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In this review, the authors examine the molecular and cellular events that …


Biology Articles » Health and Medicine » Illnesses and Pathobiology » Cellular and molecular aspects of gastric cancer » Molecular mechanisms of gastric carcinogenesis

Molecular mechanisms of gastric carcinogenesis
- Cellular and molecular aspects of gastric cancer



The bacterial, environmental and host genetic factors discussed above influence the development of gastric carcinoma. In the following section, we discuss the molecular mechanisms underlying the disease. These include abnormalities of oncogenes, tumour suppressor genes, cell adhesion molecules and cell cycle regulators. Additionally genetic instability and alterations in growth factors and cytokines contribute to the complex pathways involved in gastric carcinogenesis. Differences exist in the pathways leading to diffuse- and intestinal-type gastric carcinoma, and these are summarised in Figures 3 and 4.


Oncogenes

Many proto-oncogenes are activated in gastric carcinoma, with variations between the differing histological subtypes. The c-met gene, encoding a receptor for hepatocyte growth factor/scatter factor is amplified in 19% of intestinal-type and 39% of diffuse-type gastric cancers[80]. The majority of gastric carcinomas express two different c-met transcripts, of 7.0 kb and 6.0 kb. Expression of the 6.0kb transcript correlates well with prognostic factors such as tumour staging, depth of tumour invasion and lymph node metastasis[81]. The K-sam (KATO-III cell-derived stomach cancer amplified) oncogene is also frequently activated in gastric carcinomas, and has at least four transcriptional variants[82]. One of these, Type II, encodes a receptor for keratinocyte growth factor. K-sam is preferentially amplified in 33% of advanced diffuse or scirrhous -type gastric carcinomas but not in intestinal-type cancers[83]. Over-expression of this gene in gastric carcinoma is associated with a poorer prognosis.

Another proto-oncogene, c-erbB2, is preferentially amplified in 20% of intestinal-type gastric cancers but this is not a feature of the diffuse-type[84]. Over-expression of this gene is also correlated with poorer prognosis and liver metastases[85,86]. Mutations of K-ras are seen in intestinal-type gastric adenocarcinomas and the precursor lesions intestinal metaplasia and adenomas[87-89]. The incidence of this mutation is low and it is not a feature of diffuse-type carcinomas.


Tumour suppressor genes

The tumour suppressor gene p53 is frequently inactivated in gastric carcinoma by loss of heterozygosity (LOH), missense mutations and frame shift deletions. This occurs in over 60% of gastric cancers, regardless of the histological subtype, and is frequently observed in precursor lesions such as intestinal metaplasia, dysplasia and adenomas[88,90-94]. Mutations commonly occur at A:T sites in intestinal-type carcinomas, with GC-AT transitions being common in diffuse-type carcinomas[91]. These GC-AT transitions can be caused by carcinogenic N-nitrosamines that are found in several foodstuffs and can be produced from dietary amines and nitrates in the acidic gastric environment[95,96]. Mutations in the codon 72 of exon 4 of the p53 gene have recently been associated with an increased risk of distal gastric cancer[97]. LOH of p73, a tumour suppressor gene related to p53 is detected in 38% of gastric cancers, and alterations of this gene are predominant features of foveolar-type gastric cancers with pS2 expression[98]. pS2 is a gastric-specific trefoil factor normally expressed in gastric foveolar epithelial cells. Inactivation of the pS2 gene results in dysplasia, adenoma and adenocarcinomas in mice[99,100]. Reduction or loss of the pS2 gene by DNA methylation in the promoter region occurs in intestinal metaplasia and gastric adenomas, suggesting this process may be important at an early stage in intestinal-type gastric carcinoma development[101].

Mutations of the tumour suppressor gene APC, involved in familial polyposis coli, are also observed in intestinal-type gastric carcinoma[102]. Although APC gene missense mutations are common in the intestinal subtype, occurring in over 50% of cases, they are not involved in diffuse-type cancers. Somatic mutations of the APC gene are observed in 20%-40% of gastric adenomas and 6% of intestinal metaplasias[103,104]. The expression of β-catenin, which acts as an oncogene, is enhanced by APC inactivation.

A further tumour suppressor is nuclear retinoic acid receptor β (RARβ). Hypermethylation of this gene with reduced expression is observed in 64% of intestinal gastric cancers but this is not observed in the diffuse subtype[105]. Additional tumour suppressor gene alterations include those affecting distinct chromosomal loci. LOH at 1q and 7q are frequently associated with intestinal-type cancers while 1p is commonly affected in advanced diffuse cancers[88]. LOH of the bcl-2 gene is also frequently observed in intestinal-type cancers[106].

