胃癌侵襲性的分子生物標記：OPN-uPA-MMPs Pathway分子的表現與變異

Abstract

研究背景及目的： 胃癌的治療以切除治療為主，對於早期胃癌而言，可以內視鏡黏膜切除術進行切除，非早期胃癌則可用手術切除、內視鏡次黏膜切開術、腹腔鏡切除術加以處理。不論是內視鏡切除、腹腔鏡切除或者手術切除處理，均面臨著同樣的困難，就是很難精確地在術前評估患者的腫瘤侵襲狀況，包括胃癌患者的T staging以及N staging，均很難藉由目前的影像學檢查在術前進行精確的評估。由於術前的胃癌分期評估有其極限，因此有學者主張應該盡其可能地將胃組織及淋巴結清除，然而這種作法的效益仍然沒有定論，甚至有文獻發現這種切除手術會增加患者術後的死亡率。尤其是在面對日益老化的人口時，採取最不具侵襲性的手術已經成為一種趨勢，然而採取最不具侵襲性的手術卻面臨著可能切除不足的危險。因此，如何在術前對於胃癌患者的腫瘤侵襲程度有較為精準的評估，從而避免實施切除過度或者切除不足的手術，已經成為目前胃癌相關研究中最重要的議題之一。 隨著分子生物學的突飛猛進，對於胃癌的發生、侵襲以及轉移，有了許多不一樣的看法。近年來，胞外基質(extracellular matrix)的角色則開始受到許多矚目，腫瘤細胞與其週遭的組織細胞之對話，以及其間的訊息傳遞、對於腫瘤細胞的週期、細胞凋亡、細胞移動性、以及新生血管的形成、等，在腫瘤的形成、侵襲以及轉移過程中，均扮演著重要的角色。對於胞外基質的分子生物學研究結果，讓臨床醫師開始思索這些分子在臨床上做為癌症生物標記的可能性。 Matrix metalloproteinases (MMPs)是一個酵素家族，其主要作用在於分解胞外基質。除了分解胞外基質的作用外，MMPs還參與許多與腫瘤相關的病理生理機轉，包括腫瘤細胞的生長、分化、細胞凋零、細胞移動、侵襲、血管新生、免疫監控以及遠處轉移。而Osteopontin (OPN)是一個胞外基質相關蛋白，對於許多癌症的發生及轉移均扮演著重要的角色，OPN可以經由uPA (urokinase type plasminogen activator)來調控MMPs的表現及活性。本研究以OPN-uPA-MMPs這個訊息傳遞鏈上的相關因子，做為研究的核心，系統性地評估這些因子的表現以及基因的變異，是否可以做為胃癌的發生、侵襲、以及存活的生物指標。 第一部份：MMP-9的血中濃度做為胃癌的生物指標 MMP-9的血中濃度是一個相當有潛力的胃癌生物指標，本研究的主要目的在於比較MMP-9的血漿濃度與血清濃度，做為評估胃癌的發生、侵襲、以及存活的可行性。 我們以114位胃癌患者以及87位健康對照組的血清及血漿檢體，分別以酵素連結免疫吸附法(enzyme-linked immunosorbent assay, ELISA)，以及同功酵素圖譜(zymography)來測定MMP-9的血中濃度以及其活性。測定的結果分別以胃癌的發生、臨床病理特徵、患者的預後進行分析。每位胃癌患者至少追蹤五年以上。 我們發現在相同的胃癌患者，MMP-9的血清濃度顯著高於MMP-9的血漿濃度，在血漿及血清中，MMP-9的濃度均分別與MMP-9活性成顯著相關 (P值分別為0.002，0.048)。胃癌患者的MMP-9血漿濃度顯著高於健康對照組(P<0.001)，而MMP-9血清濃度在胃癌患者與健康對照組之間則沒有差異存在。以Receiver- operator characteristics (ROC)分析，MMP-9血漿濃度≧60ng/mL 的情況下，對於胃癌診斷的敏感度為82.5%，精確度為65.5%。升高的MMP-9 血漿濃度(≧60ng/mL)與胃癌患者的淋巴結轉移(OR=3.43, P=0.019)、淋巴侵襲(OR = 7.58, P=0.009)、以及血管侵襲(OR=4.14, P=0.033)之間，有顯著統計相關存在。MMP-9血漿濃度升高的胃癌患者，存活率較差(P=0.038)。而MMP-9 的血清濃度則與胃癌患者的侵襲性以及存活率之間沒有統計相關存在。 根據我們的研究結果，MMP-9 的血漿濃度、而非血清濃度，有機會成為預測胃癌的發生、侵襲、以及存活的生物指標。 第二部份：OPN 的血漿濃度做為胃癌的生物指標 除了胃癌之外，OPN 在其他癌症的診斷及預後價值已經被肯定，本研究的目的在於評估OPN 的血漿濃度做為評估胃癌的發生、侵襲、以及存活的可行性。 我們以132 位胃癌患者以及93 位健康對照組的血漿檢體進行研究，以酵素連結免疫吸附法來測定OPN 血漿濃度。以定量反轉錄聚合酶鏈式反應(Real-time quantitative RT-PCR)，以及免疫組織染色檢查(immunohistochemical staining，IHS)來測定胃癌組織中的OPN 表現量。測定的結果分別與胃癌的發生、臨床病理特徵、患者的預後進行分析。 我們發現在相同的胃癌患者，胃癌組織的OPN mRNA 表現要遠高於其鄰近的正常胃部組織，免疫組織染色則發現大多數的OPN 蛋白質表現在胃癌細胞上。