Aik T. Ooi

Novel stem/progenitor cell population from murine tracheal submucosal gland ducts with multipotent regenerative potential.

The airway epithelium is in direct contact with the environment and therefore constantly at risk for injury. Basal cells (BCs) have been found to repair the surface epithelium (SE), but the contribution of other stem cell populations to airway epithelial repair has not been identified. We demonstrated that airway submucosal gland (SMG) duct cells, in addition to BCs, survived severe hypoxic‐ischemic injury. We developed a method to isolate duct cells from the airway. In vitro and in vivo models were used to compare the self‐renewal and differentiation potential of duct cells and BCs. We found that only duct cells were capable of regenerating SMG tubules and ducts, as well as the SE overlying the SMGs. SMG duct cells are therefore a multipotent stem cell for airway epithelial repair This is of importance to the field of lung regeneration as determining the repairing cell populations could lead to the identification of novel therapeutic targets and cell‐based therapies for patients with airway diseases. STEM CELLS 2011;29:1283–1293

Presence of a putative tumor-initiating progenitor cell population predicts poor prognosis in smokers with non-small cell lung cancer.

Smoking is the most important known risk factor for the development of lung cancer. Tobacco exposure results in chronic inflammation, tissue injury, and repair. A recent hypothesis argues for a stem/progenitor cell involved in airway epithelial repair that may be a tumor-initiating cell in lung cancer and which may be associated with recurrence and metastasis. We used immunostaining, quantitative real-time PCR, Western blots, and lung cancer tissue microarrays to identify subpopulations of airway epithelial stem/progenitor cells under steady-state conditions, normal repair, aberrant repair with premalignant lesions and lung cancer, and their correlation with injury and prognosis. We identified a population of keratin 14 (K14)-expressing progenitor epithelial cells that was involved in repair after injury. Dysregulated repair resulted in the persistence of K14+ cells in the airway epithelium in potentially premalignant lesions. The presence of K14+ progenitor airway epithelial cells in NSCLC predicted a poor prognosis, and this predictive value was strongest in smokers, in which it also correlated with metastasis. This suggests that reparative K14+ progenitor cells may be tumor-initiating cells in this subgroup of smokers with NSCLC.

Isolation and In Vitro Characterization of Basal and Submucosal Gland Duct Stem/Progenitor Cells from Human Proximal Airways

Basal cells and submucosal gland (SMG) duct cells have been isolated and shown to be stem/progenitor cell populations for the murine airway epithelium. However, methods for the isolation of basal and SMG duct cells from human airways have not been defined. We used an optimized two‐step enzyme digestion protocol to strip the surface epithelium from tracheal specimens separate from SMG cells, and we then sorted the basal and duct stem/progenitors using fluorescence‐activated cell sorting. We used nerve growth factor receptor, as well as a combination of CD166 and CD44, to sort basal cells and also used CD166 to isolate SMG duct cells. Sorted stem/progenitor cells were cultured to characterize their self‐renewal and differentiation ability. Both basal and SMG duct cells grew into spheres. Immunostaining of the spheres showed mostly dense spheres with little to no central lumen. The spheres expressed cytokeratins 5 and 14, with some mucus‐ and serous‐secreting cells. The sphere‐forming efficiency and the rate of growth of the spheres varied widely between patient samples and correlated with the degree of hyperplasia of the epithelium. We found that only aldehyde dehydrogenase (ALDH)hi basal and duct cells were capable of sphere formation. Global inhibition of ALDH, as well as specific inhibition of the ALDH2 isoform, inhibited self‐renewal of both basal and duct cells, thereby producing fewer and smaller spheres. In conclusion, we have developed methods to isolate basal and SMG duct cells from the surface epithelium and SMGs of human tracheas and have developed an in vitro model to characterize their self‐renewal and differentiation.

