https://scholars.lib.ntu.edu.tw/handle/123456789/145130
標題: | 黏蛋白型O-醣基化在大腸直腸癌進展和轉移中扮演之角色 Roles of mucin-type O-glycosylation in colorectal cancer progression and metastasis |
作者: | 洪基翔 Hung, Ji-Shiang |
關鍵字: | 蛋白質醣基化;醣化基因;Tn抗原;T抗原;大腸直腸癌;癌症轉移;protein glycosylation;glycogene;Tn antigen;T antigen;colorectal cancer;cancer metastasis | 公開日期: | 2016 | 摘要: | 背景及目的 大腸直腸癌是全世界最常見及最致命的惡性疾病之一。雖然目前的治療方式已經顯著地延長第四期大腸直腸癌患者的存活,已經有遠端轉移的大腸直腸癌通常還是無法治癒。這個難題讓我們提出以下的疑問:目前全身性的大腸直腸癌治療方法,是否瞄準真正關鍵的腫瘤生長和轉移的分子機制? 黏蛋白種類O-醣基化是蛋白質最常見的轉譯後修飾之一,並且和許多重要的生物功能有關。細胞表面分子異常的醣基化和細胞的惡性轉變相關。和腫瘤有關的Tn及T抗原屬於腫瘤胚胎醣類抗原,常見於大腸癌、乳癌、前列腺癌及軟巢癌組織中。這些腫瘤胚胎抗原極有潛力成為癌症診斷、影像檢查及治療的標的。然而,目前關於調控Tn及T抗原的醣化基因和其功能還不是瞭解的很清楚。 這個研究重要的地方在於:(1)能幫助瞭解大腸直腸癌中異常的醣基因表現及其和病人預後的關係;(2)能闡明癌細胞的惡性表現是如何受到醣化基因的調控;以及(3)所得到的資訊可以幫助研發診斷及治療癌症的試劑與新藥。 研究對象與方法 1. 為了要發現可以調控大腸癌細胞上T及Tn抗原的醣基因,我們用即時聚合酶鏈鎖反應來分析正常及癌化的大腸直腸組織裡不同的醣基因表現。找到有不同表現的醣基因後,再比較其表現量與病人預後的關聯性。那些和預後有相關的醣基因將會被選殖出來。 2. 為了確定是否這些選殖出來的醣基因和Tn及T抗原的表現有關,這些醣基因將會被轉殖到當腸直腸癌細胞株裡,然後以流式細胞儀分析癌細胞上Tn及T抗原的表現變化。此外,為了後續的實驗,我們選殖了這些基因並製作了其重組蛋白,並製造了這些基因的多株及單株抗體。 3. 為了研究這些醣基因的功能,我們使用了過度表現和siRNA敲除技術操作大腸癌細胞株後,以細胞增生、凋亡、粘附、遷移、侵襲和集落形成測定法來檢驗癌細胞的惡性表現程度。並進行動物實驗來了解這些醣基因對癌細胞在生物體內的轉移能力所造成的影響。 結果 1. 來自台大醫院(NTUH)病人的配對組織以西方點墨法進行分析。與相鄰的非腫瘤組織相比,大腸直腸癌組織的C1GALT1有過度呈現的情況。C1GALT1的免疫組織化學分析結果指出,與正常的相對應組織相比,67.8%(59/87)的大腸直腸腫瘤有較高的C1GALT1呈現。此外,Kaplan-Meier生存分析顯示,與沒有C1GALT1過度呈現(T≤N)的大腸直腸癌病人相比,有C1GALT1過度呈現(T > N)的病人,其累積的存活率數據顯著地較低。另外,我們還觀察到,C1GALT1的呈現與T抗原的呈現有相關性,而後者是由PNA染色所檢測(圖6)。這些發現指出,C1GALT1的呈現在大腸直腸腫瘤中經常是過度呈現的情況,而其過度呈現與不佳的預後有相關性。 2. 在HCT116和SW480細胞中,C1GALT1的過度呈現會些微增加細胞的存活力,而減少C1GALT1則會些微抑制細胞存活力。transwell和matrigel侵襲實驗的結果顯示,在HCT116和SW480細胞,C1GALT1的過度呈現會顯著地增強細胞的遷移和侵襲。相反地,減少C1GALT1會抑制HCT116和SW620細胞的遷移和侵襲。這些發現指出,C1GALT1能調節大腸癌細胞的惡性行為。動物實驗的結果也顯示出,C1GALT1的過度表現會增加SW480轉移到肺部的數量,而抑制C1GALT1則會減少SW620轉移到肺部的數量。這個結果表示C1GALT1的表現能調控腫瘤在生物體內的生長和轉移能力。 3. 我們發現,C1GALT1可以調節bFGF所誘導的惡性表型。因為FGFR2在大腸直腸癌的意義已經明確地證實了,因此,我們研究了C1GALT1是否能調控FGFR2的醣基化和活動力。我們發現,在HCT116、SW480、和SW620細胞中,只有少量的內源性FGFR2會被凝集素PNA所減少,這指出,FGFR2上只有少量的Ť抗原。有趣的是,以神經氨酸酶處理後,PNA很容易就可以減少FGFR2,這指出,FGFR2帶有唾液酸T抗原。此外,我們發現,FGFR2也可以被凝集素VVA所減少,這指出,FGFR2上有Tn的存在。以神經氨酸酶去除唾液酸後,會增加凝集素VVA與FGFR2的結合,這指出,FGFR2上有唾液酸Tn的存在。這些結果強烈指出,在大腸癌細胞上,FGFR2上飾有O-聚醣。 我們接下來研究,C1GALT1是否可以修飾FGFR2上的O-聚醣。在HCT116和SW480中,C1GALT1的過度呈現會降低VVA與FGFR2的結合;而在HCT116和SW620細胞中,減少C1GALT1會增加VVA與FGFR2的結合。這些結果指出,在大腸癌細胞中,C1GALT1能夠調控FGFR2上的O-聚醣結構。此外,在HCT116和SW480細胞中,C1GALT1的過度呈現會增加FGFR2和ERK 1/2的磷酸化,而此磷酸化是由bFGF誘導的。相反地,HCT116和SW620細胞中,在以bFGF處理後,減少C1GALT1會抑制FGFR2和ERK 1/2的磷酸化。這些結果指出,在大腸癌細胞中,C1GALT1能修飾FGFR2上的O-聚醣並調控bFGF所誘導的FGFR2活性。 結論 我們發現C1GALT1在大腸腫瘤中過度表現,且其過度表現與大腸直腸腫瘤病人的較差存活期有關。C1GALT1的過度表現會藉由修改FGFR2的O-醣基化來影響其活性,增強了大腸癌細胞侵襲的潛力和類幹細胞的特性。相反地,在體外和體內試驗中,C1GALT1的降低會抑制這些惡性特質。這些發現,對O-醣基化在大腸直腸癌中的相應角色開啟了全新的視野,並指出C1GALT1有望成為治療大腸直腸癌的治療標靶。 Background and objective Colorectal cancer is one of the most common and deadliest malignant disease worldwide. Although current treatment modalities have dramatically improved the survival of patients with stage IV colorectal cancer, colorectal cancers that spread to distant organs are usually not curable. This problem raises questions as to whether current systemic anti-colorectal cancer treatments are targeting molecular mechanisms that are truly critical to cancer growth and metastasis. Mucin-type O-glycosylation is one of