The RUNX gene family is composed of three members, RUNX1/AML1, RUNX2 and RUNX3[107]. It also encodes the DNA-binding α subunits of the Runt domain transcription factor polyomavirus enhancer-binding protein 2 (PEBP2)/core-binding factor (CBF), which is a heterodimeric transcription factor. Of the RUNX family, RUNX3 is involved in gastric carcinogenesis, being necessary for the suppression of cell proliferation in the gastric epithelium. The gastric epithelium of RUNX3 knockout mice exhibits hyperplasia, reduced rate of apoptosis and reduced sensitivity to TGFβ1, suggesting the tumour suppressor activity of RUNX3 operates downstream of the TGFβ signalling pathways. In humans, loss of RUNX3 by hypermethylation of the promoter CpG island is observed in several different cancers, including 64% of gastric carcinomas. RUNX3 methylation is also a feature of 8% of chronic gastritis, 28% of intestinal metaplasia and 27% of gastric adenomas[108]. This suggests RUNX3 is a target for epigenetic gene silencing in gastric carcinogenesis[109,110].

Other genes that appear to be affected in gastric carcinogenesis include the FHIT gene and loss of heterozygosity at the DCC locus, which is a feature of intestinal-type cancers[88,111]. Promoter hypomethylation of a novel cancer/testis antigen gene CAGE has recently been described in 35% of chronic gastritis and 78% of gastric cancer[112]. Histone H4 is progressively deacetylated during the development of gastric cancer, and this is a common event in both intestinal-type and diffuse type cancers[113].


Cell-adhesion molecules and metastasis-related genes

Cell adhesion molecules may act as tumour suppressors, with mutations in the E-cadherin gene occurring preferentially in 50% of diffuse type gastric carcinoma[114]. This homophilic cell adhesion molecule belongs to a family of cell-cell adhesion molecules with an important role in intercellular adhesion by establishing cell polarity, maintaining tissue morphology and cellular differentiation in normal cells[115,116]. E-cadherin binds to the actin cytoskeleton via a series of catenin proteins[117]. Therefore changes in E-cadherin expression have a direct effect on cell adhesion and therefore plays an important step in cancer development. E-cadherin mutations affecting exons 8 or 9 induce the scattered morphology, decreased cellular adhesion and increased cellular motility of diffuse gastric cancers[118]. Mutations in β-catenin and -catenin have also been observed in gastric cancer cell lines, and together with E-cadherin mutations appear to be involved in the development and progression of diffuse and schirrhous-type cancers[119-121].

Abnormal CD44 transcripts are frequently associated with gastric carcinomas and metastatic deposits, with the pattern of these abnormal transcripts varying between the intestinal and diffuse subtypes[122]. All gastric cancer cell lines and tissues exhibit abnormal CD44 transcripts containing the intron 9 sequence[123]. This feature is also observed in 60% of gastric intestinal metaplasias but is absent from normal mucosa[124]. Osteopontin (OPN), a protein ligand of CD44, is overexpressed in 73% of gastric carcinomas and when co-expressed with CD44v9 correlates lymphatic invasion and metastasis[125,126]. Reduced expression of nm23, involved in c-myc transcriptional activation, and galectin-3, a galactoside-binding protein, are also implicated in metastatic gastric carcinoma[127,128].


Cell-cycle regulators

The cell-cycle regulator, cyclin E, is amplified in 15%-20% of gastric carcinomas that are associated with its overexpression. Gene amplification or overexpression of cyclin E are associated with aggressiveness and lymph node metastasis[129]. The expression of the CDK inhibitor p27 that binds to a wide variety of cyclin/CDK complexes and inhibits kinase activity is frequently reduced in advanced gastric carcinoma while being preserved the majority of gastric adenomas and early cancers[130]. Reduced p27 expression correlates with tumour invasion and nodal metastasis. This reduction in p27 occurs at a post-translational level, and results not from genetic abnormalities but rather from ubiquitin-mediated proteosomal degradation[131]. A family of E2F transcription factors is an important target of cyclin/CDKs at the G1/s transition. Overexpression of E2F is observed in 40% of primary gastric cancers, and this tends to be co-expressed with cyclin E[132]. Gene amplification and abnormal expression of the E2F gene may permit the development of gastric cancer.


Micosatellite and chromosomal instability

Microsatellite instability (MSI) is a hallmark of the DNA mismatch repair deficiency that is one of the pathways of gastric carcinogenesis. Microsatellites are short DNA sequence repeats that are scattered throughout the human genome and occur in nearly every case of gastric cancer associated with germline mutations of the mismatch repair (MMR) genes hMSH2, hMLH1, hPMS1, hPMS2, and MSH6/GTBP[133,134]. Errors that occur in DNA mismatch repair mechanisms in tumour cells can cause expansion and contraction of these repeats. MSI due to epigenetic inactivation of hMLH1 is found in 15%-39% of sporadic intestinal-type cancer, 70% of which are associated with loss of hMLH1 by hypermethylation of the promoter[135,136]. Such intestinal type cancers with MSI often occur in older patients and arise in the antrum. They are associated with lymphocyte infiltration, multiple tumours and a potentially favourable prognosis. Meanwhile MSI of the D1S191 locus is found in 26% of intestinal metaplasia and 46% of intestinal type gastric cancer. An identical pattern of this MSI of D1S191 is observed in adjacent intestinal metaplasia and intestinal type cancer that suggests the sequential development from the former to the latter[137]. Diffuse type cancers with MSI are more commonly observed in younger patients and have no germline mutations of hMLH1 and hMSH2, with no alteration in BAT-RII[138]. However, these cancers are frequently associated with LOH on chromosome 17q21 including the BRCA1 gene.

