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

Robert Klopfleisch,*,‡ 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-Straβe 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 identified 21 proteins with significant 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 identified 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: klopfleisch.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 profiles 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 benefit 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 difficult 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, 6380–6391
Published on Web 10/11/2010

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. Freiburg. HE-stained sections were digitized with the T3 Scan Scope Aperio (Aperio. The cause of death could be related to distant metastases to the lung in four dogs. West Highland White Terrier./. 12. positive. All tissues were analyzed using the Avidin-Biotinylin-complex method. Twelve highly malignant. Goat antirabbit IgG (pab././. Diaminobenzidine tetrahydrochloride (Sigma Aldrich. Eight micrometer sections were cut from the frozen tumor. Munich. LN-) were also diagnosed as highly malignant. estrogen receptor. quantitative RT-PCR was used to analyze whether these proteins were transcriptionally differentially regulated in metastatic and nonmetastatic CMTs. dilution 1:700). Freiburg. pH 7. Primary specific antibodies for estrogen receptor (cat.Comparative Proteomics of Canine Mammary Tumors research articles histology tumor size (cm) lymph node status ERBB2 status ER/PR status Table 1./. All dogs without lymph node metastases had a postoperative survival time of more than two years without evidence of lymph node or distant metastases. The data reveal significant overlaps yet clear differences between CMTs and human breast cancer. Germany): Cy3 for the metastasizing samples (LN+). In contrast. The lysate was ultrasound treated twice for two minutes each and then centrifuged at 4500 rpm for one minute.6) and incubated at 4 °C overnight after blocking with 50% goat serum in TBS (30 min at room temperature). 2010 6381 . for metastatic potential. No. dilution 1:700) were diluted in Trisbuffered saline (TBS. no. USA). Vector. Macrodissection and Sample Preparation. progesterone receptor (PR)./. Germany. Tissues were evaluated in ten random 400× magnification fields in three paraffin sections and graded negative if less than 10% of the cells of the relevant tissue were immunohistochemically positive. consecutive. Germany). Germany). Jena. and ERBB2 (c-erbB2./. The supernatant was collected and stored at -80 °C until analysis. As a negative control. 50 mM. -. WHWT./. +. invasive. 1 in 200.6 months on average (range 2-6 months). DCS. progesterone receptor. Material and Methods Clinical Tumor Samples. DAKO GmbH. Normal canine mammary gland tissue served as a positive control. and the percentage of tumor cells was analyzed by Axiovision 2. and 4% CHAPS (3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate)./. Germany. Germany) was used as chromogen. and Cy2 for the internal standard. An amount of 50 µg of protein was labeled with 400 pmol of the Journal of Proteome Research • Vol.27. Du ¨ren. Germany). Furthermore.0 software (Zeiss. All tumor samples were snap frozen in liquid nitrogen immediately after surgical removal. Heidelberg. no. Hamburg. Six tumors (cases 1-6. Germany). RNA integrity and quantity were analyzed using the 2100 Bioanalyzer (cutoff RIN > 8. 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 + + + + + + - + + + + + + + + + + + + . Characterization of Dogs with Metastasizing and Nonmetastasizing Canine Mammary Carcinomasa dog no. ER. Agilent. The internal standard contained equal amounts of all protein lysates./. EI629C01. negative. In addition. progesterone receptor (PI633C01. Germany. macrodissected tissue sections of all tumor probes were used to isolate mRNA for reverse transcription. erythroblastic leukemia viral oncogene homologue 2. Waldbronn./. while the other two were not submitted for necropsy or thoracic radiographs. invasive canine mammary carcinomas were included in the study (Table 1)./. Protein concentrations (range 3.4 µg/µL) were measured with the 2-D Quant Kit (GE Healthcare.1-13. Malignancy-associated and prognostic protein expression patterns are not available and therefore not applied in routine veterinary diagnostics.30 The aim of this first explorative study on the proteome of CMTs was to identify metastasis-associated protein expression patterns in CMTs. DCS. Using two-dimensional differential gel electrophoresis (2DDIGE) and matrix-assisted laser desorption/ionization-timeof-flight-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. and slides were counterstained with hematoxylin. Only tissue samples with a tumor content of more than 70% were extracted in 1 mL of lysis buffer containing 7 M urea. 9. ERBB2. None of the patients received any chemotherapeutic treatment prior to surgery. quantitative PCR (RT-qPCR) analysis using Nucleospin RNA II (MN. Hamburg. and ERBB2 expression were evaluated immunohistochemically to make results in the canine tumors comparable to studies on human breast cancer. A0485. Estrogen receptor (ER). Vista. Protein extracts were labeled with CyDyes (GE Healthcare. PR. postoperative survival times of dogs with LN+ was 3. 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. BA1000) was used as a secondary antibody. simple carcinomas with a diameter larger than the average of the LN+ but had no lymph node metastases as confirmed by five serial sections (every 100 µm) of the regional lymph node. sections were incubated with an irrelevant immune serum instead of the first antibody with all other procedures alike. cat. dilution 1:400). Cy5 for the nonmetastasizing samples (LN-). 2 M thiourea. Lymph node status was also associated with marked differences in postoperative survival. The key clinicopathological features of all cases are listed in Table 1. Histology and Immunohistochemistry. The other six tumors (cases 7-12. 2D-DIGE and Data Analysis./- a CA: canine mammary carcinoma.

