探討內皮細胞特異分子ESM1在食道腺癌中調控細胞增生與血管新生的角色

Abstract

世界十大癌症死因之一的食道癌(Esophageal cancer)在發生初期通常沒有明顯的症狀，以至於患者吞嚥困難、嘔吐及胸痛等症狀明顯的晚期才被診斷；此外目前的治療方式多以手術、化療與放療，但往往患者預後不佳且復發率極高，使得食道癌患者的五年存活率不到20%。其中，食道腺癌(Esophageal Adenocarcinoma)，發生在靠近胃部的食道下段上皮組織的腺體細胞癌化，在過去三十年間，食道腺癌的發生率逐漸攀升為歐美國家最為常見的食道癌類型。內皮細胞特異性分子(Endothelial cell specific molecule-1, ESM1)是許多組織及器官內皮細胞分泌的蛋白聚醣(Proteoglycan)，過去研究指出，許多癌症腫瘤形成時，ESM1會大量的表現，並對於癌細胞的惡性、腫瘤生長以及轉移有著高度的關聯性。我們從臨床統計資料庫The Cancer Genome Atlas (TCGA)中發現，食道腺癌的進展與ESM1的表現量有著顯著的關係。因此我們利用食道腺癌細胞株FLO-1、OE-33以及OE-19，探討ESM1對於食道腺癌特性的改變。FLO-1在ESM1大量表現的情況下，細胞的增生加快；而在OE-19中抑制ESM1表現時，可顯著降低細胞的生長。進一步使用Signal peptide缺失以及Glycosylation site (S137) 變異的ESM1確認，發現到ESM1分泌及Glycosylation的缺失，會導致細胞增生的能力下降。此外以NSG小鼠動物模式中觀察，確認ESM1能夠促進腫瘤的生長。將腫瘤進行免疫組織化學染色以及資料庫GSEA的結果判斷，食道腺癌腫瘤的血管生成能力可能受到ESM1的調控，因此我們進一步利用小動脈細胞株HMEC-1以及動物模式，進行血管新生的相關實驗。細胞模式與動物模式的實驗結果都顯示ESM1的大量表現的確能夠增加血管生成的能力，造成腫瘤的惡化與發展加劇，點出ESM1作為食道腺癌的生物標記可能性。

Esophageal cancer is one of the top ten causes of cancer death worldwide. Due to it has no obvious symptoms during the early stage of the disease, patients had usually been diagnosed lately while swallow difficulty, vomit, and chest pain happened. The main treatment strategies including tumor resection, radiation therapy, and chemotherapy. However, esophageal cancer still has a bad prognosis and a high disease recurrence rate. The 5-years survival rate of esophageal cancer was less than 20%. In the past thirty years, the incidence of esophageal adenocarcinoma (EAC), which occurred from gland cells of the gastrointestinal tract, grew rapidly and became the most prevalent type of esophageal cancer in the Western Countries. Endothelial cell-specific molecule-1 (ESM1) is a secreted proteoglycan that is involved in angiogenesis in several tissues and organs. Previous reports indicated that ESM1 was overexpressed in many cancer types and influenced the malignancy of cancer cells, tumor growth, and metastasis. We observed a significant relationship between ESM1 and EAC development by analyzing the TCGA database. According to this information, we used three EAC cell lines, FLO-1, OE-19, and OE-33, as our in vitro models to investigate the role of ESM1 in EAC malignancy. Our study showed that ESM1 overexpression in FLO-1 cells significantly promotes its cell proliferation. In contrast, the knockdown of ESM in OE-19 cells inhibits its cell proliferation. Moreover, we confirmed the importance of secretory and glycosylated ESM1 in the regulation of EAC proliferation by using two ESM1 constructs, ESM1 without signal peptide (ESM1wo) and glycosylation point mutation (ESM1-S137A). In the xenograft model, ESM1 can also promote in vivo tumor growth of EAC cells. Furthermore, based on the GSEA analysis of TCGA EAC dataset and IHC analysis of xenografts in mouse models, we speculated that the ESM1 may also promote EAC tumor development by its angiogenic effects. We further confirmed in vitro and in vivo angiogenic potential of ESM1 in EAC conditioned media with HMEC-1 and plug assays respectively. Collectively, based on our experimental results, ESM1 may promote EAC malignancies through not only provides proliferation benefit to cancer cells but also help to establish tumor vasculature by inducing angiogenesis. These results revealed that ESM1 might be a potential therapeutic target for EAC.