Background
Gastric cancer (or gastric carcinoma) is one of the most common malignancies in the world. Disease development in gastric cancer is a multi-stage process that involves more than one gene. Disease factors ultimately act on different genes at different stages, causing a change in gene structure and expression levels that lead to the development of gastric carcinogenesis. Loss of function of an anti-oncogene may occur through various channels. In addition to deletions and mutations, aberrant changes in DNA methylation are considered as the third mechanism leading to anti-oncogene inactivation [
1,
2], which plays an essential role in tumor development. CpG islands, which are CpG-rich sequences located in the upstream promoter region of a housekeeping gene, are regions at which cytosine methylation does not generally occur, although stimulation by certain factors leads to its methylation. Consequently, DNA expression is inhibited in this region [
3]. The inactivation of many tumor-associated genes such as P16, THBS1, TIMP3, hMLH1, and MGMT are related to the methylation of their respective promoter regions [
4‐
8]. Tissue inhibitor of metalloproteinase-3 (TIMP3) is related to tumor development, particularly antagonizing the activity of matrix metalloproteinases as well as inhibiting tumor growth, angiogenesis, invasion, and metastasis [
9].
In this work, we detected the methylation of the TIMP3 gene promoter CpG island and protein expression in normal gastric mucosa tissues, gastric cancer tissues, and metastatic lymph nodes of 78 patients with gastric cancer using methylation-specific PCR (MSP) and immunohistochemistry. We explored the correlation of gene promoter methylation with its corresponding protein expression and then analyzed the relationship of TIMP3 gene CpG island abnormal methylation with the development, as well as clinical and pathological features, of gastric adenocarcinoma.
Discussion
The formation of malignancy is multi-factorial and occurs in multiple stages. The biological characteristics of malignant tumors include invasive growth and metastasis. The mechanism behind tumor development involves abnormal changes in gene structure and function in the interior of the cell. Modern theorists consider that the process of tumor formation comprises two major mechanisms: the first one is at the genetic level, i.e., gene mutation causes DNA nucleotide sequence changes; and the second one is at the epigenetic level. Previous studies have focused on changes in gene expression that are inherited through meiosis and do not involve a change in DNA sequence but affect the expression and gene regulating function of DNA, mainly by chemical modification. This mechanism is gaining increased attention from researchers of tumor formation processes [
15,
16].
In the DNA of tumor tissues, abberant methylations are often observed in gene promoter regions, including hypomethylation in the oncogene. This gene is closely related to cell proliferation cycle and hypermethylation in tumor suppressor genes. These abnormal methylation changes can activate oncogenes [
17‐
19] and inhibit the mRNA transcription of tumor suppressor genes [
20], leading to gene inactivation. Aberrant methylation in DNA occurs through the presence of highly methylated CpG islands in the 5′ end of a promoter control region. The TIMP family has four protein members: TIMP-1, 2, 3, and 4. TIMP-1, 2, and 4 are soluble secretory proteins. By contrast, TIMP-3 is a non-soluble protein that combines with the extracellular matrix, is located in the outer cell membrane [
21], and can be closely connected with the basilar membrane. The function of TIMPs is the inhibition of tumor necrosis factor-α (TNF-α)-converting enzymes and the induction of programmed cell death through the stable cell surface TNF-α receptor [
22]. The induction of apoptosis through TIMP occurs by two pathways: MMP dependent and independent. Although TIMP-3 and 4 have a high affinity for MMP-2, the proteins effectively inhibit MT1-MMP, thereby activating the MMP-2 zymogen process and inhibiting tumor cell growth and metastasis. Smith et al. [
23] found that the expression of TIMP-3 in human colon cancer cell lines are rare in mitosis. Most cells are arrested in the G1 phase, although the application of TNF-α antibody decreases mitotic arrest by 70%.
To explore the relationship between TIMP3 and gastric cancer, as well as its mechanism in gastric inactivation, we detected TIMP3 gene 5′ CpG island hypermethylation in normal gastric mucosa, gastric carcinoma, and metastatic lymph nodes. We found that in >85% of all gastric cancer tissues, the TIMP3 gene 5′ CpG island exhibited aberrant methylation that was significantly higher (
P < 0.01) than that of the normal gastric tissue group, which implied that TIMP3 gene promoter CpG island methylation reduced TIMP3 gene expression and was thus the main tumor suppressor-inactivation mechanism in gastric cancer. Gagnon et al. [
24] reported that in all MCF-7 lung cancer cell lines with TIMP3 mRNA expression loss and TIMP3 gene promoter CpG island methylation, demethylation was induced by 5-AZA-CdR. TIMP3 expression before and after 5-AZA-CdR induction was assessed by RT-PCR, which showed that demethylation resulted in TIMP3 gene re-expression, consistent with our results.
