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Tandem Duplication of KIT Exon 11 Influences the Proteome of Canine Mast Cell Tumours
J. Comp. Path. 2013, Vol. 148, 318e322
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
NEOPLASTIC DISEASE
Tandem Duplication of KIT Exon 11 Influences the Proteome of Canine Mast Cell Tumours P. Schlieben*, A. Meyer*, C. Weise†, A. Bondzio‡, A. D. Gruber* and R. Klopfleisch* * Department of Veterinary Pathology, Freie Universit€at Berlin, Robert-von-Ostertag-Straße 15, 14163, † Institute of Chemistry and Biochemistry, Freie Universit€at Berlin, Thielallee 63, 14195 and ‡ Department of Veterinary Biochemistry, Freie Universit€at Berlin, Oertzenweg 19b, 14163 Berlin, Germany
Summary Mutations with permanent activation of the stem cell factor receptor KIT have been identified as one potential cause for canine cutaneous mast cell tumours (MCTs). The exact changes in global gene expression patterns associated with permanent activation of KIT in these tumours are unknown. The present study compares, by the use of two dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization timeof-flight mass spectrometry, the proteomes of canine MCTs, with and without KIT exon 11 tandem duplication. Fifteen differentially expressed proteins were identified in mutated MCTs. These are mainly involved in cytoskeleton structure and cell motility (ACTR2, ACTB and CAPPA1), cell signalling (ARHGDIA) and lipid metabolism (ALOX15 and ACSBG4), or are serum proteins. The results therefore support the notion that KIT mutation is associated with changes in the proteome of affected cells with a major effect on the composition of the cytoskeletal proteome and cell motility proteins. No overlaps were identified when the results were compared with a recent study on the proteomic differences between low- and high-grade tumours, suggesting that KIT-mutated tumours may be regarded as a separate entity of high-grade tumours with potential relevance to therapeutic strategies. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: dog; KIT; mast cell tumours; proteomic analysis
Several mutations in the stem cell factor receptor KIT have been identified as potential causes of canine cutaneous mast cell tumours (MCTs) (London et al., 1996). Of these, tandem duplication at exon 11 is the best analysed and the most relevant KIT mutation (London et al., 1999; Zemke et al., 2002; Letard et al., 2008). This mutation causes a conformational change in the juxtamembrane domain of the receptor and autophosphorylation of its tyrosine kinase domain, independent from the presence of its ligand, the stem cell factor (Pryer et al., 2003). The resulting permanent KIT activation leads to increased MCT cell proliferation and survival by influencing the vast majority of the active canine genome (Pryer et al., 2003; Masson and Ronnstrand, 2009; Correspondence to: R. Klopfleisch (e-mail: robert.klopfleisch@fu-berlin. de). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2012.07.006
Klopfleisch et al., 2012). This mutation is more prevalent in grade III canine MCTs with an incidence of 17e35% when compared with approximately 8% in lower grade MCTs (Downing et al., 2002; Letard et al., 2008). The signalling cascade of the KIT pathway has been well established in several species and it is accepted that permanent activation by KIT mutation leads to increased proliferation of canine MCT cells (Webster et al., 2007; Nurmio et al., 2008; Pittoni et al., 2011). Nevertheless, changes in the global gene expression patterns associated with tandem duplication in KIT exon 11 are unknown. High- and low-grade MCTs differ in their global protein expression patterns independent of their KIT mutation status (Schlieben et al., in press). Major expression differences are restricted to 13 proteins Ó 2012 Elsevier Ltd. All rights reserved.
