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SATB1 expression in gastric mucosa in relation to Helicobacter pylori infection and family history of gastric cancer
· Advances in Medical Sciences · Vol. 57(2) · 2012 · pp 237-243 · DOI: 10.2478/v10039-012-0049-z © Medical University of Bialystok, Poland
SATB1 expression in gastric mucosa in relation to Helicobacter pylori infection and family history of gastric cancer Zuk K1, Peczek L1, Stec-Michalska K 2, Medrek M2, Nawrot B1* 1 Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences, Lodz, Poland 2 Department of Gastroenterology, Medical University of Lodz, Lodz, Poland
* CORRESPONDING AUTHOR: Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences, 112 Sienkiewicza Str., 90-363 Lodz, Poland Tel: +48-42-6816970, Fax: +48-42-6815483 [email protected] (Barbara Nawrot)
Received 12.04.2012 Accepted 22.08.2012 Advances in Medical Sciences Vol. 57(2) · 2012 · pp 237-243 DOI: 10.2478/v10039-012-0049-z © Medical University of Bialystok, Poland
ABSTRACT Purpose: SATB1 protein, the altered levels of which are observed in tumour tissues, acts as a global regulator of gene expression. The aim of the study was to investigate the expression level of the SATB1 gene in gastric mucosa of dyspeptic patients in relation to the H. pylori infection, the family history of gastric cancer (FHGC), and histopathological changes. Material and methods: The study comprised 64 patients with dyspeptic symptoms. Group I – 28 control patients (10 H. pylori positive) without the FHGC. Group II – 36 patients (16 H. pylori positive) with the FHGC. The samples with normal mucosa (NM) or chronic superficial gastritis (CSG) were used for further analysis. qRT-PCR was used to determine the level of mRNA of SATB1. Results: The dominant histopathological changes in group I were NM and CSG. Specimens from group II have demonstrated an increasing frequency of atrophy (A) and intestinal metaplasia (IM). The A and IM specimens have shown increase of expression of the SATB1 and were excluded from further evaluation. In corpus samples of group II patients, the amount of SATB1 mRNA was higher than in antrum samples, regardless of H. pylori infection. The presence of bacterium resulted in the elevated SATB1 expression in corpus samples of group II patients only, while the genetic factor down-regulated SATB1 gene in the antrum samples of the H. pylori negative individuals. Conclusions: The expression of SATB1 gene correlates with histological changes and is altered by the selected environmental and hereditary factors, and the observed changes may have an impact on the development of gastric cancer. Key words: gene expression, SATB1, Helicobacter pylori, family history of gastric cancer, gastric cancer
INTRODUCTION Gastric cancer (GC) is a wide-spread disease, being the fourth most common cancer and the second leading cause of cancer associated death worldwide [1]. About 10% of all cases are hereditary, and very low percentage of these cases (1-3%) has been characterized as a single hereditary syndrome – Hereditary Diffuse Gastric Cancer (HDGC) [1]. This disease is an autosomal-dominant, inherited cancer syndrome, in which affected individuals develop diffuse-type gastric cancer at young age. This syndrome has
mutations in the E-cadherin gene (CDH1), and is the only one forcing the hereditary gastric cancer with the defined genetic basis. The inactivating mutations in the CDH1 gene have been identified in 30% to 50% of HDGC patients [2]. Epidemiologically, gastric adenocarcinoma occurs more often in the antrum than in the corpus [3]. GC is the result of a sequence of histopathological changes known as the Correa’s Cascade. The first step of the cascade, which may result in gastric cancer, is the gastritis, typically caused by infection with Helicobacter pylori (H. pylori) bacterium. In this cascade, chronic gastritis evolves to atrophy and, next, to intestinal metaplasia, which are preneoplastic lesions
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[4]. However, not all patients with the intestinal metaplasia develop adenocarcinoma. Premalignant lesions that precede invasive cancers, in which many of the genetic abnormalities and phenotypic characteristics of invasive cancer are present, are not fully understood yet [4]. SATB1 (special AT-rich binding protein 1), altered levels of which are observed in tumour tissues, acts as a global regulator of gene expression. It binds selectively to the nuclear matrix/scaffold-associating DNAs (MARs/SARs) [5]. SATB1 is one of the best characterised MAR-binding proteins that participate in maintenance of the chromatin architecture [6,7]. It acts as a „docking site” for several chromatin remodelling enzymes and regulates gene expression by recruiting co-repressors (HDAC) or co-activators (HAT). On the other hand, the activity of SATB1 is modulated through interactions with other proteins. Phosphorylation of SATB1 down-regulates, while acetylation up-regulates gene expression at global level [8]. It is suggested that through its contrasting activities, SATB1 regulates over 10% of genes [8]. Patani et al. has shown that in breast tissues the levels of mRNA of SATB1 were higher in malignant tissues compared to normal ones and that the SATB1 expression increased with increasing TNM stage and tumour progression [9]. Other studies on breast cancer have shown that knockdown of SATB1 in highly aggressive MDA-MB-231 cancer cells altered the expression of more than 1,000 genes, and reversed tumorigenesis. On the other hand, ectopic SATB1 expression in non-aggressive SKBR3 cells led to the gene expression patterns consistent with aggressive-tumour phenotypes [10]. A new mechanism of tumour progression exerted by SATB1 was suggested, in which SATB1 re-programmes chromatin and the transcription profiles of breast tumours, promoting in that way cancer growth and metastasis. Zhou et al. [11] have suggested that the elevated expression of SATB1 gene may be associated with the development and lymph node metastasis of NSCLC (non-small cell lung cancer) and, therefore, may have prognostic significance. They have shown that the level of SATB1 mRNA was 13-fold higher in NSCLC tissues than in normal lung tissues. Moreover, in metastatic and non-metastatic NSCLC the expression of SATB1 was ca. 24 and 6 times higher, respectively, than in normal lung tissue. The SATB1 mRNA expression is significantly up-regulated in gastric cancer and is an independent prognostic marker for this disease [12,13]. Also, it was proved that the SATB1
mRNA level corresponds to the protein level determined by immunohistochemistry [12]. We have asked the question whether the expression of SATB1 gene in gastric mucosa of dyspeptic patients is altered by environmental and hereditary factors, which are considered to be important in the development of gastric cancer [14]. We have already shown that Helicobacter pylori infection and the FHGC significantly affect the expression of some tumour suppressor genes including FHIT [15,16], HINT1 [17], and SSTR3 [18]. The decreased level of these proteins in gastric mucosa of FHGC dyspeptic patients indicates that the defence mechanisms in the gastric mucosa are disabled and those patients are more prone to the development of gastric cancer than control individuals (without FHGC). Also, H. pylori infection is thought to promote the development of gastric cancer [19]. In the present study we have searched for the relation between the expression level of SATB1 gene and H. pylori infection, the family history of gastric cancer (FHGC) and histopathological changes in gastric mucosa of patients with dyspepsia.
