Background: Despite focused research in conventional therapies and considerable advances in the understanding of the molecular carcinogenesis of head and neck squamous cell carcinoma (HNSCC), the 5-year survival rate for patients with advanced disease remains ∼15–20%. The major causes of HNSCC-related deaths are cervical node and distant metastasis. E-cadherin has a key role in epithelial intercellular adhesion and its downregulation is a hallmark of epithelial–mesenchymal transition (EMT), which is associated with invasion, metastasis, and poor prognosis. Epithelial–mesenchymal transition is the major mechanism responsible for mediating invasiveness and metastasis of epithelial cancers. Recently, we reported the role of E-cadherin transcriptional repressors in the inflammation-induced promotion of EMT in HNSCC, which is mediated by COX-2. These findings suggest that therapies targeting the cyclooxygenase pathway may diminish the propensity for tumour metastasis in HNSCC by blocking the PGE2-mediated induction of E-cadherin transcriptional repressors. Methods: Herein, we evaluate the efficacy of the COX-2 inhibitor, apricoxib, in HNSCC cell lines. Apricoxib is effective in preventing tumour cell growth in three-dimensional, and anchorage-independent growth assays, as well as decreasing the capacity for tumour cell migration. Results: Herein, we evaluate the efficacy of the COX-2 inhibitor, apricoxib, in HNSCC cell lines. Apricoxib is effective in preventing tumour cell growth in three-dimensional, and anchorage-independent growth assays, as well as decreasing the capacity for tumour cell migration. Treatment of HNSCC cells with apricoxib also causes greater upregulation of E-cadherin and Muc1 expression and downregulation of vimentin, as compared with celecoxib treatment. This has significant implications for targeted chemoprevention and anti-cancer therapy because E-cadherin expression has been implicated as a marker of sensitivity to epidermal growth factor receptor tyrosine kinase inhibitor and other therapies. We show for the first time the molecular mechanisms underlying the efficacy of apricoxib in HNSCC cells. Conclusion: In addition to reversing EMT via inhibition of COX-2, apricoxib upregulates 15-prostaglandin dehydrogenase and the prostaglandin transporter, thereby reducing the levels of active PGE2 by both suppressing its synthesis and increasing its catabolism. These findings have significant implications for metastasis and tumour progression in HNSCC.

Cyclooxygenase-2 (COX-2) is frequently overexpressed in human cancers and contributes to the malignant phenotype. Our data indicate unphosphorylated signal transducers and activators of transcription 6 (STAT6) may transcriptionally upregulate COX-2 expression and protect against apoptosis in NSCLC cells. In A427 and H2122, NSCLC cell lines that constitutively express COX-2, only unphosphorylated STAT6 was detectable by western blot, thus, all of the following STAT6-dependent effects are attributed to the unphosphorylated protein. In both cell lines, small-interfering RNA-mediated knockdown of STAT6 or stable expression of dominant-negative STAT6 decreased COX-2 expression. In contrast, transfection with a phosphorylation-deficient mutant STAT6 increased COX-2 levels. Immunofluorescent staining revealed the presence of STAT6 in H2122 nuclei, suggesting a direct role in gene regulation for the unphosphorylated protein. Consistent with this hypothesis, unphosphorylated STAT6 increased luciferase expression from a COX-2 promoter reporter construct. STAT6 co-immunoprecipitated with the transcriptional co-activator, p300, and chromatin immunoprecipitation assays demonstrated that these proteins bind a consensus STAT6 binding site located within the COX-2 promoter. STAT6 DNA-binding specificity was confirmed by electrophoretic mobility shift assay. As COX-2 over-expression has been clearly linked to apoptosis resistance and other hallmarks of malignancy, these findings suggest a novel role of unphosphorylated STAT6 in the pathogenesis of non-small cell lung cancer.

