Oncogene (2006) 25, 7650–7661 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ONCOGENOMICS Proteomic identification of the wt-p53-regulated tumor cell secretome FW Khwaja1,2, P Svoboda3, M Reed3, J Pohl3 , B Pyrzynska1,2 and EG Van Meir1,2 1Laboratory of Molecular Neuro-Oncology, Department of Neurosurgery, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA; 2Department of Hematology/Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA and 3Emory University Microchemical and Proteomics Facility, Emory University School of Medicine, Atlanta, GA, USA Tumor–stroma interactions play a major role in tumor contributes to the neoplasm (Hanahan and Weinberg, development, maintenance and progression. Yet little is 2000) and that the tumor–stroma interactions are known on how the genetic alterations that underlie cell an active process initiated by transforming events transformation elicit cell extrinsic changes modulating (Bhowmick and Moses, 2005; Taieb et al., 2006). heterotypic cell interactions. We hypothesized that these Consequently, we need to understand these tumor– events involve a modification in the complement of stroma interactions to develop more effective therapies. secreted proteins by the cell, acting as mediators of We hypothesized that loss of tumor-suppressor function intercellular communication. To test this hypothesis, we during cell transformation may have cell extrinsic effects examined the role of wt-p53, a major tumor suppressor, on through the modulation of secreted factors. We focused the tumor microenvironment through its regulation of on p53, as it is frequently mutated in cancer and is secreted factors. Using a combination of 2-DE and cICAT a transcription factor that can directly control the proteomic techniques, we found a total of 111 secreted synthesis of a large number of proteins (Harris and proteins, 39 of which showed enhanced and 21 inhibited Levine, 2005). secretion in response to wt-p53 expression. The majority Tumor-suppressive p53 is best known for its role in of these were not direct targets of p53 transcription factor maintaining genomic integrity by controlling cell cycle activity, suggesting a novel role for wt-p53 in the control progression and cell survival in response to DNA of intracellular protein trafficking and/or secreted protein damage (Steele and Lane, 2005). Nevertheless, some stability. Evidence for p53-controlled post-translational studies have suggested that p53 can influence the modifications on nine secreted proteins was also found. tumor microenvironment through suppression of angio- These findings will enhance our understanding of wt-p53 genesis and tumor invasion (Van Meir et al., 1994; modulated interactions of the tumor with its environment. Zigrino et al., 2005). These processes might be influ- Oncogene (2006) 25, 7650–7661. doi:10.1038/sj.onc.1209969; enced by p53 through two mechanisms; the induced published online 9 October 2006 secretion of inhibitory factors (Van Meir et al., 1994) or the negative regulation of secreted protumorigenic Keywords: p53; proteomics; secretion; glioma; brain can- proteins (Chiarugi et al., 1998; Sun et al., 2000). While cer; two-dimensional electrophoresis p53-regulated intracellular proteins are well studied, the extracellular ones have not been systematically analysed. Identification of the p53 controlled secreted proteins will clarify how p53 loss in tumors may lead to the altered regulation and response of the tumor cells to their Introduction environment. To examine the regulation of p53 on the cell’s Traditionally, cancer formation is thought of as a cell secretome we identified secreted proteins by p53-null autonomous process driven by mutations in genes that tumor cells in the presence or absence of reconstituted ONCOGENOMICS increase cell proliferation and survival, where a tumor is wt-p53 expression. This is the first comprehensive study primarily composed of transformed cells. Increasing of how p53 plays a role in the process of transformation evidence suggests that the tumor microenvironment also through its manipulation of the tumor microenviron- ment. Our studies identified 50 new secreted proteins Correspondence: Dr EG Van Meir, Laboratory of Molecular Neuro- controlled by p53. These proteins have known roles in Oncology, Department of Neurosurgery, Winship Cancer Institute, cancer-related processes that are dependent on hetero- Emory University, 1365C Clifton Rd, NE, C5078, Atlanta, GA 30322, USA. typic cell–cell communication such as immune response, E-mail: [email protected] angiogenesis, extracellular matrix (ECM) interaction, FWK and EGVM designed and interpreted experiments and wrote the and cell survival. Many of these proteins are secreted manuscript. FWK