Kostyantyn Krysan

Cyclooxygenase-2–Dependent Regulation of E-Cadherin: Prostaglandin E2 Induces Transcriptional Repressors ZEB1 and Snail in Non–Small Cell Lung Cancer

Elevated tumor cyclooxygenase-2 (COX-2) expression is associated with tumor invasion, metastasis, and poor prognosis in non-small cell lung cancer (NSCLC). Here, we report that COX-2-dependent pathways contribute to the modulation of E-cadherin expression in NSCLC. First, whereas genetically modified COX-2-sense (COX-2-S) NSCLC cells expressed low E-cadherin and showed diminished capacity for cellular aggregation, genetic or pharmacologic inhibition of tumor COX-2 led to increased E-cadherin expression and resulted in augmented homotypic cellular aggregation among NSCLC cells in vitro. An inverse relationship between COX-2 and E-cadherin was shown in situ by double immunohistochemical staining of human lung adenocarcinoma tissue sections. Second, treatment of NSCLC cells with exogenous prostaglandin E(2) (PGE(2)) significantly decreased the expression of E-cadherin, whereas treatment of COX-2-S cells with celecoxib (1 mumol/L) led to increased E-cadherin expression. Third, the transcriptional suppressors of E-cadherin, ZEB1 and Snail, were up-regulated in COX-2-S cells or PGE(2)-treated NSCLC cells but decreased in COX-2-antisense cells. PGE(2) exposure led to enhanced ZEB1 and Snail binding at the chromatin level as determined by chromatin immunoprecipitation assays. Small interfering RNA-mediated knockdown of ZEB1 or Snail interrupted the capacity of PGE(2) to down-regulate E-cadherin. Fourth, an inverse relationship between E-cadherin and ZEB1 and a direct relationship between COX-2 and ZEB1 were shown by immunohistochemical staining of human lung adenocarcinoma tissue sections. These findings indicate that PGE(2), in autocrine or paracrine fashion, modulates transcriptional repressors of E-cadherin and thereby regulates COX-2-dependent E-cadherin expression in NSCLC. Thus, blocking PGE(2) production or activity may contribute to both prevention and treatment of NSCLC.

Prostaglandin E2 Activates Mitogen-Activated Protein Kinase/Erk Pathway Signaling and Cell Proliferation in Non–Small Cell Lung Cancer Cells in an Epidermal Growth Factor Receptor–Independent Manner

Cyclooxygenase 2 (COX-2) overexpression is found in a wide variety of human cancers and is linked to all stages of tumorigenesis. Elevated tumor COX-2 expression is associated with increased angiogenesis, tumor invasion, suppression of host immunity and promotes tumor cell resistance to apoptosis. Previous reports have linked the COX-2 product prostaglandin E2 (PGE2) to the abnormal activation of the mitogen-activated protein kinase/Erk kinase pathway. Here we show that PGE2 is able to rapidly stimulate Erk phosphorylation in a subset of non-small cell lung cancer (NSCLC) cell lines. This effect is not evident in bronchial epithelial cells. In contrast to previous reports in colon cancer, we found that Erk activation as well as cellular proliferation induced by PGE2 was not inhibited by pretreatment of the cells with epidermal growth factor receptor (EGFR) inhibitors. Activation of the Erk pathway by PGE2 was also resistant to src kinase inhibitors but sensitive to the protein kinase C inhibition. PGE2 effects are mediated through four G protein-coupled receptors. Selective inhibition of EP receptors revealed the possible involvement of Ca2+-dependent signaling in PGE2-mediated activation of Erk. Our data indicate the presence of an EGFR-independent activation of the mitogen-activated protein kinase/Erk pathway by PGE2 in NSCLC cells. These findings provide evidence for the possible link between tumor COX-2 overexpression and elevated Erk-mediated cancer cell proliferation and migration. Importantly, these findings suggest that COX-2 overexpression may contribute to EGFR inhibitor resistance in NSCLC.

COX-2-dependent stabilization of survivin in non-small cell lung cancer

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.

Cyclooxygenase-2-Dependent Expression of Angiogenic CXC Chemokines ENA-78/CXC Ligand (CXCL) 5 and Interleukin-8/CXCL8 in Human Non-Small Cell Lung Cancer

Elevated tumor cyclooxygenase (COX)-2 activity plays a multifaceted role in non-small cell lung cancer (NSCLC). To elucidate the role of COX-2 in the in vitro and in vivo expression of two known NSCLC angiogenic peptides, CXC ligand (CXCL) 8 and CXCL5, we studied two COX-2 gene-modified NSCLC cell lines, A549 and H157. COX-2 overexpression enhanced the in vitro expression of both CXCL8 and CXCL5. In contrast, specific COX-2 inhibition decreased the production of both peptides as well as nuclear translocation of nuclear factor κB. In a severe combined immunodeficient mouse model of human NSCLC, the enhanced tumor growth of COX-2-overexpressing tumors was inhibited by neutralizing anti-CXCL5 and anti-CXCL8 antisera. We conclude that COX-2 contributes to the progression of NSCLC tumorigenesis by enhancing the expression of angiogenic chemokines CXCL8 and CXCL5.

