Praca oryginalna Original paper
Analysis of mitogen-activated protein kinase
(MAP2K2) gene polymorphisms in relation to the
occurrence of adenocarcinoma during aging in dogs
1)
BARTOSZ KEMPISTY*, **, KATARZYNA ZAORSKA*, DOROTA BUKOWSKA***,
MARCIN NOWAK****, KATARZYNA WOJTANOWICZ-MARKIEWICZ***, SZYMON POROWSKI*, EDYTA OCIEPA*, PAWEŁ ANTOSIK***, KLAUS-PETER BRÜSSOW*****,
MAŁGORZATA BRUSKA**, MICHAŁ NOWICKI*, MACIEJ ZABEL*, ****** *Department of Histology and Embryology, **Department of Anatomy, Medicine Faculty I,
Poznan University of Medical Sciences, Święcickiego 6 St., 60-781 Poznan, Poland
***Institute of Veterinary, Faculty of Animal Breeding and Biology, Poznan University of Life Sciences, Wojska Polskiego 52 St., 60-637 Poznan, Poland
****Department of Pathology, Faculty of Veterinary Medicine, Wroclaw University of Life Sciences, C. K. Norwida 31 St., 50-375 Wrocław, Poland
*****Institute of Reproductive Biology, Department of Experimental Reproductive Biology, Leibniz Institute for Farm Animal Biology, Dummerstorf, Germany
******Department of Histology and Embryology, Wroclaw Medical University, Chalubinskiego 6a St., 50-368 Wrocław, Poland
Received 09.04.2014 Accepted 14.11.2014
1) Supported by the Polish National Centre of Science (Grant No. 5279/B/P01/2011/40).
Kempisty B., Zaorska K., Bukowska D., Nowak M., Wojtanowicz-Markiewicz K., Porowski S., Ociepa E., Antosik P., Brüssow K.-P., Bruska M., Nowicki M., Zabel M.
Analysis of mitogen-activated protein kinase (MAP2K2) gene polymorphisms in relation to the occurrence of adenocarcinoma during aging in dogs
Summary
In the most recent years adenocarcinoma has been found to be the most common cancer that occurs in both humans and animals. It is well recognized that the induction of carcinogenesis and/or cancer growth and development is often associated with aging. Moreover, the molecular basis of carcinogenesis involves disturbances in expression of genes or mutation in genes that play a crucial role in regulating cell division cycle and inter- or intracellular signaling pathways. MAP2K2 belongs to the large family of kinases that are described as “checkpoints” of cell division and signaling, and therefore may be involved in carcinogenesis.
In this study, blood samples were obtained from 22 female dogs diagnosed with mammary tumors. Moreover, blood samples were obtained 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) individuals. 36 bitches diagnosed because of other reasons served as controls.
After Sanger sequencing analysis we found 17 single nucleotide variations, of which 3 were situated in exons (exon 2, 3 and 11), 2 other in 5’UTR non-coding region and the remaining 12 in splice regions of introns. Some of the polymorphisms, such as g.C-81T, could have higher probability of being involved in tumor development, also in correlation with aging. Furthermore, both variants c.A384G and g.T9144C were associated with strong risk factors of tumor occurrence and aging.
In conclusion, it may be suggested that some of the MAP2K2 gene polymorphisms may be recognized as markers for occurrence of adenocarcinoma in dogs. This also showed that possible disruption in expression of MAP2K2 protein kinase may lead to the induction of carcinogenesis, since it plays a crucial role in regulating the cell division cycle and cell signaling.
The development, growth and progression of cancer are associated with dysfunction of the cell division cycle that is regulated by multiple genes and by expression of proteins involved in many biochemical and metabolic pathways (8, 15, 18). Recent findings have demonstrated that proteins belonging to protein kinases family are in most cases responsible for pro-cesses related to cells growth and development (1, 4, 17). Moreover, protein kinases are crucial regulators in normal cell signaling, since they up-regulate key processes such as cell proliferation, differentiation and migration. It has been clearly demonstrated in the case of several proteins kinases dysregulation of their expression through mutation, polymorphisms and/or chromosomal translocation results in the activation of several improper pathways and subsequent uncon-trolled transfer of cell signals from extracellular matrix. It was also found that dysfunction of protein kinases expression is associated with induction of carcinogen-esis in humans; however most recent findings have also shown a similar level of dysfunction in develop-ment of canine and feline cancers (13, 16, 19). If the mutation occurs in the protein kinase gene sequence, it often leads to alterations in the protein structure. As a result, phosphorylation occurs in the absence of an appropriate stimulus.
