Analysis of Mitogen-Activated Protein Kinase (MAP2K2) Gene Polymorphisms in Relation to the Occurrence of Adenocarcinoma During Aging in Dogs1)

46 Med. Weter. 2016, 72 (1), 46-52

Praca oryginalna Original paper Analysis of mitogen-activated protein kinase (MAP2K2) gene polymorphisms in relation to the occurrence of adenocarcinoma during aging in dogs1)

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

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. Keywords: mutation, cancer, Canis familiaris

1) Supported by the Polish National Centre of Science (Grant No. 5279/B/P01/2011/40). Med. Weter. 2016, 72 (1), 46-52 47

The development, growth and progression of cancer pathway. This pathway is responsible for the regula- are associated with dysfunction of the cell division tion of normal cell division through a series of protein cycle that is regulated by multiple genes and by phosphorylations, transition of signals into the nucleus expression of proteins involved in many biochemical and induction of transcription of target genes (5, 9). and metabolic pathways (8, 15, 18). Recent findings The second one involves phosphatidyl inositol-3 kinase have demonstrated that proteins belonging to protein (PI3K) and its pathway that regulates cell survival, kinases family are in most cases responsible for pro- cycling and growth (6, 7). cesses related to cells growth and development (1, 4, The MAP2K2 protein belongs to the first group 17). Moreover, protein kinases are crucial regulators of signal transduction kinases that regulate crucial in normal cell signaling, since they up-regulate key steps for cell cycle control. Therefore, dysfunction of processes such as cell proliferation, differentiation this pathway often leads to the induction of carcino- and migration. It has been clearly demonstrated in the genesis. Moreover, it was found that disturbances in case of several proteins kinases dysregulation of their signal transduction pathways may be a direct reason expression through mutation, polymorphisms and/or of induction/inhibition of apoptosis, which is sig- chromosomal translocation results in the activation nificantly associated with aging. The aim of this study of several improper pathways and subsequent uncon- was to analyze the MAP2K2 gene sequence in order trolled transfer of cell signals from extracellular matrix. to find the mutation and/or polymorphisms that may It was also found that dysfunction of protein kinases be responsible for adenocarcinoma in domestic dogs expression is associated with induction of carcinogen- (Canis familiaris) during aging. esis in humans; however most recent findings have Material and methods also shown a similar level of dysfunction in development of canine and feline cancers (13, 16, 19). If the Subjects and samples collection. Blood samples were mutation occurs in the protein kinase gene sequence, obtained from 22 female dogs diagnosed with mammary it often leads to alterations in the protein structure. As tumors during surgery in the Small Animal Clinic, Univer- a result, phosphorylation occurs in the absence of an sity of Life Sciences, Poznan, Poland. Blood was collected appropriate stimulus. during standard surgery procedures. The 36 bitches diagnosed for other reasons (routine control of health-blood col- The signal from the extracellular matrix is trans- lection, following ovariohysterectomy) served as controls. ferred via receptors for tyrosine kinase (RTKs) and All examined dogs were mongrels. They were divided into is passed through the cytoplasm into the cell nucleus 3 subgroups differing in age, according to the classification with the activation of series of intermediates that are by Jugdutt et al. (10): geriatric (> 5 to 10 years old; n = 15), also phosphorylated. It was found that two specific mature adult (> 2 to 5 years old; n = 10) and young (from cytoplasmic pathways are dysregulated in cancer 1 to 2 years old; n = 11) ones. Blood was collected from cells, which leads to cancer progression. The first one the jugular vein into vials with EDTA and frozen at –80°C includes the RAS-RAF-MEK-ERK-p38-JNK signaling until further analyses.