2% SDS. To confirm the proteome analysis. Differences in expression between LN+ and LNsamples were analyzed by the Student’s t test with p-values < 0. No. and an equal volume of 2× sample buffer (7 M urea. Only spots present in all gels and with a LN+/LN.5 µL of Brilliant SYBR Green QPCR Master Mix (Stratagene. Germany) prepared in Tris-buffered saline containing 0. and FC values <0. Peptides were obtained by trypsin in-gel digestion as described previously. amplificationbased threshold. Germany) equipped with a Smart beam laser.(∆Ct LN+ . Western Blot Analysis. pH 3-7 (GE Healthcare. Exact primer concentrations and PCR time and temperature conditions were determined during initial optimization runs. The reactions were carried out in 96-well polypropylene plates covered with optical caps (Stratagene. Germany) for a total of 50 kVh at 20 °C. Germany) scanning at the Cy dyes’ respective wavelengths. 2% DTT. One missed tryptic cleavage was considered. Germany). 2010 Klopfleisch et al. emission 670 nm for DyLight 649) at a resolution of 100 µm. USA). quantitative PCR (RT-qPCR) and data analyses were performed using the MX 3000P Quantitative PCR System and MX Pro software (Stratagene. applying the adaptive baseline.31 The protein digests were measured in the reflector mode using R-cyano-4-hydroxycinnamic acid (CHCA) as matrix. The plates contained triplicates of each cDNA sample and no-template controls with water instead of cDNA templates. followed by 40 cycles of 30 s at 95 °C. 12. emission 520 nm for DyLight 488 and excitation 633 nm. and tubulopapillary. 40 µg of sample protein and a prestained protein-weight marker (Fermentas. The search was restricted to mammalian sequences only.02% bromphenol blue. known contamination peaks such as those of keratin and autoproteolytic products were excluded for a peptide mass fingerprint database search with the Mascot server (www. Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction. and 300 mA for five hours. 2% Pharmalyte IPG buffer. Specificity of amplification products was confirmed by melting curve and sequence analyses. Freiburg. 0. Berlin. Protein spots were visualized with the Typhoon 9400 fluorescence scanner (GE Healthcare.67 in spot intensity were selected for protein identification by MS. Fragment-ion spectra of selected peptides were acquired in the LIFT mode.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-). 0. Quantification of Target Gene Expression. Germany) in Tris-glycine buffer with 20% (v/v) methanol. and moving average algorithm enhancement. Gels loaded with 350 µg of protein were silver-stained for spot picking and subsequent MS analysis. solid.com) in the NCBInr database.8) containing 100 mg of DTT and equilibrated for 15 min with equilibration buffer containing 250 mg of iodoacetamide. Netherlands) with 300 nM of each primer. GE Healthcare.04% bromphenol blue) was added. USA). Cut off values for FC were set at >1. ∆Ct between the geometric mean of the three RGs and the GOI in LN+ (∆Ct LN+) and LN. and 30 s at 72 °C. Membranes were saturated with 5% (w/v) nonfat milk powder (Roth. Proteins were identified from gel spots by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) using an Ultraflex-II TOF/TOF instrument (Bruker Daltonics. The separate CyDyes labeling reactions were finally combined. the IPG strips were first equilibrated for 15 min with the equilibration buffer (6 M urea. 30% glycerol. Germany). The cDNA of all samples was amplified on the same plate for every primer pair to ensure equal amplification conditions. 