is 17% wider than the catalytic site of COX-1 channel because of the substitution of isoleucine at position 523 in COX-1 to valine at position 509 in COX-2. The wider catalytic site of COX-2 allows the binding of bulkier substrates and COX-2 selective inhibitors [35]. The catalytic domain contains several functional amino acids, including Tyr-371 a conserved residue that is essential for catalysis [34]. The peroxidase active side is shaped as a cleft away from the membrane of the ER lumen [34]. At the end of the C-terminus, COX-2 contains an instability motif of 27 amino acids, which is involved in regulation of COX-2 degradation, and a four amino acid sequence that constitutes the ER retention signal [36]. Similar to COX-1, COX-2 undergoes to post-translational modifications consisting mainly in N-glycosylation at several asparagine residues [37]. There are at least four potential N-glycosylation sites (Asp53, Asp130, Asp396, and Asp580) distributed on the catalytic domain of COX-2. N-glycosylation at Asp53, Asp130 and Asp396 regulates the folding of COX-2 into an active conformation of 72 kDa [37]. Nglycosylation of Asp580 is variable, and when present it gives rise to a COX-2 protein of 74 kDa [37]. The significance of N-glycosylation at Asp580 is not well defined. However, Sevigny et al. have detected an increase of COX-2 catalytic activity associated with the removal of Nglycosylation at Asp580 [38]. Whether tumor cells preferentially express an isoform of COX-2 that is not glycosylated at Asp580 is not known. In addition to N-glycosylation, COX-2 undergoes to S-nitrosylation by nitric oxide and nitric oxide synthase. Kim et al. elegantly demonstrated a physical interaction between the inducible nitric oxide synthase (iNOS) and COX-2 resulting in COX-2 S-nitrosylation at cysteine 256 [39]. Importantly, this modification enhances COX-2 catalytic activity [39]. Given that the production of nitric oxide by iNOS increases during inflammatory processes, the interaction between iNOS and COX-2 could represent a relevant mechanism of COX-2 activation during inflammation-driven tumorigenesis.

2.3. Intracellular localization As shown in Fig. 2, COX-2 is mainly located in the lumen of the ER, where the oxidizing environment favors its proper dimerization, and in the nuclear envelops [40]. The nuclear localization of COX-2 is consistent with its regulatory role in mitogenesis. The presence of nuclear receptors, which have been shown to mediate some of the mitogenic effects of COX-2-derived prostaglandins, further underscore the functional relevance of COX-2 nuclear localization [41]. There is evidence that, in tumor cells, COX-2 localizes in the mitochondria and lipid bodies [42,43] (Fig. 2). The intracellular location of COX-2 to the mitochondria plays an important role in the protection against oxidative stress-induced apoptosis [42], while the COX-2 location in the lipid bodies critically influences tumor growth,

likely by functioning as an additional intracellular source for the continuous supply of prostaglandins [43].

2.4. Substrates Arachidonic acid, a 20-carbon chain polyunsaturated fatty acid that contains four cis double bonds at positions 5, 8, 11, and 14, is the main substrate of COX-2 [44] (Table 1). Free arachidonic acid is released from the sn-2 position of membrane phospholipids upon agonist-dependent activation of cellular phospholipases [44]. Once released, arachidonic acid binds to the Ser516 in the catalytic loop of COX-2 [34]. The subsequent cyclooxygenase reaction is initiated by oxidant- and heme-dependent activation of COX-2 at the conserved Tyr371 residue and leads to the insertion of 2 moles of oxygen into 1 mole of arachidonic acid to yield the cyclopentane hydroperoxy endoperoxide, prostaglandin G2 (PGG2) [34,44] (Fig. 2). PGG2 is then incorporated into the peroxidase site of COX-2 where it is reduced into the unstable intermediate PGH2 [34,44] (Fig. 2). PGH2 diffuses through the membranes of the ER into the cytosol where it serves as a substrate for tissue-selective PG synthases, whose activation is directly responsible for the synthesis of structurally related prostaglandins, including PGE2, prostaglandin D2 (PGD2), PGF2α, prostacyclin (PGI2), and thromboxane A2 (TXA2) (Fig. 2) [44]. Alternatively, PGH2 can be transformed into malondialdehyde, a metabolite that possesses mutagenic properties [45]. Other polyunsaturated fatty acid derivatives that contain cis double bonds at positions 8, 11 and 14 can function as COX-2 substrates (Table 1). Endocannabinoids, including 2-arachidonyl glycerol and anandaminde (arachidonoyl ethanolamide), which do not bind to COX-1, but selectively bind to COX-2 because of the larger side pocket near the base of its active site of COX-2, are oxidized by COX-2 and generate hydroxyl endoperoxides analogous to PGH2 [46]. These PGH2 analogues are good substrates for all PG synthases with the exception of TXA2 synthase [46]. COX-2-dependent oxidation of endocannabinoids generates prostaglandin derivatives that in contrast to those generated from arachidonic acid oxidation exert mainly anti-inflammatory and anti-proliferative effects [46]. Moreover, COX2 acetylated by aspirin at Ser516 is able to oxidize endocannabinoids to yield 15-HETE derivatives [47]. An additional COX-2 selective substrate is 5S-HETE, which is mainly produced from oxidation of arachidonic acid by 5-lipooxygenase, the committed pathway for the formation of leukotrienes [44]. However, because of the presence of cis double bonds at positions 8, 11 and 14, 5S-HETE can be metabolized via the COX-2 pathway [48]. The relevance of COX-2-dependent 5S-HETE oxidation to carcinogenesis remains to be determined. However, the metabolic transformation of 5S-HETE by the COX-2 pathway suggests a potentially critical cross-talk between the 5-LOX and COX-2 that could have relevant implications in oncogenesis.

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Fig. 2. Cellular localization and enzymatic activity of COX-2. Shown is COX-2 subcellular localization to the endoplasmic reticulum (ER), nucleus, mitochondria (M) and lipid bodies (LpB). Oxygenation of arachidonic acid (AA) by COX-2 in the ER is shown. AA is converted by the cyclooxygenase activity of COX-2. Two moles of oxygen are inserted into 1 mol of AA to generate PGG2. PGG2 is then reduced by the peroxidase activity of COX-2 into PGH2. PGH2 serves as a substrate for cell-specific cytosolic prostaglandin (PG) synthases, which are in turn responsible for the production of prostaglandin E2 (PGE2), prostaglandin D2 (PGDE2), prostaglandin F2α (PGF2α), prostacyclin (PGI2) and thromboxane A2 (TXA2).

In addition to utilizing lipid substrates, COX-2 can metabolize a variety of xenobiotics, including dietary, occupational, and environmental carcinogens [49]. These compounds are transformed by the peroxidase activity of COX-2 into highly reactive molecules that interact with the cell DNA to produce base adducts leading to oncogene activation or tumor suppressor inhibition [49]. COX2-dependent metabolic transformation of xenobiotics appears to play an important pathogenetic role in certain neoplastic disorders, including colon cancer and bladder cancer.

