34 Med. Weter. 2016, 72 (1), 34-40

Praca oryginalna Original paper Association between GGA1 polymorphisms and occurrence of mammary mixed tumors and aging in domestic bitches1)

WIESŁAWA KRANC*, ADRIAN CHACHUŁA**, KATARZYNA WOJTANOWICZ-MARKIEWICZ***, KATARZYNA ZAORSKA**, EDYTA OCIEPA***, ADAM PIOTROWSKI*, DOROTA BUKOWSKA***, SYLWIA CIESIÓŁKA**, SYLWIA BORYS****, HANNA PIOTROWSKA****, AGNIESZKA SKOWROŃSKA*****, MARCIN NOWAK******, PAWEŁ ANTOSIK***, KLAUS-PETER BRÜSSOW***, BARTOSZ KEMPISTY*, **, MAŁGORZATA BRUSKA*, MICHAŁ NOWICKI**, MACIEJ ZABEL**, *******

*Department of Anatomy, **Department of Histology and Embryology, Medicine Faculty I, Poznan University of Medical Sciences, Swiecickiego 6 St., 60-781 Poznan, Poland ***Institute of Veterinary Sciences, Faculty of Animal Breeding and Biology, Poznan University of Life Sciences, Wolynska 35 St., 60-637 Poznan, Poland ****Department of Toxicology, Faculty of Farmacy, Poznan University of Medical Sciences, Dojazd 30 St., 60-631 Poznan, Poland *****Department of Human Physiology, Faculty of Medical Sciences, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland ******Department of Pathology, Faculty of Veterinary Medicine, Wroclaw University of Life Sciences, C. K. Norwida 31 St., 50-375 Wrocław, Poland *******Department of Histology and Embryology, Wroclaw Medical University, 6a Chalubinskiego St., 50-368, Wroclaw, Poland

Received 13.07.2015 Accepted 03.11.2015

Kranc W., Chachuła A., Wojtanowicz-Markiewicz K., Zaorska K., Ociepa E., Piotrowski A., Bukowska D., Ciesiółka S., Borys S., Piotrowska H., Skowrońska A., Nowak M., Antosik P., Brüssow K.-P., Kempisty B., Bruska M., Nowicki M., Zabel M. Association between GGA1 gene polymorphisms and occurrence of mammary mixed tumours and aging in domestic bitches Summary In recent years the number of malignant mammary gland tumor occurrences in domestic bitches has increased. This may be related to advanced diagnostic technologies, availability of a quick diagnosis, as well as genomic changes. Still the main genetic reasons for tumor development are frequently occurring gene mutations and/ or polymorphisms. The mutations in target often lead to amino acid changes in the structure of , which may be the reason for uncontrolled cells proliferation, and induction and growth. Therefore, in this article we described the analysis of mutation/polymorphisms frequency of the GGA1 gene in association with canine malignant mammary gland tumor occurrence and aging. In this study, blood samples were obtained from 22 female dogs diagnosed with mammary tumors. Moreover, blood samples from geriatric (> 5 to 10 years old; n = 15), mature adult (> 2 to 5 years old; n = 10) and young (from 1 to 2 years old; n = 11) dogs were also collected. 36 bitches diagnosed on account of other reasons served as controls. After the Sanger sequencing analysis, 14 single nucleotide variations were identified, of which 3 were already known polymorphisms and 11 novel variations. We observed differences in frequencies for 4 polymorphisms (g.A-172T, c.T24C, c.A692G, c.C1185T) between cases and controls. Moreover, we found increased prevalence of heterozygotes and alternative alleles in 3 polymorphisms (g.A-172T, c.A692G, c.C1185T) in the tumor diagnosed group as compared to the control. Although the results were not statistically significant, it is worth mentioning the slightly different pattern of genetic segregation of alleles in the abovementioned polymorphisms in control and tumor patients. Keywords: mammary gland tumor, bitches, GGA1, canine

1) Supported by the Polish Ministry of Scientific Research and Higher Education (Grant No. 5279/B/P01/2011/40). Med. Weter. 2016, 72 (1), 34-40 35