Cyclooxygenase-2 (COX-2) overexpression is found in a wide variety of human cancers, including lung cancer. 1 Dannenberg AJ Subbaramaiah K Targeting cyclooxygenase-2 in human neoplasia: rationale and promise. Cancer Cell. 2003; 4: 431-436 Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar 2 Dubinett SM Sharma S Huang M et al. Cyclooxygenase-2 in lung cancer. Prog Exp Tumor Res. 2003; 37: 138-162 Crossref PubMed Scopus (43) Google Scholar Elevated tumor COX-2 expression is associated with increased angiogenesis, 3 Pold M Zhu LX Sharma S et al. Cyclooxygenase-2-dependent expression of angioenic CXC chemokines ENA-78/CXC Ligand (CXCL) 5 and interleukin-8/CXCL8 in human non-small cell lung cancer. Cancer Res. 2004; 64: 1853-1860 Crossref PubMed Scopus (113) Google Scholar tumor invasion, 4 Dohadwala M Luo J Zhu L et al. Non-small cell lung cancer cyclooxygenase-2-dependent invasion is mediated by CD44. J Biol Chem. 2001; 276: 20809-20812 Crossref PubMed Scopus (222) Google Scholar 5 Dohadwala M Batra RK Luo J et al. Autocrine/paracrine prostaglandin E2 production by non-small cell lung cancer cells regulates matrix metalloproteinase-2 and CD44 in cyclooxygenase-2-dependent invasion. J Biol Chem. 2002; 277: 50828-50833 Crossref PubMed Scopus (247) Google Scholar 6 Tsujii M DuBois RN Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell. 1995; 83: 493-501 Abstract Full Text PDF PubMed Scopus (2150) Google Scholar suppression of host immunity, 7 Stolina M Sharma S Lin Y et al. Specific inhibition of cyclooxygenase 2 restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis. J Immunol. 2000; 164: 361-370 Crossref PubMed Scopus (459) Google Scholar 8 Huang M Stolina M Sharma S et al. Non-small cell lung cancer cyclooxygenase-2-dependent regulation of cytokine balance in lymphocytes and macrophages: up-regulation of interleukin 10 and down-regulation of interleukin 12 production. Cancer Res. 1998; 58: 1208-1216 PubMed Google Scholar and promotion of tumor cell resistance to apoptosis. 9 Krysan K Merchant FH Zhu L et al. COX-2-dependent stabilization of survivin in non-small cell lung cancer. FASEB J. 2004; 18: 206-208 Crossref PubMed Scopus (133) Google Scholar To analyze COX-2-dependent gene expression and apoptosis, cell lines constitutively expressing COX-2 complementary DNA in sense and antisense orientations were created by transducing A549 and H157 non-small cell lung cancer (NSCLC) cell lines with retroviral constructs. We find that COX-2 overexpression and its product prostaglandin E2 (PGE2) increase the apoptosis threshold of NSCLC cells and genetic or pharmacologic inhibition of COX-2 sensitizes them to apoptotic signals. The expression of the antiapoptotic protein survivin correlated positively with COX-2 expression in parental, COX-2 sense and antisense cells. Immunohistochemical analysis of human NSCLC resection specimens revealed frequent co-expression of COX-2 and survivin. Survivin ubiquitination was significantly diminished in COX-2 sense and elevated in antisense cells, thus leading to enhanced survivin degradation in COX-2 antisense cells. A similar effect was elicited by exogenous PGE2 treatment of parental NSCLC cells. In contrast to previous studies in other cell types, survivin in NSCLC cells was expressed in a cell cycle-independent manner. Tumor development in SCID mice was assessed by inoculation of equal amounts of control and COX-2 sense or antisense NSCLC cells. Consistent with our in vitro findings, survivin levels were significantly lower in tumors that developed from COX-2 antisense cells and higher in COX-2 sense-derived tumors. Our results support the hypothesis that COX-2 overexpression and PGE2 overproduction inhibit survivin ubiquitination, which leads to its stabilization and prevents proteasomal degradation of survivin in NSCLC cells. Our findings provide evidence for the importance of COX-2 overexpression in the regulation of anti-apoptotic proteins and lung cancer cell survival.

T lymphocyte survival is critical for the development and maintenance of an effective host antitumor immune response; however, the tumor environment can negatively impact T-cell survival. Lymphocytes exposed to tumor supernatants (TSNs) were evaluated for apoptosis after mitogen stimulation. TSN was observed to significantly enhance phorbol 12myristate 13-acetate/ionomycin- and anti-CD3-stimulated lymphocyte apoptosis. Enhanced lymphocyte apoptosis was associated with an impairment of nuclear factor kappaB nuclear translocation and diminished IkappaBalpha degradation. In lymphocytes stimulated after exposure to TSNs, cytoplasmic IkappaBalpha persisted as a result of alterations in IkappaB kinase (IKK) activity. Accordingly, although there were no apparent differences in IKK component concentrations, lymphocytes preexposed to TSNs exhibited markedly reduced IKK activity. We conclude that non-small cell lung cancer-derived soluble factors promote apoptosis in activated lymphocytes by an IKK-dependent pathway.