319
Canine Mast Cell Tumour Proteome
that are associated mainly with the cellular stress response, cell motility and metastasis, iron metabolism and mast cell differentiation. The aim of the present explorative study was to identify differences between the proteome of MCTs with and without KIT exon 11 tandem duplication by employing two dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF-MS) (Westermeier and Scheibe, 2008; Galvao et al., 2011). Six canine cutaneous MCTs were included in the study. Four tumours were scored as high grade or grade III and two tumours as low grade or grade II according to the Kiupel and Patnaik grading systems, respectively (Table 1; Patnaik et al., 1984; Kiupel et al., 2011). Tumour specimens were fixed in 4% neutral buffered formalin and processed routinely for microscopical examination. Parallel samples were snap frozen in liquid nitrogen within 15 min of resection and were stored at -80 C until further use. Macrodissection and sample preparation, including protein extraction and quantification, were performed as previously described (Klopfleisch et al., 2010a; Klose et al., 2011). Polymerase chain reaction (PCR) amplification for identifying KIT exon 11 status was performed as described previously using the forward primer 50 -CCATGTATGAAGTACA GTGGAAG-30 and the reverse primer 50 -GTTCCCTAAAGTCATT GTTA CACG-30 (Jones et al., 2004; Webster et al., 2006). The PCR protocol was adapted as described by Meyer et al. (in press). Amplified PCR products were fractionated by gel electrophoresis on an ethidium bromide-stained 2.5% agarose gel. PCR products of three tumours identified tandem duplications in exon 11 (Table 1). For 2D-DIGE, 50 mg protein was labelled with 400 pmol of the respective dye according to the manufacturer’s guidelines (GE Healthcare, Freiburg, Germany). Immobilized non-linear pH gradient (IPG) strips (pH 3e7) (GE Healthcare) were used in addition to an Ettan IPGphor 3 isoelectric focus-
sing (IEF) unit (Ettan IPGphor Manifold; GE Healthcare) for a total of 50 kVh at 20 C and 75 mA/strip for IEF. Two steps of equilibration followed IEF, before strips were transferred to the top of gels for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE; 12.5%) and sealed with 0.5% low melting point agarose. The two dimensional molecular weight separation was carried out using an Ettan DALTsix electrophoresis unit (GE Healthcare) with the following running parameters: 60 mA for 1 h, 240 mA for 1 h and 300 mA for 5 h. Each protein extract was analysed initially on at least three silver-stained 2D-PAGE gels to ensure reproducibility prior to quantitative analysis by 2DDIGE. Spot visualization, detection, matching and quantification of spot intensity were performed using a Typhoon 9400 fluorescence scanner and the DeCyder 2D Software, Version 7.0 (both GE Healthcare). An unpaired Student t test with P values <0.05 considered significant was used to identify differences in protein expression between KIT-mutated and nonmutated MCTs. Only spots present in all gels and with a ratio of spot intensity of more than 1.5 between the two groups were selected for subsequent protein identification by MS. For identification of differentially expressed proteins, silver-stained gels loaded with 350 mg of protein were prepared for MS analysis (Shevchenko et al., 1996; Klopfleisch et al., 2010a). An Ultraflex-II TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a smart beam laser was used, as well as a-cyano-4hydroxycinnamic acid (CHCA) as a matrix, to measure protein digests in the reflector mode. Trypsin in-gel digestion and peptide mass fingerprint database searches were performed as described previously (Shevchenko et al., 1996; Klose et al., 2011). Fifteen differentially expressed proteins were identified in mutated MCTs when compared with nonmutated MCTs. Seven of these were up-regulated and eight were down-regulated (Table 2). Most of the up-regulated proteins in mutated MCTs are associated with cytoskeleton structure or cell motility
Table 1 Characterization of dogs and tumours Dog
1 2 3 4 5 6
Breed
Age (years)
Histological grade (Patnaik et al., 1984)
Histological grade (Kiupel et al., 2011)