Molecular Profiling of Premalignant Lesions in Lung Squamous Cell Carcinomas Identifies Mechanisms Involved in Stepwise Carcinogenesis

Lung squamous cell carcinoma (SCC) is thought to arise from premalignant lesions in the airway epithelium; therefore, studying these lesions is critical for understanding lung carcinogenesis. Previous microarray and sequencing studies designed to discover early biomarkers and therapeutic targets for lung SCC had limited success identifying key driver events in lung carcinogenesis, mostly due to the cellular heterogeneity of patient samples examined and the interindividual variability associated with difficult to obtain airway premalignant lesions and appropriate normal control samples within the same patient. We performed RNA sequencing on laser-microdissected representative cell populations along the SCC pathologic continuum of patient-matched normal basal cells, premalignant lesions, and tumor cells. We discovered transcriptomic changes and identified genomic pathways altered with initiation and progression of SCC within individual patients. We used immunofluorescent staining to confirm gene expression changes in premalignant lesions and tumor cells, including increased expression of SLC2A1, CEACAM5, and PTBP3 at the protein level and increased activation of MYC via nuclear translocation. Cytoband enrichment analysis revealed coordinated loss and gain of expression in chromosome 3p and 3q regions, respectively, during carcinogenesis. This is the first gene expression profiling study of airway premalignant lesions with patient-matched SCC tumor samples. Our results provide much needed information about the biology of premalignant lesions and the molecular changes that occur during stepwise carcinogenesis of SCC, and it highlights a novel approach for identifying some of the earliest molecular changes associated with initiation and progression of lung carcinogenesis within individual patients. Cancer Prev Res; 7(5); 487–95. ©2014 AACR.

Molecular Pathways: Targeting Cellular Energy Metabolism in Cancer via Inhibition of SLC2A1 and LDHA

Reprogramming of cellular energy metabolism is widely accepted to be one of the main hallmarks of cancer. The aberrant expression pattern of key regulators in the glycolysis pathway in cancer cells corroborates with the hypothesis that most cancer cells utilize aerobic glycolysis as their main ATP production method instead of mitochondrial oxidative phosphorylation. Overexpression of SLC2A1 and LDHA, both important regulators of the glycolysis pathway, was detected in the premalignant lesions and tumors of lung cancer patients, suggesting the involvement of these proteins in early carcinogenesis and tumor progression in cancer. Preclinical studies demonstrated that inhibiting SLC2A1 or LDHA led to diminished tumor growth in vitro and in vivo. SLC2A1 and LDHA inhibitors, when administered in combination with other chemotherapeutic agents, showed synergistic antitumor effects by resensitizing chemoresistant cancer cells to the chemotherapies. These results indicate that disrupting SLC2A1, LDHA, or other regulators in cancer cell energetics is a very promising approach for new targeted therapies. Clin Cancer Res; 21(11); 2440–4. ©2015 AACR.

Aldehyde Dehydrogenase Activity Enriches for Proximal Airway Basal Stem Cells and Promotes Their Proliferation

Both basal and submucosal gland (SMG) duct stem cells of the airway epithelium are capable of sphere formation in the in vitro sphere assay, although the efficiency at which this occurs is very low. We sought to improve this efficiency of sphere formation by identifying subpopulations of airway basal stem cells (ABSC) and SMG duct cells based on their aldehyde dehydrogenase (ALDH) activity. ALDH(hi) ABSCs and SMG duct cells were highly enriched for the population of cells that could make spheres, while the co-culture of ALDH(hi) differentiated cells with the ALDH(hi) ABSCs increased their sphere-forming efficiency. Specific ALDH agonists and antagonists were used to show that airway specific ALDH isozymes are important for ABSC proliferation. Pathway analysis of gene expression profiling of ALDH(hi) and ALDH(lo) ABSCs revealed a significant upregulation of the arachidonic acid (AA) metabolism pathway in ALDH(hi) ABSCs. We confirmed the importance of this pathway in the metabolism of proliferating ALDH(hi) ABSCs using bioenergetics studies as well as agonists and antagonists of the AA pathway. These studies could lead to the development of novel strategies for altering ABSC proliferation in the airway epithelium.

