Saswati Hazra

Pioglitazone and Rosiglitazone Decrease Prostaglandin E2 in Non–Small-Cell Lung Cancer Cells by Up-Regulating 15-Hydroxyprostaglandin Dehydrogenase

Lung cancer cells elaborate the immunosuppressive and antiapoptotic mediator prostaglandin E2 (PGE2), a product of cyclooxygenase-2 (COX-2) enzyme activity. Because peroxisome proliferator-activated receptor (PPAR)γ ligands, such as thiazolidinediones (TZDs), inhibit lung cancer cell growth, we examined the effect of the TZDs pioglitazone and rosiglitazone on PGE2 levels in non–small-cell lung cancer (NSCLC) A427 and A549 cells. Both TZDs inhibited PGE2 production in NSCLC cells via a COX-2 independent pathway. To define the mechanism underlying COX-2 independent suppression of PGE2 production, we focused on other enzymes responsible for the synthesis and degradation of PGE2. The expression of none of the three prostaglandin synthases (microsomal PGES1, PGES2 and cystosolic PGES) was down-regulated by the TZDs. It is noteworthy that 15-hydroxyprostaglandin dehydrogenase (15-PGDH), an enzyme that produces biologically inactive 15-ketoprostaglandins from active PGE2, was induced by TZDs. The TZD-mediated suppression of PGE2 concentration was significantly inhibited by small interfering RNA to 15-PGDH. Studies using dominant-negative PPARγ overexpression or 2-chloro-5-nitrobenzanilide (GW9662; a PPARγ antagonist) revealed that the suppressive effect of the TZDs on PGE2 is PPARγ-independent. Together, these findings indicate that it is possible to use a clinically available pharmacological intervention to suppress tumor-derived PGE2 by enhancing catabolism rather than blocking synthesis.

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.

Proinflammatory Mediators Upregulate Snail in Head and Neck Squamous Cell Carcinoma

Purpose: Inflammatory cytokines have been implicated in the progression of head and neck squamous cell carcinoma (HNSCC). Herein we investigate the mechanisms by which interleukin-1β (IL-1β) might contribute to Epithelial-Mesenchymal Transition (EMT) in HNSCC. Experimental Design: We evaluated the effect of IL-1β on the molecular events of EMT in surgical specimens and HNSCC cell lines. We examined the correlation with tumor histologic features, and a SCID xenograft model was used to assess the effects of Snail overexpression. Results: Cyclooxygenase-2 (COX-2)-dependent pathways contribute to the modulation of E-cadherin expression in HNSCC. An inverse relationship between COX-2 and E-cadherin was shown in situ by double immunohistochemical staining of human HNSCC tissue sections. Treatment of HNSCC cells with IL-1β caused the downregulation of E-cadherin expression and upregulation of COX-2 expression. This effect was blocked in the presence of COX-2 small hairpin RNA. IL-1β–treated HNSCC cell lines showed a significant decrease in E-cadherin mRNA and an increase in the mRNA expression of the transcriptional repressor Snail. IL-1β exposure led to enhanced Snail binding at the chromatin level. Small hairpin RNA–mediated knockdown of Snail interrupted the capacity of IL-1β to downregulate E-cadherin. In a SCID xenograft model, HNSCC Snail-overexpressing cells showed significantly increased primary and metastatic tumor burdens. Conclusions: IL-1β modulates Snail and thereby regulates COX-2–dependent E-cadherin expression in HNSCC. This is the first report indicating the role of Snail in the inflammation-induced promotion of EMT in HNSCC. This newly defined pathway for transcriptional regulation of E-cadherin in HNSCC has important implications for targeted chemoprevention and therapy. (Clin Cancer Res 2009;15(19):6018–27)

Unphosphorylated STAT6 contributes to constitutive cyclooxygenase-2 expression in human non-small cell lung cancer

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.

Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in Non-Small Cell Lung Cancer

BackgroundOur previous studies have demonstrated that transduction of human dendritic cells (DC) with adenovirus encoding secondary lymphoid chemokine, CCL21, led to secretion of biologically active CCL21 without altering DC phenotype or viability. In addition, intratumoral injections of CCL21-transduced DC into established murine lung tumors resulted in complete regression and protective anti-tumor immunity. These results have provided the rationale to generate a clinical grade adenoviral vector encoding CCL-21 for ex vivo transduction of human DC in order to assess intratumoral administration in late stage human lung cancer.MethodsIn the current study, human monocyte-derived DC were differentiated by exposure to GM-CSF and IL-4 from cryopreserved mononuclear cells obtained from healthy volunteers. Transduction with clinical grade adenoviral vector encoding CCL21 (1167 viral particles per cell) resulted in secretion of CCL21 protein.ResultsCCL21 protein production from transduced DC was detected in supernatants (24–72 hours, 3.5–6.7 ng/4–5 × 106 cells). DC transduced with the clinical grade adenoviral vector were > 88% viable (n = 16), conserved their phenotype and maintained integral biological activities including dextran uptake, production of immunostimulatory cytokines/chemokines and antigen presentation. Furthermore, supernatant from CCL21-DC induced the chemotaxis of T2 cells in vitro.ConclusionViable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved PBMC.

The Role of PPARγ in the Cyclooxygenase Pathway in Lung Cancer

Decreased expression of peroxisome proliferator activated receptor-γ (PPARγ) and high levels of the proinflammatory enzyme cyclooxygenase-2 (COX-2) have been observed in many tumor types. Both reduced (PPARγ) expression and elevated COX-2 within the tumor are associated with poor prognosis in lung cancer patients, and recent work has indicated that these signaling pathways may be interrelated. Synthetic (PPARγ) agonists such as the thiazolidinedione (TZD) class of anti-diabetic drugs can decrease COX-2 levels, inhibit growth of non-small-cell lung cancer (NSCLC) cells in vitro, and block tumor progression in xenograft models. TZDs alter the expression of COX-2 and consequent production of the protumorigenic inflammatory molecule prostaglandin E2 (PGE2) through both (PPARγ) dependent and independent mechanisms. Certain TZDs also reduce expression of PGE2 receptors or upregulate the PGE2 catabolic enzyme 15-prostaglandin dehydrogenase. As several COX-2 enzymatic products have antitumor properties and specific COX-2 inhibition has been associated with increased risk of adverse cardiac events, directly reducing the effects or concentration of PGE2 may provide a more safe and effective strategy for lung cancer treatment. Understanding the mechanisms underlying these effects may be helpful for designing anticancer therapies. This article summarizes recent research on the relationship between (PPARγ), TZDs, and the COX-2/PGE2 pathways in lung cancer.

Focusing Downstream in Lung Cancer Prevention: 15-Hydroxyprostaglandin Dehydrogenase

Deregulated inflammatory mediators and growth factors seem to act in concert with mutational events in driving malignant transformation and progression, thus contributing to the complex pulmonary environment in the lung at risk for cancer. Pulmonary diseases that are associated with the greatest

Loss of miR125a expression in a model of K-ras-dependent pulmonary premalignancy.

Understanding the molecular pathogenesis of lung cancer is necessary to identify biomarkers/targets specific to individual airway molecular profiles and to identify options for targeted chemoprevention. Herein, we identify mechanisms by which loss of microRNA (miRNA)125a-3p (miR125a) contributes to the malignant potential of human bronchial epithelial cells (HBEC) harboring an activating point mutation of the K-ras proto-oncogene (HBEC K-ras). Among other miRNAs, we identified significant miR125a loss in HBEC K-ras lines and determined that miR125a is regulated by the PEA3 transcription factor. PEA3 is upregulated in HBEC K-ras cells, and genetic knockdown of PEA3 restores miR125a expression. From a panel of inflammatory/angiogenic factors, we identified increased CXCL1 and vascular endothelial growth factor (VEGF) production by HBEC K-ras cells and determined that miR125a overexpression significantly reduces K-ras–mediated production of these tumorigenic factors. miR125a overexpression also abrogates increased proliferation of HBEC K-ras cells and suppresses anchorage-independent growth (AIG) of HBEC K-ras/P53 cells, the latter of which is CXCL1-dependent. Finally, pioglitazone increases levels of miR125a in HBEC K-ras cells via PEA3 downregulation. In addition, pioglitazone and miR125a overexpression elicit similar phenotypic responses, including suppression of both proliferation and VEGF production. Our findings implicate miR125a loss in lung carcinogenesis and lay the groundwork for future studies to determine whether miR125a is a possible biomarker for lung carcinogenesis and/or a chemoprevention target. Moreover, our studies illustrate that pharmacologic augmentation of miR125a in K-ras–mutated pulmonary epithelium effectively abrogates several deleterious downstream events associated with the mutation. Cancer Prev Res; 7(8); 845–55. ©2014 AACR.

