Cyclooxygenase (COX)-2 and its product prostaglandin (PG) E 2 underlie an immunosuppressive network that is important in the pathogenesis of non-small cell lung cancer. CD4 + CD25 + T regulatory (Treg) cells play an important role in maintenance of immunologic self-tolerance. CD4 + CD25 + Treg cell activities increase in lung cancer and appear to play a role in suppressing antitumor immune responses. Definition of the pathways controlling Treg cell activities will enhance our understanding of limitation of the host antitumor immune responses. Tumor-derived COX-2/PGE 2 induced expression of the Treg cell-specific transcription factor, Foxp3, and increased Treg cell activity. Assessment of E-prostanoid (EP) receptor requirements revealed that PGE 2 -mediated induction of Treg cell Foxp3 gene expression was significantly reduced in the absence of the EP4 receptor and ablated in the absence of the EP2 receptor expression. In vivo, COX-2 inhibition reduced Treg cell frequency and activity, attenuated Foxp3 expression in tumor-infiltrating lymphocytes, and decreased tumor burden. Transfer of Treg cells or administration of PGE 2 to mice receiving COX-2 inhibitors reversed these effects. We conclude that inhibition of COX-2/PGE 2 suppresses Treg cell activity and enhances antitumor responses. (Cancer Res 2005; 65(12): 5211-20)

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 Ecadherin, whereas treatment of COX-2-S cells with celecoxib (1 Amol/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 downregulate 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. (Cancer Res 2006; 66(10): 5338-45)

Tumor cyclooxygenase-2 (COX-2) expression is known to be associated with enhanced tumor invasiveness. In the present study, we evaluated the importance of the COX-2 product prostaglandin E2 (PGE2) and its signaling through the EP4 receptor in mediating non-small cell lung cancer (NSCLC) invasiveness. Genetic inhibition of tumor COX-2 led to diminished matrix metalloproteinase (MMP)-2, CD44, and EP4 receptor expression and invasion. Treatment of NSCLC cells with exogenous 16,16-dimethylprostaglandin E2 significantly increased EP4 receptor, CD44, and MMP-2 expression and matrigel invasion. In contrast, anti-PGE2 decreased EP4 receptor, CD44, and MMP-2 expression in NSCLC cells. EP4 receptor signaling was found to be central to this process, because antisense oligonucleotide-mediated inhibition of tumor cell EP4 receptors significantly decreased CD44 expression. In addition, agents that increased intracellular cAMP, as is typical of EP4 receptor signaling, markedly increased CD44 expression. Moreover, MMP-2-AS treatment decreased PGE2-mediated CD44 expression, and CD44-AS treatment decreased MMP-2 expression. Thus, PGE2-mediated effects through EP4 required the parallel induction of both CD44 and MMP-2 expression because genetic inhibition of either MMP-2 or CD44 expression effectively blocked PGE2-mediated invasion in NSCLC. These findings indicate that PGE2 regulates COX-2-dependent, CD44-and MMP-2-mediated invasion in NSCLC in an autocrine/ paracrine manner via EP receptor signaling. Thus, blocking PGE2 production or activity by genetic or pharmacological interventions may prove to be beneficial in chemoprevention or treatment of NSCLC.Cyclooxygenase is the rate-limiting enzyme for the production of prostaglandins and thromboxanes from free arachidonic acid (1). Two isoenzymes of cyclooxygenase (COX) 1 have now been described: a constitutive enzyme COX-1 present in most cells and tissues and an inducible enzyme COX-2 (also referred to as PGHS-2). COX-2 is known to be up-regulated in response to cytokines, growth factors, and other stimuli (1, 2). Mounting evidence documents elevated expression of COX-2 in a variety of malignancies including colon, gastric, esophageal, prostate, pancreatic, breast, and lung carcinomas (3-12). Overexpression of tumor COX-2 may be important in tumor invasion (13,14), angiogenesis (15-18), resistance to apoptosis (15, 19 -21), and suppression of host immunity (12, 22). We reported previously that COX-2 is overexpressed in human NSCLC, and the resultant high level PGE2 production mediates dysregulation of host immunity by altering the balance of interleukins 10 and 12 (12). Accordingly, specific inhibition of COX-2 or PGE2 led to significant in vivo tumor reduction in murine lung cancer models (22). Recently, other investigators have expanded on and corroborated these observations, indicating the importance of COX-2 expression in lung cancer (23-28). Elevated expression of COX-2 has been shown to increase tumor invasiveness and enhance metastatic potential (3, 29). The complex events...