Tandem duplication in exon 11
Pit bull terrier Beagle Shar Pei Staffordshire bull terriercross Beagle Boxer
16 13 13 15
III III II III
High High Low High
+ + +
7 8
III II
High Low
P. Schlieben et al.
320
Table 2 Proteins differentially expressed in KIT-mutated versus non-mutated MCTs Spot
Gene
Ratio
t-test
Accession number in NCBI database
MW [Da]*
pI*
MOWSE score
Assigned peptides
Sequence covered [%]
gij73955437 (Ca) gij73969820 (Ca) gij73958055 (Ca) gij284795268 (Ca) gij73981496 (Ca) gij73964747 (Ca) gij74003009 (Ca)
76, 599 46, 025 31, 394 59, 776 33, 135 23, 436 60, 055
5.98 6.06 5.04 5.50 5.53 5.12 5.58
156 214 72 138 138 85 96
20 26 7 11 12 7 10
37 54 28 18 53 33 17
Down-regulated proteins in KIT-mutated MCTs D1 FGG 6.90 0.0057 gij73977992 (Ca) D2 APOA1 6.28 0.013 gij73955106 (Ca) D3 AHSG 5.59 0.024 gij74003450 (Ca) D4 ALB 4.07 0.031 gij55742764 (Ca) D5 IGGC 3.65 0.00019 gij17066528 (Ca) D6 IGGD 3.16 0.012 gij17066530 (Ca) D7 ACSBG1 3.12 0.018 gij309243088 (Sus) D8 BCAS4 1.60 0.0022 gij115497804 (Bos)
50, 027 30, 163 40, 515 70, 556 52, 779 52, 169 78, 823 18, 398
5.74 5.28 5.70 5.52 6.16 6.23 7.81 4.97
176 140 81 232 105 37 76 76
16 12 1† 28 9 1† 9 6
45 37 5† 47 24 2† 12 30
Up-regulated proteins in KIT-mutated MCTs U1 ALOX15 +5.25 0.029 U2 ACTR2 +2.91 0.022 U3 ACTB +2.29 0.0013 U4 CPNE1 +2.03 0.022 U5 CAPPA1 +1.83 0.014 U6 ARHGDIA +1.69 0.049 U7 CCT5 +1.52 0.041
ALOX15, arachidonate 15-lipoxygenase; ACTR2, actin-related protein 2; ACSBG1, long chain fatty acid CoA-ligase1; ACTB, actin beta; AHSG, fetuin-A; ALB, serum albumin; APOA1, apolipoprotein A-I; ARHGDIA, Rho GDP dissociation inhibitor alpha; BCAS4, breast carcinoma-amplified sequence 4; Ca, Canis lupus familiaris; CAPPA1, capping protein (actin filament) muscle Z-line; CCT5, T-complex protein 1 epsilon subunit; CPNE1, copine-1; Da, Dalton; FGG, fibrinogen gamma chain; IGGC, immunoglobulin heavy chain C; IGGD, immunoglobulin gamma heavy chain C/D; MW, molecular weight; MOWSE, molecular weight search; pI, isoelectric point; Sus, Sus scrofa; Bos, Bos taurus. * Listed molecular weights and pI values correspond to the listed accession numbers, which sometimes belong to species other than Canis familiaris. † Proteins were identified by unique peptide sequencing using MALDI-TOF-MS/MS.
including actin-related protein 2 (ACTR2; spot U2, ratio 2.91), actin beta (ACTB; spot U3, ratio 2.29) and capping protein (actin filament) muscle Z-line (CAPPA1; spot U5, ratio 1.83). Most of the down-regulated proteins were extracellular serum proteins including fibrinogen gamma chain (FGG; spot D1, ratio 6.90), apolipoprotein A1 (APOA1; spot D2, ratio 6.28), fetuin-A (AHSG; spot D3, ratio 5.59), serum albumin (ALB; spot D4, ratio 4.07) and immunoglobulin gamma heavy chain C (IGGC; spot D5, ratio 3.65) and immunoglobulin gamma heavy chain D (IGGD; spot D6, ratio 3.16). In addition, Rho GDP dissociation inhibitor alpha (ARHGDIA; spot U6, ratio 1.69), T-complex protein 1 epsilon subunit (CCT5; spot U7, ratio 1.52), copine-1 (CPNE1; spot U4, ratio 2.03) and arachidonate 15-lipoxygenase (ALOX15; spot U1, ratio 5.25) were also up-regulated in mutated MCTs, while long chain fatty acid CoA-ligase1 (ACSBG4; spot D7, ratio 3.12) and breast carcinoma-amplified sequence 4 (BCAS4; spot D8, ratio 1.60) were identified as down-regulated proteins in mutated MCTs. Although KIT and its mutations have been the subject of intense research, details of the downstream pathways affected by increased KIT activity are mostly unknown. In the present preliminary explorative study, 15 differentially expressed proteins were
identified covering several aspects of cell metabolism. Taking into account that further investigations in a larger group of tumours are needed to confirm the findings, four major conclusions can be drawn from this study. Firstly, KIT-mutated MCTs differ significantly from non-mutated MCTs in their proteome. However, the number of these proteins was small in light of the thousands of proteins in the canine proteome. This small number may in part have caused insufficient sensitivity of the proteomic methods used to identify more subtle differences in protein expression levels. The data nevertheless indicate that only a few common changes exist in the proteome of KIT-mutated versus non-mutated MCTs. Secondly, mutated MCTs differ from non-mutated MCTs mainly in the quantitative composition of selected cytoskeletal and cell motility proteins and proteins associated with lipid metabolism and the content of serum proteins, but not receptor proteins such as CD25, as shown by immunohistochemistry (Meyer et al., in press). For instance, three proteins associated with the cytoskeleton or cell motility were upregulated in mutated MCTs. All three, ACTR2, CAPPA1 and ACTB, contribute to the overall cell shape, but also to cell motility, for instance by the formation of lamellipodes (Butler and Cooper, 2009; Zhang et al., 2009), and changes in these proteins