9. and 50 mM Tris. La Jolla.67 were accepted as reduced gene expression. Bonn. 1 min at 58 °C. Finally. Freiburg. Cycling conditions were 10 min at 95 °C.1% Tween 20 (TBST) for one hour at room temperature and then incubated with primary antibody over6382 Journal of Proteome Research • Vol. Results Histology and Immunohistochemistry of Receptor Status. Primer sequences for mRNA transcripts of the identified proteins and the housekeeper genes hypoxanthinephosphoribosyl transferase (HPRT). For each sample. Isoelectric focusing (IEF) was performed using an Ettan IPGphor 3 Isoelectric Focusing Unit (Ettan IPGphor Manifold. The primary antibody employed was rabbit polyclonal Peroxiredoxin-6 (LIFESPAN Biosciences. 75 µA/strip.matrixscience. The RT-qPCR efficiency of all assays was between 96% and 101% (data not shown) and all yielded products of the expected sequence. The second-dimension molecular weight separation was carried out using an Ettan DALTsix Electrophoresis Unit (GE Healthcare.5 for increased gene expression.ratio of more than 1. Germany) and the following running parameters for six gels: 60 mA for one hour. First.17.32 For the database search.5% SDS-PAGE gels and sealed with 0. were rehydrated with Cy-labeled samples in the dark at room temperature overnight according to the manufacturer’s guidelines. Freiburg. The relative expression of the target gene (TG) was calculated using a comparative ∆Ct-method with multiple housekeepers as previously described. night at 4 °C. 2 M thiourea. membranes were visualized with the Typhoon 9400 (GE Healthcare.5% low-melting-point agarose. results were documented as cycle threshold (CT) set to 100 relative fluorescence unit values of background subtracted qPCR fluorescence kinetics by using the MX Pro Stratagene analysis software. 2% Pharmalyte IPG Buffer. Immobilized nonlinear pH gradient (IPG) strips. 2 M thiourea.05 considered significant. invasive. Freiburg. and ribosomal protein L32 (RP32) are shown in the Supporting Information (Supplemental Table 1). Spot detection. and quantification of spot intensity were performed using the DeCyder 2D Software. Freiburg. and a mass accuracy of 50-100 ppm was used for the searches. 40 mM DTT). Bremen. Samples were complemented to 450 µL with rehydration buffer (7 M urea.34 The housekeeper genes used were selected from a panel of reference genes (RGs) according to the GeNorm algorithm. choosing appropriate wavelengths (excitation 488 nm.∆Ct LN-)). Germany). Karlsruhe. Version 7. pH 8. matching. La Jolla.0 (GE Healthcare. 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. USA) used at a 1:500 dilution.31 Protein Identification by MALDI-TOF-MS. Seattle. After IEF. Strips were transferred onto vertical 12.(∆Ct LN-) was calculated.23 Reverse transcription. Germany) were resolved by SDS-PAGE in 12% polyacrylamide gels and transferred onto PVDF (polyvinylidene difluoride) membranes (GE Healthcare. 4% CHAPS. ATP-synthase subunit 5B (A5B). Freiburg. After several washes with TBST. Amsterdam. Finally. simple mammary carcinomas with lymph node metastases (LN+) or without lymph node me- .33. The 15 µL reaction mix contained 5 µL of cDNA and 12. Germany). Freiburg. the fold change (FC) of GOI expression was calculated (FC ) 2∧ . Only primary tumors diagnosed as highly malignant. 240 mA for one hour.research articles respective dye following the protocol of the supplier. 4% CHAPS.5 or less than 0.

