融合基因AARS-MADx诱导基因组不稳定性及参与鼻咽癌发生的机制

Highly recurrent and cancer specific fusion genes have defined distinct molecular subtypes linked to unique clinicopathological features and become ideal drug targets. Fusion genes resulting from chromosomal rearrangements are critically involved in the molecular pathogenesis not only of hematological malignancies and sarcomas, but also of epithelial cancers. To screen and characterization fusion genes in nasopharyngeal carcinomas (NPC), we performed whole-transcriptome analysis by paired-end sequencing (RNA-seq) and discovered a novel fusion gene AARS-MADx in a NPC cell line C666. In the subsequent evaluation, we demonstrated that this fusion gene was recurrently occurred in NPC samples with up to 16% positive rate in 32/200 samples detected. AARS-MADx fusion gene encoded a 37 KD protein. As functional consequences, overexpression of AARS-MADx fusion gene could induce genomic instability such as increase number of micronuclei and enhance the colony formation ability of tumor cells .Thus, we propose to further identify the fusion events at chromosome level by FISH, to detect the endogenous protein level by IHC or O-Link methods in NPC cell lines and biopsies as well as the potential clinical significance in AARS-MADx positive samples, and to characterize the molecular mechanism of AARS-MADx on genomic instability and tumorigenesis of NPC. Our results will provide a molecular basis for further development of molecular diagnosis and therapy by targeting AARS-MADx in NPC.

肿瘤特异的融合基因是理想的分子诊断标志和治疗靶标，如BCR-ABL及EML-ALK。近来发现染色体重排产生的融合基因不仅在血液肿瘤及软组织肉瘤发生中起关键作用，而在部分上皮性癌中也可能起驱动作用。我们通过高通量转录组测序（RNA-seq）和生物信息学分析，在鼻咽癌细胞株C666-1中发现新的融合基因AARS-MADx。前期研究表明AARS-MADx具有肿瘤特异性，在高达16% (32/200)鼻咽癌标本中检测到其mRNA，AARS-MADx编码-37KD蛋白，外源性表达可诱导癌细胞出现基因组不稳定性表型如微核和双核的形成，并增强癌细胞克隆形成能力。因此，本项目将明确鼻咽癌组织中AARS-MADx基因融合（FISH检测）和内源性的蛋白表达（IHC或O-LINK）并探讨其临床意义，阐明AARS-MADx诱导基因组不稳定性及其致癌机制，为发展AARS-MADx作为鼻咽癌分型和治疗靶标提供依据。

通过该项目的研究，我们明确了融合基因RARS-MAD1L1在10.03%鼻咽癌和10.7%头颈肿瘤中检测率，同时在DNA水平和蛋白水平分别证实了融合基因RARS-MAD1L1的存在。过表达RARS-MAD1L1能够增强鼻咽癌的增殖、克隆形成能力和干细胞marker的表达，侧群细胞比例以及诱导放化疗的抵抗，和加剧诱导基因组不稳定。通过免疫沉淀和质谱鉴定出RARS-MAD1L1的相互作用蛋白AIMP2，它与RARS-MAD1L1相互作用，降低了FUBP1的泛素化，促进FUBP1在c-Myc启动子的结合，增强c-Myc的表达。敲低FUBP1或抑制c-Myc均能降低RARS-MAD1L1所诱导的“干性”特征。RARS-MAD1L1阳性的头颈肿瘤病人干细胞marker的表达增强。RARS-MAD1L1转基因鼠的皮肤瘤成瘤率均高于对照组，且RARS-MAD1L1转基因鼠的皮肤瘤中FUBP1和c-Myc的表达量均增高。因此，RARS-MAD1L1可能通过FUBP1/c-Myc来发挥其促进“干性”的功能。此外，通过本项目支持，我们发现包括鼻咽癌在内的多种肿瘤中存在FGFR3-TACC3融合基因，并阐明该融合基因在肿瘤发生发展中的驱动作用和机制；发现并阐明多个参与鼻咽癌发生发展的肿瘤相关基因及其作用机制；通过微病灶多点取样全基因分析，发现食管癌癌前病变和癌组织具有高度异质性，并鉴定癌变早期的基因组改变特征，为食管癌早期干预提供依据；利用大数据库，研发了鼻咽癌预后新模型，为指导鼻咽癌精准治疗奠定基础。