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研究生:李仁傑
研究生(外文):Jen-Chieh Lee
論文名稱:以次世代定序於高磷尿性間質腫瘤發現FN1-FGFR1融合基因及其可能致瘤機制與轉譯關聯性之探討
論文名稱(外文):Identification of FN1-FGFR1 Fusion Gene in Phosphaturic Mesenchymal Tumor with Next-generation Sequencing and Insights into the Plausible Tumorigenesis Mechanism and Translational Relevance
指導教授:張逸良張逸良引用關係
指導教授(外文):Yih-Leong Chang
口試委員:蔡克嵩楊榮森吳振都黃政文
口試委員(外文):Keh-Sung TsaiRong-Sen YangChen-Tu WuJenq-Wen Huang
口試日期:2015-05-08
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:病理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:55
中文關鍵詞:高磷尿性間質腫瘤腫瘤誘導骨軟化症基因轉位次世代定序
外文關鍵詞:phosphaturic mesenchymal tumortranslocationFN1FGFR1RNA sequencing
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高磷尿性間質腫瘤(phosphaturic mesenchymal tumor, PMT)是一種特殊的軟組織及骨骼腫瘤,其特色為藉由分泌包括纖維母細胞生長因子-23(FGF23)在內的抑磷素而造成低磷血症及腫瘤誘導骨軟化症(tumor-induced osteomalacia, TIO)。FGF23主要作用在腎小管,促進磷酸根排出,進而造成低磷血症,而阻礙類骨質之礦化,致使骨質脆弱、易於骨折,亦即骨軟化症。TIO之成因,以往認為可由多種不同腫瘤造成,但依照目前的認知,絕大部分TIO皆由一種具有特定組織型態之腫瘤類型所引發,亦即PMT;而此概念也獲得最新版之世界衛生組織認可,後者認定PMT為一種特定腫瘤類型。但是,PMT之腫瘤形成分子機制則仍屬未知。
本研究基於「PMT可能為一類基因轉位所造成之腫瘤」的假設,採用次世代核醣核酸(RNA)定序之方法,在4個PMT發現其中三個具有FN1-FGFR1融合基因之RNA表達。此發現經由反轉錄聚合酶連鎖反應證實,且西方墨點法亦證實此融合蛋白之表達。螢光原位雜合進一步在另外11個PMT中發現6個案例有FN1-FGFR1融合基因。整體上,15個PMT中有9個(60%)有此融合基因。本研究進一步納入第二批腫瘤,並使用涵蓋5’端FN1、5’端FGFR1、及3’端FGFR1之三色探針進行螢光原位雜合分析,兩批總共29例得到可判讀之結果,其中17例有5’端FN1與3’端FGFR1之融合;加上RNA定序之結果,仍然有60%(18/30)之腫瘤具有此融合基因。值得注意的是,這17例中有10例其三種顏色之探針訊號重疊或緊鄰,而非FGFR1基因之5’與3’分離之樣態,此現象一方面可能反映出此兩基因之染色體相對位置關係之特殊性,另一方面也警示了使用FGFR1探針進行PMT之分子診斷時必須注意的實務上考量。
本研究之發現,對於PMT之腫瘤形成機制,產生了相當直觀的啟發。FN1基因可能藉由提供其活躍之促進子來過度表達FN1-FGFR1融合蛋白,而其所編碼之蛋白(fibronectin)之寡聚合區域可能有助於C’端之FGFR1酪氨酸激酶區域之交互磷酸化與活化。至於FGFR1則是一個已知的原致癌蛋白,在腫瘤中可經由基因突變、放大、以及轉位融合的方式而活化。不同於FGFR1基因轉位之腫瘤原型(某類血液腫瘤,其FGFR1融合基因皆丟失配體結合區域、且其活化不需要配體),在PMT所發現的FN1-FGFR1融合基因皆保留至少部分之配體結合區域,暗示著其配體(例如PMT大量分泌之FGF23)之存在可能對於FN1-FGFR1融合蛋白之活化具有促進、甚至必要之作用,由此形成一個自分泌或旁分泌的迴圈,此為一個可能且直觀的腫瘤形成機制。此外,證據顯示FGFR1的訊息傳遞可導致FGF23表現量上升,因此此融合蛋白之活化,或可解釋PMT分泌FGF23導致臨床上TIO之表現的機制。
最後,本研究的發現,也可能具有臨床治療上的意義。新發展的FGFR1抑制劑以及FGF23抗體或可藉由阻斷FN1-FGFR1融合蛋白所主導之自分泌迴圈,而被用於治療無法以手術切除之PMT,發揮抑制腫瘤生長及減輕FGF23所致TIO的作用。上述假說皆需進一步實驗證實,並將以回歸於病人之治療作為最終目標。

