Disruption of Autophagy at the Maturation Step by the Carcinogen Lindane Is Associated with the Sustained Mitogen-Activated Protein Kinase/Extracellular Signal–Regulated Kinase Activity Elisabeth Corcelle, 1,2 Marielle Nebout, 1 Soumeya Bekri, 5 Nils Gauthier, 3 Paul Hofman, 4 Philippe Poujeol, 2 Patrick Fe ´nichel, 1 and Baharia Mograbi 1,4 1 Institut National de la Sante´ et de la Recherche Me´dicale, INSERM U670, IFR 50, Faculte´ de Me´decine; 2 Centre National de la Recherche Scientifique Unite´ Mixte de Recherche 6548, Faculte´ des Sciences; 3 Institut National de la Sante´ et dela Recherche Me´dicale,U627, IFR 50; 4 Institut Nationaldela Sante´ etdela Recherche Me´dicale/Re´gion Provence Alpes Co ˆte d’ Azur, ESPRI 2006/Laboratoire de Pathologie Clinique et Expe´rimentale, Ho ˆpital Pasteur, Nice, France; and 5 Groupe Appareil Digestif et Environnement (EA3234), Faculte´ de Me ´decine, Rouen, France Abstract Macroautophagy (hereafter referred to as autophagy) has emerged as a key tumor suppressor pathway. During this process, the cytosolic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes to become autolysosomes where their contents are finally degraded. Although a reduced autophagy has been shown in human tumors or in response to oncogenes and carcinogens, the underlying mechanism(s) remain(s) unknown. Here, we show that widely used carcinogen Lindane promotes vacuo- lation of Sertoli cells. By electron and immunofluorescent microscopy analyses, we showed that these structures are acid autolysosomes, containing cellular debris, and labeled by LC3, Rab7, and LAMP1, markers of autophagosomes, late endo- somes, and lysosomes, respectively. Such Lindane-induced vacuolation results from significant delay in autophagy degradation, in relation with a decline of the lysosomal activity of aryl sulfatase A. At molecular level, we show that this defect in autolysosomal maturation is independent of mammalian target of rapamycin and p38 inhibitions. Rather, the activation of the mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinase (ERK) pathway is required for Lindane to disrupt the autophagic pathway. Most importantly, we provide the first evidence that sustained activation of ERK pathway is sufficient to commit cell to autophagic vacuolation. Taken together, these findings strongly support that the aberrant sustained activation of ERK by the carcinogen Lindane disrupts the maturation of autophagosomes into functional autolysosomes. Our findings therefore suggest the possibility that high constitutive ERK activity found in all cancers may provide a malignant advantage by impeding the tumor suppressive function of autophagy. (Cancer Res 2006; 66(13): 6861-70) Introduction Cancer cells have to adjust their metabolism, growth, and survival to support malignancy. Thus, understanding how cancer cells acquire a selective growth advantage is critical for treating this disease. Of catabolic processes deregulated during carcino- genesis, autophagy has emerged as a key tumor suppressor pathway (1, 2). Physiologically, autophagy ensures in all cell types the turnover of all organelles and most of long-lived proteins by a highly ordered pathway, which begins with the formation of a double-membrane vesicle termed ‘‘autophagosome’’ that engulfs them (for review, see ref. 3). Subsequently, an autophagosome fuses with a lysosome to become an ‘‘autolysosome’’ where the content is finally degraded for the synthesis of new molecules and organelles. As a result, autophagy controls cell modeling throughout development and prevents cell aging during life. In response to environmental stresses (nutrient starvation, hypoxia, infections, etc.), this catabolic process is up-regulated to provide the supply of energy needed for cell survival and repair (3, 4). Not only being a housekeeping process, autophagy protects the cells against the accumulation of damaged organelles and genotoxic substances that would otherwise induce mutations. Consistently, a massive autophagy kills the severely damaged cells as a safeguard mechanism against cancer. Based on these features, it has been proposed that defect in this pathway would confer a selective advantage to cells, with cancer as consequence (5). Increasing evidence points to an inverse relationship between autophagy and malignant growth. Indeed, autophagy is down- regulated by oncogenic activation of the phosphatidylinositol 3-kinase pathway and inversely up-regulated by tumor suppressors, phosphatase and tensin homologue and p53 (6, 7). Most importantly, it has been shown that the key autophagy protein beclin 1 is an haploinsufficiency tumor suppressor gene in mice (1, 2) and is frequently deleted in human cancers (8). Altogether, these findings have led to the proposals that defects in autophagy may favor carcinogenesis whereas restoration of autophagy may have promising therapeutic implications in cancer. Although critical, there is no cancer therapeutic approach that specifically targets autophagy. A prerequisite for such clinical applications is a better knowledge of the mechanisms by which oncogenes and carcinogens disrupt autophagy. No evidence is indeed available on the autophagic step delayed or stopped during tumorigenesis. Furthermore, the mechanistic basis for these defects remains still elusive. Emerging evidence indicates that the formation of auto- phagosomes is regulated by multiple signaling pathways as diverse as the GTPase G i3 , the class III phosphatidylinositol 3-kinase, and the serine/threonine kinases mammalian target of rapamycin (mTOR), protein kinase C, and the MAPK p38 (9). However, these signaling pathways are activated by a myriad of stimuli; most of them are not linked to autophagy and carcinogenesis. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Baharia Mograbi, Institut National de la Sante´ et de la Recherche Me´dicale ESPRI 2006,IFR 50, Faculte´ de Me´decine, Avenue de Valombrose, 06107 Nice Cedex 02, France. Phone: 33-4-93-37-77-94; Fax: 33-4-93-37-77-52; E-mail: mograbi@hermes.unice.fr. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-3557 www.aacrjournals.org 6861 Cancer Res 2006; 66: (13). July 1, 2006 Research Article Research. on November 12, 2015. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from