中国科学院昆明动物研究所机构知识库

三阴性乳腺癌(TNBC)是一类恶性程度高的乳腺癌。相对于其他类型的乳腺癌，TNBC呈现转移率高，复发率高与预后差等特征。目前缺乏特效的靶向药物来治疗TNBC。可变剪接是指真核生物基因转录pre-mRNA之后，内含子被切除，外显子通过不同的组合方式拼接，并形成可以编码不同蛋白产物的mRNA的过程。体内异常的剪接已成为肿瘤发生发展潜在的标志物。我们的研究发现，相比于其他乳腺癌分型，TNBC呈现特殊的剪接表达谱。进一步研究发现，作为神经性退行性疾病中关键的病理性蛋白，TDP43能作为TNBC特异性剪接谱的主要调控因子。生物信息分析与临床数据发现，TDP43在TNBC中高表达，而且其高表达与病人预后差相关。在TNBC细胞系中，敲低TDP43抑制细胞生长与迁移。同时，过表达TDP43将促进永生化细胞生长与恶性程度。蛋白质谱与免疫共沉淀实验发现，TDP43能特异性与另一个剪接因子，SRSF3结合。转录组测序分析与体外报告载体实验显示TDP43与SRSF3形成的复合物能协同地调控TNBC细胞中可变剪接。类似于TDP43，敲降SRSF3同样能TNBC肿瘤进程；同时敲降这两个剪接因子将更大程度地抑制细胞生长与迁移。为了明晰TDP43与SRSF3如何调控乳腺癌进程，我们结合生物信息分析与文献报道鉴定了两个下游基因(PAR3和NUMB)的剪接事件。功能回补实验显示，敲低TDP43或SRSF3均能抑制PAR3外显子12的排除(△12PAR3)。在敲低TDP43 或 SRSF3细胞中，过表达PAR3外显子12排除形式的转录本能部分回补细胞转移能力。我们的结果揭示TNBC存在特异性剪接模式，这种剪接模式及其主要调控因子将可能作为TNBC潜在的治疗靶点。

Triple negative breast cancer (TNBC) is one breast cancer subtype with high degree of malignancy. Compared with other breast cancer subtypes, TNBC has the characteristics of high metastasis rate, high recurrence risk, and poor prognosis. Currently, there remain no specific targeted drugs for treatment of TNBC. Alternative splicing (AS) is a critical process during post-transcription in which exons from a single gene are assembled in different ways to produce several protein isoforms in eukaryotic organisms. Aberrant alternative splicing has been highlighted as a potential hallmark of cancer. Here, we found that TNBC display a unique alternative splicing pattern in comparison with those of other breast cancers. In analyzing the underlying mechanism of the distinct alternative splicing profile, TDP43, a critical gene previously implicated in neurodegenerative disease, could function as an important splicing regulator responsible for the unique splicing profile in TNBC. Further bioinformatics analysis and clinical data found that TDP43 is highly expressed in TNBC and its high expression is positively in correlation with poor prognosis. Knockdown of TDP43 inhibits tumor progression, including proliferation and metastasis both in vitro and in vivo, and overexpression of TDP43 promotes proliferation and malignancy of mammary epithelial cells. We also perform immunoprecipitation and mass spectrometry (IP-MS) and protein domain truncation assays and show that TDP43 interacts with another splicing factor, SRSF3. RNA immunoprecipitation sequencing (RIP-seq) and minigene reporter assay demonstrate that TDP43 collaborates with SRSF3 in the regulation of AS. Similar with TDP43, knockdown of SRSF3 also inhibit the progression of TNBC in vitro and in vivo. A more noticeable reduction in both cell growth and migration was observed upon the down-regulation of both TDP43 and SRSF3 than with the down-regulation of either TDP43 or SRSF3 alone. After screening the downstream splicing events of these two splicing factors, we focused on the roles of PAR3 and NUMB in TDP43- and SRSF3-dependent cancer progression. The effect of reduced metastasis upon the knockdown of TDP43 or SRSF3 is mediated by the splicing regulation of PAR3. The TDP43/SRSF3 complex and downstream PAR3 isoform are potential therapeutic targets for TNBC.