Proteomics of Genetically Engineered Mouse Mammary Tumors Identifies Fatty Acid Metabolism Members as Potential Predictive Markers for Cisplatin Resistance
2013; Elsevier BV; Volume: 12; Issue: 5 Linguagem: Inglês
10.1074/mcp.m112.024182
ISSN1535-9484
AutoresMarc O. Warmoes, Janneke E. Jaspers, Guotai Xu, Bharath Sampadi, Thang V. Pham, Jaco C. Knol, Sander R. Piersma, Epie Boven, Jos Jonkers, Sven Rottenberg, Connie R. Jiménez,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoIn contrast to various signatures that predict the prognosis of breast cancer patients, markers that predict chemotherapy response are still elusive. To detect such predictive biomarkers, we investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes who have a differential knock-out status for the breast cancer 1, early onset (Brca1) gene. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared with that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma. The analyses were performed 24 h after administration of the maximum tolerable dose of cisplatin. At this time point, drug-sensitive BRCA1-deficient tumors showed DNA damage, but cells were largely viable. By applying paired statistics and quantitative filtering, we identified highly discriminatory markers for the sensitive and resistant model. Proteins up-regulated in the sensitive model are involved in centrosome organization, chromosome condensation, homology-directed DNA repair, and nucleotide metabolism. Major discriminatory markers that were up-regulated in the resistant model were predominantly involved in fatty acid metabolism, such as fatty-acid synthase. Specific inhibition of fatty-acid synthase sensitized resistant cells to cisplatin. Our data suggest that exploring the functional link between the DNA damage response and cancer metabolism shortly after the initial treatment may be a useful strategy to predict the efficacy of cisplatin. In contrast to various signatures that predict the prognosis of breast cancer patients, markers that predict chemotherapy response are still elusive. To detect such predictive biomarkers, we investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes who have a differential knock-out status for the breast cancer 1, early onset (Brca1) gene. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared with that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma. The analyses were performed 24 h after administration of the maximum tolerable dose of cisplatin. At this time point, drug-sensitive BRCA1-deficient tumors showed DNA damage, but cells were largely viable. By applying paired statistics and quantitative filtering, we identified highly discriminatory markers for the sensitive and resistant model. Proteins up-regulated in the sensitive model are involved in centrosome organization, chromosome condensation, homology-directed DNA repair, and nucleotide metabolism. Major discriminatory markers that were up-regulated in the resistant model were predominantly involved in fatty acid metabolism, such as fatty-acid synthase. Specific inhibition of fatty-acid synthase sensitized resistant cells to cisplatin. Our data suggest that exploring the functional link between the DNA damage response and cancer metabolism shortly after the initial treatment may be a useful strategy to predict the efficacy of cisplatin. Breast cancer is a heterogeneous disease consisting of a variety of subtypes that need different treatment strategies. In contrast to several prognostic signatures for clinical outcome, markers that predict treatment efficacy have been difficult to define. Reasons to explain this failure have been discussed elsewhere (1Borst P. Wessels L. Do predictive signatures really predict response to cancer chemotherapy?.Cell Cycle. 