Using a gel-based proteomic approach, we aimed to find and validate the differentially expressed proteins in a set of gastric adenocarcinoma patients. A total of 30 different proteins were identified in the study. They belonged to different biological processes, including metabolism, development, death, response to stress, cell cycle, cell communication, transport, and cell motility. The largest group, metabolism, contained 20 proteins with at least some role in cell metabolism.
A set of these proteins has already been found in similarly conducted experiments for GC. Among those downregulated in the tumor, enoyl CoA hydratase, mitochondrial precursor[20,21], acyl-CoA dehydrogenase, NADH dehydrogenase (ubiquinone) flavoprotein 2, mitochondrial precursor, phenazine biosynthesis-like domain-containing protein isoform a, and ATP synthase subunit d, mitochondrial have been reported with the same alterations as in our study. In contrast to our observation, ATP synthase subunit d was reported to be upregulated in another study. Among the proteins upregulated in the tumor, phosphoglycerate kinase 1, pyruvate kinase isozymes M1/M2[24,25], heat shock 70 kDa protein 1A/1B[24,25], cathepsin D precursor[22,24,26], annexin A4, alpha-enolase[24,25], nicotinamide N-methyltransferase[20,27,28], annexin A5[20,29], and actin, cytoplasmic 1 in rat GC metastases have been found elsewhere with the same trend as in our study. However, other studies have reported that pyruvate kinase, alpha-enolase, and actin[26,31] have the opposite expression patterns compared to our results. This discrepancy could perhaps result from the heterogeneity that is often present in studies of human tumor tissue. In this paper, we present and discuss eight putative biomarkers for gastric carcinogensis that were identified in our study, among which seven were validated for the first time by means of immunoblotting.
Specific protein alterations
GKN1 has already been identified as a downregulated gene in GC. It is suggested to maintain gastric mucosal integrity and mediate repair after injury. The protection of the mucosal barrier is thought to be due to the ability of GKN1 to alter the distribution of specific tight-junction proteins and to stabilize perijunctional actin. It was later demonstrated to bind with F-actin in smooth muscle cells, suggesting a role in cell–cell adhesion and the assembly of actin stress fibers. Recently, GKN1 has been shown to be a modulator of apoptotic signals. It has been confirmed as a secreted protein and as being present in native and metaplastic gastric epithelium, but absent from the gastric carcinoma and the precursor lesion of intestinal metaplasia, making it a possible tumor suppressor in gastric carcinogenesis.
GKN1 expression is downregulated in Helicobacter-pylori-positive patients. In another study, a loss of GKN1 occurred, especially in the diffuse-type tumor, but was associated with a significantly worse outcome in the intestinal type. It has been found to be downregulated in GC, using 2-DE[20,23,25,27], and these results have been validated at the protein and mRNA levels. Consistently, we found that GKN1 was underexpressed in tumor tissue, using 2-DE, and we also validated the results by immunoblotting. Our result is in agreement with other reports and, like other research groups, we were also unable to find any correlation with histopathological parameters at the protein level. The overlapping of both steps of the biomarker identification with a number of other studies in this case contributed to the confidence in the approach for the analysis of additional biomarkers found in our proteomic analysis.
MRPL12 is the first cloned and characterized mammalian mitochondrial ribosomal protein encoded by the nucleus. It accumulates in cells at the mRNA and protein levels upon growth-factor stimulation. The enhanced expression later contributes to transcriptional activation. MRPL12 mRNA levels have been detected in different organs, being especially high in the colon and skeletal muscle. Besides being a component of the mitochondrial ribosome (its dimers bind the large ribosomal unit), MRPL12 binds to mitochondrial RNA polymerase and enhances transcription in vitro. It has been speculated that it may either directly couple transcription and translation by binding simultaneously to polymerase and ribosomes, or alone bind to polymerase and activate its transcriptional activity in some way. However, Litonin et al have observed no such stimulation, so further experiments are necessary to clarify the possible role of this protein in transcription.
MRPL12 is differentially expressed, and it has previously been observed by 2-DE to be overexpressed in prostate cancer and in hepatitis B virus (HBV)-associated hepatocellular carcinoma. This is not in accordance with our results because we showed that MRPL12 was downregulated in gastric adenocarcinoma. We also observed it correlation with location: higher rates of underexpression were found in the antrum. In the context of cancer, the knockdown of MRPL12 decreases mitochondrial activity, increases glycolysis and accelerates tumor growth. We speculate that it may also be the case in gastric adenocarcinoma.
PACAP was found as an expressed sequence tag from a microarray analysis where it exhibited downregulation in intestinal-type GC. The protein was found in the endoplasmic reticulum of lymphocytes, where it exhibited upregulation in the course of B-cell differentiation[49,50]. It was found to assist in the oxidative folding of Ig domains; however, both research groups were uncertain as to whether it acted as an oxidoreductase or as a chaperone. In a very recent report, PACAP was described as helping to diversify peripheral B-cell functions by regulating Ca2+ stores, antibody secretion and integrin activation.