胃癌患者的OPN 血漿濃度顯著高於健康對照組(P<0.001)，升高的OPN 血漿濃度(≧67.3 ng/mL)與胃癌患者較末期的病程(stage III: OR=3.79, P=0.004; stage IV:OR=5.69, P=0.002)、漿膜侵襲(OR=3.21, P=0.003)、淋巴結轉移(OR=3.88, P=0.001)、淋巴侵襲(OR=3.16, P=0.004)、血管侵襲(OR=4.24, P=0.001)以及肝臟轉移(OR=11.09, P=0.005)之間，有顯著統計相關存在。而且OPN 血漿濃度升高的胃癌患者，存活率較差(P<0.0001)，特別是在具有侵襲性的胃癌族群。在Cox多變數分析中，較高的OPN 血漿濃度是一個獨立的預後因子(P<0.0001)。 根據我們的研究結果，上升的OPN 血漿濃度，與胃癌的發生、侵襲、以及患者的存活率之間，有著顯著統計相關存在。OPN 血漿濃度有機會成為胃癌的診斷以及預後的生物指標。 第三部份：MMP-2 -1306 C/T 及TIMP-2 -418 G/C 做為胃癌的生物指標Matrix metalloproteinase-2 (MMP-2) -1306 C/T 以及tissue inhibitor of metalloproteinase-2 (TIMP-2) -418 G/C 的單一核甘酸多形性(single nucleotide polymorphisms, SNPs)會破壞Sp-1 結合處，而使該基因的表現量下降，本研究的目的在於評估MMP-2 及TIMP-2 單一核甘酸多形性與胃癌的發生、侵襲、以及存活率之間的相關性。 我們以240 位胃癌患者及283 位健康對照組進行研究，抽取周邊血液中白血球的DNA 進行分析，分別以PCR 直接定序(PCR-directed sequencing)以及PCR-RFLP的方法分析MMP-2 及TIMP-2 的基因型。測定的結果分別與胃癌的發生、臨床病理特徵、患者的預後進行分析。 我們發現MMP-2 及TIMP-2 的基因型與胃癌的發生之間沒有統計相關存在。帶有MMP-2 -1306 C/C 基因型的胃癌患者，有較高的機會發生淋巴侵襲(OR=2.77,P=0.01)以及血管侵襲(OR=2.93, P=0.012)。TIMP-2 -418 G/G 基因型則與漿膜侵襲(OR=1.89, P=0.009)、淋巴結轉移(OR=2.19, P=0.021)、淋巴侵襲(OR=2.87, p=0.016)以及血管侵襲(OR=2.65, p=0.033)有關。 根據我們的研究結果，MMP-2 與TIMP-2 基因型在胃癌的侵襲過程中扮演著一定的角色，但與胃癌的發生之間則沒有相關性存在。 第四部份：uPA 表現子6 C/T 及uPA 插入子7 T/C 做為胃癌的生物指標過去的研究顯示uPA 的表現與許多腫瘤的侵襲性有關，然而uPA 的單一核甘酸多形性會如何影響胃癌的發生及侵襲，則尚未被探討過。本研究的目的在於評估uPA 表現子6 C/T 及uPA 插入子7 T/C 單一核甘酸多形性與胃癌的發生、侵襲、以及存活率之間的相關性。 我們以237 位胃癌患者及242 位健康對照組進行研究，抽取周邊血液中白血球的DNA 進行分析，以PCR 直接定序的方法分析基因型。測定的結果分別與胃癌的發生、臨床病理特徵、患者的預後進行分析。 我們發現uPA 表現子6 C/T 及uPA 插入子7 T/C 的基因型與胃癌的發生之間沒有統計相關存在。帶有uPA 表現子6 T allele 的胃癌患者，有較高的機會發生淋巴侵襲(OR=1.94, P=0.047)以及血管侵襲(OR=2.59, P=0.01)。uPA 插入子7 基因型則與胃癌患的侵襲性無關。 根據我們的研究結果，uPA 表現子6 基因型在胃癌的侵襲過程中扮演著一定的角色，但與胃癌的發生之間則沒有相關性存在。 結論 相較於MMP-9 血清濃度，MMP-9 的血漿濃度是一個較為優良的胃癌生物指標，MMP-9 的血漿濃度與胃癌的發生、侵襲性以及患者的存活之間都存在著統計相關，儘管不是獨立的預後因子，MMP-9 血漿濃度仍然有機會可以成為胃癌的診斷以及預後的生物指標。相較於MMP-9 血漿濃度，OPN 的血漿濃度不僅跟胃癌的發生、侵襲以及預後有關，而且OPN 的血漿濃度還是一個獨立的預後因子，因此OPN 做為胃癌的診斷及預後因子的可行性相當高。在單一核甘酸多形性的研究上，我們發現MMP-2 -1306 C/T、TIMP-2 -418 G/C 及uPA 表現子6 C/T 與 胃癌的發生之間沒有關聯性，也不影響患者的存活率，但與胃癌的侵襲性之間存在著統計相關。 本研究的臨床應用性在於：社區族群進行大規模的胃癌篩檢、術前幫助評估胃炎患者的腫瘤侵襲程度、術後幫助評估胃癌患者的預後。對於基礎醫學的可能貢獻在於：可以提供基礎醫學研究者更為明確的研究方向、有助於生化學家與藥理學家找到新藥物的研究方向、提供流行病學者與生物資訊學者珍貴的癌症生物標記資訊。而將來繼續努力研究方向為：檢驗本研究結果的適用範圍、發展生物標記模組(biomarker modules)、發展個人化的生物標記(individualized biomarkers)、干擾因子的尋找及排除、應用於胃癌的癌前病灶等方向。

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.