2010; 9: 4836-4840Crossref PubMed Scopus (46) Google Scholar). A shortcoming of previous attempts to identify such markers may be that tumors were usually not challenged by drugs when sampled for analysis, or treatment was given a few weeks before sampling (neoadjuvant trials). Moreover, most previous studies focused on the analysis of gene expression to identify useful markers. However, differential expression of relevant factors, such as those involved in the DNA damage response, may be easier to detect shortly after chemotherapy-induced stress, and protein level readouts may provide a more direct way of assessing drug response. In this study, we aimed at detecting predictive biomarkers at the protein level by comparing the short term treatment response of platinum-sensitive versus platinum-resistant mouse mammary tumors that represent different breast cancer subtypes. As a sensitive model, we used the K14cre;Brca1F/F;p53F/F mouse model (2Liu X. Holstege H. van der Gulden H. Treur-Mulder M. Zevenhoven J. Velds A. Kerkhoven R.M. van Vliet M.H. Wessels L.F. Peterse J.L. Berns A. Jonkers J. Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12111-12116Crossref PubMed Scopus (350) Google Scholar) for BRCA1 1The abbreviations used are:BRCA1breast cancer antigen 1FASNfatty-acid synthaseCDH1cadherin-1PARP1poly(ADP-ribose) polymerase 1HRhomologous recombinationp53tumor protein p53BisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPPARperoxisome proliferator-activated receptor. 1The abbreviations used are:BRCA1breast cancer antigen 1FASNfatty-acid synthaseCDH1cadherin-1PARP1poly(ADP-ribose) polymerase 1HRhomologous recombinationp53tumor protein p53BisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPPARperoxisome proliferator-activated receptor.-deficient breast cancer. The Brca1−/−;p53−/− tumors that arise in this model include a large intragenic deletion of Brca1, and we have previously shown that these tumors are highly sensitive to cisplatin treatment (3Rottenberg S. Nygren A.O. Pajic M. van Leeuwen F.W. van der Heijden I. van de Wetering K. Liu X. de Visser K.E. Gilhuijs K.G. van Tellingen O. Schouten J.P. Jonkers J. Borst P. Selective induction of chemotherapy resistance of mammary tumors in a conditional mouse model for hereditary breast cancer.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12117-12122Crossref PubMed Scopus (240) Google Scholar). The response that we observed in this mouse model is consistent with the sensitivity of BRCA1-like breast cancer to intensive platinum-based chemotherapy in the clinic (4Vollebergh M.A. Lips E.H. Nederlof P.M. Wessels L.F. Schmidt M.K. van Beers E.H. Cornelissen S. Holtkamp M. Froklage F.E. de Vries E.G. Schrama J.G. Wesseling J. van, d. V. van T.H. de B.M. Hauptmann M. Rodenhuis S. Linn S.C. An aCGH classifier derived from BRCA1-mutated breast cancer and benefit of high-dose platinum-based chemotherapy in HER2-negative breast cancer patients.Ann. Oncol. 2010; 22: 1561-1570Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Moreover, it was recently shown that triple-negative breast cancer patients frequently respond to cisplatin treatment, especially in patients with lower BRCA1 expression (5Silver D.P. Richardson A.L. Eklund A.C. Wang Z.C. Szallasi Z. Li Q. Juul N. Leong C.O. Calogrias D. Buraimoh A. Fatima A. Gelman R.S. Ryan P.D. Tung N.M. De Nicolo A. Ganesan S. Miron A. Colin C. Sgroi D.C. Ellisen L.W. Winer E.P. Garber J.E. Efficacy of neoadjuvant cisplatin in triple-negative breast cancer.J. Clin. Oncol. 2010; 28: 1145-1153Crossref PubMed Scopus (792) Google Scholar). breast cancer antigen 1 fatty-acid synthase cadherin-1 poly(ADP-ribose) polymerase 1 homologous recombination tumor protein p53 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol peroxisome proliferator-activated receptor. breast cancer antigen 1 fatty-acid synthase cadherin-1 poly(ADP-ribose) polymerase 1 homologous recombination tumor protein p53 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol peroxisome proliferator-activated receptor. As resistant model, we chose WAPcre;Cdh1F/F;p53F/F mice. The cadherin-1 (CDH1)- and p53-deficient mammary tumors generated in these animals resemble human pleomorphic invasive lobular carcinomas (6Derksen P.W. Braumuller T.M. van der Burg E. Hornsveld M. Mesman E. Wesseling J. Krimpenfort P. Jonkers J. Mammary-specific inactivation of E-cadherin and p53 impairs functional gland development and leads to pleomorphic invasive lobular carcinoma in mice.Dis. Model. Mech. 2011; 4: 347-358Crossref PubMed Scopus (102) Google Scholar). We show here that the tumors of this model hardly respond to cisplatin. This is also consistent with the nature of invasive lobular carcinoma cancers in patients, which usually have only a modest benefit of chemotherapy as compared with invasive ductal carcinoma (7Cristofanilli M. Gonzalez-Angulo A. Sneige N. Kau S.W. Broglio K. Theriault R.L. Valero V. Buzdar A.U. Kuerer H. Buccholz T.A. Hortobagyi G.N. Invasive lobular carcinoma classic type: response to primary chemotherapy and survival outcomes.J. Clin. Oncol. 