The median lifespan of dogs varies as a function Despite the fact that this gene and its mutations have of breed, with larger breeds typically having shorter been intensively studied in humans and animals, in lifespans than smaller breeds (7). The lifespan of dogs the case of other tumors, mutations/polymorphisms is also affected by diet and environmental conditions of this gene in bitches with mammary gland mixed in which the dogs are kept. These same factors influ- tumor in relation to aging of the organism have not ence the development of breast mixed tumors. In recent been studied. The goal of this study was to investigate years, different types of tumor have been diagnosed polymorphisms and mutations of GGA1 gene. in domestic bitches, and mixed tumors are the pre- dominating cause of mortality and morbidity of female Material and methods dogs (13). Mammary gland tumor is the most common Patients and samples collection. Blood samples were neoplasma diagnosed in females dog, however, the obtained from 22 female mongrels, mature adult 2 to knowledge on the molecular carcinogenesis of canine 5-years-old dogs diagnosed with mammary mixed tumors mammary tumor (CMT) is not yet fully recognized during the surgery in the Small Animal Clinic, University (12, 13). There are differences and similarities between of Life Sciences, Poznan, Poland. Blood was collected canine and human mammary tumors at the molecular during standard surgery procedures. 36 bitches served as level. The most important thing was to develop and controls; in the good condition, clinically healthy, lab tests to define the canine genome map, which enabled the in physiological norm. They were divided into 3 subgroups identification of new molecular markers for CMT of differing age according to the classification by Jugdutt et induction, invasion, and/or progression (15). al (11): geriatric (> 5 to 10 years old; n = 15), mature adult The GGA1 belongs to GGAs family. This is a ubiq- (> 2 to 5 years old; n = 10) and young (from 1 to 2 years uitous coat , which belongs to the large pro- old; n = 11). Blood was collected from the saphenous vein/ tein family of mammalian GGAs. Each of the three jugular vein in vials with EDTA and frozen in –80°C until mammalian GGA proteins have a modular structure further analyses. Material (tumor) was fixed in parafin and formed into 60 blocks. Blocks were cut into 5 µm slices on consisting of: (a) an NH2-terminal VHS domain, (b) automatic rotary microtome (Leica RM 2255). Each slice a region of homology to the ear of the γ1-adaption was stuck on a SuperFrost Plus glass slide (Thermo Scien- submint of AP-1 related to the γ2-adaption, followed tific) and heated to 60°C for 90 minutes. After heating glass by a GAT domain, a variable domain (1, 2, 8). The slides were incubated overnight in 37°C. After incubation γ-adaptin ear homology domain contributes to GGA glass slides were stained in an automatic slide stainer (Robot functioning but is not necessary for full functioning Stainer HMS 740, Microm) with standard H + E method, that can be restored by replacing the GGA ear domain i.e.: Mayer hematoxylin for 25 min., tap water for 8 min and with the γ-adaptin ear domain. Deleting the γ-adaptin 0.1% eosin solution for 45 min. Stained slides were dried gene together with the two GGA genes exacerbates the with alcohol and covered with transparent Histofluid resin phenotype in yeast, suggesting that they function on and cover slip in automatic coverslipper (Leica CV5030). parallel regulatory pathways. Tab. 1. PCR primer pairs with thermal conditions used for the amplifi- In mammalian cells, the association of cation of GGA gene exons GGAs with the membrane is extremely unstable and it is therefore not associated Exon Sequences Annealing temp./elongation with purified clathrin-coated vesicles or with exon 1 F 5’-TAATTTTGCCCGAATGGCTA-3’ 58°C/45 sec R 5’-TTCTGTGGGCACAACCATAG-3’ any kinds of the other components of AP-1 exon 2 F 5’-GAATATGCTTGTGGGACTGGA-3’ 58°C/30 sec complex (1). The GGA1 is not associated R 5’-TCAGATCAAAGAAGGCCAAG-3’ with GGA2, but they are co-localized on exon 3 F 