Molecular Biomarkers of Gastric Cancer Invasion: Expression and Variation of the OPN-uPA-MMPs Pathway Molecules

INTRODUCTION
Gastric cancer remains a leading cause of cancer mortality, despite a worldwide decline in incidence. In Asian countries, gastric cancer is one of the most prevalent tumor and the leading cause of cancer death (Gordon D.Luk, 2005). In the Western world, more than 80% of gastric cancer patients have advanced cancer on diagnosis with poor prognosis (Roukos, 2000). Complete resection of the tumor and adjacent lymph nodes is the only proven, effective curative treatment (Kim, 1999). Unfortunately, the accuracy of current preoperative staging is limited, particularly with regard to depth of invasion, lymph node involvement and distant metastasis4. Developing new biomarkers to identify the subgroup of gastric cancer patients with invasive phenotypes will be helpful for avoiding inappropriate attempts at curative surgery.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that breakdown the extracellular matrix (ECM) (Nagase and Woessner, Jr., 1999b). MMPs not only play important roles in physiologic ECM remodeling, but are also involved in pathological conditions, including tumor progression, invasion and metastasis (la-aho and Kahari, 2005;Egeblad and Werb, 2002b;Stamenkovic, 2003). Since tissue remodeling is often reflected in body fluids, measurements of MMPs in blood or urine have been suggested as useful tools for characterizing processes that occur in tissue (Zucker, et al, 1999). Among the MMPs family, MMP-9 (also known as 92-kDa gelatinase) is a promising new non-invasive marker (Zucker, et al, 1999).
Elevated levels of serum or plasma MMP-9 have been found in a variety of malignant tumors, such as breast cancer, colon cancer, lung cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma and gastric cancer (Hayasaka, et al, 1996; Hoikkala, et al, 2005; Ruokalainen, et al, 2005; Torii, et al, 1997; Endo et al, 1997; Shen et al, 2000; (Zucker et al., 1993). Although the usefulness of MMP-9 as a tumor marker has been established, several studies measuring MMP-9 in the peripheral blood of cancer patients, using serum or plasma samples, have produced controversial results (Torii et al, 1997; Kirman et al, 2006; Decock et al, 2005). Differences in enrolled populations and study designs most likely contribute to the discrepancies. However, blood sampling and processing may also influence the concentration of MMP-9. Several reports have highlighted the influence of blood specimen collection methods on MMP-9 concentrations (Jung et al, 205; Mannello et al, 2003). MMP-9 concentration has been found to be three-fold higher in serum than in heparin plasma (Jung et al, 1998). Platelet activation or neutrophil mobilization during clotting could produce such results (Makowski et al, 2001). Although measurements of MMP-9 in blood have been suggested to be performed in heparin plasma, rather than in serum (Jung et al, 2001), recent studies have used serum MMP-9 to investigate the correlation between MMP-9 and tumor progression. It is important to investigate how the differences between plasma and serum samples influence the diagnostic and prognostic performances of MMP-9. In the present study, we first compared the effectiveness of plasma and serum MMP-9 levels as tumor markers in gastric cancer. Then we examined whether plasma and serum MMP-9 levels correlate well with gastric cancer invasive phenotypes and survival.
MMPs can be regulated by osteopontin (OPN) through OPN-uPA-MMPs pathway (Rangaswami, et al, 2006). The role of OPN in tumorigenesis can be explained by the multiple functions of OPN in cells (Rittling and Chambers, 2004). Several mechanisms have been proposed through studies using cultured cells. First, it is recognized that OPN has adhesive activity because its receptors all mediate cell adhesion. Second, the ability of cells to migrate may be directly tied to their tumorigenicity and OPN promotes the migration of diverse cells, including monocytes, macrophages and tumor cells, along OPN gradients (Denhardt et al, 2001). In addition, OPN-deficient cells are reported to be hypomotile (Zhu et al., 2004). Third, some experiments suggest that OPN inhibits apoptosis and stimulates survival and growth of cells with inducible OPN (Wu et al., 2000), or with the addition of OPN to cell culture medium (Chang et al., 2003), via an interaction with its receptor CD44 (Lin et al., 2000). Fourth, several studies have suggested that OPN increases tumor invasiveness by inducing proteinase, particularly uPA and MMPs, via complex signaling pathways, such as AP-1 activation, PI3-kiase/Akt-dependent or NIK-dependent NF-kB activation27-31. In the present study, we used real-time RT-PCR to demonstrate that OPN mRNA expression is significantly higher in gastric cancer tissues when compared with surrounding non-tumor tissues. This observation is compatible with a previous report using cDNA microarray method in which OPN is over-expressed in gastric cancer tissues5.Recent studies have consistently reported that OPN mRNA and protein expression in cancer tissues are closely related to invasion and metastasis of gastric cancer (Sun et al., 2005). However, the application of plasma OPN level as a biomarker for gastric cancer has not been investigated.
