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:
[email protected]. ‡ Institute of Veterinary Pathology, Freie Universita¨t Berlin. § Institute of Veterinary Biochemistry, Freie Universita¨t Berlin. | Institute of Chemistry and Biochemistry, Biochemistry.
6380 Journal of Proteome Research 2010, 9, 6380–6391 Published on Web 10/11/2010
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
research articles
Comparative Proteomics of Canine Mammary Tumors
Table 1. Characterization of Dogs with Metastasizing and Nonmetastasizing Canine Mammary Carcinomasa dog no.
breed
age (years)
survival time (months)
histology
tumor size (cm)
lymph node status
ERBB2 status
ER/PR status
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 first 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-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. Furthermore, quantitative RT-PCR was used to analyze whether these proteins were transcriptionally differentially regulated in metastatic and nonmetastatic CMTs. The data reveal significant 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 confirmed by five serial sections (every 100 µm) 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 specific 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 first antibody with all other procedures alike. 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. 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 µg/µL) 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 µg of protein was labeled with 400 pmol of the Journal of Proteome Research • Vol. 9, No. 12, 2010 6381
research articles respective dye following the protocol of the supplier. The separate CyDyes labeling reactions were finally 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 µL 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 manufacturer’s 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 µA/strip. After IEF, the IPG strips were first 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 five hours. Protein spots were visualized with the Typhoon 9400 fluorescence scanner (GE Healthcare, Freiburg, Germany) scanning at the Cy dyes’ respective wavelengths. Spot detection, matching, and quantification 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 Student’s t test with p-values < 0.05 considered significant. 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 identification by MS. Gels loaded with 350 µg of protein were silver-stained for spot picking and subsequent MS analysis.31 Protein Identification by MALDI-TOF-MS. 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, 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 reflector 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 fingerprint 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 confirm the proteome analysis, 40 µg 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 difluoride) 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
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Klopfleisch 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 µm. Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction. Primer sequences for mRNA transcripts of the identified 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 efficiency of all assays was between 96% and 101% (data not shown) and all yielded products of the expected sequence. The 15 µL reaction mix contained 5 µL of cDNA and 12.5 µL 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 amplified on the same plate for every primer pair to ensure equal amplification conditions. Specificity of amplification products was confirmed by melting curve and sequence analyses. For each sample, 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, applying the adaptive baseline, amplificationbased threshold, and moving average algorithm enhancement. Quantification 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 1.5 or 1.5, p < 0.05)
a
spot
identified protein
ratio LN+/LN-
t test
accession no. in NCBI
MW [Da]b
pIb
Mowse score
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-specific 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 identified by unique peptide sequencing using MALDI-TOF-MS/MS.
Table 3. Down-Regulated Proteins in Metastasizing (LN+) Canine Mammary Tumors (ratio