Table 1 COX-2 substrates. Lipids

Non-lipids

References

Arachidonic acid 2-Arachidonoyl glycerol Arachidonoyl ethanolamide 5-S-HETE

Xenobiotics

[44,49] [46] [46] [48]

3. COX-2 overexpression in solid tumors and hematological malignancies Except for the seminal vesicles, kidneys and certain areas of the brain, normal tissues under basal conditions display low or undetectable expression of COX-2 [15,16,50]. However, challenge of cells by a wide array of pro-inflammatory stimuli results in increased levels of COX-2 expression [12,51,52]. Under these conditions, the expression of COX-2 increases transiently and promptly returns to its baseline levels after termination of the stimulus. In contrast, persistent expression of COX-2 constitutes a biological hallmark of several malignancies and is associated, at least in certain tumors, with an unfavorable clinical outcome. As listed on Table 2, several human solid tumors and hematological malignancies are characterized by the presence of constitutive expression of COX-2. The significance of COX-2 overexpression in neoplastic disorders is emphasized by epidemiological studies that demonstrate a decrease in the incidence of colon cancer, prostate

M.T. Rizzo / Clinica Chimica Acta 412 (2011) 671–687 Table 2 COX-2 overexpression in malignancies. Solid tumors

Hematological tumors

References

Brain Head and neck Thyroid Lung Breast Esophagus Stomach Biliary tract Pancreas Liver Colon Ovaries Uterus Skin Prostate

Acute leukemia Chronic lymphocytic leukemia Chronic myeloid leukemia Multiple myeloma Hodgkins' lymphoma Non-Hodgkins' lymphoma

[25,76] [74,81] [75,77] [26,73,80] [65,78] [22,79] [63] [71] [23] [72] [21] [69] [70] [24] [62]

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Consistent with this possibility, upregulation of COX-2 expression has been reported in blast cells of patients with acute leukemia and chronic myeloid leukemia (CML) [76,77]. Increased levels of COX-2 have been detected in Reed–Sternberg cells, which are the predominant cellular components infiltrating the lymph nodes of patients with Hodgkin's disease [78]. Furthermore, overexpression of COX-2 represents a negative prognostic factor in certain hematological disorders. For example, COX-2 expression correlated with increased recurrence and shorter survival in patients with non-Hodgkin's lymphomas [79]. Moreover, increased levels of COX-2 in the bone marrow cells of newly diagnosed patients with multiple myeloma correlated with decreased survival, early relapse, and poor prognosis [80]. In patients with B-CLL, levels of COX-2 in B-CLL cells correlated with the expression of CD38, which has been shown to be a negative prognostic factor [81].

Solid tumors are listed according to the primary site involved.

4. Mechanisms of COX-2 contribution to tumorigenesis

cancer, breast cancer and Hodgkin's disease in chronic users of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) [53–56]. Regular administration of NSAIDs and selective COX-2 inhibitors has also been shown to decrease the mortality for sporadic colorectal cancer and to halt the progression of adenomas into adenocarcinomas in individuals with familiar adenomatous polyposis (FAP), a hereditary condition characterized by germline mutations in one allele of the APC gene [57,58]. Upon loss of function of the wild-type adenomatous polyposis coli (APC) allele, individuals with FAP, if not treated, develop hundreds of polyps in the intestinal tract that can progress in colorectal cancer [59]. Moreover, a recent prospective clinical study showed that the use of aspirin in patients diagnosed with nonmetastatic colorectal cancer is associated with a decreased risk of cancer-related mortality and improved survival [60]. The beneficial effect of aspirin was detected particularly in patients with COX-2-expressing primary tumors, suggesting that the clinical response to aspirin resulted from inhibition of COX-2 [60]. Furthermore, the recently published results from the Nurses' Health Study indicate that regular use of aspirin is associated with decreased risk of breast cancer-related death and distant recurrence in patients with stage I to III breast cancer [61]. The relevance of COX-2 expression in cancer is also highlighted by clinical studies indicating that the presence of COX-2 correlates, in certain tumors, with a more aggressive phenotype and, importantly, with an overall worse clinical prognosis. Thus, patients with prostate cancer overexpressing COX-2 have a higher incidence of metastatic disease [62]. Moreover, COX-2 is an independent poor prognostic factor in gastric cancer and non-small cell lung cancer [63,64]. Similarly, COX-2 is expressed in 40% of invasive breast cancer and its presence correlates with an increased incidence of local recurrence, distant metastasis and decreased survival rate [65,66]. Importantly, a recent comparative genome-wide expression analysis demonstrated that COX-2 was among the genes found to be upregulated in breast cancer patients with metastatic disease to the brain [67]. Furthermore, an association between COX-2 expression and atypical hyperplasia, which constitutes a risk factor for the development of breast cancer, has been recently reported [68]. The association between COX-2 expression and overall poor prognosis has also been detected in other cancers, including among others, brain tumors, ovarian and uterine cancer, tumors of the biliary tract, hepatocarcinoma, non-small cell lung cancer, head and neck tumors, thyroid tumors, and melanoma [24,25,69–75]. The majority of the studies pertaining to the contribution of COX-2 to oncogenesis have been performed in solid tumors. However, emerging evidence indicates an important role for COX-2 expression in influencing the malignant behavior of hematopoietic cells (Table 2).

In addition to the results from clinical and epidemiological investigations, evidence extrapolated from a large number of experimental studies support the notion that COX-2 plays a critical role in tumorigenesis. Studies in cultured tumor cell lines and mouse models of cancer demonstrate that overexpression of COX-2 impacts several aspects of the malignant phenotype, including deregulated cell growth and proliferation, increased ability to escape apoptosis, sustained neovascularization, increased invasive potential and metastatic dissemination, and the ability to evade the host immune surveillance [82–87]. The mechanisms whereby COX-2 contributes to tumorigenesis are complex and not well understood. COX-2 act in a cell-autonomous manner to promote the transition of premalignant cells to malignancy, or the progression of malignant cells, by inducing genetic instability via reactive oxygen species (ROS) and lipid peroxideinduced DNA mutations [88]. COX-2 also initiates oncogenic signaling networks and transcriptional programs that result in deregulation of oncoproteins or tumor suppressor genes involved in the control of cell growth and survival [89,90]. In addition to these cell-autonomous effects, COX-2 orchestrates the interactions between tumor cells and their surrounding environment. These interactions are critical for growth, survival and dissemination of tumor cells. Indeed, COX-2 plays a critical role in modifying the tumor microenvironment to create a sanctuary in which malignant cells can further flourish and progress protected from environmental stressors [91]. These microenvironment-modifying effects of COX-2 may be conducive also for the neoplastic transformation of premalignant cells. Expression of COX-2 in stromal cells, fibroblasts, and vascular cells stimulates the release of growth factors, angiogenic proteins, and chemokines, which act upon adjacent tumor cells to activate promitogenic, prosurvival, proangiogenic and proinvasive signaling pathways, and gene programs [92–95]. Importantly, COX-2 can also promote the recruitment of innate and adaptive immune cells that are responsible for tumor cell evasion from the host immune surveillance [96,97]. This mechanism is especially relevant to the pathogenesis of certain lymphoid malignancies characterized by overexpression of COX-2. The relevance of COX-2 to tumorigenesis is further substantiated by the findings that COX-2 is a target of several oncogenes, tumor suppressor genes, growth factors, oncogenic pathways, and DNAdamaging events implicated in tumor development and progression [7,98–101]. Moreover, emerging evidence indicates a critical link among COX-2, hypoxia, tumor development, and metastasis [102]. Increased COX-2 expression in response to hypoxic changes in the microenvironment allows tumor cells to escape cell death and resist the cytotoxic consequences of radiation therapy or chemotherapy [103,104].