Dendritic cells (DCs) serve as professional antigen-presenting cells and are pivotal in the host immune response to tumor antigens. To define the pathways limiting DC function in the tumor microenvironment, we assessed the impact of tumor cyclooxygenase (COX)-2 expression on DC activities. Bone marrow-derived DCs were cultured in either tumor supernatant (TSN) or TSN from COX-2-inhibited tumors. After culture, DCs were pulsed with tumor-specific peptides, and their ability to generate antitumor immune responses was assessed following injection into established murine lung cancer. In vitro, DC phenotype, alloreactivity, antigen processing and presentation, and interleukin (IL)-10 and IL-12 secretion were evaluated. DCs cultured in TSN failed to generate antitumor immune responses and caused immunosuppressive effects that correlated with enhanced tumor growth. However, genetic or pharmacological inhibition of tumor COX-2 expression restored DC function and effective antitumor immune responses. Functional analyses indicated that TSN causes a decrement in DC capacity to (a) process and present antigens, (b) induce alloreactivity, and (c) secrete IL-12. Whereas TSN DCs showed a significant reduction in cell surface expression of CD11c, DEC-205, MHC class I antigen, MHC class II antigen, CD80, and CD86 as well as a reduction in the transporter-associated proteins, transporter associated with antigen processing 1 and 2, the changes in phenotype and function were not evident when DCs were cultured in supernatant from COX-2-inhibited tumors. We conclude that inhibition of tumor COX-2 expression or activity can prevent tumor-induced suppression of DC activities.

Elevated tumor cyclooxygenase 2 (COX-2) expression is associated with increased angiogenesis, tumor invasion and promotion of tumor cell resistance to apoptosis. The mechanism(s) by which COX-2 exerts its cytoprotective effects are not completely understood but may be due to an imbalance of pro- and anti-apoptotic gene expression. To analyze COX-2-dependent gene expression and apoptosis, we created cell lines constitutively expressing COX-2 cDNA in sense and antisense orientations. Whereas COX-2 sense cells have significantly heightened resistance to radiation and drug-induced apoptosis, COX-2 antisense cells are highly sensitive to apoptosis induction. We found that the expression of the anti-apoptotic protein survivin correlated positively with COX-2 expression. A COX-2-dependent modulation of survivin ubiquitination led to its stabilization in COX-2 overexpressing cells, and this effect was replicated by exogenous PGE2 treatment of parental tumor cells. In contrast to previous studies in other cell types, in nonsmall cell lung cancer cells survivin was expressed in a cell cycle-independent manner. When established in SCID mice in vivo, COX-2 antisense-derived tumors had significantly decreased survivin levels while COX-2 sense-derived tumors demonstrated elevated levels compared with controls. In accord with these findings, survivin and COX-2 were frequently upregulated and co-expressed in human lung cancers in situ.

Based on studies by our group and others, we hypothesized that IL-7 may possess antifibrotic activities in an IFN-gamma-dependent and independent manner. Here, we have evaluated the antifibrotic therapeutic potential of IL-7 in both in vitro and in vivo pulmonary fibrosis models. IL-7 inhibited both TGF-beta production and signaling in fibroblasts and required an intact JAK1/STAT1 signal transduction pathway. IL-7-mediated inhibition of TGF-beta signaling was found to be associated with an increase in Smad7, a major inhibitory regulator in the SMAD family. In the presence of IL-7, Smad7 dominant negative fibroblasts restored TGF-beta-induced collagen synthesis, indicating that an IL-7-mediated increase in Smad7 suppressed TGF-beta signaling. Consistent with these in vitro findings, recombinant IL-7 decreased bleomycin-induced pulmonary fibrosis in vivo, independent of IFN-gamma. The antifibrotic activities of IL-7 merit further basic and clinical investigation for the treatment of pulmonary fibrosis.

