Wnt and Hedgehog Are Critical Mediators of Cigarette Smoke-Induced Lung Cancer

BACKGROUND: Lung cancer is the leading cause of cancer death in the world, and greater than 90% of lung cancers are cigarette smoke-related. Current treatment options are inadequate, because the molecular basis of cigarette-induced lung cancer is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that human primary or immortalized bronchial epithelial cells exposed to cigarette smoke for eight days in culture rapidly proliferate, show anchorage-independent growth, and form tumors in nude mice. Using this model of the early stages of smoke-induced tumorigenesis, we examined the molecular changes leading to lung cancer. We observed that the embryonic signaling pathways mediated by Hedgehog and Wnt are activated by smoke. Pharmacological inhibition of these pathways blocked the transformed phenotype. CONCLUSIONS/SIGNIFICANCE: These experiments provide a model in which the early stages of smoke-induced tumorigenesis can be elicited, and should permit us to identify molecular changes driving this process. Results obtained so far indicate that smoke-induced lung tumors are driven by activation of two embryonic regulatory pathways, Hedgehog (Hh) and Wnt. Based on the current and emerging availability of drugs to inhibit Hh and Wnt signaling, it is possible that an understanding of the role of Hh and Wnt in lung cancer pathogenesis will lead to the development of new therapies

Effects of cigarette smoke condensate on proliferation and wound closure of bronchial epithelial cells in vitro: role of glutathione

BACKGROUND: Increased airway epithelial proliferation is frequently observed in smokers. To elucidate the molecular mechanisms leading to these epithelial changes, we studied the effect of cigarette smoke condensate (CSC) on cell proliferation, wound closure and mitogen activated protein kinase (MAPK) activation. We also studied whether modulation of intracellular glutathione/thiol levels could attenuate CSC-induced cell proliferation. METHODS: Cells of the bronchial epithelial cell line NCI-H292 and subcultures of primary bronchial epithelial cells were used for the present study. The effect of CSC on epithelial proliferation was assessed using 5-bromo-2-deoxyuridine (BrdU) incorporation. Modulation of epithelial wound repair was studied by analysis of closure of 3 mm circular scrape wounds during 72 hours of culture. Wound closure was calculated from digital images obtained at 24 h intervals. Activation of mitogen-activated protein kinases was assessed by Western blotting using phospho-specific antibodies. RESULTS: At low concentrations CSC increased proliferation of NCI-H292 cells, whereas high concentrations were inhibitory as a result of cytotoxicity. Low concentrations of CSC also increased epithelial wound closure of both NCI-H292 and PBEC, whereas at high concentrations closure was inhibited. At low, mitogenic concentrations, CSC caused persistent activation of ERK1/2, a MAPK involved in cell proliferation. Inhibition of cell proliferation by high concentrations of CSC was associated with activation of the pro-apoptotic MAP kinases p38 and JNK. Modulation of intracellular glutathione (GSH)/thiol levels using N-acetyl-L-cysteine, GSH or buthionine sulphoximine (BSO), demonstrated that both the stimulatory and the inhibitory effects of CSC were regulated in part by intracellular GSH levels. CONCLUSION: These results indicate that CSC may increase cell proliferation and wound closure dependent on the local concentration of cigarette smoke and the anti-oxidant status. These findings are consistent with increased epithelial proliferation in smokers, and may provide further insight in the development of lung cancer

CAFET Algorithm Reveals Wnt/PCP Signature in Lung Squamous Cell Carcinoma

We analyzed the gene expression patterns of 138 Non-Small Cell Lung Cancer (NSCLC) samples and developed a new algorithm called Coverage Analysis with Fisher’s Exact Test (CAFET) to identify molecular pathways that are differentially activated in squamous cell carcinoma (SCC) and adenocarcinoma (AC) subtypes. Analysis of the lung cancer samples demonstrated hierarchical clustering according to the histological subtype and revealed a strong enrichment for the Wnt signaling pathway components in the cluster consisting predominantly of SCC samples. The specific gene expression pattern observed correlated with enhanced activation of the Wnt Planar Cell Polarity (PCP) pathway and inhibition of the canonical Wnt signaling branch. Further real time RT-PCR follow-up with additional primary tumor samples and lung cancer cell lines confirmed enrichment of Wnt/PCP pathway associated genes in the SCC subtype. Dysregulation of the canonical Wnt pathway, characterized by increased levels of β-catenin and epigenetic silencing of negative regulators, has been reported in adenocarcinoma of the lung. Our results suggest that SCC and AC utilize different branches of the Wnt pathway during oncogenesis