Tab. 1. PCR primer pairs used for the amplification of exons ofMAP2K2 gene Exon Sequences Annealing temp. Length of the amplicon Length of noncoding sequences exon 1 F 5’-TAGGTCTCCGCCCCTTTC-3’ 58°C 580 bp (394 bp of 5’UTR/92 bp of exon 1/ 488 bp R 5’- TGCATCTTCTCCTCTCAGGTG-3’ 94 bp of intron 1 exon 2 F 5’- GTCCTTCCAGCCACACATCT-3’ 58°C 458 bp (123 bp of intron 1/211 bp of exon 2/ 247 bp R 5’- AGCCAATCAGAAAGCACAGG-3’ 124 bp of intron 2) exon 3 F 5’- TTGGTTGCTGTCTTGAGCAC-3’ 58°C 345 bp (108 bp of intron 2/147 bp of exon 3/ 198 bp R 5’- TTGGAAATAAGCGTGCTGTG-3’ 90 bp of intron 3) exon 4 F 5’- GCTTCGCTCTCATACCTGCT-3’ 59°C 297 bp (116 bp of intron 3/78 bp of exon 4/ 219 bp R 5’- GGGGGCAGGTTAAGGATAAA-3’ 103 bp of intron 4) exon 5, exon 6 F 5’-ACGCTATGCTCACCTCACCT-3’ 60°C 527 bp (127 bp of intron 4/52 bp of exon 5/ 350 bp R 5’- AACCGCTGCTAGAATTTGGA-3’ 89 bp of intron 5/125 bp of exon 6/134 bp of intron 6) exon 7 F 5’- GAACAGTGTCGGTGACAGGA-3’ 58°C 434 bp (106 bp of intron 6/214 bp of exon 7/ 220 bp R 5’- GACAGGTGGTGCTGAGAGG-3’ 114 bp of intron 7) exon 8 F 5’- TGGTTTCCCAAGAAAGCAAG-3’ 58°C 291 bp (122 bp of intron 7/74 bp of exon 8/ 217 bp R 5’- CCATGTGGAATTCCCTTCC-3’ 95 bp of intron 8) exon 9 F 5’- AAAGTGCTGTGCTGTCCTCA-3’ 59°C 266 bp (108 bp of intron 8/62 bp of exon 9/ 204 bp R 5’- GTGGGCAACTATCCCAGGAG-3’ 96 bp of intron 9) exon 10 F 5’- CTGGCCTATTACGGCTCAAA-3’ 59°C 268 bp (89 bp of intron 9/46 bp of exon 10/ 222 bp R 5’- CCCCACTAATGGAGTCAGGA-3’ 133 bp of intron 10) exon 11 F 5’- CTCTGGCTCTGGCTTCCTGT-3’ 62°C 350 bp (99 bp of intron 10/111 bp of exon 11/ 239 bp R 5’- AGTGTCCACTGCGGGGTAT-3’ 140 bp of 3’UTR) 48 Med. Weter. 2016, 72 (1), 46-52

Molecular analyses. Genomic DNA was extracted from Tab. 2. Single nucleotide variations identified in MAP2K2 whole peripheral blood using QIAamp DNA Blood Mini gene in studied subjects Kit (Qiagen), according to the manufacturer’s instructions. Name of Localization nt change aa change DNA was suspended in 100 µl of Qiagen elution buffer and variation stored at –20°C. Polymerase chain reaction (PCR) was used 1 g.G-236C 5’UTR G > C – to amplify the MAP2K2 gene. All 11 exons were amplified, 2 g.C-81T 5’UTR C > T – including approximately 60-bp flanking regions of each exon. The sequences of primers used are listed in Table 1. 3 c.C210T exon 2 C > T syn: D70D (GAC > GAT) The reactions were carried out in a total volume of 4 g.T9144C intron 2 T > C – 12.5 µl, containing: 10 × Taq DNA Polymerase buffer with 5 g.G9145T intron 2 G > T –

