Gastric cancer is one of the major contributors to cancer-related deaths worldwide with estimated 989 600 new cases and 738 000 deaths in 2008.1,2 It is believed that complex interplay of genetic and environmental factors triggers the accumulation of numerous genetic and epigenetic alterations in cells, resulting in deregulation of normal cell functions and disruption of stomach linen homeostasis.3-6 Individual genetic factors probably contribute to aberrant processes in the genesis of malignant phenotype. Among them, single nucleotide polymorphisms (SNPs) and other genetic variants play an important role as the main genetic elements in the aetiology of several complex diseases, including gastric cancer.7-11 In gastric carcinogenesis this is further supported by the fact that only a small proportion of individuals exposed to the known environmental risk factors develop adenocarcinoma.5,10 Therefore, there is continuing interest for determining simple genetic tests for identifying individuals at high risk for the development of gastric tumours and for identifying patients with high risk for recurrence in order to ensure improved and early diagnosis as well as better survival of patients.
A majority of gastric cancer patients show chromosomal instability (CIN) resulting in aneuploidy.4,12,13 It has been suggested that tumour cells acquire aberrant chromosome numbers and other chromosomal defects as a result of deregulation of mechanisms responsible for maintaining the chromosomal number stability, such as spindle assembly checkpoint and chromosome segregation.14,15However, mutations in mitotic genes are rare, due to the fact that severe defects of these genes would trigger cell death by cell-surveillance early in the development.14-17 Studies revealed that subtle changes in mitotic segregation genes, controlling chromatids separation or regulating the progress of mitosis, could be prime candidates for inducing the slow process of accumulation of genetic changes, leading to CIN.15,18-20 The novel hypothesis is further supported by the fact that this process is slow, and explains the late onset of sporadic epithelial cancers21,22, as well as heterogeneous mutation load observed in different sections of tumours from individual patients.
The multidomain protein kinase BUB1B (BUB1-related kinase, known as MAD3 in yeast) plays a central role in the process of spindle assembly checkpoint (SAC), which prevents defects in the segregation of sister chromatids by delaying their separation until all chromatids have achieved correct attachments to the mitotic spindle.23,24 BUB1B is part of the mitotic checkpoint complex (MCC), which together with BUB3, MAD2 and CDC20 inhibits the anaphase-promoting complex/cyclosome (APC/C), delaying the onset of anaphase and ensuring proper chromosome segregation.25 The protein BUB1B has also been localized to the kinetochores and is important for stabilizing the kinetochore-microtubule interactions and chromosome alignment.26 A dual specificity protein kinase TTK (alias MPS1) is crucial for the spindle assembly checkpoint, for chromosome biorientation on the mitotic spindle and for ensuring accurate chromosome segregation.27,28 Inhibitor and chemical genetics studies showed that TTK activity facilitates the conformational activation of MAD2 from open to closed form (C-MAD2) capable of CDC20 binding and inhibition, thus delaying the onset of anaphase.29 TTK is probably implicated in the recruitment of the MAD1-C-MAD2 complex to kinetochores and during mitosis its activity is continuously required to recruit O-Mad2 to the Mad1-C-Mad2 core.30 Furthermore, TTK is required for CENP-E recruitment, whose activity is essential for metaphase chromosome alignment.30
In the present study we examined polymorphisms rs151658 (C>G) in
The study population (n = 284) consisted of 108 Slovenian patients with gastric cancer and 176 control subjects who at the time of peripheral blood extraction did not have cancer. Tumour and corresponding non-tumour tissues at least 7 cm away from the edge of the adenocarcinoma were collected from patients who were admitted to the Clinical Department for Abdominal Surgery at the University Medical Centre Ljubljana and Department for Pathology at the Institute of Oncology Ljubljana during the years 2000-2008. Samples were macrodissected by pathologist, frozen in liquid nitrogen and stored at −70°C. Comprehensive medical data were obtained from registries and pathologist’s evaluation. The following clinico-histopathological parameters were recorded: tumour differentiation (grade), location, blood and lymphatic vessel invasion (vascular invasion, perineural invasion), occurrence of tumour cells in the lymphatic vessels (lymphatic invasion), depth of invasion (pT), lymph node involvement (pN), and presence of distant metastases (pM). The gastric cancer cases were classified into diffuse type (n = 46) and intestinal (n = 58) according to Lauren classification. The mean age ± standard deviation (SD) of patients was 66.12 ± 12.02 (range, 33-87 years), and the percentage of men was 63.0%. Cases lost to follow-up (n = 6) and those, who died within 30 days after surgery (n = 2), were excluded from survival analyses. The control population was randomly selected during the years 1999-2007 and shared the ethnic and geographic background of the gastric cancer patients. The research was approved by the National Medical Ethics Committee of the Republic of Slovenia and confidentiality of personal medical data as well as other data relating to individual identification has been assured in accordance with the Helsinki Declaration.
