Multifaceted roles of cyclooxygenase-2 in lung cancer

Drug Resistance Updates 7 (2004) 169–184

Multifaceted roles of cyclooxygenase-2 in lung cancer Karen Riedl a , Kostyantyn Krysan a , Mehis Põld a , Harnisha Dalwadi a , Nathalie Heuze-Vourc’h a , Mariam Dohadwala a , Ming Liu a , Xiaoyan Cui a , Robert Figlin a , Jenny T. Mao a , Robert Strieter a , Sherven Sharma a,b,c , Steven M. Dubinett a,b,c,∗ a

UCLA Lung Cancer Research Program, Department of Medicine, 37-131 CHS, David Geffen School of Medicine at UCLA, 10833 LeConte Avenue, Los Angeles, CA 90095-1690, USA b Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA c Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, CA, USA Received 24 March 2004; received in revised form 13 April 2004; accepted 14 April 2004

Abstract Lung cancer is the leading cause of cancer death in the United States. Although the low 5-year survival rate (under 15%) has changed minimally in the last 25 years, new agents and combinations of agents that target tumor proliferation, invasion, and survival may lead to improvement in patient outcomes. There is evidence that cyclooxygenase-2 (COX-2) is overexpressed in lung cancer and promotes tumor proliferation, invasion, angiogenesis, and resistance to apoptosis. COX-2 inhibitors have been found to inhibit tumor growth in animal models and have demonstrated responses when combined with conventional therapy in phase II clinical trials. Further understanding of the mechanisms involved in COX-2-mediated tumorigenesis and its interaction with other molecules in lung cancer may lead to improved therapeutic strategies for this disease. In addition, delineation of how COX-2-dependent genes modulate the malignant phenotype will provide novel insights in lung cancer pathogenesis. © 2004 Elsevier Ltd. All rights reserved. Keywords: Cyclooxygenase; Lung cancer; Celecoxib; Clinical trials

1. Introduction

2. Cyclooxygenase-2 in lung cancer

Lung cancer is the leading cause of cancer-related death in the United States and is responsible for more deaths than prostate, colon and breast cancers combined (Jemal et al., 2003). With the existing therapeutic efforts, patients with lung cancer have a 5-year survival rate of less than 15%. This statistic has changed minimally in the last 25 years, underscoring the need for new therapeutic strategies. Understanding the molecular mechanisms involved in the pathogenesis of lung cancer can provide opportunities to develop innovative therapies for non-small cell lung cancer (NSCLC) (Dy and Adjei, 2002a, 2002b). These therapies target molecular pathways essential to tumor proliferation, angiogenesis and apoptosis (Hirsch et al., 2002). Cyclooxygenase (COX)-2 is one of the novel targets under evaluation for lung cancer therapy and chemoprevention (Dubinett et al., 2003; Dannenberg and Subbaramaiah, 2003).

COX is the rate-limiting enzyme for the production of prostaglandins (PGs) and thromboxanes from free arachidonic acid (Herschman, 1996). The enzyme is bifunctional, with fatty acid COX, producing PGG2 from arachidonic acid, and PG hydroperoxidase activities, converting PGG2 to PGH2 . Two isoforms of COX have been described, each with unique properties. COX-1 is constitutively expressed in most cells and tissues, while COX-2 is inducible and expressed in response to cytokines, growth factors and other stimuli (Dubois et al., 1998; Smith et al., 2000). The COX metabolite, PGE2 , in turn exerts its effects through G protein-coupled receptors—EP1, EP2, EP3 and EP4. COX-2 has been reported to be constitutively overexpressed in a variety of malignancies (Chan et al., 1999; Liu et al., 2000; Shamma et al., 2000; Sheng et al., 2000; Soslow et al., 2000; Yip-Schneider et al., 2000) and is frequently constitutively elevated in human NSCLC (Hida et al., 1998b; Hosomi et al., 2000; Huang et al., 1998; Wolff et al., 1998). In 1998 three studies provided the initial documentation of the constitutive overexpression of COX-2 in human NSCLC (Hida et al., 1998b; Huang et al., 1998; Wolff et al., 1998).

∗

Corresponding author. Tel.: +1 310 794 6566; fax: +1 310 267 2829. E-mail address: [email protected] (S.M. Dubinett).

1368-7646/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.drup.2004.04.003

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In the first report, Huang et al. (1998) utilized antibodies specific for human COX-2 to evaluate NSCLC and normal adjacent lung resection specimens by immunohistochemistry. All of the 15 tumor specimens (8 adenocarcinomas and 7 squamous cell carcinomas) showed cytoplasmic staining for COX-2 in tumor cells. In contrast, adjacent normal lung showed no COX-2 staining in the alveolar lining epithelium, but demonstrated positive cytoplasmic staining often in alveolar macrophages and occasionally in bronchiolar epithelium. In a subsequent study by Wolff et al. (1998) immunohistochemistry showed COX-2 staining in 19 of 21 adenocarcinomas and in all 11 squamous cell carcinomas studied. The level of staining appeared to be less in squamous cell carcinomas than in the adenocarcinomas. Four small cell lung cancer specimens were reported to stain with a relatively weak intensity. Interestingly, abnormal alveolar epithelium in lung sections from patients with asbestosis or idiopathic fibrosing alveolitis expressed COX-2 protein. Patients with these pulmonary fibrotic disorders have an increased incidence of lung cancer (Bouros et al., 2002; Hillerdal and Henderson, 1997). Hida et al. (1998b) reported that COX-2 overexpression was seen in approximately 70% of lung adenocarcinomas. In addition, COX-2 expression was documented in one-third of atypical adenomatous hyperplasias and carcinoma in situ. This study also reported a greater proportion of lung cancer cells staining positively in lymph node metastases compared to the corresponding primary tumor (Hida et al., 1998b). COX-2 activity can be detected throughout the progression of a pre-malignant lesion to the metastatic phenotype (Hida et al., 1998b). Higher COX-2 expression was observed in lung cancer lymph node metastasis compared to primary adenocarcinoma (Hida et al., 1998b). Other studies have corroborated and expanded on the initial findings documenting the importance of COX-2 expression in lung cancer (Achiwa et al., 1999; Brabender et al., 2002; Hasturk et al., 2002; Hosomi et al., 2000; Ochiai et al., 1999; Soslow et al., 2000; Watkins et al., 1999; Wolff et al., 1998). Khuri et al. (2001) reported that COX-2 overexpression detected by in situ hybridization with riboprobes appears to portend a shorter survival among patients with early stage NSCLC. COX-2 expression in specimens from 160 patients with stage I NSCLC was evaluated. The strength of COX-2 expression was associated with both a decreased overall survival rate (P = 0.001) and a diminished disease-free survival rate (P = 0.022). A preliminary report by West et al. (2002b) using immunohistochemistry assessment of COX-2 also suggests more aggressive behavior in NSCLC and decreased survival in those expressing COX-2. Another group reported that COX-2 expression assessed by immunohistochemistry was associated with poor prognosis, independent of TNM stage in surgically resected NSCLC (Kim et al., 2003). These reports, together with studies documenting an increase in COX-2 expression in precursor lesions (Hosomi et al., 2000; Wolff et al., 1998), suggest the involvement of COX-2 in the pathogenesis of lung cancer.

