Proteome of Metastatic Canine Mammary Carcinomas: Similarities to and Differences from Human Breast Cancer

Robert Klopeisch,*, Patricia Klose, Christoph Weise,| Angelika Bondzio, Gerd Multhaup,| Ralf Einspanier, and Achim D. Gruber
Institute of Veterinary Pathology, Freie Universita t Berlin, Robert-von-Ostertag-Strae 15, 14163 Berlin, Germany, Institute of Veterinary Biochemistry, Freie Universita t Berlin, Oertzenweg 19b, 14163 Berlin, Germany, and Institute of Chemistry and Biochemistry, Biochemistry, Thielallee 63, 14195 Berlin, Germany
Received June 29, 2010

Mammary tumors are a major health threat to women and female dogs. In both, metastasis of the primary tumor to distant organs is the most common cause of tumor-related death. Nevertheless, the molecular mechanisms of tumor metastasis are far from being understood, and it is still unknown why some human and canine carcinomas metastasize and others do not. Using 2D-DIGE and MALDITOF-MS we identied 21 proteins with signicant changes (fold change >1.5; p < 0.05) in protein expression between metastasizing (n ) 6) and nonmetastasizing (n ) 6) canine mammary carcinomas. Quantitative RT-PCR was used to identify transcriptional or post-transcriptional regulation of protein expression. Up-regulated proteins in metastatic carcinomas included proliferating cell nuclear antigen, ferritin light chain, bomapin, tropomyosin 3, thioredoxin-containing domain C5, adenosin, ornithine aminotransferase, coronin 1A, RAN-binding protein 1,3-phosphoglycerate dehydrogenase, and eukaryotic translation elongation factor 1. Down-regulated proteins in metastatic carcinomas included calretinin, myosin, light chain 2, peroxiredoxin 6, maspin, ibrinogen beta chain, vinculin, isocitrate dehydrogenase 1, tropomyosin 1, annexin A5, and Rho GTPase activating protein 1. Interestingly, 19 of these 21 proteins have been described with a malignancy-associated expression in human breast cancer and other human cancer types before. Further investigations are now necessary to test whether these markers are of prognostic value for canine mammary carcinomas and whether their expression is directly involved in canine mammary carcinogenesis or represent solely a secondary reactive phenotype.
Keywords: Canine mammary tumor breast cancer metastasis 2D-DIGE scavenger cell adhesion invasion

Introduction
Mammary gland tumors are a major threat to women and female dogs. In both species, metastasis of the primary tumor to distant organs is the most common cause of tumor-related death in affected individuals.1-3 Nevertheless, the molecular mechanisms of mammary tumor metastasis are far from being understood, and it is still enigmatic why some mammary carcinomas metastasize and others do not. Several explorative proteome studies therefore have tried to uncover protein expression patterns associated with metastasis in human breast cancer. By this approach, the expression of some relevant proteins could be related with a distinctively aggressive behavior of tumor cells.3,4 For instance, tropomyosin (TPM) 1 and 3 and the mammary serine protease inhibitor (maspin) have been identied to be differentially expressed in malignant human breast cancer by proteome analyses.2,3,5-9
R.K. and P.K. contributed equally. * Corresponding author. Tel.: (+49) 30 83862445. Fax: (+49) 30 838 62522. E-mail: klopeisch.robert@vetmed.fu-berlin.de. Institute of Veterinary Pathology, Freie Universita t Berlin. Institute of Veterinary Biochemistry, Freie Universita t Berlin. | Institute of Chemistry and Biochemistry, Biochemistry.

Canine mammary tumors (CMTs) have repeatedly been proposed as a model for human breast cancer, and there are several overlaps in malignancy-associated gene expression proles between the two.10-23 However, other important features of human breast cancer carcinogenesis including malignancy-associated BRCA mutations and the impact of steroid receptor expression on prognosis differ widely between the human and CMTs.24,25 One major benet of dogs as cancer models is the spontaneous CMT development with a compressed course of cancer progression when compared to humans but a longer therapeutic window when compared to rodent models.10,12 A complete characterization of the molecular mechanisms of the CMT carcinogenesis would therefore contribute to both understanding of this tumor in dogs and the characterization of a valuable comparative model for the human disease. The diagnostic and prognostic differentiation of both malignant human and CMTs with and without potential for metastatic spread to distant organs is difcult before metastases are large enough to be visualized.26-29 In canines, histological features of invasion at the tumor edges or metastases to the regional lymph nodes are the most important prognostic factors
10.1021/pr100671c 2010 American Chemical Society

6380 Journal of Proteome Research 2010, 9, 63806391

Published on Web 10/11/2010

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histology tumor size (cm) lymph node status ERBB2 status ER/PR status

Table 1. Characterization of Dogs with Metastasizing and Nonmetastasizing Canine Mammary Carcinomasa
dog no. breed age (years) survival time (months)

1 2 3 4 5 6 7 8 9 10 11 12

rottweiler dachshund retriever WHWT yorkshire terrier retriever irish setter shorthaired pointer dachshund shepherd dog mixture shepherd dog mixture great dane

10 13 11 12 8 11 7 12 15 15 11 10

6 3 5 2 4 2 >24 >24 >24 >24 >24 >24

CA CA CA CA CA CA CA CA CA CA CA CA

3 2 1 1 2 3 3 15 3 7 9 7

+ + + + + + -

+ + + + + + + + + + + +

- /- /- /- /- /- /- /- /- /- /- /- /-

a CA: canine mammary carcinoma; +, positive; -, negative; WHWT, West Highland White Terrier; ERBB2, erythroblastic leukemia viral oncogene homologue 2; ER, estrogen receptor; PR, progesterone receptor.

for metastatic potential. Malignancy-associated and prognostic protein expression patterns are not available and therefore not applied in routine veterinary diagnostics.27,30 The aim of this rst explorative study on the proteome of CMTs was to identify metastasis-associated protein expression patterns in CMTs. We therefore compared two groups of invasive canine mammary carcinomas with similar histological features of the primary tumor mass that either had or had not metastasized to the regional lymph node by the time of surgery. Using two-dimensional differential gel electrophoresis (2DDIGE) and matrix-assisted laser desorption/ionization-timeof-ight-mass spectrometry (MALDI-TOF-MS) we were able to identify a metastasis-associated protein expression pattern that in some aspects overlaps with proteome signatures in human breast cancer. Furthermore, quantitative RT-PCR was used to analyze whether these proteins were transcriptionally differentially regulated in metastatic and nonmetastatic CMTs. The data reveal signicant overlaps yet clear differences between CMTs and human breast cancer.

Material and Methods

Clinical Tumor Samples. Twelve highly malignant, invasive canine mammary carcinomas were included in the study (Table 1). Six tumors (cases 1-6; LN+) had metastasized to the regional lymph nodes at the time of surgery as seen in hematoxylin-eosin (HE)-stained sections of the primary tumor and the regional lymph node. The other six tumors (cases 7-12; LN-) were also diagnosed as highly malignant, invasive, simple carcinomas with a diameter larger than the average of the LN+ but had no lymph node metastases as conrmed by ve serial sections (every 100 m) of the regional lymph node. All tumor samples were snap frozen in liquid nitrogen immediately after surgical removal. Lymph node status was also associated with marked differences in postoperative survival. All dogs without lymph node metastases had a postoperative survival time of more than two years without evidence of lymph node or distant metastases. In contrast, postoperative survival times of dogs with LN+ was 3.6 months on average (range 2-6 months). The cause of death could be related to distant metastases to the lung in four dogs, while the other two were not submitted for necropsy or thoracic radiographs. None of the patients received any chemotherapeutic treatment prior to surgery. The key clinicopathological features of all cases are listed in Table 1. Histology and Immunohistochemistry. Estrogen receptor (ER), progesterone receptor (PR), and ERBB2 expression were evaluated immunohistochemically to make results in the canine