the most common post-translational modifications of proteins and is associated with many important biological functions. Aberrant glycosylation of cell surface molecules is associated with malignant transformation. Tumor-associated Tn and T antigens are oncofetal carbohydrate antigens and present in colon, breast, prostate, and ovarian cancer. They are promising targets for cancer diagnosis, imaging, and therapeutics. However, the glycogenes responsible for regulating Tn and T antigens expression and their biological functions remain largely unknown. This research is significant in that: (1) it contributes to the understanding of altered glycogene expression in colorectal cancer and the association between their expression level and the patient prognosis; (2) it provides the knowledge about what and how the cancer cell behaviors are regulated by glycogenes; and (3) the gained information will allow us to develop diagnostic and therapeutic reagents for human cancers. Materials and methods 1. To identify novel glycogenes which can regulate Tn and T antigen expression in colorectal cancer cells, the differential expression of glycogenes in normal colorectal and cancerous tissues from colorectal cancer patients were analyzed by real-time RT-PCR. After the glycogenes with altered expression in colorectal cancer were found, the relation of expression level and the patient prognosis were examined. The glycogenes which have impact on patient’s survival were cloned. 2. To confirm whether these glycogenes can regulate Tn or T antigen expression, colon cancer cell lines were transfected with these genes. Then Tn and T antigen expression on cell surfaces was analyzed by flow cytometry. In addition, we cloned these genes and made their recombinant proteins as well as generated their polyclonal and monoclonal antibodies for subsequent experiments. 3. To investigate the function of these glycogenes in vitro, overexpression and siRNA knockdown techniques were used. Cell-based assays, including cell proliferation, apoptosis, adhesion, migration, invasion, and colony formation assays were performed. Animal study was also conducted to reveal the effect of the glycogenes on cancer metastasis in vivo. Results 1. Paired tissues from patients of the National Taiwan University Hospital (NTUH) were analyzed by Western blotting. C1GALT1 expression was found to be overexpressed in colorectal cancer tissues compared with adjacent non-tumor tissues. The results from immunohistochemistry of C1GALT1 indicated that 67.8% (59/87) of colorectal tumors showed higher C1GALT1 expression than their normal counterpart tissues. Moreover, a Kaplan-Meier survival analysis showed that the cumulative survival rate of colorectal cancer patients with C1GALT1 overexpression (T > N) was significantly lower than the patients without C1GALT1 overexpression (T ≤ N). In addition, we also observed that C1GALT1 expression is positively associated with T antigen expression as revealed by PNA staining. These findings suggest that C1GALT1 expression is frequently overexpressed in colorectal tumors and its overexpression is associated with poor survival. 