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Chronic inflammation constitutes the main driving force behind the oncogenic effects of COX-2 [105]. The relevance of the link among COX-2, chronic inflammation and tumorigenesis is emphasized by the overexpression of COX-2 in chronic inflammatory diseases that have an increased risk of transforming into tumors [106–108]. Moreover, according to recent statistics, 25% of human cancers develop on a background of chronic inflammation, supporting that notion that chronic inflammation constitutes a risk factor for cancer development [109]. However, there is evidence that, at least in certain experimental cancer models, COX-2 exerts tumorigenic effects with no overt signs of inflammation [110]. This notion is further supported by the clinical findings that COX-2 overexpression contributes to the development of sporadic colon cancer, which does not always develop as a consequence of chronic inflammation [109]. According to the conventional model of tumorigenesis, normal cells acquire a malignant phenotype in a multistep fashion [111]. A first genetic “hit” must be accompanied by additional genetic insults that lead to activation of oncogenes and disruption of tumor suppressor genes. These effects combined with cycles of clonal selection give rise to a population of premalignant cells that in the presence of a favorable microenvironment progress into malignant cells and further into their metastatic dissemination [111]. Evidence extrapolated from a large number of experimental studies indicates that COX-2 influences each step of tumorigenesis, from tumor initiation, to tumor promotion and tumor progression, as detailed below. 4.1. Contribution of COX-2 to tumor initiation Studies in colon cancer provided the first evidence linking COX-2 to tumorigenesis [21,112,113]. However, the first demonstration that COX-2 is sufficient to induce neoplastic transformation was provided by the elegant studies of Liu et al. [110]. In this seminal study, the murine mammary tumor viral promoter (MMTV) was employed to selectively induce the expression of COX-2 in the mammary glands of multiparous female transgenes during pregnancy and lactation. The authors observed that induction of COX-2 in the mammary gland initially resulted in focal mammary gland hyperplasia that, following repeated cycles of pregnancy and lactation, progressed into breast adenocarcinoma and eventually into metastatic disease. These findings implied for the first time that the sole expression of COX-2 is sufficient to initiate tumor formation. Notably, in this model the tumor-initiating effects of COX-2 were not accompanied by overt signs of inflammation. Additionally, COX-2 was strongly expressed in the mammary gland epithelium, but not in the adjacent stromal cells. Moreover, protein levels of the antiapoptotic protein Bcl-2 were increased in the mammary tumor tissues, while levels of the proapoptotic protein Bax and Bcl-XL were decreased. PGE2 and PGF2α concentrations were also increased, suggesting their involvement in the protumorigenic effects of COX-2. Although a straightforward extrapolation of the COX-2 tumor-initiating effects described in the mouse model of Liu et al. to human breast cancer development is difficult, additional evidence from human studies indicate that COX-2 is causally involved in the early stages of mammary neoplastic transformation. Thus, Crawford et al. demonstrated that breast tissues from healthy women contained epithelial mammary cells that expressed COX-2 along with p16INK promoter hypermethylation. Importantly, these cells have a high predisposition to transform into malignant cells, suggesting that COX-2 plays a tumor-initiating role in human breast cancer [114]. A recent study by Colby and colleagues provides additional insights into the role of COX-2 in tumor initiation [115]. Expression of a COX-2 construct that was directed to the ductal cells of the exocrine pancreas by a bovine keratin 5 promoter resulted in the spontaneous occurrence of pancreatic ductal adenocarcinomas after 6–8 months. In this model, tumor development was clearly preceded by a chronic inflammatory response, which was detected at 3–4

weeks and accompanied by an early infiltration of inflammatory cells, followed by neovascularization and ductal cell metaplastic transformation that gradually progressed into dysplasia and ultimately adenocarcinoma. Remarkably, in this model, COX-2 expression was not sufficient to induce metastases unless tumor cells were subjected to additional insults. Moreover, the tumor-initiating effects of COX-2 coincided with the onset of the inflammatory response, which was initiated and maintained by COX-2, thus underscoring the link between chronic inflammation and the tumor-initiating effects of COX-2. The link among chronic inflammation, COX-2 and tumor development is also supported by studies performed in cultured cells and animal models of gastrointestinal cancers. In these studies, chronic exposure of gastric or esophageal epithelial cells to duodenal or gastric juice initiates a local inflammatory response that gradually and progressively evolves into atypical hyperplasia, followed by lowgrade dysplasia, high-grade dysplasia and ultimately cancer even in the absence of carcinogens [116–118]. Under these experimental conditions, overexpression of COX-2 is detected early on during the initial inflammatory response and contributes to the metaplasia– neoplasia transition. Clinical evidence further supports the role of COX-2-driven inflammation in the metaplasia–dysplasia–carcinoma sequence. Thus, Barrett's esophagus, a precancerous condition in which chronic gastroesophageal reflux induces inflammation of the squamous esophageal mucosa and its replacement with metaplastic epithelium, is characterized by overexpression of COX-2 [119]. Importantly, these patients have an increased risk of developing esophageal adenocarcinoma. Similarly, chronic infection of the gastric mucosa with Helicobacter pylori (H. pylori) is associated with enhanced levels of COX-2 protein and increased risk of developing gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma [120–122]. The pathogenic role of COX-2 overexpression in precancerous conditions of the gastrointestinal tract has been challenged by recent studies showing that inhibition of COX-2 accelerates the progression of H. pylori gastritis into preneoplasia. Consistent with possibility, exogenous administration of PGE2 protected against the progression of H. pylori gastritis into preneoplasia via its immunosuppressive effects on CD4+ T cells [123]. This protective effect of COX-2 against neoplastic transformation is reminiscent of the protective effect of COX-2 during inflammation observed in the COX-2 knockout mice. These results substantiate the complexity surrounding the contribution of COX-2 to tumorigenesis and underscore the importance of understanding the cellular context in which COX-2 is overexpressed as this likely influences its biological outcome. The mechanisms underlying the tumor-initiating effects of COX-2 are not well defined. The aforementioned studies suggest at least two distinct possibilities. In the absence of overt signs of inflammation, COX-2 can function in a cell-autonomous manner and transform normal epithelial cells by conferring resistance to apoptosis. The consequent increase in cell survival allows preneoplastic cells to be exposed to additional mutagenic insults and thus to fully acquire a malignant phenotype. COX-2 itself can induce genomic instability and mutagenic effects via the production of reactive oxygen species and lipid endoperoxide products [88]. Alternatively, the chronic inflammatory process initiated and maintained by COX-2 can constitute the determinant for the tumor-initiating effects of COX-2. Stimulation of angiogenesis by COX-2 not only contributes to stimulation and progression of inflammation, but plays also a pivotal role in driving the growth of transformed cells during chronic inflammation. However, it should be emphasized that the tumor-initiating effects of COX-2 are largely dictated by the intervention of microenvironmental factors. Consistent with this possibility, the sole expression of COX-2 is not sufficient to cause tumors in other experimental cancer models or in normal tissues that constitutively expressed COX-2 under physiological conditions.

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4.2. Contribution of COX-2 to tumor promotion Overwhelming data from the current literature support the notion that COX-2 acts as a tumor promoter. Consistent with this possibility, experimental models of skin and gastric cancers indicate that although the sole expression of COX-2 is not sufficient to trigger tumor development, it dramatically accelerates the oncogenic effect of dietary carcinogens, environmental toxins and genetic mutations. Consistent with this notion, transgenic mice, in which overexpression of COX-2 is selectively directed to the skin by the keratin 5 promoter, do not develop spontaneous skin tumors [124]. However, these transgenes are more susceptible to the procarcinogenic effect of 9,10dimethylbenz(a)anthracene (DMBA), a compound that alone induces experimental skin cancers, as demonstrated by the higher number of skin tumors detected in the COX-2 transgenic mice compared to wildtype mice after exposure to DMBA [124]. On the other hand, transgenic mice that overexpress COX-2 under the control of the keratin 4 promoter do not develop skin tumors when treated with TPA [125]. However, when antracine is employed as a tumor promoter the number of skin tumors in the COX-2 transgenes is higher than those in the wild-type animals [125]. Moreover, tumors in the COX-2 transgenes are histologically more aggressive that those found in the wild-type animals [125]. These results support the concept that COX-2 exerts tumor-promoting effects in a context-dependent manner. A similar scenario has been described in models of colorectal tumors. Thus, transgenic mice that overexpress COX-2 in colonic epithelial cells do not develop spontaneous tumors [126]. However, treatment of these transgenic animals with the mutagen azoxymethane results in increased number of neoplastic colonic lesions than in the wild-type animals [126]. The tumor-promoting effects of COX-2 are also relevant to colitis-associated colon cancer. Consistent with this possibility, nimesulide, a COX-2 inhibitor, halted the development of colonic tumors in mice treated with azoxymethane and dextran sulfate sodium [127]. However, recent work of Ishikawa et al. demonstrated that neither COX-2 nor COX-1 are required for the development of colon cancer in a mouse model of colitis-associated colon cancer [128]. Studies of Oshima et al. in the APCΔ716 knockout mouse, a model of human FAP, provided the first genetic evidence to support the role of COX-2 as a tumor promoter [113]. In this model, intestinal polyps larger than 2 mm in diameter and all colonic polyps express significant levels of COX-2 protein. When APCΔ716 mice were crossed with COX-2 knockout mice a dramatic decrease in the number and size of polyps was detected in the APC mutants crossed with COX-2 wild-type mice. Similar results were obtained when APCΔ716 mice were subjected to treatment with a selective pharmacological inhibitor of COX-2 or with sulindac, a dual COX-1 and COX-2 inhibitor [113]. Similarly, COX-2 knockout in the APCMin/+ mice, which develop spontaneous adenomas due to a truncation mutation in the APC gene, also resulted in a marked decrease in the number of polyps [112]. In this model, pharmacological and genetic inhibition of COX-1 also decreased the number and size of polyps raising important questions about the potential oncogenic effects of COX-1 in colorectal cancer. Together, these results suggest that polyp formation and growth requires cooperation the between COX-1 and COX-2. Thus, while COX-1 contributes to the initial polyp formation, COX-2 is subsequently activated to further support polyp growth. Interestingly, in both models, COX-2 was expressed primarily in the stromal cell compartment rather than in neoplastic epithelial cells, suggesting that COX-2 promotes tumorigenesis in a paracrine and cell non-autonomous manner. Overexpression of COX-2 in the stromal compartment creates a proinflammatory environment, which then exerts paracrine effects on the adenoma cells promoting their transition into neoplastic cells. Stimulation of angiogenesis by COX-2 overexpressing stromal cells constitutes an important mechanism involved in polyps' growth, as suggested by the findings that in the APCΔ716 mice levels of COX-2