Furthermore, a recent study implicates a common polymorphism in the cox-2 gene with an increased risk of lung cancer (Campa et al., 2004). Epidemiological studies that indicate a decreased incidence of lung cancer in subjects who regularly use aspirin have been interpreted as supporting this hypothesis (Schreinemachers and Everson, 1994). Mounting evidence indicates that tumor COX-2 activity has a multifaceted role in conferring the malignant and metastatic phenotypes. Although multiple genetic alterations are necessary for lung cancer invasion and metastasis, COX-2 may be a central element in orchestrating this process (Achiwa et al., 1999; Hida et al., 1998b; Hosomi et al., 2000; Huang et al., 1998; Wolff et al., 1998). Studies indicate that overexpression of COX-2 is associated with apoptosis resistance (Hsu et al., 2000; Liu et al., 1998; Sheng et al., 1998; Tsujii and Dubois, 1995), angiogenesis (Gately, 2000; Leahy et al., 2000; Liu et al., 2000; Masferrer et al., 2000; Uefuji et al., 2000), decreased host immunity (Huang et al., 1998; Stolina et al., 2000), and enhanced invasion and metastasis (Tsujii et al., 1997). Thus, COX-2 can impact multiple mechanistic pathways and mechanisms in lung cancer carcinogenesis. COX-2 activities are particularly relevant in the pathogenesis of lung cancer for the following reasons: COX-2 can activate tobacco smoke carcinogens such as benzo[a]pyrene (B[a]P) (Wiese et al., 2001; Eling et al., 1990). One example of this activity is the capacity of COX-2 to catalyze the conversion of B[a]P-7,8-dihydrodiol to B[a]P-diol epoxide which binds to DNA (Ho and Lee, 2002). Thus, the capacity of COX-2 to activate environmental carcinogens including polycyclic hydrocarbons suggests that it plays an important role in tobacco-induced carcinogenesis (Wiese et al., 2001). In addition, B[a]P itself has also been demonstrated to potentially upregulate epithelial cell COX-2 expression and PGE2 production (Kelley et al., 1997). COX-2 is readily inducible by a variety of stimuli that are often present in the pulmonary microenvironment of those at risk for lung cancer. These stimuli include TGF-␤, IL-1␤, hypoxia, B[a]P, and epidermal growth factor (EGF) (Smith et al., 2000). Whereas IL-10 can downregulate COX-2 in host inflammatory cells, this capacity for IL-10 signaling is lost in human NSCLC (Berg et al., 2001; Heuze-Vourc’h et al., 2003). In addition to growth factor and cytokine induction of COX-2, the enzyme may be constitutively upregulated by virtue of oncogene expression and mutational events in lung cancer development. For example, wild-type, but not mutant p53, suppresses COX-2 transcription, thus suggesting that p53 status in lung cancer may be one of the determinants of COX-2 expression (Subbaramaiah et al., 1999). Other mutations associated with elevated COX-2 expression include those of K-ras and ␤-catenin (Bissonnette et al., 2000; Fujita et al., 2000; Gilhooly and Rose, 1999; Subbaramaiah et al., 1996). Wardlaw et al. (2002) documented a critical role for the CCAAT/enhancer-binding protein (C/EBP) and activating transcription factor/cAMP response element-binding protein (ATF/CREB) in the regulation of basal COX-2 expression in

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murine lung carcinomas. It was suggested that C/EBP and ATF/CREB may serve as new targets for downregulating COX-2 expression in lung cancer (Wardlaw et al., 2002).

3. COX-2 regulation of immunity in lung cancer Lung cancer cells elaborate immunosuppressive mediators including type 2 cytokines, PGE2 and transforming growth factor-␤ (TGF-␤) that may interfere directly with cell-mediated, anti-tumor immune responses (Huang et al., 1996, 1998, 1995; Neuner et al., 2001). In addition to producing their own suppressive factors, tumor cells may also direct surrounding inflammatory cells to release suppressive cytokines in the tumor milieu (Alleva et al., 1994; Huang et al., 1996). Tumor-derived PGE2 is one mediator that orchestrates an imbalance in the production of suppressive and immune potentiating cytokines by lymphocytes and macrophages in the tumor environment (Huang et al., 1998; Stolina et al., 2000). 3.1. Regulation of cytokine balance Studies by Huang et al. (1998) showing COX-2 overexpression in human lung cancer found that tumor-derived, high-level PGE2 production mediated dysregulation of host immunity by altering the balance of interleukin (IL)-10 and IL-12. IL-10 and IL-12 are critical regulatory elements of cell-mediated immunity. While IL-10 inhibits cellular immunity, IL-12 induces type 1 cytokine production and effective cell-mediated immunity (D’Andrea et al., 1993; Wu et al., 1993). IL-10 overproduction at the tumor site has been implicated in tumor-mediated immunosuppression (Hagenbaugh et al., 1997; Halak et al., 1999; Kim et al., 1995; Qin et al., 1997; Sharma et al., 1999) enhanced angiogenesis (Hatanaka et al., 2001), and appears to be an indicator of poor prognosis in NSCLC (De Vita et al., 2000; Hatanaka et al., 2000; Naruke et al., 2001; Urosevic et al., 2001). In contrast, IL-12 is critical for effective anti-tumor immunity (Bianchi et al., 1996; Colombo et al., 1996). Tumor models indicate that the tumor-bearing state induces lymphocyte and macrophage IL-10 production but inhibits macrophage IL-12 (Handel-Fernandez et al., 1997; Stolina et al., 2000). Thus, whereas IL-12 is the key inducer of type 1 cytokines, IL-10 production at the tumor site may inhibit type 1 cytokine production and cell-mediated anti-tumor immunity. Importantly, lung cancer-derived PGE2 has been found to induce a 10- to 100-fold increase in lymphocyte IL-10 production (Huang et al., 1996). It was hypothesized that high-level PGE2 production by lung tumor cells is dependent on tumor COX-2 expression. PGE2 production by A549 NSCLC cells was found to be elevated up to 50-fold in response to IL-1␤. Reversal of IL-1␤-induced PGE2 production in A549 cells was achieved by specific pharmacologic or antisense oligonucleotide inhibition of COX-2 activity or expression. In contrast,