MMP-2, also know as gelatinase A or 72 kDa collagenase IV, is a member of the MMP family which degrades gelatine and type IV collagen (Yu, et al. 2002). In contrast to other MMPs, MMP-2 is broadly, often constitutively, expressed by a large number of cell types and overexpressed in a wide variety of human cancers, including gastric, lung, prostate, ovarian and bladder cancers (Murray, et al., 1998; Miao, et al., 2003; Zhang, et al., 2005b; Zhou, et al., 2004; Upadhyay, et al, 1999; Vasala, et al., 2003; Davidson, et al., 1999b; Brown, et al, 1993). Human MMP-2 promoter has been shown to contain several cis-acting regulatory elements. Among them, a -1306 C→T transition interrupts Sp1 binding site and consequently diminishes promoter activity (Price, et al, 2001). Transient transfection experiments have shown that MMP-2 expression is ~1.4-2 fold higher with the C allele than with the T allele (Price, et al, 2001). The importance of Sp-1 binding activity in MMP-2 expression has also been reported in other MMP-2 promoter deletion or site-directed mutagenesis studies(Qin et al., 1999;Pan and Hung, 2002). These results suggest that patients with MMP-2 -1306 C/C genotype have higher MMP-2 expression than patients with C/T or T/T
genotype. Recently, Miao and colleagues reported that -1306C/T is associated with gastric cardia adenocarcinoma risk (Miao, et al, 2003). Subjects with the C/C genotype had greater than three-fold risk for developing gastric cardia adenocarcinoma when compared with those with the variant C/T or T/T genotype (Miao, et al, 2003).
The activity of MMP-2 is not only regulated by transcriptional regulation, but also by tissue inhibitors of metalloproteinases (TIMPs), which can form complexes
either with latent or activated MMPs (Gomez et al., 1997a;Kahari and Saarialho-Kere, 1999a). Among the TIMP family, TIMP-2 is particularly interesting due to its dual
functions of regulating MMP-2 activity (Howard et al., 1991b;Wang et al., 2000b) and its controversial effects on tumour progression(Egeblad andWerb, 2002a). TIMP-2 has been reported to be greater than 10 fold more effective than TIMP-1 in the inhibition of MMP-2 activation(Howard et al., 1991c). On the other hand, TIMP-2 has been found to be required for efficient activation of pro-MMP-2 in vivo (Wang et al., 2000a). ATIMP-2 promoter polymorphism (-418 G →C) has been identified, which is also located in the consensus sequence for the Sp-1 binding site (De Clerck et al., 1994a). Although the functional significance of this polymorphism is still unknown, it is reasonable to postulate that it interrupts the Sp-1 binding site and decreases TIMP-2 gene transcription, leading to MMP-2 and TIMP-2 imbalance (Hirano et al., 2001a).
MMP-2 promoter polymorphism has been found to be associated with susceptibility to gastric cancer (Miao et al.,2003). However, there have been no studies conducted to elucidate the associations between MMP-2 polymorphism and gastric cancer invasive phenotype and survival. If MMP-2 polymorphism influences susceptibility to gastric cancer, it may also affect tumour progression and patient survival. As for TIMP-2 polymorphism, no studies have been conducted on gastric cancer patients. In the present study, we hypothesized that in gastric cancer, MMP-2 and TIMP-2 polymorphisms not only correlate well with susceptibility, but also with invasive phenotype and survival. A hospital-based case-control study was conducted to access this hypothesis.
Urokinase type plasminogen activator (uPA), a member of the plasminogen activator (PA) family, converts plasminogen into plasmin, which can activate some prometalloproteinases and degrade the extracellular matrix (ECM) (Saksela and
Rifkin, 1988). uPA is produced in both normal and malignant cells and plays important roles not only in tissue remodeling of normal cells, but also in degradation of ECM and destruction of the basement membrane of malignant cells (Dano et al., 1985). Involvement of uPA in diverse physiologic and pathologic processes, including inflammation (Gyetko et al., 1996), fibrinolysis (Myohanen and Vaheri, 2004), tumor growth stimulation (Blasi, 1993), invasion (Nekarda et al., 1994b), angiogenesis (Bacharach et al., 1992) and metastasis (Crowley et al., 1993), has been reported in recent years. In vitro studies have demonstrated that uPA activity inhibition results in the suppression of tumor progression and reduction of metastasis (Holst-Hansen et al., 1996). Clinically, poorer outcomes have been correlated with higher uPA expression
in many types of tumors, including gastric cancer (Nekarda et al., 1994a;Yonemura et al., 1995;Heiss et al., 1995). Elevated uPA levels in gastric cancer tissue have been
found to be associated with lymph node metastasis, venous invasion, serosal involvement and poor prognosis (Nekarda et al., 1994a;Heiss et al., 1995;Yonemura et al., 1995). Plasma uPA levels tend to be significantly increased in gastric cancer patients (Herszenyi et al., 2000).
The roles of uPA expression in tumor occurrence, invasion and prognosis have been well established. However, how uPA genetic polymorphisms influence the occurrence and outcomes of tumors has not been widely investigated (Przybylowska
et al., 2002). Two polymorphisms of the uPA gene have been described. Yoshimoto et al. reported a C→T transition in the nucleotide sequence of exon 6 encoding the kringle domain. The C →T transition results in Pro (CCG) to Leu (CTG) replacement at amino-acid position 121, which may alter the whole tertiary structure of uPA and be directly or indirectly involved in the activity of uPA (Yoshimoto et al., 1996). Conne et al. reported a T→C substitution in intron 7, located 7 bp upstream of the splicing acceptor site, which may be involved in the nuclear mRNA splicing process (Conne et al., 1997). In the present study, we investigated whether uPA exon 6 C/T and intron 7 T/C polymorphisms correlate well with gastric cancer susceptibility, invasion and survival.