黏液蛋白第20型在人類子宮內膜癌中的表現與功能

Abstract

研究背景：在台灣，子宮內膜癌是第二常見的婦科癌症，其發生率還有逐年增加的趨勢。雖然近年醫學對於癌症治療有長足的進步，但是目前對於晚期或是復發性子宮內膜癌的治療效果仍是非常差。為了進一步改善患者的預後，醫師需要闡明影響病人存活的危險因子以及開發新的治療策略。 近年的研究發現黏液蛋白（mucin）在腫瘤癌化及侵襲過程中都占有重要角色。因此，許多腫瘤都可以見到黏液蛋白的過度表現與病人較差的存活率有相關性，包括乳癌、卵巢癌、前列腺癌、大腸癌和肺癌都是如此。黏液蛋白第20型（MUC20）是一個近期發現的細胞膜結合型黏液蛋白，其大量表現於腎臟、中量表現於胎盤、大腸、肺、前列腺和肝等器官中。黏液蛋白第20型在腎細胞中的作用已被證明是肝細胞生長因子（HGF）所誘導之Grb2‒Ras訊息傳導路徑的負向調節器。 研究目的：雖然黏液蛋白在許多腫瘤的惡性過程中都扮演著關鍵的作用，並且已被作為診斷標誌和可行的治療標的，然而，黏液蛋白第20型在子宮內膜癌中的所扮演的角色仍是未知。 研究方法：本研究首先利用免疫組織化學方法來分析97個子宮內膜癌組織和16個正常內膜組織檢體中的黏液蛋白第20型表現量和臨床表徵的相關性。其後，利用兩種子宮內膜癌細胞株HEC-1A和RL95-2來分析黏液蛋白第20型表現量改變對癌細胞惡性型態的影響，包括進行體外環境中癌細胞的生長、移動、和侵襲能力測試，以及進行重度聯合免疫缺陷（SCID）小鼠體內的腫瘤生長能力試驗。西方墨點法則用來分析黏液蛋白第20型所調控的訊息傳導路徑。 研究結果：本研究顯示在癌組織檢體中具有黏液蛋白第20型過量表現的比例明顯高於正常組織。再以癌組織做分析，黏液蛋白第20型過量表現又與較差預後的組織學型別（第2型子宮內膜癌）明顯相關。利用多變數分析方法更確定了黏液蛋白第20型的過量表現為影響病人存活的獨立預後因子。在癌細胞株中過度表現黏液蛋白第20型顯著的促進癌細胞的生長、移動、和侵襲能力，以及增加腫瘤在小鼠體內的生長速度。再者，黏液蛋白第20型所促進的侵襲能力可以被表皮生長因子接受器（EGFR）的抑制劑erlotinib所明顯抑制，以及過度表現黏液蛋白第20型會顯著增強表皮生長因子（EGF）所媒介之移動和侵襲能力，這代表了表皮生長因子接受器在黏液蛋白第20型所影響的惡性型態中扮演了關鍵的角色。此外，過度表現黏液蛋白第20型會明顯加強表皮生長因子所誘導的表皮生長因子接受器和其下游訊號傳導與轉錄活化因子3（STAT3）的磷酸化，而利用STAT3 的抑制劑Stattic同樣可以顯著抑制黏液蛋白第20型所調控的侵襲行為。 研究結論：本研究發現黏液蛋白第20型是子宮內膜癌的新型預後因子，而黏液蛋白第20型過量表現會藉由活化EGFR‒STAT3 訊息傳導路徑來增強表皮生長因子所媒介的侵襲行為。此研究結果將助於未來研究人類子宮內膜癌的臨床診斷、發病機制、與治療模式。

Background: Endometrial cancer (EC) is the second most common gynecologic cancer in Taiwan, and its incidence has increased in recent years. Although there has been considerable progress in the treatment of malignancy over past decade, the survival rate of advanced and recurrent EC remains poor. To further improve on outcome for patients with this disease, physicians need to identify risk factors for poor survival and develop new treatment strategies. Recent studies have shown that mucins play important roles in carcinogenesis and tumor invasion. Thus, a relationship between mucins overexpression and poor survival was found in many human tumors, including breast, ovarian, prostatic, colonic, and lung carcinomas. MUC20, a recently identified membrane-bound mucin, is highly expressed in kidney and moderately in placenta, colon, lung, prostate, and liver. MUC20 in kidney cells has been demonstrated to be a negative regulator in the hepatocyte growth factor (HGF)-induced Grb2‒Ras pathway. Objective: Although mucins play a critical role in the malignancy of various tumors and have been identified as diagnostic markers and as attractive therapeutic targets, however, the role of MUC20 in EC is still unknown. Methods: The relationship between MUC20 expression and clinical characteristics of EC was analyzed in 97 EC tumors and 16 normal tissues by immunohistochemistry. Effects of MUC20 on EC cells, HEC-1A and RL95-2, were examined by in vitro cell growth, migration, and invasion assays, as well as in vivo tumor growth in severe combined immunodeficiency (SCID) mouse model. Western blotting was performed to analyze signaling pathways modulated by MUC20. Results: MUC20 expression was significantly higher in EC tumors compared with the normal tissue. High levels of MUC20 expression in EC tumors were correlated with an unfavorable histologic subtype (type II EC). Furthermore, MUC20 was an independent prognostic factor for poor survival as evaluated by multivariate analyses. Overexpression of MUC20 in EC cells significantly enhanced cell growth, migration, and invasion, as well as tumor growth in vivo. The MUC20-enhanced invasive behavior was significantly blocked by erlotinib, an epidermal growth factor receptor (EGFR) inhibitor. Moreover, MUC20 overexpression enhanced EGF-mediated migration and invasion, suggesting a critical role of EGFR in MUC20-mediated effects. We found that MUC20 overexpression could enhance EGF-induced phosphorylation of EGFR and signal transducer and activator of transcription 3 (STAT3). Inhibition of the STAT3 activity by its inhibitor Stattic significantly suppressed the MUC20-enhanced invasive behavior. Conclusions: MUC20 is a novel prognostic factor for EC and its overexpression enhances EGF-triggered invasive behavior through activation of EGFR‒STAT3 pathway. These findings may assist to the diagnosis, pathogenesis, and therapy of human EC.