321
Canine Mast Cell Tumour Proteome
may contribute to the pleomorphism of cells from high-grade MCTs. In addition, the dysregulation of expression of these three genes facilitates tumour cell invasion and metastasis in human and canine tumours (Yarar et al., 2002; Altschuler and Willison, 2008; Nomura et al., 2008; Klopfleisch et al., 2010c; Carnell and Insall, 2011; Sun et al., 2011; Yokotsuka et al., 2011). The changes in the expression level of proteins involved in lipid metabolism or exocytosis of lipid membrane-bound vesicles including ALOX15, CPNE1 and ACSBG1 (Skawran et al., 2008; Kerjaschki et al., 2011) may be associated with a different metabolism of lipid mediators or eicosanoids, which are major secretory products of normal mast cells and involved in inflammation and haemodynamic regulation, and have been described for metastatic canine mammary tumours (Klopfleisch et al., 2010b). Thirdly, the significantly decreased levels of serum proteins, including FGG, APOA1, ALB, IGGC and IGGD, in the mutated MCTs are remarkable. As a possible explanation, we speculate that these changes may be associated with a different extracellular matrix composition in these tumours. Nonmutated MCTs may thus induce increased vascular dilation or permeability when compared with the mutated tumours, possibly due to increased secretion of vasoactive factors. This would, however, imply that non-mutated MCTs are more activated or better differentiated than mutated MCTs, an assumption that is not overtly supported by the histological appearance of the tumours in the groups. Fourthly, there is apparent overlap of the proteins identified in this study with the proteins identified by comparison of low- versus high-grade MCTs (Schlieben et al., in press). This may support the hypothesis that high-grade MCTs with KIT exon 11 tandem duplications should be viewed as a separate entity within canine high-grade MCTs, which differ in their proteome composition from non-mutated high-grade MCTs. These specific differences in protein expression levels are of potential interest for evidence-based design of therapeutic strategies for high-grade MCTs with and without KIT mutations.
Conflict of Interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
Acknowledgements We thank M. Sch€ arig and P. Schulze for technical support. This project was supported by the German
Research foundation (DFG, KL2240). The present study is part of the doctoral thesis of P. Schlieben.
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Letard S, Yang Y, Hanssens K, Palmerini F, Leventhal PS et al. (2008) Gain-of-function mutations in the extracellular domain of KIT are common in canine mast cell tumors. Molecular Cancer Research, 6, 1137e1145. London CA, Galli SJ, Yuuki T, Hu ZQ, Helfand SC et al. (1999) Spontaneous canine mast cell tumors express tandem duplications in the proto-oncogene c-kit. Experimental Haematology, 27, 689e697. London CA, Kisseberth WC, Galli SJ, Geissler EN, Helfand SC (1996) Expression of stem cell factor receptor (c-kit) by the malignant mast cells from spontaneous canine mast cell tumours. Journal Comparative Pathology, 115, 399e414. Masson K, Ronnstrand L (2009) Oncogenic signaling from the hematopoietic growth factor receptors c-Kit and Flt3. Cell Signalling, 21, 1717e1726. Meyer A, Gruber AD, Klopfleisch R. CD25 is expressed by canine cutaneous mast cell tumors but not by cutaneous connective tissue mast cells. Veterinary Pathology. in press. Nomura H, Uzawa K, Ishigami T, Kouzu Y, Koike H et al. (2008) Clinical significance of gelsolin-like actin-capping protein expression in oral carcinogenesis: an immunohistochemical study of premalignant and malignant lesions of the oral cavity. BMC Cancer, 8, 39. Nurmio M, Kallio J, Toppari J, Jahnukainen K (2008) Adult reproductive functions after early postnatal inhibition by imatinib of the two receptor tyrosine kinases, c-kit and PDGFR, in the rat testis. Reproductive Toxicology, 25, 442e446. Patnaik AK, Ehler WJ, MacEwen EG (1984) Canine cutaneous mast cell tumor: morphologic grading and survival time in 83 dogs. Veterinary Pathology, 21, 469e474. Pittoni P, Piconese S, Tripodo C, Colombo MP (2011) Tumor-intrinsic and -extrinsic roles of c-Kit: mast cells as the primary off-target of tyrosine kinase inhibitors. Oncogene, 30, 757e769. Pryer NK, Lee LB, Zadovaskaya R, Yu X, Sukbuntherng J et al. (2003) Proof of target for SU11654: inhibition of KIT phosphorylation in canine mast cell tumors. Clinical Cancer Research, 9, 5729e5734. Schlieben P, Meyer A, Weise C, Bondzio A, Einspanier R et al. Differences in the proteome of high-grade versus low-grade canine cutaneous mast cell tumours. Veterinary Journal. in press. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-
