Abstract CN12-03: The inflammation-EMT-cancer initiating cell axis in the pathogenesis of lung cancer: Breaking ground in the pursuit of targeted chemoprevention
Authors: Walser Tonya C. , Krysan Kostyantyn , Gardner Brian K. , Hong Longsheng , Fishbein Michael C. , Goodglick Lee , Ooi Aik , Gomperts Brigitte N. , Larsen Jill E. , Minna John D. , Dubinett Steven M. 
Published: January 2011

Abstract
Chronic inflammation is a risk factor for numerous malignancies. Three lectures will be given. In the first, the link between obesity, inflammation, and risk of post-menopausal breast cancer will be discussed. The potential importance of cyclooxygenase-derived prostaglandin E2 as a determinant of aromatase expression, estrogen synthesis, and breast cancer risk will be reviewed. The second lecture will focus on the link between inflammation, stromal cells, and gastrointestinal cancers. Chronic inflammation of the gastrointestinal tract results in recruitment of bone marrow-derived cells to the incipient cancer site. The importance of bone marrow-derived fibroblasts in the progression of cancer will be discussed. The final presentation will focus on linkage of inflammation, epithelial-mesenchymal transition (EMT), and stem cell biology in the pathogenesis of lung cancer. Recent studies suggest that zinc-finger transcriptional repressors of E-cadherin are induced by inflammatory responses in early lung cancer development. Convergence of inflammation-induced EMT, acquisition of stem cell traits, and activation of malignant phenotypes to yield lung cancer initiating cells will be described.
Inflammation. Smoking results in inflammation and a field of chronic injury of the respiratory tract that undergoes constant repair. Pulmonary diseases associated with the greatest risk for lung cancer, such as chronic obstructive pulmonary disease (COPD), are also characterized by abundant and dysregulated inflammation. Most patients who smoke or have COPD develop regions of airway epithelial dysplasia which are considered to be premalignant lesions. Recent molecular findings support the stepwise lung carcinogenesis model in which chronic inflammation and injury lead to dysregulated repair by stem/progenitor cells. Subsequent genetic and epigenetic alterations result in expansion of the premalignant field which can persist even after smoking cessation. The inflammatory events and dysregulated repair programs operative in stem/progenitor cells in the developing tumor microenvironment require further characterization. Delineation of these and other molecular events precipitating lung cancer development will facilitate targeted chemoprevention.
EMT. While the transcriptional repressors Snail, Slug, Zeb, and Twist are known to contribute to the progression of established tumors, they are increasingly recognized for their role in neoplastic transformation. Weinberg and colleagues were the first to report that induction of EMT in immortalized human mammary epithelial cells leads to acquisition of mesenchymal traits and expression of stem cell markers. More recently, LBX1, which directs expression of Snail and Zeb, was noted to morphologically transform mammary epithelial cells and to expand the CD44+CD24- cancer stem cell subpopulation. In a study of pancreatic and colon cancers, Zeb promoted tumorigenicity by repressing “stemness”-inhibiting miRNAs. A similar role for inflammation-induced EMT and stem cells in lung cancer initiation has not been described to date.
Cancer stem cells. The cancer stem cell (CSC) model of tumor development and progression refers to the presence of a small subset of cells in the tumor that have stem cell properties and are responsible for tumor initiation, progression, and metastasis via their aberrant self-renewal, differentiation, and repair mechanisms. These cancer initiating cells are associated with poor prognosis, relapse, and recurrence of numerous malignancies. The CSC model and therapeutic resistance fits well with the natural history of lung cancer given its high incidence of recurrence and metastasis. Identification and transcriptional profiling of unique and phenotypically defined lung cancer-initiating cells that are responsible for initiation, progression, and therapeutic resistance of non-small cell lung cancer (NSCLC) would yield important new chemotherapeutic targets.
Inflammation-EMT-cancer initiating cell axis in the pathogenesis of lung cancer. We hypothesize that patients with chronic inflammation have stem/progenitor cell populations that participate in the stepwise progression of disease from the field of injury to lung cancer. We anticipate that profiling these stem/progenitor cell populations during lung carcinogenesis will identify genetic/proteomic signatures specific for patients at greatest risk for developing lung cancer. Our preliminary data demonstrate stem/progenitor cells in the proximal and distal airway epithelium and implicate Snail as a novel biomarker for tumor-initiating stem cells.
Detection of Snail in premalignant NSCLC lesions. Human squamous cell carcinoma (SCC) and adenocarcinoma (ADC) both over express Snail compared to normal lung tissues, and high Snail expression portends poor prognosis among NSCLC patients. Likewise, premalignant squamous metaplasia (SM; SCC precursor) and atypical adenomatous hyperplasia (AAH; ADC precursor) lesions overexpress Snail compared to normal lung tissues, indicating that Snail is upregulated during early lung cancer development. Snail is also overexpressed in both the proximal and distal airways of COPD-involved lungs, as well as in premalignant lesions within those COPD-involved lungs, implicating the transcription factor in the earliest pulmonary carcinogenic events. The following laboratory-based studies were part of our initial attempt to delineate Snail-dependent events driving the initiation of lung cancer.
Snail-induced cancer-initiating cells. For the investigation of early events in lung carcinogenesis, we utilized progenitor cells resident to human bronchial epithelium (HBEC). We found that overexpression of the transcription factor Snail is a key event in transformation of bronchial epithelial cells into cancer initiating cells. Specifically, Snail represses epithelial markers, including E-cadherin, induces numerous mesenchymal markers, promotes migration and invasion, and enables anchorage-independent cell growth. Additionally, we found that Snail overexpression drives expansion of an HBEC stem cell subpopulation characterized by high CD44 and low CD24 expression. Further investigation of the Snail overexpressing HBEC cells using the Affymetrix U133 Plus 2.0 microarray indicated that a large number of established stem cell markers were significantly upregulated. Our findings suggest a link between Snail expression, induction of EMT, and gain of stem cell properties during early lung carcinogenesis. Via fluorescence activated cell sorting, we determined that the Snail-positive subpopulation responsible for anchorage-independent growth was also phenotypically ALDH+CD44+CD24-. We hypothesize that the additional Snail-mediated cancer-associated phenotypes that we have identified, including increased migration, invasion, and proliferation, apoptosis-resistance, and expression changes in oncogenes and tumor suppressors, facilitate malignant conversion of the Snail-primed ALDH+CD44+CD24- pulmonary stem cell subset. Taken together, our preliminary data suggest that inflammation-induced Snail overexpresssion represents a novel molecular signaling event driving the initiation of NSCLC.
Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN12-03.