Meredith A. Tennis

Wnt signaling pathway and lung disease.

The Wnt pathway plays an important role in development and in regulating adult stem cell systems. A variety of cellular processes is mediated by Wnt signaling, which includes cellular proliferation, differentiation, survival, apoptosis, and cell motility. Loss of regulation of these pathways can lead to tumorigenesis, and the Wnt pathway has been implicated in the development of several types of cancers, including colon, lung, leukemia, breast, thyroid, and prostate. The Wnt pathway has also been associated with other lung diseases such as interstitial lung disease (ILD) and asthma. Our increasing understanding of the Wnt pathway offers great hope that new molecular-based screening tests and pharmaceutical agents that selectively target this pathway will be developed to diagnose and treat these diseases in the future.

Sprouty-4 Inhibits Transformed Cell Growth, Migration and Invasion, and Epithelial-Mesenchymal Transition, and Is Regulated by Wnt7A through PPARγ in Non-Small Cell Lung Cancer

Sprouty proteins are potent receptor tyrosine kinase inhibitors that antagonize growth factor signaling and are involved in lung development. However, little is known about the regulation or targets of Sprouty-4 (Spry4) in lung cancer. Our study aimed to determine the role of Spry4 in non–small cell lung cancer (NSCLC). We found that Spry4 mRNA expression was decreased in NSCLC cell lines and in dysplastic lung cell lines compared with a nontransformed cell line, suggesting that Spry4 has tumor-suppressing activity. When Spry4 was stably transfected into H157 and H2122 NSCLC cell lines, decreased migration and invasion were observed. Matrix metalloproteinase-9 activity was decreased, and the expression of matrix metalloproteinase inhibitors TIMP1 and CD82 were increased. Stable expression of Spry4 led to reduced cell growth and reduced anchorage-independent growth in NSCLC cell lines, along with upregulation of tumor suppressors p53 and p21. Changes in epithelial and mesenchymal markers indicated that Spry4 expression induces a reversal of the epithelial to mesenchymal transition characteristic of tumor cells. Treatment of a nontransformed lung epithelial cell line with short hairpin RNA to Spry4 led to the decreased expression of epithelial markers and increased cell growth, supporting the concept of Spry4 acting as a tumor suppressor. We showed that the activity of the Spry4 promoter is increased by Wnt7A/Fzd9 signaling through peroxisome proliferator–activated receptor γ. These data present previously undescribed targets of Spry4 and suggest that Spry4 is a downstream target of Wnt7A/Fzd 9 signaling. Spry4 may have efficacy in the treatment of NSCLC. Mol Cancer Res; 8(6); 833–43. ©2010 AACR.

Depletion of Tumor-Associated Macrophages Slows the Growth of Chemically Induced Mouse Lung Adenocarcinomas

Chronic inflammation is a risk factor for lung cancer, and low-dose aspirin intake reduces lung cancer risk. However, the roles that specific inflammatory cells and their products play in lung carcinogenesis have yet to be fully elucidated. In mice, alveolar macrophage numbers increase as lung tumors progress, and pulmonary macrophage programing changes within 2 weeks of carcinogen exposure. To examine how macrophages specifically affect lung tumor progression, they were depleted in mice bearing urethane-induced lung tumors using clodronate-encapsulated liposomes. Alveolar macrophage populations decreased to ≤50% of control levels after 4–6 weeks of liposomal clodronate treatment. Tumor burden decreased by 50% compared to vehicle treated mice, and tumor cell proliferation, as measured by Ki67 staining, was also attenuated. Pulmonary fluid levels of insulin-like growth factor-I, CXCL1, IL-6, and CCL2 diminished with clodronate liposome treatment. Tumor-associated macrophages expressed markers of both M1 and M2 programing in vehicle and clodronate liposome-treated mice. Mice lacking CCR2 (the receptor for macrophage chemotactic factor CCL2) had comparable numbers of alveolar macrophages and showed no difference in tumor growth rates when compared to similarly treated wild-type mice suggesting that while CCL2 may recruit macrophages to lung tumor microenvironments, redundant pathways can compensate when CCL2/CCR2 signaling is inactivated. Depletion of pulmonary macrophages rather than inhibition of their recruitment may be an advantageous strategy for attenuating lung cancer progression.

Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer

Wnt7a is known to be a tumor suppressor that is lost in NSCLC, but no mechanism of loss has been established. Methylation of promoter regions has been established as a common mechanism of loss of tumor suppressor expression in NSCLC. We previously demonstrated that loss of Wnt7a in non-transformed lung epithelial cell lines led to increased cell growth, altered 3-D culture growth, and increased migration. The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity. We treated H157 and H1299 NSCLC cell lines with 5-Aza-2′-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway. When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased. Together these data suggest that in NSCLC, Wnt7a is lost by methylation in a subset of tumors and that this methylation is maintained by DNMT1. Restoration of Wnt7a expression through demethylation could be an important therapeutic approach in the treatment of NSCLC.

