- 學位論文
β1,4-N-acetylgalactosaminyltransferase III 控制EGFR調控之大腸直腸癌細胞幹性
β1,4-N-acetylgalactosaminyltransferase III modulates cancer stemness through EGFR signaling pathway in colon cancer cells
摘要
β1,4-N-acetylgalactosaminyltransferase III (B4GALNT3) 具有轉移GalNAc到所有N-glycans 和 O-glycans末端GlcNAc-β的功能，是一個合成GalNAcβ1-4GlcNAc (LacdiNAc) 醣結構的酵素。在我們過去的研究發現，過度表現B4GALNT3能增加大腸直腸癌細胞的惡性程度，但其作用機轉並不明確。我們發現與正常的大腸比較，B4GALNT3在大腸直腸癌的表現量是增加，而且與病人較差的存活率有關。在大腸直腸癌細胞株HCT116和SW480中過度表現B4GALNT3後，會增加EGF所誘導的轉移與入侵能力。抑制B4GALNT3在大腸直腸癌細胞株HCT15和HT29的表現後，會減弱EGF所誘導的轉移與入侵能力，B4GALNT3所引起的細胞爬行能力會被EGFR抑制劑erlotinib減弱。而且過度表現B4GALNT3後也會增加EGF所誘導細胞球體形成能力。反之，抑制B4GALNT3表現後，這些惡性的細胞表現型也會減弱。Wisteria floribunda agglutinin (WFA) 沉澱試驗發現，B4GALNT3的表現可以改變EGFR上N-glycans的LacdiNAc表現，增加B4GALNT3在細胞中的表現會增強活化的EGFR表現及其下游的訊息傳遞。然而，增加B4GALNT3表現後，不會對HGF所引導的遷移能力產生影響，也不影響MET的醣類結構，而且B4GALNT3的表現量對bFGF所引導的細胞球體形成能力也不會產生影響。我們的結果說明B4GALNT3能透過修飾EGFR的醣化及控制其訊息傳遞來調控癌細胞的幹性及入侵能力，我們認為抑制B4GALNT3的表現具有治療大腸直腸癌的潛力。
並列摘要
β1,4-N-acetylgalactosaminyltransferase III (B4GALNT3) catalyzes the transfer of GalNAc to any nonreducing terminal GlcNAc-β on N- and O-glycans, resulting in the synthesis of GalNAcβ1-4GlcNAc (LacdiNAc) structure. Although we previously showed that B4GALNT3 overexpression increased colon cancer cell malignant phenotypes in vitro and tumorigenesis in vivo, the underlying mechanism is poorly understood. Immunohistochemistry showed that B4GALNT3 was upregulated in colorectal tumors compared with their surrounding non-tumor colorectal tissues and was associated with poor survival of colorectal cancer patients. B4GALNT3 overexpression enhanced EGF-induced migration and invasion in HCT116 and SW480 cells. Knockdown of B4GALNT3 reduced EGF-induced migration and invasion in HCT115 and HT29 cells. The effects of B4GALNT3 on migration and invasion induced by 10% FBS were blocked with erlotinib, an EGFR inhibitor. Moreover, overexpression of B4GALNT3 increased EGF-induced sphere formation, whereas B4GALNT3 knockdown reduced cell malignant phenotypes. Wisteria floribunda agglutinin (WFA) pull down assays showed that B4GALNT3 primarily decorated the LacdiNAc structure on the N-glycans of EGFR. B4GALNT3 overexpressing cells enhanced EGF-induced phosphorylation of EGFR and its downstream signaling pathway. Overexpression of B4GALNT3 in colon cancer cells altered neither the HGF-induced motility and glycosylation of MET nor the bFGF-induced sphere forming ability. Our results suggest that B4GALNT3 regulates cancer stemness and the invasive properties of colon cancer cells through modifying EGFR glycosylation and signaling. Our findings provide a novel strategy for colorectal cancer therapy by inhibiting B4GALNT3.