5’-ACTCTTTTCCCCTTCGCTTC-3’ 58°C/30 sec perinuclear membranes, which correspond R 5’-TCAATTATGCCACAAAACAAACA-3’ to trans elements of the Golgi stack and the exon 4 F 5’-TCCAAATGTGGTTTCTGGTG-3’ 57°C/30 sec trans-Golgi network. Moreover, the AP-1 R 5’-GGGAAGACAGCATCTTCTCAT-3’ contains bound casein kinase-2 that phos- exon 5 F 5’-TGTTGAGCTTTAAACCATACTTCC-3’ 61°C/30 sec phorylated GGA1 and GGA3, thereby caus- R 5’-GCAAGCATCTGATCCTCCAT-3’ ing autoinhibition. It was demonstrated that exon 6 F 5’-CACTTAAGGGTTCATGGATGC-3’ 62°C/30 sec this autoinhibition could induce the directed R 5’-AGCACTCATCAGTCAATTGAAAA-3’ exon 7 F 5’-CACATGTGTGTTTTGTAATGCTG-3’ 58°C/30 sec transfer of mannose 6-phosphate receptors R 5’-GGGAAAGTAAATGGCACGAA-3’ from the GGAs to AP-1 (2). Doray et al. (2) exon 8, exon 9 F 5’-ACCAGGCAAATGAATTCCAC-3’ 58°C/30 sec concluded that GGAs and the AP-1 complex R 5’-TTGATTTTTCTGTAATGAGGCTTT-3’ interact to package mannose 6-phosphate exon 10 F 5’-TGCATAAAAACAGCAGTGTCAG-3’ 59°C/30 sec receptors into AP-1-containing coated ves- R 5’-AAGGCAAAATAATCAATAGCCACT-3’ icles, because mannose 6-phosphate recep- exon 11 F 5’-TGAGTATAACCCATTGCAGAGAAA-3’ 58°C/45 sec tors, defective in binding to GGAs, were R 5’-TTCGTGCTGCTTTTGTTGAC-3’ poorly incorporated into adaptor protein exon 12 F 5’-GCACTAGTGGATTTTGTAAATCATCA-3’ 58°C/30 sec complex containing clathrin coated vesicles. R 5’-TCTCCAAGGTTTGGAAGGAA-3’ 36 Med. Weter. 2016, 72 (1), 34-40

Molecular analysis. Genomic DNA was extracted from Tab. 2. Single nucleotide variations identified in theGGA gene whole peripheral blood using QIAamp DNA Blood Mini in the studied subjects Kit (Qiagen), according to the manufacturer’s instructions. Name of variation Localization nt change aa change DNA was resuspended in 100 µl of Qiagen elution buffer 1 rs8932443 5’UTR T/– – and stored in –20°C. 12 exons of the GGA gene (exon 1 – exon 12) were amplified in the polymerase chain reac- 2 g.A-172T 5’UTR A > T – tion (PCR) including up to 70-bp flanking regions of every 3 g.G-50T 5’UTR G > T – exon. The sequences of the primers used with thermal and 4 c.T24C exon 1 T > C syn: G8G (GGT > GGC) time conditions are listed in Tab. 1. The reactions were car- 5 c.T522C exon 5 T > C syn: C174C (TTT > TTC) ried out in a total volume of 12.5 µl containing: 10 × Taq 6 g.A43540G intron 5 A > G – DNA Polymerase buffer with MgCl2, 5 × GC-rich solu- tion, 0.24 mM dNTPs, 0.5 µM of the primers, 1 unit of Taq 7 g.T43580A intron 5 T > A – Polymerase (Roche) and 40-60 ng of genomic DNA. The 8 rs8602105 exon 6 C > T syn: H208H (CAC > CAT) PCR cycle conditions were: an initial denaturation at 94°C 9 c.A692G exon 7 A > G non: E231G (GAA > GGA) for 4 min, followed by 35 cycles of denaturation at 95°C 10 g.T171934C intron 7 T > C – for 30 sec, annealing at temperatures shown in Table 1 for 1 min and elongation at 72°C for 30/45 sec, with a final 11 g.A172092C intron 8 A > C – extension at 72°C for 7 min. PCR products were purified 12 g.T172127C intron 8 T > C – using membrane plates (Millipore) and used as templates 13 c.C1185T exon 11 C > T syn: D395D (GAC > GAT) in PCR-sequencing reamplification. The latter reaction was 14 rs23382375 exon 12 G > A syn: Q427Q (GAG > GAA) performed in Veriti 96 well Thermal Cycler using BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) Explanations: (g. = genomic; c. = coding; syn = synonymous; and one of the specific primers (Forward or Reverse). Ream- nt = nucleotide; aa = amino acid) plification products were purified with EDTA and ethanol precipitation and separated by electrophoresis using ABI non-synonymous and changed the amino acid sequence 3130 sequencer (Applied Biosystems). of the protein. Statistical analysis. Genotypes obtained in the study Genotype and allele frequencies determined fre- were aligned with the reference sequences from the Ensembl quencies of alleles and genotypes for all 14 variants. database. Single nucleotide variations