tumors comparable to studies on human breast cancer. All tissues were analyzed using the Avidin-Biotinylin-complex method. Primary specic antibodies for estrogen receptor (cat. no. EI629C01, DCS, Hamburg, Germany, dilution 1:400), progesterone receptor (PI633C01, DCS, Hamburg, Germany, dilution 1:700), and ERBB2 (c-erbB2, cat. no. A0485, DAKO GmbH, Heidelberg, Germany, dilution 1:700) were diluted in Trisbuffered saline (TBS, 50 mM, pH 7.6) and incubated at 4 C overnight after blocking with 50% goat serum in TBS (30 min at room temperature). Goat antirabbit IgG (pab, 1 in 200; Vector, BA1000) was used as a secondary antibody. Diaminobenzidine tetrahydrochloride (Sigma Aldrich, Munich, Germany) was used as chromogen, and slides were counterstained with hematoxylin. Normal canine mammary gland tissue served as a positive control. As a negative control, sections were incubated with an irrelevant immune serum instead of the rst antibody with all other procedures alike. Tissues were evaluated in ten random 400 magnication elds in three parafn sections and graded negative if less than 10% of the cells of the relevant tissue were immunohistochemically positive. Macrodissection and Sample Preparation. Eight micrometer sections were cut from the frozen tumor. HE-stained sections were digitized with the T3 Scan Scope Aperio (Aperio, Vista, USA), and the percentage of tumor cells was analyzed by Axiovision 2.0 software (Zeiss, Jena, Germany). Only tissue samples with a tumor content of more than 70% were extracted in 1 mL of lysis buffer containing 7 M urea, 2 M thiourea, and 4% CHAPS (3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate). The lysate was ultrasound treated twice for two minutes each and then centrifuged at 4500 rpm for one minute. The supernatant was collected and stored at -80 C until analysis. Protein concentrations (range 3.1-13.4 g/L) were measured with the 2-D Quant Kit (GE Healthcare, Freiburg, Germany). In addition, consecutive, macrodissected tissue sections of all tumor probes were used to isolate mRNA for reverse transcription, quantitative PCR (RT-qPCR) analysis using Nucleospin RNA II (MN, Du ren, Germany). RNA integrity and quantity were analyzed using the 2100 Bioanalyzer (cutoff RIN > 8, Agilent, Waldbronn, Germany). 2D-DIGE and Data Analysis. Protein extracts were labeled with CyDyes (GE Healthcare, Freiburg, Germany): Cy3 for the metastasizing samples (LN+), Cy5 for the nonmetastasizing samples (LN-), and Cy2 for the internal standard. The internal standard contained equal amounts of all protein lysates. An amount of 50 g of protein was labeled with 400 pmol of the
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respective dye following the protocol of the supplier. The separate CyDyes labeling reactions were nally combined, and an equal volume of 2 sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2% Pharmalyte IPG Buffer, 2% DTT, 0,04% bromphenol blue) was added. Samples were complemented to 450 L with rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2% Pharmalyte IPG buffer, 40 mM DTT). Immobilized nonlinear pH gradient (IPG) strips, pH 3-7 (GE Healthcare, Freiburg, Germany), were rehydrated with Cy-labeled samples in the dark at room temperature overnight according to the manufacturers guidelines. Isoelectric focusing (IEF) was performed using an Ettan IPGphor 3 Isoelectric Focusing Unit (Ettan IPGphor Manifold; GE Healthcare, Freiburg, Germany) for a total of 50 kVh at 20 C, 75 A/strip. After IEF, the IPG strips were rst equilibrated for 15 min with the equilibration buffer (6 M urea, 30% glycerol, 2% SDS, and 50 mM Tris, 0,02% bromphenol blue, pH 8.8) containing 100 mg of DTT and equilibrated for 15 min with equilibration buffer containing 250 mg of iodoacetamide. Strips were transferred onto vertical 12.5% SDS-PAGE gels and sealed with 0.5% low-melting-point agarose. The second-dimension molecular weight separation was carried out using an Ettan DALTsix Electrophoresis Unit (GE Healthcare, Freiburg, Germany) and the following running parameters for six gels: 60 mA for one hour, 240 mA for one hour, and 300 mA for ve hours. Protein spots were visualized with the Typhoon 9400 uorescence scanner (GE Healthcare, Freiburg, Germany) scanning at the Cy dyes respective wavelengths. Spot detection, matching, and quantication of spot intensity were performed using the DeCyder 2D Software, Version 7.0 (GE Healthcare, Freiburg, Germany). Differences in expression between LN+ and LNsamples were analyzed by the Students t test with p-values < 0.05 considered signicant. Only spots present in all gels and with a LN+/LN- ratio of more than 1.5 or less than 0.67 in spot intensity were selected for protein identication by MS. Gels loaded with 350 g of protein were silver-stained for spot picking and subsequent MS analysis.31 Protein Identication by MALDI-TOF-MS. Proteins were identied from gel spots by matrix-assisted laser desorption ionization-time-of-ight-mass spectrometry (MALDI-TOF-MS) using an Ultraex-II TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a Smart beam laser. Peptides were obtained by trypsin in-gel digestion as described previously.31 The protein digests were measured in the reector mode using R-cyano-4-hydroxycinnamic acid (CHCA) as matrix. Fragment-ion spectra of selected peptides were acquired in the LIFT mode.32 For the database search, known contamination peaks such as those of keratin and autoproteolytic products were excluded for a peptide mass ngerprint database search with the Mascot server (www.matrixscience.com) in the NCBInr database. The search was restricted to mammalian sequences only. One missed tryptic cleavage was considered, and a mass accuracy of 50-100 ppm was used for the searches. Western Blot Analysis. To conrm the proteome analysis, 40 g of sample protein and a prestained protein-weight marker (Fermentas, Berlin, Germany) were resolved by SDS-PAGE in 12% polyacrylamide gels and transferred onto PVDF (polyvinylidene diuoride) membranes (GE Healthcare, Freiburg, Germany) in Tris-glycine buffer with 20% (v/v) methanol. Membranes were saturated with 5% (w/v) nonfat milk powder (Roth, Karlsruhe, Germany) prepared in Tris-buffered saline containing 0.1% Tween 20 (TBST) for one hour at room temperature and then incubated with primary antibody over6382 Journal of Proteome Research Vol. 9, No. 12, 2010

Klopeisch et al.
night at 4 C. The primary antibody employed was rabbit polyclonal Peroxiredoxin-6 (LIFESPAN Biosciences, Seattle, USA) used at a 1:500 dilution. After several washes with TBST, the membranes were incubated for two hours in TBST containing 3% BSA and antirabbit DyLight 488 conjugate (1:20000) and antimouse DyLight 649-conjugate (1:7000) as secondary antibodies (Perbio Science, Bonn, Germany). Finally, membranes were visualized with the Typhoon 9400 (GE Healthcare, Freiburg, Germany), choosing appropriate wavelengths (excitation 488 nm, emission 520 nm for DyLight 488 and excitation 633 nm, emission 670 nm for DyLight 649) at a resolution of 100 m. Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction. Primer sequences for mRNA transcripts of the identied proteins and the housekeeper genes hypoxanthinephosphoribosyl transferase (HPRT), ATP-synthase subunit 5B (A5B), and ribosomal protein L32 (RP32) are shown in the Supporting Information (Supplemental Table 1).23 Reverse transcription, quantitative PCR (RT-qPCR) and data analyses were performed using the MX 3000P Quantitative PCR System and MX Pro software (Stratagene, La Jolla, USA). The reactions were carried out in 96-well polypropylene plates covered with optical caps (Stratagene, La Jolla, USA). The plates contained triplicates of each cDNA sample and no-template controls with water instead of cDNA templates. Exact primer concentrations and PCR time and temperature conditions were determined during initial optimization runs. The RT-qPCR efciency of all assays was between 96% and 101% (data not shown) and all yielded products of the expected sequence. The 15 L reaction mix contained 5 L of cDNA and 12.5 L of Brilliant SYBR Green QPCR Master Mix (Stratagene, Amsterdam, Netherlands) with 300 nM of each primer. Cycling conditions were 10 min at 95 C, followed by 40 cycles of 30 s at 95 C, 1 min at 58 C, and 30 s at 72 C. The cDNA of all samples was amplied on the same plate for every primer pair to ensure equal amplication conditions. Specicity of amplication products was conrmed by melting curve and sequence analyses. For each sample, results were documented as cycle threshold (CT) set to 100 relative uorescence unit values of background subtracted qPCR uorescence kinetics by using the MX Pro Stratagene analysis software, applying the adaptive baseline, amplicationbased threshold, and moving average algorithm enhancement. Quantication of Target Gene Expression. The relative expression of the target gene (TG) was calculated using a comparative Ct-method with multiple housekeepers as previously described.17,33,34 The housekeeper genes used were selected from a panel of reference genes (RGs) according to the GeNorm algorithm.35 Data are presented as fold change in gene expression level of the gene of interest (GOI) in metastasizing carcinomas (LN+) normalized to the housekeepers and to the similarly normalized GOI expression levels in nonmetastasizing carcinomas (LN-). First, Ct between the geometric mean of the three RGs and the GOI in LN+ (Ct LN+) and LN- (Ct LN-) was calculated. Finally, the fold change (FC) of GOI expression was calculated (FC ) 2 - (Ct LN+ - Ct LN-)). Cut off values for FC were set at >1.5 for increased gene expression, and FC values <0.67 were accepted as reduced gene expression.

Results
Histology and Immunohistochemistry of Receptor Status. Only primary tumors diagnosed as highly malignant, invasive, solid, and tubulopapillary, simple mammary carcinomas with lymph node metastases (LN+) or without lymph node me-

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Figure 1. Canine mammary carcinoma, case no. 3. All carcinomas were characterized by inltrative growth, anisocytosis, anisokaryosis, and numerous mitotic gures (arrows). Hematoxylin/Eosinstain. Magnication 1:400.

Figure 3. Canine mammary carcinoma, case no. 5. Immunohistochemistry against estrogen receptor alpha. More than 90% of the tumor of metastasizing and nonmetastasizing carcinomas had no estrogen receptor alpha expression. ABC-method, Hematoxylin-counterstain. Magnication 1:300.

Figure 2. Lymph node metastases of a canine mammary carcinoma in the inguinal lymph node, case no. 3. Case nos. 1-6 had metastatic carcinoma cells in the regional lymph node, while case nos. 7-12 were free of lymph node metastases. Hematoxylin/ Eosin-stain. Magnication 1:400.

