Despite the progressive decrease observed in the past fifty years, gastric cancer (GC) is the fourth of the world rankings incidence of various types of cancer and is the second as a cause of cancer-related death. There is distinct geographical variation in gastric cancer incidence with the highest rates reported from Japan, Korea and Eastern Asia. Other high incidence areas are Eastern Europe and parts of Latin America, while Western Europe, Africa, Australia and the US generally have low incidence rates. In the last decade there has been a downward trend in the incidence and mortality from this cancer. The reasons are to be found in the improvement of food both as regards its preservation procedures and the variability in the diet and for the decrease of infection by Helicobacter
The most important epigenetic alterations are confined within the chromatin structure like chromatin remodeling, DNA methylation and histone modification. These molecular alterations are generally common in gastric cancer, independently from its classification (gastric or intestinal). Also genetic polymorphism represents a possible endogenous cause of cancer risk. However, it must be considered that genetic polymorphisms might influence the efficacy of gastric cancer therapy and the toxicity of anticancer drugs. Although the worldwide decline in incidence and recent diagnostic and therapeutic advances provided excellent survival for patients with early gastric cancer, the prognosis of patients with advances cancer is still poor. Over the past 15 years, integrated research, including genetic polymorphism and global analysis of gene expression has clarified detailed molecular mechanisms and the role of genetic and epigenetic abnormalities of cancer-related genes in the course of development and progression of gastric cancer. This review describes an outline of the molecular pathway of stomach carcinogenesis, as signaling pathways, H.
2. Risk factors and classification
The general decrease of gastric cancer frequency in developed countries is attributed to the changes in dietary habits and food preservation methods . Chronic inflammation with gastric atrophy was shown to be the most important pathological entity with hypochlorhydria being the most important physiological abnormality. Alcohol and smoking are also thought to contribute to the etiology. Achlorhydria, pernicious anaemia and blood group A are also associated with a higher risk of gastric malignancy. However, following the discovery of H.
GC is characterized by two distinct histological type of adenocarcinoma (intestinal type and a diffuse type) each having different epidemiological and pathophysiological features . The intestinal-type generally evolves through a relatively well-defined multistep process that starts from chronic gastritis and progresses to chronic atrophy, intestinal metaplasia and dysplasia [5, 6]. It is associated with H.
The pathogenesis of gastric cancer remains poorly understood although it is evident that several environmental factors, such as H.
Among the targets of CagA strain there is also RUNX3, a tumor suppressor in many tissues and frequently inactivated in gastric cancer. Recently, it has been shown that H.
4. Molecular pathology
The bacterial, environmental and host genetic factors discussed above influence the development of gastric carcinoma. Genomic, proteomic and biotechnology could allow the identification of novel genes and molecules specifically up-regulated or down-regulated in gastric cancer. Advances in our understanding of the genetic and molecular bases of gastric cancer lead to improved diagnosis, personalized medicine and prevention of gastric cancer.
In the following section, we discuss some of the molecular mechanisms underlying the molecular pathway of stomach carcinogenesis and of the biological and clinical roles of recently identified genes involved in gastric cancer.