The signal from the extracellular matrix is trans-ferred via receptors for tyrosine kinase (RTKs) and is passed through the cytoplasm into the cell nucleus with the activation of series of intermediates that are also phosphorylated. It was found that two specific cytoplasmic pathways are dysregulated in cancer cells, which leads to cancer progression. The first one includes the RAS-RAF-MEK-ERK-p38-JNK signaling
pathway. This pathway is responsible for the regula-tion of normal cell division through a series of protein phosphorylations, transition of signals into the nucleus and induction of transcription of target genes (5, 9). The second one involves phosphatidyl inositol-3 kinase (PI3K) and its pathway that regulates cell survival, cycling and growth (6, 7).
The MAP2K2 protein belongs to the first group of signal transduction kinases that regulate crucial steps for cell cycle control. Therefore, dysfunction of this pathway often leads to the induction of carcino-genesis. Moreover, it was found that disturbances in signal transduction pathways may be a direct reason of induction/inhibition of apoptosis, which is sig-nificantly associated with aging. The aim of this study was to analyze the MAP2K2 gene sequence in order to find the mutation and/or polymorphisms that may be responsible for adenocarcinoma in domestic dogs (Canis familiaris) during aging.
Material and methods
Subjects and samples collection. Blood samples were obtained from 22 female dogs diagnosed with mammary tumors during surgery in the Small Animal Clinic, Univer-sity of Life Sciences, Poznan, Poland. Blood was collected during standard surgery procedures. The 36 bitches diag-nosed for other reasons (routine control of health-blood col-lection, following ovariohysterectomy) served as controls. All examined dogs were mongrels. They were divided into 3 subgroups differing in age, according to the classification by Jugdutt et al. (10): 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) ones. Blood was collected from the jugular vein into vials with EDTA and frozen at –80°C until further analyses.
Tab. 1. PCR primer pairs used for the amplification of exons of MAP2K2 gene
Exon Sequences Annealing temp. Length of the amplicon Length of noncoding sequences
exon 1 F 5’-TAGGTCTCCGCCCCTTTC-3’
R 5’- TGCATCTTCTCCTCTCAGGTG-3’ 58°C 580 bp (394 bp of 5’UTR/92 bp of exon 1/94 bp of intron 1 488 bp
exon 2 F 5’- GTCCTTCCAGCCACACATCT-3’
R 5’- AGCCAATCAGAAAGCACAGG-3’ 58°C 458 bp (123 bp of intron 1/211 bp of exon 2/124 bp of intron 2) 247 bp
exon 3 F 5’- TTGGTTGCTGTCTTGAGCAC-3’
R 5’- TTGGAAATAAGCGTGCTGTG-3’ 58°C 345 bp (108 bp of intron 2/147 bp of exon 3/90 bp of intron 3) 198 bp
exon 4 F 5’- GCTTCGCTCTCATACCTGCT-3’
R 5’- GGGGGCAGGTTAAGGATAAA-3’ 59°C 297 bp (116 bp of intron 3/78 bp of exon 4/103 bp of intron 4) 219 bp
exon 5, exon 6 F 5’-ACGCTATGCTCACCTCACCT-3’
R 5’- AACCGCTGCTAGAATTTGGA-3’ 60°C 527 bp (127 bp of intron 4/52 bp of exon 5/89 bp of intron 5/125 bp of exon 6/134 bp of intron 6)
350 bp
exon 7 F 5’- GAACAGTGTCGGTGACAGGA-3’
R 5’- GACAGGTGGTGCTGAGAGG-3’ 58°C 434 bp (106 bp of intron 6/214 bp of exon 7/114 bp of intron 7) 220 bp
exon 8 F 5’- TGGTTTCCCAAGAAAGCAAG-3’
R 5’- CCATGTGGAATTCCCTTCC-3’ 58°C 291 bp (122 bp of intron 7/74 bp of exon 8/95 bp of intron 8) 217 bp
exon 9 F 5’- AAAGTGCTGTGCTGTCCTCA-3’
R 5’- GTGGGCAACTATCCCAGGAG-3’ 59°C 266 bp (108 bp of intron 8/62 bp of exon 9/96 bp of intron 9) 204 bp
exon 10 F 5’- CTGGCCTATTACGGCTCAAA-3’