case no. Figure 5A. 9. ERBB2 was expressed in all LN+ and LN. All three cell proliferation.tumors (LN+/LN.B). 5.ratio >1. In such cases. while case nos. Spots U8 and U11 were identified as the cell proliferationassociated proteins Ran/TC4-binding protein (RANBP1) and eukaryotic elongation factor-1 delta (EEF1D). ABC-method. Lymph node metastases of a canine mammary carcinoma in the inguinal lymph node. Immunohistochemistry against estrogen receptor alpha. case no.5 or <0. The majority of tumor cells in 11/13 carcinomas had strong ERBB2 expression. Canine mammary carcinoma. 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). and ten were down-regulated (Table 3) in LN+ (Figure 5A. Canine mammary carcinoma. case no. Case nos. Figure 4.tumors (Figure 4).B). Figure 2. Hematoxylin-counterstain. Immunohistochemistry against ERBB2. Magnification 1:400. however. the score was well beyond the significance threshold proposed by Mascot (p < 0.64). anisokaryosis. case no. anisokaryosis. Magnification 1:300. None of the tumors had ER/PR expression as shown by immunohistochemistry (Figure 3). anisocytosis. In contrast. The proliferating cell nuclear antigen (PCNA. No. Hematoxylin/ Eosin-stain. p < 0. Three proteins could not be identified by database searches for canine or other mammalian protein sequences. All carcinomas were characterized by infiltrative growth. Hematoxylin/Eosinstain. LN+) and nonmetastasizing tumors (cases 7-12. Identification of Differentially Expressed Proteins. In some cases. Canine mammary carcinoma.05). 7-12 were free of lymph node metastases.and division-associated proteins identified had increased expression levels in LN+ when compared to LN(Table 2. anisocytosis. Differences in protein expression patterns between metastasizing canine mammary tumors (cases 1-6. 3. LN-) were analyzed using twodimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Journal of Proteome Research • Vol. spot U1) had the highest up-regulation (ratio 2. Figure 3. Metastatic tumors (dogs 1 to 6) in addition had metastatic tumor cells in surrounding lymph vessels and the regional lymph node (Figure 2). Cell Proliferation-Associated Proteins. Twenty-one proteins were identified which were significantly differentially expressed in LN+ when compared to LN. 2010 6383 . Magnification 1:400.Comparative Proteomics of Canine Mammary Tumors research articles Figure 1. top-scoring results were obtained for proteins from mammalian species other than dog (Canis familiariz). 3. 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. Hematoxylin-counterstain. All carcinomas were characterized by infiltrative growth of tumor cells into the surrounding tissues. tastases (LN-) were included in this study. 12. 1-6 had metastatic carcinoma cells in the regional lymph node.05). Magnification 1:300. Eleven of them were up-regulated (Table 2).67. More than 90% of the tumor of metastasizing and nonmetastasizing carcinomas had no estrogen receptor alpha expression. and numerous mitotic figures (arrows). 7. ABC-method. and a high mitotic rate (Figure 1). In all cases.

Myosin light chain 2.57 5.003 0.06 5. Furthermore. This was confirmed by Western blot analysis. In contrast. Figure 5A.008 0.049 0. Discussion Several proteins with impact on metastatic spread have been identified in human breast cancer cells. Up-Regulated Proteins in Metastasizing (LN+) Canine Mammary Tumors (Ratio >1. Fibrinogen beta chain. VCL. Isocitrate dehydrogenase1. TPM1. light polypeptide.57 1. Fibrinogen beta chain.5-fold (Table 4).003 0. TPM1.B). The scavenger protein thioredoxin domain-containing protein 5 (TXNDC5.042 0. Tropomyosin.012 0. PCNA.05) spot identified protein ratio LN+/LNt test accession no. Vinculin. c Proteins were identified peptide sequencing using MALDI-TOF-MS/MS.69 4.71 4. cov.05 1.64 2. Scavenger Proteins. Rho-GTPaseactivating protein (ARHGAP. PRDX6.) gi|73953587 (Ca.) gi|73995897 (Ca. Ornithine aminotransferase. spot U2) also had increased expression in LN+ samples (Table 2.09 6. spot U3) was upregulated in LN+ samples (Table 2.B). in NCBI MW [Da]b pIb Mowse score a Klopfleisch et al. The two identified serpins with differential expression in LN+ were oppositely regulated. Thioredoxin domain containing 5. Adenosine deaminase (ADA.56 1.) gi|74000375 (Ca. spot D4) showed decreased expression in LN+ samples. Vinculin.11 5.07 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. All except one (MASPIN) protein with decreased expression levels in LN+ also had decreased mRNA expression levels in LN+ when compared to LN.99 6. Ran-specific GTPase-activating protein.B). ANXA5. Figure 