Recently, a novel tissue-specific protein, gastrokine1 (GKN1), has been isolated from gastric mucosa cells of several mammalian species, including mouse . The human GKN1 gene has been localized in a 6 kb region of the chromosome 2p13 and contains 6 exons . GKN1 is found within the granules just under the apical plasma membrane, suggesting that it is a secreted rather than a membrane protein. GKN1 has been hypothesized to play an important role in maintaining the integrity of the gastric mucosa and mediating repair after injury. Oien et al.  demonstrated, by Northern blotting, that GKN1 mRNA was abundant only in normal human stomach, in all areas (cardia, body and antrum), but absent in gastric adenocarcinomas; gastro-oesophageal adenocarcinoma cell line and other normal and tumor gastro-intestinal tissues. Therefore, there is a transcriptional silencing of GKN1 gene in gastric cancer. Rippa et al.  recently demonstrated, by means of proteomic technology, that GKN1 protein is reduced in patients with
Our group has been studying the effect of GKN1 on gastric cancer cell lines (AGS and MKN28). We found by cytofluorimetry, Western blot and RT-PCR that overexpression of GKN1 in these cell lines stimulated the expression of Fas receptor. Moreover, compared to control cells, a significant increase of apoptosis, evaluated by TUNEL, was observed when GKN1 transfected cells were treated with a monoclonal antibody (IgM) anti-Fas. The activation of Fas expression was also observed by the overexpression of GKN1 in other cancer cell lines. GKN1-overexpressing gastric cancer cells exposed to FasL induced the activation of caspase-3 was as evaluated by Western blot and fluorescence assays . In addition, MTT assay showed that recombinant GKN1 reduced cell proliferation of gastric cancer cells (AGS) compared to human embryonic kidney cell line (HEK 293) and non-gastric cancer cells, human lung epidermoid carcinoma cell line (H1355). Our data represent the first report for GKN1 as modulator of apoptotic signals and suggest that GKN1 might play an important role for tissue repair during the early stages of neoplastic transformation. In fact, it was seen that individuals with a lower expression of the protein have an increased risk to develop gastric diseases .
Finally, regarding the mechanism by which
4.2 E–cadherin and the Wnt system
The signal transduction pathway called Wnt is a central mechanism for regulating gene expression and is highly conserved in vertebrates and invertebrates. It includes a large family of ligands and plays a key role in many cellular processes, which ranging from regulation of embryogenesis control processes the proliferation of mature cells. In particular, it seems crucially involved in the processes of differentiation and proliferation of stem cell elements . Central to these mechanisms is the process of regulation of expression of the β-catenin, an intracellular protein able to communicate on the surface of the cell with the system of cadherine, but also to act as nuclear transcription factor, including the Wnt/Wingless, epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF) signaling pathways [37-40].
The Wnt signalling pathway can be activated trough the binding of Wnt ligands to their receptors Frizzled (Fz) and low-density lipoprotein receptor-related protein LRP5 and LRP6 (Figure 2). The binding induces an activation signal direct protein Dishevelled (DSH) and axin, that once activated, inhibit GSK3 kinase. Normally GSK3 phosphorylates β-catenin as part of a multiprotein complex that includes GSK3, APC and axin. The phosphorylation triggers the degradation of β-catenin through the process of ubiquitination (Figure 2, left panel). It follows that the inhibition Wnt-induced GSK3 interferes with the process of degradation of β-catenin and causes its cytoplasmic accumulation in a non-complexed form . As consequence, β-catenin goes into the nucleus and regulates target gene transcription through association with the transcription factor TCF/ LEF (lymphoid enhancer binding factor) (Figure 2, right panel).
The translocation into the nucleus of β-catenin eventually leads to transcription of several genes including protagonists of carcinogenesis known protooncogenes such as c-myc and cyclin D1. In this regard, it is interesting that GSK3 can be inhibited through the PI3K/AKT pathway after toxin VacA stimulation . This is a further element to support the correlation between infection by H.
Another potential candidate in the molecular carcinogenesis process of stomach cancers is represented by RUNX3, one of the first identified members of the RUNX family (mammalian Runt related genes) . The RUNX gene family is composed of three members, RUNX1/AML1, RUNX2 and RUNX3 . These are genes coding for a group of closely related proteins with DNA binding function. In humans, loss of RUNX3 by hypermethylation of the promoter CpG islands is observed in several different cancers, including 64% of gastric carcinomas . This loss reaches 90% in patients with gastric cancer in advanced stage. Gastric epithelium of RUNX3 knockout mice exhibits hyperplasia, reduced rate of apoptosis and reduced sensitivity to TGFβ1, thus suggesting that the tumour suppressor activity of RUNX3 operates downstream of the TGFβ signaling pathways. RUNX3 methylation is also a feature of 8% of chronic gastritis, 28% of intestinal metaplasia and 27% of gastric adenomas. These observations suggest RUNX3 is a target for epigenetic gene silencing in gastric carcinogenesis [59, 60]. Another element of great interest that correlates RUNX3 to H.
