Oncogene (2012) 31, 3202–3212 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc SHORT COMMUNICATION Cathepsin D is partly endocytosed by the LRP1 receptor and inhibits LRP1-regulated intramembrane proteolysis D Derocq1,2,3,4, C Pre´bois1,2,3,4, M Beaujouin1,2,3,4, V Laurent-Matha1,2,3,4, S Pattingre1,2,3,4, GK Smith5 and E Liaudet-Coopman1,2,3,4 1IRCM, Institut de Recherche en Cance´rologie de Montpellier, Montpellier, France; 2INSERM U896, Montpellier, France; 3Universite´ Montpellier1, Montpellier, France; 4CRLC Val d’Aurelle Paul Lamarque, Montpellier, France and 5Screening and Compound Profiling, GlaxoSmithKline, Inc., Research Triangle Park, NC, USA The aspartic protease cathepsin-D (cath-D) is a marker of epithelial breast cancer cells (Vetvicka et al., 1994; poor prognosis in breast cancer that is overexpressed and Rochefort and Liaudet-Coopman, 1999; Liaudet-Coop- hypersecreted by human breast cancer cells. Secreted pro- man et al., 2006). This overproduction in breast cancer cath-D binds to the extracellular domain of the b-chain of is correlated with a poor prognosis (Ferrandina et al., the LDL receptor-related protein-1 (LRP1) in fibroblasts. 1997; Foekens et al., 1999; Rodriguez et al., 2005). The LRP1 receptor has an 85-kDa transmembrane b-chain Human cath-D is synthesized as a 52-kDa precursor and a noncovalently attached 515-kDa extracellular a-chain. that is rapidly converted in the endosomes to form an LRP1 acts by (1) internalizing many ligands via its a-chain, active, 48-kDa, single-chain intermediate, and then in (2) activating signaling pathways by phosphorylating the the lysosomes into the fully active mature protease, LRP1b-chain tyrosine and (3) modulating gene transcription composed of a 34-kDa heavy chain and a 14-kDa light by regulated intramembrane proteolysis (RIP) of its b-chain. chain (Gieselmann et al., 1985). The overexpression of LRP1 RIP involves two cleavages: the first liberates the cath-D in breast cancer cells leads to the hypersecretion LRP1 ectodomain to give a membrane-associated form, of the 52-kDa pro-cath-D into the extracellular envir- LRP1b-CTF, and the second generates the LRP1b-intra- onment (Vignon et al., 1986; Capony et al., 1989; Fusek cellular domain, LRP1b-ICD, that modulates gene tran- and Vetvicka, 1994). Cath-D affects both the cancer scription. Here, we investigated the endocytosis of pro-cath- cells and the stromal cells of the tumor microenviron- D by LRP1 and the effect of pro-cath-D/LRP1b interaction ment. Human pre-pro-cath-D cDNA transfected in on LRP1b tyrosine phosphorylation and/or LRP1b RIP. cancer cells promotes cancer cell proliferation, tumor Our results indicate that pro-cath-D was partially endocy- angiogenesis, and tumor growth and metastasis (Glondu tosedbyLRP1infibroblasts.However,pro-cath-Dand et al., 2001; Berchem et al., 2002). Human pre-pro-cath- ectopic cath-D did not stimulate phosphorylation of the D cDNA transfected in cath-DÀ/ÀMEF (mouse embryonic LRP1b-chain tyrosine. Interestingly, ectopic cath-D and its fibroblast) cells induces fibroblast outgrowth (Laurent- catalytically inactive D231Ncath-D, and pro-D231Ncath-D all Matha et al., 2005). Inhibition of cath-D expression significantly inhibited LRP1 RIP by preventing LRP1b- in breast cancer cells decreases tumor growth and CTF production. Thus, cath-D inhibits LRP1 RIP indepen- metastasis (Glondu et al., 2002; Ohri et al., 2007; dently of its catalytic activity by blocking the first cleavage. Vashishta et al., 2007). Human secreted pro-cath-D As cath-D triggers fibroblast outgrowth by LRP1, we stimulates breast cancer cell proliferation (Vignon et al., propose that cath-D modulates the growth of fibroblasts by 1986; Fusek and Vetvicka, 1994; Vetvicka et al., 1994; inhibiting LRP1 RIP in the breast tumor microenvironment. Ohri et al., 2008), fibroblast outgrowth (Laurent-Matha Oncogene (2012) 31, 3202–3212; doi:10.1038/onc.2011.501; et al., 2005) and angiogenesis (Hu et al., 2008). We have published online 14 November 2011 shown that a mutated catalytically inactive version of cath-D (D231N) is still mitogenic for tumor cells and Keywords: cancer; cathepsin D; LRP1; RIP; endocyto- fibroblasts (Glondu et al., 2001; Berchem et al., 2002; sis; tyrosine phosphorylation Laurent-Matha et al., 2005). We recently discovered that pro-cath-D is the first ligand that binds to the extracellular domain of the b-chain of the LDL receptor-related protein-1 (LRP1) in fibroblasts (Beau- Introduction jouin et al., 2010). We also showed that cath-D promotes LRP1-dependent fibroblast outgrowth by a The lysosomal aspartic protease cathepsin-D (cath-D) is mechanism that is independent of its catalytic activity overexpressed and abundantly secreted by human (Beaujouin et al., 2010). The LRP1 receptor consists of an 85-kDa transmem- Correspondence: Dr E Liaudet-Coopman, INSERM