5A. c Proteins were identified by unique peptide sequencing using MALDI-TOF-MS/MS. D7) that is involved in the citric acid cycle had decreased expression levels in LN+ (Table 3. light polypeptide. annexin A5 (ANXA5. b Listed molecular weights and pI values correspond to the listed accession numbers.6. Elongation factor 1-delta. Tropomyosin.22 0. CALB2.B). Figure 5A.82 6. spot D10). b Listed molecular weights and pI values correspond to the listed accession numbers. spot D8) had a decreased expression in LN+ samples (Table 3.05)a spot identified protein protein ratio LN+/LNt test accession no.52 0.68 6. Figure 5A. Figure 5A. 12.037 0. Ran-specific GTPase-activating protein.B). MYL2.) gi|74005281 (Ca.64 0.59 0. Serin Protease Inhibitors (Serpins). Proliferating cell nuclear antigen. spot U7) showed increased expression in LN+ samples (Tables 2 and 3.0039 0. Figure 5A.51 1.65 1. spot D3) had decreased expression in LN+ samples (Table 3. Cell Adhesion and Motility Proteins. Only two of the 11 up-regulated proteins in LN+ samples (PCNA.33 0. CORO1A) also had upregulated mRNA expression of more than 1. TXNDC5.) gi|73998800 (Ca.) gi|134085902 (Ca) 31616 19915 25133 42532 55590 87669 47180 32553 35978 50611 5. Thioredoxin domain containing 5. BOMAPIN) in LN+ samples had corresponding mRNA expression below 0. Figure 5A.73 4.B).B).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.57 2. RTqPCR was used to analyze whether differences in protein expression levels in the two tumor groups are based on transcriptional regulation.02 0. ARHGAP1. spot U5) are both involved in amino acid metabolism and were up-regulated in LN+ (Table 2).0046 0. Similarly.94 4.) gi|73981259 (Ca.044 0. Ornithine aminotransferase.76 1.00058 0. No.3.67. [%] U1 U2 U3 U4 U5 U6 U7 U8 U9 U10 U11 PCNA FTL TXNDC5 ADA OAT CORO1A BOMAPIN RANBP1 TPM3 PGDH EEF1D 2.60 0. Figure 5A.B). D-3-phosphoglycerate dehydrogenase. Ferritin.44 6. EEF1D. which sometimes belong to species other than Canis familiariz.029 0.84 5. TXNDC5. OAT. showing lack of PRDX6 expression in LN+ samples (Figure 6).) gi|73945839 (Ca. 2010 3-phosphoglycerate dehydrogenase (PGDH. PCNA. OAT. Isocitrate dehydrogenase1. ANXA5. Rho GTPase activating protein 1. 3 of the 11 up-regulated proteins (ADA.19 5. In contrast.B). D-3-phosphoglycerate dehydrogenase. Calretinin.031 0. The cytoskeletal proteins vinculin (VCL. Elongation factor 1-delta.013 gi|4505641 (Ho. spot D5) had decreased expression levels in LN+ when compared to LN(Table 3.) gi|109069575 (Ma. cov. CALB2. Miscellaneous Proteins.) gi|73992038 (Ca. FTL. IDH1. 6384 Journal of Proteome Research • Vol. spot U9) and the actin-binding protein coronin 1A (CORO1A.59 0.67-fold.5. spot U6) were up-regulated in LN+ (Table 2. FGB.015 gi|29636 (Ho) gi|188586 (Ho) gi|4758638 (Ho) gi|149721154 (Eq) gi|73977986 (Ca. suggesting a posttranscriptional regulation of their protein expression (Table 4).00023 0. the scavenger protein peroxiredoxin 6 (PRDX6.) 29092 20139 44433 41338 48753 55228 45060 24180 26632 57308 30731 4. Ferritin. MYL2.47 6. ferritin light polypeptide (FTL.) gi|73958508 (Ca. Rho GTPase activating protein 1. Proliferating cell nuclear antigen. RT-qPCR failed to detect FGB mRNA in both tumor groups (Table 3). Down-Regulated Proteins in Metastasizing (LN+) Canine Mammary Tumors (ratio <0. PRDX6.66 6.49 0. assigned peptides sequ. While serpin B5 (maspin.74 1. TPM3.samples.51 0.0013 0. Myosin light chain 2. Identification of mRNA Ratios between LN+ and LN-. the cell motility associated proteins myosin light chain 2 (MYL2. spot U4) had increased expression levels in LN+ (Table 2. Peroxiredoxin 6. spot D1) were down-regulated in LN+ (Table 3. Adenosine deaminase. PGDH.) gi|73961101 (Ca. FGB. RANBP1. Actually. Calretinin. VCL.) gi|57100553 (Ca. Adenosine deaminase.23 0. p < 0.75 6.042 0. spot U10) and ornithine aminotransferase (OAT. which sometimes belong to species other than Canis familiariz. [%] D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 CALB2 MYL2 PRDX6 MASPIN FGB VCL IDH1 TPM1 ANXA5 ARHGAP1 0. spot D9). Tropomyosin 1. spot D6) and the actin-binding protein tropomyosin 1 (TPM1. Figure 5A. Figure 5A. ARHGAP1.61 1. Annexin A5. PGDH. and fibrinogen beta chain (FGB. Furthermore.research articles Table 2. in NCBI MW [Da]b pIb Mowse score assigned peptides sequ.0051 0. TPM3. Tropomyosin 1. p < 0.77 6.) gi|73974692 (Ca. EEF1D. spot D2) and calretinin (CALB2.B). Isocitrate dehyrogenase 1 protein (IDH1.00 5.019 0. serpin B10 (bomapin. RANBP1. tropomyosin 3 (TPM3. Peroxiredoxin 6.36 These . Annexin A5. OAT. 9.40 0. IDH1.) gi|66864897 (Ca. Table 3. FTL.