stained polyacrylamide gels. Analytical Chemistry, 68, 850e858. Skawran B, Steinemann D, Becker T, Buurman R, Flik J et al. (2008) Loss of 13q is associated with genes involved in cell cycle and proliferation in dedifferentiated hepatocellular carcinoma. Modern Pathology, 21, 1479e1489. Sun D, Zhou M, Kowolik CM, Trisal V, Huang Q et al. (2011) Differential expression patterns of capping protein, protein phosphatase 1, and casein kinase 1 may serve as diagnostic markers for malignant melanoma. Melanoma Research, 21, 335e343. Webster JD, Yuzbasiyan-Gurkan V, Kaneene JB, Miller R, Resau JH et al. (2006) The role of c-KIT in tumorigenesis: evaluation in canine cutaneous mast cell tumors. Neoplasia, 8, 104e111. Webster JD, Yuzbasiyan-Gurkan V, Miller RA, Kaneene JB, Kiupel M (2007) Cellular proliferation in canine cutaneous mast cell tumors: associations with cKIT and its role in prognostication. Veterinary Pathology, 44, 298e308. Westermeier R, Scheibe B (2008) Difference gel electrophoresis based on lys/cys tagging. Methods in Molecular Biology, 424, 73e85. Yarar D, D’Alessio JA, Jeng RL, Welch MD (2002) Motility determinants in WASP family proteins. Molecular Biology of the Cell, 13, 4045e4059. Yokotsuka M, Iwaya K, Saito T, Pandiella A, Tsuboi R et al. (2011) Overexpression of HER2 signaling to WAVE2-Arp2/3 complex activates MMP-independent migration in breast cancer. Breast Cancer Research and Treatment, 126, 311e318. Zemke D, Yamini B, Yuzbasiyan-Gurkan V (2002) Mutations in the juxtamembrane domain of c-KIT are associated with higher grade mast cell tumors in dogs. Veterinary Pathology, 39, 529e535. Zhang R, Zhou L, Li Q, Liu J, Yao W et al. (2009) Up-regulation of two actin-associated proteins prompts pulmonary artery smooth muscle cell migration under hypoxia. American Journal of Respiratory Cellular and Molecular Biology, 41, 467e475.
May 9th, 2012 ½ Received, Accepted, July 17th, 2012
Available online at www.sciencedirect.com
www.elsevier.com/locate/jcpa
NEOPLASTIC DISEASE
Tandem Duplication of KIT Exon 11 Influences the Proteome of Canine Mast Cell Tumours P. Schlieben*, A. Meyer*, C. Weise†, A. Bondzio‡, A. D. Gruber* and R. Klopfleisch* * Department of Veterinary Pathology, Freie Universit€at Berlin, Robert-von-Ostertag-Straße 15, 14163, † Institute of Chemistry and Biochemistry, Freie Universit€at Berlin, Thielallee 63, 14195 and ‡ Department of Veterinary Biochemistry, Freie Universit€at Berlin, Oertzenweg 19b, 14163 Berlin, Germany
Summary Mutations with permanent activation of the stem cell factor receptor KIT have been identified as one potential cause for canine cutaneous mast cell tumours (MCTs). The exact changes in global gene expression patterns associated with permanent activation of KIT in these tumours are unknown. The present study compares, by the use of two dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization timeof-flight mass spectrometry, the proteomes of canine MCTs, with and without KIT exon 11 tandem duplication. Fifteen differentially expressed proteins were identified in mutated MCTs. These are mainly involved in cytoskeleton structure and cell motility (ACTR2, ACTB and CAPPA1), cell signalling (ARHGDIA) and lipid metabolism (ALOX15 and ACSBG4), or are serum proteins. The results therefore support the notion that KIT mutation is associated with changes in the proteome of affected cells with a major effect on the composition of the cytoskeletal proteome and cell motility proteins. No overlaps were identified when the results were compared with a recent study on the proteomic differences between low- and high-grade tumours, suggesting that KIT-mutated tumours may be regarded as a separate entity of high-grade tumours with potential relevance to therapeutic strategies. Ó 2012 Elsevier Ltd. All rights reserved. Keywords: dog; KIT; mast cell tumours; proteomic analysis
Several mutations in the stem cell factor receptor KIT have been identified as potential causes of canine cutaneous mast cell tumours (MCTs) (London et al., 1996). Of these, tandem duplication at exon 11 is the best analysed and the most relevant KIT mutation (London et al., 1999; Zemke et al., 2002; Letard et al., 2008). This mutation causes a conformational change in the juxtamembrane domain of the receptor and autophosphorylation of its tyrosine kinase domain, independent from the presence of its ligand, the stem cell factor (Pryer et al., 2003). The resulting permanent KIT activation leads to increased MCT cell proliferation and survival by influencing the vast majority of the active canine genome (Pryer et al., 2003; Masson and Ronnstrand, 2009; Correspondence to: R. Klopfleisch (e-mail: robert.klopfleisch@fu-berlin. de). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2012.07.006