U896, IRCM Val brane b-chain and a noncovalently attached 515-kDa d’Aurelle-Paul Lamarque, 34298 Montpellier Cedex 5, France. E-mail: [email protected] extracellular a-chain (Strickland and Ranganathan, 2003; Received 24 July 2011; revised 21 September 2011; accepted 22 Lillis et al.,2005;Montelet al., 2007). The b-chain has an September 2011; published online 14 November 2011 extracellular domain, a transmembrane region and a Cathepsin D, endocytosis and LRP1 RIP D Derocq et al 3203 cytoplasmic tail. The extracellular a-chain contains bind- b-chain of the LRP1 receptor in fibroblasts (Beaujouin ing sites for numerous structurally diverse ligands, et al., 2010), and as LRP1 is an endocytic receptor, we including lipoprotein particles, proteases and protease- investigated the role of LRP1 in the internalization of inhibitor complexes, extracellular matrix proteins, cyto- secreted pro-cath-D in fibroblasts (Figure 1). We first kines and growth factors. LRP1 has a well-defined role as analyzed the endocytosis of S35-radiolabelled pro-cath-D a scavenger receptor mediating the endocytosis of 440 secreted by cancer cells in human mammary fibroblasts different extracellular ligands that bind to its a-chain. It (HMF) in which endogenous LRP1 synthesis had been delivers most, but not all, of these ligands to lysosomes for blocked by RNA interference (Figure 1A). Internalized degradation (Herz and Strickland, 2001; Gonias et al., labelled 52-kDa pro-cath-D was first transformed into a 2004; Emonard et al., 2005; May et al., 2007). It has also 48-kDa endosomal intermediate and then into the 34-kDa been shown that LRP1 is involvedinsignaltransduction lysosomal mature enzyme (Figure 1A, panel a, lane 1) by phosphorylation of the tyrosine in the cytoplasmic (Capony et al., 1987). Excess M6P prevented pro-cath-D NPXY motifs of its b-chain and modulation of signaling from binding to its M6P receptors and inhibited the pathways such as the MAP kinase pathway (Barnes et al., internalization of pro-cath-D by 82% (Figure 1A, panel a, 2001, 2003; Boucher et al., 2002; Boucher and Gotthardt, compare lanes 1 and 3). Thus, 18% of pro-cath-D was 2004; Loukinova et al., 2002; Yang et al., 2004; Newton taken up by HMF fibroblasts in a manner that was et al., 2005; Hu et al., 2006). More recent studies have independent of the M6P receptors (Figure 1A, panel a, shown that LRP1 influences gene transcription by lane 3), as shown for mouse fibroblasts (Laurent-Matha regulated intramembrane proteolysis (RIP) of its b-chain et al., 2005). Inhibiting LRP1 synthesis with siRNA (May et al., 2002; Kinoshita et al., 2003; von Arnim et al., (Figure 1A, panel b) led to a 50% decrease in reminiscent 2005; Zurhove et al., 2008). RIP is a process that involves pro-cath-D internalization (Figure 1A, panel a, compare two cleavages, as described for Notch (De Strooper et al., lanes 3 and 4). There was also a decrease in pro-cath-D 1999), APP (amyloid precursor protein) (De Strooper endocytosis in LRP1-silenced fibroblasts (Figure 1A, et al., 1998) and LRP6 (Mi and Johnson, 2007). The first panel d) when uptake was measured over 18 h cleavage, performed by metalloproteinases (Quinn et al., (Figure 1A, panel c, compare lanes 3 and 4, and panel e 1999; Rozanov et al., 2004; Liu et al., 2009; Selvais et al., for quantification). We then investigated pro-cath-D 2009, 2011) and by the membrane-associated b-secretase, internalization by LRP1 using MEF that lacked M6P BACE1 (von Arnim et al., 2005), occurs in the extra- receptors (Figure 1B). Silencing LRP1 in RM6PÀ/ÀMEF cellular region, close to the plasma membrane, and leads to cells (Figure 1B, panel b) partly decreased pro-cath-D shedding of the LRP1b ectodomain. This produces the endocytosis over 3 h (Figure 1B, panel a, left) and 18 h membrane-associated carboxyl-terminal fragment, (Figure 1B, panel a, right, and panel c for quantification). LRP1b-CTF,whichisthencleavedbyg-secretases within Re-expression of the LRP1b chain alone in LRP1- its transmembrane domain (Hass et al., 2009). The silenced RM6PÀ/ÀMEF cells caused accumulation of 52- LRP1b-intracellular domain, LRP1b-ICD, is released into kDa pro-cath-D (Figure 1B, panel a, right and left). We the cytosol, where it may interact with signaling proteins, obtained further evidence by examining pro-cath-D translocate to the nucleus and control gene transcription endocytosis in LRP1À/ÀMEF cells re-transfected with full- (May et al., 2002; Kinoshita et al., 2003; von Arnim et al., length LRP1 (clone B-41) or with the LRP1b chaininthe 2005; Zurhove et al., 2008). We have now investigated the presence of M6P (Figure 1C). Re-expressing full-length mechanisms by which cath-D affects the behavior of LRP1 in LRP1À/ÀMEF cells (Figure 1C, panel b) stimulated LRP1. We studied the endocytosis of pro-cath-D by LRP1 pro-cath-D endocytosis (Figure 1C, panel a).