Human telomerase reverse transcriptase (hTERT) is an important determinant of telomerase activity, the enzyme that catalyses the telomere DNA synthesis. The majority of intestinal carcinomas have shortened telomere length, high levels of telomerase activity and a significant expression of hTERT[139]. Over 50% of intestinal metaplasias express low levels of telomerase activity, equivalent to 10% of the activity in gastric carcinoma[140]. hTERT is unregulated at an early stage in gastric carcinogenesis and H pylori may act as a trigger factor for hyperplasia in hTERT positive “stem cells” in intestinal metaplasia[139].


Growth factors and cytokines

Gastric cancer cells express a wide array of growth factors and cytokines that act via autocrine, paracrine and juxtacrine mechanisms. Again the expression of these mediators varies depending on the histological subtype. These interactions are summarised in Figure 5. The EGF family, which includes EGF, TGFα, IGF II and bFGF, are commonly overexpressed in intestinal-type carcinoma. Meanwhile TGFβ, IGF II and bFGF are predominantly overexpressed in the diffuse subtype[101]. Co-expression of EGF/TGFα, EGFR and cripto correlates well with the biological malignancy, as these factors induce metalloproteinases[141,142]. Overexpression of cripto is frequently associated with intestinal metaplasia and gastric adenoma[143]. Gastric cancer cells express neutrophilin-1 (NRP-1), a co-receptor for VEGF receptor 2 endothelial cells[144]. EGF induces both NRP-1 and VEGF expression, suggesting that regulation of NRP-1 expression in gastric cancer is intimately associated with the EGF/EGFR system.

Interleukin-1α is produced by inflammatory cells and also gastric cancer cells. It acts as an autocrine growth factor for gastric carcinoma cells and is important in EGF and EGF receptor expression[145]. The interplay between IL-1α and the EGF/receptor system acts to stimulate gastric cancer growth. IL-6 also acts in an autocrine fashion to stimulate gastric cancer cells. Il-1α and IL-6 both stimulate the expression of each other by tumour cells{167}. IL-8, a member of the CXC family of chemokines has numerous roles in gastric carcinogenesis with over 80% of gastric tumours expressing both this cytokine and its receptor[146,147]. IL-8 enhances expression of EGF receptor, type IV collagenase (metalloproteinase (MMP)-9), VEGF and IL-8 mRNA itself by gastric cancer cells, while reducing E-cadherin mRNA expression.

The negative growth factor TGF is frequently overexpressed in gastric carcinoma, particularly diffuse type carcinomas with diffusely productive fibrosis[148]. Angiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and IL-8 are produced by tumour cells and result in neovascularisation within gastric carcinoma tissue. VEGF promotes angiogenesis and progression of gastric carcinomas, particularly those of the intestinal subtype, while bFGF is has a stronger association for diffuse gastric carcinoma[149,150]. HGF/SF (hepatocyte growth factor/scatter factor) is produced by stimulated stromal cells such as fibroblasts, and functions in a paracrine manner as a morphogen or motogen[101].

It can be seen that several of the molecular mecha-nisms are distinct for intestinal- and diffuse-type gastric carcinoma development, while some are common to both. Regarding intestinal-type carcinogenesis, there are three possible routes leading to carcinoma development. Firstly, progression through the pre-cancerous lesions of intestinal metaplasia to adenoma and finally carcinoma. Secondly, intestinal metaplasia may proceed directly to carcinoma. The third route involves the development of de novo gastric carcinoma with no preceding stage.


Summary of Molecular Mechanisms involved in gastric carcinogenesis

The first two pathways are summarised in Figure 3 and Figure 4. Genetic instability and hyperplasia of hTERT positive stem cells precede replication error at the DS191 locus, DNA hypermethylation at the D17S5 locus, pS2 loss, RARβ loss, RUNX3 loss, CD44 abnormal transcripts and p53 mutation, all of which accumulate in 30% of incomplete intestinal metaplasia. All of these epigenetic and genetic changes are common events in intestinal-type cancers.

An adenoma to carcinoma sequence is observed in around 20% of gastric adenomas with APC mutations. Molecular events associated with this sequence are loss of heterozygosity and mutation of p53, reduced p27 expression, loss of RUNX3, over-expression of cyclin E and abnormal c-met transcription. The resulting advanced intestinal-type gastric carcinomas frequently exhibit DCC loss, APC mutations, 1qLOH, loss of p27, reduced TGFβ receptor expression, reduced nm23 and c-erbB2 gene amplification. The “de novo” pathway of gastric carcinoma development involves LOH and abnormal expression of p73 exhibited in the development of foveolar-type gastric cancers. Meanwhile, diffuse-type gastric carcinogenesis involves LOH at chromosome 17p, MOH or mutation of p53, RUNX3 loss and mutation or loss of E-cadherin. Several of the above molecular events may be present in mixed gastric cancers that have both intestinal and diffuse components.


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