2005; 23: 41-48Crossref PubMed Scopus (308) Google Scholar). Platinum agents induce DNA damage by forming inter- and intrastrand DNA cross-links. The repair of DNA-platinum adducts involves several repair pathways including the Fanconi anemia pathway, nucleotide excision repair, and homologous recombination (HR) (8Deans A.J. West S.C. DNA interstrand cross-link repair and cancer.Nat. Rev. Cancer. 2011; 11: 467-480Crossref PubMed Scopus (703) Google Scholar). Because BRCA1 is an important player in the HR pathway, which results in error-free repair of double strand breaks, it is not unexpected that BRCA1-deficient tumors respond to platinum. Multiple cisplatin resistance mechanisms have been put forward (9Galluzzi L. Senovilla L. Vitale I. Michels J. Martins I. Kepp O. Castedo M. Kroemer G. Molecular mechanisms of cisplatin resistance.Oncogene. 2012; 31: 1869-1883Crossref PubMed Scopus (1760) Google Scholar), of which reactivation of the HR pathway by genetic restoration of BRCA1 function is found to be a clinically relevant cisplatin resistance mechanism (10Borst P. Rottenberg S. Jonkers J. How do real tumors become resistant to cisplatin?.Cell Cycle. 2008; 7: 1353-1359Crossref PubMed Scopus (179) Google Scholar). Unfortunately, the precise BRCA1 status or HR activity of tumor cells is frequently not known for breast cancer patients. Early treatment resistance and response proteins that assess HR competence, both in familial and sporadic breast cancers, could therefore aid in selecting patients for platinum-based chemotherapy. In addition, identification of (druggable) predictive markers of resistant tumors might help to identify patients that need an alternative treatment. In this study, we found that major discriminatory proteins after treatment with cisplatin are involved in fatty acid metabolism and signaling. These proteins include the following: FASN, which is known as a central player in de novo fatty acid synthesis; fatty acid-binding protein 4 (FABP4), a major transporter of fatty acids; and γ-synuclein, a protein that has hypothesized lipid binding properties. Our data suggest that the analysis of fatty acid metabolism may be a useful readout to predict platinum resistance early after initial treatment. All chemicals, unless otherwise specified, were obtained from Sigma-Aldrich. HPLC solvents, LC-MS grade water, acetonitrile, and formic acid were obtained from Biosolve (Biosolve B.V., Valkenswaard, The Netherlands). Porcine sequence-grade modified trypsin was obtained from Promega (Promega Benelux B.V., Leiden, The Netherlands). The generation of Cdh1−/−;p53−/−(WEP) or Brca1−/−;p53−/−(KB1P) mammary tumors has been described previously (2Liu X. Holstege H. van der Gulden H. Treur-Mulder M. Zevenhoven J. Velds A. Kerkhoven R.M. van Vliet M.H. Wessels L.F. Peterse J.L. Berns A. Jonkers J. Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12111-12116Crossref PubMed Scopus (350) Google Scholar, 6Derksen P.W. Braumuller T.M. van der Burg E. Hornsveld M. Mesman E. Wesseling J. Krimpenfort P. Jonkers J. Mammary-specific inactivation of E-cadherin and p53 impairs functional gland development and leads to pleomorphic invasive lobular carcinoma in mice.Dis. Model. Mech. 2011; 4: 347-358Crossref PubMed Scopus (102) Google Scholar, 11Derksen P.W. Liu X. Saridin F. van der Gulden H. Zevenhoven J. Evers B. van Beijnum J.R. Griffioen A.W. Vink J. Krimpenfort P. Peterse J.L. Cardiff R.D. Berns A. Jonkers J. Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis.Cancer Cell. 2006; 10: 437-449Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Orthotopic transplantation of tumors into syngeneic mice and treatment with cisplatin were performed as reported previously (3Rottenberg S. Nygren A.O. Pajic M. van Leeuwen F.W. van der Heijden I. van de Wetering K. Liu X. de Visser K.E. Gilhuijs K.G. van Tellingen O. Schouten J.P. Jonkers J. Borst P. Selective induction of chemotherapy resistance of mammary tumors in a conditional mouse model for hereditary breast cancer.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12117-12122Crossref PubMed Scopus (240) Google Scholar). Tumor samples for the proteomic analysis were snap-frozen and stored at −80°C until use. All animal experiments were approved by The Netherlands Cancer Institute ethical review committee. The Cdh1−/−;p53−/− cell line (KEP11) was derived from a primary tumor that arose in a K14cre;Cdh1F/F;Trp53F/F mouse, and the cells were cultured as described (11Derksen P.W. Liu X. Saridin F. van der Gulden H. Zevenhoven J. Evers B. van Beijnum J.R. Griffioen A.W. Vink J. Krimpenfort P. Peterse J.L. Cardiff R.D. Berns A. Jonkers J. Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis.Cancer Cell. 2006; 10: 437-449Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). KEP11 cells were transduced with pLKO-puro short hairpin RNA (shRNA) lentiviruses obtained from Mission library clones (Sigma-Aldrich). To target Fasn, we used TRCN0000075704 (shRNA#1) and TRCN0000075707 (shRNA#2). After selection with 3 μg/ml puromycin, 8000 cells per well were seeded in 6-well plates and assayed for clonal growth in the presence of cisplatin. 1 day after seeding, cells were incubated for 24 h with 2, 2.25, or 2.5 μm cisplatin. Surviving colonies were visualized using Leishman stain 6, 8, or 9 days after starting treatment. The efficacy of Fasn inhibition was determined by quantitative RT-PCR using the LightCycler® 480 SYBR Green I Master reagents according to the manufacturer's protocol (Roche Applied Science, catalog number 4707516001). To amplify mouse hypoxanthine-guanine phosphoribosyltransferase Hprt or Fasn cDNA, the following primers were used (5′ to 3′): Hprt_for (CTGGTGAAAAGGACCTCTCG) and Hprt_rev (TGAAGTACTCATTATAGTCAAGGGCA); Fasn_for (ATTGTCGCTCTGAGGCTGTTG) and Fasn_rev (TTGCTCCTTGCTGCCATCTG). To measure cell proliferation, ∼2000 KEP11-derived cells were seeded into 96-well plates. At the indicated time points, each well was refreshed by 150 μl of fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (0.5 mg/ml, Sigma-Aldrich) and incubated for another 4 h at 37°C. Then the medium was removed, and 150 μl of DMSO was added into each well to dissolve the resultant formazan crystals. Cell growth was determined by the absorbance detected at 490 nm using a microplate reader (Tecan, Infinite M200PRO). For homogenization, we cut into smaller parts an ∼20-mg piece of tumor tissue into a bath of liquid nitrogen. The proteins in the breast tumor tissue samples were solubilized in 800 μl of 1× reducing SDS Sample Buffer (containing 62.5 mm Tris-HCl, 2% w/v SDS, 10% v/v glycerol, and 0.0025% bromphenol blue, 100 mm DDT, pH 6.8) using a Pellet Pestles micro-grinder system (Kontes Glassware, Vineland, NJ). Subsequently, the proteins were denatured by heating at 100°C for 10 min. Any insoluble debris was removed by centrifugation for 15 min at maximum speed (16.1 relative central force) in a bench top centrifuge. Proteins were fractionated using one-dimensional SDS-PAGE. 25 μl of each homogenized sample (containing about 50 μg of protein) was loaded into a well of a pre-cast 4–12% NuPAGE (w/v) BisTris 1.5-mm minigel (Invitrogen). The stacking gel contained 4% (w/v) acrylamide/BisTris. Electrophoresis was carried out at 200 V in NuPAGE MES SDS running buffer (50 mm Tris base, 50 mm MES, 0.1% w/v SDS, 1 mm EDTA, pH 7.3) until the dye front reached the end of the gel. Following electrophoresis, gels were fixed with a solution of 50% ethanol and 3% phosphoric acid. Staining was carried out in a solution of 34% methanol, 3% phosphoric acid, 15% ammonium sulfate, and 0.1% Coomassie Blue G-250 (Bio-Rad) with subsequent destaining in milli-Q water. In-gel digestion and nanoLC-FT-MS for the 12 tumors from the discovery experiment were performed as described previously (12Warmoes M. Jaspers J.E. Pham T.V. Piersma S.R. Oudgenoeg G. Massink M.P. Waisfisz Q. Rottenberg S. Boven E. Jonkers J. Jimenez C.R. Proteomics of mouse BRCA1-deficient mammary tumors identifies DNA repair proteins with diagnostic and prognostic value in human breast cancer.Mol. Cell. Proteomics. 