R 5’- CCCCACTAATGGAGTCAGGA-3’ 59°C 268 bp (89 bp of intron 9/46 bp of exon 10/133 bp of intron 10) 222 bp
exon 11 F 5’- CTCTGGCTCTGGCTTCCTGT-3’
Molecular analyses. Genomic DNA was extracted from whole peripheral blood using QIAamp DNA Blood Mini Kit (Qiagen), according to the manufacturer’s instructions. DNA was suspended in 100 µl of Qiagen elution buffer and stored at –20°C. Polymerase chain reaction (PCR) was used to amplify the MAP2K2 gene. All 11 exons were amplified, including approximately 60-bp flanking regions of each exon. The sequences of primers used are listed in Table 1.
The reactions were carried out in a total volume of 12.5 µl, containing: 10 × Taq DNA Polymerase buffer with MgCl2, 5 × GC-rich solution, 0.24 mM dNTPs, 0.5 µM of the primers, 1 unit of Taq Polymerase (Roche) and 40-60 ng of genomic DNA. The PCR cycle conditions were as fol-lows: an initial denaturation at 94°C for 4 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at temperatures shown in Table 1 for 1 min and extension at 72°C for 1 min, with a final extension at 72°C for 7 min. PCR products were purified using membrane plates (Milli-pore) and used as templates in PCR-sequencing reamplifica-tion. The latter reaction was performed in the same Veriti 96 well Thermal Cycler as for the PCR reaction, using BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) and one of the specific primers (forward or reverse one). Reamplification products were purified with EDTA and ethanol precipitation and separated by electrophoresis using ABI 3130 sequencer (Applied Biosystems).
Statistical analyses. Genotypes obtained in the study were aligned with the reference sequences from Ensembl database. Single nucleotide variations were assessed and calculation of the chi-square test for deviation from Hardy-Weinberg equilibrium (HWE) was performed. Genotype and allele frequencies were evaluated and compared between study and control groups using the Fisher’s exact test. Odds ratio values (OR) were also evaluated and p < 0.05 was considered to indicate statistically significant differences. Additionally, we used Haploview 3.2 software to obtain
MAP2K2 gene structure. Linkage disequilibrium values
(LD) were calculated as R2 value and the Gabriel et al.
algorithm was used.
Results and discussion
In total, we performed Sanger sequencing of 11 exons with approximately 60-bp non-coding splicing regions of the MAP2K2 gene in 58 subjects. Upon alignment of obtained and reference sequences we identified 17 single nucleotide variations, of which 3 were situated in exons (exon 2, 3 and 11), 2 others in 5’UTR non-coding region and the remaining 12 – in splice regions of introns. Numbering of nucleotides was carried out with reference to the first nt in the first coding triplet, AUG in exon1. All single nucleotide variations identified in the study are listed in Table 2.
All of the nucleotide changes were substitutions and were biallelic. None of the 3 substitutions in exon 2, 3 and 11, respectively, changed the amino acid sequence. Frequencies of alleles and genotypes for all 17 variants were determined. Distribution of almost all MAP2K2 genotypes was consistent with HWE, except for varia-tions 1, 2, 5, 6 and 12 for the subjects group, as they
were homozygous for all the tumor cases. Differences in genotype and allele frequencies between study and control groups and between individual control subgroups indicated two putative risk alleles and two others with a putative protective role. Frequencies and OR values for those variations are shown in Table 3.