Klopfleisch et al., 2012). This mutation is more prevalent in grade III canine MCTs with an incidence of 17e35% when compared with approximately 8% in lower grade MCTs (Downing et al., 2002; Letard et al., 2008). The signalling cascade of the KIT pathway has been well established in several species and it is accepted that permanent activation by KIT mutation leads to increased proliferation of canine MCT cells (Webster et al., 2007; Nurmio et al., 2008; Pittoni et al., 2011). Nevertheless, changes in the global gene expression patterns associated with tandem duplication in KIT exon 11 are unknown. High- and low-grade MCTs differ in their global protein expression patterns independent of their KIT mutation status (Schlieben et al., in press). Major expression differences are restricted to 13 proteins Ó 2012 Elsevier Ltd. All rights reserved.
319
Canine Mast Cell Tumour Proteome
that are associated mainly with the cellular stress response, cell motility and metastasis, iron metabolism and mast cell differentiation. The aim of the present explorative study was to identify differences between the proteome of MCTs with and without KIT exon 11 tandem duplication by employing two dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF-MS) (Westermeier and Scheibe, 2008; Galvao et al., 2011). Six canine cutaneous MCTs were included in the study. Four tumours were scored as high grade or grade III and two tumours as low grade or grade II according to the Kiupel and Patnaik grading systems, respectively (Table 1; Patnaik et al., 1984; Kiupel et al., 2011). Tumour specimens were fixed in 4% neutral buffered formalin and processed routinely for microscopical examination. Parallel samples were snap frozen in liquid nitrogen within 15 min of resection and were stored at -80 C until further use. Macrodissection and sample preparation, including protein extraction and quantification, were performed as previously described (Klopfleisch et al., 2010a; Klose et al., 2011). Polymerase chain reaction (PCR) amplification for identifying KIT exon 11 status was performed as described previously using the forward primer 50 -CCATGTATGAAGTACA GTGGAAG-30 and the reverse primer 50 -GTTCCCTAAAGTCATT GTTA CACG-30 (Jones et al., 2004; Webster et al., 2006). The PCR protocol was adapted as described by Meyer et al. (in press). Amplified PCR products were fractionated by gel electrophoresis on an ethidium bromide-stained 2.5% agarose gel. PCR products of three tumours identified tandem duplications in exon 11 (Table 1). For 2D-DIGE, 50 mg protein was labelled with 400 pmol of the respective dye according to the manufacturer’s guidelines (GE Healthcare, Freiburg, Germany). Immobilized non-linear pH gradient (IPG) strips (pH 3e7) (GE Healthcare) were used in addition to an Ettan IPGphor 3 isoelectric focus-
sing (IEF) unit (Ettan IPGphor Manifold; GE Healthcare) for a total of 50 kVh at 20 C and 75 mA/strip for IEF. Two steps of equilibration followed IEF, before strips were transferred to the top of gels for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE; 12.5%) and sealed with 0.5% low melting point agarose. The two dimensional molecular weight separation was carried out using an Ettan DALTsix electrophoresis unit (GE Healthcare) with the following running parameters: 60 mA for 1 h, 240 mA for 1 h and 300 mA for 5 h. Each protein extract was analysed initially on at least three silver-stained 2D-PAGE gels to ensure reproducibility prior to quantitative analysis by 2DDIGE. Spot visualization, detection, matching and quantification of spot intensity were performed using a Typhoon 9400 fluorescence scanner and the DeCyder 2D Software, Version 7.0 (both GE Healthcare). An unpaired Student t test with P values <0.05 considered significant was used to identify differences in protein expression between KIT-mutated and nonmutated MCTs. Only spots present in all gels and with a ratio of spot intensity of more than 1.5 between the two groups were selected for subsequent protein identification by MS. For identification of differentially expressed proteins, silver-stained gels loaded with 350 mg of protein were prepared for MS analysis (Shevchenko et al., 1996; Klopfleisch et al., 2010a). An Ultraflex-II TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a smart beam laser was used, as well as a-cyano-4hydroxycinnamic acid (CHCA) as a matrix, to measure protein digests in the reflector mode. Trypsin in-gel digestion and peptide mass fingerprint database searches were performed as described previously (Shevchenko et al., 1996; Klose et al., 2011). Fifteen differentially expressed proteins were identified in mutated MCTs when compared with nonmutated MCTs. Seven of these were up-regulated and eight were down-regulated (Table 2). Most of the up-regulated proteins in mutated MCTs are associated with cytoskeleton structure or cell motility
Table 1 Characterization of dogs and tumours Dog
1 2 3 4 5 6
Breed
Age (years)
Histological grade (Patnaik et al., 1984)
Histological grade (Kiupel et al., 2011)