MATERIALS AND METHODS Patients
The study comprised 64 ethnically homogenous patients with functional gastrological disorders classified according to ROME III criteria, including postprandial distress syndrome and epigastric pain syndrome [20]. They were divided into two groups. Group I consisted of 28 control patients without a family history of gastric cancer, 10 of them were H. pylori positive (the UB13C test confirmed by the rapid urease test). Group II consisted of 36 patients with the FHGC (at least one member of a given family being a first degree relative who died of gastric cancer before the age of 60, in another family member a cancer of different organ had been diagnosed), 18 of them were H. pylori positive individuals. No data on the type of gastric cancer were collected. Patients below the age of 60 were non-smokers and in the past month did not take any non-steroid anti-inflammatory drugs (NSAIDs), inhibitors of proton pump (IPPs), or H 2 blockers. The study was conducted in accordance with the Helsinki Declaration and with principles of the Good Clinical Practice. This study was approved by the Ethical Commission of the Medical
Table 1. Characteristics of patients enrolled to the studies – antrum (a) and corpus (c); F – female, M – male. Selection criterion Group I - patients (28) without the family history of gastric cancer
Group II – patients (36) with the family history of gastric cancer
H. pylori infection
Gender (F/M)
Age mean ± SD, range (years)
Hp(-)
18 (6/12)
33.6 ± 11.5 (32.8 ± 15.7/34.0 ± 9.5)
Hp(+)
10 (3/7)
34.6 ± 10.9 (34.9 ± 10.7/34.0 ± 13.9)
Hp(-)
18 (8/10)
40.9 ± 14.1 (49.4 ± 13.8/34.2 ± 10.8)
Hp(+)
18 (11/7)
45.1 ± 13.0 (48.2 ± 10.6/36.8 ± 16.8)
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University of Lodz, Poland (approval number RNN/399/07/ KB, as of 24.07.2007). Each patient, before being enrolled into the research program, was acquainted with the aim of the study and gave conscious, written consent to participate in the study. The characteristics of the patients is given in Tab. 1.
Biopsies
All patients attending an open access endoscopy service for the investigation of dyspepsia had mucosal biopsies taken from the antrum and corpus. Two samples from each topographic site (from the antrum and from the corpus) were taken for determination of the level of mRNA of the SATB1. The initial total number of samples for molecular biology screening was 256, 112 – from group I and 144 from group II patients. The remaining samples, one from the prepyloric part, taken 3-5 cm proximally from the pylorus, and one from the corpus, taken 5-8 cm distally from the cardia, were immersed in 10% formalin and submitted for histology. The total number of samples for histopathology screening was 128, 56 – from group I and 72 from group II patients. Sections were stained with haematoxylin and eosin, alcian blue-periodic acid Schiff, and modified Giemsa. Only cases with two adequately sized biopsies of, both, the antral and corpus mucosa, were accepted for histological assessment.
Isolation of total RNA from biopsy specimens
The biopsy specimens were placed separately in properly labeled sterile tubes and washed three times in PBS buffer without Ca2+ and Mg2+ ions. After the washing, the specimens were transferred to a manual homogenizer and 1 mL of lysing reagent (TriPure Isolation Reagent, Boehringer Mannheim) was added. The material was placed in Eppendorf tubes and stored for further studies frozen at -70°C for 2-4 weeks. After de-freezing, total RNA was isolated from the homogenate (according to the protocol of the manufacturer of TriPure Isolation Reagent) and used for determination of the level of mRNA of the selected proteins. The level of SATB1 and GAPDH expression in the biopsy specimen was evaluated by real time quantitative RT-PCR. Samples were analyzed blind to the family history of gastric cancer status. Primers listed in Tab.2 were designed with the help of the Primer3 program (http://frodo.wi.mit.edu/cgibin/ primer3/primer3_www.egi). Total RNA (200 ng/2.0 mL) isolated from the sample and solutions of each of the primers
(1 mL of 3.0 mM) were added to the enzymatic reaction mixture (6.0 mL) containing LC RT-PCR Reaction Mix SYBR Green I (2 mL), LC RT-PCR Enzyme Mix (0.2 mL), MgCl2 (5 mM, 0.8 mL) stock solution and PCR-grade water (Roche Applied Science). Amplification conditions included reverse transcription of the template RNA at 55°C for 10 min, and deactivation of the reverse transcriptase at 95°C for 30 s. cDNA amplification procedure included denaturation (95°C for 0 s), annealing (56°C for 15 s) and extension (72°C for 13 s). The products were identified by the thermal dissociation method and electrophoresis in 2% agarose gel (not shown). The values of the melting temperatures (Tm ± SD) for amplification products of GAPDH and of SATB1 genes were in the range of 84.86 ± 0.27 and 82.41 ± 0.39°C, respectively. The level of SATB1 mRNA was normalized to the level of mRNA of the control GAPDH house-keeping protein (SATB1/GAPDH).