There were high odds ratio values indicating more than 6-fold higher incidence of the allele T presence in the control than in the tumor group for variation of g.C-81T. Although without statistical significance, there was also more than 7- and 8-fold higher preva-lence of the allele T in the subgroups of young and adult bitches, respectively, in comparison with the geriatric subgroup. The results of the genotype CT were paral-lel to those of the alparal-lele T. Similarly, there was over a 3-fold higher prevalence of the alternative allele T and genotype CT in the control than in the tumor group for variation of g.C9176T. Moreover, prevalence of both allele T and genotype CT was the highest in the young subgroup and it was 5-fold higher (for the allele T and the genotype CT) than in the adult subgroup and 7-fold (for the allele T) and 8-fold (for the genotype CT) higher than in the geriatric subgroup. There was also higher prevalence of an alternative allele T and heterozygote GT for variation g.G15523T in the con-trol than in the tumor group as well as in the young and adult subgroups than in the geriatric subgroup. However, the odds ratio values were approximately twice lower than in the two previously described varia-tions and the results were not statistically significant. Tab. 2. Single nucleotide variations identified in MAP2K2 gene in studied subjects
Name of
variation Localization nt change aa change
1 g.G-236C 5’UTR G > C –
2 g.C-81T 5’UTR C > T –
3 c.C210T exon 2 C > T syn: D70D (GAC > GAT)
4 g.T9144C intron 2 T > C –
5 g.G9145T intron 2 G > T –
6 g.C9176T intron 2 C > T –
7 c.A384G exon 3 A > G syn: P128P (CCA > CCG)
8 g.G13590T intron 3 G > T – 9 g.G13778C intron 4 G > C – 10 g.T14399C intron 5 T > C – 11 g.T14657C intron 6 T > C – 12 g.G15523T intron7 G > T – 13 g.C17391T intron7 C > T – 14 g.C19971T intron 9 C > T – 15 g.C20086T intron 10 C > T – 16 g.C21727G intron 10 C > G –
17 c.C1188T exon 11 C > T syn: S396S (AGC > AGT)
Explanations: g. = genomic; c. = coding; syn = synonymous; nt = nucleotide; aa = amino acid
Tab. 3. Frequencies and odds ratio values for chosen single nucleotide variations for MAP2K2 gene
Variation Tumor patients Controls p value OR (95% CI)
g.C-81T Genotype frequency (n = 22) (n = 36) CC 22 (1.0) 32 (0.89) CT 0 4 (0.11) C vs. T 0.2274 6.2 (0.3-121.6) subgroup Y (n = 11) CC 9 (0.82) CT 2 (0.18) Y vs.G 0.1904 8.2 (0.4-188.8) subgroup A (n = 10) CC 8 (0.8) CT 2 (0.2) A vs. G 0.169 9.1 (0.4-212.7) subgroup G (n = 15) CC 15 (1.0) CT 0 Allele frequency C 44 (1.0) 68 (0.94) T 0 4 (0.06) C vs. T 0.2401 5.9 (0.3-111.3) subgroup Y C 20 (0.91) T 2 (0.09) Y vs.G 0.2027 7.4 (0.03-163.1) subgroup A C 18 (0.9) T 2 (0.1) A vs. G 0.181 8.2 (0.4-181.3) subgroup G C 30 (1.0) T 0 g.T9144C Genotype frequency (n = 22) (n = 36) CT 2 (0.09) 6 (0.17) CC 20 (0.91) 27 (0.75) T vs. C 0.1496 3.4 (0.7-17.1) TT 0 3 (0.08) subgroup Y (n = 11) CT 3 (0.27) CC 7 (0.64) G vs.Y 0.1827 3.7 (0.5-25.6) TT 1 (0.09) subgroup A (n = 10) CT 1 (0.1) CC 7 (0.7) G vs. A 0.3181 2.8 (0.4-20.8) TT 2 (0.2) subgroup G (n = 15) CT 2 (0.13) CC 13 (0.87) TT 0 Allele frequency T 2 (0.05) 12 (0.17) C 42 (0.95) 60 (0.83) T vs. C 0.0692 4.2 (0.9-19.8) subgroup Y T 5 (0.23) C 17 (0.77) G vs.Y 0.1123 4.1 (0.7-23.6) subgroup A T 5 (0.25) C 15 (0.75) G vs. A 0.0855 4.7 (0.8-27) subgroup G T 2 (0.07) C 28 (0.93)
