Discussion
The Ras family comprises three members including K-Ras, H-Ras and N-Ras that encode proteins known as p21, which play a key role in transducing growth signals from the cell surface to the cell nucleus. The activation of Ras signaling, such as the ‘Ras-RAF-MEK-ERK’ signaling cascade, causes cell proliferation, differentiation and survival. The role of the Ras gene in the development of malignant tumors has always been a focus of research. Currently, it is known that the aberrant activation of Ras signaling causes persistent cell proliferation and leads to tumorigenesis (8).
Ras genes are well investigated due to their frequent activation and mutation in human cancers. Abnormal Ras signaling activation is mainly caused by mutations. Ras mutations lead to constantly activated Ras signaling in the absence of extracellular signals. Activating mutations of Ras are found in 20–25% of all human tumors and in up to 90% of certain tumor types. While mutations of H-Ras and N-Ras do not frequently occur, high levels of K-Ras mutations are found in leukemias, colon, pancreatic and lung cancer (9). However, in the absence of a mutation, it is difficult to explain the observed high Ras signaling. For example, in some tumor entities, including in gastric cancer, although Ras mutations are seldom detected, Ras activity is still unusually high (10–11). The underlying mechanisms regarding this remain largely unknown.
Findings of other studies have shown a novel mechanism for Ras activity regulation besides point mutation (4–6). Ras has two structural conformations, ‘on’ and ‘off’. When Ras is bound to the nucleotide guanosine diphosphate (GDP), it is in an ‘off’ state, whereas Ras bound to guanosine triphosphate (GTP) is in an ‘on’ state. The activation and deactivation of Ras is controlled by GAPs and guanine nucleotide exchange factors (GEFs) (12). The GAPs have GTP enzyme activity, which facilitate converting Ras from an active GTP-bound state to an inactive GDP-bound state. Inversely, the GEFs change Ras from an inactive GDP-bound state to an active GTP-bound state. The balance between GEF and GAP activity determines the guanine nucleotide status of Ras, thereby regulating Ras activity. Since Ras GAPs switch off Ras signaling transduction pathways, the genes encoding these RAS GAPs are considered to be potential tumor suppressor genes. A number of studies have indicated that certain members of the Ras GAP family are indeed tumor suppressors, including neurofibromin (NF1) and DAB2IP (13–17).
The RASAL1 gene, which is located on chromosome 12 (12q23-q24), has an overall length of 1100 bp and a structure mainly consisting of exon 1 and 2 and Ca2+-binding sites. As a newly discovered gene, it was proved to regulate the activity of the Ras signal transduction pathway (4). The RASAL1 gene possesses the characteristics of Ras GAPs, which enhance the intrinsic Ras-GTPase activity through hydrolyzing GTP into GDP, and thus is involved in cell differentiation, generation and apoptosis. Jin et al found that RASAL1 is expressed in almost all normal tissues, including in the heart, kidney, gastrointestinal tract, pancreas, lung, prostate and bone marrow, suggesting that RASAL1 genes possess a significant role in maintaining various normal human physiological functions (4). In recent studies, RASAL1 was found to be downregulated in a variety of tumor tissues, including naso-pharyngeal, breast, lung, liver and esophageal cancer and lymphoma. By studying 152 patient colorectal cancer tissues and 18 types of colon cancer cells, Ohta et al found that the expression of RASAL1 decreased markedly in colorectal cancer cells that contained the wild-type K-Ras gene, but did not decrease in those colorectal cancer cells with a mutant K-Ras gene (5). RASAL1 expression was detected in 46.9% (30/64) of adenocarcinoma, 17.4% (8/46) of large adenoma and none of the small adenoma samples (0/42). Based on the above study, RASAL1 was found to inhibit tumor progression by downregulating the Ras signal activity. It was also found that the ectopic expression of RASAL1 in transfected colorectal cancer cells in culture promoted Ras inactivation, which was confirmed by the depression of ERK, the downstream effector in the Ras signaling transduction pathway, as well as the suppression of the malignant phenotype in colorectal cancer cells. In their study, Calvisi et al also found that in the absence of Ras mutations, the downregulation of RASAL1, as well as other Ras GAPs (DAB2IP and NF1), resulted in the unrestrained activation of Ras signaling in the presence of wild-type Ras in human hepatocarcinogenesis (12), but it is unknown whether or not this also occurs in gastric cancer.
In our study, using immunohistochemistry, it was found that of 50 cases of gastric cancer tissues 12 cases were (−), 23 cases (+), 13 cases (++) and 2 cases (+++); whereas in 50 cases of normal gastric tissues 16 cases were (++) and 34 cases were (+++). This finding indicates that the RASAL1 expression is significantly reduced in gastric cancer tissues as well. Furthermore, we found that a reduced RASAL1 expression was associated with tumor size, differentiation degree, invasion depth and lymph node metastasis, but not with age and gender. RT-PCR and western blotting were used to detect the expression of RASAL1 in three types of gastric adenocarcinoma cell lines, including well-, moderately and poorly differentiated adenocarcinoma cells. The results confirmed that RASAL1 expression decreased in the three types of gastric adenocarcinoma cells compared with the normal gastric epithelial cell line, and the expression level was correlated with the differentiation: the poorest differentiation had the lowest expression. In a recent study, Seto et al investigated 10 gastric cancer cell lines by immunoblotting, and found that RASAL1 expression was reduced in 6 out of the 10 cell lines (18). These authors also reported that the immunohistochemical analyses in primary gastric tumors revealed that the RASAL1 expression was reduced in 23 out of 48 (48%) of the gastric cancers cell lines, but in none of the adenomas (0/10). These results suggest that RASAL1 is important in the tumorigenesis and development of gastric carcinoma. The results of our study have shed light on the pathogenesis of gastric carcinoma, and are a new therapeutic target for gastric carcinoma treatment.
Epigenetic silencing has been found to be the key mechanism responsible for the downregulated expression of the RASAL1 gene in tumors. Evidence indicates that CpG island methylation in the promoter of the RASAL1 gene may induce silencing in multiple tumors, including in esophageal cancer, nasopharyngeal carcinoma and colorectal cancer cells (4). Simultaneously, other studies have demonstrated that in colon cancer cells, the gene methylation of RASAL1 was reversed by using the methylation inhibitor 5-acetazolamide-2-cytosine deoxyriboside (5-Aza-CdR) (19). Kolfschoten et al, however, found another important mechanism affecting RASAL1 expression. Since RASAL1 is a transcription target of the anti-oncogene pituitary homeobox 1 (PITX1), RASAL1 expression may be downregulated by a decrease in PITX1 gene expression in human gastric carcinogenesis (20–21).
In conclusion, our findings demonstrate that the expression of RASAL1 is markedly decreased in gastric carcinoma tissues and cell lines, and is associated with gastric carcinoma differentiation degree and progression, suggesting that it is important in the development of gastric carcinoma. The mechanisms that contribute to the downregulated expression of RASAL1 in gastric cancer require further investigation.