Evaluation of Clinical Colon Carcinoma Using Activity-Based Proteomic Profiling Melissa C

Clinical Proteomics Journal Copyright © Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN 1542-6416/04/01:301–311/$25.00 Original Article Evaluation of Clinical Colon Carcinoma Using Activity-Based Proteomic Profiling Melissa C. Zhang,1 Jiangyue Wu,1 Kristin G. Ardlie,2 and Emme C. K. Lin1,∗ 1ActivX Biosciences, Inc., La Jolla, CA; and 2Genomics Collaborative, Inc., Cambridge, MA expression. We identified one tumor-specific Abstract activity by mass spectrometry to be fibroblast The evaluation of clinical tumor tissues is a activation protein (FAP), an integral membrane valuable approach to discovering novel drug serine protease that has been reported to be pre- targets because of the direct relevance of human sent only in tumor stroma or during wound samples. We used activity-based proteomic pro- healing and absent in normal tissues. FAP activ- filing (ABPP) to study the differences in serine ity was further found to be approximately hydrolase activities from 12 matched pairs of twofold higher in stage III relative to stage II clinical normal and tumor colon tissues. Unlike colon cancer, suggestive of a role in tumor pro- traditional proteomics or measures of mRNA gression. We were also able to identify other abundance, ABPP actually quantifies enzymatic proteins, some of which had not been previ- activities, a characteristic crucial for drug target- ously linked to cancer, which had higher activity ing. Several serine hydrolases were differentially in tumors. Our results demonstrate the applica- active in tumor vs normal tissues, despite a lack bility of ABPP for the efficient identification of of obvious corresponding alterations in protein multiple clinical disease targets. *Author to whom all correspondence and reprint requests should be addressed: Emme C. K. Lin, ActivX Biosciences, Inc., 11025 N. Torrey Pines Road, La Jolla, CA 92037. E-mail: [email protected] 301 302 _______________________________________________________________________________ Zhang et al. Key Words: Tumor; activity; proteomics; colorectal conditions are well suited for evaluating clin- adenocarcinoma; serine hydrolase. ical tissue specimens, which are frequently of limited quantities but provide the most rele- Introduction vant source of human disease targets. The lifetime probability of developing From the analysis of 12 pairs (tumor and cancer is one in two for men and one in three adjacent normal) of clinical colon tissues, we for women (1). As cancer remains the second found the activities of 12 of 22 soluble serine leading cause of death behind heart disease, hydrolases and 3 of 22 membrane serine the search for novel drug targets continues. hydrolases to be significantly increased in Common methods for evaluating normal tumor vs normal tissues. In contrast, three vs tumor tissues include two-dimensional proteins in the membrane fraction had signif- (2-D) gel electrophoresis and microarray, icantly decreased activity. Following mass which measure protein and mRNA levels, spectrometry identification, a number of pro- respectively. These approaches search for dif- teins were found that had previously been ferences on a global scale; however, mRNA studied in relation to cancer. Fibroblast ac- levels do not always correlate to protein abun- tivation protein (FAP) was one such tumor- dance, and protein abundance does not specific activity, and, interestingly enough, always reflect enzyme activity. Zymogen pro- by using ABPP we found a trend of increased forms, inhibitors, and protein-protein interac- FAP activity with more advanced tumors. We tions can all regulate enzymatic activity. In also report the identification of other protein order to increase the probability of finding a activities that had not been previously linked druggable cancer target, one approach is to with cancer, such as the intracellular isoforms find the differences in the “business end” of of platelet activating factor acetylhydrolase proteins, that is, in the protein activities that (PAF-AH), phospholipases with emerging are upregulated in tumor tissues. roles in inflammatory disease. The identifica- Here, we present an activity-based pro- tion of cancer-associated proteins validates teomic profiling (ABPP) approach for drug ABPP as a rapid means of tumor target dis- target discovery in clinical colorectal car- covery and implicates the other activities as cinoma. We used a fluorescently tagged, having significant roles. activity-based chemical probe to comprehen- sively scan the large serine hydrolase super- Materials and Methods family. This family comprises Ͼ1000 members and includes proteases, lipases, esterases, and Tissues amidases, among others. In addition, there are Matched primary tumors and adjacent still many serine