Sugimoto H, Nakamura M, Yoda H, Hiraoka K, Shinohara K, Sang M, Fujiwara K, Shimozato O, Nagase H, Ozaki T
Laboratory of DNA Damage Signaling, Chiba Cancer Center Research Institute , Chuou-ku, Chiba, Japan.
Laboratory of Cancer Genetics, Chiba Cancer Center Research Institute , Chuou-ku, Chiba, Japan.
Cell Death Discov. 2015 Aug 10;1:15010. doi: 10.1038/cddiscovery.2015.10. eCollection 2015.
Runt-related transcription factor 2 (RUNX2) has been considered to be one of master regulators for osteoblast differentiation and bone formation. Recently, we have described that RUNX2 attenuates p53/TAp73-dependent cell death of human osteosarcoma U2OS cells bearing wild-type p53 in response to adriamycin. In this study, we have asked whether RUNX2 silencing could enhance gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells. Under our experimental conditions, GEM treatment increased the expression level of p53 family TAp63, whereas RUNX2 was reduced following GEM exposure, indicating that there exists an inverse relationship between the expression level of TAp63 and RUNX2 following GEM exposure. To assess whether TAp63 could be involved in the regulation of GEM sensitivity of AsPC-1 cells, small interfering RNA-mediated knockdown of TAp63 was performed. As expected, silencing of TAp63 significantly prohibited GEM-dependent cell death as compared with GEM-treated non-silencing cells. As TAp63 was negatively regulated by RUNX2, we sought to examine whether RUNX2 knockdown could enhance the sensitivity to GEM. Expression analysis demonstrated that depletion of RUNX2 apparently stimulates the expression of TAp63, as well as proteolytic cleavage of poly ADP ribose polymerase (PARP) after GEM exposure, and further augmented GEM-mediated induction of p53/TAp63-target genes, such as p21 (WAF1) , PUMA and NOXA, relative to GEM-treated control-transfected cells, implying that RUNX2 has a critical role in the regulation of GEM resistance through the downregulation of TAp63. Notably, ablation of TAp63 gave a decrease in number of γH2AX-positive cells in response to GEM relative to control-transfected cells following GEM exposure. Consistently, GEM-dependent phosphorylation of ataxia telangiectasia-mutated protein was remarkably impaired in TAp63 knockdown cells. Collectively, our present findings strongly suggest that RUNX2-mediated repression of TAp63 contributes at least in part to GEM resistance of AsPC-1 cells, and thus silencing of RUNX2 may be a novel strategy to enhance the efficacy of GEM in p53-deficient pancreatic cancer cells.
runt相关转录因子2（RUNX2）被认为是成骨细胞分化和骨形成的主要调节因子之一。最近，我们描述了RUNX2可减弱阿霉素诱导的、携带野生型p53的人骨肉瘤U2OS细胞中p53/TAp73依赖性细胞死亡。在本研究中，我们探讨了RUNX2基因沉默是否能增强p53缺陷型人胰腺癌AsPC-1细胞对吉西他滨（GEM）的敏感性。在我们的实验条件下，GEM处理可增加p53家族TAp63的表达水平，而GEM处理后RUNX2表达降低，这表明GEM处理后TAp63和RUNX2的表达水平呈负相关。为评估TAp63是否参与AsPC-1细胞对GEM敏感性的调节，我们进行了小干扰RNA介导的TAp63基因敲低实验。正如预期的那样，与GEM处理的非沉默细胞相比，TAp63基因沉默显著抑制了GEM依赖性细胞死亡。由于TAp63受RUNX2负调控，我们试图研究RUNX2基因敲低是否能增强对GEM的敏感性。表达分析表明，RUNX2基因缺失明显刺激了TAp63的表达，以及GEM处理后聚ADP核糖聚合酶（PARP）的蛋白水解切割，相对于GEM处理的对照转染细胞，GEM介导的p53/TAp63靶基因如p21（WAF1）、PUMA和NOXA的诱导进一步增强，这意味着RUNX2通过下调TAp63在GEM耐药调节中起关键作用。值得注意的是，与GEM处理后对照转染细胞相比，TAp63缺失使GEM诱导的γH2AX阳性细胞数量减少。一致地，TAp63基因敲低细胞中GEM依赖性的共济失调毛细血管扩张突变蛋白磷酸化明显受损。总的来说，我们目前的研究结果强烈表明，RUNX2介导的TAp63抑制至少部分促成了AsPC-1细胞对GEM的耐药性，因此RUNX2基因沉默可能是增强GEM对p53缺陷型胰腺癌细胞疗效的一种新策略。
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