Statistical analysis
All results are given as a ratio of SATB1 to GAPDH products (SATB1/GAPDH). We used the multi-way table and Pearson Chi-square to determine the differences in frequency of histopathological changes depending on family history of gastric cancer. The Shapiro-Wilk W test was used to analyze the normality of the data distribution. The differences between two independent groups of data were calculated by the use of the non-parametric Mann-Whitney U test or by a parametric test – the t Student test. All statistical analyses were done using Statistica ver. 8.0 software (StatSoft Inc., Tulsa, OK, USA). A value of p < 0.05 was considered to be statistically significant. On the graphs, the median is marked as a horizontal line (central tendency) and variation statistics – a box – the lower and upper quartiles (the 25th and 75th percentiles of the distribution). Whiskers outside of the box represent min-max values.
RESULTS Analysis of bioptates in respect to histopathological changes
The percentage distribution of superficial gastritis, atrophy and intestinal metaplasia in the mucosa samples was analyzed by histopathological assessment. The samples collected from
Table 2. Primers used for amplification of SATB1 and GAPDH genes (F – forward, R – reverse, bp – base pair). Amplified gene
Accession number
SATB1
NM_002971.3 and NM_001131010.1
GAPDH
NM_002046.3
Primer sequence F: 5’ - ATG GGA AAC CAG AGA ACA A - 3’ R: 5’ - AGC TCG CAC AAC CAT C - 3’ F: 5’ - CAT CAT CTC TGC CCC CTC TG - 3’ R: 5’ - TCC ACG ATA CCA AAG TTG TC - 3’
Amplicon size (bp) 142
159
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Table 3. Histopathological examination of gastric mucosa samples. Total number of biopsies – 128, 56 - from group I and 72 from group II. Histopathological changes are described as NM normal mucosa, CSG - chronic superficial gastritis, A - atrophy, IM - intestinal metaplasia. Histological changes (%) Selection criterion
Bioptates from patients of group I (without the FHGC)
NM/CSG A
IM
54 (96.4)
2 (3.6)
0 (0.0)
14 (19.4)
10 (13.9)
Bioptates from patients of group II 48 (66.7) (with the FHGC)
both topographical sites of the stomach (the antrum and the corpus) irrespective of H. pylori infection were divided into two pools, for those from the control patients of group I and for those from patients of group II with FHGC. Thus, the only criterion of samples selection was the family history of gastric cancer. The results for these two pools show the numbers of samples with the normal mucosa and chronic superficial gastritis as well as those with either atrophy or intestinal metaplasia (Tab. 3). The dominant histopathological changes in group I samples are those with the normal mucosa and chronic superficial gastritis (> 96%). Specimens from the group of patient with FHGC (group II) have demonstrated an increasing frequency of atrophy (from 3.6% to 19.4%) and intestinal metaplasia (from 0.0% to 13.9%). All these results were statistically significant (p = 0.00005).
The relation between SATB1 mRNA levels and histology
Due to the remarkable number of samples with atrophy and intestinal metaplasia in group II of the patients, the respective samples from analogous topographical sites (taken for molecular biology screening) were subjected to analysis of the relation between the expression level of the SATB1 gene and the histopathology changes. The samples from group II patients (2 from the antrum and 2 from the corpus) were Figure 1. Comparison of the level of expression of SATB1 gene in samples with atrophy and intestinal metaplasia (A and IM, 26 samples) to samples with normal mucosa and chronic superficial gastritis (NM and CSG, 102 samples).
divided into those representing normal and superficial gastritis mucosa and representing mucosa with atrophy and intestinal metaplasia. The level of SATB1 mRNA was analyzed in both groups of samples. The amount of the mRNA of SATB1 in each biopsy specimen was determined by the real time RTPCR and normalized to the amount of mRNA of GAPDH. For each sample the amplification reaction was repeated twice and the resultant data were used for the calculation of a median. The amounts of SATB1 mRNA obtained in this analysis were compared for both analyzed groups of samples. As shown in Fig. 1, the samples with atrophy and intestinal metaplasia have shown statistically significant (p = 0.048580) increase of expression of the SATB1 gene in comparison to the samples from normal mucosa and chronic superficial gastritis. Due to the elevated level of SATB1 mRNA in the tissue samples with atrophy and intestinal metaplasia, bioptates with these changes were excluded from further evaluation. Thus, from each screened group of patients only the samples with normal mucosa as well as with chronic superficial gastritis were used for further analysis. We decided that the following study will be carried out on a homogenous group of patients with NM/GSC changes (54 bioptates versus 48 bioptates from gr. I and II, respectively). In contrary, the number of bioptates with A/IM changes was not well balanced – 2 bioptates versus 24 bioptates in gr. I and II, respectively.
Expression of SATB1 gene in relation to stomach topography
The selected tissue samples from group I and group II patients (with NM and CSG histopathological changes) were screened for the level of SATB1 mRNA in relation to the topographic site of the stomach. The analysis was performed separately for the control group and group II samples (H. pylori positive and H. pylori negative). This analysis has shown that in control Figure 2. Expression of SATB1 gene in relation to stomach topography. The level of SATB1 mRNA is compared between samples from the control individuals (group I, upper graph) and from group II (lower graph) H. pylori negative (Hp(-)) and H. pylori positive (Hp(+)) depending of stomach topography (antrum versus corpus). The numbers of the samples of each investigated sub-group are shown under the plots.
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samples the level of SATB1 mRNA is similar in the antrum and the corpus, independently of H. pylori infection (Fig. 2, upper graphs). However, the level of the SATB1 mRNA is statistically higher in the corpus of group II samples, both H. pylori negative and H. pylori positive (Fig. 2, lower graphs).