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specific COX-1 inhibition was not effective. Consistent with these findings, IL-1␤ induced COX-2 mRNA expression and protein production in A549 cells. Specific inhibition of COX-2 abrogated the capacity of IL-1␤-stimulated A549 cells to induce IL-10 in lymphocytes and macrophages. Furthermore, specific inhibition of A549 COX-2 reversed the tumor-derived PGE2 -dependent inhibition of macrophage IL-12 production when whole blood was cultured in tumor supernatants. These results indicate that lung tumor-derived PGE2 plays a pivotal role in promoting lymphocyte and macrophage IL-10 induction, while simultaneously inhibiting macrophage IL-12 production (Huang et al., 1998). Thus, these studies demonstrated functional COX-2 expression by NSCLC cells and the definition of a pathway whereby tumor COX-2 expression and high-level PGE2 production mediate profound alteration in cytokine balance in the lung cancer microenvironment (Huang et al., 1998). To evaluate lung tumor COX-2 modulation of anti-tumor immunity in vivo, Stolina et al. (2000) studied the effect of specific genetic or pharmacological inhibition of COX-2 in a murine Lewis lung carcinoma (3LL) model. Specific inhibition of COX-2 led to significant tumor reduction in vivo in murine lung cancer models. Both specific genetic or pharmacologic inhibition of COX-2 led to marked lymphocytic infiltration of the tumor and reduced tumor growth. Treatment of mice with anti-PGE2 mAb replicated the growth reduction seen in tumor-bearing mice treated with COX-2 inhibitors. COX-2 inhibition was accompanied by a significant decrement in IL-10 and a concomitant restoration of IL-12 production by antigen-presenting cells (APCs). Because PGE2 is a potent inducer of IL-10, it was hypothesized that COX-2 inhibition led to anti-tumor responses by downregulating production of this potent immunosuppressive cytokine. In support of this concept, transfer of IL-10 transgenic T lymphocytes that overexpress IL-10 under control of the IL-2 promoter (Hagenbaugh et al., 1997; Sharma et al., 1999) reversed the COX-2 inhibitor-induced anti-tumor response (Stolina et al., 2000). In normal cells, a negative feedback loop regulates COX-2 expression (Molina-Holgado et al., 2002; Moore et al., 2001; Pomini et al., 1999). Elevated levels of IL-10 inhibit induced COX-2 expression, thereby limiting PGE2 production in normal cells. Heuze-Vourc’h et al. (2003) recently demonstrated that IL-10 cannot downregulate COX-2 expression and subsequent PGE2 production in NSCLC cells. Unresponsiveness of COX-2 to IL-10 in NSCLC may be caused by the lack of surface expression of IL-10R␣, the IL-10 binding subunit, in these cells. Deficiency of an IL-10-mediated COX-2 regulatory feedback loop may contribute to COX-2 overexpression and the maintenance of high levels of PGE2 in the lung tumor microenvironment, thereby promoting the malignant phenotype. In contrast to NSCLC, normal lung epithelial cells displayed cell surface IL-10R␣, suggesting that NSCLC responsiveness to IL-10 may be regulated through modulation of cell surface IL-10R␣ expression occurring during malignant transformation.

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3.2. Regulation of APC function Anti-tumor immune responses require the coordinate activities of lymphocyte effectors and professional antigen-presenting cells (APC) (Huang et al., 1994). Dendritic cells (DCs) are professional APC that are pivotal participants in the initiation of T-cell responses (Banchereau and Steinman, 1998). DCs acquire Ag in the periphery and subsequently transport it to lymphoid organs where they prime specific immune responses (Banchereau and Steinman, 1998). The tumor microenvironment can adversely affect DC maturation and function (Almand et al., 2000). Studies indicate that COX-2 metabolites can play a major role in tumor-induced inhibition of DC differentiation (Sombroek et al., 2002). Prostanoids have been found to mediate these effects by both IL-10-dependent (Harizi et al., 2002, p. 76) and independent pathways (Sombroek et al., 2002). Tumor-derived PGE2 has been shown to inhibit DC differentiation and function in an EP2 receptor-dependent manner (Yang et al., 2003). To define the pathways limiting DC function in the tumor environment, bone marrow-derived DCs were cultured in murine lung cancer tumor supernatants (TSN; Sharma et al., 2003). Although pulsed with tumor-specific peptides these DCs were incapable of generating anti-tumor immune responses in vivo. When injected into established murine lung cancer, DCs generated in TSN caused immunosuppressive effects that correlated with enhanced tumor growth. Genetic or pharmacological inhibition of murine lung cancer COX-2 expression restored DC function and effective anti-tumor immune responses. Functional analyses indicated that TSN caused a decrease in DC capacity to: (a) process and present antigens; (b) induce alloreactivity; (c) secrete IL-12. These limitations in DC activity were prevented when DCs were cultured in supernatant from COX-2-inhibited tumors. Whereas DCs cultured in TSN showed a significant reduction in cell surface expression of CD11c, DEC205, MHC class I, MHC class II, CD80, CD86, as well as a reduction in the transporter-associated proteins, TAP1 and TAP2, these changes were not evident when DCs were cultured in TSN from COX-2-inhibited tumors. Thus, inhibition of COX-2 expression or activity can prevent tumor-induced suppression of DC activities.

4. COX-2 regulation of angiogenesis in lung cancer In vivo expansion and maintenance of a functional vascular network serving the tumor is required for propagation, invasion and subsequent metastasis (Folkman, 1995). Thus, angiogenesis is requisite for tumor growth (Folkman, 2001) and has been specifically implicated in the pathogenesis and prognosis of lung cancer (D’Amico et al., 1999; Dazzi et al., 1999; Duarte et al., 1998; Fontanini et al., 1995; Giatromanolaki et al., 1996; Harpole et al.,

