Identification of metastasis-associated proteins in ovarian cancer cell line by proteomics approach

卵巢癌是全球女性致死率最高之婦科癌症之一。大多數的惡性卵巢腫瘤為上皮細胞型態，並且大部分病患在被診斷出時都已經發生癌症轉移的現象。然而病人在接受手術切除及化療後，仍然具有很高的復發率，晚期病人平均存活期更是只有四年。卵巢上皮細胞癌 (epithelial ovarian cancers)，根據組織型態可以分為四大類：漿液型 (serous)、黏液型 (mucinous)、子宮內膜樣 (endometrioid) 和透明細胞型(clear cell)，其中子宮內膜樣約占其中10 ~ 20 %。實驗室先前從病患腫瘤組織中分離出一細胞株─OVTW59，並且運用Transwell assay將細胞株根據侵襲能力細分，原始分離出可少量穿過Transwell的細胞株命名為P0，能通過四次Transwell篩選的細胞株名命為P4。並且從先前微陣列 (cDNA microarray) 分析，發現抑癌基因insulin-like growth factor binding protein-3 (IGFBP-3) 於P0及P4細胞當中有差異表現，並且IGFBP-3的表現量與病人的預後、存活率呈現負相關。 為了釐清與癌症轉移相關之蛋白參與卵巢癌轉移的機制，於是我們以二維電泳將P0及P4細胞的蛋白體展開，並尋找其中差異表現的蛋白質，並以液相層析─質譜儀鑑定身分。再運用生物資訊軟體─Ingenuity Pathway Analysis (IPA) 建構P0及P4細胞差異表現之蛋白質的網絡，及分析其可能扮演的角色。從IPA的分析結果，我們選定與腫瘤細胞株侵襲性有關的Calreticulin做後續的研究。從西方墨點法(Western blotting) 的實驗結果發現Calreticulin的表現確實與卵巢癌細胞株的侵襲性呈現正相關。此外，我們藉由大量表現及抑制生成Calreticulin，證實Calreticulin的確會影響卵巢癌細胞株細胞遷移 (migration)、侵襲 (invasion) 及分泌基質金屬蛋白酶 (matrix metalloproteinase-2/9, MMP-2/9) 的能力。然而IGFBP-3、Calreticulin和MMP-2/9及與細胞遷移相關蛋白之間的調控機制，仍有待後續研究釐清。Ovarian cancer is the most lethal gynecological cancer among women worldwide. The majority of malignant ovarian tumors are epithelial type and metastasis has occurred in most patients at the time of diagnosis. Subsequent recurrence rate is high after complete surgery and chemotherapy. Survival is at about 4 years for patients with advanced ovarian cancer. Based on the morphological criteria, epithelial ovarian cancers (EOC) are classified into four histological subtypes: serous, mucinous, endometrioid and clear cell. The endometrioid carcinoma (EC) subtype represents about 10 to 20 % of EOC. In our previous study, we have established an ovarian endometrioid carcinoma cell line OVTW59. The original lines were designated as P0, and the highly invasive subline P4 was isolated from 4 rounds of invasion selection. By cDNA microarray analysis, we have demonstrated that insulin-like growth factor binding protein-3 (IGFBP-3) is an invasion suppressor gene. Patients with low serum IGFBP-3 level are correlated with poor survival. In this study, we want to identify metastasis-associated proteins from ovarian endometrioid carcinoma cell line OVTW59. We used 2D-electrophoresis to compare the protein profiles between the original cell line P0 and the highly invasive subline P4, and potential candidates were subsequently identified by LC-MS/MS. Then we built up a potential network involving those differentially expressed proteins by Ingenuity Pathway Analysis. We found a novel metastasis-associated protein, calreticulin. Western blotting demonstrated that calreticulin expression level is highly correlated with cancer invasion. By overexpressing and knocking down Calreticulin, we found that Calreticulin regulates cell migration, invasion, and MMP-2/9 secretion. However, the regulatory mechanism among IGFBP-3, Calreticulin, MMP-2/9, and cell migration-associated proteins remains to be further elucidated