Genomic DNA from gastric tumour and nontumour tissues was extracted using a Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) and QuickGene™ DNA Tissue Kit S (Fujifilm Corporation, Tokyo, Japan) on QuickGene-810 DNA isolation system (Fujifilm) according to manufacturer’s protocol. Genomic DNA from control population was extracted from peripheral blood samples using Wizard® Genomic DNA Purification Kit (Promega) following the manufacturer’s protocol. The DNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc.). Genotyping for polymorphism rs151658 (C>G) in
A total of 21 paired gastric adenocarcinoma (GA) and adjacent control tissue samples were ground with a mortar and pestle in liquid nitrogen and lysed with 7 mol/L urea, 2 mol/L thiourea, 40 g/L CHAPS, with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). For every 10 mg tissue, 50 μl lysis buffer was added. After sonication on ice (3 × 10 s), the samples were incubated for 1 h on ice with occasional vortexing, and then centrifuged at 20,000 × g for 1 h at 4°C. The supernatants were collected and the protein concentrations were determined using the commercial Bradford reagent (Thermo Fisher Scientific, Waltham, MA, USA) with BSA used as the standard. Immunoblot analysis was performed on 42 samples. A total of 30 μg protein per sample was loaded onto 10% gels, separated using SDS-PAGE, and transferred onto PDVF membranes (Millipore, Billerica, MA, USA), which were then blocked in 50 g/L skimmed milk 1 h. The primary antibody was used in the following dilution: anti-Mpsl (anti-TTK) antibody, 1 pg/ml (ab11108, Abcam, Cambridge, UK). Horseradish peroxidase-conjugated secondary antibody was used in the following dilution: goat anti-mouse antibody, 1:5000 (115-035-062, Jackson ImmunoResearch, Newmarket, Suffolk, UK). The proteins were revealed by chemiluminescence using LAS-4000 CCD camera (Fujifilm, Tokyo, Japan). The blots were then quantified with Multi Gauge software (Fujifilm) and the intensities were normalized to Ponceau-S-stained membranes, to allow for loading and transfer variations.
Statistical evaluation of the genotyping data was carried out using the χ2 or Fischer’s exact tests to compare the groups regarding genotype frequencies. Hardy-Weinberg (HW) equilibrium was calculated with an online program (
To assess the statistical significance of altered protein abundance in the immunoblotting (as the tumour vs. non-tumour paired samples), non-parametric Wilcoxon signed-rank test was used. The tests were double-sided and the values with p < 0.05 with a confidence level of 95% were considered to be statistically significant. To assess the correlation of the altered protein abundance from the immunoblotting with the histopathological parameters, repeated measures ANOVA was used. The values with p < 0.05 were considered to be statistically significant. Bonferroni post-tests were used to determine where the differences were significant. All of the analyses were performed using Microsoft Office Excel 2007 (Microsoft Corporation, Washington, USA) and GraphPad Prism 5 (GraphPad Software, Inc., California, USA).
In order to functionally evaluate intronic polymorphisms we identified their effect in the context of polymorphic biological sequences on protein binding motifs. We used web-based software PROMO, which is part of the ALGGENE web-server.32,33 The search for putative binding sites was performed using the following parameters: human species, all motifs, and all factors. The data for comparisons of genotype frequencies in European populations of examined SNPs in this study was extracted from the 1000 Genomes Project data platform using a specific version of the Ensembl browser (
The clinical information and demographic characteristics of selected patients with gastric cancer in this study are summarized in Table 1. At the end of a period of up to 11 years of follow-up, a total of 69 patients out of 100 have died.