MgCl2, 5 × GC-rich solution, 0.24 mM dNTPs, 0.5 µM of 6 g.C9176T intron 2 C > T – the primers, 1 unit of Taq Polymerase (Roche) and 40-60 ng of genomic DNA. The PCR cycle conditions were as fol- 7 c.A384G exon 3 A > G syn: P128P (CCA > CCG) lows: an initial denaturation at 94°C for 4 min, followed 8 g.G13590T intron 3 G > T – by 35 cycles of denaturation at 95°C for 30 sec, annealing 9 g.G13778C intron 4 G > C – at temperatures shown in Table 1 for 1 min and extension 10 g.T14399C intron 5 T > C – at 72°C for 1 min, with a final extension at 72°C for 7 min. 11 g.T14657C intron 6 T > C – PCR products were purified using membrane plates (Milli- pore) and used as templates in PCR-sequencing reamplifica- 12 g.G15523T intron7 G > T – tion. The latter reaction was performed in the same Veriti 96 13 g.C17391T intron7 C > T – well Thermal Cycler as for the PCR reaction, using BigDye 14 g.C19971T intron 9 C > T – Terminator v3.1 Cycle Sequencing Kit (Life Technologies) 15 g.C20086T intron 10 C > T – and one of the specific primers (forward or reverse one). Reamplification products were purified with EDTA and 16 g.C21727G intron 10 C > G – ethanol precipitation and separated by electrophoresis using 17 c.C1188T exon 11 C > T syn: S396S (AGC > AGT) ABI 3130 sequencer (Applied Biosystems). Explanations: g. = genomic; c. = coding; syn = synonymous; Statistical analyses. Genotypes obtained in the study nt = nucleotide; aa = amino acid 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- were homozygous for all the tumor cases. Differences Weinberg equilibrium (HWE) was performed. Genotype and in genotype and allele frequencies between study allele frequencies were evaluated and compared between and control groups and between individual control study and control groups using the Fisher’s exact test. Odds subgroups indicated two putative risk alleles and two ratio values (OR) were also evaluated and p < 0.05 was others with a putative protective role. Frequencies and considered to indicate statistically significant differences. OR values for those variations are shown in Table 3. Additionally, we used Haploview 3.2 software to obtain There were high odds ratio values indicating more MAP2K2 gene structure. Linkage disequilibrium values than 6-fold higher incidence of the allele T presence (LD) were calculated as R2 value and the Gabriel et al. in the control than in the tumor group for variation of algorithm was used. g.C-81T. Although without statistical significance, there was also more than 7- and 8-fold higher preva- Results and discussion lence of the allele T in the subgroups of young and adult In total, we performed Sanger sequencing of 11 bitches, respectively, in comparison with the geriatric exons with approximately 60-bp non-coding splicing subgroup. The results of the genotype CT were paral- regions of the MAP2K2 gene in 58 subjects. Upon lel to those of the allele T. Similarly, there was over alignment of obtained and reference sequences we a 3-fold higher prevalence of the alternative allele T identified 17 single nucleotide variations, of which and genotype CT in the control than in the tumor group 3 were situated in exons (exon 2, 3 and 11), 2 others for variation of g.C9176T. Moreover, prevalence of in 5’UTR non-coding region and the remaining 12 – in both allele T and genotype CT was the highest in the splice regions of introns. Numbering of nucleotides young subgroup and it was 5-fold higher (for the allele was carried out with reference to the first nt in the first T and the genotype CT) than in the adult subgroup and coding triplet, AUG in exon1. All single nucleotide 7-fold (for the allele T) and 8-fold (for the genotype variations identified in the study are listed in Table 2. CT) higher than in the geriatric subgroup. There was All of the nucleotide changes were substitutions and also higher prevalence of an alternative allele T and were biallelic. None of the 3 substitutions in exon 2, 3 heterozygote GT for variation g.G15523T in the con- and 11, respectively, changed the amino acid sequence. trol than in the tumor group as well as in the young Frequencies of alleles and genotypes for all 17 variants and adult subgroups than in the geriatric subgroup. were determined. Distribution of almost all MAP2K2 However, the odds ratio values were approximately genotypes was consistent with HWE, except for varia- twice lower than in the two previously described variations 1, 2, 5, 6 and 12 for the subjects group, as they tions and the results were not statistically significant. Med. Weter. 2016, 72 (1), 46-52 49

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) 50 Med. Weter. 2016, 72 (1), 46-52

Tab. 3. Continuation 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) Med. Weter. 2016, 72 (1), 46-52 51