Up-regulated proteins (Table 2) in LN+ are indicated in green. little is known on proteins involved in the metastatic spread of canine mammary tumors (CMTs). 2010 6385 .14. 12. 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. proteins are involved in various cellular functions such as cell division. and angiogenesis. a comparative model of the human disease. Isoelectric focusing pH 3-7. 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. Differentially expressed proteins in nonmetastasizing canine mammary carcinomas (LN+) (A) when compared to metastasizing canine mammary carcinomas (LN-) (B). cell adhesion. No.Comparative Proteomics of Canine Mammary Tumors research articles Figure 5.20.37. while other aspects differed. However. The present study therefore compared the proteome of metastatic and nonmetastatic canine mammary carcinomas in an explorative approach to Journal of Proteome Research • Vol. 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. down-regulated proteins (Table 3) in red. 9.38 In general. matrix invasion. 1.18. 24 cm wide gel. 2D-DIGE of macrodissected tissue lysates of dog no. and prediction of metastatic spread is difficult by histology before metastases are large enough to be visualized. In contrast.15.

oncogenic transformation. PCNA is located in the nucleus and acts as a cofactor of DNA polymerase delta and thereby increases DNA replication.research articles Klopfleisch et al.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.62 1.38 0. 6) and a pool of all LN+ (dog nos.51 1.53 0. 12. Proteins with Proliferation Activity Are Up-Regulated in Metastatic Canine Mammary Tumors. All single LN+ (dog nos.22. While Those with Cell Motility Are Overexpressed in Metastatic Canine Mammary Tumors.05 0. a cellular feature well-known for several metastatic tumors.44 Up-regulation of RANBP1 therefore 6386 Journal of Proteome Research • Vol. Transcriptional down-regulation of vinculin (VCL) in LN+ is obviously in line with this general assumption.40 0. 10.36 A correlation between EEF1D overexpression. 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.27 1.41 In addition. 5.46. 9. Increased proliferation and uncontrolled cell division are common features of malignant human and canine mammary tumors. Figure 6. and its expression is associated with reduced cell motility. the proteinexpression level and grade of cell adhesion were directly correlated.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.a 0. In these models.54 results in unstable mitotic microtubules and decreased apoptosis when failure of mitotic progression is detected. 4. and invasive growth of breast cancer and other tumor cells has been reported in several studies.43 RANBP1 acts as a negative regulator of chromosome condensation by inhibiting RCC1 (regulator of chromosome condensation 1)-stimulated guanine nucleotide release from RAN.5. 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. it is a potential tumor suppressor.36.82 0.45 Eukaryotic elongation factor-1 delta (EEF1D) was the third up-regulated protein directly associated with cell proliferation in LN+ tumors.51 Paxillin. 9. is also down-regulated in canine mammary tumors when compared to a normal mammary gland.43 RANBP1 directly interacts with GTP-charged RAN which is involved in mitosis and apoptosis.: no mRNA expression detected in the tissue samples. 11) and a pool of all LN.53 LN+ samples had decreased expression of tropomyosin 1 (TPM1) protein but increased protein expression of tropomyosin 3 (TPM3).99 1.47 Prevention of E-cadherinmediated cell-cell adhesion by increased EEF1D protein expression levels may represent a possible molecular mechanism for this observation.63 0. 1.72 n.49 Up-regulation of EEF1D therefore indicates not only increased proliferation but also decreased cell-cell adhesion in canine mammary tumors. EEF1D promotes cell-cycle progression by delivery of aminoacyl tRNAs to the ribosome and functions as a guanine nucleotide exchange factor. No. another focal adhesion complex protein.50 Furthermore.d.43.39. Ratio of mRNA Expression Levels of the Differentially Expressed Proteins in Metastasizing (LN+) and Nonmetastasizing (LN-) Canine Mammary Carcinomas spot identified 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 1.20.28 0.09 0. Tropomyosins are also cytoskeletal actin-binding proteins which are involved in the contractile system of several cell types. 2010 . 1-6) have decreased PRDX6 expression levels when compared to single LN.(dog nos.d. loss of cell-cell adhesions and enhanced cellular motility are features of metastasizing tumors.91 1. Table 4.59 0. based on the observation that restoration of vinculin in SV-40-transformed murine fibroblasts and pancreatic carcinoma cells had a suppressive effect of the tumorigenic ability of these cells.62 0.42 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. identify proteins involved in metastatic spread and potential new metastasis markers. 8. It modulates the dynamics of cell-cell and cell-matrix adhesion. Proteins Associated with Cell Adhesion Are DownRegulated.49 0.48 This is also in line with the significant down-regulation of E-cadherin in invasive canine mammary tumors.58 0. 7-12). VCL is a cytoskeletal protein with functions in the focal adhesion complex.40 The significantly higher expression of proliferating cell nuclear antigen (PCNA) in metastasizing carcinomas (LN+) when compared to nonmetastasizing canine mammary carcinomas (LN-) in the present study confirms this general observation.(dog nos. 1. 2. As mentioned above.38 0. Western blot analysis of PRDX6 protein expression levels in metastasizing CMT (LN+) and nonmetastasizing CMT (LN-).