Figure 3. Expression of SATB1 gene in relation to Helicobacter pylori infection. The level of SATB1 mRNA is compared in the antrum and corpus samples from control (group I, upper graph) and from group II patients (lower graph) depending on H. pylori infection. The numbers of the samples of each investigated subgroup are shown under the plots.
Expression of SATB1 gene in relation to the H. pylori infection An influence of Helicobacter pylori infection on the expression level of the SATB1 gene was checked in the screened samples from patients of group I and group II. The upper plot of Fig. 3 shows comparison of SATB1 mRNA in control samples from the antrum and from the corpus without and with Helicobacter pylori infection. No statistically significant differences are observed between Hp(-) and Hp(+) samples in this group of patients. In contrast, the results of analysis of an influence of the H. pylori infection on the level of SATB1 mRNA in FHGC samples have shown statistically significant increase of the SATB1 mRNA in the corpus samples after bacterial infection (p = 0.0146) (lower graph of Fig. 3).
Expression of SATB1 in relation to the family history of gastric cancer
To answer the question whether hereditary factors have any influence on the level of expression of SATB1 gene, the level of the SATB1 mRNA in the control samples was compared with the FHGC samples. All the samples were divided for those H. pylori negative and H. pylori positive. Interestingly, statistically significant decrease of SATB1 expression level was noticed in the antrum samples with FHGC (the upper plot of Fig. 4) and increase of the SATB1 expression level was noticed in the corpus H. pylori positive samples with the FHGC (the lower plot of Fig. 4).
Figure 4. Influence of family history of gastric cancer on the expression of the SATB1 gene in stomach mucosa of patients from group I and group II (without and with a family history of cancer, respectively). The upper plot represents results for H. pylori negative patients, and the lower plot for H. pylori positive patients. Comparison between the control and the FHGC samples is done for the antrum and corpus samples.
DISCUSSION There are numerous reports showing elevated levels of SATB1 mRNA in malignant tissues compared to normal tissues [9-11], and these data suggest a contribution of SATB1 to cancer development. In our study we had been interested in ethiopathogenesis of gastric cancer which is associated with multiple environmental and hereditary factors, including Helicobacter pylori infection and the family history of gastric cancer. We have previously demonstrated that these two factors affect the expression profile of the selected tumour suppressor genes as FHIT [15,16], HINT1 [17], and SSTR3 [18], the levels of which are down-regulated in malignant tissues and cells. The decreased levels of these proteins in gastric mucosa of FHGC dyspeptic patients, in comparison to control individuals (without FHGC), indicate that defence mechanisms in the gastric mucosa are disabled and those patients may be more prone to development of gastric
cancer. Since SATB1 is suggested to be a prognostic gene for monitoring the development of a cancer, we have thought that its expression may also be sensitive to the environmental and hereditary factors investigated by us. The present study clearly demonstrates that SATB1 mRNA level is correlated to several screened factors. First, we demonstrate that SATB1 expression depends on the histopathological changes of gastric mucosa (Fig. 1). This issue is important in relation to the development of a gastric cancer, which is the last step of the Correa’s Cascade. Our study has shown that the expression level of the SATB1 is elevated in stomach mucosa of patients with more advanced
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histopathological changes, as atrophy and intestinal metaplasia (which are preneoplastic lesions [4]), in comparison to normal mucosa and those with chronic superficial gastritis, the initial steps of Correa’s Cascade. This result suggests that increased expression of the global regulator is already switched on, as in preneoplastic lesions, what may result in development of gastric carcinoma with high levels of SATB1. Moreover, the expression of SATB1 gene in gastric mucosa of dyspeptic patients is altered by the family history of gastric cancer, and in tissue samples from H. pylori infected patients with FHGC the level of SATB1 mRNA is higher than in the control individuals (p = 0.0068) (Fig. 4). The observed dependence, however, is statistically significant in corpus mucosa samples only. Interestingly, also infection of corpus mucosa of the FHGC patients with H. pylori, which is thought to be a first class carcinogen, results in the elevated expression level of the SATB1 in comparison to analogous samples without bacterium (p = 0.0148) (Fig. 3). An increased expression of SATB1 in the corpus mucosa of H. pylori positive patients of the group II, in comparison to analogous samples from the antrum, confirms that the corpus mucosa is more sensitive to the investigated factors. We speculate that the tissues of various morphology from both topological sites may exert different genetic response to FHGC. Another interesting data obtained within this study has shown that in gastric mucosa of patients without H. pylori infection the observed changes in SATB1 mRNA level are opposite, i.e. that level is lower at patients with FHGC, with statistical significance in the antrum samples (Fig. 4 upper graph). From the other studies we know that those patients of group II are more prone to the development of gastric cancer, despite of the absence of bacteria. Therefore, it is challenging to find a molecular mechanism by which genetic factors, as the FHGC, predispose to GC. One of the possible pathways altered by the elevated level of SATB1 is that associated with expression of c-MYC, which is the protooncogene described as one of critical elements of several carcinogenesis processes [21]. SATB1 represses expression of c-MYC [22] and the high levels of c-MYC are observed in more than 40% of gastric cancers. Its increased expression is also noticed in precancerous gastric lesions [23]. Because the expression level of the SATB1 is decreased in patients with FHGC, our hypothesis is that it may up-regulate of the c-MYC gene. Further studies to verify this hypothesis are carried out in our Laboratory.
CONCLUSIONS In conclusion, we demonstrate that the variable expression level of the SATB1 in gastric mucosa of dyspeptic patients observed for given environmental and hereditary factors, as well as for given histological changes of the stomach tissues may be attributed to the opposite functions of SATB1 as the co-repressor or co-activator of global gene expression.