On the contrary, we observed an increased incidence of the alternative allele C and homozygote CC in tumor patients in comparison 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 variations (c.C210T, g.G13778C, g.T14399C, g.C17391T, g.C20086T, g.C21727G, c.C1188T) a higher prevalence of an alter- Fig. 1. Structure of MAP2K2 gene shown as a case-control association native allele and heterozygote and/or minor Explanation: Figure 1 shows MAP2K2 gene structure displayed as a case-as- homozygote in tumor patients was noted sociation test. The relationship between every two single nucleotide variations is shown as linkage disequilibrium (LD), calculated as R2 value. Numbers in than in the control group. On the other hand, squares represent percentage of subjects in the study that were in linkage in a higher incidence of those alleles and geno- reference to two variations crossed in each square. MAP2K2 gene structure types was observed in the youngest control revealed two haplotype blocks. patients in comparison with older subjects (data not shown). Most of these variations comprised pathways from extracellular matrix into target gene a haplotype block with 82% or 100% LD between pairs sequences in the cell nucleus (3, 12, 14). It was shown of polymorphisms (Fig. 1). that normal expression and/or activity of MAP2K2 are MAP2K2 gene structure of the case-control asso- responsible for cell survival (20). Moreover, disrup- ciation study revealed 2 haplotype blocks. The same tion in the gene structure or deregulation of the protein significantly strong linkage between pairs of studied activity might be the main reason of not programmed variations was observed also in the control and tumor cells division or disturbances in signaling pathway group individually and the rate varied from 71 and 77% transduction, which finally leads to the induction of car- in tumor patients to 100% in controls (data not shown). cinogenesis and/or growth and development of cancer. Interestingly, there was a significantly stronger linkage In this study we used the animal model for investiga- between two pair of polymorphisms (g.T9144C and tions on MAP2K2 polymorphism, which would be g.T14657C; c.A384G and g.T14657C) at the level of associated with the incidence of adenocarcinoma as 71% in controls and only of 16% in tumor patients. well as in correlation with aging in dogs. It is also worth mentioning that both former varia- We did not find a statistically significant associa- tions in each pair were putative risk variants and they tion between g.C-81T polymorphism in the group of manifested 100% linkage in the association study. canines with adenocarcinoma and controls; however, The presumably stronger linkage of both variants after functional analysis we suggested that this poly- with polymorphism g.T14657C in the control group morphism could influence the folding of the mRNAs was capable of masking its putative harmful effect in during transcription and/or reflect several other biologi- those individuals. Similarly, strong linkage between cal interactions with molecules, e.g. splicing factors. variations of c.C210T and g.C17391T was observed in Moreover, our results showed that the animals with 82% of controls, while tumor patients showed linkage g.C-81T polymorphism could have a higher probability at the level of 52%. of tumor development also in correlation with aging. The induction of carcinogenesis as well as growth Similarly, there was the same trend in the allele C and and development of many cancer types, including homozygote CC frequencies for variation of c.A384G adenocarcinoma, is up- and/or down-regulated by the as for variation of g.T9144C. Moreover, the former and expression of many target gene clusters that influence the latter variations were found to be in 100% linkage cell survival or apoptosis. The mitogen-activated pro- disequilibrium and both indicated a strong risk for the teins kinase (MAP2K2) investigated in this study is incidence of both tumor development and aging. a main factor involved in regulation of cell division Until now, few data have been available which cycles. MAP2K2 activity is associated with target would indicate an association of MAP2K2 kinase protein phosphorylation pathways, recognized also as polymorphisms with carcinogenesis and aging in dogs. the crucial stage in activation of signal transduction However, information is available regarding gastro- 52 Med. Weter. 2016, 72 (1), 46-52 intestinal stromal tumors, hepatocellular carcinoma, 9. Johnson G. L., Lapadat R.: Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002, 298, 1911-1912. and/or lung cancer in humans, which were associated 10. Jugdutt B. I., Jelani A., Palaniyappan A., Idikio H., Uweira R. E., Menon V., with MAP2K2 gene polymorphisms. In such a study, Jugdutt C. E.: Aging-related early changes in markers of ventricular and matrix Kang et al. (11) performed mutation screening on 22 remodeling after reperfused ST-segment elevation myocardial infarction in the freshly frozen gastrointestinal stromal tumors using canine model: effect of early therapy with an angiotensin II type 1 receptor blocker. Circulation 2010, 122, 341-351. multiplex oncogene screening panel with mass spec- 11. Kang G., Lee J., Jang K. T., Beadling C., Corless C. L., Heinrich M. C., Park troscopy. They detected 390 known mutations across J. O., Kang W. K., Park C. K., Kim K. M.: Multiplex mutation screening by 30 genes, most of which included several known genes mass spectrometry in gastrointestinal stromal tumours. Pathology 2012, 44, 460-464. fully recognized as the cell cycle division markers, 12. Lee S. Y., Shin S. J., Kim H. S.: ERK1/2 activation mediated by the nutlin- and which were crucial for intracellular cell signaling 3-induced mitochondrial translocation of p53. Int. J. Oncol. 