Clinicopathological characteristics of patients with gastric cancer
Parameter | Number of patients (%) |
---|---|
Age (years ± standard deviation) (n = 108) | 66.12 ± 12.02 |
Gender (n = 105) | |
Male | 68 (63.0) |
Age (years ± standard deviation) (n = 66) | 65.07 ± 12.03 |
Female | 40 (37.0) |
Age (years ± standard deviation) (n = 39) | 67.90 ± 11.94 |
Lauren’s classification (n = 104) | |
Intestinal | 58 (55.8) |
Diffuse | 46 (44.2) |
Location (n = 101) | |
Upper | 40 (39.6) |
Lower | 34 (33.7) |
Mixed | 27 (26.7) |
Grade/differentiation (n = 105) | |
Well | 9 (8.6) |
Moderate | 24 (22.9) |
Poor | 72 (68.6) |
Vascular invasion (n = 80) | |
Present | 27 (33.8) |
Not present | 53 (66.3) |
Perineural invasion (n = 95) | |
Present | 44 (46.3) |
Not present | 51 (53.7) |
Lymphatic invasion (60) | |
Present | 53 (88.3) |
Not present | 7 (11.7) |
pN (n = 105) | |
0 | 24 (22.9) |
1-2 | 15 (14.3) |
3-6 | 20 (19.0) |
> 7 | 46 (43.8) |
pT (n = 105) | |
Muscularis propria | 6 (3.7) |
Subserosa | 50 (42.6) |
Serosa | 49 (36.1) |
pN = number of positive regional lymph nodes; pT = tumour invasion
The overall 5-year survival was 33.5%. No statistically significant association between tested genetic variations and survival was observed (p > 0.05). Univariate survival analysis showed that only Lauren’s classification and lymph node involvement (pN) were significant prognostic factors. Diffuse type predicted shorter 5-year survival (logrank test, χ2 = 5.516, p = 0.019) with overall mean estimate of survival for patients with intestinal type 64.67 months ± 7.74 (SE) (CI = 49.50-79.84) and 39.73 months ± 6.83 (SE) (CI = 26.75-53.11) for patients with diffuse type of gastric cancer. Regarding the parameter pN, patients with 7 or more positive lymph nodes had shorter survival time of 21.93 months ± 4.30 (SE) with CI = 13.5030-36 (log-rank test, χ2 = 34.169, p = 0.000). Multivariate analysis was performed for the same set of patients with complete clinical data sets. Cox regression model included both significant variables, pN and tumour classification. The enter method showed significant improvement (p < 0.05) if both parameters were entered into the model (Table 2).
Multivariate survival analysis of clinic-pathological variables in gastric cancer patients
Variable | B Predicted change in the hazard for a unit increase in the predictor. | SE (B) | OR (95% CI) | P |
---|---|---|---|---|
pN | 0.670 | 0.127 | 1.954 (1.525-2.504) | 0.000 |
Lauren’s classification | 0.591 | 0.246 | 1.807 (1.116-2.925) | 0.016 |
CI = confidence interval; OR = odds ratio; pN = number of positive regional lymph nodes; SE = standard error
Associations between clinicopathological parameters and genotypes of SNPs are presented in Table 3. Statistical analysis revealed a weakly significant association for rs151658 genotypes C/G and risk of developing diffuse type of gastric cancer, and genotype G/G and risk of developing intestinal type of cancer (p = 0.049). Similar results were obtained for both male and female populations of patients (p = 0.047 and p = 0.024, respectively). Genotype A/A of rs1801376 polymorphism was significantly associated with higher risk of developing the diffuse type of gastric cancer in total and female populations of patients (p = 0.007). Interestingly, A/G genotype was under-represented in populations with diffuse type of gastric cancer. Genotype A/A of this polymorphism was also associated with the invasion of tumour cells into subserosa layer of stomach in male population (p = 0.009). A/A genotype was also associated with tumour location, namely, A/A frequencies were increased in patients with tumours disseminated across the whole stomach (p = 0.035).
