李明亭臺灣大學：生化科學研究所陳世勳Chen, Shih-HsunShih-HsunChen2007-11-262018-07-062007-11-262018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/52755癌細胞的入侵與轉移，始終是治癒癌症最大的難題。之前文獻顯示，腫瘤的Fibronectin (FN) 表現量與癌細胞入侵能力及Matrix Mmetalloproteinases (MMPs) 的釋放量有關。為了癌症轉移的研究，實驗室已建立一套完整系統: 利用Boyden chamber assay從母代A431癌細胞 (A431P) 經三次循環篩選出更具入侵能力的細胞株 (A431III)。同時A431III也被發現會分泌更多的MMP-9以及具有較強的移動、貼附與延展能力。然而，這些特性增強的原因仍處於未知的階段。 此篇研究，我們觀察到A431III內生性FN的表現量與細胞外分泌量有大量提高的趨勢。利用siRNA技術來抑制FN表現量的情況下，顯示A431III的高入侵能力有完全被抑制的現象，其傷口癒合能力也有明顯減弱的趨勢。 Tissue transglutaminase (TG2) 傳統被認為是催化蛋白與蛋白間連接的酵素，最近被發現與腫瘤生成有相關性。2000年一篇報告顯示，TG2被發現具有另一項新的功能: 在細胞外扮演FN與Integrin相互連結的一個重要酵素。因此我們想進一步去研究A431P與A431III中TG2的表現量以及TG2與FN、Integrin的作用能力是否有所差異。實驗顯示，與大部分報告指出TG2隨著癌症轉移惡化而表現量減少的結果相反，TG2在較強入侵力A431III的表現量約是A431P的四倍。此外，TG2、FN及Integrin三者間的相互作用程度，在A431III中都比A431P要高出許多。利用siRNA來抑制TG2表現量的情況下，我們發現A431III原本較高的MMP-9分泌量、FN-Integrin作用力、FAK phosphorylation以及入侵、貼附、延展、移動能力都有降低的趨勢，甚至降到A431P的程度。 綜合以上結果，我們推測: A431III中TG2上升所增強的FN-Integrin交互作用，會活化FAK等相關的訊息傳遞，促使細胞入侵能力的上升。此外也顯示，本實驗室利用Boyden chamber assay所篩選出更具入侵力的癌細胞株，將會是一個研究癌症轉移相關機制的有利工具與系統。The development of invasion and metastasis is a major impediment to the successful treatment of cancer. An increase in fibronectin (FN) expression in human tumor has been shown to be correlated with a high potential of tumor cell invasion and elevated matrix metalloproteinases (MMPs) secretion. We have previously shown that human A431 III sub-line isolated from the parental A431 cell (A431P) using Boyden chamber secreted higher levels of MMP-9 than A431P. The A431III cells exhibit elevated migratory, adherent, and spreading characteristics. However, the molecular mechanisms of highly metastatic features in A431III cells have not been fully understood. In this study, we observed that the amounts of endogenous FN in A431III sub-line were significantly higher than those in A431P cells. Moreover, we found that knock-down of endogenous FN by siRNA resulted in totally inhibition of the invasive potential and decreased the ability of wound healing in A431III cells. Since tissue transglutaminase (TG2) has been found to play an important role in tumor progression and could also act as a coreceptor for integrin-mediated binding of cells to FN, we further planed to investigate the roles of TG2 and its relationship with integrins in the potentials of cancer cell invasion and metastasis in highly invasive A431 cancer cell models. On contrary to other previous studies that showed that a decrease of TG2 expression and activity was accompanied by the increasing metastatic potential. Interestingly, our study showed that TG2 levels were significantly increased in A431III cells compared to A431P cells. Furthermore, the interactions of FN, TG2, and integrin β1/β3 were markedly higher in A431III than in A431P cells. After the knockdown of TG2, the invasive ability, MMP-9 secretion, the interaction between FN and integrin β1, FAK phosphorylation, and highly metastasis-related appearances of A431III were decreased to the levels as that of A431P cells. Thus, our results indicate that increased expression of TG2 could enhance association of FN with integrins and trigger integrin-mediated outside-in signaling, at least in part leading to enhancement of the metastatic potentials in A431III cells. Our data suggest that the highly invasive A431III sub-line would be an excellent model for investigating the mechanism of tumor metastasis through FN and TG2-mediated signaling.Contents I Figure contents IV --------------------------------------------------------------------------------------- 中文摘要 1 Abstract 2 --------------------------------------------------------------------------------------- Introduction 4 Invasion and metastasis of cancer cells 4 The role of Matrix Metalloproteinases in invasion and metastasis 5 Integrins signaling in cancer invasion and metastasis 6 The role of Focal adhesion kinase (FAK) in integrin-mediated signaling 8 The role of Fibronectin in cancer 9 The role of Tissue Transglutaminase in cancer biology 12 The model of metastatic variants selected from primary