In the present study, all 78 cases of normal gastric tissue were positive for TIMP3 protein expression. Among these 78 cases, only 28 (35.9%) were positive for methylation. Among the 20 patients with early gastric cancer, 9 were positive for TIMP3 protein expression and 17 (85%) were positive for methylation. Among the 58 cases of advanced gastric cancer, 21 were positive for TIMP3 protein expression and 52 (89.7%) were positive for methylation. Among the 78 cases of metastatic lymph nodes, 12 were positive for TIMP3 protein expression and 77 cases (98.7%) were positive for methylation. Increased methylation rate was significant among the early cancer, advanced cancer, and metastatic lymph node samples compared with normal tissue samples (
P < 0.01), confirming that the loss of TIMP3 protein expression was closely related to TIMP3 gene promoter CpG island methylation. Feng HF et al. [
25] reported the same results in JAR and JEG-3 choriocarcinoma cell lines. Therefore, our study confirmed that TIMP3 gene promoter CpG island methylation was the main reason for the reduced TIMP3 protein expression in gastric cancer.
Among the 58 advanced gastric cancer samples, 52 were positive for TIMP3 promoter CpG island methylation and 21 were positive for TIMP3 protein expression. This finding suggested that apart from CpG island methylation, other factors such as genetic variation caused the absence of TIMP3 gene expression. In our experiments, 34 samples were positive for methylation, and unmethylation was usually interpreted as follows: incomplete methylation status of genes may exist, and if the tumor tissue is mixed with non-tumor cells, a positive PCR product for unmethylation (by MSP with unmethylated primer) may be observed. TIMP3 protein expression of the sample was negative, so these samples were defined as methylation positive.
In China, the 5-year survival rate of gastric carcinoma patients is only about 40% after resection. The poor prognosis is associated with extensive local invasion and/or regional lymph node metastasis [
26]. Local recurrence remains as a major cause of cancer-related deaths after resection in gastric cancer patients [
27]. Therefore, reliable criteria for the prediction of recurrence and identification of tumors must be established to understand molecular and cellular processes, as well as discover new and possible therapeutic molecular targets [
28].
Kerbel [
29] reported that subcloning tumor cells with metastatic potential have a growth advantage in the early stage of primary foci. The reason for such growth advantage is that the metastatic subclone cell population may secrete a particular factor or local cell growth factor, thereby causing a special reaction that results in selective growth. Detection of molecular marker levels in primary tumor specimens may reflect the general metastatic characteristics of the whole tumor. In this group, the methylation rates were only 35.9% for the normal gastric tissues, 85% and 89.7% for the early and advanced cancers, respectively, 98.7% for metastatic lymph nodes. Consistently high TIMP3 protein expression was observed only in normal gastric tissues. Early and advanced gastric cancers exhibited focal weak expression, and metastatic lymph nodes showed almost no positive expression. This finding suggested that TIMP3 methylation increased with tumor progression and that protein expression gradually decreased. Consequently, the metastatic potential and growth advantage of cells gradually increased, ultimately resulting in metastasis. Yegnasubramanian et al. [
30] reported that tumor metastasis was related to TIMP3 methylation in prostate cancer cell lines as revealed by the real-time MSP of samples from 73 patients with early prostate cancer, 91 patients with metastatic prostate cancer, and 25 cases with prostatic tissue. This result implied a relationship between CpG methylation and the grading and progress of prostate cancer. TIMP3 gene methylation reportedly plays an important role in the metastatic process, and the detection of TIMP3 CpG methylation has some practical value in predicting the metastatic potential of the primary tumor.
TIMP3 has a high frequency of genetic variation in many types of human malignant tumors, including mutations, deletions, methylation, chromosomal translocation, and so on [
31]. These mutations result in the loss of the normal function of a tumor suppressor gene, namely, the inhibition of cell proliferation (i.e., gene inactivation) and are closely related to the development of cancer [
32]. However, few studies have been conducted on TIMP3 gene modification in primary gastric cancer, and the results of these studies show discrepant results from a low gene deletion rate to no mutation at all. Accordingly, other inactivation mechanisms of gastric cancer involving TIMP3 gene have been investigated [
33]. Thus, the complexity of tumor pathogenesis is not only a reflection of genetic change by mutation or deletion but also a reflection of epigenetic alterations, such as DNA methylation. An in-depth study of the relationship between DNA methylation and gene expression, as well as its corresponding mechanism, is recommended because the results can guide the clinical treatment of cancer.
Competing interest
The authors hereby declare that they have no competing interests with one another.
Authors’ contributions
ZG and JZ carried out the pathological and PCR examinations, DD and ZG analyzed the data, and ZG drafted the manuscript. All authors read and approved the final manuscript.