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標題: | 纖維母細胞生長因子受器第二型-第三型異型二聚體在頭頸癌上的重要性 FGFR2-FGFR3 heterodimer:functional significance in head and neck cancer cells |
作者: | Jian-Wei Wu 吳建緯 |
指導教授: | 蔡丰喬(Feng-Chiao Tsai) |
共同指導教授: | 涂熊林(Hsiung-Lin Tu) |
關鍵字: | 頭頸癌,FGFR2,FGFR3,細胞遷移,異型二聚體, Head and neck cancer,FGFR2,FGFR3,Cell migration,Heterodimer, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 纖維母細胞生長因子受器(Fibroblast growth factor receptor, FGFR)是種在細胞遷移上扮演著重要角色的膜蛋白,FGFR家族有著不同的成員(FGFR1-4)與亞型(Isoform IIIb IIIc)。其活化方式是同成員的FGFR結合形成FGFR homodimer,並促使下游訊息傳遞,而也有文獻指出不同成員的FGFR能結合形成FGFR heterodimer,但對於FGFR heterodimer的功能目前仍是未知。 頭頸部鱗狀上皮細胞癌是種高致死率且容易發生轉移的癌症,許多研究指出FGFR的功能異常與頭頸癌之間有高度相關的關係,然而目前卻沒有以FGFR作為標靶的治療藥物,為了探索FGFR如何調控頭頸癌細胞的遷移,我們進行傷口癒合實驗(Wound healing assay),發現過度表達FGFR3IIIb會降低細胞遷移的速度,此外頭頸癌細胞是種表現FGFR2為主的細胞株,基於以上的結果,我們推測過度表達的FGFR3或許能與FGFR2結合形成FGFR2-3 heterodimer,並抑制FGFR2活化所產生的訊息傳遞。 為了驗證假說,我們以生物物理光學技術(Fluorescence cross-correlation spectroscopy, FCCS)以及生物化學技術(Co-immunoprecipitation, Co-IP)證實FGFR2-3 heterodimer的存在。為了進一步了解FGFR2-3 heterodimer的功能,讓表現FGFR2為主的頭頸癌細胞過度表達FGFR3來形成FGFR2-3 heterodimer,再藉由FGF7的刺激來觀察FGFR2-3 heterodimer在MAPK、AKT、STAT pathway上的影響。免疫螢光染色(Immunofluorescence, IF)結果顯示FGFR2-3 heterodimer不會啟動MAPK pathway,而西方墨點法(Western blot, WB)顯示FGFR2-3 heterodimer不會啟動AKT pathway,因此初步結果指出FGFR2-3 heterodimer會干擾MAPK與AKT pathway的訊息傳遞。 為了更進一步探討FGFR2-3 heterodimer對下游訊息傳遞的影響,我造了點突變(K508R)、失去膜內區域(∆Intra)與失去穿膜區域(∆Trans)的FGFR3質體(FGFR3 mutants),文獻指出K508R點突變會使FGFR3失去活性,缺少膜內區域的FGFR3-∆Intra也沒有活性,而FGFR3-K508R與FGFR3-∆Intra保留穿膜區域,預期會留在膜上與FGFR2形成Heterodimer來干擾訊息傳遞,因此我們將FGFR3-K508R與FGFR3-∆Intra作為負向對照組;而文獻指出失去穿膜區域會使FGFR3被分泌到細胞外,但細胞外的FGFR3-∆Trans是否能與膜上的FGFR2形成Heterodimer來抑制FGFR2的訊息傳遞仍是未知,因此想探索FGFR3-∆Trans作為調控FGFR2活性蛋白藥物的可能性,而膜質分離的結果顯示所有FGFR3 mutants都位於膜上,推測FGFR3-∆Trans能與其他FGFR形成Heterodimer而留在膜上,期望能透過FGFR3-K508R與FGFR3-∆Intra來解開FGFR2-3 heterodimer的功能以及透過FGFR3-∆Trans找出對付頭頸癌或FGFR2相關疾病的對策。 Fibroblast growth factor receptor (FGFR), a kind of receptor tyrosine kinase (RTK) with 4 members (FGFR1-4) and two isoforms (IIIb IIIc), plays an important role in cellular migration. It is well-known that FGFRs of same member will form homodimer to trigger downstream signal transduction; however, some reports also indicated that different FGFRs would form heterodimer. So far, the function of FGFR2-3 heterodimer still remains unclear. Head and neck squamous cell carcinoma (HNSCC) is a kind of cancer with high mortality and high metastasis. Many reports pointed out that aberrant FGFR signaling is highly correlated with HNSCC. However, there is still no effective treatment targeting FGFR. To explore how FGFR regulates HNSCC migration, we conducted wound healing assay and found that overexpression of FGFR3IIIb would decrease migration speed of SAS cells, one of HNSCC cell lines. Besides, we confirmed that