were assessed and Distribution of all GGA genotypes was consistent with calculation of the chi-square test for deviation from the HWE. Frequencies and OR values for chosen varia- Hardy-Weinberg equilibrium (HWE) was performed. The tions are shown in Tab. 3. There were differences in genotype and allele frequencies were evaluated and com- the genotype and allele frequencies between the tumor pared between study and control groups using the Fisher’s and control group for heterozygote, alternative homo- exact test. Odds ratio values (OR) were also evaluated and zygote and for alternative allele for 4 polymorphisms p < 0.05 was considered statistically significant. Addition- (g.A-172T, c.T24C, c.A692G, c.C1185T). Although they ally, we used Haploview 3.2 software to obtain the GGA were not statistically significant or were on the bound- gene structure. Linkage disequilibrium values (LD) were ary of statistical significance, they could suggest an calculated as R2 value and the Gabriel et al algorithm was inclination to risk or protective variants with reference used. to a larger group of subjects. Much higher prevalence of heterozygotes and alternative alleles was observed in Results and discussion 3 polymorphisms (g.A-172T, c.A692G, c.C1185T) in the In total, Sanger sequencing of 12 exons (exon 1 to tumor group in comparison with the control group. The exon 12) with approximately 70-bp non-coding splicing odds ratio values were quite high (see Tab. 3); however regions of the GGA gene in 58 subjects was performed. the results for all of the variations were not statistically Upon alignment of obtained and reference sequences 14 significant or were on the boundary of significance. single nucleotide variations were identified, of which 3 Very high OR values for both heterozygote AT and for were already known polymorphisms with determined the alternative allele T was observed in polymorphism rs numbers (rs8932443 in 5’UTR region, rs8602105 g.A-172T for the tumor group in comparison with the in exon 6, rs23382375 in exon 12) and 11 novel varia- control group (OR = 8.9 for the AT genotype, p = 0.1647; tions. Of those 11, 2 were localized in the 5’UTR region, OR = 8.5 for the T allele, p = 0.1698). There were no 4 in exons (6, 8) and 5 in introns and splicing regions differences in the genotype and allele frequencies among (2 variations in intron 5 and 8, 1 in intron 7). The num- all 3 control subgroups, as the wild allele A comprised bering of nucleotides was carried with reference to the all genotypes in that group. Therefore, the more than first nt in the first coding triplet AUG in exon 1. All 8-fold higher incidence of the T allele in tumor patients single nucleotide variations identified in the study are could indicate a possible risk allele for tumor presence. listed in Tab. 2. There were high OR values for heterozygote or alter- Eleven of the nucleotide changes were substitutions native homozygote and alternative allele in the other two and were biallelic and 1 was in/del type of variation. Of putative risk variants for the tumor group in comparison all 6 variations in coding regions, 5 were synonymous with the control group, and differences were also seen and did not change the amino acid sequence and 1 was in control subgroups. In polymorphism c.A692G there Med. Weter. 2016, 72 (1), 34-40 37

Tab. 3. Frequencies and odds ratio values for chosen single nucleotide variations for the GGA gene Variation Tumor patients Controls p value OR (95% CI) g.A-172T Genotype frequency (n = 22) (n = 36) AA 20 (0.91) 36 (1.0) AT 2 (0.09) 0 T vs. C 0.1647 8.9 (0.4-194.5) subgroup Y (n = 11) AA 11 (1.0) subgroup A (n = 10) AA 10 (1.0) subgroup G (n = 15) AA 15 (1.0) Allele frequency A 42 (0.95) 72 (1.0) T 2 (0.05) 0 T vs. C 0.1698 8.5 (0.4-182) subgroup Y A 22 (1.0) subgroup A A 20 (1.0) subgroup G A 30 (1.0) c.T24C Genotype frequency (n = 22) (n = 36) TT 12 (0.54) 15 (0.42) CT 7 (0.32) 15 (0.42) C vs. T 0.4544 1.5 (0.5-4.7) CC 3 (0.14) 6 (0.16) subgroup Y (n = 11) TT 3 (0.27) CT 6 (0.55) CC 2 (0.18) subgroup A (n = 10) TT 6 (0.6) CT 3 (0.3) Y vs. A 0.2621 2.8 (0.5-16.9) CC 1 (0.1) Y vs. A 0.5974 2 (0.2-26.2) subgroup G (n = 15) TT 6 (0.4) CT 6 (0.4) Y vs. G 0.464 1.8 (0.4-8.7) CC 3 (0.2) Allele frequency T 31 (0.7) 45 (0.625) C 13 (0.3) 27 (0.375) C vs. T 0.3828 1.4 (0.6-3.2) subgroup Y T 12 (0.55) C 10 (0.45) subgroup A T 15 (0.75) C 5 (0.25) Y vs. A 0.172 2.5 (0.7-9.3) subgroup G T 18 (0.6) C 12 (0.4) 38 Med. Weter. 2016, 72 (1), 34-40