hydrolases whose functions normal colon tissues from 12 patients diag- are unknown. The advantage of the ABPP nosed with colorectal adenocarcinoma were method is that one can use crude tissue lysates selected from samples residing in the Global to rapidly scan for and quantify alterations in RepositoryTM at Genomics Collaborative, Inc. protein activities. Other advantages over tra- (Cambridge, MA). All samples were obtained ditional protein screening methods include the from human subjects with full informed con- requirement for only small quantities of tissue sent. The study set consisted of tumor and (20 ␮g is used per gel lane), high sensitivity normal tissue pairs from patients with stage II (low femtomole levels of active protein can be (n = 6) and stage III (n = 6; lymph node detected) (2), and the ability to study both sol- involvement) diagnoses. Staging was estab- uble and membrane protein activities. These lished by pathologists at the site of collection Clinical Proteomics________________________________________________________________ Volume 1, 2004 Activity-Based Clinical Proteomic Profiling _____________________________________________________ 303 and confirmed by pathologists at Genomics quenched by adding an equal volume of 2ϫ Collaborative Inc. The median percentage of reducing SDS-PAGE loading buffer and heat- carcinoma in the tumor tissues was 78%, with ing at 95°C for 5 min. Equal protein amounts 0% in the normal tissues. Following procure- (20 ␮g/lane) were loaded onto 12% SDS- ment from surgical specimens, tissues were PAGE gels. Gels were directly scanned for flash frozen and stored in liquid nitrogen until fluorescence using a 605-nm bandpass filter used. for detection on the Hitachi FMBIO IIe Tissues were processed by crushing them (Miraibio, Alameda, CA). Fluorescence was into approx 1-mm pieces under liquid nitro- measured using the accompanying Image- gen, using a ceramic mortar and pestle. Ap- Analysis software for each distinct activity proximately 50–100 mg wet weight of each band on the gel and expressed as fluores- tissue was homogenized in 50 mM Tris buffer cence volume units. at pH 7.4, using an Omni mechanical homog- enizer (Omni International, Warrenton, VA). Protein Identification Each lysate was briefly sonicated and the Tissue lysates (1–3 mg) were labeled with unlysed material was pelleted by centrifuga- AX5070, subsequently denatured by heating to tion at 1100g for 15 min. The crude super- 65°C for 10 min with 8 M urea and 10 mM natant was then subjected to centrifugation at dithiothreitol, and modified with 40 mM 110,000g for 1 h at 4°C. The resultant high- iodoacetamide. The lysate was then applied to speed supernatant containing cytosolic pro- a Bio-Rad PD10 gel exclusion column to elim- teins was collected as the soluble fraction. The inate excess free probe and to exchange the membrane pellet was solubilized by soni- buffer for 2 M urea and 20 mM ammonium cation in 50 mM Tris buffer at pH 7.4 with bicarbonate. The eluate was adjusted to con- 0.1% Triton X-100. Protein concentrations were tain 1% SDS, and heated at 65°C for 5 min assayed using the Dc protein kit (Bio-Rad Lab- before adding an equal volume of 2ϫ binding oratories, Hercules, CA). Approximately 1 and buffer (2% Triton X-100, 1% Tergitol type NP40, 1.5% of the original tissue weight were recov- 300 mM NaCl, 2 mM EDTA, 20 mM Tris, pH ered in the soluble and membrane protein 7.4). This was then incubated with Affi-Gel Hz fractions, respectively. Hydrazide Gel (Bio-Rad) coupled to anti- rhodamine monoclonal antibodies custom gen- Proteome Labeling and Analysis erated through Rockland Immunochemicals The fluorophosphonate TAMRA (AX5070) (Gilbertsville, PA). The AX5070-labeled pro- probe was synthesized as previously de- teins were eluted in nonreducing SDS-PAGE scribed (2) and was solubilized in dimethyl loading buffer and resolved on SDS-PAGE. sulfoxide (DMSO). To label tissue lysates, we After scanning for fluorescence, the protein added AX5070 probe to each tissue lysate (1 bands of interest were excised and prepared mg/mL) to a final concentration of 2 ␮M and for tryptic digestion. Peptides were analyzed incubated it for 1 h at 25°C. One hour has by nano-liquid chromatography/ tandem mass been shown to provide complete end-point spectrometry (LC/MS/MS), on a combina- labeling. Final DMSO concentrations were tion system of an Agilent 1100 capillary <5% of total volume. Membrane proteomes HPLC/Micro Auto-sampler (Agilent Technolo- were deglycosylated with PNGase F (New gies, Palo Alto, CA) and an LCQ DecaXP ion England Biolabs, Beverly, MA) after probe trap mass spectrometer (Thermo Finnigan, San labeling in order to sharpen bands and to get Jose, CA). Data generated were analyzed baseline molecular weights. Reactions

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