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Integrated Cellular and Plasma Proteomics of Contrasting B-Cell Cancers Reveals Common, Unique and Systemic Signatures*□S

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Integrated Cellular and Plasma Proteomics of Contrasting B-Cell Cancers Reveals Common, Unique and Systemic Signatures*□S crossmark Research © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Integrated Cellular and Plasma Proteomics of Contrasting B-cell Cancers Reveals Common, Unique and Systemic Signatures*□S Harvey E. Johnston‡§, Matthew J. Carter‡, Kerry L. Cox‡, Melanie Dunscombe‡, Antigoni Manousopoulou§¶, Paul A. Townsendʈ, Spiros D. Garbis§¶, and Mark S. Cragg‡** Approximately 800,000 leukemia and lymphoma cases are both subunits of the interleukin-5 (IL5) receptor. IL5 treat- diagnosed worldwide each year. Burkitt’s lymphoma (BL) ment promoted E␮-TCL1 tumor proliferation, suggesting and chronic lymphocytic leukemia (CLL) are examples of an amplification of IL5-induced AKT signaling by TCL1. contrasting B-cell cancers; BL is a highly aggressive Tumor plasma contained a substantial tumor lysis signa- Downloaded from lymphoid tumor, frequently affecting children, whereas ture, most prominent in E␮-myc plasma, whereas E␮- CLL typically presents as an indolent, slow-progressing TCL1 plasma contained signatures of immune-response, leukemia affecting the elderly. The B-cell-specific overex- inflammation and microenvironment interactions, with pression of the myc and TCL1 oncogenes in mice induce putative biomarkers in early-stage cancer. These findings spontaneous malignancies modeling BL and CLL, respec- provide a detailed characterization of contrasting B-cell http://www.mcponline.org/ tively. Quantitative mass spectrometry proteomics and tumor models, identifying common and specific tumor isobaric labeling were employed to examine the biology mechanisms. Integrated plasma proteomics allowed the underpinning contrasting E␮-myc and E␮-TCL1 B-cell tu- dissection of a systemic response and a tumor lysis sig- mors. Additionally, the plasma proteome was evaluated nature present in early- and late-stage cancers, respec- using subproteome enrichment to interrogate biomarker tively. Overall, this study suggests common B-cell cancer emergence and the systemic effects of tumor burden. signatures exist and illustrates the potential of the further Over 10,000 proteins were identified (q<0.01) of which evaluation of B-cell cancer subtypes by integrative 8270 cellular and 2095 plasma proteins were quantita- proteomics. Molecular & Cellular Proteomics 16: by guest on September 10, 2018 tively profiled. A common B-cell tumor signature of 695 10.1074/mcp.M116.063511, 386–406, 2017. overexpressed proteins highlighted ribosome biogenesis, cell-cycle promotion and chromosome segregation. E␮- myc tumors overexpressed several methylating enzymes Burkitt’s lymphoma (BL)1 and chronic lymphocytic leukemia ␮ and underexpressed many cytoskeletal components. E - (CLL) represent the extremes of B cell cancers; BL is highly TCL1 tumors specifically overexpressed ER stress re- aggressive and frequently affects children, whereas CLL typ- sponse proteins and signaling components in addition to ically presents as an indolent, slow-progressing leukemia in the elderly (1, 2). From the ‡Antibody and Vaccine Group, Cancer Sciences Unit, BL is a hallmark myc-driven tumor; induced by chromo- Faculty of Medicine, General Hospital, University of Southampton, somal translocation of the transcription factor myc to immu- Southampton SO16 6YD, UK; §Centre for Proteomic Research, Insti- noglobulin (Ig) enhancers (3). Myc influences ϳ15% of the tute for Life Sciences, University of Southampton, Highfield Campus, genome, regulating processes such as cell proliferation, me- Southampton, SO17 1BJ, UK; ¶Clinical and Experimental Sciences tabolism, adhesion, angiogenesis and de-differentiation (4– Unit, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, UK; ʈMolecular and Clinical Cancer Sciences, Paterson 6). Such neoplastic-like traits make myc an aggressive onco- Building, Manchester Cancer Research Centre, Manchester Aca- gene, dysregulated or overexpressed in most cancers (7). demic Health Science Centre, University of Manchester, Manchester, Formal proof of its oncogenic properties were demonstrated M20 4BX when human myc was placed into the ␮ Ig heavy chain Received August 27, 2016, and in revised form, December 19, 2016 enhancer (E␮) region of the mouse. These ‘E␮-myc’ mice Published, MCP Papers in Press, January 4, 2017, DOI 10.1074/ mcp.M116.063511 Author contributions: Conceptualization: H.E.J., P.A.T., S.D.G., and 1 The abbreviations used are: BL, Burkitt’s lymphoma; CLL, M.S.C. Methodology: H.E.J. Validation: H.E.J. and M.J.C. Formal Chronic lymphocytic leukaemia; DEP, Differentially expressed pro- analysis: H.E.J. Investigation: H.E.J. Resources: H.E.J., M.D., K.L.C., tein; E␮, ␮ Ig heavy chain enhancer; Ig, Immunoglobulin; iTRAQ, A.M., and S.D.G. Writing – Original draft: H.E.J. and M.S.C. Writing – Isobaric tags for relative and absolute quantitation; Rs, Regulation ϩ Review & Editing: H.E.J., M.S.C., and M.J.C. Visualization: H.E.J. score (mean of log2(ratios)/(SD of log2(ratios) 1)); SEC, Size exclu- Supervision: M.S.C., P.A.T., and S.D.G. Funding Acquisition: M.S.C., sion chromatography; SuPrE, Subproteome enrichment; TMT, Tan- P.A.T., and H.E.J. dem mass tags; WT, Wild type. 386 Molecular & Cellular Proteomics 16.3 Cellular and Plasma Proteomics of B-cell Tumors produced high-penetrance lymphomas within around 100 (WT) mice. The resulting proteomes have been interrogated to days, recapitulating several molecular and pathological as- identify common and tumor-specific signatures and to under- pects of BL (3, 8–10). stand the combined cellular and extracellular characteristics In contrast, CLL is a relatively indolent B-cell cancer, pre- of B-cell tumors. senting with a CD5ϩCD19ϩ leukemia (11, 12). Up to 90% of CLL cases express TCL1—a protein involved in lymphocyte MATERIALS AND METHODS development (13–15). TCL1 is suggested to promote cell sur- Materials—Tris(hydroxymethyl)aminomethane (TRIS), SDS, vival and proliferation by amplification of AKT phosphoryla- Na2EDTA, NH4Cl, NaHCO3, sodium deoxycholate (DOC), tion, induced by growth factor-, cytokine- and B-cell receptor- guanidine hydrochloride, glycine, HPLC and LC-MS grade induced PI3K signaling (16, 17). The E␮-TCL1 mouse, ACN and formic acid (FA) and 100 ␮m cell sieves were pur- overexpressing TCL1 in B-cells again through the ␮ enhancer, chased from Fisher Scientific, Loughborough, UK. Tween20 was developed as a potential model of CLL (18). E␮-TCL1 (tween), sodium heparin, propidium iodide (PI), carboxyfluo- mice recapitulate the expanded bone marrow, splenic and rescein succinimidyl ester (CFSE), asparagine, 2-mercapto- circulatory CD5ϩ B-cell populations of CLL, detectable from ethanol, octylphenoxypolyethoxyethanol (IgePalCA630), triton around 3–5 months of age. The disease progresses to lethal x-100, protease inhibitors, ponceau S, acetic acid, methyl splenomegaly and leukemia at ϳ12 months (18, 19). Currently methanethiosulfonate (MMTS), tris(2-carboxyethyl)phosphine it is regarded as one of the most useful preclinical models of (TCEP), triethylammonium bicarbonate (TEAB), DMSO, hy- Downloaded from CLL (20, 21). droxylamine and ammonium hydroxide (NH4OH) were pur- Together, these models provide the opportunity to study chased from Sigma, St. Louis, MO. oncogenesis mediated by two contrasting oncogenes at op- Dulbecco’s modified Eagle’s medium (DMEM), glutamine, posing ends of the proliferative spectrum. E␮-myc tumors, pyruvate, penicillin and streptomycin were purchased from like BL, are highly aggressive, have rapid onset and form Life Technologies, Carlsbad, CA. Mouse B-cell isolation kits http://www.mcponline.org/ lymphoid tumors, whereas E␮-TCL1 mice, like CLL, present a and magnetic cell sorting columns were purchased from relatively indolent and slow-developing leukemia with sec- Miltenyi Biotech, Bergisch Gladbach, Germany. Twenty- ondary lymphoid organ involvement (3, 18). three-gauge needles and 1-ml syringes were purchased from In addition to cellular characterizations of tumors, plasma BD Biosciences, Franklin Lakes, NJ, Interleukin 5 (IL5) recom- analysis holds the potential to identify additional biological binant protein from Peprotech, Rocky Hill, NJ, Immobilon 0.45 signatures arising during oncogenesis. In particular, extracel- ␮m pore PVDF membrane from Millipore, Billerica, MA and lular fluids can provide insight into the tumor-host dialogue nonfat milk from Marvel, Birmingham, UK. 30ϫ DTT and 3ϫ by guest on September 10, 2018 between the immune system and micro-environment and re- red loading buffer were purchased for Cell Signaling Technol- port on the metabolic and homeostatic aberrations that tu- ogies, Danvers, MA. Enhanced chemiluminescence reagents, mors display. Further, combining cellular and plasma charac- tandem mass tags (TMT) 10-plex isobaric labeling reagents terization can provide greater insight into the mechanisms and 2 kDa Mw cut-off Slide-A-Lyzer dialysis cassettes and by which any biomarkers of disease may be entering the proteomics grade lys-c were purchased from Thermo Scien- circulation. tific, Waltham, MA. Proteomics grade trypsin was purchased Liquid chromatography coupled with mass spectrometry from Roche, Basel, Switzerland. Isobaric tags for relative and (LC-MS) proteomics currently provides the best means of absolute quantitation (iTRAQ) 8-plex reagents were pur- establishing global differential protein expression profiles of chased from ABSciex, Framingham, MA. Antibodies are de- cellular and plasma samples. These approaches are evolving tailed in Supplementary
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