Tab. 3. Continuation Variation Tumor patients Controls p value OR (95% CI) c.A692G Genotype frequency (n = 22) (n = 36) AA 19 (0.86) 33 (0.92) AG 2 (0.09) 3 (0.08) T vs. C 0.9206 1.1 (0.2-7.2) GG 1 (0.05) 0 T vs. C 0.3254 5.1 (0.2-130.6) subgroup Y (n = 11) AA 11 (1.0) subgroup A (n = 10) AA 9 (0.9) AG 1 (0.1) A vs. Y 0.4456 3.6 (0.1-99.9) subgroup G (n = 15) AA 13 (0.87) AG 2 (0.13) G vs. Y 0.3652 4.3 (0.2-98.1) Allele frequency A 40 (0.91) 69 (0.96) G 4 (0.09) 3 (0.04) T vs. C 0.2912 2.3 (0.5-10.8) subgroup Y A 22 (1.0) subgroup A A 19 (0.95) G 1 (0.05) A vs. Y 0.455 3.5 (0.1-90) subgroup G A 28 (0.93) G 2 (0.07) G vs. Y 0.3832 4 (0.2-86.4) c.C1185T Genotype frequency (n = 22) (n = 36) CC 18 (0.82) 35 (0.97) CT 4 (0.18) 1 (0.03) T vs. C 0.0757 7.8 (0.8-74.8) subgroup Y (n = 11) CC 11 (1.0) CT 0 G vs. Y 0.6059 2.4 (0.1-64.1) subgroup A (n = 10) CC 10 (1.0) CT 0 G vs. A 0.6447 2.2 (0.1-58.8) subgroup G (n = 15) CC 14 (0.93) CT 1 (0.07) Allele frequency C 40 (0.91) 71 (0.99) T 4 (0.09) 1 (0.01) T vs. C 0.0843 7.1 (0.8-65.7) subgroup Y C 22 (1.0) T 0 G vs. Y 0.6174 2.3 (0.1-58.6) subgroup A C 20 (1.0) T 0 G vs. A 0.6577 2.1 (0.1-53.8) subgroup G C 29 (0.97) T 1 (0.03) Explanations: (Y = young; A = adult; G = geriatric) Med. Weter. 2016, 72 (1), 34-40 39 was a 5-fold higher incidence of the alternative homo- zygote GG in the tumor group (OR = 5.1, p = 0.3254) and also higher prevalence of the heterozygote AG in more advanced age subgroups: adult and geriatric sub- groups in comparison with the young subgroup (OR = 3.6, p = 0.4456 for adults vs. young and OR = 4.3, p = 0.3652 for geriatrics vs. young). The results for the allele frequency were similar. High OR values for the G allele were observed both for the tumor group in comparison with controls (OR = 2.3, p = 0.2912) and in adults and geriatrics in comparison with young subjects (OR = 3.5, p = 0.4550 for adults vs. young and OR = 4, p = 0.3832 for geriatrics vs. young). Moreover, single nucleotide substitution in this variation changed the amino acid sequence of the protein from polar glutamic acid into non-polar hydrophobic glycine, which could change the protein activity. That could strongly indicate not only the alternative allele G as a risk variant but also higher Fig. 1. Structure of GGA gene shown as a case-control asso- incidence of tumor presence with reference to the age ciation of a subject. type and allele frequency between tumor and control Moreover, differences in allele and genotype frequen- groups. Moreover, the OR values for the rest of identified cies in polymorphism c.C1185T between study groups variations did not differ between the study groups and suggest it to be a risk variant for tumor incidence. were close to the neutral ‚1’ value. Very high OR values for both heterozygote CT and Haploview analysis. The GGA gene structure of the alternative allele T were observed for the tumor group case-control association study revealed no haplotype in comparison with the control group (OR = 7.8, p = blocks, according to the Gabriel et al. (4) algorithm 0.0757 for the CT genotype and OR = 7.1, p = 0.0843 (Fig. 1). There was a high linkage between variations: for the T allele). Moreover, there was over a 2-fold c.T522C and c.A692G (in 58% of all subjects studied) higher prevalence of heterozygote CT and the T allele in and between variations: g.T43580A and g.T172127C Geriatric subjects in comparison with two younger age (in 100% of all subjects studied). Those values did not subgroups. The nucleotide substitution did not change differ between control and tumor groups separately. the amino acid sequence of the protein; however, the Interestingly, 100% LD between g.T43580A and nucleotide change itself could influence the folding of g.T172127C was seen only in the control group, as the mRNA and its biological interactions with other both variations were not differentiated and comprised molecules, e.g. splicing factors. Therefore, similarly to only wild homozygotes in all tumor patients. It is worth polymorphism c. A692G, the presence of the alternative mentioning that there was a 30% linkage between varia- allele T in variation c.C1185T could suggest it to be tions: g.G-50T and rs8602105 and between c.T24C and a risk allele for tumor presence and higher incidence of rs8602105 in tumor patients, while no such association tumorigenesis with age. was seen in the control group (data not shown). In contrast, slightly higher prevalence of the hetero- The GGA1 gene and its pathogenic influence on zygote CT and alternative allele C were observed in the organisms has not been widely studied. Several stud- control group in comparison with tumor patients. Also, ies about GGA1 in humans were conducted, but till there were higher OR values for the heterozygote and now only limited information has been available about C allele prevalence in young patients with reference its role in diseases (2, 19). A study was conducted to older age subgroups. Although the results were not about consensus coding sequences of the human breast statistically significant and the OR values were lower and colorectal and determined the sequence of well- than in the case of the putative risk variants described annotated human protein-coding genes in two common above, the higher incidence of the C allele in the control human tumor types (19). 189 genes were identified, group could suggest a putative protective variant with which were mutated at significant frequency. Most of reference to the larger study group. these genes were not known to be altered in tumors and The results in polymorphism g.G-50T were inconclu- are predicted to affect a wide range of cellular functions, sive. There was higher prevalence of the heterozygote including transcription, adhesion, and invasion (21). It GT and the alternative allele T in young subjects in has been reported that genes coding proteins responsible comparison with older age subgroups (OR = 5.5 for the for intercellular trafficking like OTOF, LRBA, AEGP, GT genotype, p = 0.2891 and OR = 5 for the T allele, PLEKHA8, LOC283849, SORL1, KTN1 and GGA1 p = 0.3085 for young vs. adults and OR = 3.1 for the are significantly mutated in the breast (20). It has been GT genotype, p = 0.3816 and OR = 2.9 for the T allele, proposed to call these genes candidate genes, because p = 0.3976 for young vs. geriatrics; data not shown). their influence is not well-known (19). In the same year However, we did not observe any differences in geno- the study about GGA1 and its influence on pathogenesis 40 Med. Weter. 2016, 72 (1), 34-40 of Alzheimer Disease was conducted and it was reported 2. Doray B., Ghosh P., Griffith J., Geuze H. J., Kornfeld .:S Cooperation of GGAs and AP-1 in packaging MPRs at the trans-Golgi network. Science 2002, 297, that the GGA1 protein could be involved in AD patho- 1700-1703. genesis (21). Whilst GGA1 in humans is analyzed in 3. Einem B. von, Wahler A., Schips T., Serrano-Pozo A., Proepper C., Boeckers T. M., Rueck A., Wirth T., Hyman B. T., Danzer K. M., Thal D. R., Arnim von several studies, there is an incomparably lower amount C. A.: The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA) Proteins of studies about GGA1 in animals. Alter Amyloid-β Precursor Protein (APP) Processing through