Comparison of clinic-pathological features and genotypes
Parameter | Subject | Variant/Genotype | P | |||
---|---|---|---|---|---|---|
GG | CG | CC | ||||
Lauren’s classification | Intestinal | Total | 17 | 21 | 20 | |
Diffuse | 5 | 25 | 16 | |||
Intestinal | Male | 9 | 10 | 14 | ||
Diffuse | 5 | 20 | 8 | |||
Intestinal | Female | 8 | 11 | 6 | ||
Diffuse | 0 | 5 | 8 | |||
AA | AG | GG | ||||
Intestinal | Total | 18 | 33 | 7 | ||
Diffuse | 28 | 13 | 5 | χ2=9.951 | ||
Intestinal | Male | 15 | 14 | 4 | 0.472 F=1.836 | |
Diffuse | 20 | 9 | 4 | |||
Intestinal | Female | 3 | 19 | 3 | ||
Diffuse | 8 | 4 | 1 | |||
CC | CT | TT | ||||
Tumour differentiation | Well | Total | 1 | 4 | 4 | |
Moderate | 7 | 9 | 7 | |||
Poor | 23 | 39 | 7 | |||
Well | Male | 0 | 2 | 3 | ||
Moderate | 6 | 7 | 2 | 0.139 F=6.439 | ||
Poor | 19 | 20 | 6 | |||
Well | Female | 1 | 2 | 1 | ||
Moderate | 1 | 2 | 5 | |||
Poor | 19 | 20 | 6 | |||
AA | AG | GG | ||||
pT | Muscularispropria | Total | 1 | 4 | 1 | 0.232 F=5.250 |
Subserosa | 27 | 18 | 5 | |||
Serosa | 18 | 25 | 6 | |||
Muscularispropria | Male | 0 | 3 | 1 | ||
Subserosa | 22 | 6 | 2 | |||
Serosa | 13 | 14 | 5 | |||
Muscularispropria | Female | 1 | 1 | 0 | 0.816 F=1.967 | |
Subserosa | 5 | 12 | 3 | |||
Serosa | 5 | 11 | 1 | |||
AA | AG | GG | ||||
Tumour | Upper | Total | 14 | 23 | 3 | |
location | Lower | 13 | 15 | 6 | ||
Whole | 18 | 6 | 3 |
pT = tumour invasion
Genotype T/T of rs1031963 was associated with well differentiated tumours in total population (p = 0.035); however, when we stratified it into female and male populations, we observed a significant association of this genotype with moderately differentiated tumours in the female population (p = 0.004). Clinico-pathological features lymph node involvement (pN), depth of invasion (pT), vascular invasion, perineural invasion and lymphatic invasion did not show significant associations with investigated polymorphisms.
The analyses of genotype frequencies in selected SNPs between cases and controls are shown in Tables 4 and 5. The frequencies of all genotypes in cases and control groups were in Hardy-Weinberg equilibrium.