ACKNOWLEDGEMENTS
The study was supported by the National Science Center in Poland, Project No MNiSW 402 307336 to B.N. Authors state they have no commercial associations or patent licenses that might result in a conflict of interest with the work presented in the submitted paper.
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SATB1 expression in gastric mucosa in relation to Helicobacter pylori infection and family history of gastric cancer Zuk K1, Peczek L1, Stec-Michalska K 2, Medrek M2, Nawrot B1* 1 Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences, Lodz, Poland 2 Department of Gastroenterology, Medical University of Lodz, Lodz, Poland
* CORRESPONDING AUTHOR: Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences, 112 Sienkiewicza Str., 90-363 Lodz, Poland Tel: +48-42-6816970, Fax: +48-42-6815483 [email protected] (Barbara Nawrot)
Received 12.04.2012 Accepted 22.08.2012 Advances in Medical Sciences Vol. 57(2) · 2012 · pp 237-243 DOI: 10.2478/v10039-012-0049-z © Medical University of Bialystok, Poland
ABSTRACT Purpose: SATB1 protein, the altered levels of which are observed in tumour tissues, acts as a global regulator of gene expression. The aim of the study was to investigate the expression level of the SATB1 gene in gastric mucosa of dyspeptic patients in relation to the H. pylori infection, the family history of gastric cancer (FHGC), and histopathological changes. Material and methods: The study comprised 64 patients with dyspeptic symptoms. Group I – 28 control patients (10 H. pylori positive) without the FHGC. Group II – 36 patients (16 H. pylori positive) with the FHGC. The samples with normal mucosa (NM) or chronic superficial gastritis (CSG) were used for further analysis. qRT-PCR was used to determine the level of mRNA of SATB1. Results: The dominant histopathological changes in group I were NM and CSG. Specimens from group II have demonstrated an increasing frequency of atrophy (A) and intestinal metaplasia (IM). The A and IM specimens have shown increase of expression of the SATB1 and were excluded from further evaluation. In corpus samples of group II patients, the amount of SATB1 mRNA was higher than in antrum samples, regardless of H. pylori infection. The presence of bacterium resulted in the elevated SATB1 expression in corpus samples of group II patients only, while the genetic factor down-regulated SATB1 gene in the antrum samples of the H. pylori negative individuals. Conclusions: The expression of SATB1 gene correlates with histological changes and is altered by the selected environmental and hereditary factors, and the observed changes may have an impact on the development of gastric cancer. Key words: gene expression, SATB1, Helicobacter pylori, family history of gastric cancer, gastric cancer
INTRODUCTION Gastric cancer (GC) is a wide-spread disease, being the fourth most common cancer and the second leading cause of cancer associated death worldwide [1]. About 10% of all cases are hereditary, and very low percentage of these cases (1-3%) has been characterized as a single hereditary syndrome – Hereditary Diffuse Gastric Cancer (HDGC) [1]. This disease is an autosomal-dominant, inherited cancer syndrome, in which affected individuals develop diffuse-type gastric cancer at young age. This syndrome has
mutations in the E-cadherin gene (CDH1), and is the only one forcing the hereditary gastric cancer with the defined genetic basis. The inactivating mutations in the CDH1 gene have been identified in 30% to 50% of HDGC patients [2]. Epidemiologically, gastric adenocarcinoma occurs more often in the antrum than in the corpus [3]. GC is the result of a sequence of histopathological changes known as the Correa’s Cascade. The first step of the cascade, which may result in gastric cancer, is the gastritis, typically caused by infection with Helicobacter pylori (H. pylori) bacterium. In this cascade, chronic gastritis evolves to atrophy and, next, to intestinal metaplasia, which are preneoplastic lesions
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[4]. However, not all patients with the intestinal metaplasia develop adenocarcinoma. Premalignant lesions that precede invasive cancers, in which many of the genetic abnormalities and phenotypic characteristics of invasive cancer are present, are not fully understood yet [4]. SATB1 (special AT-rich binding protein 1), altered levels of which are observed in tumour tissues, acts as a global regulator of gene expression. It binds selectively to the nuclear matrix/scaffold-associating DNAs (MARs/SARs) [5]. SATB1 is one of the best characterised MAR-binding proteins that participate in maintenance of the chromatin architecture [6,7]. It acts as a „docking site” for several chromatin remodelling enzymes and regulates gene expression by recruiting co-repressors (HDAC) or co-activators (HAT). On the other hand, the activity of SATB1 is modulated through interactions with other proteins. Phosphorylation of SATB1 down-regulates, while acetylation up-regulates gene expression at global level [8]. It is suggested that through its contrasting activities, SATB1 regulates over 10% of genes [8]. Patani et al. has shown that in breast tissues the levels of mRNA of SATB1 were higher in malignant tissues compared to normal ones and that the SATB1 expression increased with increasing TNM stage and tumour progression [9]. Other studies on breast cancer have shown that knockdown of SATB1 in highly aggressive MDA-MB-231 cancer cells altered the expression of more than 1,000 genes, and reversed tumorigenesis. On the other hand, ectopic SATB1 expression in non-aggressive SKBR3 cells led to the gene expression patterns consistent with aggressive-tumour phenotypes [10]. A new mechanism of tumour progression exerted by SATB1 was suggested, in which SATB1 re-programmes chromatin and the transcription profiles of breast tumours, promoting in that way cancer growth and metastasis. Zhou et al. [11] have suggested that the elevated expression of SATB1 gene may be associated with the development and lymph node metastasis of NSCLC (non-small cell lung cancer) and, therefore, may have prognostic significance. They have shown that the level of SATB1 mRNA was 13-fold higher in NSCLC tissues than in normal lung tissues. Moreover, in metastatic and non-metastatic NSCLC the expression of SATB1 was ca. 24 and 6 times higher, respectively, than in normal lung tissue. The SATB1 mRNA expression is significantly up-regulated in gastric cancer and is an independent prognostic marker for this disease [12,13]. Also, it was proved that the SATB1
mRNA level corresponds to the protein level determined by immunohistochemistry [12]. We have asked the question whether the expression of SATB1 gene in gastric mucosa of dyspeptic patients is altered by environmental and hereditary factors, which are considered to be important in the development of gastric cancer [14]. We have already shown that Helicobacter pylori infection and the FHGC significantly affect the expression of some tumour suppressor genes including FHIT [15,16], HINT1 [17], and SSTR3 [18]. The decreased level of these proteins in gastric mucosa of FHGC dyspeptic patients indicates that the defence mechanisms in the gastric mucosa are disabled and those patients are more prone to the development of gastric cancer than control individuals (without FHGC). Also, H. pylori infection is thought to promote the development of gastric cancer [19]. In the present study we have searched for the relation between the expression level of SATB1 gene and H. pylori infection, the family history of gastric cancer (FHGC) and histopathological changes in gastric mucosa of patients with dyspepsia.