tumor 15 --------------------------------------------------------------------------------------- Materials and methods 26 Materials 26 Determination of protein concentration of cell lysates, membrane fraction, and conditioned media 26 Western blot 27 Detection of extracellular transglutaminase activity 27 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) 28 Transfection of siRNAs 29 Cell growth experiments 29 In vitro invasion assay 30 Migration assay 30 Cell attachment and spreading assay 31 Wound-healing assay 31 Gelatin zymography 31 Co-immunoprecipitation 32 Immunofluorescence and confocal microscopy 33 Statistical analysis 33 --------------------------------------------------------------------------------------- Results 34 Up-regulated FN expression was found in highly invasive A431 sub-line 34 Effect of FN on the invasiveness of A431P and A431III cells 34 Induction of MMP-9 by endogenous FN may lead to highly invasiveness of A431III 35 Increased mobility of more invasive A431 was highly correlated with up-regulated endogenous FN expression 36 The increased interaction of integrin, FN, and TG2 was found in more invasive A431 cells 37 Colocalization of TG2 and FN in A431P and A431III cells 38 TG2 expression increased in highly invasive A431III sub-line 38 Increased TG2 expression in A431III enhanced cell migratory ability 39 Increased TG2 expression in A431III modulated cell adhesion and spreading 40 TG2 promoted tumor cell invasive potential 40 Increased MMP-9 secretion of A431III was correlated with an increased TG2 expression in A431III cells 41 An increase of surface TG2 enhanced integrin-FN interaction, FAK phosphorylation, and a metastatic potential in A431 sub-line 41 Effect of TG2 and Fn siRNAs on the growth of A431P and A431III 42 --------------------------------------------------------------------------------------- Discussion 55 --------------------------------------------------------------------------------------- Reference 62 Figure contents Fig. I A scheme of metastatic cascade 17 Fig. II A scheme of tumor progression 17 Fig. III The protein structure of MMPs 19 Fig. IV Invasion and migration regulated by integrin-mediated signals 21 Fig. V Three types of modules in FN structure 21 Fig. VI A scheme of FN domain 22 Fig. VII The model of FN-integrin interaction 22 Fig. VIII A scheme of TG2 diverse functions of TG2 23 Fig. IX The model of TG2 association with integrin and FN 23 Fig. X Four distinct domains of TG2 24 Fig. XI TG2 involvement in FN-integrin mediated signaling 25 Fig. XII The development of metastatic potential within primary tumors 25 Table I The Matrix Metalloproteinase family 18 Table II The Family of MMPs 20 Table III Various integrin heterocomplexes in human tumor cells 20 Table IV The family of TGs 24 Fig. 1 Expression and secretion of FN in A431P and A431III cells 43 Fig. 2 The amounts of endogenous FN might be responsible for the invasiveness of A431 cells through MMPs activation. 44 Fig. 3 Increase of FN in highly invasive A431 cells may enhance their migratory ability 45 Fig. 4 The increased interaction of integrin, FN, and TG2 was found in A431III cells. 46 Fig. 5 Localization of TG2 with FN on the surface of A431P and A431III cells 47 Fig. 6 Expression and activity of TG2 in A431P and A431III cells 48 Fig. 7 Elevated mobility in A431III was correlated with increased TG2 expression 49 Fig. 8 Higher attachment and spreading ability in more invasive A431 was mediated by TG2 50 Fig. 9 Highly invasive ability of A431III may be attributed to increased TG2 expression 51 Fig. 10 Higher MMP-9 secretion of A431III was correlated with increased TG2 expression 52 Fig. 11 The higher interaction between FN and integrin and elevated FAK phosphorylation might be mediated by TG2 53 Fig. 12 The proposed model for functional roles of TG2 and Fn in the promotion of A431 cancer cell invasion and migration. 542434895 bytesapplication/pdfen-US癌症轉移纖維連接蛋白素轉麩胺酵素2A431FibronectinTissue Transglutaminase[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52755/1/ntu-95-R93b46019-1.pdf