Cyclo-oxygenase (COX)-2 is frequently overexpressed in non-small cell lung cancer (NSCLC). Previous studies have implicated a number of cytokines and growth factors in the regulation of COX-2 expression, and COX-2 itself modulates the expression of a variety of genes. 1 Hayashi Y Kobayashi M Kuwata H et al. Interferon-γ and interleukin 4 inhibit interleukin 1 beta-induced delayed prostaglandin E(2) generation through suppression of cyclooxygenase-2 expression in human fibroblasts. Cytokine. 2000; 12: 603-612 Crossref PubMed Scopus (32) Google Scholar 2 Huang ZF Massey JB Via DP Differential regulation of cyclo-oxygenase-2 (COX-2) mRNA stability by interleukin-1 β (IL-1 β) and tumor necrosis factor-α (TNF-α) in human in vitro differentiated macrophages. Biochem Pharmacol. 2000; 59: 187-194 Crossref PubMed Scopus (107) Google Scholar 3 Saha D Datta PK Sheng H et al. Synergistic induction of cyclo-oxygenase-2 by transforming growth factor-β1 and epidermal growth factor inhibits apoptosis in epithelial cells. Neoplasia. 1999; 6: 508-517 Abstract Full Text PDF Scopus (79) Google Scholar 4 Fong CY Pang L Holland E et al. TGF-β1 stimulates IL-8 release, COX-2 expression, and PGE(2) release in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2000; 279: 201-207 PubMed Google Scholar 5 Sato T Nakajima H Fujio K et al. Enhancement of prostaglandin E2 production by epidermal growth factor requires the coordinate activation of cytosolic phospholipase A2 and cyclo-oxygenase-2 in human squamous carcinoma A431 cells. Prostaglandins. 1997; 53: 355-369 Crossref PubMed Scopus (45) Google Scholar Our previous studies show that prostaglandin E2, the major product of COX-2 activity, suppresses the T-cell–mediated immunity in lung cancer. 6 Stolina M Sharma S Lin Y et al. Specific inhibition of cyclo-oxygenase-2 restores antitumor reactivity by altering the balance of IL-10 and IL-12 synthesis. J Immunol. 2000; 164: 361-370 Crossref PubMed Scopus (460) Google Scholar 7 Huang M Stolina M Sharma S et al. Non-small cell lung cancer cyclo-oxygenase-2-dependent regulation of cytokine balance in lymphocytes and macrophages: up-regulation of interleukin-10 and down-regulation of interleukin-12 production. Cancer Res. 1998; 58: 1208-1216 PubMed Google Scholar In addition to its effects on cell-mediated immunity, tumor overexpression of COX-2 appears to be involved in enhanced invasion, promotion of angiogenesis, and resistance to apoptosis. 8 Tsujii M DuBois RN Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell. 1995; 83: 493-501 Abstract Full Text PDF PubMed Scopus (2150) Google Scholar 9 Ding XZ Tong WG Adrian TE Blockade of cyclo-oxygenase-2 inhibits proliferation and induces apoptosis in human pancreatic cancer cells. Anticancer Res. 2000; 20: 2625-2631 PubMed Google Scholar 10 Masferrer JL Leahy KM Koki AT et al. Antiangiogenic and antitumor activities of cyclo-oxygenase-2 inhibitors. Cancer Res. 2000; 60: 1306-1311 PubMed Google Scholar 11 Liu XH Kirschenbaum A Yao S et al. Inhibition of cyclo-oxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo. J Urol. 2000; 164: 820-825 Abstract Full Text Full Text PDF PubMed Google Scholar Thus, there is mounting evidence indicating the importance of COX-2 in NSCLC. However, the knowledge about the changes in gene expression caused by the overexpression of COX-2 in NSCLC is fragmentary. In order to elucidate the COX-2–induced changes in NSCLC, we have conducted a microarray study using the Human Cancer Specific GeneChip (Affymetrix; Santa Clara, CA). In our study, we compared the expression of approximately 1,700 genes in the human lung adenocarcinoma cell line A549, transduced with COX-2, to A549 cells with either no or very little COX-2 expression. As a result, we have determined that the A549 cells of high COX-2 content express lower levels of insulin-like growth factor binding protein (IGFBP)-3 messenger RNA and, as determined by IGFBP-3 enzyme-linked immunosorbent assay, lower levels of free soluble IGFBP-3 protein. Thus, the amount of free soluble A549 inversely correlated with the expression level of COX-2. Previous studies have shown that IGFBP-3 antagonizes the mitogenicity of insulin-like growth factor (IGF)-1 and IGF-2. 12 Grimberg A Cohen P Role of insulin-like growth factors and their binding proteins in growth control and carcinogenesis. J Cell Physiol. 2000; 183: 1-9 Crossref PubMed Scopus (463) Google Scholar 13 Baxter RC Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities. Am J Physiol Endocrinol Metab. 2000; 278: 967-976 PubMed Google Scholar In addition, IGFBP-3 also has an IGF-independent tumor suppressor activity. 14 Butt AJ Firth SM King MA et al. Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells. J Biol Chem. 2000; 275: 39174-39181 Crossref PubMed Scopus (195) Google Scholar 15 Hochscheid R Jaques G Wegmann B Transfection of human insulin-like growth factor-binding protein-3 gene inhibits cell growth and tumorigenicity: a cell culture model for lung cancer. J Endocrinol. 2000; 166: 553-563 Crossref PubMed Scopus (48) Google Scholar 16 Williams AC Collard TJ Perks CM et al. Increased p53-dependent apoptosis by the insulin-like growth factor binding protein IGFBP-3 in human colonic adenoma-derived cells. Cancer Res. 2000; 60: 22-27 PubMed Google Scholar Therefore, we hypothesize that COX-2–induced suppression of IGFBP-3 in lung cancer cells is one of the elements leading to increased tumorigenicity. This is the first study to implicate IGFBP-3 as a gene modulated by COX-2 overexpression in cancer cells.