2012; M111.013334Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In short, processed gel lanes were cut in 10 equal bands, after which they were in-gel digested with trypsin. Extracted peptides from each band were separated on a C18 column for subsequent MS/MS analysis. Cell lysates from the FASN knockdown and control experiments were applied to a one-dimensional SDS-polyacrylamide gel. Proteins were allowed to enter the stacking gel, and the voltage was switched off when the proteins were just in the running gel. The samples on gel were processed as a single gel band and were in-gel digested with trypsin. After vacuum centrifugation, the peptide extract was filtered through a 0.45-μm low protein-binding PVDF membrane (Millipore) to remove particles. Extracted peptides were separated on a 75-μm × 20-cm custom-packed Reprosil C18 aqua column (1.9 μm, 120 Å) in a 150-min gradient (5–32% acetonitrile + 0.5% acetic acid at 300 nl/min) using a U3000 RSLC high pressure nano-LC (Dionex). Eluting peptides were measured on line by a Q Exactive mass spectrometer (ThermoFisher Scientific) operating in data-dependent acquisition mode. Peptides were ionized using a stainless steel emitter at a potential of +2 kV (ThermoScientific). Intact peptide ions were detected at a resolution of 35,000 and fragment ions at a resolution of 17,500; the MS mass range was 350–1500 Da. AGC target settings for MS were 3E6 charges and for MS/MS 2E5 charges. Peptides were selected for higher energy C-trap dissociation fragmentation at an underfill ratio of 1% and a quadrupole isolation window of 1.5 Da, peptides were fragmented at a normalized collision energy of 30. QE raw files were searched against the International Protein Index mouse 3.68 database (56,729 entries, released December 18, 2009) using MaxQuant 1.2.2.5 (13Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9154) Google Scholar). Data was filtered at 1% false discovery rate at both the peptide and protein levels. MS/MS spectra were searched against the mouse International Protein Index database 3.31 (56,555 entries, released August 17, 2007) using Sequest (version 27, revision 12), which is part of the BioWorks 3.3 data analysis package (Thermo Fisher, San Jose, CA). MS/MS spectra were searched with a maximum allowed deviation of 10 ppm for the precursor mass and 1 atomic mass unit for fragment masses. Methionine oxidation and cysteine carboxamidomethylation were allowed modifications; two missed cleavages were allowed, and the minimum number of tryptic termini was one. After database searching the DTA and OUT, files were imported into Scaffold version 1.07 (Proteome software, Portland, OR). Scaffold was used to organize the gel band data and to validate peptide identifications using the Peptide Prophet algorithm (14Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3621) Google Scholar, 15Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3886) Google Scholar). Only identifications with a probability >95% were retained. Subsequently, the Protein Prophet algorithm was applied, and protein identifications with a probability of >99% with two peptides or more were retained. The false discovery rate for the detected proteins using this workflow is on average around 0.5%, and it was not calculated again (16Albrethsen J. Knol J.C. Piersma S.R. Pham T.V. de Wit M. Mongera S. Carvalho B. Verheul H.M. Fijneman R.J. Meijer G.A. Jimenez C.R. Subnuclear proteomics in colorectal cancer: identification of proteins enriched in the nuclear matrix fraction and regulation in adenoma to carcinoma progression.Mol. Cell. Proteomics. 