performed experiments with the help of PS, MR through receptor-mediated nonclassical secretory path- and JP for the MS analyses. BP performed the microarrays and Northern blot. All authors read the manuscript. ways. These results are important to advance our Received 10 May 2006; revised 26 July 2006; accepted 27 July 2006; understanding on how tumor–stroma interactions con- published online 9 October 2006 tribute to cancer progression. wt-p53-regulated secreted proteins in glioma cells FW Khwaja et al 7651 Results expression using two complementary proteomic techni- ques: two-dimensional gel electrophoresis (2-DE) and To identify p53-regulated secreted proteins involved in cleavable isotope-coded affinity tag (cICAT). the cell–cell communication events important for human cell transformation, we selected the LN-Z308 cell line as it was derived from a malignant human glioma that lost 2-DE of the tumor cell secretome both p53 alleles in vivo by well-characterized genetic The secreted proteins were separated by 2-DE analysis events suggesting selective pressure for their loss using nonlinear pH range of 3–10 and linear range of (Albertoni et al., 1998). Reactivating wt-p53 function 4–7 in triplicates to ensure reproducibility (Go¨ rg et al., in these cells would revert or restore the release of p53- 2004). The proteins were visualized by silver staining regulated secreted proteins and allow their identification and analysed using ImageMaster software. As a further in the conditioned media (CM). To this purpose, we precaution against artefacts, we profiled both 2024 and used isogenic clones of LN-Z308 with tetracycline- WT11 clones and only proteins found secreted in both inducible (2024 p53 tet-on) (Albertoni et al., 2002) and were retained. repressable (WT11 p53 tet off) (Van Meir et al., 1994) The protein spots were next excised from the gel, wt-p53 expression (Figure 1). These cells undergo subjected to in-gel digestion with trypsin, and identi- growth arrest but not apoptosis in response to p53 fied using matrix-assisted laser desorption/ionization (Van Meir et al., 1995) and show induction of the cell time-of-flight (MALDI-TOF/TOF) MS/MS analysis. cycle inhibitor p21 upon p53 induction (Figure 1a). We found on average >150 spots on each gel Using this system, we generated differential profiles of representing 68 individual proteins (Figure 1b; Tables the cell lines’ secretome with and without wt-p53 1–3). A semiquantitative analysis of this differential ac 2024 WT11 --LNZ308 ++- Dox --+ - + wtp53 2024 2024+wt p53 p53 p21 Actin b MW 2024 - wt p53 2024 + wt p53 110 X-linked factor 75 KIAA0548 MMP-2 α-catenin BCL6 repressor MIF Saposin ADAM-10 KIAA0828 50 PG-M PG-M PEDF PAI-1 NF1 SPARC CYR61 33 Gal-1 Gal-1 OPN Gal-3 Transgelin 2 VEGF FGF-4 RTVP-1 25 α-2HS IGFBP6 β-2M 15 BDNF TIMP-3 14-3-3 10 Thioredoxin MT2A NTF1 TPM4-ALK oncoprotein TGF-β pH 4 pH 9 pH 4 pH 9 Figure 1 Representative 2-DE gels of secreted proteins from glioma cells with inducible wt-p53 expression. (a) Two p53-null clones (WT11 and 2024) with dox-inducible wt-p53 expression were used (See Materials and methods). Western blot shows wt-p53 induction, and downstream activation of the p21 cell cycle inhibitor, 48 h postinduction in serum-free media. (b) Secreted proteins found in the CM from uninduced (left) and wt-p53 induced (right) 2024 cells were analysed by 2-DE analysis using IEF strips pH3-10 NL and 12.5% SDS–Page. Protein spots circled indicate proteins with enhanced secretion (right) or reduced (left) in response to wt-p53. Samples were run in triplicate and location of representative proteins is indicated. Black arrow shows p53-induced post-translational modification of Gal-1. White arrow shows the location of KIAA0828. (c) Enlargement showing acidic shift of Gal 1 in CM from wt p53 expressing cells (arrows). Oncogene Oncogene 7652 Table 1 Secreted proteins with enhanced accumulation in CM upon p53 induction 2024 WT11 Protein name Access. no. MW pI mRNA 2-DE H:L s.d. H:L s.d. Functional category Mode of secretion Post-translational requirement Galectin-3 gi:4504983 26.19 8.58 1.05 C 1.60 0.12 1.53 0.10 Adhesion and matrix Vesicle-mediated; Glycosylation/ interactions; apoptosis ectocytosis phosphorylation Lysyl oxidase-like pro- gi:4959425 71.10 6.32 1.05 1.65 0.07 1.65 0.00 Adhesion and matrix Classical Glycosylation wt-p53-regulated secreted proteins in glioma cells tein 2 interactions b-Galactosidase binding gi:12804557 14.72 5.33 C 1.36 0.18 1.27 0.12 Cell proliferation reg- Vesicle-mediated; Glycosylation lectin ulation ectocytosis Nm23 protein gi:35068 20.41 7.06 1.04 2.50 0.00 Cell proliferation/dif- Unknown non- Phosphorylation ferentiation classical pathway X-linked brain specific gi:21322252