Elevated tumor cyclooxygenase (COX-2) expression is associated with increased angiogenesis, tumor invasion, and suppression of host immunity. We have previously shown that genetic inhibition of tumor COX-2 expression reverses the immunosuppression induced by non-small cell lung cancer (NSCLC). To assess the impact of COX-2 expression in lung cancer invasiveness, 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. COX-2-S clones expressed significantly more COX-2 protein, produced 10-fold more prostaglandin E2, and demonstrated an enhanced invasive capacity compared with control vector-transduced or parental cells. CD44, the cell surface receptor for hyaluronate, 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 suggest that a COX-2-mediated, CD44-dependent pathway is operative in NSCLC invasion. Because tumor COX-2 expression appears to have a multifaceted role in conferring the malignant phenotype, COX-2 may be an important target for gene or pharmacologic therapy in NSCLC.

Secondary lymphoid tissue chemokine (SLC, also referred to as Exodus 2 or 6Ckine) is a recently identified high endothelial-derived CC chemokine. The ability of SLC to chemoattract both Th1 lymphocytes and dendritic cells formed the rationale to evaluate this chemokine in cancer immunotherapy. Intratumoral injection of recombinant SLC evidenced potent antitumor responses and led to complete tumor eradication in 40% of treated mice. SLC-mediated antitumor responses were lymphocyte dependent as evidenced by the fact that this therapy did not alter tumor growth in SCID mice. Studies performed in CD4 and CD8 knockout mice also revealed a requirement for both CD4 and CD8 lymphocyte subsets for SLC-mediated tumor regression. In immunocompetent mice, intratumoral SLC injection led to a significant increase in CD4 and CD8 T lymphocytes and dendritic cells, infiltrating both the tumor and the draining lymph nodes. These cell infiltrates were accompanied by the enhanced elaboration of Th1 cytokines and chemokines monokine induced by IFN-γ and IFN-γ-inducible protein 10 but a concomitant decrease in immunosuppressive cytokines at the tumor site. In response to irradiated autologous tumor, splenic and lymph node-derived cells from SLC-treated tumor-bearing mice secreted significantly more IFN-γ, GM-CSF, and IL-12 and reduced levels of IL-10 than did diluent-treated tumor-bearing mice. After stimulation with irradiated autologous tumor, lymph node-derived lymphocytes from SLC-treated tumor-bearing mice demonstrated enhanced cytolytic capacity, suggesting the generation of systemic immune responses. These findings provide a strong rationale for further evaluation of SLC in tumor immunity and its use in cancer immunotherapy.

In two murine lung cancer models adenoviral interleukin 7-transduced dendritic cells (DC-AdIL-7) were administered intratumorally, resulting in complete tumor regression. Intratumoral DC-AdIL-7 therapy was as effective as DCs pulsed with specific tumor peptide antigens. Comparison with other intratumoral therapies including recombinant IL-7, AdIL-7 vector alone, unmodified DCs, IL-7-transduced fibroblasts, or DCs pulsed with tumor lysates revealed DC-AdIL-7 therapy to be superior in achieving antitumor responses and augmenting immunogenicity. Mice with complete tumor eradication as a result of either DC-AdIL-7 or AdIL-7 therapy were rechallenged with parental tumor cells 30 days or more after complete tumor eradication. All the DC-AdIL-7-treated mice completely rejected a secondary rechallenge, whereas the AdIL-7-treated mice had sustained antitumor effects in only 20-25% of the mice. DC-AdIL-7 therapy was more effective than AdIL-7 in achieving systemic antitumor responses and enhancing immunogenicity. After complete tumor eradication, those mice treated with DC-AdIL-7 evidenced significantly greater release of splenocyte GM-CSF and IFN-gamma than did controls or AdIL-7-treated mice. After intratumoral injection, gene-modified DCs trafficked from the tumor to lymph node sites and spleen. DCs were detected in nodal tissues for up to 7 days after intratumoral injection. We report that intratumoral DC-AdIL-7 leads to significant systemic immune responses and potent antitumor effects in murine lung cancer models.