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標題: | 肝細胞癌過度表現磷脂肌醇聚糖3之致癌性與類胰島素生長激素訊息傳導路徑研究 Hepatocellular carcinoma overexpressed Glypican-3-Mediated Oncogenesis Involves the Insulin-like Growth Factor Signaling Pathway |
作者: | Wei Cheng 鄭威 |
指導教授: | 許輝吉 |
關鍵字: | 磷脂肌醇聚糖3,第二型類胰島素生長激素,類胰島素生長激素一型受器,肝細胞癌, glypican-3 (GPC3),insulin-like growth factor II (IGF2),insulin-like growth factor I receptor (IGF-1R),hepatocellular carcinoma (HCC), |
出版年 : | 2010 |
學位: | 博士 |
摘要: | Glypican-3 (GPC3) 磷脂肌醇聚糖3是一種可經由糖基磷脂酰肌醇glycosyl-phosphatidylinositol (GPI) 鍵連結在細胞膜上的糖蛋白。且是導致Simpson- Golabi - Behmel增生綜合症的突變基因。之前我們經由北方墨點試驗發現在超過70% 肝細胞癌中會大量表現GPC3。Glypican - 3(GPC3)GPC3在肝細胞癌,威姆氏瘤 (Wilms’ tumor),肝母細胞癌,及絨毛膜癌中呈現過度表現,且可促進癌細胞的生長。GPC3曾被報導過與Wnt,Hedgehog,BMPs及類胰島素生長激素(IGF)的路徑有關,但真正的機轉仍不清楚。在本研究中,我們進一步闡明了GPC3引發腫瘤的可能機轉。
我們發現GPC3的表現可促成NIH3T3細胞株細胞的生長快速,細胞的核質比增加,細胞可在洋菜膠中長成細胞團塊,且可在祼鼠體內會長成腫瘤,而無GPC3表現的載體NIH3T3細胞則無此等現象。在表現GPC3之PLC-PRF-5細胞中亦會有細胞生長快速的現象。反之,在GPC3 調降(knockdown)的HuH – 7肝細胞癌細胞株會減少其致癌性。 我們發現GPC3可經由IGF2刺激IGF-1R及其下游ERK造成這些分子的磷酸化。在GPC3 調降的肝癌細胞其IGF-1R和ERK會減少磷酸化。因此顯示GPC3是經由IGF2及其受體,以及隨後激活的胰島素樣生長因子信號通路而具有致癌性。我們亦發現GPC3可經由其N端富含脯氨酸的區域(proline-rich domain)與第2型類胰島素生長激素(IGF2)及類胰島素生長激素一型受器(IGF-1 receptor (IGF-1R))接合並共同促進肝癌細胞的生長,而此區域亦會影下游胞外信號調節激酶(ERK)的活化及AP-1的活性。 GPC3是一可分泌的分子,我們發現GPC3轉染的乳癌細胞株MCF7細胞的conditioned media可促進MCF7及HA22T/VGH細胞的細胞團塊生長。這個新的發現對GPC3在未來肝癌的癌症治療發展可能是很重要的。 Glypican-3 (gpc3) belongs to the glycosylphosphatidylinositol (GPI)-anchored glypican family and responsible for Simpson-Golabi-Behmel overgrowth syndrome, an X-linked condition characterized by pre-and postnatal overgrowth with visceral and skeletal anomalies. Previously, we have shown that GPC3 is overexpressed in more than 70% hepatocellular carcinoma (HCC) by Northern Blot analysis. In addition to HCC, GPC3 overexpression was also reported in certain types of human cancers, including Wilms' tumor, hepatoblastoma, melanoma, and choriocarcinoma. GPC3 can promote the growth of cancer cells. GPC3 has been linked to Wnt, Hedgehog, BMPs and insulin-like growth factor (IGF) pathway, but the molecular mechanism remains unclear. In this study, we attempted to elucidate the mechanisms for GPC3-mediated oncogenesis. We demonstrated that ectopic overexpression of GPC3 in NIH3T3 cells led to cancer cell phenotypes including growth in serum-free medium, forming colonies in soft agar, and formation of in vivo malignant tumors in nude mice. GPC3-expressing PLC-PRF-5 cells enhanced cell growth in low serum. On the other hand, GPC3 knockdown decreased the oncogenic capability of HuH-7 cells. GPC3 stimulated the phosphorylation of IGF-1R and the downstream signaling molecule ERK in an IGF2-dependent fashion. Also, GPC3 knockdown in HCC HuH-7 cells decreased the phosphorylation of IGF-1R, IRS-1 and ERK. Therefore, we conclude that GPC3 confers oncogenecity through the interaction between IGF2 and its receptor, and the subsequent activation of the IGF-signaling pathway. We further demonstrated that GPC3 bound specifically through its N-terminal proline-rich region to both insulin-like growth factor (IGF)-II and IGF-1R. This proline-rich region was also important for the downstream ERK activation and AP-1 activity. GPC3 is a secretory protein. We demonstrated that the conditioned medium of MCF7 cells after transient transfection with GPC3 promoted colony growth of MCF7 and HA22T/VGH cells in soft agar. These findings are novel in the current understanding of the role of GPC3 in HCC and can be important in future developments of cancer therapy. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44700 |
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