Division of Drug Discovery and Safety, LACDR, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
J Exp Clin Cancer Res. 2021 Mar 1;40(1):82. doi: 10.1186/s13046-021-01863-4.
Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic opportunities. Recently, splicing factors have gained attention as potential targets for cancer treatment. Here we systematically evaluated the role of RNA splicing factors in TNBC cell proliferation.
In this study, we performed an RNAi screen targeting 244 individual splicing factors to systematically evaluate their role in TNBC cell proliferation. For top candidates, mechanistic insight was gained using amongst others western blot, PCR, FACS, molecular imaging and cloning. Pulldown followed by mass spectrometry were used to determine protein-protein interactions and patient-derived RNA sequencing data was used relate splicing factor expression levels to proliferation markers.
We identified nine splicing factors, including SNRPD2, SNRPD3 and NHP2L1, of which depletion inhibited proliferation in two TNBC cell lines by deregulation of sister chromatid cohesion (SCC) via increased sororin intron 1 retention and down-regulation of SMC1, MAU2 and ESPL1. Protein-protein interaction analysis of SNRPD2, SNRPD3 and NHP2L1 identified that seven out of the nine identified splicing factors belong to the same spliceosome complex including novel component SUN2 that was also critical for efficient sororin splicing. Finally, sororin transcript levels are highly correlated to various proliferation markers in BC patients.
We systematically determined splicing factors that control proliferation of breast cancer cells through a mechanism that involves effective sororin splicing and thereby appropriate sister chromatid cohesion. Moreover, we identified SUN2 as an important new spliceosome complex interacting protein that is critical in this process. We anticipate that deregulating sororin levels through targeting of the relevant splicing factors might be a potential strategy to treat TNBC.
三阴性乳腺癌（TNBC）是一种侵袭性的乳腺癌亚型，治疗选择有限。最近，剪接因子作为癌症治疗的潜在靶点受到关注。在这里，我们系统地评估了 RNA 剪接因子在 TNBC 细胞增殖中的作用。
在这项研究中，我们针对 244 个单独的剪接因子进行了 RNAi 筛选，以系统地评估它们在 TNBC 细胞增殖中的作用。对于顶级候选物，我们通过 Western blot、PCR、FACS、分子成像和克隆等方法获得了机制上的见解。下拉实验结合质谱分析用于确定蛋白质-蛋白质相互作用，并且利用患者衍生的 RNA 测序数据将剪接因子表达水平与增殖标志物相关联。
我们鉴定了九个剪接因子，包括 SNRPD2、SNRPD3 和 NHP2L1，其中这些因子的耗竭通过增加 sororin 内含子 1 的保留和下调 SMC1、MAU2 和 ESPL1 来破坏姐妹染色单体的粘连（SCC），从而抑制了两种 TNBC 细胞系的增殖。SNRPD2、SNRPD3 和 NHP2L1 的蛋白质-蛋白质相互作用分析表明，在鉴定出的九个剪接因子中，有七个属于同一个剪接体复合物，包括对 sororin 有效剪接至关重要的 novel 成分 SUN2。最后，sororin 转录本水平与 BC 患者的各种增殖标志物高度相关。
我们通过涉及有效 sororin 剪接和适当的姐妹染色单体粘连的机制，系统地确定了控制乳腺癌细胞增殖的剪接因子。此外，我们确定 SUN2 是一个重要的新剪接体复合物相互作用蛋白，在这个过程中是关键的。我们预计通过靶向相关剪接因子来调节 sororin 水平可能是治疗 TNBC 的一种潜在策略。