4.4 Genomic instability
In gastric cancer the loss of genomic stability represents a key molecular step that occurs early in the carcinogenesis process and creates a permissive environment for the accumulation of genetic and epigenetic alterations in tumor suppressor genes and oncogenes. It iswidely accepted that gastric cancer can follow at least two major genomic instability pathways, chromosome instability (CIN) and microsatellite instability (MSI). CIN is defined as the loss of chromosomal material during dysfunctional chromosome replication, repair or segregation . MSI, which results from an erroneous DNA mismatch repair system, has been well known to be involved in the carcinogenesis of hereditary nonpolyposis colon cancers and some of sporadic colorectal cancers [64, 65]. A variable fraction from 15% to 50% of sporadic gastric cancer ischaracterized by MSI as a result of genetic inactivation or mainly Mismatch Repair (MMR) genes epigenetics, including hMLH1 and hMSH2. The inactivation of MMR genes is not itself a transforming event and additional genetic changes are required for progression to malignancy. In particular, in gastric cancer MSI is observed with the presence of mutations in repetitive sequences of genes involved in the regulation of cell growth (TGF-βRII, IGF-IIR), in apoptosis (BAX) and DNA repair (hMSH6, hMSH3). These mutations can alter the gene expression and give an advantage in cell growth and in clonal expansion. GC with MSI represents a tumor subset with clinico-pathological specific features. In particular, MSI GC is an intestinal gastric cancer, with antral location, low prevalence of vascular invasion or lymph node infiltration and better prognosis.
5. The molecular aspect of gastric cancer as rational for new therapeutic targeted strategies
In recent years it has strengthened the tendency to identify therapeutic strategies different from classical chemotherapeutic approach. The reviewed signaling pathways are relevant contributors for gastric carcinogenesis and encompass a multitude of potential therapeutic targets. In particular, there are growing efforts designed to identify the molecular mechanisms whose inhibition can significantly reduce the clinical aggressiveness of tumor malignacy. This innovative approach has achieved major successes in rare neoplastic diseases such as chronic myeloid leukemia and gastrointestinal stromal tumors (GIST) where it was possible to effectively inhibit constitutionally activated receptor tyrosine kinase such as the KIT gene [66, 67]. Except for the inhibition of HER2 in breast carcinoma, the same success has not yet been achieved in other tumors hence the need to further investigate on the existence of new potential molecular targets. Under this aspect, GC is not an exception indeed it represents a disease for the possible application of targeted therapies.
5.1 HER 2
HER2 (Human Epidermal Growth Factor Receptor 2) also known as Neu, ErbB-2, CD340 (cluster of differentiation 340) or p185 is part of a large family of receptors of tyrosine kinase activities. Along with HER1, also known as EGFR, up-regulation of HER2 is an important event in molecular carcinogenesis of many cancers. Ligand binding to EGFR extracellular domain leads to its activation, with subsequent homodimerization leading to the phosphorylation of its intracellular tyrosine kinase domain. This will initiate a series of intracellular signals, including activation of the central Ras/Raf/mitogen activated protein kinases (MAPK) signaling pathway (Figure 3). Up to now, the best model known is constituted by breast carcinoma in which HER2 is amplified in about 20% of cases . This amplification is correlated to an increase of the expression of the protein and thus in growth advantage . The prognostic negative role played by the amplification of HER2 in breast cancer is balanced by the possibility to interfere with that oncogenetic mechanism through the use of molecular therapies targeted with humanized monoclonal antibodies (i.e. Trastuzumab) . On the basis of the results obtained in the treatment of breast cancer and counting on the fact that gastric carcinomas show amplification of HER2 in approximately 20% of cases, clinical trials have been designed that demonstrated significant improvement in progression-free survival disease for patients with HER2 overexpression, treated with trastuzumab, particularly when associated to conventional chemotherapy [71-73]. It is interesting to note how the aberrations of HER2 are practically exclusive of gastric (and cardial) adenocarcinomas of intestinal type, while they are decidedly not represented in the forms of the diffuse type. As in the case of E-cadherin alterations, there is a correlation between morphological and molecular mechanism in the diffuse forms. The EGFR/MAPK pathway has also shown to be activated in gastric carcinomas with microsatellite instability .