Abstract 3425: Interleukin-27 inhibits epithelial mesenchymal transition in lung carcinogenesis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Interleukin-27 (IL-27), the newest member of the IL-6/IL-12 family, is secreted by antigen presenting cells and has anti-tumor activity through its activation of the JAK-STAT pathway. Transcriptional factors STAT1 and STAT3 have been implicated in the balance of tumor suppressor and oncogenic functions. Epithelial mesenchymal transition (EMT) is a process whereby cells undergo changes from a highly polarized epithelial phenotype to a mesenchymal, migratory phenotype, which plays an important role in tumor metastasis. The role of IL-27 in EMT was evaluated in lung carcinogenesis. Human bronchial epithelial cells (HBECs), genetically modified HBEC to over-express Snail and K-ras (HBEC-Snail, HBEC-K-ras), and non-small cell lung cancer (NSCLC) lines (A549, H2122) were treated with IL-27 at multiple time points. Phosphorylation of STAT1 and STAT3 measured by Western Blot demonstrated activation of both pathways in these cell lines treated with IL-27. However, the STAT1 pathway was attenuated in HBEC-K-ras cells exposed to IL-27. Western analysis of markers for epithelial to mesenchymal transition (EMT) (E-cadherin, γ-catenin, N-cadherin, Vimentin, and Snail) showed up-regulation of E-cadherin and γ-catenin, important for the epithelial phenotype, which peaked 8 hours after IL-27 exposure, while markers important for the mesenchymal phenotype (N-cadherin, Vimentin, and Snail) were all down-regulated at similar time points. The addition of a STAT3 inhibitor to IL-27 treatment led to continued down-regulation of Vimentin beyond 8 hours. A wound healing functional assay to study cell migration after IL-27 treatment of cancer cell lines for up to 48 hours, demonstrated overall decrease in cell migration. In summary, IL-27 inhibits epithelial mesenchymal transition in NSCLCs and oncogenically manipulated HBECs through the regulation of STAT1 and STAT3 pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3425. doi:10.1158/1538-7445.AM2011-3425

Abstract B57: Modulation of prostaglandin E2 by statins in human bronchial epithelial cells harboring K-ras mutation: The potential advantage of combination therapy

Statin drugs, such as lovastatin and simvastatin, block cholesterol synthesis by inhibiting the function of HMG-CoA reductase in the cellular cholesterol biosynthesis pathway. In a recent large case-control study, statins reduced the risk of lung cancer suggesting the utility of statins for lung cancer chemoprevention. In our studies, while both lovastatin and simvastatin dose-dependently decreased proliferation in both histologically normal and K-ras mutated human bronchial epithelial cells (HBECs), it also unexpectedly increased expression of PGE2 in mutated HBECs. The statin-mediated upregulation of PGE2 is most pronounced in K-ras mutated HBECs compared to P53 deleted or EGFR mutated HBECs. Treatment with the cholesterol precursor, mevalonic acid, inhibited statin mediated up-regulation of PGE2 indicating that the effect of the statins on PGE2 is sterol-dependent. We found that the suppression of proliferation by statins is also sterol dependent. Statins up-regulate the production of PGE2 by up-regulating mRNA and protein expression of COX-2. The addition of the MEK inhibitor (U0126) revealed the potential involvement of the ERK pathway for statin-mediated upregulation of PGE2 in HBECs. The combination of statins and a low dose of celecoxib (0.1 micro molar), a COX-2 inhibitor, suppressed the increased production of PGE2 by K-ras mutated HBECs. Studies are underway to determine the effect of statin-mediated upregulation of PGE2 on apoptosis resistance and invasion in K-ras mutated HBECs. Our studies suggest that the combination of statins with a low dose of celecoxib overcomes the upregulation of PGE2 induced by statins alone. Thus the combination represents a potentially effective lung cancer chemopreventative regimen worthy of further investigation. Citation Information: Cancer Prev Res 2010;3(12 Suppl):B57.