MATERIALS AND METHODS Patients
The study comprised 64 ethnically homogenous patients with functional gastrological disorders classified according to ROME III criteria, including postprandial distress syndrome and epigastric pain syndrome [20]. They were divided into two groups. Group I consisted of 28 control patients without a family history of gastric cancer, 10 of them were H. pylori positive (the UB13C test confirmed by the rapid urease test). Group II consisted of 36 patients with the FHGC (at least one member of a given family being a first degree relative who died of gastric cancer before the age of 60, in another family member a cancer of different organ had been diagnosed), 18 of them were H. pylori positive individuals. No data on the type of gastric cancer were collected. Patients below the age of 60 were non-smokers and in the past month did not take any non-steroid anti-inflammatory drugs (NSAIDs), inhibitors of proton pump (IPPs), or H 2 blockers. The study was conducted in accordance with the Helsinki Declaration and with principles of the Good Clinical Practice. This study was approved by the Ethical Commission of the Medical
Table 1. Characteristics of patients enrolled to the studies – antrum (a) and corpus (c); F – female, M – male. Selection criterion Group I - patients (28) without the family history of gastric cancer
Group II – patients (36) with the family history of gastric cancer
H. pylori infection
Gender (F/M)
Age mean ± SD, range (years)
Hp(-)
18 (6/12)
33.6 ± 11.5 (32.8 ± 15.7/34.0 ± 9.5)
Hp(+)
10 (3/7)
34.6 ± 10.9 (34.9 ± 10.7/34.0 ± 13.9)
Hp(-)
18 (8/10)
40.9 ± 14.1 (49.4 ± 13.8/34.2 ± 10.8)
Hp(+)
18 (11/7)
45.1 ± 13.0 (48.2 ± 10.6/36.8 ± 16.8)
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University of Lodz, Poland (approval number RNN/399/07/ KB, as of 24.07.2007). Each patient, before being enrolled into the research program, was acquainted with the aim of the study and gave conscious, written consent to participate in the study. The characteristics of the patients is given in Tab. 1.
Biopsies
All patients attending an open access endoscopy service for the investigation of dyspepsia had mucosal biopsies taken from the antrum and corpus. Two samples from each topographic site (from the antrum and from the corpus) were taken for determination of the level of mRNA of the SATB1. The initial total number of samples for molecular biology screening was 256, 112 – from group I and 144 from group II patients. The remaining samples, one from the prepyloric part, taken 3-5 cm proximally from the pylorus, and one from the corpus, taken 5-8 cm distally from the cardia, were immersed in 10% formalin and submitted for histology. The total number of samples for histopathology screening was 128, 56 – from group I and 72 from group II patients. Sections were stained with haematoxylin and eosin, alcian blue-periodic acid Schiff, and modified Giemsa. Only cases with two adequately sized biopsies of, both, the antral and corpus mucosa, were accepted for histological assessment.
Isolation of total RNA from biopsy specimens
The biopsy specimens were placed separately in properly labeled sterile tubes and washed three times in PBS buffer without Ca2+ and Mg2+ ions. After the washing, the specimens were transferred to a manual homogenizer and 1 mL of lysing reagent (TriPure Isolation Reagent, Boehringer Mannheim) was added. The material was placed in Eppendorf tubes and stored for further studies frozen at -70°C for 2-4 weeks. After de-freezing, total RNA was isolated from the homogenate (according to the protocol of the manufacturer of TriPure Isolation Reagent) and used for determination of the level of mRNA of the selected proteins. The level of SATB1 and GAPDH expression in the biopsy specimen was evaluated by real time quantitative RT-PCR. Samples were analyzed blind to the family history of gastric cancer status. Primers listed in Tab.2 were designed with the help of the Primer3 program (http://frodo.wi.mit.edu/cgibin/ primer3/primer3_www.egi). Total RNA (200 ng/2.0 mL) isolated from the sample and solutions of each of the primers
(1 mL of 3.0 mM) were added to the enzymatic reaction mixture (6.0 mL) containing LC RT-PCR Reaction Mix SYBR Green I (2 mL), LC RT-PCR Enzyme Mix (0.2 mL), MgCl2 (5 mM, 0.8 mL) stock solution and PCR-grade water (Roche Applied Science). Amplification conditions included reverse transcription of the template RNA at 55°C for 10 min, and deactivation of the reverse transcriptase at 95°C for 30 s. cDNA amplification procedure included denaturation (95°C for 0 s), annealing (56°C for 15 s) and extension (72°C for 13 s). The products were identified by the thermal dissociation method and electrophoresis in 2% agarose gel (not shown). The values of the melting temperatures (Tm ± SD) for amplification products of GAPDH and of SATB1 genes were in the range of 84.86 ± 0.27 and 82.41 ± 0.39°C, respectively. The level of SATB1 mRNA was normalized to the level of mRNA of the control GAPDH house-keeping protein (SATB1/GAPDH).
