The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation Christelle Forcet*, Xin Ye†‡, Laure Granger*, Ve´ ronique Corset*, Hwain Shin†‡, Dale E. Bredesen†‡§, and Patrick Mehlen*§ *Apoptosis͞Differentiation Laboratory, Molecular and Cellular Genetic Center, Centre National de la Recherche Scientifique Unite´Mixte de Recherche 5534, University of Lyon, 69622 Villeurbanne, France; †Buck Institute for Age Research, Novato, CA 94945; and ‡Neuroscience Department, University of California, San Diego, CA 92093 Edited by H. Robert Horvitz, Massachusetts Institute of Technology, Cambridge, MA, and approved December 28, 2000 (received for review August 9, 2000) The expression of DCC (deleted in colorectal cancer) is often The link between the putative role of DCC as a tumor markedly reduced in colorectal and other cancers. However, the suppressor and the ability of DCC to bind netrin-1 and to rarity of point mutations identified in DCC coding sequences and mediate axon guidance was, however, not at all clear. Recently, the lack of a tumor predisposition phenotype in DCC hemizygous we have shown that DCC is a dependence receptor (13) and mice have raised questions about its role as a tumor suppressor. therefore functionally related to other dependence receptors DCC also mediates axon guidance and functions as a dependence such as p75NTR, the common neurotrophin receptor, the andro- receptor; such receptors create cellular states of dependence on gen receptor, and RET (14–17). Such receptors create cellular their respective ligands by inducing apoptosis when unoccupied by states of dependence on their respective ligands by inducing ligand. We now show that DCC drives cell death independently apoptosis when unoccupied by ligand but inhibiting apoptosis in of both the mitochondria-dependent pathway and the death the presence of ligand (13–16). Hence, we have shown that the receptor͞caspase-8 pathway. Moreover, we demonstrate that DCC expression of DCC induces apoptosis in the absence of netrin-1, interacts with both caspase-3 and caspase-9 and drives the acti- but in the presence of netrin-1, DCC is antiapoptotic. Further- vation of caspase-3 through caspase-9 without a requirement for more, DCC was demonstrated to be a caspase substrate, with the cytochrome c or Apaf-1. Hence, DCC defines an additional pathway major site of cleavage at Asp-1290. The caspase cleavage of DCC for the apoptosome-independent caspase activation. was shown to be required for DCC to exert its proapoptotic effect, just as it has been shown for the androgen receptor and ogelstein and his colleagues (1) have shown that the devel- RET (13, 16, 17). Functionally, therefore, DCC serves as a Vopment of colonic carcinoma from normal colonic epithe- caspase amplifier in the absence of ligand via exposure of a lium is associated with the mutation of a specific set of genes. proapoptotic domain lying in the amino-terminal region of the Allelic deletions (loss of heterozygosity) on chromosome 18q in intracellular domain, proximal to the cleavage site. more than 70% of primary colorectal tumors prompted the We investigated the mechanism by which DCC induces cell search for a tumor suppressor gene at that locus. This search led death when cleaved by caspases. We demonstrate that DCC to the cloning of a putative cell-surface receptor, DCC (deleted induces apoptosis in a caspase-9-dependent pathway, yet by a in colorectal cancer) (1). DCC expression was then shown to be mechanism that is independent of the intrinsic (mitochondria- markedly reduced in more than 50% of colorectal tumors. dependent) apoptotic pathway. We also show that DCC recruits Moreover, the loss of DCC expression is not restricted to colon caspase-3 and caspase-9, resulting in the activation of caspase-3 carcinoma but has been observed in other tumor types, including via caspase-9. Hence, DCC defines an additional pathway for the carcinoma of the stomach, pancreas, esophagus, prostate, blad- apoptosome-independent caspase activation. der, breast, male germ cell tumors, neuroblastomas, gliomas, and Methods some leukemias (2, 3). However, proof that DCC is a tumor suppressor gene remains inconclusive (4, 5). Cells, Transfection Procedures, and Constructs. The neuroblastoma cell line IMR32 constitutively expressing DCC was from ECA cell DCC encodes an approximately 200-kDa type I membrane Ϫ͞Ϫ protein of 1,447 amino acids, which displays homology in its lines. The Apaf1 SAK-2 cells were obtained from P. Gruss, extracellular domain with cell adhesion molecules (2), suggesting Max Planck Institute for Biophysical Chemistry, Go¨ttingen, Ger- that DCC may play a role in cell–cell or cell–matrix interactions (6). many, and transient transfection was performed by using Lipo- fectamineϩ (Life Technologies, Grand Island, NY) according to However, DCC-mediated cell aggregation has not been firmly Ϫ͞Ϫ established (7). Recently, Tessier-Lavigne and collaborators (8, 