Phosphaturic mesenchymal tumors (PMT) are distinctive soft tissue and bone tumors that typically cause hypophosphatemia and tumor-induced osteomalacia (TIO) through secretion of phosphatonins such as fibroblast growth factor 23 (FGF23). FGF23 acts on renal tubules and promotes renal wasting of phosphate. The resultant hypophosphatemia then hinders osteoid mineralization and renders the skeletal system fragile and prone to fracture, hence osteomalacia. Although TIO was previously thought to be caused by a variety of different tumor types, it is now understood that PMT underlie the overwhelming majority of cases of TIO. As such, PMT has recently been accepted by the World Health Organization as a formal tumor entity. However, the genetic basis and oncogenic pathways underlying its tumorigenesis remain obscure.
In this study, we employed the next-generation RNA sequencing technique and identified a novel FN1-FGFR1 fusion gene in 3 out of 4 PMTs. The fusion transcripts and proteins were subsequently confirmed with RT-PCR and western blotting analyses, respectively. Fluorescence in situ hybridization (FISH) analysis showed 6 cases with FN1-FGFR1 fusion, out of an additional group of 11 PMTs studied. Overall, 9 out of 15 PMTs (60%) were positive for this fusion. An expanded group of 30 cases were then analyzed with a tri-color 5’-FN1/5’-FGFR1/3’-FGFR1 probe set. Again, 60% (18/30) of PMT harbored FN1-FGFR1 fusion. Of note, 10 of the 17 cases with FISH-confirmed FN1-FGFR1 fusion had the three probes fused together instead of break-apart of the 5’ and 3’ ends of FGFR1, a phenomenon that likely reflected the unusual relative chromosomal orientation of the fusion partners, with practical relevance in molecular diagnosis.
The current findings provide immediate insights into the tumorigenesis of PMT. The FN1 gene probably provides its constitutively active promoter to over-express the FN1-FGFR1 fusion protein, while the oligomerization domains of the encoded protein (fibronectin) may facilitate the activation of the FGFR1 kinase domain. Fibroblast growth factor receptor 1 (FGFR1) is a well-known proto-oncoprotein, which is activated in tumors through mutations, amplifications, and translocations. Interestingly, unlike the prototypical leukemia-inducing FGFR1 fusion genes which are ligand-independent, the FN1-FGFR1 chimeric protein was predicted to preserve its ligand-binding domains, suggesting a requirement for, or an advantage of, the presence of its ligands (such as FGF23 highly secreted by the tumor cells) in the activation of the chimeric receptor tyrosine kinase, thus effecting an autocrine or paracrine mechanism of tumorigenesis. Furthermore, as FGFR1 signaling has been shown to lead to the upregulation of FGF23, the fusion gene may also account for the over-expression of FGF23, hence the clinical manifestation of TIO.
Finally, the current findings may also have therapeutic relevance. Emerging fibroblast growth factor receptor inhibitors and anti-FGF23 antibody may serve to block the FN1-FGFR1 autocrine loop and ameliorate the FGF23-mediated TIO in cases with inoperable PMT. Further studies are warranted to confirm our hypotheses, with an ultimate goal to benefit the patients.

