2015-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656504摘要：「高磷尿性間質腫瘤」(PMT)是一罕見軟組織及骨腫瘤，其藉由分泌FGF23來造成低磷血症與骨骼軟化症。其致瘤之基因機轉直到最近才有所突破，係由本研究團隊率先以全mRNA次世代定序配合螢光原位雜合(FISH)發現了60% (9/15)的PMT具有FN1-FGFR1融合基因。推測FN1基因可能提供具持續活性的啟動子，及該蛋白的聚合結構域，使3’端FGFR1過度表現並聚合而活化。有趣的是，此融合蛋白具有FGFR1的受體接受部位，似乎暗示FGF (尤其高度表現的FGF23) 的存在將有助於FN1-FGFR1活化，因而形成一個自分泌迴圈。本研究將著手闡釋PMT的致瘤機轉，特別著重FN1-FGFR1的功能角色。我們使用CRISPR/Cas9系統進行基因組編輯，創造FN1與FGFR1之基因融合，試圖將正常人類纖維母細胞癌化；進而在該細胞檢測FGF23之表現，融合蛋白之磷酸化，以及檢測融合蛋白與細胞對於FGF23之刺激是否敏感。另外也將給予FGFR1抑制劑以及anti-FGF23抗體，來測試這些試劑可能的臨床用途。同時，我們收集了總共約70例PMT，將進行FISH來更深入了解FN1-FGFR1存在的比例，並將使用鄰位連接分析法來探索其可能病理診斷用途，尤其用在FISH容易失敗的脫鈣後骨骼檢體。最後，針對40%缺乏FN1-FGFR1融合基因 (但仍常表現FGFR1) 的案例，我們將再次使用次世代定序，包括針對FGFR1-4等基因全長進行深度DNA定序，或者全mRNA定序，來試圖發現新的基因融合或突變。<br> Abstract: Phosphaturic mesenchymal tumors (PMT) are uncommon, distinctive soft tissue and bone tumors that typically cause hypophosphataemia and tumor-induced osteomalacia (TIO) through secretion of phosphatonins such as fibroblast growth factor 23 (FGF23). 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. The genetic basis and oncogenic pathways underlying its tumorigenesis remained obscure until immediately recently, when we first identified a novel FN1-FGFR1 fusion gene in 60% (9/15) PMTs by whole transcriptome sequencing in conjunction with fluorescence in situ hybridization analysis. We have proposed that the FN1 gene possibly provides its constitutively active promoter and the encoded protein’s oligomerization domains to over-express and facilitate the activation of the FGFR1 kinase domain. 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, thereby to achieve an autocrine or paracrine mechanism of tumorigenesis.In this study, the investigators set to elucidate the tumorigenic mechanism of PMT, with emphasis on the functional roles of FN1-FGFR1 gene. Fusion gene is created by CRISPR/Cas9-mediated genome editing in human fibroblasts with an effort to transform the cells. The transformed cells are tested for FGF23 expression, FN1-FGFR1 chimeric protein phosphorylation, and their sensitivity to, as well as the biological consequences of, FGF23 stimulation. With therapeutic implications in mind, FGFR1 inhibitors and an anti-FGF23 antibody are tested for their in vitro anti-tumor potency. Meanwhile, fluorescence in situ hybridization is performed to characterize the fusion gene on an expanded cohort of 60-70 cases, and proximity ligation assay to explore its potential utility as a diagnostic adjuvant (especially in decalcified samples where FISH is prone to failure). Finally, next-generation sequencing, including deep DNA sequencing targeting the full-length FGFR1-4 genes and whole transcriptome sequencing, is conducted to discover alternative fusions/mutations in the roughly 40% cases which lack FN1-FGFR1 fusion (which still usually express FGFR1). This work is expected to substantially redefine our understanding of PMT, from the molecular basis of tumorigenesis to the therapeutic options.高磷尿性間質腫瘤致瘤機轉FN1基因FGFR1基因纖維母細胞生長因子23 (FGF23)基因組編輯次世代定序phosphaturic mesenchymal tumortumorigenesisFN1FGFR1FGF23CRISPRnext-generation sequencing