Sensitization to the Lysosomal Cell Death Pathway by Oncogene- Induced Down-regulation of Lysosome-Associated Membrane Proteins 1 and 2 Nicole Fehrenbacher, 1 Lone Bastholm, 2 Thomas Kirkegaard-Sørensen, 1 Bo Rafn, 1 Trine Bøttzauw, 1 Christina Nielsen, 1 Ekkehard Weber, 3 Senji Shirasawa, 4 Tuula Kallunki, 1 and Marja Ja ¨a ¨ttela ¨ 1 1 Apoptosis Department and Centre for Genotoxic Stress Response, Institute for Cancer Biology, Danish Cancer Society; 2 Institute of Molecular Pathology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 3 Institute of Physiological Chemistry, Medical Faculty, Martin-Luther-UniversityHalle-Wittenberg,Halle, Germany; and 4 Department of Cell Biology, School of Medicine, Fukuoka University, Fukuoka, Japan Abstract Expression and activity of lysosomal cysteine cathepsins correlate with the metastatic capacity and aggressiveness of tumors. Here, we show that transformation of murine embryonic fibroblasts with v-H-ras or c-src Y527F changes the distribution, density, and ultrastructure of the lysosomes, decreases the levels of lysosome-associated membrane pro- teins (LAMP-1 and LAMP-2) in an extracellular signal- regulated kinase (ERK)- and cathepsin-dependent manner, and sensitizes the cells to lysosomal cell death pathways induced by various anticancer drugs (i.e., cisplatin, etoposide, doxorubicin, and siramesine). Importantly, K-ras and erbb2 elicitasimilarERK-mediatedactivationofcysteinecathepsins, cathepsin-dependent down-regulation of LAMPs, and in- creased drug sensitivity in human colon and breast carcinoma cells, respectively. Notably, reconstitution of LAMP levels by ectopic expression or by cathepsin inhibitors protects trans- formed cells against the lysosomal cell death pathway. Furthermore,knockdownofeither lamp1 or lamp2 issufficient to sensitize the cells to siramesine-induced cell death and photo-oxidation–induced lysosomal destabilization. Thus, the transformation-associated ERK-mediated up-regulation of cysteine cathepsin expression and activity leads to a decrease in the levels of LAMPs, which in turn contributes to the enhanced sensitivity of transformed cells to drugs that trigger lysosomal membrane permeabilization. These data indicate that aggressive cancers with high cysteine cathepsin levels are especially sensitive to lysosomal cell death pathways and encourage the further development of lysosome-targeting compounds for cancer therapy. [CancerRes2008;68(16):6623–33] Introduction Lysosomes are highly dynamic cytosolic organelles that receive membrane traffic input from the biosynthetic (trans -Golgi network),endocytic,andautophagicpathways(1,2).Theycontain more than 50 hydrolases that can process all the major macro- molecules of the cell to breakdown products available for metabolic reuse (3, 4). Cathepsin proteases are among the best- studied lysosomal hydrolases. They are maximally active at the acidic pH of lysosomes (pH 4–5). However, many of them can be activeattheneutralpHoutsidelysosomes,albeitwithadecreased efficacy and/or altered specificity (5). For example, transformation and tumor environment enhance the expression of lysosomal cysteine cathepsins and increase their secretion into the extracel- lular space (6). Once outside the tumor cells, cathepsins stimulate angiogenesis, tumor growth, and invasion in murine cancer models, thereby enhancing cancer progression (7, 8). On the other hand, the leakage of cathepsins into the cytosol can trigger either necrotic or apoptotic cell death pathways (4, 9). Thus, the maintenance of lysosomal membrane integrity is of utmost importance for the cell survival. Cancer cells harbor several acquired changes that help them to avoid spontaneous and therapy-induced apoptosis (10, 11). Thus, the lysosomal cell death pathways characterized by an early lysosomal membrane permeabilization and the subsequent translocation of cathepsins into the cytosol have awoken increasing interest among cancer researchers. Such lysosomal cell death pathways can be induced by death receptors of tumor necrosis factor (TNF) receptor family, p53 tumor suppressor protein, oxidative stress, microtubule-stabilizing and microtubule- destabilizing agents, siramesine, etoposide, staurosporine, etc. (12–19). Once in the cytosol, cathepsins, particularly cysteine cathepsins B and L and aspartate cathepsin D, can initiate the intrinsic apoptosis pathway possibly via a cleavage-mediated activation of proapoptotic Bcl-2 family proteins (20). Importantly, cytosolic cathepsins can also trigger caspase-independent and Bcl-2–insensitive cell death pathways even in highly apoptosis- resistantcancercells(4,21).Forexample,siramesine,apromising anticancer drug presently under preclinical development, is a lysosomotropic detergent that accumulates in lysosomes and directly destabilizes them, leading to the translocation of cathepsins into the cytosol- and cathepsin-dependent cell death pathway even in the presence of the antiapoptotic Bcl-2 protein (17, 22). The signaling pathways leading to the lysosomal leakage induced by most other lysosome-destabilizing agents are poorly understood. Depending on the cell type, TNF-induced permeabi- lizationoflysosomesandthefollowingcelldeathcanoccureither independent of caspases or via a pathway involving both caspase- 8 and caspase-9 (13, 18). Free radicals, sphingosine, iron, phospholipases, and cathepsin B are among the suggested caspase-independent mediators of the lysosomal destabilization (4, 9, 23). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for N. Fehrenbacher: Departments of Medicine, Cell Biology and Pharmacology, New York University Cancer Institute, New York, NY 10016. Requests for reprints: Marja Ja ¨a ¨ttela ¨, Apoptosis Department and Centre for Genotoxic Stress Response, Institute for Cancer Biology, Danish Cancer Society, DK- 2100 Copenhagen, Denmark. Phone: 45-35257318; Fax: 45-35257721; E-mail: mj@cancer.dk. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-0463 www.aacrjournals.org 6623 Cancer Res 2008; 68: (16). August 15, 2008 Research Article Downloaded from http://aacrjournals.org/cancerres/article-pdf/68/16/6623/2592353/6623.pdf by guest on 15 October 2023