Figure 4. Canine mammary carcinoma, case no. 7. Immunohistochemistry against ERBB2. The majority of tumor cells in 11/13 carcinomas had strong ERBB2 expression. ABC-method, Hematoxylin-counterstain. Magnication 1:300.

tastases (LN-) were included in this study. All carcinomas were characterized by inltrative growth of tumor cells into the surrounding tissues, anisocytosis, anisokaryosis, and a high mitotic rate (Figure 1). Metastatic tumors (dogs 1 to 6) in addition had metastatic tumor cells in surrounding lymph vessels and the regional lymph node (Figure 2). None of the tumors had ER/PR expression as shown by immunohistochemistry (Figure 3). In contrast, ERBB2 was expressed in all LN+ and LN- tumors (Figure 4). Identication of Differentially Expressed Proteins. Differences in protein expression patterns between metastasizing canine mammary tumors (cases 1-6, LN+) and nonmetastasizing tumors (cases 7-12, LN-) were analyzed using twodimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization-time-of-ight-mass spectrometry (MALDI-TOF-MS). Twenty-one proteins were identied which were signicantly differentially expressed in LN+ when compared to LN- tumors (LN+/LN- ratio >1.5 or <0.67, p < 0.05). Eleven of them were up-regulated (Table 2), and ten were down-regulated (Table 3) in LN+ (Figure 5A,B).

In some cases, top-scoring results were obtained for proteins from mammalian species other than dog (Canis familiariz). This can most likely be explained by the fact that the orthologous dog sequences are not present in the database used for the search (NCBInr). In such cases, the Mascot MOWSE scores tended to be comparably low because peptide masses can be matched only for those peptides where the sequences are identical between species. In all cases, however, the score was well beyond the signicance threshold proposed by Mascot (p < 0.05). Three proteins could not be identied by database searches for canine or other mammalian protein sequences. Cell Proliferation-Associated Proteins. All three cell proliferation- and division-associated proteins identied had increased expression levels in LN+ when compared to LN(Table 2; Figure 5A,B). The proliferating cell nuclear antigen (PCNA, spot U1) had the highest up-regulation (ratio 2.64). Spots U8 and U11 were identied as the cell proliferationassociated proteins Ran/TC4-binding protein (RANBP1) and eukaryotic elongation factor-1 delta (EEF1D).
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Table 2. Up-Regulated Proteins in Metastasizing (LN+) Canine Mammary Tumors (Ratio >1.5, p < 0.05)
spot identied protein ratio LN+/LNt test accession no. in NCBI MW [Da]b pIb Mowse score
a

Klopeisch et al.

assigned peptides

sequ. cov. [%]

U1 U2 U3 U4 U5 U6 U7 U8 U9 U10 U11

PCNA FTL TXNDC5 ADA OAT CORO1A BOMAPIN RANBP1 TPM3 PGDH EEF1D

2.64 2.57 2.05 1.76 1.74 1.65 1.61 1.57 1.56 1.51 1.51

0.037 0.0039 0.019 0.012 0.003 0.003 0.042 0.008 0.0051 0.042 0.013

gi|4505641 (Ho.) gi|66864897 (Ca.) gi|109069575 (Ma.) gi|73992038 (Ca.) gi|73998800 (Ca.) gi|73958508 (Ca.) gi|73945839 (Ca.) gi|73995897 (Ca.) gi|73961101 (Ca.) gi|73981259 (Ca.) gi|73974692 (Ca.)

29092 20139 44433 41338 48753 55228 45060 24180 26632 57308 30731

4.57 5.66 6.11 5.47 6.44 6.84 5.73 4.94 4.75 6.19 5.25

112 93 80 56 37 22 68 67 111 189 92

9 8 7 1c 1c 1c 1c 1c 16 15 9

32 42 14 3c 2c 1c 5c 6c 47 23 24

a Abbreviations: ADA, Adenosine deaminase; ANXA5, Annexin A5; ARHGAP1, Rho GTPase activating protein 1; CALB2, Calretinin; EEF1D, Elongation factor 1-delta; FGB, Fibrinogen beta chain; FTL, Ferritin, light polypeptide; IDH1, Isocitrate dehydrogenase1; MYL2, Myosin light chain 2; OAT, Ornithine aminotransferase; PCNA, Proliferating cell nuclear antigen; PGDH, D-3-phosphoglycerate dehydrogenase; PRDX6, Peroxiredoxin 6; RANBP1, Ran-specic GTPase-activating protein; TPM1, Tropomyosin 1; TPM3, Tropomyosin; TXNDC5, Thioredoxin domain containing 5; VCL, Vinculin. b Listed molecular weights and pI values correspond to the listed accession numbers, which sometimes belong to species other than Canis familiariz. c Proteins were identied by unique peptide sequencing using MALDI-TOF-MS/MS.

Table 3. Down-Regulated Proteins in Metastasizing (LN+) Canine Mammary Tumors (ratio <0.67, p < 0.05)a
spot identied protein protein ratio LN+/LNt test accession no. in NCBI MW [Da]b pIb Mowse score assigned peptides sequ. cov. [%]

D1 D2 D3 D4 D5 D6 D7 D8 D9 D10

CALB2 MYL2 PRDX6 MASPIN FGB VCL IDH1 TPM1 ANXA5 ARHGAP1

0.23 0.22 0.33 0.40 0.49 0.52 0.59 0.59 0.60 0.64

0.02 0.00058 0.0013 0.049 0.044 0.0046 0.029 0.031 0.00023 0.015

gi|29636 (Ho) gi|188586 (Ho) gi|4758638 (Ho) gi|149721154 (Eq) gi|73977986 (Ca.) gi|73953587 (Ca.) gi|74005281 (Ca.) gi|74000375 (Ca.) gi|57100553 (Ca.) gi|134085902 (Ca)

31616 19915 25133 42532 55590 87669 47180 32553 35978 50611

5.06 5.09 6.00 5.68 6.77 6.82 6.69 4.71 4.99 6.07

51 35 43 82 51 60 35 131 38 79

1c 1c 1c 9 1c 9 1c 13 1c 9

3c 5c 3c 28 2c 15 1c 29 3c 21

a Abbreviations: ADA, Adenosine deaminase; ANXA5, Annexin A5; ARHGAP1, Rho GTPase activating protein 1; CALB2, Calretinin; EEF1D, Elongation factor 1-delta; FGB, Fibrinogen beta chain; FTL, Ferritin, light polypeptide; IDH1, Isocitrate dehydrogenase1; MYL2, Myosin light chain 2; OAT, Ornithine aminotransferase; PCNA, Proliferating cell nuclear antigen; PGDH, D-3-phosphoglycerate dehydrogenase; PRDX6, Peroxiredoxin 6; RANBP1, Ran-specic GTPase-activating protein; TPM1, Tropomyosin 1; TPM3, Tropomyosin; TXNDC5, Thioredoxin domain containing 5; VCL, Vinculin. b Listed molecular weights and pI values correspond to the listed accession numbers, which sometimes belong to species other than Canis familiariz. c Proteins were identied peptide sequencing using MALDI-TOF-MS/MS.

Cell Adhesion and Motility Proteins. The cytoskeletal proteins vinculin (VCL, spot D6) and the actin-binding protein tropomyosin 1 (TPM1, spot D8) had a decreased expression in LN+ samples (Table 3; Figure 5A,B). Similarly, the cell motility associated proteins myosin light chain 2 (MYL2, spot D2) and calretinin (CALB2, spot D1) were down-regulated in LN+ (Table 3; Figure 5A,B). In contrast, tropomyosin 3 (TPM3, spot U9) and the actin-binding protein coronin 1A (CORO1A, spot U6) were up-regulated in LN+ (Table 2; Figure 5A,B). Serin Protease Inhibitors (Serpins). The two identied serpins with differential expression in LN+ were oppositely regulated. While serpin B5 (maspin, spot D4) showed decreased expression in LN+ samples, serpin B10 (bomapin, spot U7) showed increased expression in LN+ samples (Tables 2 and 3; Figure 5A,B). Scavenger Proteins. The scavenger protein thioredoxin domain-containing protein 5 (TXNDC5, spot U3) was upregulated in LN+ samples (Table 2; Figure 5A,B). Furthermore, ferritin light polypeptide (FTL, spot U2) also had increased expression in LN+ samples (Table 2; Figure 5A,B). In contrast, the scavenger protein peroxiredoxin 6 (PRDX6, spot D3) had decreased expression in LN+ samples (Table 3; Figure 5A,B). This was conrmed by Western blot analysis, showing lack of PRDX6 expression in LN+ samples (Figure 6). Miscellaneous Proteins. Isocitrate dehyrogenase 1 protein (IDH1, D7) that is involved in the citric acid cycle had decreased expression levels in LN+ (Table 3, Figure 5A,B). Furthermore,
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3-phosphoglycerate dehydrogenase (PGDH, spot U10) and ornithine aminotransferase (OAT, spot U5) are both involved in amino acid metabolism and were up-regulated in LN+ (Table 2). Adenosine deaminase (ADA, spot U4) had increased expression levels in LN+ (Table 2; Figure 5A,B). Rho-GTPaseactivating protein (ARHGAP, spot D10), annexin A5 (ANXA5, spot D9), and brinogen beta chain (FGB, spot D5) had decreased expression levels in LN+ when compared to LN(Table 3; Figure 5A,B). Identication of mRNA Ratios between LN+ and LN-. RTqPCR was used to analyze whether differences in protein expression levels in the two tumor groups are based on transcriptional regulation. Only two of the 11 up-regulated proteins in LN+ samples (PCNA, CORO1A) also had upregulated mRNA expression of more than 1.5-fold (Table 4). Actually, 3 of the 11 up-regulated proteins (ADA, OAT, BOMAPIN) in LN+ samples had corresponding mRNA expression below 0.67-fold, suggesting a posttranscriptional regulation of their protein expression (Table 4). All except one (MASPIN) protein with decreased expression levels in LN+ also had decreased mRNA expression levels in LN+ when compared to LN- samples. RT-qPCR failed to detect FGB mRNA in both tumor groups (Table 3).