Variation Tumor patients Controls p value OR (95% CI) g.C9176T Genotype frequency (n = 22) (n = 36) CC 22 (1.0) 34 (0.94) CT 0 2 (0.06) C vs. T 0.4523 3.3 (0.2-71.1) subgroup Y (n = 11) CC 9 (0.82) CT 2 (0.18) Y vs. A 0.2891 5.5 (0.2-130.4) subgroup A (n = 10) CC 10 (1.0) CT 0 subgroup G (n = 15) CC 15 (1.0) CT 0 Y vs. G 0.1904 8.2 (0.4-188.9) Allele frequency C 44 (1.0) 70 (0.97) T 0 2 (0.03) C vs. T 0.4616 3.2 (0.1-67.3) subgroup Y C 20 (0.91) T 2 (0.09) Y vs. A 0.3085 5 (0.2-110.7) subgroup A C 20 (1.0) T 0 subgroup G C 30 (1.0) T 0 Y vs. G 0.2027 7.4 (0.3-163.1)
c.A384G Genotype frequency (n = 22) (n = 36)
AA 0 3 (0.08) AG 2 (0.09) 6 (0.17) GG 20 (0.91) 27 (0.75) T vs. C 0.1496 3.3 (0.7-17.1) subgroup Y (n = 11) AA 1 (0.09) AG 3 (0.27) GG 7 (0.64) G vs.Y 0.1827 3.7 (0.5-25.6) subgroup A (n = 10) AA 2 (0.2) AG 1 (0.1) GG 7 (0.7) G vs. A 0.3181 2.8 (0.4-20.8) subgroup G (n = 15) AA 0 AG 2 (0.13) GG 13 (0.87) Allele frequency A 2 (0.05) 12 (0.17) G 42 (0.95) 60 (0.83) T vs. C 0.0692 4.2 (0.9-19.8) subgroup Y A 5 (0.23) G 17 (0.77) G vs.Y 0.1123 4.1 (0.7-23.6) subgroup A A 5 (0.25) G 15 (0.75) G vs. A 0.0855 4.7 (0.8-27) subgroup G A 2 (0.07) G 28 (0.93) Explanations: (Y = young; A = adult; G = geriatric)
On the contrary, we observed an increased incidence of the alternative allele C and homozygote CC in tumor patients in com-parison with the control group for variation of g.T9144C. There was approximately 3- and over 4-fold higher prevalence of the genotype CC and the allele C, respectively, in the geriatric subgroup than in both young and adult subgroups. The results were on the boundary of statistical significance. This could reflect the relatively small numerical force of all subgroups, although it might indicate a strong trend in allele and genotype distribution in larger popula-tions. However, some of the results were also inconclusive. In case of several varia-tions (c.C210T, g.G13778C, g.T14399C, g.C17391T, g.C20086T, g.C21727G, c.C1188T) a higher prevalence of an alter-native allele and heterozygote and/or minor homozygote in tumor patients was noted than in the control group. On the other hand, a higher incidence of those alleles and geno-types was observed in the youngest control patients in comparison with older subjects
(data not shown). Most of these variations comprised a haplotype block with 82% or 100% LD between pairs of polymorphisms (Fig. 1).
MAP2K2 gene structure of the case-control
asso-ciation study revealed 2 haplotype blocks. The same significantly strong linkage between pairs of studied variations was observed also in the control and tumor group individually and the rate varied from 71 and 77% in tumor patients to 100% in controls (data not shown). Interestingly, there was a significantly stronger linkage between two pair of polymorphisms (g.T9144C and g.T14657C; c.A384G and g.T14657C) at the level of 71% in controls and only of 16% in tumor patients. It is also worth mentioning that both former varia-tions in each pair were putative risk variants and they manifested 100% linkage in the association study. The presumably stronger linkage of both variants with polymorphism g.T14657C in the control group was capable of masking its putative harmful effect in those individuals. Similarly, strong linkage between variations of c.C210T and g.C17391T was observed in 82% of controls, while tumor patients showed linkage at the level of 52%.