1996; Macchiarini et al., 1992). Several growth factors and cytokines have been implicated in tumor-related angiogenesis in lung cancer including vascular endothelial growth factor (VEGF), transforming growth factors ␣ and ␤, basic fibroblast growth factor (FGF) (Guddo et al., 1999; Ohta et al., 1999; Wikstrom et al., 1998; Koukourakis et al., 1997) and chemokines such as CXCL8 (IL-8) (Arenberg et al., 1996; Pold et al., 2004). Tumor suppressor genes, oncogenes and immune responses have been implicated in complex regulation of these proteins involved in the angiogenic process (Ambs et al., 1998; Konishi et al., 2000; Ravi et al., 2000). COX-2 has been shown to promote angiogenesis in vitro (Daniel et al., 1999; Tsujii et al., 1998) and in vivo (Masferrer et al., 2000; Williams et al., 2000). Masferrer et al. (2000) found that FGF-2-induced angiogenesis is dependent on COX-2 expression. Consistent with findings from other groups (Stolina et al., 2000; Williams et al., 2000), Masferrer et al. found that the COX-2 inhibitor celecoxib significantly decreased tumor growth in the Lewis lung carcinoma (LLC) model. In this model, predominant COX-2 expression in the tumor-associated vasculature was noted. These findings suggested that COX-2-derived PGs contribute to tumor growth by inducing neovascularization. In a study by Amano et al. (2003) angiogenesis and tumor growth were suppressed in EP3 knockout mice in a LLC model. Thus, COX-2 inhibition may contribute to the anti-tumor response by downregulating angiogenic activities in lung cancer. To assess the role of host-derived COX-2 in the LLC model, Williams et al. (2000) studied lung cancer growth in COX-2−/− mice. In contrast to C57BL/6 wild-type or COX-1−/− mice in which LLC tumors developed and grew rapidly, tumor growth in COX-2−/− mice was significantly attenuated. Compared to wild-type or COX-1−/− mice, fibroblasts from COX-2−/− mice were found to have a greater than 90% reduction in VEGF. These findings strongly support the concept that host stromal elements can enhance tumor growth, promoting a ’landscape’ in which vasculature is maintained and expanded (Hanahan and Weinberg, 2000; Kinzler and Vogelstein, 1998; Leahy et al., 2002). Thus, in addition to the tumor cells’ contribution, host-derived COX-2 appears to regulate important angiogenic mediators in the tumor milieu by contributing products, such as PGs, whose paracrine effects promote tumor growth. Recent studies (Leahy et al., 2002) add further support and insight into the contribution of stromal elements in COX-2-dependent tumor growth. Regardless of tumor cell expression of COX-2, the neovascular cells associated with tumors consistently demonstrated heightened COX-2 expression (Koki et al., 1999; Leahy et al., 2002). In both cornea and tumor angiogenic models, COX-2 was found to be expressed in vascular endothelial cells (Leahy et al., 2002). In keeping with these findings, specific COX-2 inhibition significantly limited angiogenesis. These pre-clinical studies are consistent with studies in human NSCLC (Marrogi et al., 2000; Ermert et al., 2003). Immunostaining for COX-2 correlated positively with VEGF

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status in human NSCLC sections (Marrogi et al., 2000), and evaluation of COX isoenzymes and downstream enzymes revealed increased COX-2 expression with strong immunostaining in endothelial cells of vessels found near or within the tumor (Ermert et al., 2003). Pold et al. (2004) demonstrated COX-2-dependent expression of ELR+ angiogenic chemokines CXCL5 and CXCL8 in vitro and in vivo in NSCLC. To evaluate the role of COX-2 expression of CXCL5 and CXCL8, NSCLC cell lines were transduced with a retroviral vector expressing the human COX-2 cDNA in the sense (COX-2-S) and antisense (COX-2-AS) orientations. CXCL5 and CXCL8 were significantly increased in NSCLC cell lines overexpressing COX-2, whereas CXCL5 and CXCL8 was suppressed in COX-2-AS cells or following pharmacologic inhibition of COX-2 in COX-2-S cell lines. In a SCID mouse model using COX-2-S and COX-2-AS cells, tumor growth was inhibited with genetic or pharmacologic inhibition of COX-2. In addition, CXCL5 and CXCL8 were decreased with inhibition of COX-2. Treatment with anti-CXCL5 or anti-CXCL8 neutralizing antibodies resulted in a significant decrease in tumor growth. Moreover, neutralization of PGE2 led to a decrease in CXCL5 and CXCL8 production. Consistent with previous reports, inhibition of NF-␬B suppressed the expression of CXCL8 (Bancroft et al., 2002) and CXCL5. These studies establish the role of angiogenic chemokines, CXCL5 and CXCL8, in NSCLC through COX-2 activation of NF-␬B nuclear translocation.

5. COX-2 regulation of invasion in lung cancer The complex events associated with tumor cell invasion include the active movement of cells across the extracellular matrix and spread to distant organ sites (Chambers et al., 2002). To assess the impact of COX-2 expression in lung cancer invasiveness, COX-2-S and COX-2-AS NSCLC cell lines were compared (Dohadwala et al., 2001). COX-2-S clones expressed significantly more COX-2 protein, produced 10-fold more PGE2 and demonstrated an enhanced invasive capacity compared to control vector-transduced or parental cells. CD44 is a cell surface receptor for hyaluronate, a major glycosaminoglycan component of extracellular matrix. Adhesion to extracellular matrix, a critical initial step in the metastatic process, has been found to be CD44-dependent in several malignancies (Bartolazzi et al., 1994; Lamb et al., 1997; Seiter et al., 1993; Yu and Stamenkovic, 1999). CD44 was overexpressed in COX-2-S cells, and specific blockade of CD44 significantly decreased tumor cell invasion. In contrast, COX-2-AS clones had a very limited capacity for invasion and showed diminished expression of CD44. These findings indicate that a COX-2-mediated, CD44-dependent pathway is involved in NSCLC invasion (Dohadwala et al., 2001). Subsequent studies focused on the role of tumor-derived PGE2 in modulating COX-2-dependent NSCLC invasion

