N-deacetylase/N-sulfotransferase 4表現於大腸直腸癌細胞對巨噬細胞分化和血管新生之調控

Abstract

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。已知NDST4參與Heparan sulfate proteoglycans (HSPGs) 之生合成過程，而HSPGs在發育、生長和炎症反應、腫瘤發生等不同生理和病理機制皆扮演重要角色。本論文將探討NDST4對於大腸直腸癌腫瘤微環境之巨噬細胞分化和血管新生的調控。首先利用THP-1及U937細胞建立巨噬細胞分化之細胞模型，藉由M0、M1和M2亞型得的細胞形態、標的基因和細胞表面抗原確認其分化。接續以表現NDST4之大腸直腸癌細胞或其條件培養液與M0細胞共培養，結果顯示：表現NDST4之大腸直腸癌細胞及其條件培養液可促進巨噬細胞分化成M1亞型。此外，利用先前研究之異種移植腫瘤組織切片經CD68染色，結果顯示NDST4表現之大腸直腸癌細胞所形成之腫瘤，其腫瘤周圍巨噬細胞 (tumor-associated macrophage) 的數量較多。另一方面，以不同濃度doxycycline誘導大腸直腸癌細胞表現NDST4，確認NDST4表現會抑制細胞內和分泌至細胞外的促血管新生因子urokinase-type plasminogen activator (uPA) 的mRNA及蛋白表現。檢測我們收集的51對大腸直腸癌檢體，腫瘤的uPA表現量明顯高於其成對的正常黏膜組織 ; 利用不同來源之大腸直腸癌資料庫分析，顯示預後較差的病患其腫瘤uPA表現量明顯較高，NDST4表現量則明顯較低。最後，藉由人類細胞激素晶片分析，發現誘導表現NDST4之大腸直腸癌細胞的條件培養液，其Serpin E1、macrophage migration inhibitory factor (MIF)、IL-8、CXCL12及CXCL1含量下降。綜合以上，推測大腸直腸癌細胞表現NDST4可能藉由減少上述調節因子，進而影響腫瘤微環境之巨噬細胞分化及血管新生。

In previous loss of heterozygosity (LOH) study, we explore N-deacetylase/N-sulfotransferase 4 (NDST4) as a novel tumor suppressor gene (TSG) at chromosome 4q25-q28.2 in colorectal cancer (CRC). NDST4 is one of the pivotal enzymes responsible for heparan sulfate biosynthesis on a core protein to form heparan sulfate proteoglycans (HSPGs), which play important roles in development、 inflammation and tumorigenesis. We have proposed that loss of NDST4 function might impair the biosynthesis of specific HSPGs leading to tumor-promoting inflammation and tumor progression. In the study, we further aimed to investigate NDST4-mediated modulation of macrophage polarization and angiogenesis in CRC. First, macrophage differentiation model were established with THP-1 and U937 cells. Macrophage subtype were determined by morphological change and surface marker expression measured by qRT-PCR and flow cytometry. CRC cells with inducible NDST4 expression, including a single stable clone (HCT116/NDST4-C5) and a mixed line (HCT116/NDST4-Mix), were established in previous study. CRC cells expressing NDST4 and the conditioned media harvested could promte M0 cells differentiation into M1-polarized macrophages. Meanwhile, previously established xenogrft tumor tissue sections were used to perform immunohistochemistry staining of CD68 (M0). The results revealed larger numbers of tumor-associated macrophage in NDST4 expressing xenograft tumor. In the second part, NDST4 expression in CRC cells decrease the gene expression and protein secretion of urokinase-type plasminogen activator (uPA). In addition, uPA upregulation was observed in 51 primary tumor collected when compared to their matched normal mucosa. By using 4 public CRC datasets, the uPA expression is signigicantly higher in the tumors of high risk group than that in the tumors of low risk group. In contrast, the NDST4 expression is significantly lower in the tumors of high risk group. The conditioned medium from CRC cells expressing NDST4 exhibited a decreased level of Serpin E1、MIF、IL-8、CXCL12 and CXCL1 by cytokine array analysis. Taken together, NDST4 expression in CRC cells might decrease the synthesis and/or secretion of the mediators, and these modulate macrophage differentiation and tumor angiogenesis in tumor microenvironment.