Statistical analysis
All results are given as a ratio of SATB1 to GAPDH products (SATB1/GAPDH). We used the multi-way table and Pearson Chi-square to determine the differences in frequency of histopathological changes depending on family history of gastric cancer. The Shapiro-Wilk W test was used to analyze the normality of the data distribution. The differences between two independent groups of data were calculated by the use of the non-parametric Mann-Whitney U test or by a parametric test – the t Student test. All statistical analyses were done using Statistica ver. 8.0 software (StatSoft Inc., Tulsa, OK, USA). A value of p < 0.05 was considered to be statistically significant. On the graphs, the median is marked as a horizontal line (central tendency) and variation statistics – a box – the lower and upper quartiles (the 25th and 75th percentiles of the distribution). Whiskers outside of the box represent min-max values.
RESULTS Analysis of bioptates in respect to histopathological changes
The percentage distribution of superficial gastritis, atrophy and intestinal metaplasia in the mucosa samples was analyzed by histopathological assessment. The samples collected from
Table 2. Primers used for amplification of SATB1 and GAPDH genes (F – forward, R – reverse, bp – base pair). Amplified gene
Accession number
SATB1
NM_002971.3 and NM_001131010.1
GAPDH
NM_002046.3
Primer sequence F: 5’ - ATG GGA AAC CAG AGA ACA A - 3’ R: 5’ - AGC TCG CAC AAC CAT C - 3’ F: 5’ - CAT CAT CTC TGC CCC CTC TG - 3’ R: 5’ - TCC ACG ATA CCA AAG TTG TC - 3’
Amplicon size (bp) 142
159
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Table 3. Histopathological examination of gastric mucosa samples. Total number of biopsies – 128, 56 - from group I and 72 from group II. Histopathological changes are described as NM normal mucosa, CSG - chronic superficial gastritis, A - atrophy, IM - intestinal metaplasia. Histological changes (%) Selection criterion
Bioptates from patients of group I (without the FHGC)
NM/CSG A
IM
54 (96.4)
2 (3.6)
0 (0.0)
14 (19.4)
10 (13.9)
Bioptates from patients of group II 48 (66.7) (with the FHGC)
both topographical sites of the stomach (the antrum and the corpus) irrespective of H. pylori infection were divided into two pools, for those from the control patients of group I and for those from patients of group II with FHGC. Thus, the only criterion of samples selection was the family history of gastric cancer. The results for these two pools show the numbers of samples with the normal mucosa and chronic superficial gastritis as well as those with either atrophy or intestinal metaplasia (Tab. 3). The dominant histopathological changes in group I samples are those with the normal mucosa and chronic superficial gastritis (> 96%). Specimens from the group of patient with FHGC (group II) have demonstrated an increasing frequency of atrophy (from 3.6% to 19.4%) and intestinal metaplasia (from 0.0% to 13.9%). All these results were statistically significant (p = 0.00005).
The relation between SATB1 mRNA levels and histology
Due to the remarkable number of samples with atrophy and intestinal metaplasia in group II of the patients, the respective samples from analogous topographical sites (taken for molecular biology screening) were subjected to analysis of the relation between the expression level of the SATB1 gene and the histopathology changes. The samples from group II patients (2 from the antrum and 2 from the corpus) were Figure 1. Comparison of the level of expression of SATB1 gene in samples with atrophy and intestinal metaplasia (A and IM, 26 samples) to samples with normal mucosa and chronic superficial gastritis (NM and CSG, 102 samples).
divided into those representing normal and superficial gastritis mucosa and representing mucosa with atrophy and intestinal metaplasia. The level of SATB1 mRNA was analyzed in both groups of samples. The amount of the mRNA of SATB1 in each biopsy specimen was determined by the real time RTPCR and normalized to the amount of mRNA of GAPDH. For each sample the amplification reaction was repeated twice and the resultant data were used for the calculation of a median. The amounts of SATB1 mRNA obtained in this analysis were compared for both analyzed groups of samples. As shown in Fig. 1, the samples with atrophy and intestinal metaplasia have shown statistically significant (p = 0.048580) increase of expression of the SATB1 gene in comparison to the samples from normal mucosa and chronic superficial gastritis. Due to the elevated level of SATB1 mRNA in the tissue samples with atrophy and intestinal metaplasia, bioptates with these changes were excluded from further evaluation. Thus, from each screened group of patients only the samples with normal mucosa as well as with chronic superficial gastritis were used for further analysis. We decided that the following study will be carried out on a homogenous group of patients with NM/GSC changes (54 bioptates versus 48 bioptates from gr. I and II, respectively). In contrary, the number of bioptates with A/IM changes was not well balanced – 2 bioptates versus 24 bioptates in gr. I and II, respectively.
Expression of SATB1 gene in relation to stomach topography
The selected tissue samples from group I and group II patients (with NM and CSG histopathological changes) were screened for the level of SATB1 mRNA in relation to the topographic site of the stomach. The analysis was performed separately for the control group and group II samples (H. pylori positive and H. pylori negative). This analysis has shown that in control Figure 2. Expression of SATB1 gene in relation to stomach topography. The level of SATB1 mRNA is compared between samples from the control individuals (group I, upper graph) and from group II (lower graph) H. pylori negative (Hp(-)) and H. pylori positive (Hp(+)) depending of stomach topography (antrum versus corpus). The numbers of the samples of each investigated sub-group are shown under the plots.
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samples the level of SATB1 mRNA is similar in the antrum and the corpus, independently of H. pylori infection (Fig. 2, upper graphs). However, the level of the SATB1 mRNA is statistically higher in the corpus of group II samples, both H. pylori negative and H. pylori positive (Fig. 2, lower graphs).
