Mad2 and BubR1 modulates tumourigenesis and paclitaxel response in MKN45 gastric cancer cells

Aneuploidy and chromosomal instability (CIN) are common features of gastric cancer (GC), but their contribution to carcinogenesis and antitumour therapy response is still poorly understood. Failures in the mitotic checkpoint induced by changes in expression levels of the spindle assembly checkpoint (SAC) proteins cause the missegregation of chromosomes in mitosis as well as aneuploidy. To evaluate the possible contribution of SAC to GC, we analyzed the expression levels of proteins of the mitotic checkpoint complex in a cohort of GC cell lines. We found that the central SAC proteins, Mad2 and BubR1, were the more prominently expressed members in disseminated GC cell lines. Silencing of Mad2 and BubR1 in MKN45 and ST2957 cells decreased their cell proliferation, migration and invasion abilities, indicating that Mad2 and BubR1 could contribute to cellular transformation and tumor progression in GC. We next evaluated whether silencing of SAC proteins could affect the response to microtubule poisons. We discovered that paclitaxel treatment increased cell survival in MKN45 cells interfered for Mad2 or BubR1 expression. However, apoptosis (assessed by caspase-3 activation, PARP proteolysis and levels of antiapoptotic Bcl 2-family members), the DNA damage response (assessed by H2Ax phosphorylation) and exit from mitosis (assessed by Cyclin B degradation and Cdk1 regulation) were activated equally between cells, independently of Mad2 or BubR1-protein levels. In contrast, we observed that the silencing of Mad2 or BubR1 in MKN45 cells showed the induction of a senescence-like phenotype accompanied by cell enlargement, increased senescence-associated β-galactosidase activity and increased IL-6 and IL-8 expression. In addition, the senescent phenotype is highly increased after treatment with PTX, indicating that senescence could prevent tumorigenesis in GC. In conclusion, the results presented here suggest that Mad2 and BubR1 could be used as prognostic markers of tumor progression and new pharmacological targets in the treatment for GC.

染色体非整倍体（aneuploidy）与染色体不稳定性（chromosomal instability, CIN）是胃癌（gastric cancer, GC）的常见特征，但二者对肿瘤发生及抗肿瘤治疗应答的贡献仍未得到充分阐明。由纺锤体装配检查点（spindle assembly checkpoint, SAC）蛋白表达水平改变诱导的有丝分裂检查点功能异常，会引发有丝分裂过程中染色体错配分离，进而导致染色体非整倍体。为评估SAC对胃癌的潜在贡献，我们对一组胃癌细胞系的有丝分裂检查点复合物蛋白表达水平进行了分析。研究发现，核心SAC蛋白Mad2与BubR1在播散性胃癌细胞系中呈高表达。在MKN45与ST2957细胞中沉默Mad2与BubR1的表达，会降低细胞的增殖、迁移与侵袭能力，这提示Mad2与BubR1可能参与胃癌的细胞转化与肿瘤进展过程。我们进一步评估了SAC蛋白沉默是否会影响细胞对微管毒素的应答。结果发现，紫杉醇（paclitaxel）处理可提高Mad2或BubR1表达被干扰的MKN45细胞的存活率。然而，细胞凋亡（通过半胱氨酸天冬氨酸蛋白酶-3（caspase-3）活化、多聚ADP核糖聚合酶（PARP）蛋白水解及抗凋亡Bcl-2家族成员水平进行评估）、DNA损伤应答（通过H2Ax磷酸化水平进行评估）以及有丝分裂退出（通过细胞周期蛋白B（Cyclin B）降解与细胞周期蛋白依赖性激酶1（Cdk1）调控水平进行评估）的激活程度在各组细胞间并无显著差异，且不受Mad2或BubR1蛋白水平的影响。与之相反，我们观察到在MKN45细胞中沉默Mad2或BubR1可诱导出类衰老表型，伴随细胞体积增大、衰老相关β-半乳糖苷酶（senescence-associated β-galactosidase）活性升高，以及白细胞介素-6（IL-6）与白细胞介素-8（IL-8）表达上调。此外，经紫杉醇（简称PTX）处理后，这种衰老表型会显著增强，这提示衰老可能抑制胃癌的肿瘤发生。综上，本研究结果表明，Mad2与BubR1可作为胃癌肿瘤进展的预后标志物，同时也可作为胃癌治疗的新型药理学靶点。