SAS is a FGFR2-dominant cell line with lower expression of FGFR3. Based on these evidence, we inferred that FGFR3 may bind with FGFR2 to form FGFR2-3 heterodimer and the function of FGFR2-3 heterodimer is to block signal transduction of FGFR2. To verify the hypothesis, we utilized Fluorescence cross-correlation spectroscopy (FCCS) and Co-immunoprecipitation (Co-IP) to prove the existence of FGFR2-3 heterodimer. To explore the function of FGFR2-3 heterodimer, we made FGFR2-dominant SAS overexpressing FGFR3 to form FGFR2-3 heterodimer. With stimulation of FGF7, FGFR2-specific ligand, the effect of FGFR2-3 heterodimer on MAPK, AKT, and STAT pathway was observed by Immunofluorescence (IF) and Western blot (WB). IF showed that FGFR2-3 heterodimer will not activate MAPK pathway. WB indicated that FGFR2-3 heterodimer will not activate AKT pathway. Therefore, preliminary result demonstrated that FGFR2-3 heterodimer would interfere with signal transduction. To further figure out whether FGFR2-3 heterodimer blocks signal transduction, I made 3 different truncated-FGFR3 plasmids (FGFR3 mutants), point mutation(K508R) FGFR3, intracellular-domain-truncated FGFR3(FGFR3-∆Intra), and transmembrane-domain-truncated FGFR3(FGFR3-∆Trans). It is reported that K508R point mutation would lead to abrogation of kinase activity of FGFR3. Deletion of intracellular domain would also abolish kinase activity of FGFR3. Because of reservation of transmembrane domain on FGFR3-K508R and FGFR3-∆Intra, we speculate they would be located on membrane and form heterodimer with FGFR2 to block signal transduction. As a result, FGFR3-K508R and FGFR3-∆Intra are regarded as negative control. Some reports pointed out that FGFR3 lacking transmembrane domain would be secreted from cell, but whether FGFR3-∆Trans form heterodimer with FGFR2 to regulate signal transduction remains elusive. To investigate whether FGFR3-∆Trans acts as drug to regulate signal transduction of FGFR2, membrane/cytosol fractionation was conducted. The result showed that all FGFR3 mutants are located on membrane. We inferred that FGFR3-∆Trans could bind with other FGFRs to remain on membrane. We expected that some FGFR3 mutants may bind with FGFR2 to interfere with signal transduction. Membrane/cytosol fractionation demonstrated that all FGFR mutants are located on membrane. With the help of these FGFR3 mutants, we hope to unravel the mystery of FGFR2-3 heterodimer. With the help of these FGFR3 mutants, we hope to unravel the effect of FGFR2-3 heterodimer on different pathways and find out the therapeutic strategies for HNSCC or FGFR2-associated diseases. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68028 |
DOI: | 10.6342/NTU202003818 |
全文授權: | 有償授權 |
顯示於系所單位: | 藥理學科所 |
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