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expression showed a correlation with expression levels of two known angiogenic proteins, VEGF and bFGF [113]. The role of COX-2 as a tumor promoter in colon cancer development is also substantiated by clinical findings. For example, COX-2 is not expressed in the normal colonic epithelium, but its expression is detected in 50% of patients with adenomas and 85% of patients with sporadic colon cancer [21]. However, in colorectal cancer the expression of COX-2 is confined mostly in the epithelial neoplastic cells [129]. 4.3. Contribution of COX-2 to metastatic disease A role for COX-2 in metastases was first implicated by the studies of Tsujii et al. In these studies, forced expression of COX-2 in cultured colon cancer cell lines correlated with increased invasive and metastatic behavior [86]. Subsequent studies in cultured tumor cells and mouse models of breast and lung cancers corroborated the contribution of COX-2 to metastatic disease [130,131]. Notably, expression profiling analysis of low and highly metastatic lung cancer cells demonstrated that COX-2 was among the genes upregulated in the highly metastatic clones [132]. Moreover, inhibition of COX-2 decreased the invasive properties of the highly metastatic lung cancer clones [132]. Similarly, gene expression profiling by microarray analysis revealed that COX-2 was among the genes found to be upregulated in invasive breast cancer cells [133]. The mechanisms involved in dissemination and colonization of tumor cells from their primary site to distant organs are still poorly understood. However, the spread of cancer cells requires a sequential series of events characterized by increased motility and invasive properties, invasion of the peripheral and lymphatic circulation, homing and colonization of distant organs [134]. Interestingly, emerging evidence indicates that cancer cells in their primary site can harbor molecular signatures of a metastatic phenotype even in the absence of detectable distant metastases [134]. Therefore, identifying early markers of a metastatic phenotype in tumor cells prior to their dissemination to distant organs is critical for increasing the success of therapeutic interventions for metastatic disease. There is ample evidence that COX-2 influences each step of the metastatic cascade. Studies in breast cancer have shown that COX-2 enhances motility and invasion via several mechanisms, including stimulation of the epithelial–mesenchymal transition (EMT), downregulation of E-cadherin expression and upregulation of proteolytic enzymes involved in the degradation of the membrane basement [85,135–139]. The relevance of these latter effects is emphasized by recent microarray findings that show an association between COX-2 and elevated expression of metalloprotease-2 in invasive breast cancer [140]. In addition to influencing motility and invasion, COX-2 positively impacts survival of tumor cells during metastasis by preventing anoikis, a form of cell death caused by cell detachment from the matrix [141]. Moreover, COX-2 overexpression facilitates the hematogenous and lymphatic dissemination of tumor cells [142–144]. Despite improved chemotherapy regiments and the introduction of new biological agents the majority of cancer patients die of metastatic disease [145]. Therefore, a full appreciation of the molecular mechanisms whereby COX-2 regulates metastatic responses of tumor cells is fundamental to our ability of identifying novel molecular targets and signaling networks that can be exploited to control or prevent metastatic dissemination of tumor cells. 5. Mechanisms regulating COX-2 expression and activity in tumor cells Increased levels of COX-2 in tumor cells are initiated and maintained by complex mechanisms, many aspects of which are still not well understood. However, the evidence available hitherto indicates that COX-2 protein levels are regulated by a wide array of

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Table 3 Regulation of COX-2 expression in tumors cells. Modes

Mechanisms

References

Transcriptional

Activation by transcription factors Tumor suppressor gene inactivation Inhibition of transcription silencing Increased mRNA stability Translational silencers MicroRNAs Suicide inactivation ERAD pathway

[32,98–100,146–152] [148,164–166] [169,170] [175–179] [180] [181] [185] [185–187]

Translational

Degradation

ERAD: endoplasmic reticulum associated degradation.

stimuli that impinge upon its transcription, translation or degradation (Table 3). 5.1. Regulation of COX-2 transcription Transcriptional activation of COX-2 plays a major role in regulation of COX-2 protein levels in tumor cells. The 5'-flanking region of the COX-2 promoter contains cis regulatory elements that constitute putative consensus sequences for the binding of transcription factors, which either independently, synergistically or in cooperation with coactivators, increase COX-2 transcription in response to a wide array of stimuli, including oncogenic proteins, tumor suppressors, hypoxia, radiation therapy, chemotherapy, viral agents, and protein growth factors [32,98–100,103,104,146–152]. These stimuli elicit the activation of diverse signaling pathways that converge in the nucleus to phosphorylate target transcription factors, which are ultimately responsible for the increase in COX-2 transcription [153]. The involvement of distinct transcription factors in regulation of COX-2 gene expression is stimulus-, cell type-, and context-dependent. Studies performed in epithelial cell lines transformed by oncogenic Ras or v-Src demonstrated the critical involvement of cjun/AP-1 in stimulation of COX-2 transcription [154,155]. Viral oncoproteins, including human papilloma virus (HPV) oncoproteins E5, E6, and E7, which are implicated in HPV-mediated carcinogenesis, activate COX-2 transcription via the cooperation of NF-kB and AP-1 [156]. In breast cancer cells, NFAT cooperates with AP-1 in binding to the COX-2 promoter, leading to increased transcription and protein synthesis of COX-2 [157]. Other studies have shown cooperation between AP-1 and PEA3 (a member of the Ets family of transcription factors) for modulation of COX-2 transcription in breast cancer cells [158], while members of the C/EBP family of transcription mediate stimulation of COX-2 transcription in pancreatic cancer cells [159]. Certain environmental toxins also stimulate COX-2 transcription. In this respect, binding of AhR, a basic helix–loop–helix transcription factor, to a putative xenobiotic response element found in the COX-2 promoter results in stimulation of COX-2 transcription [160,161]. Signaling pathways activated by hypoxia are also important regulators of COX-2 transcription via the binding of the hypoxia inducible factor 1 to the H3 region of the COX-2 promoter [162]. An additional important aspect of COX-2 transcriptional regulation is the presence of a positive feedback loop initiated by the release of PGE2, which leads to cAMP/PKA-induced phosphorylation of CREB followed by its interaction with the CRE binding site of the COX-2 promoter [163]. This mechanism of COX-2 regulation has important implications in tumorigenesis as it ensures that lasting levels of COX-2 are maintained in tumor cells or in the tumor microenvironment. Mutational inactivation of tumor suppressor genes can lead to stimulation of COX-2 transcription. In cell lines and experimental models of colon cancer, mutations or deletions of the APC tumor suppressor gene result in loss of APC protein and consequent constitutive activation of the Wnt pathway [98]. This leads to stabilization of β-catenin in the cytosol and its subsequent translocation in the nucleus where it interacts with the T-cell factor/