Cyclooxygenase 2-Dependent Expression of Survivin Is Critical for Apoptosis Resistance in Non-Small Cell Lung Cancer

Elevated tumor cyclooxygenase 2 (COX-2) expression is associated with increased angiogenesis, tumor invasion, and promotion of tumor cell resistance to apoptosis. In our previous studies using non-small cell lung cancer (NSCLC) cell lines constitutively expressing COX-2 cDNA in sense and antisense orientations, we demonstrated that constitutive overexpression of COX-2 leads to stabilization of the inhibitor of apoptosis protein survivin resulting in the elevated apoptosis resistance of COX-2–overexpressing cells. Genetic or pharmacologic suppression of COX-2 activity increased proteasomal degradation of survivin and cellular response to apoptosis induction. Our data show that expression of survivin in non-small cell lung cancer cells can be significantly down-regulated by RNA interference. Whereas COX-2–overexpressing NSCLC cells have significantly higher apoptosis resistance than the parental cells, inhibition of survivin expression by small interfering RNA decreases apoptosis resistance to the level of the parental non-small cell lung cancer. We conclude that COX-2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer.

A Phase I Trial to Determine the Optimal Biological Dose of Celecoxib when Combined with Erlotinib in Advanced Non–Small Cell Lung Cancer

Purpose: Overexpression of cyclooxygenase-2 (COX-2) activates extracellular signal-regulated kinase/mitogen-activated protein kinase signaling in an epidermal growth factor receptor (EGFR) tyrosine kinase inhibition (TKI)–resistant manner. Because preclinical data indicated that tumor COX-2 expression caused resistance to EGFR TKI, a phase I trial to establish the optimal biological dose (OBD), defined as the maximal decrease in urinary prostaglandin E-M (PGE-M), and toxicity profile of the combination of celecoxib and erlotinib in advanced non–small cell lung cancer was done. Experimental Design: Twenty-two subjects with stage IIIB and/or IV non–small cell lung cancer received increasing doses of celecoxib from 200 to 800 mg twice daily (bid) and a fixed dose of erlotinib. Primary end points included evaluation of toxicity and determination of the OBD of celecoxib when combined with erlotinib. Secondary end points investigate exploratory biological markers and clinical response. Results: Twenty-two subjects were enrolled, and 21 were evaluable for the determination of the OBD, toxicity, and response. Rash and skin-related effects were the most commonly reported toxicities and occurred in 86%. There were no dose-limiting toxicities and no cardiovascular toxicities related to study treatment. All subjects were evaluated on intent to treat. Seven patients showed partial responses (33%), and five patients developed stable disease (24%). Responses were seen in patients both with and without EGFR-activating mutations. A significant decline in urinary PGE-M was shown after 8 weeks of treatment, with an OBD of celecoxib of 600 mg bid. Conclusions: This study defines the OBD of celecoxib when combined with a fixed dose of EGFR TKI. These results show objective responses with an acceptable toxicity profile. Future trials using COX-2 inhibition strategies should use the OBD of celecoxib at 600 mg bid.

Cyclooxygenase-2-Dependent Activation of Signal Transducer and Activator of Transcription 3 by Interleukin-6 in Non–Small Cell Lung Cancer

Purpose: Cyclooxygenase-2 (COX-2), phosphorylated signal transducers and activators of transcription 3 (STAT3), and interleukin-6 (IL-6) are elevated in non–small cell lung cancer (NSCLC). These molecules affect numerous cellular pathways, including angiogenesis and apoptosis resistance, and, therefore, may act in concert in NSCLC. Experimental Design: We examined IL-6 and phosphorylated STAT3 in COX-2-overexpressing [COX-2 sense-oriented (COX-2-S)] NSCLC cells and control cells. The effect of IL-6, STAT3, phosphatidylinositol 3-kinase, and mitogen-activated protein/extracellular signal-regulated kinase kinase on vascular endothelial growth factor (VEGF) production and apoptosis resistance was assessed in COX-2-overexpresing cells. Results: We report that NSCLC cells overexpressing COX-2 (COX-2-S) have increased IL-6 and phosphorylated STAT3 expression compared with control cells. IL-6 induced expression of VEGF in NSCLC cells. Moreover, blocking IL-6, mitogen-activated protein/extracellular signal-regulated kinase kinase, or phosphatidylinositol 3-kinase decreased VEGF production in COX-2-S cells. The addition of IL-6 to NSCLC cells resulted in increased apoptosis resistance. Furthermore, the inhibition of STAT3 or IL-6 induced apoptosis and reduced survivin expression, a member of the inhibitor of apoptosis protein family in COX-2-S cells. Conclusions: Overall, these findings suggest a novel pathway in which COX-2 activates STAT3 by inducing IL-6 expression. This pathway could contribute to tumor formation by promoting survivin-dependent apoptosis resistance and VEGF production. These findings provide a rationale for the future development of STAT3, IL-6, and/or COX-2-targeted therapies for the treatment of lung cancer.