BCL6 degradation caused by the interaction with the C-terminus of pro-HB-EGF induces cyclin D2 expression in gastric cancers

BCL6 is a transcriptional repressor that has important functions in lymphocyte differentiation and lymphomagenesis, but there have been no reports of BCL6 expression in gastric cancers. In the present study, we investigated the BCL6 function in gastric cancers. Treatment with TPA resulted in BCL6 degradation and cyclin D2 upregulation. This phenomenon was inhibited by the suppression of the nuclear translocation of HB-EGF-CTF (C-terminal fragment of pro-HB-EGF). The HB-EGF-CTF nuclear translocation leads to the interaction of BCL6 with HB-EGF-CTF and the nuclear export of BCL6, and after that BCL6 degradation was mediated by ubiquitin/proteasome pathway. Real-time RT–PCR and siRNA targeting BCL6 revealed that BCL6 suppresses cyclin D2 expression. Our data indicate that BCL6 interacts with nuclear-translocated HB-EGF-CTF and that the nuclear export and degradation of BCL6 induces cyclin D2 upregulation. We performed immunohistochemical analyses of BCL6, HB-EGF and cyclin D2 in human gastric cancers. The inverse correlation between BCL6 and cyclin D2 was also found in HB-EGF-positive human gastric cancers. BCL6 degradation caused by the HB-EGF-CTF also might induce cyclin D2 expression in human gastric cancers. Inhibition of HB-EGF-CTF nuclear translocation and maintenance of BCL6 function are important for the regulation of gastric cancer progression

Microbial Patterns Signaling via Toll-Like Receptors 2 and 5 Contribute to Epithelial Repair, Growth and Survival

Epithelial cells (ECs) continuously interact with microorganisms and detect their presence via different pattern-recognition receptors (PRRs) including Toll-like receptors (TLRs). Ligation of epithelial TLRs by pathogens is usually associated with the induction of pro-inflammatory mediators and antimicrobial factors. In this study, using human airway ECs as a model, we found that detection of microbial patterns via epithelial TLRs directly regulates tissue homeostasis. Staphylococcus aureus (S. aureus) and microbial patterns signaling via TLR2 and TLR5 induce a set of non-immune epithelial responses including cell migration, wound repair, proliferation, and survival of primary and cancerous ECs. Using small interfering RNA (siRNA) gene targeting, receptor-tyrosine kinase microarray and inhibition studies, we determined that TLR and the epidermal growth factor receptor (EGFR) mediate the stimulating effect of microbial patterns on epithelial repair. Microbial patterns signaling via Toll-like receptors 2 and 5 contribute to epithelial repair, growth and survival. This effect is independent of hematopoietic and other cells as well as inflammatory cytokines suggesting that epithelia are able to regulate their integrity in an autonomous non-inflammatory manner by sensing microbes directly via TLRs

Lung epithelium as a sentinel and effector system in pneumonia – molecular mechanisms of pathogen recognition and signal transduction

Pneumonia, a common disease caused by a great diversity of infectious agents is responsible for enormous morbidity and mortality worldwide. The bronchial and lung epithelium comprises a large surface between host and environment and is attacked as a primary target during lung infection. Besides acting as a mechanical barrier, recent evidence suggests that the lung epithelium functions as an important sentinel system against pathogens. Equipped with transmembranous and cytosolic pathogen-sensing pattern recognition receptors the epithelium detects invading pathogens. A complex signalling results in epithelial cell activation, which essentially participates in initiation and orchestration of the subsequent innate and adaptive immune response. In this review we summarize recent progress in research focussing on molecular mechanisms of pathogen detection, host cell signal transduction, and subsequent activation of lung epithelial cells by pathogens and their virulence factors and point to open questions. The analysis of lung epithelial function in the host response in pneumonia may pave the way to the development of innovative highly needed therapeutics in pneumonia in addition to antibiotics