Interaction of Recently, a significant advance has been made in the Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1). PLoS One. 2015, 10:e0129047. doi: 10.1371/journal.pone.0129047 understanding of molecular events of the mammary 4. Gabriel S. B., Schaffner S. F., Nguyen H., Moore J. M., Roy J., Blumenstiel B., gland tumor in humans and dogs (7, 16, 17). This is cru- Higgins J., DeFelice M., Lochner A., Faggart M., Liu-Cordero S. N., Rotimi C., Adeyemo A., Cooper R., Ward R., Lander E. S., Daly M. J., Altshuler D.: The cial for the development of novel effective therapeutic structure of haplotype blocks in the . Science 2002, 296, 2225- agents and strategies. Grüntzig (5) in his own study tried -2229, doi: 10.1126/science1069424 5. Grüntzig K., Graf R., Hässig M., Welle M., Meier D., Lott G., Erni D., Schenker to answer the Swiss canine registry and classified the N. S., Guscetti F., Boo G., Axhausen K., Fabrikant S., Folkers G., Pospischil A.: dog according to the breed, age, sex, neuter status, and The Swiss Canine Registry: a retrospective study on the occurrence of tumours place of residence. Among all dogs diagnosed, 23.5% in dogs in Switzerland from 1955 to 2008. J. Comp. Pathol. 2015, 152, 161-171. doi: 10.1016/j.jcpa.2015.02.005 related to mammary gland tumors (5, 13). The most 6. Head E.: A canine model of human aging and Alzheimer’s disease. Biochim. common group of dogs suffering are middle-age bitches Biophys. Acta. 2013, 1832, 1384-1389. 7. Hirst J., Lindsay M. R., Robinson M. S.: GGAs: Roles of the Different Domains (9, 18, 20). In recent years, an increasing number of and Comparison with AP-1 and Clathrin. Mol. Biol. Cell 2001, 12, 3573-3588. canine molecular tools have been developed, not only on 8. Jensen-Jarolim E., Fazekas J., Singer J., Hofstetter G., Oida K., Matsuda H., genomic, RNA and protein levels. These studies explain Tanaka A.: Crosstalk of carcinoembryonic antigen and transforming growth factor-β via their receptors: comparing human and canine. Immunol. Immunother. specific aspects of canine carcinogenesis, particularly of 2015, 64, 531-537. doi:10.1007/s00262-015-1684-6 the mammary gland. 9. Joshi A., Garg H., Nagashima K., Bonifacino J. S., Freed E. O.: GGA and ARF proteins MODULATE retrovirus assembly and relase. Mol. Cell 2008, 25; 30, The GGAs family are proteins of monomeric clathrin 227-238. doi: 10.1016/j.molcel.2008.03.015 adaptors primarily localized at the trans-Golgi network; 10. Joshi A., Nagashima K., Freedcorre E. O.: Defects in cellular sorting and moreover, they have been reported in late endosomes. retroviral assembly induced by GGA overexpression. BMC Cell Biol. 2009, 29, 10:72. Published online 2009 29. doi: 10.1186/1471-2121-10-72 GGAs contain four domains: VHS (an N-terminal 11. Jugdutt B. I., Jelani A., Palaniyappan A., Idikio H., Uweira R. E., Menon V., Vps27, Hrs, STAM homology), a domain related to Jugdutt C. E.: Aging-related early changes in markers of ventricular and matrix remodeling after reperfused ST-segment elevation myocardial in- cargo proteins with protein receptors (cation-dependent farction in the canine model: effect of early therapy with an angiotensin II mannose 6-phosphate receptors CD-MPR); GAT domain type 1 receptor blocker. Circulation 2010, 27, 122, 341-351. doi: 10.1161/ CIRCULATIONAHA.110.948190 that binds Arfs, ubiquitin, and Tsg101 (GGA and Tom) 12. Kempisty B., Zaorska K., Bukowska D., Nowak M., Ciesiółka S., Wojtanowicz- is responsible for recruitment of GGA proteins to the Markiewic K., Jopek K., Bryja A., Antosik P., Brüssow K.-P., Bruska M., membrane via interaction with GTP-bound Arf; a hinge Nowicki M., Zabel M.: Association between polymorphic variants of RAF1 gene with occurrence of mammary tumor and aging in canines. Drug Disc. Develop. region that recruits clathrin; and the last domain: GAE Deliv. 