miR-17-92/p38a Dysregulation Enhances Wnt SignalingandSelectsLgr6þCancerStem-likeCells during Lung Adenocarcinoma Progression

Defining the molecular and cellular roots of lung cancer relapse after initial treatment remains an imperative to improve survival. Here we report that the lung stem cell marker Lgr6 becomes enriched in non–small cell lung cancer (NSCLC) cells during malignant progression. Lgr6þ NSCLC cells displayed self-renewal and differentiation properties along with a higher tumorigenic potential. Mechanistic investigations suggested that a defective repression of the miR-1792 gene cluster was responsible for evolution of a selection for outgrowth of Lgr6þ NSCLC cells. High levels of expression of miR-19 family members were found to target and downregulate levels of p38a kinase, providing a specific survival signal for Lgr6þ cells as mediated by increased Wnt/ß-catenin activity. Our results identify a specific stem-like cell population in NSCLC with increased malignant potential, the elucidation of which may enable earlier prognosis and possibly the development of more effective targeted treatments. Cancer Res; 76(13); 4012–22. 2016 AACR. Introduction Lung cancer is the major cause of cancer-related deaths in western countries (1). Early detection and surgery have proven to be the most effective ways to improve survival (2). Important advances have beenmade in the last 20 years,with thedetectionof oncogenes and mutations linked to many lung cancer types. However, therapies applied using drugs directed to those molecules or related pathways have failed to significantly increase survival, especially in the most prevalent cancer types, for example, lung adenocarcinoma (3–5). Cancer relapse after radiotherapy and chemotherapy is the essential reason for reduced survival (6). Previously, efforts have focused on investigating the cells and mechanisms involved in the resistance and later virulence of lung cancers after therapeutic treatments (7, 8). Recent theories in cancer research consider the existence of cell populations with stem cell–like properties in many cancer types (9, 10). These cancer stem cells would be responsible for tumor maintenance and would harbor the potential to colonize other tissues as metastatic cells (11, 12). These cells would also be especially resistant to drugs, and they would be selected during tumor progression, generating more aggressive and metastatic disease (13, 14). In this article, we have studied the cellular and molecular mechanisms behind the development and progression of human lung adenocarcinoma. We have previously characterized a population of human lung cells with stem cell potential to differentiate into all bronchioalveolar lineages while being able to selfrenew. These epithelial cells express the stem cell marker Lgr6 (Leucine-rich repeat-containing G protein–coupled receptor 6). Lgr6, and its homologous receptors, are amplifiers of Wnt signaling and are involved in maintaining stem cell self-renewal (15). We have analyzed the contribution of Lgr6þ cells to human lung adenocarcinomas.Wehaveobserved that cells-expressing Lgr6 are enriched from early to late stages of human lung adenocarcinomas malignancy. These cells showed a disruption in the balance between the p38a and miR-17-92 pathways. p53 mutation prevents its repression of miR-17-92 expression (16). Cross-talk between signaling pathways and its role in cancer has been extensively reported elsewhere (17, 18). Reduced p38a promotes activation of Wnt signaling and, in human lung cancer samples, we observed a correlation between a deficiency in p38a protein and increase of Wnt signaling, promoting Lgr6þ cells during cancer progression. These findings provide a new tool for early prognosis of human lung adenocarcinomas. We demonstrate how a novelspecific population of cells, and their regulatory mechanisms, is linked and contribute to better understanding of the malignant progression of lung adenocarcinomas. This coadjuvant process, in parallel to oncogenic mutations, helps select cells with a higher potential to perpetuate (self-renewal) and manipulate the microenvironment, facilitating colonization of foreign tissues (metastasis). Materials and Methods Isolation and culture of human lung adenocarcinoma cells Cultures were established from freshly isolated lung adenocarcinoma cells obtained from cancer patients undergoing tumor Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge,Cambridge, UnitedKingdom. Translational Cell and Tissue Research, Department of Imaging and Pathology, KU Leuven, Belgium. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Juan-Jose Ventura, KU Leuven, Minderbroedersstraat 12, blok Q, Leuven 3000, Belgium. Phone: 320-1633-6615; Fax: 320-1633-6615; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-15-3302 2016 American Association for Cancer Research. Cancer Research Cancer Res; 76(13) July 1, 2016 4012 on November 15, 2017. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst May 4, 2016; DOI: 10.1158/0008-5472.CAN-15-3302