Discussion
Several proteins with impact on metastatic spread have been identied in human breast cancer cells.3,6,36 These

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Figure 5. Differentially expressed proteins in nonmetastasizing canine mammary carcinomas (LN+) (A) when compared to metastasizing canine mammary carcinomas (LN-) (B). Up-regulated proteins (Table 2) in LN+ are indicated in green, down-regulated proteins (Table 3) in red. 2D-DIGE of macrodissected tissue lysates of dog no. 1. Isoelectric focusing pH 3-7, 24 cm wide gel.

proteins are involved in various cellular functions such as cell division, cell adhesion, matrix invasion, and angiogenesis. In contrast, little is known on proteins involved in the metastatic spread of canine mammary tumors (CMTs), a comparative model of the human disease, and prediction of metastatic spread is difcult by histology before metastases are large enough to be visualized. However, several mostly immunohistochemistry-based studies found many overlaps in protein expression between human and canine mammary tumors indicating a similar molecular

carcinogenesis in some aspects, while other aspects differed.14,15,18,20,37,38 In general, all of these studies on canine mammary tumor protein expression were based on the hypothesis that mammary tumors show similar protein expression patterns in both species. This biased approach to canine mammary tumors may however hinder an objective view on the nature of this tumor type and the decryption of its molecular carcinogenesis. The present study therefore compared the proteome of metastatic and nonmetastatic canine mammary carcinomas in an explorative approach to
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Klopeisch et al.

Figure 6. Western blot analysis of PRDX6 protein expression levels in metastasizing CMT (LN+) and nonmetastasizing CMT (LN-). All single LN+ (dog nos. 1, 4, 5, 6) and a pool of all LN+ (dog nos. 1-6) have decreased PRDX6 expression levels when compared to single LN- (dog nos. 8, 9, 10, 11) and a pool of all LN- (dog nos. 7-12). Table 4. Ratio of mRNA Expression Levels of the Differentially Expressed Proteins in Metastasizing (LN+) and Nonmetastasizing (LN-) Canine Mammary Carcinomas
spot identied protein mRNA ratio LN+/LN-

U1 U2 U3 U4 U5 U6 U7 U8 U9 U10 U11 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10

a

PCNA FTL TXNDC5 ADA OAT CORO1A BOMAPIN RANBP1 TPM3 PGDH EEF1D CALB2 MYL2 PRDX6 MASPIN FGB VCL IDH1 TPM1 ANXA5 ARHGAP1

1.99 1.27 1.38 0.62 0.62 1.53 0.51 1.40 0.82 0.91 1.28 0.05 0.09 0.63 0.72 n.d.a 0.49 0.59 0.38 0.58 0.54

results in unstable mitotic microtubules and decreased apoptosis when failure of mitotic progression is detected.43 Recent studies also hypothesize that an interaction between RANBP1 and psoriasin (S100A7) also contributes to breast cancer cell invasiveness by a so far unknown mechanism.45 Eukaryotic elongation factor-1 delta (EEF1D) was the third up-regulated protein directly associated with cell proliferation in LN+ tumors. EEF1D promotes cell-cycle progression by delivery of aminoacyl tRNAs to the ribosome and functions as a guanine nucleotide exchange factor.36 A correlation between EEF1D overexpression, oncogenic transformation, and invasive growth of breast cancer and other tumor cells has been reported in several studies.36,46,47 Prevention of E-cadherinmediated cell-cell adhesion by increased EEF1D protein expression levels may represent a possible molecular mechanism for this observation.48 This is also in line with the signicant down-regulation of E-cadherin in invasive canine mammary tumors.49 Up-regulation of EEF1D therefore indicates not only increased proliferation but also decreased cell-cell adhesion in canine mammary tumors, a cellular feature well-known for several metastatic tumors. 2. Proteins Associated with Cell Adhesion Are DownRegulated, While Those with Cell Motility Are Overexpressed in Metastatic Canine Mammary Tumors. As mentioned above, loss of cell-cell adhesions and enhanced cellular motility are features of metastasizing tumors. Transcriptional down-regulation of vinculin (VCL) in LN+ is obviously in line with this general assumption. VCL is a cytoskeletal protein with functions in the focal adhesion complex. It modulates the dynamics of cell-cell and cell-matrix adhesion, and its expression is associated with reduced cell motility.50 Furthermore, it is a potential tumor suppressor, based on the observation that restoration of vinculin in SV-40-transformed murine broblasts and pancreatic carcinoma cells had a suppressive effect of the tumorigenic ability of these cells. In these models, the proteinexpression level and grade of cell adhesion were directly correlated.51 Paxillin, another focal adhesion complex protein, is also down-regulated in canine mammary tumors when compared to a normal mammary gland.52 The focal adhesion complex may therefore play a major role in metastatic progression of canine mammary tumors similar to that for human breast cancer. Tropomyosins are also cytoskeletal actin-binding proteins which are involved in the contractile system of several cell types.5,53 LN+ samples had decreased expression of tropomyosin 1 (TPM1) protein but increased protein expression of tropomyosin 3 (TPM3). This opposite regulation of the two tropomyosins is surprising but has been described in other cancer types before and is related to their diverging func-

n.d.: no mRNA expression detected in the tissue samples.

identify proteins involved in metastatic spread and potential new metastasis markers. 1. Proteins with Proliferation Activity Are Up-Regulated in Metastatic Canine Mammary Tumors. Increased proliferation and uncontrolled cell division are common features of malignant human and canine mammary tumors.20,22,39,40 The signicantly higher expression of proliferating cell nuclear antigen (PCNA) in metastasizing carcinomas (LN+) when compared to nonmetastasizing canine mammary carcinomas (LN-) in the present study conrms this general observation. PCNA is located in the nucleus and acts as a cofactor of DNA polymerase delta and thereby increases DNA replication.41 In addition, PCNA also plays a role in RAD6-dependent DNA repair in response to DNA damage and inhibition of apoptosis via negative regulation of the tyrosine kinase c-abl stability.42 Up-regulation of the Ran/TC4-binding protein (RANBP1) indicates increased mitotic activity and an antiapoptotic state in metastatic canine mammary carcinomas similar to several other human tumors including breast cancer.43 RANBP1 directly interacts with GTP-charged RAN which is involved in mitosis and apoptosis.43 RANBP1 acts as a negative regulator of chromosome condensation by inhibiting RCC1 (regulator of chromosome condensation 1)-stimulated guanine nucleotide release from RAN.43,44 Up-regulation of RANBP1 therefore
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Comparative Proteomics of Canine Mammary Tumors