A patent application has disclosed the use of PACAP as a universal marker of different types of cancer, GC included. However, they claim that increased concentrations of the protein and/or its fragments are associated with cancer, whereas we discovered just the opposite for GC. The same was reported by Huang et al in a 2-DE experiment (although this was without validation), by Hasegawa et al and Katoh et al. However, in the latter two cases, this was at the non-protein level. We validated our results by immunoblotting. Due to its downregulation in the tumor, we strongly support the previous speculation that PACAP might be a candidate tumor-suppressor gene.
GSTM3 is a glutathione S-transferase (GST) that belongs to the mu class. It is rather unusual; it is not only about 70% identical in its protein sequence to the other mu-class transferases, but it is also considerably shorter and transcribed in the reverse orientation.
Several polymorphisms have been found in GSTs, which can alter the susceptibility to carcinogens and toxins and influence the toxicity and efficacy of drug treatment. They have been studied in relation to several cancers, GC included. For example, Martinez and colleagues have found no association with the GSTM3 genotype and GC risk, whereas Tatemichi and co-workers have described a possible association between GSTM3 polymorphisms and Ig titer levels in serum against H. pylori.
GSTM3 is found in several normal tissues, including the stomach. A comparison of the differential GSTM3 expression in cancers is made rather difficult by the fact that many studies have focused on the whole GST family or class, but not on individual isoforms. For instance, antral GST enzyme activity has been found to be significantly lower in the stomach of H. pylori-infected patients, but the contributions of separate isoforms has not been studied. For GSTM3 specifically, the gene is highly expressed in a subgroup of patients with head and neck squamous cell carcinoma. The protein is upregulated in neuroblastoma and in polycystic ovary syndrome. We found that GSTM3 was downregulated in gastric adenocarcinoma. Our results are in agreement with a study reporting downregulation in the seminomatous germ cell tumor, where the changes were also reflected at the transcriptional level. This reduced expression could be indicative of the decreased detoxification capacity of tumor cells.
Septins belong to a family of conserved GTP-binding proteins. They have been implicated in many cellular processes. SEPT2 is thought to be involved in cytokinesis, as well as chromosome congression and segregation[65,66]. Its fibers appear to contact actin bundles and focal adhesion complexes physically, thereby linking it to a functional interaction with actin-based cytoskeletal systems in interphase cells. It has also been found in the microtubule spindle during metaphase and is proposed to form a mitotic scaffold for different effectors to coordinate cytokinesis with chromosome congression and segregation. Several other binding partners and functions have been proposed for SEPT2, such as the DNA damage response, the regulation of the efficiency of vesicular transport, and FCγR-mediated phagocytosis. Recently, it has been reported that SEPT2 is part of a diffusion barrier between the primary cilia and the cell and it is essential for retaining receptor-signaling pathways in primary cilia. Also, in response to physiological and pathological stimuli, SEPT2 redistributes and its interaction with actin increases, which allows for the dynamic modulation of the airway epithelial barrier function. Despite all the progress, the exact molecular mechanisms, cellular, and physiological functions of septins are still poorly understood and interactome studies could help.
Septins have been linked to diseases such as neurodegeneration and cancer. It is proposed that altered SEPT2 expression can lead to disordered chromosomal dynamics and underlie the development of the aneuploidy common to cancers. SEPT2, among others, has been found to be a fusion partner of the mixed lineage leukemia (MLL) gene in therapy-related acute myeloid leukemia. Such fusion is associated with downregulation of SEPT2 and MLL in myeloid neoplasia. It has also been shown to be downregulated in glioblastoma. On the other hand, it has been identified as upregulated in hepatoma carcinoma cells, where its phosphorylation on Ser218 by casein kinase 2 has been determined as crucial for hepatoma carcinoma cell proliferation. In a 2-DE experiment, SEPT2 was, in agreement with our results, determined to be upregulated in GC; however, no validation was carried out. The same expression pattern was found in renal cell carcinoma and in late-stage human colon cancer tissue, and it is abundantly expressed in several brain tumors and brain-tumor cell lines. Taken as a whole, these results suggest its possible role as an oncogene.
Ubiquitination is a post-translational modification carried out in several steps, one of them being conjugation of an activated ubiquitin to an ubiquitin-conjugating enzyme (E2) via a highly conserved catalytic cysteine residue. By directly influencing the type of lysine used to label the substrates, they influence the fate of the substrates. One of the E2s is UBE2N, which acts as part of a complex that enables the formation of the non-canonical Lys63-mediated polyubiquitin chains. As opposed to Lys48-mediated ones, these do not target proteasome degradation but mediate other processes. Among other functions, it has been shown that UBE2N in a complex with Mms2 functions via Lys63-mediated polyubiquitination in DNA repair, whereas in a complex with Uev1A, it functions in activating nuclear factor-κB signaling[78,80]. Both of the partner proteins, however, are dispensable for the RNF8-dependent propagation of DNA damage signals via ubiquitination, although there are some questions as to the importance of this activity.
