探讨FRS2α基因扩增在高分化脂肪肉瘤/去分化脂肪肉瘤中的作用

Well-differentiated liposarcoma/dedifferentiated liposarcoma (WDLS)/ (WDLS/DDLS) are the most common types of liposarcoma, characterized by amplified or overexpressed genes within chromosome 12q13-15, including MDM2, CDK4, and others. Our previous study showed that FRS2α gene is also consistently amplified in WDLS/DDLS and FRS2α signaling should be further investigated as a potential therapeutic target for liposarcoma. The aim of this study was to demonstrate the mechanisms of the amplification of FRS2α, activation of FRS2α/PI3K/Akt and FRS2α/Ras/Erk in WDLS/DDLS. Six human DDLS cell lines, three human WDLS cell lines, one human preadipocytic cell line, and one mouse preadipocyte cell line were employed in this study. The transfection of siRNA knockdown of FRS2α,FGFR,PI3K,Erk to WDLS/DDLS cells was used to evaluate the role on the growth state and adpocytic differentiation of the neoplastic cells. Knockdown of FRS2α,FGFR,PI3K,Erk to WDLS/DDLS cells by TALEN method was used to evaluate the role on the growth state and adpocytic differentiation of the neoplastic cells. A vector expressing FRS2α was transfected into preadipocytes to observe its effect on tumor formation.

高分化脂肪肉瘤/去分化脂肪肉瘤(well-differentiated liposarcoma/dedifferentiated liposarcoma,WDLS/DDLS) 是最常见的一类脂肪肉瘤类型。本研究的目的在于研究FRS2α基因扩增在WDLS/DDLS中的调控机制，以探讨FRS2α 能否成为其潜在靶点。本研究用人DDLS细胞6系、人WDLS细胞系3系、人及小鼠前脂肪细胞系各1系作为研究对象。通过RNAi技术建立FRS2α、FGFR、PI3K、Erk缺失的WDLS/DDLS细胞模型并建立裸鼠移植模型等实验观察是否能抑制肿瘤生长并促进脂肪分化；通过TALEN技术敲除肿瘤细胞内的FRS2α、FGFR、PI3K、Erk等基因并建立裸鼠移植模型等实验观察是否能抑制肿瘤生长并促进脂肪分化；将FRS2α基因转染入前脂肪细胞观察FRS2α过度表达增加是否能促进肿瘤形成。

高分化脂肪肉瘤/去分化脂肪肉瘤(well-differentiated liposarcoma/dedifferentiated liposarcoma,WDL/DDL) 是最常见的脂肪肉瘤类型。本研究的目的在于研究FRS2α基因在WDL/DDL中是否存在稳定扩增及其在该类肿瘤发生发展中的调控机制，以探讨FRS2α 能否成为该类脂肪肉瘤的潜在靶点。主要结果：①对40例WDL/DDL进行SNP/CNV microarray研究，发现WDL/DDL在12q15上存在2个稳定扩增的区域，一个扩增区域涵盖MDM2基因，另一个区域涵盖FRS2基因，所有脂肪瘤与正常脂肪组织均未检出该区域的扩增；②用荧光原位杂交（fluorescence in situ hybridization, FISH）证实了FRS2在WDL/DDL（136/146, 93%）的肿瘤组织及细胞株(6/6)中均有高水平扩增，而脂肪瘤及其他肿瘤中FRS2基因拷贝数正常；③Real-time PCR、Immunoblotting及免疫组化结果显示FRS2（163/182，90%）及phospho-FRS2（168/182，92%）高表达于WDL/DDL肿瘤组织；④免疫组化结果示Akt（148/182，81%）及Erk（166/182，91%）高表达于WDL/DDL肿瘤组织；⑤用FGFR抑制剂NVP-BGJ-398处理DDL细胞株，结果显示处理后其相应的下游分子FRS2α、PI3K、Erk等在RNA及蛋白水平也显著下调，肿瘤细胞生长受到显著抑制；⑥分别通过ShRNA与CRISPR/CAS技术敲除肿瘤细胞内的FRS2α、PI3K、Erk等基因并建立裸鼠移植模型等实验观察，发现肿瘤生长受到显著抑制，其相应的下游分子在RNA及蛋白水平也显著下调；⑦将FRS2α基因转染入前脂肪细胞，发现FRS2α过度表达能促进肿瘤形成。本课题组的研究结果提示在WDL/DDL这类肿瘤中除了存在MDM2与CDK4等基因的高水平扩增以外，还存在FRS2基因的稳定扩增，FRS2α/PI3K/Akt通路和/或FRS2α/Ras/Erk通路的激活在该类肿瘤的发生发展中起着重要作用。系统研究FRS2α基因扩增以及其下游通路在该类肿瘤中的作用机制对于该类肿瘤的辅助诊断、特别是靶向治疗具有重要意义。