tions. TPM1 is a tumor suppressor inducing anoikis (detachment-induced apoptosis) in breast cancer cells. Thus, the down-regulation of TPM1 enables neoplastic cells to survive outside their normal microenvironment and thereby supports metastasis.5 In contrast, TPM3 overexpression activates epithelial-mesenchymal transition in hepatocellular carcinomas and contributes to tumor cell dissemination in ERBB2-positive breast cancer.3,56 Tropomyosin expression levels have not been analyzed in canine tissues before but obviously are similarly regulated in human breast cancer. Coronin1A (CORO1A) is another actin-binding protein that was also transcriptionally up-regulated in LN+ samples. Coronins are highly conserved regulators of the actin cytoskeleton and are enriched at the leading edge of migrating cells.57,58 This inuence of coronin expression on cell migration may explain why invasive subgroups of several tumor types like hepatocellular carcinomas and melanomas have increased coronin expression levels when compared to benign tumors and nonneoplastic cells.59-61 CORO1A expression levels have not been analyzed in canine mammary tumors before but according to this study may be a potential marker of invasive potential and an interesting protein to be analyzed in canine mammary tumors in more detail. The increased expression of adenosine deaminase (ADA) in LN+ samples may also contribute to their metastatic spread although the protein is not directly associated with cell-cell adhesion. However, increased cytoplasmic levels of ADA or treatment with exogenous ADA signicantly decrease adhesion and increase invasiveness of human lymphoma cells, and a similar mechanism may be true for canine mammary tumors.62 Interestingly, increased ADA protein expression levels in LN+ samples were associated with decreased mRNA expression levels, indicating a posttranscriptional up-regulation of this enzyme. 3. Maspin versus Bomapin: Regulators of Matrix Invasion Are Differentially Expressed in Metastasizing Carcinomas. Serin protease inhibitors (Serpins) are proteins with a similar structure and the ability to inhibit serine proteases. Serpin activity regulates different intracellular and extracellular processes such as cellular differentiation, tumor suppression, apoptosis, and extracellular matrix modulation and cell migration.8 Surprisingly, the two serpins identied in the present study were regulated in opposite directions in LN+ samples. Maspin had decreased expression levels, while bomapin was up-regulated in LN+ when compared to LN- samples. Maspin (mammary serine protease inhibitor; SERPINB5) is a well-described tumor suppressor in several tissue types including the mammary gland, prostate, and squamous epithelium. Down-regulation of maspin is associated with malignant behavior and metastatic spread of breast, prostate, and pancreatic cancer.6,7,63 Especially in human breast cancer, it is down-regulated in invasive breast carcinomas when compared to normal human mammary epithelial cells and plays an important role in tumor invasion and metastasis.7,8,64 The only immunohistochemical study available on maspin expression in canine mammary tumors identied no differences of maspin protein expression in malignant epithelial cells when compared to benign cells.6 However, analysis of mRNA expression levels in canine mammary tumors at different stages of malignant progression has pointed toward a decreased expression that was, however, not signicant.17 According to the results of this study, maspin may indeed be involved in metastatic spread of canine mammary tumors, but the differ5,54,55

research articles
ences in protein expression levels seem too weak to be used as a routine immunohistochemical marker for metastatic potential of canine mammary tumors. The expression of bomapin (serpin peptidase inhibitor; SERPINB10) has not been analyzed in canine mammary tumors before, and its function in physiology and disease in general is mostly unknown. So far it is clear that bomapin expression has a cytoprotective effect against tumor necrosis factor alpha (TNFR)-induced cell death.65 Furthermore, a genetic variation in the bomapin gene has been identied in human patients with prostate cancer, and its expression is increased in a lymphoma cell line when compared to non-neoplastic leucocytes.66,67 Similar to the situation in human cancer, bomapin was up-regulated in LN+ samples. 4. Scavenger Proteins May Protect Cells with Metastatic Potential from Hypoxia and Oxidative Stress. Invasive, rapid growth of tumors induces oxidative stress due to insufcient vascularization. Oxidative stress in turn is associated with generation of free radicals and lipid peroxidation products that contribute to genetic instability and cell death. To survive and metastasize metastatic tumor cells have to adapt to this hypoxic, radical-inducing environment. In the present study, metastatic cells had a signicantly increased expression of thioredoxin domain-containing protein 5 (TXNDC5) when compared to LN- samples. TXNDC5 is located in the endoplasmic reticulum and known to inhibit apoptosis by chaperone activity on hypoxia-induced antiapoptotic molecules.68 Expression of thioredoxins has not been analyzed in canine mammary tumors before, but in humans a similar increased expression was found in poorly differentiated hepatocellular and colorectal carcinomas.69 Surprisingly, peroxiredoxin 6 (PRDX6), a protein with strong antioxidant properties, was transcriptionally down-regulated in LN+ tumors. Peroxiredoxins are present in various cellular compartments and reduce peroxides to the corresponding alcohol or water.70 Furthermore, the phospholipase A2 activity of peroxiredoxin 6 promotes invasion and metastasis of lung cancer cells.70-72 So far, expression levels of peroxiredoxins like PRDX6 have not been described in canine tumors but are upregulated in metastatic human breast and lung cancers.72 Down-regulation of PRDX6 is associated with resistance to chemotherapy of breast cancer cells.73 Nevertheless, downregulation of PRDX6 in LN+ tumors was consistent with immunoblotting and may indicate that its function seems to differ in the canine tumors when compared to their human counterpart, and the relevance of their expression is unclear at the moment. Free metal ions are known to contribute to the formation of reactive oxygen species like H2O2 and are supposed to be involved in breast cancer carcinogenesis.74 Ferritin (FTL) sequesters Fe(II) from Fenton-like reactions in which the spontaneous oxidation to Fe(III) donates single electrons to transform innocuous reactive oxygen species into highly toxic ones.75 In the present study, LN+ tumors contained increased protein levels of ferritin, and this may contribute to the resistance of the metastatic cells to hypoxic stresses. Interestingly, low cytosolic ferritin protein levels have been associated with a good prognosis in lymph node-negative human breast cancer when compared to lymph node-positive cancer, and a similar association between prognosis and ferritin expression seems to exist in canine mammary tumors.76 Isocitrate dehyrogenase-1 (IDH1) protein, an inducer of the HIF-1 pathway, was signicantly transcriptionally downJournal of Proteome Research Vol. 9, No. 12, 2010 6387

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Klopeisch et al.

Table 5. Comparison of Protein Expression Pattern in Metastatic Canine Mammary Tumors with Human Breast Cancer and Other Human Cancer Typesa
protein gene expression in human breast cancer gene expression in other human cancer types

ADA CORO1A EEF1D FTL PGDH

OAT PCNA RANBP1 SERPINB10 TPM3 TXNDC5

Proteins with Increased Expression in LN+ CMT decreased in lymphomas associated with decreased adhesion increased in invasive melanomas and hepatocellular carcinomas59-61 47 increased in mammary cancer cells increased in esophageal carcinomas36 increased expression in breast cancer with prognostic increased in esophageal cancer80 74,76 potential increased enzymatic activity indicates poor prognosis decreased in colorectal cancer in breast cancer,81 decreased in breast cancer, potential tumor suppressor82 increased serum levels in patients with hepatocellular carcinomas83 2 increased in rapidly proliferating breast cancer cells increased in multiple malignant cancer types increased in breast cancer cells45 increased in many cancer types43 increased in acute myeloid leukemia,65,67 genetic variations in prostate cancer66 3 increased in metastatic breast cancer cells increased in hepatocellular carcinoma56 increased in colorectal cancer,69 tumor promoter in gastric carcinoma84 Proteins with Decreased Expression in LN+ CMT decreased in nasopharyngeal cancer cells85 expression induces motility and metastatic spread of esophageal squamous cell carcinoma,86 down-regulation in metastatic lung cancer87 increased in a breast cancer cell line79 decreased in glioma cell line77 decreased in invasive breast cancer7,8,63,64 decreased in metastatic oral squamous carcinoma88 decreased in oral squamous cell carcinoma53 decreased in breast cancer2,5 decreased in colorectal cancer89 increased in metastatic breast cancer cells3 expression promotes metastasis of lung cancer90 decreased in breast cancer metastases4 decreased in metastatic colorectal cancer91

ANXA5 ARHGAP1

IDH1 MASPIN MYL2 TPM1 PRDX6 VCL

a

LN+ CMT: metastasizing canine mammary tumors.

regulated in LN+. Decreased IDH1 levels in turn increase cellular levels of hypoxia-inducible factor 1 alpha (HIF-1alpha), thereby supporting tumor growth in hypoxic microenvironments.77 Recent studies characterized IDH1 as a tumor suppressor gene that contains a prognostically relevant mutation in the majority of human glioblastomas.78 In this tumor type, mutation of IDH1 leads to functional changes but unaltered expression levels. In contrast, decreased IDH1 protein levels have been identied in malignant bladder cancer, while an in vitro selected highly metastatic breast cancer cell line had an increased IDH1 expression level.79 5. Protein Expression Patterns in Metastatic Canine Mammary Tumors Resemble Human Breast Cancer Protein Expression in Several Aspects. A literature search identied several overlaps in protein expression patterns in metastatic canine mammary tumors with human breast cancer and other human cancers (Table 5). Nineteen of the 21 proteins differentially expressed in metastatic canine mammary tumors also have a malignancy- or metastasis-associated expression pattern in different human cancers. Moreover, nine of the proteins have a similar malignancy-associated protein expression in canine mammary tumors when compared with human breast cancer (Table 5). These included relevant tumor markers like MASPIN, PCNA, PGDH, and TPM1/3. Our ndings therefore suggest that several aspects of mammary carcinogenesis are similar in both species and support the model character of canine mammary tumors for the human disease. Two of the proteins (IDH1, PRDX6) had an opposite regulation in malignant canine and
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breast cancer cells (Table 5). The exact causes of these expression differences are unknown, but the in vitro setting in the IDH1 studies may represent one possible cause.77,79 Despite all similarities, 12 of the proteins differentially regulated in metastatic canine mammary tumors have no known relevance in human breast cancer as of yet, and this may reect the differences between the two species. However, these 12 proteins may also be interesting candidates to be analyzed in metastatic human breast cancer.

Conclusion
In conclusion, comparison of the proteomic prole of metastasizing versus nonmetastasizing canine mammary tumors identied 21 proteins that had signicantly different expression levels in the two tumor types. Most of the proteins are involved in cellular functions relevant for metastatic spread like cell adhesion, extracellular matrix modulation, and hypoxia resistance. Interestingly, most of the proteins are similarly dysregulated in human cancer. The ndings therefore support the value of canine mammary tumors as a potential comparative model for human cancer. Nevertheless, further studies are needed to evaluate whether these proteins and their expression levels are drivers and initial events or mere passengers and secondary phenotypes of the metastatic process in canine mammary tumors and whether their expression levels are of prognostic relevance.

