title: Quantitative mass spectrometry identifies drug targets in cancer stem cell-containing side population.
abstract: A multifaceted approach is presented as a general strategy to identify new drug targets in a breast cancer stem cell-containing side population. The approach we have utilized combines side population cell sorting and stable isotope labeling by amino acids in cell culture with mass spectrometry to compare and identify proteins with differential expression profiles between side population cells, know to be enriched in cancer stem cells, and nonside population cells, which are depleted in cancer stem cells, for two breast cancer cell lines, MCF7 and MDA-MB231. Almost 900 proteins were quantified, and several important proteins in cell cycle control and differentiation were found to be upregulated in the cancer stem cell-containing side population. Most interestingly, a splice isoform of pyruvate kinase M2 as well as peroxiredoxin 6 were found to be downregulated. The differential levels of three of these proteins, thymosin beta4 (TB4), proliferation-associated protein 2G4, and SIAH-interacting protein, were validated using Western blot. Furthermore, functional validation provided clear evidence that elevated TB4 expression contributes to drug resistance in the stem cell population. Small interfering RNA silencing of TB4 led to a loss of chemoresistance in two separate breast cancer populations. These proteins likely contribute to resistance in the cancer stem cell-containing side population, and their altered expression in a tumor causes clinical resistance to chemotherapy. The ability to perform quantitative mass spectrometry has enabled the identification of a series of proteins that could serve as future therapeutic targets.
authors.affiliation: The Skaggs Institute for Chemical Biology and Department of Chemistry and Immunology, La Jolla, California 92037, USA.
authors.forename: Sebastian C J
authors.initials: SC
authors.lastname: Steiniger
authors.forename: Judith A
authors.initials: JA
authors.lastname: Coppinger
authors.forename: Jörg A
authors.initials: JA
authors.lastname: Krüger
authors.forename: John
authors.initials: J
authors.lastname: Yates
authors.forename: Kim D
authors.initials: KD
authors.lastname: Janda
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins; D004354:Drug Screening Assays, Antitumor / Q000295:instrumentation / Q000379:methods*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D016513:Mice, SCID; D014411:Neoplastic Stem Cells / Q000166:cytology*; D009687:Nuclear Proteins; D054465:Peroxiredoxin VI / Q000096:biosynthesis; D011770:Pyruvate Kinase / Q000096:biosynthesis; D016601:RNA-Binding Proteins; D013947:Thymosin / Q000096:biosynthesis
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C101633:PA2G4 protein, human; C095938:Pa2g4 protein, mouse; D016601:RNA-Binding Proteins; D013947:Thymosin; D054465:Peroxiredoxin VI; D011770:Pyruvate Kinase
references.citation: J Exp Med. 1996 Apr 1;183(4):1797-806
references.pmid: 8666936
references.citation: Int J Cancer. 1991 Jul 9;48(5):672-7
references.pmid: 2071228
references.citation: Nat Med. 1997 Jul;3(7):730-7
references.pmid: 9212098
references.citation: Am J Pathol. 1999 Sep;155(3):799-804
references.pmid: 10487837
references.citation: Nature. 1963 Jul 6;199:79-80
references.pmid: 14047954
references.citation: J Am Chem Soc. 2004 Dec 8;126(48):15656-7
references.pmid: 15571383
references.citation: Cancer Res. 2005 Jul 1;65(13):5506-11
references.pmid: 15994920
references.citation: Exp Cell Res. 2006 May 15;312(9):1651-7
references.pmid: 16515784
references.citation: Cell. 2006 Jun 2;125(5):929-41
references.pmid: 16751102
references.citation: Eur J Cancer. 2006 Jun;42(9):1293-7
references.pmid: 16644203
references.citation: Blood. 2006 Dec 1;108(12):3906-12
references.pmid: 16912222
references.citation: Nat Rev Mol Cell Biol. 2006 Dec;7(12):952-8
references.pmid: 17139335
references.citation: Nat Methods. 2007 Mar;4(3):207-14
references.pmid: 17327847
references.citation: Cancer Cell. 2007 Mar;11(3):259-73
references.pmid: 17349583
references.citation: Anal Chem. 2007 Apr 15;79(8):3056-64
references.pmid: 17367114
references.citation: Curr Pharm Biotechnol. 2007 Jun;8(3):167-75
references.pmid: 17584089
references.citation: Science. 2007 Jul 20;317(5836):337
references.pmid: 17641192
references.citation: Nat Rev Cancer. 2007 Oct;7(10):791-9
references.pmid: 17851544
references.citation: Mol Cell Proteomics. 2007 Oct;6(10):1771-7
references.pmid: 17627935
references.citation: Mol Cell Biochem. 2007 Dec;306(1-2):201-12
references.pmid: 17660947
references.citation: Mol Cancer Res. 2007 Dec;5(12):1254-62
references.pmid: 18171983
references.citation: Cancer Res. 2008 Feb 1;68(3):800-7
references.pmid: 18245481
references.citation: Breast Cancer Res. 2007;9(6):R76
references.pmid: 17980029
references.citation: Nature. 2008 Mar 13;452(7184):230-3
references.pmid: 18337823
references.citation: Nat Methods. 2008 Apr;5(4):319-22
references.pmid: 18345006
references.citation: Proc Natl Acad Sci U S A. 1990 Apr;87(7):2536-40
references.pmid: 2320573
references.citation: Int J Oncol. 2002 Sep;21(3):499-507
references.pmid: 12168092
references.citation: Anal Chem. 2002 Nov 1;74(21):5593-9
references.pmid: 12433093
references.citation: Gene Expr Patterns. 2002 Nov;2(1-2):27-34
references.pmid: 12617833
references.citation: Br J Cancer. 2000 Feb;82(3):683-90
references.pmid: 10682683
references.citation: Nature. 2000 Aug 3;406(6795):532-5
references.pmid: 10952316
references.citation: Nat Biotechnol. 2001 Mar;19(3):242-7
references.pmid: 11231557
references.citation: Nat Med. 2001 Sep;7(9):1028-34
references.pmid: 11533706
references.citation: Nature. 2001 Nov 1;414(6859):105-11
references.pmid: 11689955
references.citation: Oncogene. 2003 May 22;22(21):3297-306
references.pmid: 12761500
references.citation: Arch Med Res. 2003 Jul-Aug;34(4):315-24
references.pmid: 12957530
references.citation: Proc Natl Acad Sci U S A. 2004 Jan 20;101(3):781-6
references.pmid: 14711994
references.citation: Mol Cell Proteomics. 2004 Jul;3(7):729-35
references.pmid: 15102926
references.citation: Cancer Chemother Rep. 1968 Feb;52(2):251-5
references.pmid: 5646852
references.citation: Cancer Res. 1968 Nov;28(11):2187-96
references.pmid: 5723963
references.citation: Cell. 1996 Oct 18;87(2):159-70
references.pmid: 8861899