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(Dohadwala et al., 2002). PGE2 , produced at heightened levels in COX-2 overexpressing tumor cells, affects target cells through interaction with G-protein-coupled EP receptors of four distinct subtypes. The pathways whereby autocrine/paracrine production of PGE2 could impact the invasive phenotype via EP receptor signaling in NSCLC have been studied. In addition to CD44, matrix metalloproteinase (MMP) production may be critical in lung cancer invasion. CD44 is known to induce co-clustering with MMPs and can therefore promote MMP activity, tumor invasion and angiogenesis (Yu and Stamenkovic, 1999, 2000). Antibody-mediated blockade of tumor-derived PGE2 decreased CD44 and MMP-2 expression as well as invasion. In addition, exposure of NSCLC cells to exogenous PGE2 upregulates CD44, EP4 receptor and MMP-2 expression and potently enhances invasion (Dohadwala et al., 2002). These studies indicate an important autocrine/paracrine role for PGE2 in the regulation of CD44 and MMP-2-dependent invasion in human NSCLC. They are also consistent with investigations in monocytes in which PGE2 was found to activate membrane type 1-MMP (MT1-MMP) and thereby promote activation of MMP-2 (Shankavaram et al., 2001). The role of MT1-MMP in the COX-2-dependent regulation of NSCLC cells has not yet been investigated. In contrast to the mechanisms described in the studies above, Pan et al. (2001) reported that NSAIDs, including the COX-2 inhibitor NS-398, could inhibit MMP-2 transcription in lung cancer cells by suppressing promoter activity. In the evaluation of a highly metastatic human lung cancer cell line, Kozaki et al. (2001) found that the level of COX-2 expression correlated with motility and invasion in vitro as well as metastatic potential in vivo. In accord with these findings, COX-2 inhibitors were found to decrease lung cancer metastases in vivo (Kozaki et al., 2001). In related studies, the expression of COX-2 and laminin 5 were found to be frequently co-expressed in early stage lung adenocarcinomas (Niki et al., 2002). Laminin 5, an extracellular matrix protein involved in cell migration and invasion, has been found to be frequently expressed in several different malignancies (Pyke et al., 1994, 1995; Skyldberg et al., 1999; Sordat et al., 1998). Immunohistochemical analysis of 102 lung adenocarcinomas which were ≤2 cm in size revealed that COX-2 and laminin 5 were expressed in 95 and 80%, respectively. An overall significant correlation was found between expression levels of COX-2 and laminin 5 and frequent co-expression of these proteins was noted at the lung adenocarcinoma invasive front (Niki et al., 2002). Common pathways may induce these proteins; epidermal growth factor receptor (EGFR) signaling has been shown to induce COX-2 (Coffey et al., 1997) as well as laminin 5 (Niki et al., 2002). In fact, in squamous carcinoma cell lines the expression levels of laminin 5 correlates with gene amplification of EGFR (Ono et al., 2002). The assertion that EGFR signaling is a common upstream regulator of COX-2 and laminin 5 is supported by the fact that lung adenocarcinomas that overexpress EGFR and erB-2 have been shown

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to have higher levels of COX-2 and laminin 5 compared to those without concomitant overexpression of the receptors (Niki et al., 2002). Another possibility not yet assessed is that COX-2-derived PGs may modulate laminin 5 levels. Alternatively, as suggested by recent studies (Buchanan et al., 2003; Pai et al., 2002), PGE2 has the capacity to transactivate EGFR and thus may impact laminin 5 expression as well as other proteins via this pathway to stimulate cancer cell migration and invasion. Another mechanism of metastasis and invasion in lung cancer includes lymphangiogenesis or the formation of new lymphatic vessels. A recent study by Su et al. (2004) demonstrated that human lung adenocarcinoma cells that overexpressed COX-2 had significantly increased vegf-c gene expression which has been associated with lymphangiogenesis in animal models (Stacker et al., 2001). The induction of vegf-c was shown to occur through PGE2 activation of the Her2/neu tyrosine kinase signaling pathway via the EP1 receptor. Furthermore, immunohistochemical evaluation of 59 lung adenocarcinoma tissue samples revealed a correlation between COX-2 overexpression, vegf-c and lymphatic vessel density. This study provides further evidence for the interactions between COX-2 and human epidermal growth factor signaling in the development and progression of lung cancer.

6. COX-2 regulation of apoptosis in lung cancer Dysregulation of apoptosis is intimately involved in carcinogenesis and a broad variety of anti-cancer agents mediate their effects by induction of apoptosis (Ferreira et al., 2002). Thus, heightened resistance to apoptosis may be responsible for drug or radiation resistance in lung cancer therapy. Apoptosis induction has been widely investigated and consistently supported in studies that seek to define the potential anti-neoplastic mechanisms of COX-2 inhibition. In the landmark studies of Tsujii and Dubois (1995) forced expression of COX-2 was found to increase Bcl-2 expression and resistance to apoptosis. Subsequent studies in a variety of tumors suggest that both Bcl-2-dependent (Liu et al., 1998) and independent (Hsu et al., 2000) pathways may be operative. COX-2-selective inhibitors have been shown to induce apoptosis in several different types of tumors (Erickson et al., 1999; Hara et al., 1997; Liu et al., 1998; Sawaoka et al., 1998; Sheng et al., 1998) including lung cancer (Chang and Weng, 2001; Hida et al., 2000; Yao et al., 2000). Lin et al. (2001) found that either overexpression of COX-2 or exposure to PGE2 can increase the apoptosis threshold in lung adenocarcinoma cells by upregulation of the mcl-1 gene in a PI3K/Akt-dependent manner (West et al., 2002a). A recent study by Krysan et al. (2004a) evaluated the role of the anti-apoptotic protein, survivin, in NSCLC cell lines overexpressing COX-2. NSCLC cells that overexpress COX-2 were found to have a significantly increased resistance to radiation and drug-induced apoptosis. Survivin levels

strongly correlated with COX-2 expression. Heightened tumor COX-2 expression led to decreased ubiquitination and stabilization of survivin, an effect replicated with exogenous PGE2 treatment. This relationship was also seen in vivo in an established tumor model. Similarly, in 15 human NSCLC samples, immunostaining and ELISA of COX-2 and survivin revealed a strong correlation of co-expression. The practical implications of apoptosis regulation by NSAIDs include their potential use in combination with chemotherapy and radiation therapy. Thus, cells overexpressing COX-2 have been found to resist apoptosis (Tsujii and Dubois, 1995) and COX-2 inhibition can promote apoptosis in these cells (Chang and Weng, 2001; Crew et al., 2000; Dannenberg et al., 2001; Elder et al., 1997; Hara et al., 1997; Hsu et al., 2000; Hsueh et al., 2000; Liu et al., 1998; Sheng et al., 1998; Yao et al., 2000). Hida et al. (2000) found that the COX-2 inhibitor nimesulide can induce apoptosis in NSCLC cell lines. In vitro evaluation of nimesulide as an adjunct to chemotherapy revealed that the IC50 values of various anticancer agents including etoposide and cisplatin were significantly reduced (Hida et al., 2000). These in vitro studies have been validated in vivo (Hida et al., 2002). A distinct benefit of combining COX-2 inhibition with chemotherapy is the possibility of limiting chemotherapy-induced COX-2 expression by tumor cells. Subbaramaiah et al. (2000) found that microtubule-interfering agents such as taxol can stimulate COX-2 transcription via ERK and p38 MAP kinase pathways. Thus, tumor cells that escape apoptosis induction by microtubule-interfering agents, such as taxol, may become promoters of angiogenesis, invasion, apoptosis resistance and immune dysregulation as a function heightened COX-2 expression. These findings are consistent with those of Moos et al. (1999) and Cassidy et al. (2002) who found that taxanes and their analogues increased macrophage COX-2 expression. The latter studies present a potential pathway for paracrine PGE2 production to negatively impact lung cancer chemotherapy regimens and provide a rationale for the clinical evaluation of COX-2 inhibitors in combination with microtubule-interfering agents for NSCLC. Radiation-induced apoptosis can be significantly enhanced by COX-2 inhibitors (Milas et al., 1999) and this increase in apoptosis has been demonstrated in lung cancer models (Hida et al., 2000; Pyo et al., 2001). Milas et al. (1990, 1991) were the first to suggest that NSAID-induced radiation responsiveness may be related to neovascularization and host immune competence. Several studies have now documented that COX-2 inhibitors potentiate radiation therapy in model systems by enhancing radiation-induced apoptosis (Kishi et al., 2000; Milas, 2001; Petersen et al., 2000; Shah et al., 2002). While certain COX-2 inhibitors primarily induce apoptosis, others may predominantly induce growth arrest (Thun et al., 2002). In addition, an individual NSAID may induce anti-tumor effects via different mechanistic pathways in different types of tumors. For example, sulindac, a