The role of prostacyclin in lung cancer

Prostanoids are bioactive lipids that interact with 7-membrane-spanning G-protein-coupled receptors on target cells to impart their biologic effects. They include prostaglandins, prostacyclin, and thromboxane. Prostanoids are widely distributed; mediate several diverse biologic effects like platelet aggregation and smooth-muscle contraction; and are known to be involved in allergies, acquired immunity, and cancer metastasis. Prostanoids have also been associated with breast and endometrial cancer promotion, and with the inhibition of melanoma. The role of prostanoids in the development of lung disease has been poorly understood. In particular, prostacyclin possesses significant anti-inflammatory and antimetastatic properties and is the main product of cyclooxygenase-2 activity in the lung. In fact, the balance of the various members of the prostanoids family, specifically the prostaglandins PGE(2) and prostacyclin (PGI(2)), seems to play an increasingly important role in the development of lung cancer. Gaining a better understanding of prostanoids and their associated pathways is critical to the future development of molecular-based and pharmaceutical treatments of lung disease.

Prostacyclin and EMT Pathway Markers for Monitoring Response to Lung Cancer Chemoprevention

Lung cancer is the leading cause of cancer death worldwide and global burden could be reduced through targeted application of chemoprevention. The development of squamous lung carcinoma has been linked with persistent, high-grade bronchial dysplasia. Bronchial histology improved in former smokers in a chemoprevention trial with the prostacyclin analogue iloprost. Prostacyclin acts through peroxisome proliferator-activated receptor gamma (PPARγ) to reverse epithelial to mesenchymal transition and promote anticancer signaling. We hypothesized that the prostacyclin signaling pathway and EMT could provide response markers for prostacyclin chemoprevention of lung cancer. Human bronchial epithelial cells were treated with cigarette smoke condensate (CSC) or iloprost for 2 weeks, CSC for 16 weeks, or CSC for 4 weeks followed by 4 weeks of CSC and/or iloprost, and RNA was extracted. Wild-type or prostacyclin synthase transgenic mice were exposed to 1 week of cigarette smoke or one injection of urethane, and RNA was extracted from the lungs. We measured potential markers of prostacyclin and iloprost efficacy in these models. We identified a panel of markers altered by tobacco carcinogens and inversely affected by prostacyclin, including PPARγ, 15PGDH, CES1, COX-2, ECADHERIN, SNAIL, VIMENTIN, CRB3, MIR34c, and MIR221. These data introduce a panel of potential markers for monitoring interception of bronchial dysplasia progression during chemoprevention with prostacyclin. Chemoprevention is a promising approach to reduce lung cancer mortality in a high-risk population. Identifying markers for targeted use is critical for success in future clinical trials of prostacyclin for lung cancer chemoprevention. Cancer Prev Res; 11(10); 643–54. ©2018 AACR.

Corrigendum: Depletion of Tumor-Associated Macrophages Slows the Growth of Chemically Induced Mouse Lung Adenocarcinomas

[This corrects the article on p. 587 in vol. 5, PMID: 25505466.].

Abstract 233: Prostacyclin analogs, iloprost and treprostinil, differentially influence proliferation of lung tumor cells

Lung cancer will kill approximately 160,000 Americans this year–more than the next four cancer subtypes (breast, prostate, colorectal, and pancreatic) combined. Even if lung cancer is diagnosed at an early stage, the 5 year survival rate is 52.2%. The poor survival rates for early stage disease suggests that chemopreventive interventions in high-risk populations are necessary. We found that prostacyclin (prostaglandin I 2 or PGI 2 ) synthase (PGIS) overexpression retards lung tumor growth in preclinical chemical carcinogen and smoking models, and treatment with iloprost (a PGI 2 analog currently approved for treatment of pulmonary hypertension) inhibits lung adenocarcinoma formation in these same preclinical models. Oral iloprost also improves bronchial dysplasia (precursor lesions to squamous cell lung cancer) in former smokers. Mechanistic in vitro and in vivo studies using genetically modified mice showed that PGI 2 and iloprost exert their chemopreventive effects through activation of the peroxisome proliferator-activated receptor γ (PPARγ) pathway rather than the classic PGI 2 receptor, IP 1 , and that this activation requires frizzled 9 (Fzd9) activity. Treprostinil, a next generation PGI 2 analog also approved to treat pulmonary hypertension, activates IP 1 as well, but its ability to activate PPARγ/Fzd9 has yet to be fully characterized. In vitro studies show that in addition to binding to IP, iloprost and treprostinil differentially activate other prostaglandin (PG) receptors suggesting that they may have different chemopreventive efficacies. Molecular modeling of iloprost and treprostinil binding to PPARγ indicates these molecules may induce different PPARγ conformations which could affect binding of co-activators/repressors or interaction with the retinoic acid receptor (which is required for induction of gene transcription). While iloprost does not affect mouse lung tumor cell proliferation in vitro, treprostinil and prostaglandin E 2 actually increase proliferation 2-5 fold in a dose dependent manner. Alveolar macrophage conditioned media also increases mouse lung tumor cell proliferation, and this increase is enhanced if macrophages are treated with treprostinil or PGE 2 prior to media harvest. If macrophages are pretreated with iloprost, either alone or in combination with PGE 2 or treprostinil, this increased proliferation is attenuated suggesting that iloprost may be exerting its chemopreventive effects directly on tumor cells and by modulating macrophage production of pro-growth factors. Macrophage IL-6 production decreased almost 80% with iloprost pre-treatment while little effect was seen with treprostinil pre-treatment. Comparisons between iloprost and treprostinil may indicate new agents/targets for lung cancer chemoprevention. Citation Format: Lori D. Dwyer-Nield, Gregory Hickey, Meredith A. Tennis, Kevin S. Choo, Donald S. Backos, Robert L. Keith. Prostacyclin analogs, iloprost and treprostinil, differentially influence proliferation of lung tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 233. doi:10.1158/1538-7445.AM2014-233

