探讨分化良好的脂肪肉瘤向去分化脂肪肉瘤转化的机制

Atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) can undego a type of malignant progression termed dedifferentiated liposarcoma (DDL), leading to more aggressive behaviour. The aim of this study was to demonstrate the mechanisms responsible for this progression. Three human ALT/WDL cell lines, five human DDL cell lines, and one mouse preadipocytic cell were employed in this study. A vector expressing ASK1 or JUN was transfected into ALT/WDL cells to observe its effect on adpocytic differentiation. The transfection of siRNA knockdown of ASK1 or JUN to ASK1- or JUN- amplified DDL cells was used to evaluate the role on the differentiation state of the neoplastic cells. In addition, we injected DDL cells into ASK1- or JUN- knockout mice, observing the effect on adpocytic differentiation of the tumors. At last, we injected preadipocytic cells into nude mice, evaluating whether the neoplastic cells harboring the high-level amplification of ASK1 gene could be selected. The goal of the study is to provide the clue for targeted gene theray to DDL.

非典型脂肪瘤性肿瘤/分化良好的脂肪肉瘤(atypical lipomatous tumor/well-differentiated liposarcoma,ALT/WDL)可进展为去分化脂肪肉瘤(dedifferentiated liposarcoma,DDL)，失去脂肪分化。本研究目的在于探讨引起这一转化的分子机制。用人DDL细胞5系、人ALT/WDL细胞系3系、小鼠前脂肪细胞系1系作为研究对象。将ASK1或JUN基因转染入ALT/WDL细胞观察ASK1或JUN表达增加是否能抑制脂肪分化；通过RNAi技术建立ASK1及JUN缺失的DDL细胞模型并建立裸鼠移植模型等实验观察抑制ASK1与JUN是否能促进脂肪分化；观察DDL细胞植入ASK1或JUN基因敲除小鼠后移植瘤的脂肪分化情况；将小鼠前脂肪细胞植入裸鼠体内模拟脂肪肉瘤在体内的自然筛选及演进过程。

非典型脂肪瘤性肿瘤/高分化脂肪肉瘤(atypical lipomatous tumor/well-differentiated liposarcoma,ALT/WDL) ，可进展为预后差的去分化脂肪肉瘤(dedifferentiated liposarcoma,DDL)，失去脂肪分化。本研究目的在于探讨引起这一转化的分子机制。主要结果：①对24例DDL及23例ALT/WDL进行CGH array研究 ，发现24例DDL中20例除了有12q13-15区域的扩增以外，还有11例含有1p32区的扩增、9例有6q23区的扩增； 23例ALT/WDL中均无1p32或6q23区的扩增；②对9例 DDL进行SNP/CNV microarray研究发现所有病例（9/9）均有ASK1基因扩增，而无6q23扩增的肿瘤未能检测出该基因的扩增；③用荧光原位杂杂交（fluorescence in situ hybridization, FISH）证实了ASK1在有6q23扩增的DDL（临床样本）的DDL区域（9/9，100%）及ALT/WDL 区域（6/6，100%）和DDL细胞系（DDL6Q）均有高水平扩增；而在单纯的ALT/WDL病例（包括临床样本和细胞系）、缺乏6q23的DDL临床组织样本以及DDL细胞系中ASK1拷贝数正常；④用Real-time PCR、Western Blot和免疫组织化学等方法对肿瘤临床样本和细胞系均进行了检测，结果显示6q23扩增的肿瘤中ASK1存在高水平表达，进一步支持ASK1为6q23区扩增的靶基因并可能为导致脂肪肉瘤失去分化的重要原因；⑤用ASK1抑制剂thioredoxin (TRX)处理DDL细胞，结果显示TRX处理7天后，细胞形态出现了显著改变，细胞内脂质进行性聚集；⑥对100例ALT/WDL及97例DDL进行了FRS2的FISH检测，结果显示ALT/WDL及DDL中共有172例（172/197, 87.3%）出现基因扩增，包括ALT/WDL83例（83/100, 83.0%）和DDL89例（89/97, 91.8%）。本课题的数据提示DDL有向脂肪细胞分化的倾向但最终被阻断，而这一过程很可能与6q23上的ASK1基因的扩增和过度表达有关，因此有深入研究的必要。