9) the manufacturer’s instructions. The caspase-9 cells were have suggested that DCC may function as a component of a obtained from R. Flavell, Yale Univ. School of Medicine, New Haven, CT, and transient transfection was performed by using receptor complex that mediates the effects of the axonal chemoat- ϩ tractant netrin-1. The role of DCC in mediating growth cone Lipofectamine (Life Technologies). The 293-EBNA-netrin-1 extension has been supported by the analysis of the DCC knockout producing netrin-1 were obtained from M. Tessier-Lavigne, Univ. mice, which display abnormal brain development (4). However, the of California San Francisco. Transient transfection of colorectal cell signaling transduction of netrin-1 through DCC that results in axon line REGb or of human embryonic kidney 293T cells was per- outgrowth is mainly unknown. In response to netrin-1 binding, formed as described (13) by using the following plasmids. pDCC- DCC has been shown to interact with other netrin-1 receptors like UNC5H (i.e., three members UNC5H1, -2, and -3) (10) or the This paper was submitted directly (Track II) to the PNAS office. adenosine A2b receptor shown to transduce cAMP production Abbreviation: TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP end labeling. upon netrin-1 binding (11). Recently, it also has been proposed that §To whom reprint requests should be addressed. E-mail: [email protected] or Frazzled, the Drosophila ortholog of DCC, is not, in certain [email protected]. circumstances, a transducing receptor but rather a carrier for the The publication costs of this article were defrayed in part by page charge payment. This cue netrin-1 that allows netrin-1 distribution in specific regions of article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. the nervous system (12). §1734 solely to indicate this fact. 3416–3421 ͉ PNAS ͉ March 13, 2001 ͉ vol. 98 ͉ no. 6 www.pnas.org͞cgi͞doi͞10.1073͞pnas.051378298 Downloaded by guest on October 1, 2021 CMV-S, pDCC-CMV.D1290N, and pGNET1myc have been de- 100 ␮l of lysate, and protein-A Sepharose was used to pull them scribed (13). pBabe-p35 was a gift of P. Friesen, Univ. of Wisconsin, out. A similar immunodepletion of caspase-9 was performed by Madison. pcDNA-Crma was a gift of David Pickup, Duke Univ. using caspase-9 antibody (PharMingen). Medical Center, Durham, NC. pcDNA-Bcl2 was obtained by cloning the 1,855-bp cDNA fragment of Bcl-2 (18) into pcDNA-3.1. Results pcDNA-casp3, pcDNA-casp6, pcDNA-casp7, pcDNA-casp8, and DCC-Induced Apoptosis via Caspase-9. As DCC cleavage by caspases pcDNA-casp9 were gifts of G. S. Salvesen, Burnham Institute, La has been shown to be required for DCC proapoptotic activity Jolla, CA. Dominant negative caspase-encoding plasmids were (13), we first assessed whether caspase inhibition is sufficient to obtained by mutating the active site cysteines to alanine via a block DCC-induced cell death. Baculovirus protein p35, a potent Quikchange strategy (Stratagene). pcDNA-DNcasp3, pcDNA- and general caspase inhibitor (22), was expressed in the presence DNcasp6, pcDNA-DNcasp7, pcDNA-DNcasp8, and pcDNA- of DCC in human embryonic kidney 293T cells, and cell death DNcasp9 encode, respectively, the dominant negative mutants for was measured by trypan blue staining. Fig. 1A shows the caspase-3, -6, -7, -8, and -9. complete suppression of DCC-induced cell death in the presence of p35, confirming the role of caspases in DCC-induced apo- Immunoblotting and Immunoprecipitation. One-dimensional immu- ptosis. A similar block of cell death was observed when the noblots by using antibodies raised against DCC (Oncogene caspase inhibitor zVAD-fmk was used (Fig. 1C). Hence, caspase Research Products, Cambridge, MA), Flag M2 epitope (Sigma), activation is required for DCC-induced cell death. and caspase-3 and caspase-9 (PharMingen) were performed as To specify what caspase was involved, we either coexpressed described (19). Coimmunoprecipitations were performed on DCC with dominant negative (catalytic) mutants of caspases or cotransfected 293T or IMR32 lysed in 50 mM Hepes, pH 7.6͞125 expressed DCC in the presence of caspase inhibitors. Coexpression mM NaCl͞5 mM EDTA͞0.1% Nonidet P-40, by using a of the dominant negative mutant of caspase-8 failed to block caspase-3, caspase-9, or Flag M2 antibody and protein-A Sepha- DCC-induced apoptosis (Fig. 1B). Similarly, the addition of the rose (Sigma). DCC interaction with caspases was monitored by caspase-8 inhibitor IETD-fmk showed no effect on cell death immunoblot by using anti-DCC antibody. induction by DCC (Fig. 1C). The caspase-8 inhibitor CrmA was also unable to block DCC-induced cell death (not shown). Hence, Cell Death Analysis and Caspase Activity Measurement. Cell death caspase-8 is not required for DCC-induced cell death. The coex- was analyzed by using trypan blue as described in Rabizadeh et al. pression of the dominant negative mutants of caspase-6 and -7 also (20).