and a similar association between prognosis and ferritin expression seems to exist in canine mammary tumors.Comparative Proteomics of Canine Mammary Tumors tions. TPM1 is a tumor suppressor inducing anoikis (detachment-induced apoptosis) in breast cancer cells. was transcriptionally down-regulated in LN+ tumors. TPM3 overexpression activates epithelial-mesenchymal transition in hepatocellular carcinomas and contributes to tumor cell dissemination in ERBB2-positive breast cancer. TXNDC5 is located in the endoplasmic reticulum and known to inhibit apoptosis by chaperone activity on hypoxia-induced antiapoptotic molecules. radical-inducing environment. rapid growth of tumors induces oxidative stress due to insufficient vascularization.68 Expression of thioredoxins has not been analyzed in canine mammary tumors before. but the differ5. Thus. maspin may indeed be involved in metastatic spread of canine mammary tumors. Coronin1A (CORO1A) is another actin-binding protein that was also transcriptionally up-regulated in LN+ samples. 12.75 In the present study. and this may contribute to the resistance of the metastatic cells to hypoxic stresses. Down-regulation of maspin is associated with malignant behavior and metastatic spread of breast. the two serpins identified in the present study were regulated in opposite directions in LN+ samples. the down-regulation of TPM1 enables neoplastic cells to survive outside their normal microenvironment and thereby supports metastasis.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. prostate. increased cytoplasmic levels of ADA or treatment with exogenous ADA significantly decrease adhesion and increase invasiveness of human lymphoma cells. and pancreatic cancer.62 Interestingly. analysis of mRNA expression levels in canine mammary tumors at different stages of malignant progression has pointed toward a decreased expression that was. bomapin was up-regulated in LN+ samples.17 According to the results of this study.7. expression levels of peroxiredoxins like PRDX6 have not been described in canine tumors but are upregulated in metastatic human breast and lung cancers. tumor suppression. and extracellular matrix modulation and cell migration. LN+ tumors contained increased protein levels of ferritin. indicating a posttranscriptional up-regulation of this enzyme.65 Furthermore.63 Especially in human breast cancer. Coronins are highly conserved regulators of the actin cytoskeleton and are enriched at the leading edge of migrating cells.8 Surprisingly. the phospholipase A2 activity of peroxiredoxin 6 promotes invasion and metastasis of lung cancer cells.76 Isocitrate dehyrogenase-1 (IDH1) protein. Serpin activity regulates different intracellular and extracellular processes such as cellular differentiation.64 The only immunohistochemical study available on maspin expression in canine mammary tumors identified no differences of maspin protein expression in malignant epithelial cells when compared to benign cells.samples.5 In contrast.8. increased ADA protein expression levels in LN+ samples were associated with decreased mRNA expression levels. 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. not significant. 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. while bomapin was up-regulated in LN+ when compared to LN. metastatic cells had a significantly increased expression of thioredoxin domain-containing protein 5 (TXNDC5) when compared to LN. So far it is clear that bomapin expression has a cytoprotective effect against tumor necrosis factor alpha (TNFR)-induced cell death. Serin protease inhibitors (Serpins) are proteins with a similar structure and the ability to inhibit serine proteases. SERPINB5) is a well-described tumor suppressor in several tissue types including the mammary gland. and its function in physiology and disease in general is mostly unknown. and squamous epithelium. 9. Interestingly. The expression of bomapin (serpin peptidase inhibitor. and its expression is increased in a lymphoma cell line when compared to non-neoplastic leucocytes.3. Maspin had decreased expression levels. In the present study. Invasive. and a similar mechanism may be true for canine mammary tumors. 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.58 This influence 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. No.67 Similar to the situation in human cancer.54.59-61 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. was significantly transcriptionally downJournal of Proteome Research • Vol.70 Furthermore.70-72 So far. and the relevance of their expression is unclear at the moment. a protein with strong antioxidant properties. an inducer of the HIF-1 pathway.56 Tropomyosin expression levels have not been analyzed in canine tissues before but obviously are similarly regulated in human breast cancer. 