Tandem duplication in exon 11
Pit bull terrier Beagle Shar Pei Staffordshire bull terriercross Beagle Boxer
16 13 13 15
III III II III
High High Low High
+ + +
7 8
III II
High Low
P. Schlieben et al.
320
Table 2 Proteins differentially expressed in KIT-mutated versus non-mutated MCTs Spot
Gene
Ratio
t-test
Accession number in NCBI database
MW [Da]*
pI*
MOWSE score
Assigned peptides
Sequence covered [%]
gij73955437 (Ca) gij73969820 (Ca) gij73958055 (Ca) gij284795268 (Ca) gij73981496 (Ca) gij73964747 (Ca) gij74003009 (Ca)
76, 599 46, 025 31, 394 59, 776 33, 135 23, 436 60, 055
5.98 6.06 5.04 5.50 5.53 5.12 5.58
156 214 72 138 138 85 96
20 26 7 11 12 7 10
37 54 28 18 53 33 17
Down-regulated proteins in KIT-mutated MCTs D1 FGG 6.90 0.0057 gij73977992 (Ca) D2 APOA1 6.28 0.013 gij73955106 (Ca) D3 AHSG 5.59 0.024 gij74003450 (Ca) D4 ALB 4.07 0.031 gij55742764 (Ca) D5 IGGC 3.65 0.00019 gij17066528 (Ca) D6 IGGD 3.16 0.012 gij17066530 (Ca) D7 ACSBG1 3.12 0.018 gij309243088 (Sus) D8 BCAS4 1.60 0.0022 gij115497804 (Bos)
50, 027 30, 163 40, 515 70, 556 52, 779 52, 169 78, 823 18, 398
5.74 5.28 5.70 5.52 6.16 6.23 7.81 4.97
176 140 81 232 105 37 76 76
16 12 1† 28 9 1† 9 6
45 37 5† 47 24 2† 12 30
Up-regulated proteins in KIT-mutated MCTs U1 ALOX15 +5.25 0.029 U2 ACTR2 +2.91 0.022 U3 ACTB +2.29 0.0013 U4 CPNE1 +2.03 0.022 U5 CAPPA1 +1.83 0.014 U6 ARHGDIA +1.69 0.049 U7 CCT5 +1.52 0.041
ALOX15, arachidonate 15-lipoxygenase; ACTR2, actin-related protein 2; ACSBG1, long chain fatty acid CoA-ligase1; ACTB, actin beta; AHSG, fetuin-A; ALB, serum albumin; APOA1, apolipoprotein A-I; ARHGDIA, Rho GDP dissociation inhibitor alpha; BCAS4, breast carcinoma-amplified sequence 4; Ca, Canis lupus familiaris; CAPPA1, capping protein (actin filament) muscle Z-line; CCT5, T-complex protein 1 epsilon subunit; CPNE1, copine-1; Da, Dalton; FGG, fibrinogen gamma chain; IGGC, immunoglobulin heavy chain C; IGGD, immunoglobulin gamma heavy chain C/D; MW, molecular weight; MOWSE, molecular weight search; pI, isoelectric point; Sus, Sus scrofa; Bos, Bos taurus. * Listed molecular weights and pI values correspond to the listed accession numbers, which sometimes belong to species other than Canis familiaris. † Proteins were identified by unique peptide sequencing using MALDI-TOF-MS/MS.