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research articles
(18) Klopeisch, R.; Schutze, M.; Linzmann, H.; Brunnberg, L.; Gruber, A. D. Increased Derlin-1 Expression in Metastases of Canine Mammary Adenocarcinomas. J. Comp. Pathol. 2009. (19) Wang, J.; Hua, H.; Ran, Y.; Zhang, H.; Liu, W.; Yang, Z.; Jiang, Y. Derlin-1 is overexpressed in human breast carcinoma and protects cancer cells from endoplasmic reticulum stress-induced apoptosis. Breast Cancer Res. 2008, 10 (1), R7. (20) Klopeisch, R.; Schutze, M.; Gruber, A. D. Loss of p27 expression in canine mammary tumors and their metastases. Res. Vet. Sci. 2010, 88 (2), 300303. (21) Viglietto, G.; Motti, M. L.; Fusco, A. Understanding p27(kip1) deregulation in cancer: down-regulation or mislocalization. Cell Cycle 2002, 1 (6), 394400. (22) Klopeisch, R.; Gruber, A. D. Differential expression of cell cycle regulators p21, p27 and p53 in metastasizing canine mammary adenocarcinomas versus normal mammary glands. Res. Vet. Sci. 2009, 87 (1), 916. (23) Klopeisch, R.; Gruber, A. D. Increased Expression of BRCA2 and RAD51 in Lymph Node Metastases of Canine Mammary Adenocarcinomas. Vet. Pathol. 2009, 46 (3), 41622. (24) Misdorp, W. Tumors of the mammary gland. In Tumours in Domestic Animals, 4th ed.; Meuten, D. J., Ed.; Iowa State Press: IA, 2002; pp 575-606. (25) de Las Mulas, J. M.; Millan, Y.; Dios, R. A prospective analysis of immunohistochemically determined estrogen receptor alpha and progesterone receptor expression and host and tumor factors as predictors of disease-free period in mammary tumors of the dog. Vet. Pathol. 2005, 42 (2), 20012. (26) Kitchell, B. E. a. L., A. S. Diseases of the mammary glands. In Handbook of Small Animal Practice, 3rd ed.; Morgan, R. V., Ed., W.B. Saunders: Philadelphia, 1997; pp 615-625. (27) Misdorp, W.; Else, R. W.; Hellme n, E.; Lipscomb, T. P. Histological classication of mammary tumors of the dog and the cat; Armed Forces Institute of Pathology: WA, 1999; Vol. 7, pp 9-22. (28) Misdorp, W.; Hart, A. A. Canine mammary cancer. I. Prognosis. J. Small Anim. Pract. 1979, 20 (7), 38594. (29) Benjamin, S. A.; Lee, A. C.; Saunders, W. J. Classication and behavior of canine mammary epithelial neoplasms based on lifespan observations in beagles. Vet. Pathol. 1999, 36 (5), 42336. (30) Misdorp, W.; Hart, A. A. Prognostic factors in canine mammary cancer. J. Natl. Cancer Inst. 1976, 56 (4), 77986. (31) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 1996, 68 (5), 8508. (32) Suckau, D.; Resemann, A.; Schuerenberg, M.; Hufnagel, P.; Franzen, J.; Holle, A. A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Anal. Bioanal. Chem. 2003, 376 (7), 95265. (33) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25 (4), 4028. (34) Vandesompele, J.; De Preter, K.; Pattyn, F.; Poppe, B.; Van Roy, N.; De Paepe, A.; Speleman, F. Accurate normalization of realtime quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002, 3 (7), RESEARCH0034. (35) Etschmann, B.; Wilcken, B.; Stoevesand, K.; von der Schulenburg, A.; Sterner-Kock, A. Selection of reference genes for quantitative real-time PCR analysis in canine mammary tumors using the GeNorm algorithm. Vet. Pathol. 2006, 43 (6), 93442. (36) Ogawa, K.; Utsunomiya, T.; Mimori, K.; Tanaka, Y.; Tanaka, F.; Inoue, H.; Murayama, S.; Mori, M. Clinical signicance of elongation factor-1 delta mRNA expression in oesophageal carcinoma. Br. J. Cancer 2004, 91 (2), 2826. (37) Klopeisch, R.; Klose, P.; da Costa, A.; Brunnberg, L.; Gruber, A. D. HEPACAM1 and 2 are differentially regulated in canine mammary adenomas and carcinomas and its lymph node metastases. BMC Vet. Res. 2010, 6, 15. (38) Munson, L.; Moresco, A. Comparative pathology of mammary gland cancers in domestic and wild animals. Breast Dis. 2007, 28, 721. (39) Pena, L. L.; Nieto, A. I.; Perez-Alenza, D.; Cuesta, P.; Castano, M. Immunohistochemical detection of Ki-67 and PCNA in canine mammary tumors: relationship to clinical and pathologic variables. J. Vet. Diagn. Invest. 1998, 10 (3), 23746. (40) Leonardi, E.; Girlando, S.; Serio, G.; Mauri, F. A.; Perrone, G.; Scampini, S.; Dalla Palma, P.; Barbareschi, M. PCNA and Ki67 expression in breast carcinoma: correlations with clinical and biological variables. J. Clin. Pathol. 1992, 45 (5), 4169. (41) Moldovan, G. L.; Pfander, B.; Jentsch, S. PCNA, the maestro of the replication fork. Cell 2007, 129 (4), 66579. (42) He, X.; Wei, C.; Song, T.; Yuan, J.; Zhang, Y.; Ma, Q.; Shi, W.; Zhong, H. Proliferating cell nuclear antigen destabilizes c-Abl tyrosine

Acknowledgment. We thank Monika Scha rig and Petra Schulze for excellent technical assistance. The project was supported by the Deutsche Forschungsgemeinschaft (DFG, KL2240). The present study is part of a PhD-thesis and was supported by the Dahlem Research School (DRS). Supporting Information Available: Supplemental Tables 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. References
(1) Schulz, D. M.; Bollner, C.; Thomas, G.; Atkinson, M.; Esposito, I.; Hoer, H.; Aubele, M. Identication of differentially expressed proteins in triple-negative breast carcinomas using DIGE and mass spectrometry. J. Proteome Res. 2009, 8 (7), 34308. (2) Hondermarck, H.; Vercoutter-Edouart, A. S.; Revillion, F.; Lemoine, J.; el-Yazidi-Belkoura, I.; Nurcombe, V.; Peyrat, J. P. Proteomics of breast cancer for marker discovery and signal pathway proling. Proteomics 2001, 1 (10), 121632. (3) Li, D. Q.; Wang, L.; Fei, F.; Hou, Y. F.; Luo, J. M.; Zeng, R.; Wu, J.; Lu, J. S.; Di, G. H.; Ou, Z. L.; Xia, Q. C.; Shen, Z. Z.; Shao, Z. M. Identication of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry. Proteomics 2006, 6 (11), 335268. (4) Bartkowiak, K.; Wieczorek, M.; Buck, F.; Harder, S.; Moldenhauer, J.; Effenberger, K. E.; Pantel, K.; Peter-Katalinic, J.; Brandt, B. H. Two-Dimensional Differential Gel Electrophoresis of a Cell Line Derived from a Breast Cancer Micrometastasis Revealed a Stem/ Progenitor Cell Protein Prole. J. Proteome Res. 2009, 8, 20042014. (5) Raval, G. N.; Bharadwaj, S.; Levine, E. A.; Willingham, M. C.; Geary, R. L.; Kute, T.; Prasad, G. L. Loss of expression of tropomyosin-1, a novel class II tumor suppressor that induces anoikis, in primary breast tumors. Oncogene 2003, 22 (40), 6194203. (6) Espinosa de los Monteros, A.; Millan, M. Y.; Ramirez, G. A.; Ordas, J.; Reymundo, C.; Martin de las Mulas, J. Expression of maspin in mammary gland tumors of the dog. Vet. Pathol. 2005, 42 (3), 250 7. (7) Sager, R.; Sheng, S.; Pemberton, P.; Hendrix, M. J. Maspin. A tumor suppressing serpin. Adv. Exp. Med. Biol. 1997, 425, 7788. (8) Stark, A. M.; Schem, C.; Maass, N.; Hugo, H. H.; Jonat, W.; Mehdorn, H. M.; Held-Feindt, J. Expression of metastasis suppressor gene maspin is reduced in breast cancer brain metastases and correlates with the estrogen receptor status. Neurol. Res. 2010, 32, 303-308. (9) Toillon, R. A.; Lagadec, C.; Page, A.; Chopin, V.; Sautiere, P. E.; Ricort, J. M.; Lemoine, J.; Zhang, M.; Hondermarck, H.; Le Bourhis, X. Proteomics demonstration that normal breast epithelial cells can induce apoptosis of breast cancer cells through insulin-like growth factor-binding protein-3 and maspin. Mol. Cell. Proteomics 2007, 6 (7), 123947. (10) Khanna, C.; Hunter, K. Modeling metastasis in vivo. Carcinogenesis 2005, 26 (3), 51323. (11) Vail, D. M.; MacEwen, E. G. Spontaneously occurring tumors of companion animals as models for human cancer. Cancer Invest. 2000, 18 (8), 78192. (12) Khanna, C.; Lindblad-Toh, K.; Vail, D.; London, C.; Bergman, P.; Barber, L.; Breen, M.; Kitchell, B.; McNeil, E.; Modiano, J. F.; Niemi, S.; Comstock, K. E.; Ostrander, E.; Westmoreland, S.; Withrow, S. The dog as a cancer model. Nat. Biotechnol. 2006, 24 (9), 10656. (13) Benson, J. R. Role of transforming growth factor beta in breast carcinogenesis. Lancet Oncol. 2004, 5 (4), 22939. (14) Klopeisch, R.; Schutze, M.; Gruber, A. D. Downregulation of transforming growth factor beta (TGFbeta) and latent TGFbeta binding protein (LTBP)-4 expression in late stage canine mammary tumours. Vet. J. 2009. (15) Klopeisch, R.; Schutze, M.; Gruber, A. D. RAD51 protein expression is increased in canine mammary carcinomas. Vet. Pathol. 47, (1), 98-101. (16) Maacke, H.; Opitz, S.; Jost, K.; Hamdorf, W.; Henning, W.; Kruger, S.; Feller, A. C.; Lopens, A.; Diedrich, K.; Schwinger, E.; Sturzbecher, H. W. Over-expression of wild-type Rad51 correlates with histological grading of invasive ductal breast cancer. Int. J. Cancer 2000, 88 (6), 90713. (17) Klopeisch, R.; Gruber, A. D. Derlin-1 and stanniocalcin-1 are differentially regulated in metastasizing canine mammary adenocarcinomas. J. Comp. Pathol. 2009, 141 (2-3), 11320.