Distribution of genotype frequencies of
Variants | Genotype | Cases (n) | Controls (n) | HWE (cases) | HWE (controls) | |
---|---|---|---|---|---|---|
GG | 24 | 55 | χ2=3,628 0.163 | χ2=1.08 0.299 | χ2=2.87 0.090 | |
CG | 48 | 76 | ||||
C/C | 36 | 44 | ||||
CC | 32 | 48 | χ2=1.059 0.589 | χ2=0.345 0.557 | χ2=0.035 0.852 | |
CT | 54 | 89 | ||||
TT | 18 | 39 | ||||
AA | 47 | 89 | χ2=2.291 0.318 | χ2=0.021 0.885 | χ2=0.005 0.941 | |
AG | 49 | 69 | ||||
GG | 12 | 13 | ||||
AA | 36 | 49 | χ2=1.186 0.553 | χ2=1.530 0.216 | χ2=0.040 0.841 | |
AG | 24 | 39 | ||||
GG | 8 | 7 | ||||
AA | 11 | 40 | F=6.955 | χ2=3.352 0.067 | χ2=0.013 0.909 | |
AG | 25 | 30 | ||||
GG | 4 | 6 |
F = Fisher statistics; HWE = Hardy-Weinberg Equilibrium;
Odds ratios for
Genotype model | Cases (n)/Control group (n) | OR (95% CI) p value with Yates correction | P | PY |
---|---|---|---|---|
Dominant | 72/120 | 0.900 | χ2=0.149 | χ2=0.062 |
TT+CT vs. CC | vs. 32/48 | (0.527-1.536) | 0.699 | 0.803 |
Recessive | 18/36 | 0.797 | χ2=0.507 | χ2=0.309 |
TT vs. CT+CC | vs. 86/137 | (0.426-1.491) | 0.476 | 0.578 |
Heterozygous | 54/89 | 0.910 | χ2=0.108 | χ2=0.035 |
CT vs. CC | vs. 32/48 | (0.520-1.594) | 0.742 | 0.853 |
Dominant | 41/101 | 0.364 | χ2=9.848 | χ2=8.834 |
TT+CT vs. CC | vs. 26/26 | (0.192-0.691) | ||
Recessive | 11/26 | 0.763 | χ2=0.467 | χ2=0.241 |
TT vs. CT+CC | vs. 56/101 | (0.351-1.659) | 0.494 | 0.623 |
Heterozygous | 30/75 | 0.400 | χ2=6.959 | χ2=6.057 |
CT vs. CC | vs. 26/26 | (0.201-0.797) | ||
Dominant | 31/57 | 1.994 | χ2=1.862 | χ2=1.281 |
TT+CT vs. CC | vs. 6/22 | (0.731-5.437) | 0.172 | 0.258 |
Recessive | 7/13 | 1.185 | χ2=0.107 | χ2=0.004 |
TT vs. CT+CC | vs. 30/66 | (0.429-3.269) | 0.743 | 0.949 |
Heterozygous | 24/44 | 2.000 | χ2=1.775 | χ2=1.188 |
CT vs. CC | vs. 6/22 | (0.714-5.606) | 0.183 | 0.276 |
Dominant | 61/82 | 1.409 | χ2=1.927 | χ2=1.601 |
GG+AG vs. AA | vs. 47/89 | (0.868-2.287) | 0.165 | 0.206 |
Recessive | 12/13 | 1.519 | χ2=0.999 | χ2=0.615 |
GG vs. AA+AG | vs. 96/158 | (0.666-3.465) | 0.318 | 0.433 |
Heterozygous | 49/69 | 1.345 | χ2=1.304 | χ2=1.025 |
AG vs. AA | vs. 47/89 | (0.808-2.237) | 0.253 | 0.311 |
Dominant | 32/46 | 0.947 | χ2=0.029 | χ2=0.0002 |
GG+AG vs. AA | vs. 36/49 | (0.508-1.766) | 0.864 | 0.990 |
Recessive | 8/7 | 1.676 | χ2=0.917 | χ2=0.466 |
GG vs. AA+AG | vs. 60/88 | (0.577-4.868) | 0.338 | 0.495 |
Heterozygous | 24/39 | 0.838 | χ2=0.272 | χ2=0.124 |
AG vs. AA | vs. 36/49 | (0.430-1.630) | 0.602 | 0.725 |
Dominant | 29/36 | 2.929 | χ2=6.719 | χ2=5.737 |
GG+AG vs. AA | vs. 11/40 | (1.281-6.700) | ||
Recessive | 4/6 | 1.296 | χ2=0.147 | χ2=0.001 |
GG vs. AA+AG | vs. 36/70 | (0.344-4.889) | 0.701 | 0.971 |
Heterozygous | 25/30 | 3.030 | χ2=6.732 | χ2=5.709 |
AG vs. AA | vs. 11/40 | (1.292-7.108) |
χ2 = chi-square statistics; OR = odds ratio; CI = confidence interval
The tested polymorphisms did not show significant differences between gastric cancer patients and control group. In contrast, when we stratified the population for gender, we found significant association between
Comparisons of the SNPs’ genotype frequencies between our test groups and European populations are presented in Figure 3. Genotype frequencies of rs151658, rs1031963 and rs1801376 in our groups of populations showed significant differences from European population (Table 6). The frequency of rs151658 C/C genotype was higher than expected in the Slovenian population of patients compared to total European population (p = 0.015). Similarly, we observed more rs1031963 C/C genotypes in the male population of Slovenian patients with gastric cancer (p = 0.042) compared with total European population and European population stratified for males. The rs1801376 A/G genotype was higher and A/A genotype was under-represented in female population of patients with gastric cancer compared to the total European population (p = 0.034) and female European population (p = 0.014).