lymphocyte enhancer (TCF/LEL) transcriptional factors, thereby promoting activation of various genes implicated in oncogenesis, including COX-2 [164]. The relevance of the link among APC, COX-2 and colon cancer is substantiated by the findings that constitutive activation of the Wnt pathway, resulting from mutations or deletions of APC, constitutes the initiating factor that drives tumor formation in patients with sporadic colorectal cancer or with FAP [165,166]. Other tumor suppressors, such as p53, also contribute to regulation of COX-2 transcription [148]. While the mechanisms by which p53 mutations lead to enhanced COX-2 transcription are not well understood, wildtype p53 negatively regulates COX-2 transcription by interfering with the binding of the TATA binding protein to the promoter region of COX-2 [148]. There is evidence that constitutive expression of COX-2 in tumor cells also results from inhibition of transcriptional silencing, an epigenetic mechanism that selectively repress gene expression in mammalian cells. The most common epigenetic silencing occurs as a consequence of DNA methylation, which consists of the attachment of a methyl group to the C-5 of cytosine by a DNA methylase [167]. DNA methylation prevents the subsequent binding of transcription factors to the promoter region and therefore leads to gene inactivation [167]. Normal cells acquire specific patterns of DNA methylation during embryonic development, which are then maintained during adult life [167]. In contrast, aberrant DNA methylation patterns, caused by either global hypomethylation or hypermethylation in the CpG islands, are often detected in tumor cells [168]. In human gastric tumors, overexpression of COX-2 has been linked to COX-2 hypermethylation [169]. Moreover, patterns of COX-2 hypomethylation have been detected in precancerous conditions such as H. piloryinduced gastritis, suggesting that hypomethylation of COX-2 DNA constitutes an early epigenetic mechanism used by tumor cells to increase and maintain elevated levels of COX-2 protein [170]. 5.2. Regulation of COX-2 translation Aberrant regulation of COX-2 at the post-transcriptional level is currently recognized as an important mechanism that promotes and/ or maintains constitutive expression of COX-2 in malignant cells [171,172]. The 3'-UTR of the COX-2 promoter contains several AREs, which are recognized by various RNA binding proteins that control both mRNA decay and protein translation [33,173]. In normal cells levels of COX-2 protein are likely kept low because of the interaction of AREs with certain RNA binding proteins that regulate mRNA decay. In contrast, in tumor cells, the interaction of RNA-binding proteins with AREs within the COX-2 promoter increases COX-2 mRNA stability and translation. Among the RNA-binding proteins identified hitherto, the heterogeneous nuclear ribonucleoprotein U (HuR) [174], a member of the ELAV (embryonic lethal abnormal vision) mRNA binding proteins, plays a critical role in enhancing expression of COX2 protein in tumor cells [175,176]. In a study of patients with ovarian carcinoma, the expression of COX-2 and HuR were found to be independent poor prognostic factors [177]. The mechanisms underlying activation and binding of HuR to the AREs within the COX-2 promoter are not well understood. However, HuR reportedly facilitates the binding of β-catenin to a putative β-catenin responsive element found in the proximal region of the 3'-UTR of COX-2, with subsequent increase of COX-2 mRNA stabilization [178]. A recent study carried out in fibroblasts demonstrated a novel regulatory mechanism that involves stabilization of mRNA COX-2 [179]. In these studies, TGF-beta increases COX-2 protein expression via upregulation of the heterogeneous nuclear ribonucleoprotein A/B [179]. Studies of Dixon et al. have also demonstrated that the trans-acting ARE-binding factor TIA-1, which has been shown to inhibit translation of certain mRNAs during environmental stresses, also acts as a transcriptional silencer upon its binding to the AREs within the COX-2 promoter [180]. Thus, downregulation of TIA-1 could represent a

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mechanism by which tumor cells increase COX-2 protein levels to escape hostile microenvironment conditions or chemotherapy-induced death. Another mechanism involved in the aberrant regulation of COX-2 expression in tumor cells involves dysregulation of microRNAs (miRNAs) [181]. These small single-stranded non coding RNAs regulate gene expression by degrading or repressing translation through Watson–Crick base pairing in the 3'-UTR of their target mRNA or in conjunction with sequence-specific mRNA-binding proteins [181]. Recent studies have implicated miRNAs in a wide array of biological processes, including tumor development and metastasis [182]. Moreover, aberrant expression of certain miRNAs contributes to regulation of COX-2 gene expression during normal and pathological processes [183,184]. Thus, Strillacci et al. demonstrated the involvement of miR-101 in binding to and inhibiting COX-2 translation in cultured colon cancer cells [184]. Importantly, analyses of tissue samples from patients with colorectal cancer demonstrated that miR101 expression inversely correlated with COX-2 expression and metastatic disease to the liver [184]. 5.3. Regulation of COX-2 degradation The mechanisms that regulate COX-2 degradation in tumor cells have been largely unexplored. Similar to COX-1, COX-2 undergoes irreversible suicide inactivation of its catalytic activity, which occurs in vitro and likely also occurs in vivo, following the generation of heme and tyrosyl radical intermediates during the cyclooxygenase and peroxidase reactions [185]. The resulting structurally damaged COX-2 can be then degraded via a proteosomal independent protein degradation pathway. An additional system that regulates COX-2 degradation involves the endoplasmic reticulum-associated degradation (ERAD) pathway [185,186]. ERAD-mediated COX-2 degradation involves the transport of COX-2 from the ER to the cytosol, where it undergoes ubiquitination and proteolysis by the 26S proteosome [185,186]. Interestingly, while the ERAD pathway mainly removes misfolded or structurally damaged proteins, COX-2 is degraded via the ERAD pathway in its native state. N-Glycosylation of Asn594 within the 27-amino acid instability sequence located in the C-terminal of COX-2 is critical for ERAD-dependent degradation (Fig. 1). Despite recent advances on the mechanisms responsible for COX-2 degradation, its relevance to oncogenesis remains ill defined. A recent study suggests that caveolin-1, a membrane protein whose expression is downregulated in several tumors, facilitates the degradation of COX-2 via the ERAD pathway [187]. Therefore, downregulation of caveolin-1 in tumor cells could contribute to the maintenance of elevated levels of COX-2 protein by preventing its degradation. Future studies to further address this interesting possibility are warranted. 6. Oncogenic signaling pathways activated by COX-2 An additional strategy for blocking the effects of COX-2 on malignant cell behavior is to interfere with the downstream signaling pathways that are activated as a result of COX-2 overexpression. PGE2 is the most abundant among the prostaglandins produced by COX-2 overexpressing tumors [188–191]. The critical role of PGE2 in oncogenesis is further substantiated by the findings that certain tumors display, in concert with COX-2 overexpression, alterations in the expression and activity of other enzymes involved in PGE2 biosynthesis and degradation [192–194]. For example, we detected constitutive expression of the microsomal prostaglandin synthase-1 (mPGES-1), the rate-limiting enzyme for the production of PGE2, along with COX-2 expression, in the human glioma cell line U87-MG [195]. Constitutive expression of mPGES-1 and COX-2 in U87-MG cells was accompanied by increased basal concentrations of PGE2 as compared to those detected in primary human astrocytes, which expressed neither mPGES-1 nor COX-2 under resting conditions [195].

