FGFR1基因在鱗狀上皮性鼻咽癌之功能分析

Abstract

鼻咽癌(Nasopharyngeal Cancer)是發生於鼻咽腔黏膜上皮細胞的癌症，好發的地區及人種主要是在中國南方的華人，而在歐美卻少見，根據2012年衛生署統計資料，鼻咽癌是臺灣地區第十常見的癌症。本論文針對造成鼻咽癌發生的基因進行研究，在先前的研究中，我們使用cDNA微陣列分析（cDNA microarray analysis），去比較各個鼻咽癌(NPC)細胞株與正常鼻咽黏膜上皮(NNM)細胞株之間的各個基因表現量，發現在鼻咽癌細胞株中的纖維母細胞生長因子受體(FGFR1)有顯著的低表現，相較之下，在各種正常鼻黏膜上皮細胞中之FGFR1則可見高度表現。本篇研究首先以87個NPC和10個NNM檢體進行IHC染色，我們發現在98%的NPC檢體中，可以觀察到癌細胞皆低表現FGFR1，而在85%的NNM檢體中則可以觀察到FGFR1的高表現。為了進一步探討FGFR1基因在鼻咽癌中所扮演的調控角色，本篇利用實驗室建立的TW01N1人類鼻咽癌細胞株，以qPCR和WB確定FGFR1基因在鼻咽癌細胞株和正常鼻黏膜細胞株之間的表現量差異，再進一步建構pcDNA6.2-FGFR1質體，並轉染到TW01N1-NPC細胞株，使其高表現FGFR1，藉此觀察基因在鼻咽癌病理機制上所扮演的角色及其功能。實驗結果發現，轉染FGFR1的TW01N1鼻咽癌細胞株，的確增加FGFR1的mRNA和蛋白質表現，接著在體外培養的細胞實驗中，發現到FGFR1基因表現的增加，不但會影響細胞型態的表現，並且會降低NPC-TW01N1細胞株的生長、移動、侵犯的功能。透過FGF配體活化FGFR1，使磷酸化FGFR1活化下游訊號路徑，例如FAK、MEK/ERK-MAPK、PI3K/Akt訊號路徑，實驗結果發現在磷酸化FGFR1高表現下，這些促使腫瘤生長的訊號傳遞因子有減少的現象，而進一步影響細胞生長、分化、移動，甚至是影響細胞型態，可以推測FGFR1高表現與抑制鼻咽癌細胞生長具相關性。另外在FGF配體活化的情況下，合併以EMT相關的抗體N-cadherin、E-cadherin、Snail和vimentin觀察各個蛋白表現，我們發現這些EMT相關蛋白在FGFR1低表現的NPC-TW01N1細胞株中會有vimentin 表現高的情況，代表細胞偏向間質細胞的特性，有增加細胞移動、侵犯的能力，而高表現FGFR1的TW01N1轉染細胞株則有傾向於E-cadherin高表現，且vimentin表現明顯降低，表示轉化為上皮性細胞，細胞生長減緩。動物實驗的部分，我們也同樣發現，將NOD/SCID小鼠打入FGFR1高表現的NPC-TW01N1細胞，腫瘤生長的速度會明顯減緩，之後在進行WB及IHC的實驗中我們也看到與細胞實驗中一致的結果。由體外實驗以及動物實驗的結果推論，鼻咽癌細胞中FGFR1基因的表現，在鼻咽癌腫瘤的生成過程中扮演一種抑制癌細胞生長的角色。

Nasopharyngeal carcinoma (NPC) is a tumor arising from the epithelial cells that cover the surface and lining the nasopharynx, the incidence is much higher in the Southern China population than in the Western world. According to the statistical data from the Department of Health at 2012, NPC is the 10th commonly seen cancer in Taiwan. The purpose of this research was to find out the genes associated with NPC pathogenesis. In the previous study, we used different NPC cell lines and normal nasal mucosal epithelial (NNM) cell lines to compared the mRNA expression levels by using cDNA microarray. We found that fibroblast growth factor receptor 1 (FGFR1) expression was significantly lower in NPC cell lines and higher in NNM cell lines. In this study, first we used immunohistochemical staining of 87 NPC biopsy specimens and 10 hNNM biopsy specimens. We found that most of the cases revealed low expression of FGFR1 protein in 98% of NPC biopsy specimens, and high expression FGFR1 protein in 85% of normal nasal polyps cases. To further identify the role of FGFR1 in NPC cell line, we used NPC-TW01N1 cell lines to compare their mRNA and protein level of FGFR1 gene between NPC and NNM cell lines. To study the role of FGFR1 in the molecular pathogenesis of NPC and its functions, we constructed a stable pcDNA6.2-FGFR1 transfected NPC-TW01N1 cell line, which could be induced to overexpression FGFR1 protein. From this cell line we found that tumor cells could be up-regulated to express FGFR1 mRNA and protein remarkably. FGFR1 gene could regulate not only the morphological change but also tumor cell migration, proliferation and invasion. On the other hand, when we used FGF ligand to active FGFR1, a variety of signaling proteins are phosphorylated in response to FGF stimulation, including FAK、MEK/ERK-MAPK、PI3K/Akt, leading to stimulation of intracellular signaling pathways that control cell proliferation, differentiation, migration, survival and cell shape. The results show that phosphorylated FGFR1 expression can decrease the expression of those signaling factors. Due to the morphological change in transfectants, we expect that the EMT may happen in the overexpression FGFR1 transfectant. Among EMT specific markers, we found the upregulation of epithelium marker E-cadherin and downregulation of mesenchymal marker vimentin. However, in NOD/SCID mice bearing FGFR1 transfected NPC xenograft, the tumor growth was significantly decreased; when FGFR1 expression levels and localization in tumor masses were verified by using Western blotting and IHC, it conformed to the result of in vitro assay. Taken together, our findings strongly suggest that fibroblast growth factor receptor 1 (FGFR1) may play as a suppressor role in the process of NPC development.