Silencing the Snail-Dependent RNA Splice Regulator ESRP1 Drives Malignant Transformation of Human Pulmonary Epithelial Cells

Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated, including in non-small cell lung cancer (NSCLC). Here, we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo Snail-mediated transformation relied upon silencing of the tumor-suppressive RNA splicing regulatory protein ESRP1. In clinical specimens of NSCLC, ESRP1 loss was documented in Snail-expressing premalignant pulmonary lesions. Mechanistic investigations showed that Snail drives malignant progression in an ALDH+CD44+CD24- pulmonary stem cell subset in which ESRP1 and stemness-repressing microRNAs are inhibited. Collectively, our results show how ESRP1 loss is a critical event in lung carcinogenesis, and they identify new candidate directions for targeted therapy of NSCLC.Significance: This study defines a Snail-ESRP1 cancer axis that is crucial for human lung carcinogenesis, with implications for new intervention strategies and translational opportunities. Cancer Res; 78(8); 1986-99. ©2018 AACR.

Abstract 1063: Examining early events in carcinogenesis by profiling premalignant lesions in the airways of patients with lung squamous cell carcinomas.

Little is known about the mechanisms involved in the initiation and progression of lung cancer, even though lung cancer contributes to the most cancer-related deaths in the world. Lung squamous cell carcinoma (SCC) is the second most common histological subtype, and often arises centrally from a large airway, usually a bronchus. It is believed that squamous lung cancer develops through a series of genetic and epigenetic changes that alter the normal epithelium from squamous metaplasia, to dysplasia, then carcinoma in situ, and finally to invasive SCC. Although there have been studies devoted to discovering the genetic and molecular changes observed in the tumor, few studies have contributed to the investigation of squamous metaplasia or dysplasia. To better understand carcinogenesis in the lung, especially those steps involved in the early and precancerous stages, a precise study of the biology of premalignant lesions is needed. To achieve this, we used laser capture microdissection to specifically isolate normal airway basal cells, premalignant cells, and tumor regions within individual patients with squamous lung cancer. The RNAs obtained were successfully sequenced using Next Generation Sequencing. Using two different statistical models of linear mixed effects and edgeR (empirical analysis of digital gene expression data in R), we identified a list of genes that were differentially expressed in the premalignant lesions and in tumors when compared to the normal basal cells. We were able to validate at RNA and protein levels several of the genes identified to have higher expression in premalignant lesions and tumors than in normal basal cells. We performed a gene network analysis and found that some of these genes are known to be involved in the development of other squamous tumors, cell cycle progression, cell death and survival, and cellular assembly, while some genes are uncharacterized or novel to cancer development. We also discovered that, even though TP53 RNA expression was not significantly different among normal, premalignant, and tumor cells, several proteins that were upregulated in the premalignant lesions were known to target TP53 protein for degradation, cytoplasmic localization, or disruption of its function. This suggests that the disruption of TP53 pathway began at the premalignant stage of carcinogenesis, and persisted through the formation of invasive carcinoma. This is the first comprehensive gene expression profiling of airway premalignant lesions and allows us to study the biology behind these lesions in the setting of carcinogenesis. Citation Format: Aik T. Ooi, Adam Gower, Kelvin Zhang, Jessica Vick, Longsheng Hong, Michael Fishbein, Brian Nagao, W Dean Wallace, David Elashoff, Marc Lenburg, Steven Dubinett, Avrum Spira, Brigitte Gomperts. Examining early events in carcinogenesis by profiling premalignant lesions in the airways of patients with lung squamous cell carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1063. doi:10.1158/1538-7445.AM2013-1063

Abstract CN12-03: The inflammation-EMT-cancer initiating cell axis in the pathogenesis of lung cancer: Breaking ground in the pursuit of targeted chemoprevention

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.