including actin-related protein 2 (ACTR2; spot U2, ratio 2.91), actin beta (ACTB; spot U3, ratio 2.29) and capping protein (actin filament) muscle Z-line (CAPPA1; spot U5, ratio 1.83). Most of the down-regulated proteins were extracellular serum proteins including fibrinogen gamma chain (FGG; spot D1, ratio 6.90), apolipoprotein A1 (APOA1; spot D2, ratio 6.28), fetuin-A (AHSG; spot D3, ratio 5.59), serum albumin (ALB; spot D4, ratio 4.07) and immunoglobulin gamma heavy chain C (IGGC; spot D5, ratio 3.65) and immunoglobulin gamma heavy chain D (IGGD; spot D6, ratio 3.16). In addition, Rho GDP dissociation inhibitor alpha (ARHGDIA; spot U6, ratio 1.69), T-complex protein 1 epsilon subunit (CCT5; spot U7, ratio 1.52), copine-1 (CPNE1; spot U4, ratio 2.03) and arachidonate 15-lipoxygenase (ALOX15; spot U1, ratio 5.25) were also up-regulated in mutated MCTs, while long chain fatty acid CoA-ligase1 (ACSBG4; spot D7, ratio 3.12) and breast carcinoma-amplified sequence 4 (BCAS4; spot D8, ratio 1.60) were identified as down-regulated proteins in mutated MCTs. Although KIT and its mutations have been the subject of intense research, details of the downstream pathways affected by increased KIT activity are mostly unknown. In the present preliminary explorative study, 15 differentially expressed proteins were
identified covering several aspects of cell metabolism. Taking into account that further investigations in a larger group of tumours are needed to confirm the findings, four major conclusions can be drawn from this study. Firstly, KIT-mutated MCTs differ significantly from non-mutated MCTs in their proteome. However, the number of these proteins was small in light of the thousands of proteins in the canine proteome. This small number may in part have caused insufficient sensitivity of the proteomic methods used to identify more subtle differences in protein expression levels. The data nevertheless indicate that only a few common changes exist in the proteome of KIT-mutated versus non-mutated MCTs. Secondly, mutated MCTs differ from non-mutated MCTs mainly in the quantitative composition of selected cytoskeletal and cell motility proteins and proteins associated with lipid metabolism and the content of serum proteins, but not receptor proteins such as CD25, as shown by immunohistochemistry (Meyer et al., in press). For instance, three proteins associated with the cytoskeleton or cell motility were upregulated in mutated MCTs. All three, ACTR2, CAPPA1 and ACTB, contribute to the overall cell shape, but also to cell motility, for instance by the formation of lamellipodes (Butler and Cooper, 2009; Zhang et al., 2009), and changes in these proteins
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may contribute to the pleomorphism of cells from high-grade MCTs. In addition, the dysregulation of expression of these three genes facilitates tumour cell invasion and metastasis in human and canine tumours (Yarar et al., 2002; Altschuler and Willison, 2008; Nomura et al., 2008; Klopfleisch et al., 2010c; Carnell and Insall, 2011; Sun et al., 2011; Yokotsuka et al., 2011). The changes in the expression level of proteins involved in lipid metabolism or exocytosis of lipid membrane-bound vesicles including ALOX15, CPNE1 and ACSBG1 (Skawran et al., 2008; Kerjaschki et al., 2011) may be associated with a different metabolism of lipid mediators or eicosanoids, which are major secretory products of normal mast cells and involved in inflammation and haemodynamic regulation, and have been described for metastatic canine mammary tumours (Klopfleisch et al., 2010b). Thirdly, the significantly decreased levels of serum proteins, including FGG, APOA1, ALB, IGGC and IGGD, in the mutated MCTs are remarkable. As a possible explanation, we speculate that these changes may be associated with a different extracellular matrix composition in these tumours. Nonmutated MCTs may thus induce increased vascular dilation or permeability when compared with the mutated tumours, possibly due to increased secretion of vasoactive factors. This would, however, imply that non-mutated MCTs are more activated or better differentiated than mutated MCTs, an assumption that is not overtly supported by the histological appearance of the tumours in the groups. Fourthly, there is apparent overlap of the proteins identified in this study with the proteins identified by comparison of low- versus high-grade MCTs (Schlieben et al., in press). This may support the hypothesis that high-grade MCTs with KIT exon 11 tandem duplications should be viewed as a separate entity within canine high-grade MCTs, which differ in their proteome composition from non-mutated high-grade MCTs. These specific differences in protein expression levels are of potential interest for evidence-based design of therapeutic strategies for high-grade MCTs with and without KIT mutations.
Conflict of Interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.
Acknowledgements We thank M. Sch€ arig and P. Schulze for technical support. This project was supported by the German
Research foundation (DFG, KL2240). The present study is part of the doctoral thesis of P. Schlieben.
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May 9th, 2012 ½ Received, Accepted, July 17th, 2012