2010; 9: 988-1005Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). For each protein identified, the total number of MS/MS spectra detected for each protein identified (spectral counts) was exported to Excel 2003 (Microsoft, Redmond, WA). Normalization was performed as described previously (12Warmoes M. Jaspers J.E. Pham T.V. Piersma S.R. Oudgenoeg G. Massink M.P. Waisfisz Q. Rottenberg S. Boven E. Jonkers J. Jimenez C.R. Proteomics of mouse BRCA1-deficient mammary tumors identifies DNA repair proteins with diagnostic and prognostic value in human breast cancer.Mol. Cell. Proteomics. 2012; M111.013334Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 17Pham T.V. Piersma S.R. Warmoes M. Jimenez C.R. On the β-binomial model for analysis of spectral count data in label-free tandem mass spectrometry-based proteomics.Bioinformatics. 2010; 26: 363-369Crossref PubMed Scopus (134) Google Scholar). A one-sided paired β-binomial test (18Pham T.V. Jimenez C.R. An accurate paired sample test for count data.Bioinformatics. 2012; 28: i596-i602Crossref PubMed Scopus (53) Google Scholar) was applied to find proteins that showed statistically significant differences in spectral count numbers between the untreated control tumors and the cisplatin-treated tumors, and it was applied both to the BRCA1-deficient and -proficient model. Statistical testing between the two differently treated tumor models was performed using a unpaired two-sided β-binomial test (17Pham T.V. Piersma S.R. Warmoes M. Jimenez C.R. On the β-binomial model for analysis of spectral count data in label-free tandem mass spectrometry-based proteomics.Bioinformatics. 2010; 26: 363-369Crossref PubMed Scopus (134) Google Scholar). Proteins with a p value of less than 0.05 were designated as being significant. Hierarchical clustering was carried out using R statistical software. For protein clustering, the abundances were normalized to zero means and unit variance for each individual protein. Subsequently, the Euclidean distance measure was used. For sample clustering, a divergence measure between two Poisson distributions was used, preventing highly abundant proteins from dominating others in contribution to the total sample difference, as described by Albrethsen et al. (16Albrethsen J. Knol J.C. Piersma S.R. Pham T.V. de Wit M. Mongera S. Carvalho B. Verheul H.M. Fijneman R.J. Meijer G.A. Jimenez C.R. Subnuclear proteomics in colorectal cancer: identification of proteins enriched in the nuclear matrix fraction and regulation in adenoma to carcinoma progression.Mol. Cell. Proteomics. 2010; 9: 988-1005Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The Ward linkage was used. For analysis of reproducibility, we calculated the average coefficient of variation of the spectral counts from overlapping proteins of each set of three biological replicates. For STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) pathway analysis (version 9.0) (19Szklarczyk D. Franceschini A. Kuhn M. Simonovic M. Roth A. Minguez P. Doerks T. Stark M. Muller J. Bork P. Jensen L.J. von Mering C. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored.Nucleic Acids Res. 2011; 39: D561-D568Crossref PubMed Scopus (2705) Google Scholar), International Protein Index identifiers were mapped to human gene symbols, after which networks were generated and downloaded. Graphical rich networks with color intensities indicating protein fold changes were made using the Cytoscape software (20Shannon P. Markiel A. Ozier O. Baliga N.S. Wang J.T. Ramage D. Amin N. Schwikowski B. Ideker T. Cytoscape: a software environment for integrated models of biomolecular interaction networks.Genome Res. 2003; 13: 2498-2504Crossref PubMed Scopus (25321) Google Scholar) after which groups of well connected proteins were identified (21Nepusz T. Yu H. Paccanaro A. Detecting overlapping protein complexes in protein-protein interaction networks.Nat. Methods. 2012; 9: 471-472Crossref PubMed Scopus (934) Google Scholar). Gene ontology analysis was performed using the BiNGO (Biological Networks Gene Ontology, Ghent, Belgium) software (22Maere S. Heymans K. Kuiper M. BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks.Bioinformatics. 