Journal of Proteome Research Vol. 9, No. 12, 2010 6389

research articles
(43) kinase and regulates cell apoptosis in response to DNA damage. Apoptosis 2009, 14 (3), 26875. Rensen, W. M.; Roscioli, E.; Tedeschi, A.; Mangiacasale, R.; Ciciarello, M.; Di Gioia, S. A.; Lavia, P. RanBP1 downregulation sensitizes cancer cells to taxol in a caspase-3-dependent manner. Oncogene 2009, 28 (15), 174858. Milano, J., Jr.; Strayer, D. S. Effects of overexpression of Ran/TC4 mammalian cells in vitro. Exp. Cell Res. 1998, 239 (1), 319. Emberley, E. D.; Gietz, R. D.; Campbell, J. D.; HayGlass, K. T.; Murphy, L. C.; Watson, P. H. RanBPM interacts with psoriasin in vitro and their expression correlates with specic clinical features in vivo in breast cancer. BMC Cancer 2002, 2, 28. Joseph, P.; Lei, Y. X.; Whong, W. Z.; Ong, T. M. Oncogenic potential of mouse translation elongation factor-1 delta, a novel cadmiumresponsive proto-oncogene. J. Biol. Chem. 2002, 277 (8), 61316. Kolettas, E.; Lymboura, M.; Khazaie, K.; Luqmani, Y. Modulation of elongation factor-1 delta (EF-1 delta) expression by oncogenes in human epithelial cells. Anticancer Res. 1998, 18 (1A), 38592. Yang, S.; Du, J.; Wang, Z.; Yuan, W.; Qiao, Y.; Zhang, M.; Zhang, J.; Gao, S.; Yin, J.; Sun, B.; Zhu, T. BMP-6 promotes E-cadherin expression through repressing deltaEF1 in breast cancer cells. BMC Cancer 2007, 7, 211. Pinho, S. S.; Matos, A. J.; Lopes, C.; Marcos, N. T.; Carvalheira, J.; Reis, C. A.; Gartner, F. Sialyl Lewis x expression in canine malignant mammary tumours: correlation with clinicopathological features and E-Cadherin expression. BMC Cancer 2007, 7, 124. Mierke, C. T.; Kollmannsberger, P.; Zitterbart, D. P.; Diez, G.; Koch, T. M.; Marg, S.; Ziegler, W. H.; Goldmann, W. H.; Fabry, B. Vinculin facilitates cell invasion into 3D collagen matrices. J. Biol. Chem. Rodriguez Fernandez, J. L.; Geiger, B.; Salomon, D.; Sabanay, I.; Zoller, M.; Ben-Zeev, A. Suppression of tumorigenicity in transformed cells after transfection with vinculin cDNA. J. Cell Biol. 1992, 119 (2), 427-38. Scibelli, A.; dAngelo, D.; Pelagalli, A.; Tafuri, S.; Avallone, L.; Della Morte, R.; Staiano, N. Expression levels of the focal adhesionassociated proteins paxillin and p130CAS in canine and feline mammary tumors. Vet. Res. 2003, 34 (2), 193202. He, Q. Y.; Chen, J.; Kung, H. F.; Yuen, A. P.; Chiu, J. F. Identication of tumor-associated proteins in oral tongue squamous cell carcinoma by proteomics. Proteomics 2004, 4 (1), 2718. Bohling, S. D.; Jenson, S. D.; Crockett, D. K.; Schumacher, J. A.; Elenitoba-Johnson, K. S.; Lim, M. S. Analysis of gene expression prole of TPM3-ALK positive anaplastic large cell lymphoma reveals overlapping and unique patterns with that of NPM-ALK positive anaplastic large cell lymphoma. Leuk. Res. 2008, 32 (3), 38393. Choi, H. S.; Yim, S. H.; Xu, H. D.; Jung, S. H.; Shin, S. H.; Hu, H. J.; Jung, C. K.; Choi, J. Y.; Chung, Y. J. Tropomyosin3 overexpression and a potential link to epithelial-mesenchymal transition in human hepatocellular carcinoma. BMC Cancer 2010, 10, 122. Choi, H. S.; Yim, S. H.; Xu, H. D.; Jung, S. H.; Shin, S. H.; Hu, H. J.; Jung, C. K.; Choi, J. Y.; Chung, Y. J. Tropomyosin3 overexpression and a potential link to epithelial-mesenchymal transition in human hepatocellular carcinoma. BMC Cancer 2010, 10, 122. Uetrecht, A. C.; Bear, J. E. Coronins: the return of the crown. Trends Cell Biol. 2006, 16 (8), 4216. Gerisch, G.; Albrecht, R.; Heizer, C.; Hodgkinson, S.; Maniak, M. Chemoattractant-controlled accumulation of coronin at the leading edge of Dictyostelium cells monitored using a green uorescent protein-coronin fusion protein. Curr. Biol. 1995, 5 (11), 12805. Wu, L.; Peng, C. W.; Hou, J. X.; Zhang, Y. H.; Chen, C.; Chen, L. D.; Li, Y. Coronin-1C is a novel biomarker for hepatocellular carcinoma invasive progression identied by proteomics analysis and clinical validation. J. Exp. Clin. Cancer Res. 2010, 29, 17. Thal, D.; Xavier, C. P.; Rosentreter, A.; Linder, S.; Friedrichs, B.; Waha, A.; Pietsch, T.; Stumpf, M.; Noegel, A.; Clemen, C. Expression of coronin-3 (coronin-1C) in diffuse gliomas is related to malignancy. J. Pathol. 2008, 214 (4), 41524. Roadcap, D. W.; Clemen, C. S.; Bear, J. E. The role of mammalian coronins in development and disease. Subcell. Biochem. 2008, 48, 12435. Gines, S.; Marino, M.; Mallol, J.; Canela, E. I.; Morimoto, C.; Callebaut, C.; Hovanessian, A.; Casado, V.; Lluis, C.; Franco, R. Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase-CD26 interaction. Biochem. J. 2002, 361 (Pt 2), 203 9. Chen, E. I.; Florens, L.; Axelrod, F. T.; Monosov, E.; Barbas, C. F., III; Yates, J. R., III; Felding-Habermann, B.; Smith, J. W. Maspin alters the carcinoma proteome. FASEB J. 2005, 19 (9), 11234. Zou, Z.; Anisowicz, A.; Hendrix, M. J.; Thor, A.; Neveu, M.; Sheng, S.; Radi, K.; Seftor, E.; Sager, R. Maspin, a serpin with tumor(65)

