唾液酸轉移酶ST3Gal1對腫瘤生長及血管增生之影響

The effects of ST3Gal1 mediated sialylation on tumor progression and angiogenesis

楊惠玲(Hui-Ling Yeo)

指導教授 ： 陳鈴津 ； 張大慈

國立清華大學/生命科學院/分子與細胞生物研究所/博士(2018年)

摘要

唾液酸轉移酶（ST3Gal1）及它的產物 sialyl-3T ， disialyl-T 高度表現在各種腫瘤，且與病人較差的癒後能力息息相關。但唾液酸修飾對其基質蛋白的功能影響仍不是很清楚。本論文利用腫瘤細胞皮下接種至實驗鼠揭示降低 ST3Gal1 表現會抑制腫瘤的生長及腫瘤內血管的形成。另外，我們發現 Vasorin (VASN) 是唾液酸轉移酶的其中一個基質蛋白且與 TGF-β1 誘導腫瘤內新生血管形成相關。所以本論文探討 ST3Gal1 如何調控 VASN 從而影響腫瘤內血管的生成。經由質譜儀分析, VASN 被證實是 ST3Gal1 的基質蛋白，因為在VASN上的O-連接醣基化修飾，百份之八十是 sialyl-3T，disialyl-T。另外，培養液取自抑制 ST3Gal1表現的癌細胞，可以抑制人臍靜脉血管內皮細胞 （HUVEC）形成體外管柱（Tube formation），但加入 VASN 抗體能緩解這個現象。另外，過度表現去唾液酸轉移酶的癌細胞所生產的VASN，可明顯抑制 TGF-β1 誘導 Smad2 和 Smad3 磷酸化。使用神經氨酸酶（Neuraminidase）移除 α2,3-上連接的唾液酸可提高VASN 與 TGF-β1 的結合兩到三倍，因而降低了 TGF-β1 誘導腫瘤內新生血管形成。為了了解ST3Gal1，VASN 和 TGF-β1 在人類乳癌的調控關係，我們利用 114 個乳癌的病人樣本進行分析及結果顯示 ST3GAL1與TGFB1在癌細胞的表現較高並與病人癒後較差有關，尤其乳癌後期的病人愈發明顯，另外，VASN 高表現 的病人有明顯較好的癒後，但只有在TGF-β1低表現時。因為TGF-β1可刺激 ST3GAL1 基因的表現，這些發現揭示一個由TGF-β1調控唾液酸修飾的迴路，借由誘導 ST3Gal1 表現上升修飾 VASN，從而降低VASN 在癌症中的對 TGF-β1 抑制作用 。

關鍵字

唾液酸修飾 ； 唾液酸專移酶 ； 乳癌 ； 腫瘤新生血管 ； 乙型轉化生長因子

並列摘要

ST3Gal1 is key sialyltransferase which adds α2,3-linked sialic acid to substrates and generates core 1 O-glycan structure. Up-regulation of ST3Gal1 has been shown to be tumorigenic and its O-glycan products, sialyl-3T, and disialyl-T were associated with worse prognosis of various of cancer patients. Here, we demonstrated that ST3GAL1-silencing significantly reduced tumor growth along with a notable decrease in vascularity of MCF7 xenograft tumors. However, the protein substrates of ST3Gal1 implicated in tumor progression remain elusive. Here we reported Vasorin (VASN), which was shown to bind TGF-β1, to be one of the substrates of ST3Gal1, and further demonstrated that ST3Gal1-mediated O-linked sialylation of VASN affected TGF-β1 induced-angiogenesis. LC/MS/MS analysis revealed that more than 80% of O-glycans of soluble VASN are sialyl-3T and disialyl-T. Removal of sialylation on VASN by neuraminidase enhanced its binding to TGF-β1 by 2 to 3-fold and thereby dampening TGF-β1 signaling and angiogenesis, as indicated by impaired tube formation of HUVECs, suppressed angiogenesis gene expression and reduced activation of Smad2 and Smad3 in HUVEC cells. Since TGF-β1 is known to transcriptionally activate ST3Gal1, our findings illustrated a feedback regulatory loop in which TGF-β1 up-regulates ST3Gal1 to circumvent the negative impact of VASN. Kaplan Meier survival analysis showed a strong correlation among ST3GAL1, VASN and TGFB1 to relapse-free survive by using 114 fresh primary breast cancer and their adjacent normal tissues. In conclusion, our findings elucidated ST3Gal1-mediated sialylation of VASN promotes tumor progression, and these findings point to the targeting of ST3Gal1 as a new strategy for the treatment of breast cancer.

並列關鍵字

Angiogenesis ； ST3Gal1 ； Vasorin ； TGF-Beta ； Breast cancer ； Sialylation

參考文獻

1 Anscher MS, Peters WP, Reisenbichler H, Petros WP, Jirtle RL. Transforming growth β as a predictor of liver and lung fibrosis after autologous bone marrow transplantation for advanced breast cancer. N Engl J Med 1993; 328: 1592-1598.

2 Arnaoutova I, Kleinman HK. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat Protoc 2010; 5: 628-635.

3 Barcellos-Hoff MH, Akhurst RJ. Transforming growth factor-β in breast cancer: too much, too late. Breast cancer research : BCR 2009; 11: 202.

4 Bierie B, Chung CH, Parker JS, Stover DG, Cheng N, Chytil A et al. Abrogation of TGF-β signaling enhances chemokine production and correlates with prognosis in human breast cancer. J Clin Invest 2009; 119: 1571-1582.

5 Bull C, Stoel MA, den Brok MH, Adema GJ. Sialic acids sweeten a tumor's life. Cancer Res. 2014; 74: 3199-3204.