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Pharmacological and genetic inhibition of mPGES-1 blunted PGE2 production and decreased cell proliferation, consistent with a regulatory role of mPGES-1 in tumor growth [195]. Remarkably, constitutive expression of mPGES-1 was also detected in tumor tissue samples obtained from patients with gliomas relative to normal brain tissue from the same patient [195]. On the other hand, downregulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the enzyme that catalyzes the degradation of prostaglandins, has been reported in colon and cancer cell lines where it correlates with increased cell proliferation [193,194]. Once released from tumor cells or from the surrounding microenvironment, PGE2 elicits autocrine or paracrine cellular responses primarily through its binding to a family of receptors designated as EP1, EP2, EP3 and EP4, which are differentially coupled to heterotrimeric G proteins (Fig. 3) [196]. The EP1 receptor is coupled to Gq and stimulates phospholipase C activation, calcium mobilization and consequent protein kinase C activation [196]. The EP4 and EP2 receptor subtypes are coupled to Gs and stimulate intracellular cAMP production via activation of adenylcyclase [196]. The EP3 receptor couples to Gi, which lowers intracellular cAMP levels via inhibition of adenylcyclase [196]. However, splice variants of the EP3 receptor can couple to Gs, Gq or G12 [196]. The presence of distinct EP receptor subtypes, which are co-expressed in the same cell and whose expression levels often change depending on cell type and cell context, contributes to the complexity of PGE2 signaling (Fig. 3). Adding to this complexity is the ability of PGE2 to transactivate protein growth factor receptors such as EGF and HGF, via its binding to the EP receptors [197,198]. PGE2 modulation of protein growth factor receptor signaling may function as a signaling amplification loop to ensure continuous influx of stimulatory signals to tumor cells. The biological relevance of EP receptor-dependent signaling to the oncogenic effects of PGE2 is corroborated by the impairment of tumor cell growth, invasion, angiogenesis and metastatic dissemination in EP receptor knockout animals [199–201]. In addition to binding the EP receptors, PGE2 interacts with nuclear receptors, including NR4A2 and peroxisome proliferator-activated receptor-δ (PPAR-δ) [41,202]. The relevance of these interactions is substantiated by the involvement of these receptors in PGE2dependent stimulation of proliferation in colon cancer and breast cancer cells [41,203]. 6.1. PGE2-dependent signaling in tumor growth The growth-inducing effects of PGE2 are mediated by a complex circuitry of signaling pathways whose activation is cell type- and cell context-dependent (Fig. 3). Activation of the Ras/Raf-1/ERK pathway occurs downstream to the EP4 and EP2 receptors and plays an important regulatory role in the proliferative responses of several tumor cells to PGE2 [204–208]. In colon cancer cells, binding of PGE2 to the EP2 receptor stimulates the release of the EGR receptor ligand, amphiregulin, which binds to and phosphorylates the EGF receptor resulting in ERK activation and stimulation of cell growth [209]. Conversely, in non-small cell lung cancer cells activation of ERK by PGE2 does not involve transactivation of the EGF receptor and occurs via EP1-dependent activation of protein kinase C [210]. An additional pathway that plays a crucial role in mediating the proliferative responses of tumor cells to PGE2 is the Wnt pathway. In their elegant studies, Castellone and colleagues demonstrated that binding of PGE2 to the EP2 receptor results in activation of two distinct signaling pathways that converge in promoting β-catenin nuclear accumulation and activation [211]. Hence, Gβγ subunit-dependent activation of the PI3K/AKT pathway leads to inhibition of GSK-3β activity and phosphorylation of β-catenin. On the other hand, the association of Gα subunit with axin, a signaling component of the Wnt pathway, releases β-catenin from the axin-GSK-3β complex facilitating its nuclear accumulation. The relevance of Wnt activation by PGE2

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Fig. 3. EP receptor-dependent signaling pathways involved in regulation of tumor cell functions by PGE2. Binding of PGE2 to the each of the EP receptor subtypes conveys signals to downstream effectors via activation of distinct heterotrimeric G-proteins. Release of active, GTP-bound α subunit of Gs downstream to the EP2 and EP4 receptor subtypes, stimulates adenylyl cyclase activity leading to an increase in the intracellular production of cAMP following by activation of protein kinase A (PKA) and phosphorylation of target intracellular targets, including transcription factors, in addition to activation of p38-MAPK. PGE2 activates the Wnt pathway via the EP2 receptor. The released Gαs associates with axin leading to the release of β-catenin from the axin/GSK-3β complex and leading to subsequent nuclear translocation of β-catenin. On the other hand, Gβγ dimers activate phosphatidylinositol 3kinase (PI3K) and AKT resulting in inhibition of GSK-3β and phosphorylation of β-catenin. AKT can also transmit promigratory, proliferative and prosurvival signals via activation of ERK, or modulation of proteins involved in regulation of apoptosis. PGE2 also transactivates the epidermal growth factor receptor (EGFR) via the EP2/EP4 receptors. This occurs via the release of amphiregulin (AR) or via β-arrestin-dependent activation of c-Src, which in turn can directly phosphorylate the EGFR or stimulates AR release. EGRF transactivation leads to activation of the Ras/Raf/MEK/ERK pathway. Gαq, downstream to the EP1 receptor, activates the classical signaling system consisting of phospholipase C (PLC) activation, which results in cleavage of phosphatidylinositol bisphosphate into diacylglycerol (DAG) and inositol triphosphate (IP3) following by increase in the intracellular concentration of calcium (Ca++). DAG and Ca++ activate protein kinase C (PKC) following by activation of the Raf/MEK/ERK pathway and upregulation of survivin. Interaction of PGE2 with the EP3 receptor leads to activation of Gαi resulting in inhibition of adenylyl cyclase and cAMP/PKA activation. Variants of the EP3 receptor can couple to G12 and stimulate Rho and c-jun amino terminal kinase (JNK) activation. The signaling network activated downstream to the EP receptors converges at the nuclear levels, wherein activation of transcriptional factors is ultimately responsible for stimulation of tumor cell growth and survival, enhancement of neovascularization, tumor invasion and escape from the immune system.

has also been recently demonstrated in human hematopoietic stem cells. These findings raise the interesting possibility that PGE2 contributes to cancer stem cell biology [212]. We and others have shown the critical role of the cAMP/PKA pathway, activated downstream to the EP2 and EP4 receptors, in mediating PGE 2 -dependent cellular responses in tumor cells [195,208]. In mammary cells, PGE2 interacts with the EP2 receptor and activates the cAMP/PKA pathway, which ultimately stimulates mammary cell hyperplasia via the induction and release of amphiregulin [208]. In studies performed in U87-MG cells, we reported that

PGE2 stimulates cell growth via a signaling pathway that involves cAMP/PKA-dependent activation of the transcription factor CREB [195,213]. Interestingly, PKA type II is the main PKA isoform activated by PGE2 in U87-MG cells [195]. In line with our results, a recent study reported that PGE2 regulates mesenchymal stem cell growth via activation of PKA type II [214]. The contribution of the EP3 receptor subtypes to the proliferative effects of PGE2 in tumor cells is less understood. There is evidence that the EP3 receptor has a negative effect on tumor growth. Consistently, work of Macias–Perez demonstrated that activation of EP3 receptor