Klopeisch et al.
suppressing activity in human mammary epithelial cells. Science 1994, 263 (5146), 5269. Schleef, R. R.; Chuang, T. L. Protease inhibitor 10 inhibits tumor necrosis factor alpha -induced cell death. Evidence for the formation of intracellular high M(r) protease inhibitor 10-containing complexes. J. Biol. Chem. 2000, 275 (34), 263859. Shioji, G.; Ezura, Y.; Nakajima, T.; Ohgaki, K.; Fujiwara, H.; Kubota, Y.; Ichikawa, T.; Inoue, K.; Shuin, T.; Habuchi, T.; Ogawa, O.; Nishimura, T.; Emi, M. Nucleotide variations in genes encoding plasminogen activator inhibitor-2 and serine proteinase inhibitor B10 associated with prostate cancer. J. Hum. Genet. 2005, 50 (10), 50715. Ulger, C.; Toruner, G. A.; Alkan, M.; Mohammed, M.; Damani, S.; Kang, J.; Galante, A.; Aviv, H.; Soteropoulos, P.; Tolias, P. P.; Schwalb, M. N.; Dermody, J. J. Comprehensive genome-wide comparison of DNA and RNA level scan using microarray technology for identication of candidate cancer-related genes in the HL60 cell line. Cancer Genet. Cytogenet. 2003, 147 (1), 2835. Sullivan, D. C.; Huminiecki, L.; Moore, J. W.; Boyle, J. J.; Poulsom, R.; Creamer, D.; Barker, J.; Bicknell, R. EndoPDI, a novel proteindisulde isomerase-like protein that is preferentially expressed in endothelial cells acts as a stress survival factor. J. Biol. Chem. 2003, 278 (47), 4707988. Wang, Y.; Ma, Y.; Lu, B.; Xu, E.; Huang, Q.; Lai, M. Differential expression of mimecan and thioredoxin domain-containing protein 5 in colorectal adenoma and cancer: a proteomic study. Exp. Biol. Med. (Maywood, NJ, U.S.) 2007, 232 (9), 11529. Karihtala, P.; Mantyniemi, A.; Kang, S. W.; Kinnula, V. L.; Soini, Y. Peroxiredoxins in breast carcinoma. Clin. Cancer Res. 2003, 9 (9), 341824. Chang, X. Z.; Li, D. Q.; Hou, Y. F.; Wu, J.; Lu, J. S.; Di, G. H.; Jin, W.; Ou, Z. L.; Shen, Z. Z.; Shao, Z. M. Identication of the functional role of peroxiredoxin 6 in the progression of breast cancer. Breast Cancer Res. 2007, 9 (6), R76. Ho, J. N.; Lee, S. B.; Lee, S. S.; Yoon, S. H.; Kang, G. Y.; Hwang, S. G.; Um, H. D., Phospholipase A2 activity of peroxiredoxin 6 promotes invasion and metastasis of lung cancer cells. Mol. Cancer Ther. 2010, 9 (4), 825-32. Cortesi, L.; Barchetti, A.; De Matteis, E.; Rossi, E.; Della Casa, L.; Marcheselli, L.; Tazzioli, G.; Lazzaretti, M. G.; Ficarra, G.; Federico, M.; Iannone, A. Identication of protein clusters predictive of response to chemotherapy in breast cancer patients. J. Proteome Res. 2009, 8 (11), 491633. Kabat, G. C.; Rohan, T. E. Does excess iron play a role in breast carcinogenesis? An unresolved hypothesis. Cancer Causes Control 2007, 18 (10), 104753. Arosio, P.; Ingrassia, R.; Cavadini, P. Ferritins: a family of molecules for iron storage, antioxidation and more. Biochim. Biophys. Acta 2009, 1790 (7), 58999. Ricolleau, G.; Charbonnel, C.; Lode, L.; Loussouarn, D.; Joalland, M. P.; Bogumil, R.; Jourdain, S.; Minvielle, S.; Campone, M.; Deporte-Fety, R.; Campion, L.; Jezequel, P. Surface-enhanced laser desorption/ionization time of ight mass spectrometry protein proling identies ubiquitin and ferritin light chain as prognostic biomarkers in node-negative breast cancer tumors. Proteomics 2006, 6 (6), 196375. Zhao, S.; Lin, Y.; Xu, W.; Jiang, W.; Zha, Z.; Wang, P.; Yu, W.; Li, Z.; Gong, L.; Peng, Y.; Ding, J.; Lei, Q.; Guan, K. L.; Xiong, Y. Gliomaderived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha. Science 2009, 324 (5924), 2615. Yan, H.; Parsons, D. W.; Jin, G.; McLendon, R.; Rasheed, B. A.; Yuan, W.; Kos, I.; Batinic-Haberle, I.; Jones, S.; Riggins, G. J.; Friedman, H.; Friedman, A.; Reardon, D.; Herndon, J.; Kinzler, K. W.; Velculescu, V. E.; Vogelstein, B.; Bigner, D. D. IDH1 and IDH2 mutations in gliomas. N. Engl. J. Med. 2009, 360 (8), 76573. Xu, S. G.; Yan, P. J.; Shao, Z. M. Differential proteomic analysis of a highly metastatic variant of human breast cancer cells using twodimensional differential gel electrophoresis. J. Cancer Res. Clin. Oncol. 2010. Boult, J.; Roberts, K.; Brookes, M. J.; Hughes, S.; Bury, J. P.; Cross, S. S.; Anderson, G. J.; Spychal, R.; Iqbal, T.; Tselepis, C. Overexpression of cellular iron import proteins is associated with malignant progression of esophageal adenocarcinoma. Clin. Cancer Res. 2008, 14 (2), 37987. Brocklehurst, D.; Champion, A. E.; Cheek, T. R.; Dewhurst, D. G. The value of 6-phosphogluconate dehydrogenase (6-PGDH) activity as a marker of tumour cellularity and prognostic indicator in primary breast cancer. Tumor Biol. 1986, 7 (2-3), 99104. Wolf, I.; OKelly, J.; Rubinek, T.; Tong, M.; Nguyen, A.; Lin, B. T.; Tai, H. H.; Karlan, B. Y.; Koefer, H. P. 15-hydroxyprostaglandin

(44) (45)

(66)

(46) (47) (48)

(67)

(68)

(49)

(69)

(50) (51)

(70) (71)

(52)

(72)

(53) (54)

(73)

(74) (75) (76)

(55)

(56)

(57) (58)

(77)

(59)

(78)

(60)

(79)

(61) (62)

(80)

(81)

(63) (64)

(82)

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Journal of Proteome Research Vol. 9, No. 12, 2010

Comparative Proteomics of Canine Mammary Tumors

dehydrogenase is a tumor suppressor of human breast cancer. Cancer Res. 2006, 66 (15), 781823. Murayama, H.; Fukuda, Y.; Tsunekawa, S.; Ikemoto, M.; Nagata, A. Ratio of serum ornithine carbamoyltransferase to alanine aminotransferase as a potent indicator for hepatocellular carcinoma. Clin. Biochem. 2007, 40 (13-14), 107780. Zhang, L.; Hou, Y.; Li, N.; Wu, K.; Zhai, J., The inuence of TXNDC5 gene on gastric cancer cell. J. Cancer Res. Clin. Oncol. 2010. Chan, C. M.; Wong, S. C.; Lam, M. Y.; Hui, E. P.; Chan, J. K.; Lo, E. S.; Cheuk, W.; Wong, M. C.; Tsao, S. W.; Chan, A. T. Proteomic comparison of nasopharyngeal cancer cell lines C666-1 and NP69 identies down-regulation of annexin II and beta2-tubulin for nasopharyngeal carcinoma. Arch. Pathol. Lab. Med. 2008, 132 (4), 67583. Ito, T.; Shimada, Y.; Kan, T.; David, S.; Cheng, Y.; Mori, Y.; Agarwal, R.; Paun, B.; Jin, Z.; Olaru, A.; Hamilton, J. P.; Yang, J.; Abraham, J. M.; Meltzer, S. J.; Sato, F. Pituitary tumor-transforming 1 increases cell motility and promotes lymph node metastasis in esophageal squamous cell carcinoma. Cancer Res. 2008, 68 (9), 321424.

research articles
(87) Zohrabian, V. M.; Nandu, H.; Gulati, N.; Khitrov, G.; Zhao, C.; Mohan, A.; Demattia, J.; Braun, A.; Das, K.; Murali, R.; JhanwarUniyal, M. Gene expression proling of metastatic brain cancer. Oncol. Rep. 2007, 18 (2), 3218. (88) Marioni, G.; Staferi, C.; Staferi, A.; De Filippis, C.; Blandamura, S. MASPIN tumour-suppressing activity in head and neck squamous cell carcinoma: emerging evidence and therapeutic perspectives. Acta Otolaryngol. 2009, 129 (5), 47680. (89) Mlakar, V.; Berginc, G.; Volavsek, M.; Stor, Z.; Rems, M.; Glavac, D. Presence of activating KRAS mutations correlates signicantly with expression of tumour suppressor genes DCN and TPM1 in colorectal cancer. BMC Cancer 2009, 9, 282. (90) Ho, J. N.; Lee, S. B.; Lee, S. S.; Yoon, S. H.; Kang, G. Y.; Hwang, S. G.; Um, H. D. Phospholipase A2 activity of peroxiredoxin 6 promotes invasion and metastasis of lung cancer cells. Mol. Cancer Ther. 2010, 9 (4), 82532. (91) Yang, H. J.; Chen, J. Z.; Zhang, W. L.; Ding, Y. Q. Focal adhesion plaque associated cytoskeletons are involved in the invasion and metastasis of human colorectal carcinoma. Cancer Invest. 2010, 28 (2), 12734.

(83)

(84) (85)

(86)

PR100671C

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