Abstract 2356: Biomarkers of progressive squamous lung dyplasia.

Lung cancer is the leading cause of cancer death in the United States. Squamous cell lung cancers (SCC) comprise approximately 30% of lung cancer cases, and 48,000 people are projected to die this year in the U.S. from SCC. Bronchial dysplasia (BD) is a precursor of SCC, however not all BDs progress to carcinoma. Persistent or progressive BD is associated with a higher risk for the development of invasive lung cancer. We hypothesize that characterization of the difference between persistent and regressive BD will identify high risk patients and suggest novel therapeutic targets for prevention of progression to lung SCC. Gene expression analysis was used to identify differences between persistent and regressive BD. Subjects recruited to Colorado SPORE in Lung Cancer screening studies that met sputum atypia criteria and underwent multiple bronchoscopies were evaluated. Sixty-three frozen baseline biopsies showing BD by histology were classified as persistent/progressive (group 1) or regressive (group 2) according the presence or absence of BD on follow-up biopsies at the same sites. HE 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2356. doi:10.1158/1538-7445.AM2013-2356

Abstract PR03: Frizzled 9 expression is a potential marker of response to iloprost chemoprevention of lung cancer

Lung cancer remains the leading cause of cancer death in the United States and chemoprevention offers an appealing area of investigation in the face of limited therapeutic success. We recently demonstrated improvement in endobronchial histology in former smokers after oral iloprost treatment. Of the 48 patients who received iloprost in the chemoprevention trial, 23 had regressive histology and 25 had stable or progressive histology. Identifying markers that predict response to treatment will refine target populations for future trials and clinical applications. In vitro studies of NSCLC indicate that iloprost, a prostacyclin analogue, acts through the G-protein coupled receptor Frizzled 9 (Fzd9) instead of the prostacyclin receptor. We hypothesize that Fzd9 expression status predicts response to iloprost chemoprevention and that expression is modified by promoter hypermethylation. Patients who have lost Fzd9 expression will not respond to iloprost treatment. If Fzd9 is lost by methylation, a demethylating agent could restore Fzd9 expression and sensitivity to iloprost chemoprevention. In a urethane A/J mouse model of lung cancer, Fzd9 mRNA expression is reduced in lung tumors compared to matched, uninvolved lung tissue. Human lung tumors demonstrated reduced Fzd9 mRNA expression compared to matched normal lung tissue. Fzd9 expression is also decreased in human primary dysplastic cell lines, suggesting that loss of Fzd9 expression occurs early in lung tumorigenesis. Non-transforming lung epithelial cell lines (B2B or HBEC) were treated for four weeks with 1% cigarette smoke condensate. B2B cells were treated with the tobacco carcinogen NNK (10μM) for 2 hours. Both treatment conditions resulted in reduced Fzd9 mRNA expression measured by qPCR. Future work will assess Fzd9 expression in an in vitro chemoprevention and smoking model, in tissues from additional mouse models of lung cancer, and in tissues from chemoprevention trials. These initial studies suggest that Fzd9 expression is lost in early smoking-induced lesions. This study has the potential to improve iloprost lung cancer chemoprevention by allowing future trials to more effectively target high-risk patients and by providing a clinical biomarker for identification of chemoprevention candidates. This abstract is also presented as Poster A29. Citation Format: Meredith Tennis, Lori Nield, Dan Merrick, Robert Keith. Frizzled 9 expression is a potential marker of response to iloprost chemoprevention of lung cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr PR03.