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non-selective COX-2 inhibitor, acts to induce apoptosis in the human colon cancer cell line HT-29 but induces predominantly growth arrest in human lung cancer cells (Heasley et al., 1997; Shiff et al., 1995). Culture conditions, including the NSAID concentration, may also impact which mode of action predominates (Chang and Weng, 2001; Thun et al., 2002). Thus, in parallel to the induction of apoptosis, COX-2 inhibitor-induced alterations in cell cycle progression have been reported (DuBois et al., 1996; Hung et al., 2000). Mammalian cell cycle progression is governed by cyclins, cyclin-dependent kinases (CDKs) and their inhibitors (CDKIs) (Malumbres and Barbacid, 2001). By binding CDKs, cyclins activate kinase activity and promote cell cycle progression. In contrast, CDKIs, including members of the kinase inhibitor protein family which bind CDK–cyclin complexes, inhibit cell cycle progression. In a study specifically addressing cell cycle regulation by COX-2 inhibitors in human lung cancer cells, Hung et al. (2000) found that p27KIP1 was upregulated in response to the COX-2 inhibitor NS398. Normal epithelial cells, including lung epithelium, usually express high levels of p27KIP1 . However, a decrease of this tumor suppressor gene product is found in NSCLC; more than 70% of NSCLC tumors show reduced p27KIP1 and the intracellular level of this protein is predominantly regulated by translational or post-translational mechanisms (Catzavelos et al., 1999; Esposito et al., 1997; Shirane et al., 1999; Yatabe et al., 1998). Because NS398 inhibited the degradation of p27KIP1 , it was suggested that the regulation of this protein constitutes another mechanistic pathway of COX-2 inhibitor-mediated tumor growth-suppressive effects in lung cancer (Hung et al., 2000). In addition to COX-2-dependent effects, NSAIDs can also act to induce apoptosis by COX-2-independent pathways (Hawk et al., 2002; Hwang et al., 2002; Song et al., 2002). In support of the concept of COX-2-independent effects, NSAIDs induce apoptosis in cancer cells that do not express COX-2 (Grosch et al., 2001; Xu, 2002). In addition, the dose of NSAIDs used in some studies to induce apoptosis exceeds the amount required to inhibit COX-2 enzymatic activity (Hawk et al., 2002). NSAIDs could act in a COX-2-independent manner through PPAR-␦ (He et al., 1999), PPAR-␥ (Wick et al., 2002), NF␬B (Frantz and O’Neill, 1995), AP-1 (Huang et al., 1997), cytochrome c and apoptotic-inducing-factor release (Sanchez-Alcazar et al., 2003) or arachidonic acid (Cao et al., 2000). Delineation of these additional COX-2-independent pathways will facilitate the rational use of COX-2 inhibitors in lung cancer therapy and prevention (Hwang et al., 2002).

7. Clinical investigation of COX-2 inhibition in lung cancer Because pre-clinical data strongly suggest that overexpression of COX-2 plays a critical role in tumor-mediated angiogenesis, invasion and metastasis, apoptosis resistance

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and immune dysregulation, clinical trials are currently evaluating COX-2 inhibition in the context of treatment and chemoprevention of lung cancer. The following are examples of the lung cancer clinical areas under investigation. 7.1. COX-2 inhibition in lung cancer treatment (Table 1) Taxane chemotherapy has been shown to induce COX-2 and PGE2 overexpression (Cassidy et al., 2002; Moos et al., 1999; Subbaramaiah et al., 2000) by stimulating transcription and mRNA stablity (Subbaramaiah et al., 2003). In order investigate the potential additive or synergistic effects of COX-2 inhibition combined with conventional therapy for NSCLC, studies have begun assessing COX-2 inhibitors combined with chemotherapy. Johnson et al. (2003) reported preliminary results in the evaluation of COX-2 inhibition plus docetaxel in recurrent NSCLC. This phase II trial utilized celecoxib (400 mg b.i.d.) and docetaxel (75 mg/m2 i.v. every 3 weeks). There was a significant decline of intratumoral PGE2 levels following treatment. In addition, serum VEGF declined post-celecoxib treatment while endostatin levels increased. In 33 of 41 patients reported eligible for response assessment, 4 (12%) had a partial response (PR) and 8 had stable disease (SD). Thus, these preliminary data indicate that celecoxib in combination with chemotherapy can significantly decrease PGE2 within tumor tissues, suggesting that COX-2-dependent expression of genes that are deleterious to the anti-tumor response may also be decreased. Based on results of the BLOT trial of neoadjuvant paclitaxel/carboplatin alone (Pisters et al., 2000), investigators are evaluating the role of celecoxib plus paclitaxel/carboplatin in the preoperative setting. In a phase II trial, Altorki et al. (2003) assessed the role of a selective COX-2 inhibitor, celecoxib, as an adjunct to preoperative chemotherapy in patients with resectable NSCLC. Twenty-nine patients completed a phase II trial of preoperative chemotherapy and celecoxib for stage IB-IIIA NSCLC. Two cycles of paclitaxel (225 mg/m2 ) and carboplatin (area under the curve of 6) were given 3 weeks apart. Celecoxib was given orally at 400 mg b.i.d. from day 1 until the day of surgery. Surgery was performed on days 42–56. Preoperative clinical stages included patients with stage IB (16), IIB (3) or IIIA (10) disease. All 29 patients completed induction therapy and 26 patients completed preoperative celecoxib. There were two treatment-related deaths: one secondary to neutropenic sepsis after the second cycle of chemotherapy and the second due to respiratory failure one week after right pneumonectomy. Three patients discontinued celecoxib secondary to generalized skin rash, without any other significant unexpected toxicities observed. Overall response rate was 65% (48% PR, 17% CR). Twenty-eight of 29 patients were explored and resected. There were no complete pathological responses, but 7 (24%) had minimal residual microscopic disease. It was concluded that in comparison to historically