The Potential and Rationale for COX-2 Inhibitors in Lung Cancer

Cyclooxygenase-2 (COX-2) overexpression is seen in many malignancies including lung cancer. Elevated tumor prostaglandin E2 (PGE2), a major COX-2 metabolite, levels have been implicated in angiogenesis, tumor growth and invasion, apoptosis resistance and suppression of anti-tumor immunity. Recent studies also revealed that PGE2 signaling may confer cells resistant to targeted growth factor receptor therapy by cross-activation of the receptor signaling pathway downstream components. Pre-clinical studies in animal tumor models have shown tumor reduction when animals are treated with COX-2 inhibitors and have demonstrated promising results when COX-2 inhibitors were combined with chemotherapeutic drugs. Based on these observations several ongoing clinical trials are currently evaluating COX-2 inhibitors as adjuvants with chemotherapy or radiation therapy in patients with advanced non-small cell lung cancer. Further understanding of the mechanisms of COX-2 in tumorigenesis and its interaction with other cellular pathways may highlight the new diagnostic, prognostic and therapeutic markers and facilitate future development of targeted strategies for lung cancer treatment and prevention.

Inflammation and lung carcinogenesis: applying findings in prevention and treatment

Lung carcinogenesis is a complex process requiring the acquisition of genetic mutations that confer the malignant phenotype as well as epigenetic alterations that may be manipulated in the course of therapy. Inflammatory signals in the lung cancer microenvironment can promote apoptosis resistance, proliferation, invasion, metastasis, and secretion of proangiogenic and immunosuppressive factors. Here, we discuss several prototypical inflammatory mediators controlling the malignant phenotype in lung cancer. Investigation into the detailed molecular mechanisms underlying the tumor-promoting effects of inflammation in lung cancer has revealed novel potential drug targets. Cytokines, growth factors and small-molecule inflammatory mediators released in the developing tumor microenvironment pave the way for epithelial–mesenchymal transition, the shift from a polarized, epithelial phenotype to a highly motile mesenchymal phenotype that becomes dysregulated during tumor invasion. Inflammatory mediators within the tumor microenvironment are derived from neoplastic cells as well as stromal and inflammatory cells; thus, lung cancer develops in a host environment in which the deregulated inflammatory response promotes tumor progression. Inflammation-related metabolic and catabolic enzymes (prostaglandin E2 synthase, prostaglandin I2 synthase and 15-hydroxyprostaglandin dehydrogenase), cell-surface receptors (E-type prostaglandin receptors) and transcription factors (ZEB1, SNAIL, PPARs, STATs and NF-κB) are differentially expressed in lung cancer cells compared with normal lung epithelial cells and, thus, may contribute to tumor initiation and progression. These newly discovered molecular mechanisms in the pathogenesis of lung cancer provide novel opportunities for targeted therapy and prevention in lung cancer.

Prostaglandin E2-Dependent Enhancement of Tissue Inhibitors of Metalloproteinases-1 Production Limits Dendritic Cell Migration through Extracellular Matrix

Dendritic cell (DC) migration is crucial for the initiation of immune responses. The balance between metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) has been shown to modulate DC migration. PGE2, which is overproduced in a wide variety of human malignancies, has been implicated in MMP and TIMP regulation in various cells, including monocytes. In the present study, we hypothesized that tumor-derived PGE2 would affect DC migratory capacity through the extracellular matrix (ECM) by altering MMP and TIMP balance. Treatment of monocyte-derived immature DC with exogenous PGE2 induced TIMP-1 secretion but not MMP-9 production and was correlated with reduced DC migration through ECM. Because recombinant TIMP-1 replicated PGE2 inhibition of DC migration while anti-TIMP-1 neutralizing Ab reversed it, we conclude that PGE2-mediated induction of TIMP-1 was responsible for the reduced migration of PGE2-treated DC. Similarly, DC cultured for 48 h in supernatants from cyclooxygenase-2 overexpressing lung cancer cells that secrete high levels of PGE2, exhibited decreased migration through ECM. Finally, analysis of E prostanoid receptor expression and their selective inhibition revealed that the enhanced TIMP-1 secretion in PGE2-treated DC was mediated predominantly by the E prostanoid receptor 2. These findings indicate that PGE2-dependent enhancement of TIMP-1 production causes reduced migration of DC through ECM.