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variants reduces colon cancer growth in a manner dependent on G12induced activation of Rho kinase [215]. 6.2. PGE2-dependent signaling in angiogenesis We and others have previously shown that COX-2 critically influences tumor-associated angiogenesis via the release of PGE2 and VEGF [84,216]. Additional studies have shown that PGE2 stimulates angiogenesis either directly by influencing endothelial cell responses, or indirectly by inducing the release of angiogenic growth factors [217,218]. A recent study demonstrates a critical role of PGE2 in orchestrating the cooperation between cancer cells and endothelial cells and proposes a model in which PGE2 released from COX-2-expressing prostate cancer cells acts in an autocrine and paracrine manner to activate distinct signaling pathways that culminate in stimulation of angiogenesis [219]. Thus, PGE2 interaction with the endothelial EP2/EP4 receptors results in activation of AKT and NF-kB, while binding of PGE2 to EP2/EP4 receptors expressed on the tumor cells increases the production of urokinase plasminogen activator receptor (uPAR) and VEGF production [219]. These signaling events ultimately translate into stimulation of angiogenesis. Emerging evidence emphasizes the contribution of the microenvironment to the angiogenic response evoked by COX-2 and PGE2 [220–222]. Amano et al. demonstrated that PGE2 released from tumor cells impinge upon the stromal tumor microenvironment to stimulate VEGF production in a manner dependent on the EP3 receptor [220]. Recently, using a tumor implantation assay, Katoh et al. demonstrated that Lewis lung carcinoma cells stimulated stromal formation and angiogenic responses via COX-2-dependent release of PGE2 and CXCL2 [94]. These responses were markedly decreased in EP3 and EP4 knockout mice, suggesting the involvement of both receptor subtypes [94]. 6.3. PGE2-dependent signaling in tumor invasion and metastasis PGE2-dependent tumor cell migration and invasion are also dependent on the activation of signaling networks that are often cell type- and cell context-specific. The AKT/PI3-kinase pathway plays a critical role in transmitting the promigratory signals of PGE2 to tumor cells. Similar to ERK, activation of AKT by PGE2 involves transactivation of the EGF receptor but in a manner dependent on p60src [223]. Signaling through the EP4 receptor mediates PGE2induced activation of p60src in a manner dependent on β-arrestin, as reported recently by Kim et al. in A549 cells [224]. In addition to influencing cancer cell motility, PGE2 induces cellular responses that lead to an increase of tumor cell invasion, including tumor cell adhesion to the extracellular matrix, extracellular matrix degradation and adhesion of tumor cells to the vascular bed. The signaling events that mediate these responses are triggered by EP4 receptor ligation followed by upregulation of CD44 expression and increased metalloprotease-2 activity. A recent study implicates signaling events activated by PGE2 downstream to the EP1 receptor in migration and invasion of a variety of tumor cell types [210,225–227]. 6.4. PGE2-dependent signaling in tumor survival The ability of tumor cells to survive in hostile environmental conditions or escape radiation therapy and chemotherapy-induced death likely depends on several factors, but overexpression of COX-2 in tumor cells or in the tumor microenvironment could constitute an adaptive response to stressors that otherwise would lead to their demise. In addition to the involvement of ERK and AKT in mediating the antiapoptotic effects of PGE2 [83,209], several effectors of the intrinsic and extrinsic pathway of apoptosis, including bcl-2, Bim, and survivin are also regulated by PGE2 [83,228–231]. The role of the EP receptors in mediating these responses is not well defined. However,

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upregulation of survivin by PGE2 in hepatocarcinoma cells is mediated by the EP1 receptor [83], while in the HCT-116 mouse intestinal cell line, signaling via the EP2 receptor leads to EGF receptor-dependent AKT activation, which halts apoptosis by inhibiting Bax translocation to the mitochondria [231]. 6.5. PGE2-dependent signaling pathways in tumor immune tolerance Overexpression of COX-2 promotes a state of chronic inflammation that contributes to create a less immunogenic tumor microenvironment, which allows premalignant and malignant cells to escape the host immune surveillance. Several lines of evidence indicate that many of the tolerogenic effects of COX-2 in the context of malignant diseases are mediated by PGE2 [96,97,232]. The mechanisms involved in the immunosuppressive effects of PGE2 include suppression of T cell function, inhibition of dendritic cell activation, stimulation of T regulatory cell functions, and polarization of activated macrophages [233–235]. Recent studies indicate that PGE2 also stimulates the accumulation of myeloid-derived suppressor cells in the tumor microenvironment in a manner dependent on EP2 receptor signaling [236]. The downstream signaling events that mediate the immunosuppressive effects of PGE2 are not well understood. However, studies performed in CD4+T cells that were obtained from patients with Hodgkin's disease, imply a role of the tyrosine kinase lck in PGE2induced impairment of T cell function [237]. The immune responses stimulated by PGE2 have important implications for immunotherapy. Thus, recent preclinical studies in models of pancreatic and breast cancers showed that combination of COX-2 inhibitors with tumor vaccine reduced tumor growth and progression [238,239]. These findings support the rationale for the therapeutic use of COX-2 inhibitors in combination with immunotherapy to maximize cancer therapy. However, the efficacy of this approach remains to be confirmed in clinical studies. 7. Mechanism of action and antitumoral effects of COX-2 inhibitors The gastrointestinal adverse side effect associated with inhibition of COX-1 by NSAIDs, led to the development of COX-2 specific inhibitors (coxibs). Celecoxib and rofecoxib were the first coxibs to be developed, followed by etoricoxib, valdecoxib and luminaracoxib. Coxibs are slow, time-dependent inhibitors of COX-2 that irreversibly bind to specific residues in the catalytic active site of COX-2, leading to its inactivation [240]. These compounds demonstrated marked antitumoral properties in cultured tumor cell lines and tumor xenografts [241,242]. These effects were initially attributed to their ability to interfere with the production of prostanoids and their downstream signaling effectors. Subsequent studies, however, demonstrated that, in certain tumor cells, coxibs exerted antitumoral effects only when employed at concentrations higher than those required to inhibit COX-2 activity [243]. Moreover, tumor cells devoid of COX-2 expression were similarly sensitive to the antiproliferative effects of coxibs [244,245]. Together, these findings led to the hypothesis that coxibs possess COX-2-independent effects. Consistent with this possibility, other enzymes, whose expression and activity is dysregulated in tumor cells, are inhibited by coxibs [246–248]. The COX-2-independent effects of coxibs have been exploited for the development of structural analogues of coxibs that lack the adverse side effects derived from COX-2 inhibition. For example, dimethylcelecoxib, effectively suppresses proliferation and cell survival of various tumor cell lines [249,250]. However, the mechanisms of dimethylcelecoxib and other coxib analogues are still not well understood. A recent report indicates that dimethylcelecoxib inhibits the production of PGE2 [251]. The aforementioned findings underscore the need for additional experimental studies on the antitumoral

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mechanisms of dimethylcelecoxib and its analogues, prior to their introduction in clinical trials. Similar to coxibs, NSAIDs and aspirin possess antitumoral properties derived from their ability to inhibit COX enzymes. While NSAIDs are weak time-independent inhibitors of COX-1 and COX-2, aspirin potently and irreversibly inhibit COX-1 and COX-2 by acetylation of Ser530 and Ser526, respectively [252,253]. In contrast to COX-1, acetylated COX-2 can still metabolize fatty acids and generate metabolically active compounds that possess potent antiinflammatory affects [254]. However, NSAIDs and aspirin can also have COX-independent effects on tumor cells. For example, aspirin induces tumor apoptosis via activation of NF-kB [255]. Recent studies elegantly demonstrate a novel mechanism whereby sulindac, a COX-1 and COX-2 inhibitor, induces apoptosis by interacting with the retinoic receptor X alpha and inhibiting activation of AKT [256]. 8. Clinical use of COX-2 inhibitors The development of coxibs as anti-inflammatory agents, along with the paramount evidence in support of COX-2 contribution to tumorigenesis, provided the opportunity and rationale for their introduction in clinical trials, initially for chemoprevention and later for cancer therapy. Steinbach et al. first demonstrated a significant reduction in the number and size of colorectal polyps, in patients with FAP following treatment with celecoxib [58]. The efficacy of coxibs in chemoprevention for colorectal cancer was subsequently confirmed in three large randomized clinical trials: Adenoma Prevention with Celecoxib (APC), Adenomatous Polyp Prevention on Vioxx (APPROVe) and Prevention Spontaneous Adenomatous Polyps (PreSAP) [257– 259]. However, a significant increase in the incidence and severity of thrombotic events, which persisted for one year after the stopping treatment, was observed in the APPROVe trial [260]. These results led to the withdrawal of rofecoxib from the pharmaceutical market, while celecoxib is currently approved as a chemopreventive agent only for patients with FAP. However, a recent meta-analysis of independent estimate from 72 studies found no significant increase in the risk of cardiovascular effects associated with the long-term use of celecoxib unless patients had previous risk factors for cardiovascular disease [261]. While the adverse cardiovascular effects observed as a result of COX-2 inhibition have been mainly attributed to the decreased production of PGI2, additional mechanisms are also involved [262]. Thus, recent studies indicate that in the presence of celecoxib arachidonic acid is shunted towards the 5-LO pathway, producing proinflammatory mediators implicated in cardiovascular diseases [263]. Interestingly, NSAIDs also possess chemopreventive effects. In this respect, data from recently published retrospective study indicate a decrease in the risk of developing gastric cancer in patients with H. pylori-infected gastric ulcers treated with NSAIDs [264]. The rationale for using coxibs as adjuvant agents either with systemic chemotherapy or with radiation therapy, is supported by a large number of experimental studies in which COX-2 selective inhibitors sensitized tumor cells to the apoptotic and antiproliferative effects of radiation or chemotherapeutic agents [103,104] The underlying mechanisms are multiple and include downregulation of the expression and activity of the P-glycoprotein, downregulation of anti-apoptotic effectors, inhibition of prosurvival signaling pathways such as AKT and ERK, cell cycle arrest and enhancement of anti-tumor immune response [265,266]. In spite of the paramount preclinical evidence, clinical studies designed to evaluate the efficacy of celecoxib in combination with adjuvant systemic chemotherapeutic or radiation therapy have generated mixed results. Although initial pilot studies showed survival benefits in patients receiving coxibs, results from subsequent large randomized clinical studies comparing chemotherapy either alone or in combination with coxibs in non-small cell lung cancer, prostate cancer, and breast cancer, have been less promising