Comparison of
Population | N | Genotype counts | P |
---|---|---|---|
EUR | 503 | 97 (C|C) / 246 (C|G) / 160 (G|G) | χ2 = 8.391; P = 0.015 a χ2 = 11.143; P = 0.004 b NS c |
SI (total)a | 283 | 80 (C|C) / 124 (C|G) / 79 (G|G) | |
SI (cases)b | 108 | 36 (C|C) / 48 (C|G) / 24 (G|G) | |
SI (controls)c | 175 | 44 (C|C) / 76 (C|G) / 55 (G|G) | |
EUR | 503 | 125 (C|C) / 259 (C|T) / 119 (T|T) | NS d NS e χ2 = 5.715; P = 0.057 f |
EUR - male | 240 | 56 (C|C) / 124 (C|T) / 60 (T|T) | _ c _ d χ2 = 6.348; P = 0.042 f |
SI (total)d | 280 | 80 (C|C) / 143 (C|T) / 57 (T|T) | |
SI (cases - female)e | 36 | 6 (C|C) / 23 (C|T) / 7 (T|T) | |
SI (cases - male)f | 65 | 25 (C|C) / 29 (C|T) / 11 (T|T) | |
EUR | 503 | 240 (A|A) / 217 (A|G) / 46 (G|G) | NS g F = 6.569; P = 0.034 h NS 1 |
EUR - female | 263 | 135 (A|A) / 109 (A|G) / 19 (G|G) | _ g F = 8.277; P = 0.014 h _ i |
SI (total)g | 279 | 136 (A|A) / 118 (A|G) / 25 (G|G) | |
SI (cases - female)h | 40 | 11 (A|A) / 25 (A|G) / 4 (G|G) | |
SI (cases - male)i | 68 | 36 (A|A) / 24 (A|G) / 8 (G|G) |
EUR = European population; F = Fisher statistics; SI (cases) = gastric cancer patients; SI (controls) = control population; SI (total) = combined populations of patients and controls; χ2 = chi-square statistics; superscript letters indicate comparisons between European population and Slovenian populations
Immunobloting data on individual samples (Figure 1A) demonstrated statistical significance for the increased abundance of TTK (p = 0.03) in the tumour tissues. No statistically significant correlation of TTK abundance with clinical his topathological parameters or rs151658 genotypes was observed. However, some trends were observed (Figures 1B and C) for lymph node involvement (pN) and antral tumour location: TTK abundance was higher in normal tissues compared to tumour tissues when no regional nodes were invaded with tumour cells (pN = 0) and when the tumours were located at the bottom of the stomach (antrum).
To determine if different intronic polymorphisms could affect binding of transcription factors, we performed
In this study, we investigated the effects of selected polymorphisms in mitotic kinases
To assess if the above mentioned genotypes perhaps had an effect on TTK protein levels, immunoblotting was performed. While the results regarding the effect of genotypes on protein abundance remain inconclusive, it should be noted that polymorphisms usually exert low-penetrance effects, which could more profoundly affect the pathogenesis of gastric cancer in early stages; however, when the disease progresses, the mutation load and aberrant expression of other genes mask their effects. We did, however, confirm higher abundance of TTK in tumour tissues, which is in accordance with several other studies and points out the deregulation of cell cycle homeostasis, higher proliferative trend of tumour cells and weakened spindle assembly checkpoint leading to increased genome instability and aneuploidy.37,38 Furthermore, this study showed a trend of increased TTK abundance associated with the spread of cancer cells to regional lymph nodes indicating a possible link between TTK levels and metastatic potential of malignant gastric cells.
Homozygous mutations of critical spindle-assembly
In conclusion, our study provides evidence that polymorphisms in mitotic kinases