UBE2N is differentially expressed between different types of leukemia and lymphoma cell lines. It has significantly lower transcriptional expression levels in non-small-cell lung cancer and is correlated with pN and the stage of the disease. In a breast-cancer metastatic model using iTRAQ technology, UBE2N was downregulated when comparing cells with the most metastatic potential and non-metastatic cells. On the other hand, it was observed in a 2-DE experiment to be overexpressed in HBV-associated liver cancer, which is consistent with our results. It has already been shown to be differentially expressed in GC; however, it has not been validated whether it is up- or downregulated. UBE2N-dependent Lys63-mediated polyubiquitination regulates processes that often enhance cell survival in response to certain forms of stress, therefore, our result supports its implication in the regulation of similar processes in gastric cancerogenesis.
TALDO1 is an almost ubiquitous cofactor-less enzyme of the pentose phosphate pathway. Its activity is tissue specific and, in the brain, it is selectively expressed in oligodendrocytes, thus connecting it to different autoimmune diseases, such as multiple sclerosis. Its expression is developmentally controlled.
TALDO1 is the rate-limiting enzyme of the non-oxidative part of the pentose phosphate pathway that catalyzes the reversible transfer of a three-carbon unit between various sugar phosphates (from ketose to aldose sugar phosphates). It has a role in regulating the balance between the two branches of the pentose phosphate pathway and its overall output, as measured by NADPH and glutathione production, and thus influences the sensitivity to cell-death signals.
When comparing tumor and normal TALDO1, its activity is increased in neoplastic liver. Its gene is highly expressed in a subgroup of patients with squamous cell carcinoma of the head and neck. Furthermore, it is upregulated in late-stage human colon-cancer tissue and in the sera of colorectal cancer patients. In metastatic, compared to non-metastatic GC cell lines and in GC tissue, TALDO1 was overexpressed, as shown by 2-DE. However, in both studies, again, no validation was performed. All these studies are consistent with our results, which were also validated by immunoblotting. We also found that TALDO1 correlated with pN status at stages pN0 and pN3. A higher TALDO1 expression in the tumor tissue could reflect an increased metabolism of glucose for the synthesis of nucleic acids in malignant cells.
TPT1 is a ubiquitously expressed and highly conserved protein. It is associated with various cellular processes, such as cell-cycle progression, release of histamine and various interleukins, apoptosis, malignant transformation, and tumor reversion[94,95]. Very recently, it was also discovered as a glucose-regulated protein, important for the survival of pancreatic beta cells.
It has been implicated in cancer, although it is not tumor-specific. It is upregulated in various tumor tissue cell lines when compared to normal tissue cell lines, in breast and colon cancer. As for the gastric tissue, TPT1 has been reported as cDNA present in libraries only from normal gastric tissues. In our study, TPT1 was not validated as generally differentially expressed in the whole group of samples. Instead, its expression was location-correlated; TPT1 was upregulated in gastric adenocarcinoma from the cardia/gastroesophageal border. In contrast to the general worldwide decline of GC rates, an increasing incidence of gastric cardia cancer has been observed in several countries. This suggests that it is a distinct clinical entity. Therefore, it is possible that TPT1 is implemented only in gastric cardia/gastroesophageal border carcinogenesis.
Comparison of tumor and adjacent, non-tumor gastric tissues by means of proteome analysis, including differential 2-DE coupled to MS analysis, revealed 30 protein alterations. Some of the differentially expressed proteins had already been observed in GC in previous studies, which supports the reliability of our analysis. Several other proteins were found with the same trend of differential expression in other types of cancer, which could suggest that they are commonly involved in carcinogenesis. The high mortality rate from GC is due to delayed detection and surgical resection at advanced stages of the disease. A breakthrough in the early diagnosis of GC has not occurred yet and there are currently very few markers that are clinically in use; however, advances in proteomic research are facilitating the identification of novel diagnostic, prognostic, or therapeutic biomarkers. It is apparent that a collection of protein biomarkers will be necessary for reliable cancer detection and monitoring, as single biomarkers often have an inadequate predictive value. There is, therefore, a need for the expedited development of new, validated biomarkers to be added to the list of clinically relevant tumor-associated proteins in the proteome databases of gastric tissue and cell lines. To the best of our knowledge, we are the first to observe aberrant expression of MRPL12 in gastric adenocarcinoma, and, in addition, aberrant expression of PACAP, GSTM3, SEPT2, UBE2N, TALDO1 and TPT1 for the gastric cardia/esophageal border were validated in gastric adenocarcinoma, also for the first time. Future experiments are planned to use these biomarkers in the design of a combinatory microarray and to translate the obtained results to blood samples, so the proteins would ultimately be useful as biomarkers for early detection.