[267–271]. A recent randomized, placebo, controlled study in which aromatase inhibitors were given either alone or in combination with celecoxib to patients with ductal carcinoma in situ, showed no benefit in any of the outcome measures in the arm receiving celecoxib [270]. Moreover, a recent clinical trial in prostate cancer designed to correlate the efficacy of celecoxib with specific biological markers, such intratumoral concentrations of prostaglandins, COX-1 and COX-2 protein levels, and markers of angiogenesis, growth and apoptosis, demonstrated no correlation between celecoxib and any of the markers measured. These results however are not surprising in light of the findings that prostatic tumor tissues in this study had decreased levels of COX-2 and COX-1 expression compared to normal prostatic tissues [271]. Using an in vivo model of colon cancer, Yan et al. have recently demonstrated that the ability of celecoxib to have a chemopreventive effect on colon cancer is dictated by the concomitant expression levels and activity of the PGE2-degrading enzyme 15-PGDH in the colonic mucosa [272]. Thus, celecoxib was ineffective in preventing the development of colon tumors in 15-PGDH-knockout mice. These results were further corroborated by examination of the colonic mucosa from 16 individuals enrolled in the APC trial who were at high risk for developing colon cancer. Four of these individuals, who had 15-PGDH levels below the cohort mean, developed new adenomas after treatment with celecoxib [272]. These results underscore the need of examining and correlating levels of 15-PGDH in cancer patients to their response to the treatment with COX-2 inhibitors to determine whether similar mechanisms of resistance to celecoxib are responsible for treatment failure. Despite the issues with the efficacy of COX-2 inhibitors in cancer therapy, several clinical trials using celecoxib in combination with systemic chemotherapy or radiation therapy are currently ongoing [273]. Results from these trials will provide useful information in further establishing the efficacy of COX-2 inhibitors in adjuvant chemotherapy. 9. Concluding remarks and future perspectives As summarized in this review, the contribution of COX-2 to tumorigenesis is supported by paramount evidence stemming from epidemiological, clinical, and experimental studies. Several aspects of the molecular mechanisms underlying the aberrant expression of COX-2 in tumor cells have been elucidated, and key signaling pathways involved in transmitting the oncogenic and tumorpromoting effects of COX-2 in a variety of tumor cell types have been dissected. However, the apparent discrepancies between the potent antitumoral effects of COX-2 inhibitors in vitro and their lack of efficacy in the majority of the clinical trials performed to date, imply that despite the tremendous advances in our knowledge, many aspects of COX-2 contribution to cancer still elude our understanding. The complexity surrounding the role of COX-2 in tumorigenesis is also underscored by results of other studies that argue against the contribution of COX-2 to oncogenesis [123,128]. Therefore, additional basic research is needed to unequivocally determine when COX-2 is a cause or consequence of neoplastic transformation. The timing, the cell type and the intracellular localization of COX-2 in a tumor might constitute critical determinants for dictating the net biological outcome of COX-2 inhibition. In this respect, the development of fluorescent COX-2 probes as recently reported by the studies of Marnett et al. will greatly facilitate these efforts [274]. The mechanisms of COX-2 degradation in tumor cells are still not well understood, but additional research in this area could provide insights into the regulation of COX-2 in tumor cells and aid in the identification of potential new targets for therapy. While several of the signaling pathways activated by COX-2 in tumor cells have been dissected, our knowledge on the crosstalk and interactions between them is very limited. The identification of signaling networks

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associated with certain COX-2-dependent cellular outcomes is crucial because their inhibition has the potential to lead to therapeutic approaches that are more selective than inhibition of an isolated signaling pathway. Interfering with prostaglandin-dependent signaling events in tumor cells has the advantage of being a more selective approach for cancer control. In this respect, inhibition of EP receptor signaling by selective EP receptor antagonists or inhibition of mPGES1 represents an effective alternative to inhibition of COX-2, but additional information on their expression levels and their signaling interactions in human tumor tissues is needed. Finally, the translation of the findings accumulated throughout many years of experimentations in vitro and animal models should be accelerated by studies with primary tumor samples. Several lessons from the outcome of the clinical trials with COX-2 selective inhibitors in cancer therapy can be also learned and applied to future studies to achieve a better understanding of the role of COX2 in oncogenesis. COX-2 inhibitors in cancer therapy cannot be used indiscriminately, but should be tailored based on the COX-2 status of the patient's tumor. Moreover, the assumption that COX-2 expression in tumor cells correlates with response to therapy may be too simplistic, and efforts should be made to identify which subsets of COX-2 positive patients may benefit from COX-2 inhibitors. Histological analyses to determine the cell compartment (tumor vs. microenvironment) that expresses COX-2 in the primary tumors prior to therapy could also provide important insight in establishing which COX-2-expressing tumors are likely to respond to COX-2 inhibition. Similarly, the concomitant expression of enzymes involved in PGE2 degradation should also be assessed especially in patients who are not responding to the treatment with COX-2 inhibitors. In conclusion, additional basic research is needed to generate the information that clinicians will need to establish when inhibition of COX-2 can provide beneficial effects for cancer patients. Similarly, improvement in the design of clinical trials should prove useful to determine the efficacy of the current COX-2 selective inhibitors and clearly establish whether COX-2 remains a rational target for cancer therapy. Understanding the mechanisms of COX-2 contribution to oncogenesis remains an exciting area of research that will undoubtedly continue to positively impact our understanding of the complexity of tumor cell behavior. Acknowledgments I thank Christopher Brown for helping with the preparation of the figures and Elaine Bammerlin for critical reading of the manuscript. I am grateful to the students and members of my laboratory for their support. I regret having to omit the work of many colleagues because of space limitations. Work from my laboratory discussed in this manuscript was supported by a Methodist Cancer Center Award and a Clarian Value Funds Award. References [1] Otto JC, Smith WL. Prostaglandin endoperoxide synthases -1 and -2. J Lipid Mediat Cell Signal 1995;12:139–56. [2] Jarvin R, Jarvin I, Kurg R, Brash AR, Samel N. On the evolutionary origin of cyclooxygenase (COX) enzymes: characterization of marine invertebrate COX genes points to independent duplication events in vertebrate and invertebrate lineages. J Biol Chem 2004;279:13624–33. [3] Dey I, Keller K, Beley A, Chadee K. Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica. Proc Natl Acad Sci USA 2003;100:13561–6. [4] Hassid A, Levine L. Induction of fatty acid cyclo-oxygenase activity in canine kidney cells (MDCK) by benzo (a) pyrene. J Biol Chem 1977;252:6591–3. [5] Pong SS, Hong SL, Levine L. Prostaglandin production by methylcholanthrenetransformed mouse BALB/3T3. Requirement for protein synthesis. J Biol Chem 1977;252:1408–33. [6] Ohichi K, Levine L. Stimulation of prostaglandin synthesis by tumor-promoting phorbol-12, 1-diesters in canine kidney (MDCK) cells. Cycloheximide inhibits the stimulated prostaglandin synthesis, deacylation of lipids, and morphological changes. J Biol Chem 1978;253:4783–90.

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