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initiated a phase II trial using celecoxib 400 mg p.o. b.i.d. until progression plus concurrent weekly paclitaxel (50 mg/m2 )/carboplatin (AUC 2) and chest radiation therapy (63 Gy) for stage III NSCLC patients. Nine patients with stage III NSCLC had been enrolled at the time of the preliminary report. Three patients out of the nine developed grade 3 esophagitis, and one patient had grade 3 pneumonitis. Three out of five evaluable patients had objective CR or PR. Blood and urine specimens were obtained pre-, post-celecoxib and every 2 months for VEGF and PGE-M (the major urinary metabolite of PGE2 ) assays. Post-celecoxib, serum/plasma levels of VEGF from the first eight patients did not show a consistent pattern, but VEGF levels fell in the months following treatment. These preliminary data indicate that COX-2 inhibition may lead to changes in serum/plasma VEGF. Several additional phase I and II trials are also in progress to evaluate the role of COX-2 inhibition with radiation therapy at various stages of disease (Choy and Milas, 2003; Komaki et al., 2004; Liao et al., 2003; Table 1). Fig. 1. A randomized double-blind, phase II study of preoperative celecoxib/paclitaxel/carboplatin for Stage IIIA NSCLC (Altorki et al., 2003).

reported response rates, the addition of a selective COX-2 inhibitor may enhance the response to preoperative paclitaxel/carboplatin in NSCLC (Altorki et al., 2003). A randomized, double-blind phase II confirmatory trial is now underway with plans to enroll approximately 110 patients with stage IIIA disease (Fig. 1). 7.2. COX-2 inhibition combined with radiation therapy in lung cancer (Table 1) To assess the role of COX-2 inhibitors as potential radiation sensitizers in NSCLC, Carbone et al. (2002)

7.3. COX-2 inhibtion combined with EGFR inhibition in lung cancer (Table 1) Evidence that EGFR and COX-2 have related signaling pathways that can interact to regulate cellular proliferation, migration and invasion (Buchanan et al., 2003; Coffey et al., 1997; Pai et al., 2002; Alaoui-Jamali and Qiang, 2003) has triggered interest in evaluating the combination of COX-2 inhibition and EGFR inhibition in NSCLC. Krysan et al. described a potential mechanism of EGFR tyrosine kinase inhibitor (TKI) resistance in NSCLC mediated through an EGFR-independent cross-activation of the MAPK/Erk signaling pathway by PGE2 (Krysan et al., 2004b). We demonstrated that PGE2 transactivates the EGF signal transduction pathway leading to Erk kinase

Table 1 Ongoing clinical trials with COX-2 inhibition for the treatment of lung cancer Disease stage

Indication

Treatment

Phase (reference)

I resected

Adjuvant therapy (stage I/II head and neck cancers also) Neoadjuvant therapy Untreated Untreated Locally recurrent or persistent disease Progressive disease after chemotherapy or inability to tolerate chemotherapy Progression after platinum-based therapy Previously treated Inoperable early stage Adjuvant therapy Untreated Inoperable locally advanced Inoperable, untreated or previously treated with platinum-based therapy

Celecoxib or placebo

IIa

Carboplatin/paclitaxel +/− celecoxib Docetaxel + celecoxib Carboplatin/gemcitabine + celecoxib +/− zileuton Irinotecan/docetaxel + celecoxib Erlotinib + celecoxib

IIa IIa IIa I/IIa Ia

Gefitinib + celecoxib Docetaxel + celecoxib +/− radiation Celecoxib + radiation Celecoxib + radiation Celecoxib + carboplatin/paclitaxel + radiation Celecoxib + radiation Celecoxib + radiation

IIa II (Johnson et al., 2003) II (Choy and Milas, 2003) II (Choy and Milas, 2003) II (Carbone et al., 2002) I/II (Choy and Milas, 2003) I (Liao et al., 2003)

IIIA (N2+) IIIB/IV IIIB/IV IIIB/IV IIIB/IV Recurrent Recurrent I/II I/II resected III IIB/IIIA/IIIB I–IIIB a

http://www.nci.nih.gov/clinicaltrials.

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endpoints include evaluation of toxicity and determination of the optimal biologic dose of the combination at the lowest dose level demonstrating optimal biologic activity. The secondary endpoints are to investigate the biological response as defined by exploratory biological markers and clinical response rates. Three subjects will be assigned to each cohort and receive a fixed dose of erlotinib at 150 mg p.o. daily for two 4-week cycles. They will also receive celecoxib in escalating doses per cohort, starting with 200-mg p.o. b.i.d. and increasing by 100-mg doses to 400-mg p.o. b.i.d., then increasing by 200-mg doses to 800-mg p.o. b.i.d. for a total of 8 weeks. The first two cohorts have been enrolled and no unexpected toxicities have been observed. 7.4. COX-2 inhibition in chemoprevention of lung cancer Fig. 2. Inhibition of COX-2 and EGFR signaling pathways.

phosphorylation and activation in NSCLC cells whereas this transactivation was not evident in normal lung epithelial cells. PGE2 -dependent Erk phosphorylation was resistant to EGF receptor inhibitor PD153035, despite the fact that EGF-dependent Erk phosphorylation was completely abolished by PD153035. PGE2 -dependent Erk phosphorylation was EP receptor-mediated and partially abolished by protein kinase C inhibitors but not src kinase inhibitors. This mechanism of cross-talk between COX-2 and EGFR signaling pathways in lung cancer appears to be distinct to that previously described in colon cancer (Buchanan et al., 2003; Pai et al., 2002) (Fig. 2). Consistent with these findings, the coexpression of EGFR and COX-2 in human cervical cancer specimens portended a poor prognosis with increased locoregional recurrences (Kim et al., 2004). Based on these data, we have initiated a phase I, dose-escalation trial at UCLA Medical Center to investigate the optimal biologic dose of the combination of COX-2 inhibition (celecoxib) and EGFR inhibition (erlotinib, an EGFR TKI) in advanced NSCLC (Fig. 3). The study plans to enroll 21–27 subjects with stage IIIB or IV NSCLC. The primary

Fig. 3. A phase I trial of a COX-2 inhibitor (celecoxib) in combination with an EGFR inhibitor (erlotinib; OSI-774) in advanced NSCLC.

