Abstract PR-11: Snail-induced and EMT-mediated early lung cancer development: Promotion of invasion and expansion of stem cell populations

Abstract

Abstract PR-11 The pulmonary environment at risk for carcinogenesis contains inflammatory mediators capable of potently promoting epithelial-mesenchymal transition (EMT) through the induction of E-Cadherin transcriptional repressors, including Snail. EMT is often activated during cancer invasion and metastasis, but the role of Snail-mediated EMT during early lung cancer development has not been defined. Here, we report that Snail-mediated induction of EMT in immortalized human bronchial epithelial cells (HBECs) results in increased invasion and expansion of resident pulmonary stem cell populations. By western analysis, HBECs stably transduced to express Snail (HBEC-S) have up-regulated Snail and concomitantly down-regulated E-Cadherin compared to HBEC vector control cells (HBEC-V). Snail expression by HBECs results in EMT, as numerous lung epithelial markers - Occludin, Mucin 1, Mucin 5AC, Cytokeratin (Ck) 18, β-Catenin, and γ-Catenin - are down-regulated in HBEC-S compared to HBEC-V, while several mesenchymal markers - Vimentin, p63, and CD44 - are up-regulated. Microarray gene expression analysis suggests that Snail initiates a program of invasion and stem cell population expansion. Specifically, TIMPs are down-regulated, while MMPs and numerous stem cell markers are up-regulated in HBEC-S compared to HBEC-V. Confirming the gene array data, MMP-3, MMP-9, and MMP-13 are up-regulated by up to 3.5-fold in supernatants from HBEC-S compared to HBEC-V, as measured in a Luminex-based multiplex assay. HBECs are characterized as multipotential pulmonary epithelial progenitor cells, because they are largely Hoechst-, CD44+, CD24-, Ck14+, Ck5+, and ALDH+ by flow cytometry analysis. Importantly, the ALDH+ stem cell population is expanded by approximately 30% in HBEC-S compared to HBEC-V. A role for Snail in EMT, invasion, and stem cell population expansion was confirmed by studies conducted in three-dimensional (3D) cell culture models. In a 3D spheroid culture model, HBEC-V forms compact spheroidal colonies, whereas HBEC-S forms stellate cell aggregates with invasive processes. In a 3D organotypic model of squamous metaplasia, HBEC-V forms well-organized and differentiated epithelial cell layers atop a modified basement membrane. In contrast, HBEC-S epithelial cell layers are disorganized, discohesive, and exhibit invasive behavior. E-Cadherin staining is intense in HBEC-V but nearly absent in HBEC-S. Staining with markers for basal cells of the lung epithelium, Ck14 and p63, is appropriately restricted to the basal epithelial cell layer in HBEC-V cultures. In contrast, HBEC-S cultures are uniformly positive for the markers regardless of the location of the cells within the epithelium and underlying mucosa, suggesting loss of polarity and expansion of the progenitor cell population. Cells positive for CD44, a marker associated with both invasion and stem cells, are also more numerous in HBEC-S cultures compared to HBEC-V cultures. In situ, precursor lesions of human squamous cell carcinoma and adenocarcinoma overexpress Snail compared to normal lung tissues, indicating that Snail is up-regulated during early lung cancer development. Together, these findings indicate that the role of Snail in early lung cancer development includes induction of EMT, promotion of invasion, and expansion of resident pulmonary stem cell populations. Citation Information: Cancer Prev Res 2008;1(7 Suppl):PR-11.