2013, 42, 1027- during cancer development. As a result, they found that -1035. 13. Levidou G., Saetta A. A., Gigelou F., Karlou M., Papanastasiou P., Stama- mutations other than KIT and PDGFRA, also including telli A., Kavantzas N., Michalopoulos N. V., Agrogiannis G., Patsouris E., kinase MAP2K2, are rare in gastrointestinal stromal Korkolopoulou P.: ERK/pERK expression and B-raf mutations in colon cancer. However, in another study by Bansal et al. (2) adenocarcinomas: correlation with clinicopathological characteristics. World occurrence and the role of mutation of MEK (MAP/ J. Surg. Oncol. 2012, 10, 47. 14. Lim J. H., Liu Y., Reineke E., Kao H. Y.: Mitogen-activated protein kinase ERK) kinase were studied during the development of extracellular signal-regulated kinase 2 phosphorylates and promotes Pin1 human lung cancer. The authors suggested that this protein-dependent promyelocytic leukemia protein turnover. J. Biol. Chem. kinase is a main factor responsible for transforming 2011, 286, 44403-44411. 15. Mandelblatt J. S., Hurria A., McDonald B. C., Saykin A. J., Stern R. A., Van cells to neoplastic ones and, therefore, it may func- Meter J. W., McGuckin M., Traina T., Denduluri N., Turner S., Howard D., tion as a dominant oncogene playing a potentially Jacobsen P. B., Ahles T.: Thinking and Living With Cancer Study. Cognitive pivotal role in human carcinogenesis. After sequenc- effects of cancer and its treatments at the intersection of aging: what do we ing, they found an allelic variant in MEK-1 kinase know; what do we need to know? Semin. Oncol. 2013, 40, 709-725. 16. Samarakoon R., Higgins C. E., Higgins S. P., Higgins P. J.: Differential require- cDNA, nucleotide 783 G/A, (no amino acid change), ment for MEK/ERK and SMAD signaling in PAI-1 and CTGF expression in a MEK-2 kinase cDNA change (nucleotide 977 C/T response to microtubule disruption. Cell Signal. 2009, 21, 986-995. mutation leading to 298 Pro into Leu change in protein 17. Touny L. H. el, Vieira A., Mendoza A., Khanna C., Hoenerhoff M. J., Green J. E.: Combined SFK/MEK inhibition prevents metastatic outgrowth of dor- sequence), a MEK-2 kinase cDNA change nucleotide mant tumor cells. J. Clin. Invest. 2014, 124, 156-168. 537 C/T (no amino acid change), and a frequent MEK-2 18. Wasserman J. D., Zambetti G. P., Malkin D.: Towards an understanding of kinase cDNA germ line polymorphism nucleotide 744, the role of p53 in adrenocortical carcinogenesis. Mol. Cell Endocrinol. 2012, A/C (no amino acid change). However, they concluded 351, 101-110. 19. Xu F., Ito S., Hamaguchi M., Senga T.: Disruption of cell spreading by the that both MEK-1 and MEK-2 kinases polymorphisms activation of MEK/ERK pathway is dependent on AP-1 activity. Nagoya do not play a crucial role in human lung cancer devel- J. Med. Sci. 2010, 72, 139-144. opment. 20. Zhao Z., Liu Y., He H., Chen X., Chen J., Lu Y. C.: Candidate genes influenc- ing sensitivity and resistance of human glioblastoma to Semustine. Brain Res. In conclusion, we suggest that some of the MAP2K2 Bull. 2011, 86, 189-194. gene polymorphisms may serve as potential markers for adenocarcinoma, also in association with aging Corresponding author: Bartosz Kempisty PhD, ul. Święcickiego 6, 60-781 in dogs, although no data are available which would Poznań, Poland; e-mail: [email protected] indicate an important role of mutation in this gene during carcinogenesis in humans. References 1. Aoki K., Kumagai Y., Sakurai A., Komatsu N., Fujita Y., Shionyu C., Matsuda M.: Stochastic ERK activation induced by noise and cell-to-cell propagation regulates cell density-dependent proliferation. Mol. Cell. 2013, 52, 529-540. 2. Bansal A., Ramirez R. D., Minna J. D.: Mutation analysis of the coding sequences of MEK-1 and MEK-2 genes in human lung cancer cell lines. Oncogene 1997, 14, 1231-1234. 3. Chung E. J., Urick M. E., Kurshan N., Shield W. 3rd, Asano H., Smith P. D., Scroggins B. S., Burkeen J., Citrin D. E.: MEK1/2 inhibition enhances the radiosensitivity of cancer cells by downregulating survival and growth signals mediated by EGFR ligands. Int. J. Oncol. 2013, 42, 2028-2036. 4. Deschênes-Simard X., Kottakis F., Meloche S., Ferbeyre G.: ERKs in cancer: friends or foes? Cancer Res. 2014, 74, 412-419. 5. Downward J.: Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer. 2003, 3, 11-22. 6. Franke T. F., Hornik C. P., Segev L., Shostak G. A., Sugimoto C.: PI3K/Akt and apoptosis: size matters. Oncogene 2003, 22, 8983-8998. 7. Fresno Vara J. A., Casado E., de Castro J., Cejas P., Belda-Iniesta C., González-Barón M.: PI3K/Akt signalling pathway and cancer. Cancer Treat Rev. 2004, 30, 193-204. 8. Gómez-López S., Lerner R. G., Petritsch C.: Asymmetric cell division of stem and progenitor cells during homeostasis and cancer. Cell Mol. Life Sci. 2014, 71, 575-597.