Several lung cancer chemoprevention trials have not demonstrated a decrease in the incidence of lung cancer and unexpected adverse effects have been noted in other trials (Hennekens et al., 1996; Lippman et al., 2001; Omenn et al., 1996). Generally, these phase III trials were designed based on epidemiological or animal data without the use of systematic pilot phase I/II trials (Hennekens et al., 1996; Lippman et al., 2001; Omenn et al., 1996). A systematic approach is needed with well-designed pilot trials to determine feasibility and to evaluate promising chemopreventive agents. According to the field carcinogenesis theory, malignant transformation may occur throughout the respiratory epithelium at multiple independent sites simultaneously (Fong et al., 1999; Hirsch et al., 2001). Thus, systemic therapy targeting the process of tumorigenesis may reverse the progression of pre-malignancy and prevent the development of lung cancer. Pre-clinical models show that COX-2 expression is abundant in alveolar type II cells in lung cancer-sensitive mouse strains and in pre-malignant lesions (Wardlaw et al., 2000). Inhibition of COX in these models, with NSAIDs or COX-2-specific inhibitors, slows tumorigenesis (Rioux and Castonguay, 1998). COX-2 expression and PG levels appear to be key factors contributing to lung carcinogenesis. Animal models demonstrate that COX-2-specific inhibitors protect against tumorigenesis resulting from exposure to the tobacco-specific nitrosamine, NNK (Rioux and Castonguay, 1998). In addition, the overexpression of COX-2 and PG is associated with several well-established risk factors for lung cancer, including upregulation of EGFR (Kinoshita et al., 1999), epithelial cell proliferation (Murata et al., 1999), microvascular angiogenesis (Masferrer et al., 2000), and resistance to apoptosis (Hida et al., 1998a). Upregulation of PG synthesis in the lung microenvironment also produces immunosuppressive effects which may interfere with anti-tumor immunity and promote tumor growth (Huang et al., 1998; Kalinski et al., 1997; Roth and Golub, 1993; Swisher et al., 1993).

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7.5. Phase II trials of COX-2 inhibition for chemoprevention of NSCLC Two ongoing phase II trials at UCLA are evaluating the role of celecoxib in chemoprevention of NSCLC. The objective of both trials is to determine the feasibility of celecoxib treatment for chemoprevention of lung cancer in populations at high risk of developing primary or secondary lung cancers. The trials are evaluating the effect of celecoxib on cellular and molecular events associated with lung carcinogenesis, including (1) modulation of biomarkers; (2) regulation of arachidonic acid metabolism; (3) anti-tumor immunity, and (4) angiogenesis in the lung microenvironment. The safety and long-term effects of treatment will also be monitored. A single-arm, pilot trial enrolled smokers at high risk for developing lung cancer, defined as age ≥45 years and smoking history of ≥20 pack-years with or without evidence of airflow obstruction. Trial objectives were to determine the efficacy and feasibility as well as to evaluate the safety and long-term side effects of celecoxib treatment in active smokers. Subjects were treated with celecoxib at 400 mg orally twice daily for 6 months, with evaluations at 2 weeks and 6 months. Bronchoalveolar lavage (BAL) was performed before and after one month of celecoxib treatment to obtain alveolar macrophages (AM). Results show that treatment with celecoxib decreased PGE2 production in AMs recovered from smokers (Mao et al., 2003). Furthermore, IL-10 production was upregulated in smokers and addition of COX-2 inhibition abrogated this production. These findings indicate that overproduction of PGE2 and IL-10 can be inhibited in subjects at risk for lung cancer with oral celecoxib therapy. A randomized phase II trial will further evaluate celecoxib in a larger population of 180 former smokers (≥30 pack-years) with either evidence of airflow obstruction or a history of successful surgical resection for stage I NSCLC. Patients are randomized (one-to-one, double-blind, placebo-controlled, crossover design) to treatment. A chemoprevention trial evaluating COX-2 inhibition in current and former smokers is also underway at M.D. Anderson Cancer Center (Xu, 2002). Molecular epidemiologic studies are needed to identify reliable biomarkers that are highly predictive of lung cancer risk. This ‘molecular profiling’ will efficiently characterize the highest risk population for enrollment in trials of chemoprevention, permitting smaller sample size, therapeutic stratification, and shorter trial duration. In addition, reliable surrogate endpoint markers need to be identified and validated to monitor the therapeutic efficacy of lung cancer chemoprevention strategies including those evaluating COX-2 inhibition. The selection of biomarkers to be evaluated and additional targets in lung cancer prevention and therapy may be facilitated by COX-2-dependent gene discovery programs (Kozaki et al., 2001; Pold et al., 2002). The transformation to malignancy can occur throughout the respiratory epithelium. Systemic therapy targeting

molecular processes involved in lung cancer carcinogenesis, such as COX-2-specific inhibition, has the potential to slow tumorigenesis (Rioux and Castonguay, 1998). Ongoing trials of COX-2 inhibitors in chemoprevention of NSCLC will determine the feasibility of this approach as well as the effects of COX-2 inhibitors in lung carcinogenesis.

8. Conclusions and future directions COX-2 has been implicated in regulating the malignant phenotype in lung cancer. There is strong evidence for its involvement in pathways that direct immune regulation, angiogenesis, invasion and apoptosis in NSCLC. Additional evidence is mounting which connects COX-2 signaling with other signaling pathways involved in tumorigenesis. Early human trials are underway to investigate COX-2 inhibition in chemoprevention, and in combination with standard chemotherapy, radiation therapy and new biologic agents in NSCLC. Establishing the safety of this potential treatment while studying mechanisms of action will be important for the future investigation of COX-2 inhibition in lung cancer. In addition, delineation of how COX-2-dependent genes modulate the malignant phenotype will provide novel insights in lung cancer pathogenesis. The biology of COX-2 overexpression in NSCLC and its effects on tumor proliferation, blood vessel development, cell death, metastatic potential and tumor immunity, are under investigation in human trials. The importance of COX-2 inhibition in (1) chemoprevention, (2) high-risk patients in the post-surgical setting, (3) neoadjuvant therapy and, (4) combination with other targeted agents is currently being evaluated. Clinical trials in these areas are ongoing and will help to determine the importance of these diverse COX-2-dependent mechanisms in lung cancer.

Acknowledgements We thank Sandra Tran for her assistance in the preparation of this manuscript. This work is supported by the UCLA SPORE in Lung Cancer (National Institutes of Health Grant P50 CA90388) and NIH 1T32HL6699201.

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