- 學位論文
CYR61 在胃癌細胞之腹膜黏著轉移作用扮演之角色
The Role of CYR61 in Peritoneum Dissemination of Gastric Cancer Cells Adhesion
摘要
胃癌是全球發生率與死亡率都極高的癌症，超過85%的人會出現淋巴轉移的現象。而在胃癌的末期，胃癌細胞會透過各種擴散途徑進行轉移，進而影響病患的預後。尤其胃部具有豐富的淋巴系統，造就淋巴系統成為主要的轉移途徑，它最常轉移至腹腔內及鎖骨上方的淋巴系統。胃癌末期通常會出現嚴重的腹膜轉移。這引起我們想要探討胃癌之腹膜轉移的興趣。CCN family 為一群分泌型蛋白，包括六個已經確定的成員：CYR61 (cysteine-rich protein/cef-10)、CTGF (connective tissue growth factor/Fisp12)、Nov (nephroblastoma overexpressed gene)、WISP-1（Ｗnt-1 inducible secreted protein-1/elm1）、WISP-2 (亦稱rCop-1) 及WISP-3。從較早之前的實驗室研究中指出，CYR61的表現和腫瘤細胞的惡性度有相關性，胃癌病人癌化組織中CYR61 蛋白表現明顯較正常組織高，而且具淋巴轉移的病人CYR61會高度表現。而in vitro 實驗已指出，CYR61的確可以促進癌細胞的轉移。但目前我們仍不清楚究竟CYR61造成的轉移，是否會導致腹膜轉移。若是，它是藉著何種機制是值得我們進一步探討的。 從臨床報告中，我們得知在胃癌的末期，CYR61表現相對較高的病患會有較多腹膜轉移的現象。而轉移的其中一個重要步驟~癌細胞的黏附，一直在探討腹膜轉移時佔很重要的一角。而從我們的實驗中發現CYR61高度表現的腫瘤細胞會造成較多的癌細胞黏附在腹膜之上。所以我們以反轉錄聚合脢鏈鎖反應先篩選穩定高表現CYR61的胃癌細胞株中黏附因子(integrin)的表現，發現integrin alpha2/beta1會受到CYR61的調控而大量表現。以短暫轉殖 antisense-CYR61質體進入高表現CYR61的MKN45細胞株，則抑制了 integrin alpha2/beta1的蛋白表現。而且透過黏附實驗，亦證實了CYR61調控integrin alpha2/beta1 的現象也會影響到癌細胞的黏附情況。我們發現過度表現CYR61會促進癌細胞黏附在腹膜之上，反之抑制CYR61則會減少癌細胞的黏附。透過抑制型抗體抑制掉integrin alpha2/beta1的作用，亦會減少癌細胞黏附在腹膜上。最後我們在CYR61調控integrin alpha2/beta1的分子機制探討中，發現 CYR61會透過ERK激脢的活化，經由轉錄因子AP1的居中調控，調控integrin alpha2/beta1的蛋白表現量。 綜合以上的實驗結果可知，在人類胃癌細胞中，CYR61對於細胞黏附作用與侵襲能力的調控扮演著重要的角色，而且integrin alph2/beta1為其下游重要的effector。我們的結果首次證實了，在人類胃癌細胞中CYR61的正向調控integrin alpha2/beta1是經由MAPK/ERK dependent的訊息路徑。因此藉由阻斷癌細胞的黏附力，可在未來提供一個新的治療策略，造福腹膜轉移的胃癌病人。
並列摘要
Gastric cancer is the second most common cause of cancer mortality worldwide, and development of metastasis to peritoneum, lymph node, and liver is responsible for the majority of cancer related death. Metastasizing cells must traverse the endothelial lining of the lymphatic or vascular systems in order to metastasize to distant sites. When cancer cells reach the distant site, they have to adhere to the local cells, then can they process the metastasizing steps. The previous study demonstrates that expression of CYR61 was correlated with tumor malignancy and lymph mode metastasis in gastric cancer patients. We first further investigated whether CYR 61 was involved in gastric cancer-mediated peritoneum dissemination. Our clinical observation revealed that increased CYR61 protein expression was associated with peritoneum dissemination in advanced gastric cancer patients, but its functions in peritoneum metastasis are still unclear. We constructed a CYR61 expression vector and introduced into a low-invasive AGS cells and showed enhanced cell adhesion activity. Notably, integrin alpha2 was significantly elevated in CYR61-overexpressing AGS cells. A Transient transfection of antisens-CYR61 into MKN45 cells reduced CYR61-mediated cell adhesion ability. Collectively, our clinical findings have shown that there is a positive correlation between the levels of CYR61 and peritoneal dissemination in gastric cancer tissues in tumor status 3 and 4. AP-1 is a critical transcription factor involved in integrin alpha2 gene regulation in many cell types. In this study, we found that CYR61-induced the MAPK/ERK activation cascade, including AP-1 accumulation. The AP-1 activation is also blocked by antisense-AP1. Further, blockage of AP-1 activation significantly caused inhibition of cell adhesion ability. Taken together, these data suggest that CYR61 is critical for adhesion in gastric carcinoma cells, and integrin alpha2 is the major downstream effector gene in mediating the effects of CYR61. For the first time we demonstrate that CYR61 regulates integrin alpha2 gene via a MAPK/ERK pathway. Blockage of the functions of CYR61 by integrin alpha2 inhibitors may provide a new target for treating the gastric cancer patients with peritoneum dissemination.