N-deacetylase/N-sulfotransferase 4調控大腸直腸癌之血管新生因子表現的角色

The role of N-deacetylase/N-sulfotransferase 4 in regulation of angiogenic factor expression in colorectal cancer

蕭羽芩(Yu-Chin Hsiao)

指導教授 ： 楊雅倩

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803035

摘要

大腸直腸癌為我國發生率第一名之癌症，主要是由於腸道上皮細胞不正常增生，並累積基因變異，包含抑癌基因失去功能和致癌基因過度活化所致。本實驗室先前利用失異合性 (loss of heterozygosity) 研究，於人類第四號染色體4q26區域篩選出N-deacetylase/N-sulfotransferase (NDST4) 為大腸直腸癌相關之抑癌基因。目前已知NDST4參與Heparan sulfate proteoglycan (HSPG) 生合成之過程，而HSPG於人體之發育、生長、發炎反應、抵抗微生物侵襲、癌症發生等不同生理及病理機制上皆扮演重要角色。實驗室先前建立以Tet-on inducible system表現NDST4之大腸直腸癌細胞株，研究結果顯示NDST4表現可以抑制異種移植腫瘤 (xenograft tumor) 之血管新生情形，接著利用human angiogenesis array篩選NDST4表現所調控之血管新生因子，結果發現urokinase-type plasminogen activator (uPA) 表現量在NDST4表現後顯著下降，因此本論文將探討NDST4對於大腸直腸癌血管新生情形之調控機制。以不同濃度doxycycline誘導不同NDST4表現量之大腸直腸癌細胞研究顯示：NDST4表現會藉由抑制NF-κB路徑之p65蛋白磷酸化而降低促血管新生因子uPA之基因及蛋白表現；且NDST4表現亦對於uPA參與之plasminogen activator (PA) 系統內其他成員造成相同趨勢之影響。另外，檢測其他血管新生相關因子之基因表現量，發現NDST4表現可以降低促血管新生因子angiogenin、amphiregulin、VEGFA及TIMP-1基因表現。以Phorbol myristate acetate 處理細胞增加NF-κB路徑活化以提高PA系統和促血管新生相關因子之表現後，發現NDST4表現對於uPA及其他因子有更明顯之抑制程度。

關鍵字

大腸直腸癌 ； 血管新生

並列摘要

Colorectal cancer (CRC) is the top of cancer incidence in Taiwan, caused by abnormal proliferation of intestinal epithelial cells with accumulation of genetic alterations which include inactivation of tumor suppressor genes (TSGs) and activation of oncogenes. In previous loss of heterozygosity study, we identified N-deacetylase/N-sulfotransferase 4 (NDST4) gene as a novel CRC-associated TSG at 4q26. NDST4 plays a role in heparan sulfate (HS) biosynthesis, which modulates heparan sulfate proteoglycans (HSPGs) to regulate growth factors, cytokines and chemokines. Previously our lab has established Tet-On inducible NDST4-expressing human CRC HCT116 cells, and showed NDST4 expression exhibited significant inhibition of tumorigenesis with lower microvessel density in mouse xenograft tumor models. Accordingly, a Human Angiogenesis Array was used to identify the angiogenic factors regulated by NDST4 expression. The results revealed that urokinase-type plasminogen activator (uPA) was obviously decreased in the conditioned medium harvested from NDST4-expressing HCT116 cells. In the study, we further aimed to investigate the mechanism of NDST4 expression on regulating angiogenesis of CRC. Using different concentration of doxycycline to induce various levels of NDST4 expression in CRC cells showed that NDST4 could suppress gene and protein expression of pro-angiogenesis factor uPA via decrease of NF-κB p65 phosphorylation. Furthermore, NDST4 expression could show similar effect on plasminogen activator (PA) system, in which uPA is involved. On the other hand, NDST4 could also inhibit the gene expression of other angiogenesis-related factors, including angiogenein, amphiregulin, vascular endothelial growth factor A (VEGFA) and tissue inhibitor of metalloproteinase-1 (TIMP-1). After treating cells with phorbol myristate acetate, NF-κB pathway was activated, resulting in enhancement of the expression of PA system and angiogenesis-related factors in CRC cells. Then the inhibition effects of NDST4 expression on these genes were more obviously observed.

並列關鍵字

CRC ； NDST4 ； angiogenesis ； uPA ； NF-κB

參考文獻

1. Lasry, A., A. Zinger, and Y. Ben-Neriah, Inflammatory networks underlying colorectal cancer. Nat Immunol, 2016. 17(3): p. 230-40.

2. Jasperson, K. and R.W. Burt, The Genetics of Colorectal Cancer. Surg Oncol Clin N Am, 2015. 24(4): p. 683-703.

3. Half, E., D. Bercovich, and P. Rozen, Familial adenomatous polyposis. Orphanet J Rare Dis, 2009. 4: p. 22.

4. Vasen, H.F., et al., New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology, 1999. 116(6): p. 1453-6.

5. Haggar, F.A. and R.P. Boushey, Colorectal Cancer Epidemiology: Incidence, Mortality, Survival, and Risk Factors. Clin Colon Rectal Surg, 2009. 22(4): p. 191-7.