The mechanisms of angiogenesis have recently received a lot of interest in oncology and the inhibition of tumor angiogenesis has become a therapeutic option even feasible in gastric cancer . The vascular endothelial growth factor (VEGF) is a dimeric heparin-binding glycoprotein and it is characterized by the ability to exert a powerful mitogenic action on endothelial cells, promoting their growth both in the primary tumor and in metastases, in the latter case after having stimulated the migration from home neoplastic primitive and secondary levels. A very recent study has shown a direct relationship between VEGF concentrations and new blood vessel development; gastric mucosal neovascularization was also reported to be significantly higher in the antrum of patients with H.
The mammalian target of rapamycin (mTOR) pathway has become a major focus of preclinical and clinical cancer research . mTOR is a central regulatory kinase that increases the production of proteins involved in key cellular processes such as cell growth and proliferation, cell metabolism, and angiogenesis [81-83]. mTOR increases translation of proteins that drive cell growth and cell division, such as cyclin D1, and decreases translation of negative regulators of cell cycle progression . It plays a role in cellular metabolism too by stimulating the surface expression of nutrient transporters . mTOR consists in a double molecular complex (mTORC1 and mTORC2). mTORC1 is regulated by two components of the complex tuberous sclerosis (TSC1 and TSC2), which are controlled by the PI3K/AKT. mTORC2 instead regulates AKT cascade. Recently, because of their function in cell proliferation, these molecular complexes were considered ideal target for the design of drugs in oncology .
The efficiency of this approach has obtained a first success in a group of rare tumors known as PEComi . However, significant results seem to be possible in the context of solid tumors. In fact, recent data show that the activation of mTOR in gastric cancer represents a key event observed in approximately 50% of cases . Hence, the first attempts to use mTOR inhibitors to improve the efficiency of systemic therapies [89, 90].
In this review, we have summarized reports on genes, proteins and factors involved in gastric carcinogenesis based on currently available literature. Gastric carcinoma results from a complex interaction between bacterial, environmental, host-genetic and molecular mechanisms. It is evident that gastric cancer is the consequence of a multistep process involving different genetic and epigenetic changes in numerous genes. Host genetic background and environmental factors also play an important role in the pathogenesis of the disease. The majority of genetic alterations contributing to the malignant transformation were observed in growth regulatory genes, and in genes involved in cell cycle progression and arrest. In recent years, the analysis of molecular carcinogenesis gastric epithelial neoplasm has certainly provided information of great importance. It is understood that the molecular mechanisms involved in carcinogenesis of intestinal type are different from those prevailing in the development of diffuse one. The element of greater importance from a clinical point lies in the fact that the elucidation of these mechanisms is the prerequisite for exploring innovative therapeutic approaches. While the conventional forms of treatment seem to have reached the limit of effectiveness, it is possible that use of targeted therapies based on solid preclinical rational can translate into tangible clinical benefit. The reviewed signaling pathways are relevant contributors for gastric carcinogenesis and encompass a multitude of potential therapeutic targets. In addition to these signaling-related targets we included new data on
GKN1 as being involved in gastric cancer susceptibility phenotype.
This work was supported by funds from PON Ricerca e Competitivita` 2007–2013 (PON01_02782).