The Functional Role of VNN3 Gene in Nasopharyngeal Carcinoma Tumorigenesis

鼻咽癌為一種極易導致死亡的癌症，並好發於中國東南方及東南亞。而其詳細發病機制尚待闡明。儘管新療法以及早期診斷的方式不斷進步，往往卻因確診後已是晚期故其預後不良。最近的研究顯示SRY (sex-determining region Y)-box 5 (SOX5)基因在鼻咽癌中扮演一個重要的角色。當SOX5大量表現時會提高鼻咽癌腫瘤的形成且預後差。在本次研究中，我們使用cDNA微陣列分析（cDNA microarray analysis）的方法，比較鼻咽癌細胞和正常鼻咽黏膜上皮細胞之間的表現量，再經由及時定量聚合酶連鎖反應（Quantitative RT-PCR）及西方墨點法（Western Blotting）的分析發現SOX5基因在鼻咽癌細胞中的表現量有顯著增加。進一步研究SOX5基因表現量增加的分子機制時，我們發現VNN3 (vascular non-inflammatory molecular 3)在鼻咽癌細胞中亦有顯著增加的表現量，但對於SOX5所調控之相關基因如VNN3，如何去控制腫瘤的發展卻尚未被研明。為了探討VNN3基因是否會改變鼻咽癌的進程，我們首先使用qRT-PCR來分析不同鼻咽癌細胞株其VNN3基因的表達，而發現特別在NPC-TW01細胞株有高度表達。此外我們透過免疫組織染色及西方墨點法發現VNN3蛋白在多種鼻咽癌細胞株與病人檢體中表現量均增加。在本研究中，我們透過特殊培養基從NPC-TW01細胞株中分離了鼻咽癌幹細胞(NPC Cancer stem cells, CSCs)，並發現其也有較高的VNN3基因表達水平。為了進一步分析VNN3的功能在鼻咽癌症幹細胞中所扮演的角色，利用VNN3基因的干擾質體(shRNA)去轉染鼻咽癌幹細胞而發現VNN3降低了95%的基因表達。進而導致NPC癌細胞的增生、轉移、及侵襲能力等有顯著的的降低。因VNN3基因所表達之蛋白質為一種酰胺水解酶，能將pantetheine 水解為 pantothenic acid (維他命 B5)以及cysteamine，此兩者皆為抗氧化劑。故我們降低VNN3的表達時使細胞內活性氧化物 (ROS)上升，最終使細胞走入凋亡(apoptosis)。在異種移植的鼻咽癌腫瘤 (NPC xenografts)長於嚴重混和性免疫缺陷症 (sever combined immune deficiency，簡稱SCID)之老鼠的實驗中發現以抑制VNN3基因表現的shRNA所轉染的鼻咽癌幹細胞在SCID老鼠體內長到第六週時，腫瘤重量比控制組小了百分之五十一；組織病理分析時發現抑制VNN3基因表達能減緩異種移植的腫瘤細胞生長並伴隨著嚴重的細胞凋亡。總之，VNN3在鼻咽癌所扮演的重要功能是項新穎的發現，VNN3因子有很大的潛力當作鼻咽癌的分子標靶。Nasopharyngeal carcinoma (NPC) is one of the leading cause of cancer-related deaths in southeastern China and South East Asia. However, the detailed pathogenetic mechanisms of NPC remain to be elucidated. Despite novel therapies and advances in early detection, it is often diagnosed at an advanced stage and has a poor prognosis. Recent evidences showed that SRY (sex-determining region Y)-box 5 (SOX5) plays an important role in nasopharyngeal cancer progression, but little is known about the role of its associated protein such as VNN3 gene (vascular non-inflammatory molecular 3) in controlling tumor progression. Previously, our laboratory had found that SOX5 overexpression enhances tumor progression in NPC, and has a poor prognosis. In this study, we compared the differential gene expression in SOX5 over-expressed and regular expressed NPC cell lines by cDNA microarray analysis, and observed that the RNA and protein expression levels of SOX5 were dramatically increased in the SOX5 gene over-expressed NPC cells by qRT-PCR and Western blot analysis. At the same time we also found that VNN3 was significantly increased in NPC cell lines. In order to investigate that whether VNN3 gene could also alter NPC progression, we observed at first the gene expression in different NPC cell lines using qRT-PCR analysis and found that the expression of VNN3 in NPC cell line, especially in NPC-TW01 cell line was highly upregulated. Besides, we also revealed that the expression level of VNN3 protein was increased in multiple NPC cell lines and NPC biopsy specimens by immunohistochemical staining and Western bloting. In this study, we have established the NPC cancer stem cells (CSCs) from NPC-TW01 cell line by conditioned culture medium; highly expression level of VNN3 gene was found in the NPC-TW01 cancer stem cells (NPC-CSCs). To further analysis of the functional role of VNN3 in this NPC-CSCs line, the expression of VNN3 was decreased to about 95% of the control in NPC CSCs after knockdown of VNN3 by VNN3 shRNA-lentiviral infection. This in turn resulted in a significant reduction of cell proliferation, migration and invasion abilities. VNN3 is an amidohydrolase that hydrolyzes pantetheine into pantothenic acid (vitamin B5) as well as cysteamine, both them are free radical scavenger when VNN3 expression was suppressed, it caused an increase of intracellular reactive oxygen species (ROS), and cell death through apoptosis; beside the weight of NPC xenograft tumors from these lentiviral infected NPC CSCs cell lines had diminished about 51% after 6 weeks, compared with shLuc control in SCID mice. Impairment of tumor cell proliferation, accompanied with necrosis and apoptosis was also found in the NPC-CSC xenografts treated with VNN3 shRNA histopathologically. These novel findings of the functional role of VNN3 may have a potential implication in molecular targeted therapy for NPC.口試委員審定書 i 致謝 ii 中文摘要 iii Abstract v 目錄 Table of Contents vii List of Figures ix Introduction 1 1. Nasopharyngeal carcinoma (NPC) 1 2. Etiology of NPC 3 3. Molecular biomarkers and prognostic factors of NPC 6 4. SRY (sex-determining region Y)-box 5 (SOX5) 9 5. Vascular non-inflammatory molecular 3 (VNN3) 11 6. Cancer stem cell 13 Materials and Methods 16 1. Cell lines 16 2. Extraction of RNA and preparation of cDNA 17 3. Quantitative RT-PCR (qRT-PCR) 18 4. Statistical analysis of qRT-PCR results 19 5. Immunohistochemical staining 19 6. Western Blotting 21 7. Mini-plasmid purification 22 8. Amplification and Extraction of plasmids 23 9. Establishment of stable shVNN3 NPC cell lines by lentiviral vector system 23 10. MTT Assay 24 11. Scratch wound healing assay 25 12. Invasion assay 25 13. AnnexinV/DAPI double staining 26 14. Measurement of intracellular ROS levels 27 15. In vivo assay of xenograft growth 27 16. Statistical analysis 28 Results 29 1. Increase of VNN3 gene expression in SOX5 overexpressed cells 29 2. VNN3 gene expression in NNM and NPC tumor cell lines 29 3. VNN3 protein expression in NPC biopsy specimens 30 4. NPC CSCs express pluripotency-associated markers 30 5. Knockdown of VNN3 results in an decrease of SOX5 mRNA level 31 6. Functional analysis of VNN3 gene expression 32 7. Alterations in intracellular ROS production of VNN3-knockdown NPC CSCs 33 8. Functional analysis of VNN3 in SCID mice bearing NPC xenografts 34 Discussion 36 References 41 Figures 50 Tables 6