2005; 21: 3448-3449Crossref PubMed Scopus (3091) Google Scholar) on the top three most significant groups of well connected proteins identified by Cluster ONE (Clustering with Overlapping Neighborhood Expansion, Egham, UK). We have previously shown that BRCA1-deficient mammary tumors, which contain large intragenic deletions of the Brca1 and p53 genes, are highly sensitive to the maximum tolerable dose of cisplatin (3Rottenberg S. Nygren A.O. Pajic M. van Leeuwen F.W. van der Heijden I. van de Wetering K. Liu X. de Visser K.E. Gilhuijs K.G. van Tellingen O. Schouten J.P. Jonkers J. Borst P. Selective induction of chemotherapy resistance of mammary tumors in a conditional mouse model for hereditary breast cancer.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12117-12122Crossref PubMed Scopus (240) Google Scholar). When we treated CDH1-deficient tumors, however, we found that these hardly responded to the same regimen (Fig. 1A). This difference in cisplatin response between the models is not unexpected, because CDH1-deficient tumors are still capable of repairing cisplatin-induced DNA damage by homologous recombination (HR), in contrast to BRCA1-deficient tumors. In line with this, we previously observed that the CDH1-deficient tumors do not respond to treatment with the PARP inhibitor olaparib, which targets HR deficiency (23Rottenberg S. Jaspers J.E. Kersbergen A. van der Burg E. Nygren A.O. Zander S.A. Derksen P.W. de Bruin M. Zevenhoven J. Lau A. Boulter R. Cranston A. O'Connor M.J. Martin N.M. Borst P. Jonkers J. High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 17079-17084Crossref PubMed Scopus (759) Google Scholar). These contrary drug responses therefore provide an opportunity to investigate differential treatment-induced protein expression in two mouse models, which carry mammary tumors that resemble specific breast cancer subtypes. To measure proteins of viable tumor cells after treatment, we aimed at a time point when sufficient DNA damage was induced but when most drug-sensitive tumor cells had not yet entered apoptosis. Moreover, the percentage of stromal cells that eventually replace viable tumor tissue should be small. As presented in Fig. 1B, we found that 24 h after cisplatin administration most BRCA1-deficient tumor cells showed DNA damage foci (pH2AX), but only a few tumor cells showed morphological signs of cell death (e.g. pyknosis, nuclear fragmentation, or hypereosinophilic cytoplasm) or activation of caspase 3. In contrast, 48 or 96 h after treatment, the number of dying BRCA1-deficient tumor cells increased and was replaced by reactive stroma. In cisplatin-resistant (Cdh1−/−;p53−/−) tumors, the number of apoptotic or necrotic tumor cells was also low after 24 h of treatment as expected by the poor response (data not shown). Hence, the 24-h time point is appropriate to investigate differential induction of protein expression in cisplatin-sensitive versus cisplatin-resistant tumors. To identify early response biomarkers, we used three individual cisplatin-sensitive tumors (Brca1−/−;p53−/−) and three cisplatin-resistant tumors (Cdh1−/−;p53−/−) that were either treated with cisplatin or left untreated (see Fig. 2 for experimental setup). Comparative proteomics based on SDS-PAGE (see supplemental Fig. 1A for gel images) in combination with nanoLC-MS/MS identified a total of 3486 proteins in the 12 mammary tumor samples using stringent protein identification criteria (only protein identifications with a probability of >99% identified with at least two peptides of >95% in one of the samples were retained). The whole dataset of identified proteins is provided in supplemental Tables 1, and supplemental Table 2 contains the peptide identifications. The number of identified proteins in each biological group was comparable and ranged from 3104 to 3206 with good reproducibility of protein identification in the four groups: 66–75% of the proteins were identified in all three biological replicates (for Venn diagrams see supplemental Fig. 1B). Unsupervised cluster analysis using all 3486 proteins (supplemental Fig. 2) showed that CDH1-deficient tumors were clearly separated from the BRCA1-deficient ones
Referência(s)