2. Overexpression of C1GALT1 slightly increased cell viability in HCT116 and SW480 cells, whereas knockdown of C1GALT1 slightly inhibited cell viability in HCT116 and SW620 cells. We also analyzed migration and invasion by transwell and matrigel invasion assay, respectively. Results showed that overexpression of C1GALT1 significantly enhanced cell migration and invasion in HCT116 and SW480 cells. In contrast, knockdown of C1GALT1 suppressed cell migration and invasion in HCT116 and SW620 cells. These findings suggest that C1GALT1 can regulate malignant behaviors of colon cancer cells in vitro. We then conducted animal experiment and observed that overexpression of C1GALT1 increased lung metastasis of SW480 cells, whereas knockdown of C1GALT1 suppressed lung metastasis of SW620 cells. These results suggest that C1GALT1 expression is able to modulate tumor growth and metastasis in vivo. 3. We found that C1GALT1 can regulate bFGF-induced malignant phenotypes. Since the significance of FGFR2 in colorectal cancer has been clearly demonstrated, we investigated whether C1GALT1 can regulate FGFR2 glycosylation and activity. Only low amounts of endogenous FGFR2 were pulled down by PNA in HCT116, SW480, and SW620 cells, indicating small amounts of T antigens on FGFR2. Interestingly, after neuraminidase treatment, FGFR2 was easily pulled down by PNA, suggesting that FGFR2 carries sialyl T antigens. Moreover, FGFR2 could also be pulled down by VVA, indicating the presence of Tn on FGFR2. Removal of sialic acids with neuraminidase increased VVA binding to FGFR2, indicating the presence of sialyl Tn on FGFR2. These results strongly suggest that FGFR2 is decorated with O-glycans in colon cancer cells. We next investigated whether C1GALT1 can modify O-glycans on FGFR2. Overexpression of C1GALT1 decreased VVA binding to FGFR2 in HCT116 and SW480 cells, whereas knockdown of C1GALT1 increased VVA binding to FGFR2 in HCT116 and SW620 cells. These results suggest that C1GALT1 is able to modulate O-glycan structures on FGFR2 in colon cancer cells. Furthermore, overexpression of C1GALT1 increased bFGF-induced phosphorylation of FGFR2 and ERK1/2 in HCT116 and SW480 cells. Conversely, knockdown of C1GALT1 suppressed the phosphorylation of FGFR2 and ERK1/2 after bFGF treatment in HCT116 and SW620 cells. These findings suggest that C1GALT1 modifies O-glycans on FGFR2 and regulates bFGF-induced activation of FGFR2 in colon cancer cells. Conclusion We found that C1GALT1 is overexpressed in colorectal tumors and its overexpression is associated with poor survival of patients with colorectal tumors. C1GALT1 overexpression enhances the invasive potential and stem-like cell property of colon cancer cells via modifying O-glycosylation and activity of FGFR2. Conversely, C1GALT1 knockdown suppresses these malignant properties in vitro and in vivo. These findings open novel insights into the relevant role of O-glycosylation in colorectal cancer and suggest C1GALT1 as a promising therapeutic target for the treatment of colorectal cancer. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/277266 | DOI: | 10.6342/NTU201600991 | Rights: | 論文使用權限: 不同意授權 |
顯示於: | 臨床醫學研究所 |
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