論文口試委員審定書………………………………………………………………. I
誌謝…………………………………………………………………………………. II
中文摘要……………………………………………………………………………. III
英文摘要……………………………………………………………………………. V
Chapter 1 Introduction…..……………………………………………………… 1
Chapter 2 Materials and Methods………………..…………………………….. 5
2.1 Tumor samples………………………………………………….. 5
2.2 RNA sequencing and data analysis……………………………... 6
2.3 PCR and direct sequencing……………………………………... 7
2.4 Western blotting………………………………………………… 8
2.5 FISH analysis…………………………………………………… 8
2.6 Immunohistochemistry………………………………………….. 10
Chapter 3 Results………………………………………………………………... 11
3.1 RNA sequencing analysis: Fusion detection……………………. 11
3.2 RNA sequencing analysis: Expression analysis………………… 12
3.3 PCR verification of the fusion gene…………………………….. 12
3.4 Western blotting confirmation of chimeric protein expression… 13
3.5 FISH analysis characterizing the prevalence of the fusion……... 13
3.6 Immunohistochemistry demonstrating FGFR1 expression in a majority of PMT…………………………………………………
15
Chapter 4 Discussion…………………………………………………………….. 16
4.1 The past knowns and unknowns………………………………... 16
4.2 The present study and findings…………………………………. 16
4.3 The mechanistic insights and hypotheses………………………. 19
4.3.1 Roles of FN1……………………………………………….. 19
4.3.2 Roles of FGFR1……………………………………………. 20
4.3.3 Mechanisms of FGF23 upregulation……………………….. 22
4.3.4 Therapeutic implications…………………………………… 23
4.4 The future work and challenges………………………………… 24
4.4.1 In vitro fusion gene creation and cell transformation assay... 25
4.4.2 Characterization of FGF23 expression in transformed cells.. 27
4.4.3 Sensitivity of the transformed cells to FGF23 stimulation… 28
4.4.4 Response to FGFR inhibitors and anti-FGF23 antibody…... 28
4.4.5 Discovery of alternative/analogous fusions/mutations in cases without FN1-FGFR1 fusion………………………….
29
4.4.6 In vivo experiments………………………………………… 31
Chapter 5 Conclusion…..……………………………………………………….. 32
References ………………..……………………………………………………….. 33

圖目錄

Figure 1 Histological variations of PMT and FGF23 mRNA CISH……….….. 38
Figure 2 The probe maps of the FN1-FGFR1 tricolor probe…………………... 39
Figure 3 Heatmap plot with hierarchical clustering analysis of the 31-gene expression profiles in 4 PMTs and 28 non-PMT tumours……………
40
Figure 4 EdgeR MDS plot and DeSeq PCA plot of PMTs and non-PMTs……. 41
Figure 5 RT-PCR and DNA PCR (confirmation of the fusion gene)………….. 42
Figure 6 Sanger sequencing, western blot, and FISH………………………….. 43
Figure 7 Tricolor FISH………………………………………………………… 44
Figure 8 FGFR1 immunohistochemistry………………………………………. 45
Figure 9 Predicted domains retained in the chimeric protein………………….. 46
Figure 10 Illustration of chromosomal rearrangement in FN1-FGFR1 fusion….. 47
Figure 11 Proposed hypotheses of mechanism of PMT tumorigenesis…………. 48
Figure 12 Strategy of fusion gene cloning………………………………………. 49
Figure 13 Design of CRISPR-mediated genome editing………………………... 50


表目錄

Table 1 Clinical and Molecular findings of the extended group of PMTs……….. 51
Table 2 List of the samples of which the RNA sequencing data were compared... 52
Table 3 Sequences of primers used in RT-PCR and DNA PCR…………………. 53
Table 4 Fusions detected in 3 of the 4 index cases of PMT……………………… 55


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