4.samples. apoptosis. Maspin versus Bomapin: Regulators of Matrix Invasion Are Differentially Expressed in Metastasizing Carcinomas.69 Surprisingly.73 Nevertheless.57. but in humans a similar increased expression was found in poorly differentiated hepatocellular and colorectal carcinomas. 3.6.66. Peroxiredoxins are present in various cellular compartments and reduce peroxides to the corresponding alcohol or water. Scavenger Proteins May Protect Cells with Metastatic Potential from Hypoxia and Oxidative Stress. 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. 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.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. however. SERPINB10) has not been analyzed in canine mammary tumors before. 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. peroxiredoxin 6 (PRDX6). a genetic variation in the bomapin gene has been identified in human patients with prostate cancer. Maspin (mammary serine protease inhibitor.7.72 Down-regulation of PRDX6 is associated with resistance to chemotherapy of breast cancer cells. prostate.6 However. 2010 6387 . However.

extracellular matrix modulation. PCNA.77 Recent studies characterized IDH1 as a tumor suppressor gene that contains a prognostically relevant mutation in the majority of human glioblastomas. Nineteen of the 21 proteins differentially expressed in metastatic canine mammary tumors also have a malignancy. most of the proteins are similarly dysregulated in human cancer.79 5. A literature search identified several overlaps in protein expression patterns in metastatic canine mammary tumors with human breast cancer and other human cancers (Table 5). Table 5. PGDH. 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.78 In this tumor type.or metastasis-associated expression pattern in different human cancers.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. Decreased IDH1 levels in turn increase cellular levels of hypoxia-inducible factor 1 alpha (HIF-1alpha). 12. Most of the proteins are involved in cellular functions relevant for metastatic spread like cell adhesion. nine of the proteins have a similar malignancy-associated protein expression in canine mammary tumors when compared with human breast cancer (Table 5). Two of the proteins (IDH1. . decreased IDH1 protein levels have been identified in malignant bladder cancer. Conclusion In conclusion.76 potential increased enzymatic activity indicates poor prognosis decreased in colorectal cancer in breast cancer.64 decreased in metastatic oral squamous carcinoma88 decreased in oral squamous cell carcinoma53 decreased in breast cancer2. 12 of the proteins differentially regulated in metastatic canine mammary tumors have no known relevance in human breast cancer as of yet. Moreover.67 genetic variations in prostate cancer66 3 increased in metastatic breast cancer cells increased in hepatocellular carcinoma56 increased in colorectal cancer. However. The exact causes of these expression differences are unknown. regulated in LN+. these 12 proteins may also be interesting candidates to be analyzed in metastatic human breast cancer. These included relevant tumor markers like MASPIN. while an in vitro selected highly metastatic breast cancer cell line had an increased IDH1 expression level. PRDX6) had an opposite regulation in malignant canine and 6388 Journal of Proteome Research • Vol. In contrast. No. and TPM1/3.86 down-regulation in metastatic lung cancer87 increased in a breast cancer cell line79 decreased in glioma cell line77 decreased in invasive breast cancer7. comparison of the proteomic profile of metastasizing versus nonmetastasizing canine mammary tumors identified 21 proteins that had significantly different expression levels in the two tumor types. Our findings 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.8. 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.research articles Klopfleisch et al.81 decreased in breast cancer. Interestingly. thereby supporting tumor growth in hypoxic microenvironments.65. 2010 breast cancer cells (Table 5). but the in vitro setting in the IDH1 studies may represent one possible cause. and this may reflect the differences between the two species. 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.77. mutation of IDH1 leads to functional changes but unaltered expression levels. The findings therefore support the value of canine mammary tumors as a potential comparative model for human cancer. and hypoxia resistance.79 Despite all similarities.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. Nevertheless. Protein Expression Patterns in Metastatic Canine Mammary Tumors Resemble Human Breast Cancer Protein Expression in Several Aspects. 9.63.

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