The induction of carcinogenesis as well as growth and development of many cancer types, including adenocarcinoma, is up- and/or down-regulated by the expression of many target gene clusters that influence cell survival or apoptosis. The mitogen-activated pro-teins kinase (MAP2K2) investigated in this study is a main factor involved in regulation of cell division cycles. MAP2K2 activity is associated with target protein phosphorylation pathways, recognized also as the crucial stage in activation of signal transduction
pathways from extracellular matrix into target gene sequences in the cell nucleus (3, 12, 14). It was shown that normal expression and/or activity of MAP2K2 are responsible for cell survival (20). Moreover, disrup-tion in the gene structure or dereguladisrup-tion of the protein activity might be the main reason of not programmed cells division or disturbances in signaling pathway transduction, which finally leads to the induction of car-cinogenesis and/or growth and development of cancer. In this study we used the animal model for investiga-tions on MAP2K2 polymorphism, which would be associated with the incidence of adenocarcinoma as well as in correlation with aging in dogs.
We did not find a statistically significant associa-tion between g.C-81T polymorphism in the group of canines with adenocarcinoma and controls; however, after functional analysis we suggested that this poly-morphism could influence the folding of the mRNAs during transcription and/or reflect several other biologi-cal interactions with molecules, e.g. splicing factors. Moreover, our results showed that the animals with g.C-81T polymorphism could have a higher probability of tumor development also in correlation with aging.
Similarly, there was the same trend in the allele C and homozygote CC frequencies for variation of c.A384G as for variation of g.T9144C. Moreover, the former and the latter variations were found to be in 100% linkage disequilibrium and both indicated a strong risk for the incidence of both tumor development and aging.
Until now, few data have been available which would indicate an association of MAP2K2 kinase polymorphisms with carcinogenesis and aging in dogs. However, information is available regarding gastro-Fig. 1. Structure of MAP2K2 gene shown as a case-control association
Explanation: Figure 1 shows MAP2K2 gene structure displayed as a case-as-sociation test. The relationship between every two single nucleotide variations is shown as linkage disequilibrium (LD), calculated as R2 value. Numbers in
squares represent percentage of subjects in the study that were in linkage in reference to two variations crossed in each square. MAP2K2 gene structure revealed two haplotype blocks.
intestinal stromal tumors, hepatocellular carcinoma, and/or lung cancer in humans, which were associated with MAP2K2 gene polymorphisms. In such a study, Kang et al. (11) performed mutation screening on 22 freshly frozen gastrointestinal stromal tumors using multiplex oncogene screening panel with mass spec-troscopy. They detected 390 known mutations across 30 genes, most of which included several known genes fully recognized as the cell cycle division markers, and which were crucial for intracellular cell signaling during cancer development. As a result, they found that mutations other than KIT and PDGFRA, also including kinase MAP2K2, are rare in gastrointestinal stromal cancer. However, in another study by Bansal et al. (2) occurrence and the role of mutation of MEK (MAP/ ERK) kinase were studied during the development of human lung cancer. The authors suggested that this kinase is a main factor responsible for transforming cells to neoplastic ones and, therefore, it may func-tion as a dominant oncogene playing a potentially pivotal role in human carcinogenesis. After sequenc-ing, they found an allelic variant in MEK-1 kinase cDNA, nucleotide 783 G/A, (no amino acid change), a MEK-2 kinase cDNA change (nucleotide 977 C/T mutation leading to 298 Pro into Leu change in protein sequence), a MEK-2 kinase cDNA change nucleotide 537 C/T (no amino acid change), and a frequent MEK-2 kinase cDNA germ line polymorphism nucleotide 744, A/C (no amino acid change). However, they concluded that both MEK-1 and MEK-2 kinases polymorphisms do not play a crucial role in human lung cancer devel-opment.
In conclusion, we suggest that some of the MAP2K2 gene polymorphisms may serve as potential markers for adenocarcinoma, also in association with aging in dogs, although no data are available which would indicate an important role of mutation in this gene during carcinogenesis in humans.
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Corresponding author: Bartosz Kempisty PhD, ul. Święcickiego 6, 60-781 Poznań, Poland; e-mail: etok@op.pl