Figure 3. Expression of SATB1 gene in relation to Helicobacter pylori infection. The level of SATB1 mRNA is compared in the antrum and corpus samples from control (group I, upper graph) and from group II patients (lower graph) depending on H. pylori infection. The numbers of the samples of each investigated subgroup are shown under the plots.
Expression of SATB1 gene in relation to the H. pylori infection An influence of Helicobacter pylori infection on the expression level of the SATB1 gene was checked in the screened samples from patients of group I and group II. The upper plot of Fig. 3 shows comparison of SATB1 mRNA in control samples from the antrum and from the corpus without and with Helicobacter pylori infection. No statistically significant differences are observed between Hp(-) and Hp(+) samples in this group of patients. In contrast, the results of analysis of an influence of the H. pylori infection on the level of SATB1 mRNA in FHGC samples have shown statistically significant increase of the SATB1 mRNA in the corpus samples after bacterial infection (p = 0.0146) (lower graph of Fig. 3).
Expression of SATB1 in relation to the family history of gastric cancer
To answer the question whether hereditary factors have any influence on the level of expression of SATB1 gene, the level of the SATB1 mRNA in the control samples was compared with the FHGC samples. All the samples were divided for those H. pylori negative and H. pylori positive. Interestingly, statistically significant decrease of SATB1 expression level was noticed in the antrum samples with FHGC (the upper plot of Fig. 4) and increase of the SATB1 expression level was noticed in the corpus H. pylori positive samples with the FHGC (the lower plot of Fig. 4).
Figure 4. Influence of family history of gastric cancer on the expression of the SATB1 gene in stomach mucosa of patients from group I and group II (without and with a family history of cancer, respectively). The upper plot represents results for H. pylori negative patients, and the lower plot for H. pylori positive patients. Comparison between the control and the FHGC samples is done for the antrum and corpus samples.
DISCUSSION There are numerous reports showing elevated levels of SATB1 mRNA in malignant tissues compared to normal tissues [9-11], and these data suggest a contribution of SATB1 to cancer development. In our study we had been interested in ethiopathogenesis of gastric cancer which is associated with multiple environmental and hereditary factors, including Helicobacter pylori infection and the family history of gastric cancer. We have previously demonstrated that these two factors affect the expression profile of the selected tumour suppressor genes as FHIT [15,16], HINT1 [17], and SSTR3 [18], the levels of which are down-regulated in malignant tissues and cells. The decreased levels of these proteins in gastric mucosa of FHGC dyspeptic patients, in comparison to control individuals (without FHGC), indicate that defence mechanisms in the gastric mucosa are disabled and those patients may be more prone to development of gastric
cancer. Since SATB1 is suggested to be a prognostic gene for monitoring the development of a cancer, we have thought that its expression may also be sensitive to the environmental and hereditary factors investigated by us. The present study clearly demonstrates that SATB1 mRNA level is correlated to several screened factors. First, we demonstrate that SATB1 expression depends on the histopathological changes of gastric mucosa (Fig. 1). This issue is important in relation to the development of a gastric cancer, which is the last step of the Correa’s Cascade. Our study has shown that the expression level of the SATB1 is elevated in stomach mucosa of patients with more advanced
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histopathological changes, as atrophy and intestinal metaplasia (which are preneoplastic lesions [4]), in comparison to normal mucosa and those with chronic superficial gastritis, the initial steps of Correa’s Cascade. This result suggests that increased expression of the global regulator is already switched on, as in preneoplastic lesions, what may result in development of gastric carcinoma with high levels of SATB1. Moreover, the expression of SATB1 gene in gastric mucosa of dyspeptic patients is altered by the family history of gastric cancer, and in tissue samples from H. pylori infected patients with FHGC the level of SATB1 mRNA is higher than in the control individuals (p = 0.0068) (Fig. 4). The observed dependence, however, is statistically significant in corpus mucosa samples only. Interestingly, also infection of corpus mucosa of the FHGC patients with H. pylori, which is thought to be a first class carcinogen, results in the elevated expression level of the SATB1 in comparison to analogous samples without bacterium (p = 0.0148) (Fig. 3). An increased expression of SATB1 in the corpus mucosa of H. pylori positive patients of the group II, in comparison to analogous samples from the antrum, confirms that the corpus mucosa is more sensitive to the investigated factors. We speculate that the tissues of various morphology from both topological sites may exert different genetic response to FHGC. Another interesting data obtained within this study has shown that in gastric mucosa of patients without H. pylori infection the observed changes in SATB1 mRNA level are opposite, i.e. that level is lower at patients with FHGC, with statistical significance in the antrum samples (Fig. 4 upper graph). From the other studies we know that those patients of group II are more prone to the development of gastric cancer, despite of the absence of bacteria. Therefore, it is challenging to find a molecular mechanism by which genetic factors, as the FHGC, predispose to GC. One of the possible pathways altered by the elevated level of SATB1 is that associated with expression of c-MYC, which is the protooncogene described as one of critical elements of several carcinogenesis processes [21]. SATB1 represses expression of c-MYC [22] and the high levels of c-MYC are observed in more than 40% of gastric cancers. Its increased expression is also noticed in precancerous gastric lesions [23]. Because the expression level of the SATB1 is decreased in patients with FHGC, our hypothesis is that it may up-regulate of the c-MYC gene. Further studies to verify this hypothesis are carried out in our Laboratory.
CONCLUSIONS In conclusion, we demonstrate that the variable expression level of the SATB1 in gastric mucosa of dyspeptic patients observed for given environmental and hereditary factors, as well as for given histological changes of the stomach tissues may be attributed to the opposite functions of SATB1 as the co-repressor or co-activator of global gene expression.
ACKNOWLEDGEMENTS
The study was supported by the National Science Center in Poland, Project No MNiSW 402 307336 to B.N. Authors state they have no commercial associations or patent licenses that might result in a conflict of interest with the work presented in the submitted paper.
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