2014, 1, 1-8. is a C-terminal γ-adaptin ear homology – this domain 13. Klopfleisch R., von Euler H., Sarli G., Pinho S. S., Gärtner F., Gruber A. .:D Molecular carcinogenesis of canine mammary tumours: news from an old binds a few proteins, including rabaptin 5, epsinR, and disease. Vet. Pathol. 2011, 48, 98-116. γ-synergin (10). Joshi (10) in his own study on the ret- 14. Li F., Tian P., Zhang J., Kou C.: The clinical and prognostic significance of mid- rovirus proved that GGA over-expression induces the kine in breast patients. Tumour Biol. 2015, 10 doi: 10.1007/sl3277-015-3710-x 15. Rismanchi S., Muhammadnejad S., Amanpour S., Muhammadnejad A.: First formation of large swollen compartments that sequester pathological study of canine primary breast lymphoma and the description of Arf proteins. its clinicopathological characteristics as an animal model for human primary breast lymphoma. Biomed. Rep. 2014, 201, 3, 75-77. In the current study, the association between the 16. Shafiee R., Javanbakht J., Atyabi N., Kheradmand P., Kheradmand D., GGA1 and occurrence of malignant mammary tumors Bahrami A., Daraei., Khadivar F.: Diagnosis, classification and grading of canine mammary tumours as a model to study human breast: an Clinico- and aging in domestic bitches was examined. GGA1 Cytohistopathological study with environmental factors influencing public health and its properties are well-described in literature, but and medicine. Cell Int. 2013, 13, 79. doi: 10.1186/1475-2867-13-79 according to the discoveries of Sjöblom et al, Wahle et 17. Shapshak P., Duncan R., Kangueane P., Somboonwit C., Sinnott J., Commins D., Singer E., Levine A.: HIV associated dementia and HIV encephalitis II: Genes al. (19, 21) and our recent results we can assume that on 22 expressed in individually microdissected Globus pallidus our knowledge about this protein is very narrow, as it is neurons (Preliminary analysis). Bioinformation 2011, 6, 183-186. 18. Simon P. Langdon: Animal Modeling of Pathology and Studying Tumor Response proven that GGA1 takes part in pathogenesis of severe to Therapy. Curr. Drug Targets 2012, 13, 1535-1547. diseases, such as breast cancer and Alzheimer Disease. 19. Sjöblom T., Jones S., Wood L. D., Parsons D. W., Lin J., Barber T. D., It is very important to provide more studies about GGA1 Mandelker D., Leary R. J., Ptak J., Silliman N., Szabo S., Buckhaults P., Farrell C., Meeh P., Markowitz S. D., Willis J., Dawson D., Willson J. K., Gazdar to expand our knowledge of negative interactions of A. F., Hartigan J., Wu L., Liu C., Parmigiani G., Park B. H., Bachman K. E., GGA1. The GGA1 gene is conserved among a number Papadopoulos N., Vogelstein B., Kinzler K. W., Velculescu V. E.: The consensus coding sequences of human breast and colorectal s. Science 2006, 314, 268-274. of species, e.g.: chimpanzee, Rhesus Monkey, rat, mouse 20. Suer S., Misra S., Saidi L. F., Hurley J. H.: Structure of the GAT domain of and human, which is a big facilitation in research on the human GGA1: A syntaxin amino-terminal domain fold inan endosomal traffick- ing adapter. PNAS 2003, 15,100, 4451-4456. GGA1 gene and its products, as we can transfer research 21. Wahle T., Tha D. R., Sastre M., Rentmeister A., Bogdanovic N., Famulok M., results from animals to humans. Heneka M. T., Walter J.: GGA1 is expressed in the human brain and affects the generation of amyloid beta-peptide. J. Neurosci. 2006, 12838-12846. References 22. Woźna M., Kempisty B., Piotrowska H., Dorszewska J., Bukowska D., Nowicki M.: The immunological, biochemical and molecular bases of canine 1. Angelica E. C. Dell, Puertollano R., Mullins C., Aguilar R. C., Vargas J. D., senescence and carcinogenesis: a review. Vet. Med. (Praha) 57, 350-359. Hartnell L. M., Bonifacino J. S.: GGAs: A Family of ADP Ribosylation Factor- binding Proteins Related to Adaptors and Associated with the Golgi Complex. Corresponding author: mgr Wiesława Kranc, ul. Święcickiego 6, 60-781 Cell Biol. 2000, 149, 81-14994. Poznań